Methods for culturing primary cancer cells in brain organoids

Abstract

Disclosed herein are methods of producing organoids. In some aspects, the methods involve producing organoids by implanting primary cancer cells such as glioblastoma cells into brain organoids, thereby producing brain cancer organoids. The method can comprise an incubation step in which the brain organoid is incubated with primary cancer cells in a hanging liquid droplet to produce the brain cancer organoid. In some aspects, the brain cancer organoids provide tools for understanding and treating cancers of the nervous system, such as glioblastoma.

Description

METHODS FOR CULTURING PRIMARY CANCER CELLS IN BRAINORGANOIDS

[0001] This application claims priority of U.S. Provisional Patent Application No. 63 / 640,382, filed April 30, 2024, which is hereby incorporated by reference in its entirety.

[0002] The application contains a Sequence Listing in compliance with ST.26 format and is hereby incorporated by reference in its entirety. Said Sequence Listing, created on April 30, 2025 is named UCLAP0195WO.xml and is 6,333 bytes in size.FIELD

[0003] The present invention generally relates to methods for culture and analysis of cancer cells.BACKGROUND

[0004] Glioblastoma is an aggressive form of cancer and the most common primary malignant brain cancer in adults. Glioblastomas recur after surgery, and the ability of glioblastoma cells to migrate through brain tissue to distant regions outside of the tumor bulk is thought to confer resistance to standard-of-care treatment by impeding complete surgical resection. There is a need to develop improved methods of culturing and analyzing glioblastomas and other cancers in order to inform improved methods of treatment. Provided herein are solutions to these and other needs.SUMMARY

[0005] Culturing cancer cells in vitro offers the opportunity to gain insight into mechanisms of disease and potential treatments. Organoids provide a venue to culture and study cancer cells in vitro while recapitulating aspects of the natural in vivo tumor environment. Despite these advantages, improved methods of producing organoids that contain cancer cells are needed. For example, methods for improving the efficiency with which primary cancer cells can be directly incorporated into organoids are needed.

[0006] In some aspects, the present disclosure relates to methods of providing an organoid. In some aspects, the method comprises incubating a brain organoid and primary cancer cells in a hanging liquid droplet, wherein the primary cancer cells become attached to and / or incorporated into the brain organoid to form a brain cancer organoid (i.e. a brain organoid containing cancer cells). The incubation in the hanging liquid droplet can provide an efficient and cost-effective means to introduce cancer cells (e.g. primary cancer cells) directly into thebrain organoid. Also provided are organoids produced by any of the methods provided herein. In some aspects, the brain organoids can be used to monitor, study, and / or analyze the cancer. The brain organoids can also be used to screen candidate agents in order to identify agents with anti-cancer activity, such as agents that reduce metastasis, proliferation, growth, invasion, and / or migration, of cancer cells. Thus, the methods provided herein provide superior methods for producing brain cancer organoids and studying cancer cells in vitro, in comparison to certain existing methods.

[0007] Also provided herein are methods of analyzing migration of target cells, such as cancer cells, in a fused organoid comprising a first and second organoid. The methods can provide advantageous conditions for analyzing migration of the target cells and for identifying conditions and agents that can affect the migration.

[0008] In some aspects, provided herein is a method for providing an organoid, the method comprising: contacting a brain organoid with cancer cells in suspension to form a liquid droplet containing the brain organoid and cancer cells; and incubating the liquid droplet on a solid surface for a first period of time during which the liquid droplet is a hanging liquid droplet that adheres to and hangs beneath the solid surface; wherein the cancer cells become attached to and / or incorporated into the brain organoid to form a brain cancer organoid.

[0009] Also provided is a brain organoid comprising neurons and cancer cells, wherein the organoid comprises neuron-cancer cell synapses. The brain organoid may comprise colocalization of presynaptic and postsynaptic molecules and / or cells. Presynaptic molecules include SYN1 and VGLUT1. Postsynaptic molecules include PSD95 and GLUA4.

[0010] The suspension may comprise a first cell culture medium. The liquid droplet can be 10-50 microliters (pL) in volume. The droplet may be, may be at least, or may be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, or 200 pL in volume, or any derivable range therein.

[0011] The cancer cells in suspension can be provided at any suitable concentration for use in the method. For example, the cancer cells in suspension, prior to being contacted with the organoid, can be provided at a concentration of between about O.lxlO6cells / mL to about 500xl06cells per mL. The concentration of cancer cells in suspension can be at least, at most, or at or about O.lxlO6, 0.2xl06, 0.3xl06, 0.4xl06, 0.5xl06, 0.6xl06, 0.7xl06, 0.8xl06, 0.9xl06, IxlO6, 2xl06, 3xl06, 4xl06, 5xl06, 6xl06, 7xl06, 8xl06, 9xl06, 10xl0615xl06, 20xl06, 25xl06, 30xl06, 35xl06, 40xl06, 45xl06, 50xl06, 55xl06, 60xl06, 65xl06, 70xl06, 75xl06, 80xl06, 85xl06, 90xl06, 95xl06, lOOxlO6, 150xl06, 200xl06, 250xl06, 300xl06, 350xl06, 400xl06, 450xl06, 500xl06, or more, cells / mL, or any derivable range therein. The cancer cells can be provided at a concentration of approximately 50xl06cells per mL.

[0012] Any suitable volume of cancer cells in suspension can be contacted with the brain organoid. For example, the brain organoid can be contacted with at least, at most, or at or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,198, 199, or 200 pL of cancer cells in suspension.

[0013] The number of cancer cells in the brain cancer organoid, or the number of target cells in the first organoid or fused organoid, can be any suitable number of cells, such as between 100 cells to 10xl06cells. The number of cancer cells in the brain cancer organoid, or the number of target cells in the first organoid or fused organoid, can be at least, at most, or at or about, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, O.OlxlO6, 0.02xl06, 0.03xl06, 0.04xl06, 0.05xl06, 0.06xl06, 0.07xl06, 0.08xl06, 0.09xl06, O.lxlO6, 0.2xl06, 0.3xl06, 0.4xl06, 0.5xl06, 0.6xl06, 0.7xl06, 0.8xl06, 0.9xl06, IxlO6, 2xl06, 3xl06, 4xl06, 5xl06, 6xl06, 7xl06, 8xl06, 9xl06, or 10xl06, cells, or any derivable range therein. The number of cancer cells in the brain cancer organoid, or the number of target cells in the first organoid or fused organoid, can be approximately 500,000 cells.

[0014] The total number of cells in the organoid can be any suitable number of cells. For example, the organoid can comprise between about 1000 cells to about lOOxlO6cells. The organoid can comprise at least, at most, or at or about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, O.OlxlO6, 0.02xl06, 0.03xl06, 0.04xl06, 0.05xl06, 0.06xl06, 0.07xl06, 0.08xl06, 0.09xl06, O.lxlO6, 0.2xl06, 0.3xl06, 0.4xl06, 0.5xl06, 0.6xl06, 0.7xl06, 0.8xl06, 0.9xl06, IxlO6, 2xl06, 3xl06, 4xl06, 5xl06, 6xl06, 7xl06, 8xl06, 9xl06, 10xl06, 20xl06, 30xl06, 40xl06, 50xl06, 60xl06, 70xl06, 80xl06, 90xl06, or lOOxlO6cells, or any derivable range therein.

[0015] The percentage of cancer cells and / or target cells in the organoid can be any suitable percentage. For example, the percentage can be between 0.001% and about 50%. The percentage can be at least, at most, or at or about, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%, or more, or any derivable range therein.

[0016] Any two populations of cells can be provided at any suitable ratio. For example, the cancer cells and brain organoid cells may be present at any ratio. The target cells and first organoid cells may be present at any ratio. The target cells and fused organoid cells may be present at any ratio. The ratio of any of the foregoing may be any suitable ratio, for example, a ratio of about 10000: 1, 1000: 1, 100: 1, 80: 1, 40: 1, 20: 1, 10: 1, 5: 1, 1 : 1, 1 :5, 1 : 10, 1 :20, 1 :40, 1 :80, 1 : 100, 1 : 1000, or 1 : 10000 or any range derivable therein.

[0017] The duration of the first period of time can be 8-12 hours. The duration of the first period of time can be at least, at most, or at or about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or any derivable range therein. The cancer cells can comprise at least 5%, at least 10%, or at least 15% of the cells in the brain cancer organoid. At the end of the first period of time, the cancer cells can comprise at least 5%, at least 10%, or at least 15% of the cells in the brain cancer organoid. At least 5%, at least 10%, or at least 15% of the cells in the brain cancer organoid can be cancer cells or be derived from the cancer cells. At least 5%, at least 10%, or at least 15% of the cells in the brain cancer organoid can be cancer cells or can be derived from the cancer cells after the first period of time. A greater number and / or proportion of the cancer cells can become attached to and / or incorporated into the brain organoid in comparison to a control method in which the brain organoid and cancercells are not incubated in a hanging liquid droplet, and wherein the control method is the same as the non-control method with the exception that during the first period of time the liquid droplet on the solid surface is not a hanging liquid droplet. In the control method, during the first period of time, the liquid droplet can rest on top of the solid surface. In the control method, during the first period of time, the solid surface can be a well. For example, the well can be a flat-bottom, u-bottom, or v-bottom culture well. The method can further comprise or exclude transferring the brain cancer organoid to a culture condition and culturing the brain cancer organoid for a second period of time. In the culture condition, the brain cancer organoid does not need to be cultured in a hanging liquid droplet. In the culture condition, the brain cancer organoid can be submerged in media, such as a second cell culture medium, on a low adhesion substrate. The cancer cells can remain viable for at least 1 week after formation of the brain cancer organoid. The cancer cells can remain viable for at least 1, 2, 3, 4, 5, 6, 7, or more days after formation of the brain cancer organoid. The cancer cells can remain viable for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more weeks, after formation of the brain cancer organoid. The method can comprise or exclude monitoring the brain cancer organoid for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks, or longer, after formation of the brain cancer organoid. The method can comprise or exclude analyzing one or more characteristics of cells in the brain cancer organoid at least one week after formation of the brain cancer organoid, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more weeks after formation of the brain cancer organoid. The method can comprise or exclude analyzing one or more characteristics of the cancer cells in the brain cancer organoid at least one week after formation of the brain cancer organoid. The one or more characteristics can comprise or exclude growth, proliferation, survival, gene expression, invasion, and / or migration. The method can comprise or exclude determining growth of the cancer cells in the brain cancer organoid at least one week after formation of the brain cancer organoid, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more weeks after formation of the brain cancer organoid. The method can comprise or exclude determining the invasiveness of the cancer cells in the brain cancer organoid at least one week after formation of the brain cancer organoid, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more weeks after formation of the brain cancer organoid. The determining can comprise or exclude assessing cells expressing a marker specific for the cancer cells. The marker specific for the cancer cells can be a fluorescent protein.

[0018] The cell culture medium, first cell culture medium, and / or second cell culture medium may facilitate neuron-tumor synapses or neuron-cancer cell synapses. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise or excludeneuroligin-3 (NLGN3) nucleic acid or protein. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise or exclude brain-derived neurotrophic factor (BDNF) nucleic acid or protein. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise or exclude GSK2879552. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise or exclude EPZ-5676. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise or exclude Bay K 8644. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise or exclude N-methyl-d- aspartate (NMD A). The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise neuroligin-3 (NLGN3) nucleic acid or protein, brain-derived neurotrophic factor (BDNF) nucleic acid or protein, GSK2879552, EPZ-5676, Bay K 8644, and N-methyl-d-aspartate (NMD A). The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise or exclude soluble NLGN3 protein and / or soluble BDNF protein. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise or exclude DMEM. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise or exclude F-12. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise or exclude GlutaMax. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise or exclude N-2 supplement. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise or exclude lipids. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise or exclude anti-microbial components. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise or exclude Primocin. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise or exclude serum. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise or exclude heparin. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise or exclude Matrigel. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise or exclude B-27 supplement.

[0019] The organoid may be characterized by the number of pre and post-synaptic markers that co-localize with cancer cells. The fraction of cancer cells co-localizing with pre- and post- synaptic markers may be 0.01-0.1. The median of the fraction of cancer cells co-localizing with pre- and post-synaptic markers may be 0.01-0.1. The fraction of cancer cells or median of the fraction of cancer cells co-localizing with pre- and post-synaptic markers may be, may be at least, or may be at most 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13,0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1, or any derivable range therein.

[0020] The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise heparin at a concentration of 4-6 pg / ml. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise heparin at a concentration of 5 pg / mL. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise heparin at a concentration of, of at least, or of at most 1,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4,5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8,9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6,11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3,13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15,15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7,16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4,18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 pg / mL or ng / mL or mg / mL or any range derivable therein.

[0021] The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise Matrigel at a concentration of 0.5-2% vol / vol. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise Matrigel at a concentration of 1% vol / vol. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise Matrigel at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 % v / v, or any derivable range therein.

[0022] The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise GSK2879552 at a concentration of 0.5-3 pM. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise GSK2879552 at a concentration of 1 pM. The cell culture medium, first cell culture medium, and / or second cellculture medium may comprise GSK2879552 at a concentration of, of at least, or of at most 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4,5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8,9.9, or 10 pM or nM or mM, or any derivable range therein.

[0023] The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise EPZ-5676 at a concentration of 0.5-3 pM. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise EPZ-5676 at a concentration of 1 pM. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise EPZ-5676 at a concentration of, of at least, or of at most 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4,3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 pM or nM or mM, or any derivable range therein.

[0024] The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise NMDA at a concentration of 0.5-3 pM. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise NMDA at a concentration of 1 pM. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise NMDA at a concentration of, of at least, or of at most 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4,3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 pM or nM or mM, or any derivable range therein.

[0025] The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise Bay K 8644 at a concentration of 0.5-3 pM. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise Bay K 8644 at a concentration of 1 pM. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise Bay K 8644 at a concentration of, of at least, or of at most 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4,3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 pM or nM or mM, or any derivable range therein.

[0026] The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise BDNF at a concentration of 1-100 ng / mL. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise BDNF at a concentration of 10 ng / mL. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise BDNF at a concentration of, of at least, or of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, or 200 ng / mL, or ug / mL, or ng, or ug (or any derivable range therein).

[0027] The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise NLGN3 at a concentration of 10-500 ng / mL. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise NLGN3 at a concentration of 100 ng / mL. The cell culture medium, first cell culture medium, and / or second cell culture medium may comprise NLGN3 at a concentration of, of at least, or of at most 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, or 300 ng / mL, or ug / mL, or ng, or ug (or any derivable range therein).

[0028] The method can further comprise or exclude screening a candidate agent for activity in reducing cancer cell growth, proliferation, survival, and / or invasion, wherein the method comprises culturing the brain cancer organoid in the presence of the candidate agent, and wherein reduced cancer cell growth, proliferation, survival, and / or invasion as compared to the cancer cell growth, survival, proliferation, and / or invasion, respectively, in the absence of the candidate agent identifies the candidate agent as having activity in reducing cancer cell growth, proliferation, survival, and / or invasion. The activity can comprise or exclude activity in reducing cell growth, proliferation, survival, and / or invasion of the cancer cells. The activity can comprise or exclude activity in reducing cell growth, proliferation, survival, and / or invasion of cancer in a subject. The candidate agent can comprise or exclude a small molecule, oligonucleotide, antibody, peptide, or protein.

[0029] The cancer cells can be from a subject. The cancer cells can be obtained from a biological sample from a subject that has cancer. The biological sample can comprise or exclude a resected tumor, a biopsy, a needle aspirate, a section, and / or a fluid, or a component of any of the foregoing. The subject can be a human subject. The cancer cells can comprise primary cancer cells. The cancer cells can comprise glioblastoma cells. The cancer cells can be from a tumor. The subject can have a cancer of the nervous system. The cancer cells can be from a cancer of the nervous system. The cancer of the nervous system can comprise or exclude brain stem glioma, pineal astrocytic tumor, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglial tumor, mixed glioma, ependymal tumor, medulloblastoma, pineal parenchymal tumor, meningeal tumor, germ cell tumor, or craniopharyngioma. The cancer of the nervous system can be glioblastoma. The glioblastoma can comprise or exclude neural, proneural, classical, or mesenchymal glioblastoma. The cancer of the nervous system can be astrocytoma. The cancer cells can be obtained by resecting a tumor and dissociating the tumor to provide dissociated cancer cells in suspension. Prior to being contacted with the brain organoid, the cancer cells may not have been: cultured; plated; trypsinized; passaged; expanded; and / or incubated in suspension or in two-dimensional culture under conditions for proliferation and / or expansion. The cancer cells may be cells that have notbeen frozen prior to being contacted with the brain organoid. The cancer cells may be cells that have been frozen prior to being contacted with the brain organoid. The cancer cells may be ones that are not immortalized. The cancer cells may be ones that have not been immortalized in vitro.

[0030] The cancer cells can be contacted with the brain organoid within 48 hours of obtaining the cancer cells from a subject. The cancer cells can be contacted with the brain organoid within 24 hours of obtaining the cancer cells from a subject. The cancer cells can be contacted with the brain organoid within 6 hours of obtaining the cancer cells from a subject. The cancer cells can be contacted with the brain organoid within 2 hours of obtaining the cancer cells from a subject. The cancer cells can be contacted with the brain organoid within 1 hour of obtaining the cancer cells from a subject. The amount of time between obtaining the cancer cells from a subject and contacting the cancer cells with the organoid can be less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours.

[0031] The brain organoid can be generated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). The brain organoid can be generated from human embryonic stem cells (ESCs). The brain organoid can be generated from human induced pluripotent stem cells (iPSCs). In some embodiments, the cells of the brain organoid are not genetically modified. The cells of the brain organoid and / or the cancer cells can be human cells. The brain organoid cells and cancer cells can be from the same subject or from different subjects. The brain organoid can be a cortical organoid. In some aspects, provided herein is an organoid generated according to any of the methods provided herein.

[0032] In some aspects, provided herein is a method for analyzing cell migration, the method comprising: a) contacting a first organoid containing target cells with a second organoid to form a fused organoid comprising the first organoid and the second organoid; b) incubating the fused organoid for a period of time; and c) determining the location of one or more of the target cells within the fused organoid.

[0033] The target cells can be cancer cells. The first organoid can be a brain cancer organoid formed by any of the methods provided herein. The first organoid and / or the second organoid can be brain organoids. The contacting in step a) can comprise incubating the first organoid and second organoid together in a partition. The partition is a cell-culture well or dish, or any other suitable container, such as any described herein. The bottom of the partition can be slanted and the first organoid and second organoid can contact one another when pulled downwards by gravity. In some embodiments, the partition does not contain a third organoid.In some embodiments, the contacting in step a) comprises providing the first organoid and second organoid together in a composition, and wherein the composition does not comprise a third organoid. In some embodiments, the fused organoid does not comprise a third organoid.

[0034] The first organoid and second organoid can be spheroid in shape or substantially spheroid in shape. An organoid that is spheroid or substantially spheroid in shape can be any organoid that is roughly in the shape of a ball or a sphere. The spheroid shape may be somewhat distorted (e.g. stretched and not perfectly spherical), and may have aberrations such as protrusions or indentations. An organoid that is spheroid or substantially spheroid in shape may also be described as globular in shape. The organoid can be any other suitable shape, and may also be described in some aspects as a three-dimensional (3D) cell aggregate. A person having skill in the art will readily recognize the meaning of spheroid in the context of cell culture. The first and second organoids can be spheroid in shape, and fused at an interface between the first organoid and second organoid. The location of the interface can be at a furrow between the two spheroid organoids. The maximum diameter of the interface can be smaller than the maximum diameter of the first organoid and smaller than the maximum diameter of the second organoid. In some embodiments, the first organoid and the second organoid can be distinguished from one another based on morphology, genotype, and / or expression of a marker. Determining the location of the one or more target cells within the fused organoid can comprise or exclude detecting the presence of one or more target cells in the first organoid and / or the second organoid. Determining the location of the one or more target cells within the fused organoid can comprise or exclude quantifying the number or density of target cells in the first organoid and / or the second organoid. Determining the location of the one or more target cells within the fused organoid can comprise or exclude quantifying the number or density of target cells in the second organoid. Determining the location of the one or more target cells within the fused organoid can comprise or exclude detecting a marker specific for the target cells. The marker specific for the target cells can comprise or exclude an endogenous marker. The marker specific for the target cells can comprise or exclude a heterologous marker and / or can be expressed from a transgene. The marker specific for the target cells can comprise or exclude a fluorescent protein. Determining the location of the one or more target cells can comprise or exclude fluorescence microscopy, immunohistochemistry, immunofluorescence, and / or flow cytometry. The method can comprise or exclude separating the first organoid from the second organoid prior to determining the location of the one or more target cells. The first organoid and second organoid can be separated at the interface. In some embodiments, the method does not comprise separating the first organoid from the second organoid prior to determining thelocation of the one or more target cells. In some embodiments, the second organoid does not contain target cells prior to the contacting. The presence of target cells in the second organoid after the incubating in step b) can be indicative of migration and / or invasion. Increased numbers of target cells in the second organoid can be indicative of increased migration and / or invasion.

[0035] The method can comprise or exclude perturbing the fused organoid and / or the target cells and determining the effect of the perturbation on target cell migration. The effect of the perturbation on target cell migration can be determined in comparison to a control fused organoid in which the perturbation is not performed. The perturbation can comprise or exclude providing target cells with a different genetic background than the target cells in the control fused organoid. The perturbation can comprise or exclude altering gene expression in the target cells and / or fused organoid. The perturbation can comprise or exclude incubating the fused organoid and / or the target cells in the presence of a candidate agent. Reduced target cell migration in the presence of the candidate agent can identify the candidate agent as having activity in reducing cell migration and / or invasion. The target cells can be cancer cells and the reduced cell migration and / or invasion can be reduced cancer cell migration and / or invasion. The candidate agent can comprise or exclude a small molecule, oligonucleotide, antibody, peptide, or protein.

[0036] The cancer cells can comprise or exclude primary cancer cells. The cancer cells can be from a tumor. The cancer cells can be from a cancer of the nervous system. The cancer of the nervous system can comprise or exclude brain stem glioma, pineal astrocytic tumor, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglial tumor, mixed glioma, ependymal tumor, medulloblastoma, pineal parenchymal tumor, meningeal tumor, germ cell tumor, or craniopharyngioma. In some embodiments, the cancer of the nervous system comprises or excludes glioblastoma. The cancer cells can comprise or exclude glioblastoma cells. The cancer cells can be from a subject. The cancer cells can be obtained from a biological sample from a subject that has cancer. The subject can be a human subject. The cancer cells may be human cancer cells.

[0037] The methods of the disclosure may include or exclude one or more of the following steps: trypsinization, cell counting, cell scoring, tissue dissociation, cell expansion, isolation of target cells, centrifugation, incubation, incubation at 37° C, incubation in a hanging liquid droplet, incubation not in a hanging liquid droplet, incubation in a cell culture well or dish, transfer of cells and / or organoids to and / or from a hanging liquid droplet, implantation of cells into an organoid, organoid fusion, splitting of fused organoids, dissociation of organoids, monitoring and / or analysis of live organoids, harvesting organoids for analysis, incubatingorganoids with agents for screening the activity of the agents, determining the number and / or location of cancer cells and / or target cells within organoids, immunofluorescence, immunohistochemistry, fluorescence microscopy, flow cytometry, fluorescence in situ hybridization, and nucleic acid sequencing.

[0038] The organoids may be of consistent size. The organoid may have a diameter of about 0.1-20 mm, 0.5-15 mm, 1-10 mm, 5-10 mm, 6-7 mm, or 1-2 mm. The organoid may have a diameter of, of at least, or of at most 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2,I.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4,3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10,10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7,I I.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4,13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1,15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8,16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5,18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 20.1, 20.2,20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23, 23.1, 23.2, 23.3, 23.4, 23.5, 23.6,23.7, 23.8, 23.9, 24, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25, 25.1, 25.2, 25.3,25.4, 25.5, 25.6, 25.7, 25.8, 25.9, 30, 26, 26.1, 26.2, 26.3, 26.4, 26.5, 26.6, 26.7, 26.8, 26.9, 27,27.1, 27.2, 27.3, 27.4, 27.5, 27.6, 27.7, 27.8, 27.9, 28, 28.1, 28.2, 28.3, 28.4, 28.5, 28.6, 28.7,28.8, 28.9, 29, 29.1, 29.2, 29.3, 29.4, 29.5, 29.6, 29.7, 29.8, 29.9, 30, 30.1, 30.2, 30.3, 30.4,30.5, 30.6, 30.7, 30.8, 30.9, 31, 31.1, 31.2, 31.3, 31.4, 31.5, 31.6, 31.7, 31.8, 31.9, 32, 32.1,32.2, 32.3, 32.4, 32.5, 32.6, 32.7, 32.8, 32.9, 33, 33.1, 33.2, 33.3, 33.4, 33.5, 33.6, 33.7, 33.8,33.9, 34, 34.1, 34.2, 34.3, 34.4, 34.5, 34.6, 34.7, 34.8, 34.9, 35, 35.1, 35.2, 35.3, 35.4, 35.5,35.6, 35.7, 35.8, 35.9, 36, 36.1, 36.2, 36.3, 36.4, 36.5, 36.6, 36.7, 36.8, 36.9, 37, 37.1, 37.2,37.3, 37.4, 37.5, 37.6, 37.7, 37.8, 37.9, 38, 38.1, 38.2, 38.3, 38.4, 38.5, 38.6, 38.7, 38.8, 38.9, 39, 39.1, 39.2, 39.3, 39.4, 39.5, 39.6, 39.7, 39.8, 39.9, 40, 40.1, 40.2, 40.3, 40.4, 40.5, 40.6,40.7, 40.8, 40.9, 41, 41.1, 41.2, 41.3, 41.4, 41.5, 41.6, 41.7, 41.8, 41.9, 42, 42.1, 42.2, 42.3,42.4, 42.5, 42.6, 42.7, 42.8, 42.9, 43, 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, 45, 45.1, 45.2, 45.3, 45.4, 45.5, 45.6, 45.7,45.8, 45.9, 46, 46.1, 46.2, 46.3, 46.4, 46.5, 46.6, 46.7, 46.8, 46.9, 47, 47.1, 47.2, 47.3, 47.4,47.5, 47.6, 47.7, 47.8, 47.9, 48, 48.1, 48.2, 48.3, 48.4, 48.5, 48.6, 48.7, 48.8, 48.9, 49, 49.1, 49.2, 49.3, 49.4, 49.5, 49.6, 49.7, 49.8, 49.9, or 50 mm or nm, or any derivable range therein.

[0039] An organoid may be a miniaturized and simplified version of an organ produced in vitro in three dimensions that shows realistic micro-anatomy. A brain organoid can be produced from stem or progenitor cells under conditions that facilitate production of brain cells from the stem / progenitor cells. Certain details of producing organoids are described by Takebe et al. (2019); Science, Vol. 364, Issue 6444, pp. 956-959. Certain details of producing brain organoids are described by Koo et al. (2019), Mol Cells.; 42(9): 617-627. The Takebe et al. and Koo et al. references are incorporated herein by reference in their entireties. The brain organoid can be selected from neurospheres, neural aggregates, neural rossettes, cortical spheroids, cortical organoid, cerebral organoid, or whole-brain organoids. The brain organoid is preferably a cortical organoid. The brain organoid may be generated from human embryonic stem cells (ESCs), human induced pluripotent stem cells (iPSCs), or a cell line comprising ESCs or iPSCs. For example, a brain organoid can be generated by culturing human ESCs or human iPSCs in a medium comprising a Rho kinase inhibitor, a Wnt signal inhibitor and a TGFp signal inhibitor on a low adhesion substrate to generate aggregates; and culturing the aggregates in a medium comprising a Wnt signal inhibitor and a TGFp signal inhibitor and lacking a Rho kinase inhibitor, on a low adhesion substrate to generate the brain organoid. In certain such methods, the Wnt signal inhibitor comprises IWR-1 , the TGFp signal inhibitor comprises SB431542, and the Rho kinase inhibitor comprises Y-27632. Moreover, the brain organoid expresses or is genetically modified to express one or both telencephalon markers, Foxgl and Six 3.

[0040] Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art. Embodiments of the disclosure also include those described in WO2021138209, which is incorporated by reference for all purposes. It is contemplated that embodiments of WO2021138209 relating to the methods and organoid compositions may be applicable to the organoid compositions and methods of the current application.

[0041] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0042] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0043] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0044] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), “characterized by” (and any form of including, such as “characterized as”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0045] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. The phrase “consisting of’ excludes any element, step, or ingredient not specified. The phrase “consisting essentially of’ limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments described in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”

[0046] It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an embodiment set forth in the Examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary of Invention, Detailed Description of the Embodiments, Claims, and description of Figure Legends.

[0047] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that thedetailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

[0049] FIG. 1 shows a schematic illustrating an exemplary method for providing a brain cancer organoid involving incubation of a brain organoid and primary cancer cells in a hanging liquid droplet.

[0050] FIG. 2 shows an exemplary fluorescence microscopy image of GFP+ glioblastoma cells in a brain cancer organoid following incubation in hanging liquid droplet.

[0051] FIGS. 3A-3C shows results from incubation of a brain organoid and primary cancer cells in a hanging liquid droplet, a liquid droplet that is not hanging (insert), or a liquid droplet in a v-bottom well. FIG. 3A shows experimental setup for three different incubation conditions. FIG. 3B shows exemplary fluorescence microscopy images of GFP+ glioblastoma cells in brain cancer organoids following incubation. FIG. 3C shows the percentage of GFP+ glioblastoma cells in the brain cancer organoids following the incubation.

[0052] FIG. 4A shows a schematic illustrating an exemplary workflow for analyzing target cell migration in a fused organoid as described herein.

[0053] FIG. 4B shows an exemplary immunofluorescence image of a fused organoid comprising gliomasphere (GS) cells.

[0054] FIG. 5 shows results from experiments assessing GS cell migration in fused organoids following knockdown of indicated genes. Results show percentage of GS cells that migrated to the untransplanted organoid. The plots show data, in order of left to right, of #1, #2, #3, #4, #5, and #6.

[0055] FIG. 6 shows results from experiments assessing GS cell migration in fused organoids following knockdown of indicated genes. Results show fold change in migration for each condition using organoids with the PTPRZ1 knockdown background normalized to the scramble shRNA background.

[0056] FIGS. 7A-7F show exemplary fluorescence microscopy images from fused organoid migration experiments using GS005 GS cells. Each image shows a GFP+ GS cell- transplanted organoid fused with an untransplanted organoid after allowing time for migration. Knockdown conditions are indicated in tables, and percent migration quantification is shown for each exemplary image.

[0057] FIGS. 8A-8F show exemplary fluorescence microscopy images from fused organoid migration experiments using GS025 GS cells. Each image shows a GFP+ GS cell- transplanted organoid fused with an untransplanted organoid after allowing time for migration. Knockdown conditions are indicated in tables, and percent migration quantification is shown for each exemplary image.

[0058] FIG. 9A-9B. Establishment of human organoid tumor transplantation (HOTT) model. FIG. 9A: Workflow of Human Organoid Tumor Transplantation (HOTT) modeling. Tumors were obtained from either freshly resected surgical sampling or from dissection of patient xenografted mice. These tumors were then dissociated, and part of the original tumor was sequenced with single-cell or single-nuclei transcriptomics, and the rest of the tumor was transplanted onto cortical organoids via a novel hanging drop method after GFP labeling. The organoids were then maintained in different media conditions to explore various environmental conditions for 3-4 weeks before harvest. Half of the organoids were processed via fluorescent activated cell sorting (FACS) to isolate and sequence GFP+ tumor cells, GFP- organoid cells, and tumor-naive batch-matched organoids, while the other half of transplanted organoids were used for immunofluorescence and RNAscope validation. The experimental workflow was implemented on three primary tumors (indicated by the human brain logo) and 2 direct-from- patient orthotopic xenograft (DPDOX) tumors (represented by the mouse brain logo). FIG. 9B: Heterogenous tumor cell type identification via projection to published GBM meta-atlas as reference. The entire set of experimental media conditions across five tumor samples was analyzed jointly. Cell type annotation was performed by projection to a compendium of GBM published dataset (Bl), these cell types were well represented and heterogeneous GBM cell types were identified (B2), with a robust correlation with annotations from Neftel et al (B3) and annotations from Ravi et al (B4). No dramatic separation across 5 tumors (B5) or 5 culture conditions (B6) in UMAP space was detected.

[0059] FIG. 10A-10C. Transplanted tumors maintain sternness and preserve primary tumor complexity. FIG. 10A: Cellular composition of organoid transplanted GBM across various culture conditions. HOTT modeling enables robust engraftment of patient tumors, even for those that failed to be engrafted into the mouse brain (confirmation from Chris). Notably,100% success engraftment is achieved in FBS containing culture conditions. In addition, the intricate complexity of primary tumors is also preserved across all media conditions. Stacked barcharts are shown representing the fraction of each cell type identified via single-cell analysis for each tumor and condition. FIG. 10B: Sternness of transplanted primary tumors across diverse culture conditions including FBS containing media. Transplanted primary tumors across diverse culture conditions demonstrated persistence of their sternness features as measured by MKI67+, SOX2+ and NES+ immunostaining and their quantification as a fraction of the GFP+ population across multiple tumors. This sternness was observed across all media conditions, including those containing FBS. FIG. 10C: Immunofluoresence staining verification of primary tumor complexity across different culture conditions. Verification of primary tumor complexity across various culture conditions was performed using cell typespecific marker staining, including the progenitor marker HOPX, neuronal markers MEF2C and CTIP2, as well as the astrocyte marker GFAP. The accompanying bar graphs, representing multiple tumor samples, illustrate the prevalence of the most populated cell types across different culture conditions. Notably, the culture condition without FBS supplemented with EGF and bFGF (i.e., M3noFBS+HEF), preferred for gliomasphere growth, demonstrated a higher abundance of dividing cells and progenitors, while exhibiting fewer neuronal cells. This observation is further supported by single-cell RNAseq data, which indicated a higher proportion of cycling and radial glial (RG) populations (FIG. 10A).

[0060] FIG. 11A-11E HOTT derived tumor cells recapitulate the molecular features of their corresponding primary tumors. FIG. 11 A: The recapitulation of tumor cell type heterogeneity revealed by the correlation of HOTT derivation to their corresponding primary tumors. Three HOTT samples were individually clustered using the routine Seurat pipeline, and resulting clusters were merged and correlated to their corresponding primary tumor cell types annotated by projection onto the GBM meta-atlas. The correlation to their corresponding primary tumors demonstrates the recapitulation of tumor cell type heterogeneity. FIG. 11B: ssGSEA shows no substantial difference across culture conditions for the underlying gene expression programs. ssGSEA analysis was executed to determine whether fidelity to individual cell types was altered across media conditions. Ridge plots of each media condition for the enrichment score was generated, revealing no substantial differences across culture conditions for the underlying gene expression programs. FIG. 11C: HOTT cell type specific fidelity to GBM meta-atlas. The fidelity of HOTT tumor cell type to the GBM meta-atlas cell types was compared. Bubble plot, where the size of dots represents the cell number obtained by single cell transcriptome and the intensity of dots represents the predicted ID score, i.e., thesimilarity to GBM typical cell types. When compared to the first two columns representing primary tumors, the HOTT model demonstrates the best recapitulation of certain cell types including cycling, neuronal, vascular, and astrocyte, while microglia and MES were retained at relatively lower numbers and lower levels of similarity. Furthermore, culture conditions without FBS favored cycling and radial glia cells, with M3noFBS+HEF showing the most dramatic effect. These results mirror previous discoveries in FIG. 10, showing the robustness of the findings. Interestingly, OPC populations are also significantly favored in culture conditions without FBS. FIG. HD: Differential gene expression between primary and transplanted tumor. Differential gene expression analysis was performed between primary and transplanted tumors. Bar graphs of the top 10 most significant Reactome pathways were shown. Pathways related to both innate and adaptive immune systems were enriched in primary cells compared to transplanted cells, corroborating the observation of diminished immune cells in the HOTT system. FIG. HE: Histogram of prediect.ed. score shows peri-tumor non-neoplastic cells mirror developmental brain molecular features in both HOTT and published GBM dataset.

[0061] FIG. 12A-12C. HOTT uncovers bidirectional tumor - microenvironmental communication mediated by PTPRZ1. FIG. 12A: Cell composition shift of non-neoplastic cells between tumor transplanted and tumor naive organoids. When comparing non-neoplastic organoid cells from tumor-exposed organoids with batch-matched naive organoid cells, the inventors observed separation of these two samples in UMAP space across all three primary GBM experiments (left panel). Cell composition analysis (right panel) revealed a persistent shift from dividing cells and progenitors towards neuronally committed populations after exposure to GBM, suggesting that GBM may promote the differentiation of surrounding normal cells. FIG. 12B. CellChat analysis identified PTN-PTPRZ1 as the most significant cellcell interaction in tumor microenvironment, but not in normal adult brain environment. Cellcell communication in the tumor microenvironment was assessed using CellChat (Jin et al., 2021), a computational tool that enables prediction of cell interaction based on receptor - ligand relationships in a cell type specific manner. Bl) In the HOTT system, represented by LB4878 in the M3 culture condition, PTN was identified as the most significant signaling pathway (right column) with PTPRZ1 as its major receptor (left column). The PTN signaling pathway network was shown in a circle plot. Dominant sources of this signaling as both sources and targets, included neoplastic dividing cells, progenitors and radial glia, as shown in the 2D scatter plot. B2) When leveraging the same strategies to analyze a published GBM dataset, Yu et al by comparing neoplastic and non-neoplastic cells also captured in the dataset, PTN - PTPRZ1 was again identified as the top signaling between the tumor and the microenvironment. B3)However, when a similar analysis was conducted with a normal adult human brain dataset, this pathway did not emerge as a primary mediator of cell-cell interactions. Note the significantly lower PTN interaction strength observed in the 2D scatter plots within the normal adult brain as compared to the GBM TME. The interaction strength across analyses demonstrates higher scores in the neoplastic cell types and tumor samples compared to normal cell types. FIG. 12C: RNAscope revealed high percentage of PTN & PTPRZ1 double positive cells in both core and peripheral areas of primary tumors. RNAscope revealed high percentages of PTN and PTPRZ1 double positive cells in both core and peripheral areas of primary tumors. FFPE sections were derived from surgical samples removed from the tumor core (core region) and the tumor boundary (peripheral region). Neoplastic regions were identified by post-staining with NES on contiguous sections, as well as by assessing cell density based on DAPI nuclei staining. NES staining on the right is autofluorescence, staining on the left with cellular morphology is true staining. Almost all the cells are PTN and PTPRZ1 double positive, mirroring the equal incoming and outcoming interaction strength in Fig 10B 10D scatter plots. The observation of PTPRZ1 and PTN positive cells found in both the neoplastic and non-neoplastic areas of the tumor in both the core and peripheral regions supports the role of PTN-PTPRZ1 in potentially mediating tumor migration.

[0062] FIG. 13A-13F Environmental PTPRZ1 knockdown increases glioma migration. FIG. 13A: Shematic of novel organoid fusion migration assay. Workflow of a novel organoid fusion migration assay. Gliomaspheres were treated with a shRNA lentivirus that contains a scramble or PTPRZ1 -targeting sequence with a GFP reporter prior to transplantation. After transplantation using the hanging drop method, the HOTTs were allowed to fuse with a tumor- naive organoid and cultured for 3 weeks. At time of harvest, the fusions were severed with a scalpel and digested for FACS analysis. FIG. 13B: Live images of organoid fusions. Images of the HOTT fusions at 12, 18 and 25 days post fusion (dpf). GFP signal represents engrafted GS. White dotted line represents the fusion boundary. Scale bar = 750 um. FIG. 13C: PTPRZ1 knockdown of gliomaspheres and organoids. Western blot analysis of organoid and GS used for transplantation (left and middle). PTPRZ1 protein levels significantly decreased following shRNA lentivirus treatment. Real-time PCR of gliomaspheres show PTPRZ1 knockdown after shRNA was introduced, while PTN expression was unchanged (right). In the bar graph on the right, the left bar in each of the group of 2 bars is PTPRZ1 and the right bar is PTN. FIG. 13D: Schematic representation of the various transplant combinations. GS cells were treated with either a scramble shRNA or PTPRZ1 targeting shRNA and transplanted onto scramble or PTPRZ1 knockdown organoids, resulting in 4 experimental conditions. FIG. 13E:Gliomasphere migration across the fusion boundary. Box-and-whisker plots of migration rate for two patient-derived gliomasphere lines, GS005 and GS025. The migration rate was calculated by dividing the number of GFP+ cells on the tumor-naive side with the total number of GFP+ cells from both sides (n=3 replicates per line). Under identical tumor PTPRZ1 KD status, organoid PTPRZ1 KD resulted in increased GS migration towards the tumor-naive side. In the graph, the data represents, from left to right, TumorScrOrgScr, TumorScrOrgKD, TumorKI)OrgScr, and TumorKDOrgKD. FIG. 13F: Gliomasphere coverage in the organoid fusions. Immunostaining of the organoid fusions at the fusion boundary (left). Migration of GFP labeled GS cells was observed. Under identical tumor PTPRZ1 KD status, organoid PTPRZ1 KD resulted in increased GS migration towards the tumor-naive side (example labeled with white arrowheads). Scale bar = 250 um. Schematic of the binning strategy employed to quantify the distribution of GS cells was shown on the top right. The organoid fusion was divided into eight equal bins labeled Tl-4 (transplant) and N 1-4 (naive) which stem from the fusion boundary. The amount of GFP+ cells in each bin was quantified and plotted as a ridge graph as the percentage of total GFP cells present in each bin (bottom right).

[0063] FIG. 14A-14G. Environmental knockdown of PTPRZ1 alters tumor cell fate. FIG. 14A: Gliomasphere Transplants HM0X1 and MEF2C Immunostaining. Effects of tumor or environmental PTPRZ1 KD on cell fate were assessed with immunostaining of mesenchymal marker HM0X1 and neuronal marker MEF2C. Organoid KD of PTPRZ1 significantly reduces the expression of tumor HM0X1 and MEF2C. Moreover, organoid HM0X1 expression was also reduced with organoid PTPRZ1 KD, suggesting a role for PTPRZ1 in driving mesenchymal transition both intrinsically and through cell-cell communication. FIG. 14B: Quantification of HM0X1 and MEF2C Immunostaining. Violin plots depicting the quantified tumor and organoid expression of HM0X1 and MEF2C. Fold change of each experimental condition was calculated based on the average of all the data points in TumorScrOrgScr. Tumor and organoid expression was based on the ratio of GFP+ / Signal of interest+ over total GFP+ cells and GFP- / Signal of interest+ over total GFP- cells, respectively. P-value determined by two-tailed Mann-Whitney test, *p < 0.05, **p < 0.01, ***p < 0.001. Experiments were conducted in two patient-derived gliomasphere lines (GS005 and GS025) with a minimum of 3 but up to 6 replicates per line. FIG. 14C: Gliomasphere Transplants GFAP and CTIP2 Immunostaining. Effects of tumor or environmental PTPRZ1 KD on cell fate were assessed with immunostaining of astrocyte marker GFAP and deep layer neuronal marker CTIP2. Organoid KD of PTPRZ1 increased tumor GFAP and organoid GFAP expression, suggesting a role for PTPRZ1 in regulating astrocyte differentiation. In addition, under the context oforganoid PTPRZ1 KD, tumor PTPRZ1 KD significantly increased tumor CTIP2 expression. On the other hand, tumor PTPRZ1 KD increased organoid CTIP2 expression, suggesting a role for neuronal fate regulation via tumor-normal PTPRZ1 signaling. FIG. 14D: Quantification of GFAP and CTIP2 Immunostaining. Violin plots depicting the quantified tumor and organoid expression of GFAP and CTIP2. Fold change and statistical significance were calculated as described in FIG. 14B. FIG. 14E: NAZ2329 inhibits PTPRZ1 Phosphatase Activity. Western blot analysis of NAZ2329 treated GS cells at concentrations of 0 (Vehicle), 6.25, 12.5 and 25 uM. Total SRC and pY416 SRC levels were examined and the ratio of pSRC to SRC levels increased in a dose-dependent manner, validating the blockage of PTPRZ1 phosphatase activity. Actin was used as a loading control. 30 ug of protein was loaded in each lane. FIG. 14F: PTPRZ1 Inhibition enriches organoid astrocytes. Effects of cell fate driven by PTPRZ1 catalytic inhibition was investigated by immunostaining of astrocyte marker GFAP and neuronal marker CTIP2 (left). The organoid transplants were treated with a DMSO vehicle or 25 pM of NAZ2329. Violin plots showing quantification of GFAP and CTIP2 expression (right). P-value determined by two-tailed Mann-Whitney test, *p < 0.05. Experiment was conducted with three GS lines with four replicates per line. FIG. 14G: Different roles of tumor and peritumor PTPRZ1 on tumor phenotypes. Graphic model illustrating the different roles of tumor derived or peritumor derived PTPRZ1 on tumor sternness and migration. Intrinsic tumor PTPRZ1 drives tumor sternness and migration, whereas microenvironmental PTPRZ1 suppresses both. This mechanism is independent of PTPRZ1 phosphatase activity.

[0064] FIG. 15A-15E. Experimental Setup for Human Organoid Tumor Transplantation (HOTT) model (Associated with FIG. 9). FIG. 15A: Cortical organoid generation. Cortical organoids were derived from multiple lines of hESC (human embryonic stem cells). Top panel: Representative images of cortical organoids cultivated in various differentiation media, together with a growth curve presented to track their size growth. To generate cortical organoids, human embryonic stem cells were dissociated into single cell suspensions and aggregated into ultra-low attachment V-bottom plates in the presence of ROCK, WNT and TGFb inhibitors (Media 1, Ml). These organoids were introduced into Media 2 (M2) and Media 3 (M3) on Day 18 and Day 35, respectively, to further shape them towards a cortical fate. Bottom panel: IF verification illustrating the stepwise progression towards cortical fate. At Week 5, neural progenitor characteristics were evident, characterized by the presence of rosette structures and positive staining for PAX6 and Ki67. Subsequent neural differentiation was marked by the expression of the deep-layer neuronal marker CTIP2 at Week 10, followed by the appearance of the upper-layer neuronal marker SATB2 and the typical astrocytic markerGFAP at Week 15. FIG. 15B: Optimization of GBM transplantation. Three different transplantation methods of HOTT were compared using the same quantity of tumor cells. After three weeks of culturing in M3 media, all transplanted organoids were sectioned and stained for GFP (Green Fluorescent Protein), and the percentage of GFP-positive area relative to the total organoid area in the field was calculated to assess transplantation efficiency. FIG. 15C: Different culture conditions and their expected outcomes. Five different culture conditions were employed to investigate the response of GBM to various environmental perturbations, with expected outcomes outlined alongside each condition. FIG. 15D: Representative FACS plots. Following 3-4 weeks of culture in different media, FACS (Fluorescence-Activated Cell Sorting) sorting was performed to enrich GFP-labeled GBM cells. A gate was established using GFP-negative (GFP-) naive organoids (top panel), and only samples with over 1000 GFP- positive (GFP+) cells proceeded to single-cell transcriptome analysis. FIG. 15E: Representative InferCNV plot. InferCNV was used to identify neoplastic cells from non- neoplastic organoid cells, as shown by the representative InferCNV plots and feature plots to highlight neoplastic cell clusters corresponding to InferCNV clusters.

[0065] FIG. 16A-16E. Characterization of Tumor Heterogeneity in HOTT System (Associated with Figures 9 and 10). FIG. 16A: GBM meta atlas UMAP annotated with utilized public datasets. A compendium of 7 GBM published datasets was used to generate a GBM meta-atlas. These datasets were downloaded from publicly available data depositions and filtered to maintain only tumor cells as indicated by copy number variation analysis. Integration was performed using RPCA analysis (Seurat v4) and clusters were annotated for cell type. The UMAP shown here depicts the integrated analysis yielded co-mingling of all 7 datasets included. FIG. 16B: UMAPs of 5 tumor samples projected to GBM meta atlas annotated by sources (left panel) and transplanted tumor predicted. id. score (right panel). The projection of 5 HOTT GBM samples alongside their corresponding primary tumors onto the GBM meta- atlas facilitates the reduction of batch effects and technical disparities between single-cell and single-nucleus RNAseq methodologies. In projected UMAP space annotated by source (left panel), the preservation of the majority of the tumor complexity was shown with less maintenance of certain cell types such as microglia and oligodendrocytes, which are only present in primary tumor samples in red. While in projected UMAP space annotated by predicted ID score (right panel), high molecular similarity was shown between HOTT cell types and their corresponding GBM meta-atlas cell types. FIG. 16C: Cellular composition of organoid transplanted GBM using Neftel et al. and Ravi et al. annotation. Cellular composition of 5 HOTT GBM samples utilizing Neftel et al and Ravi et al annotations, complementing Fig10A, not only exhibited similar trends to those observed with the predicted ID annotation but also showed a robust correlation among the major cell types annotated using three different annotation systems. FIG. 16D: Immunofluorescence staining confirming heterogenous cell types of transplanted DPDOX tumors across different culture conditions. Additional immunofluorescence validation of the most populous cell types within transplanted DPDOX tumors across various culture conditions, with their quantification displayed in Fig 10B and IOC. FIG. 16E: Quantification of transplanted tumor cells in both immunofluorescence staining (top panel) and scRNA seq data where tumor cells were identified by inferCNV analysis (bottom panel). Additional quantification was conducted to assess tumor cell numbers in 5 HOTT samples across 5 different culture conditions. The number of GFP+ cells in immunofluorescence (IF) assays matched the tumor cell count obtained via single-cell RNAseq following identification through inferCNV features. Consistently, FBS-containing culture conditions exhibited higher tumor cell counts, whereas base media without FBS yielded the fewest tumor cells. Moreover, supplementation with growth factors (HEF and PDGF) increased tumor cell numbers.

[0066] FIG. 17A-17B. HOTT derived tumor cells recapitulate the molecular features of their corresponding primary tumors. (Associated with Figure 11). FIG. 17A: Additional Correlation heatmaps using Neftel and Vidhya annotation to further demonstrate that HOTT derived tumors recapture the heterogeneous cell types of their corresponding primary tumors. Additional heatmaps demonstrate the correlation of HOTT derived tumor cells to their corresponding primary tumors using Neftel et al and Ravi et al annotations further demonstrates the recapitulation of tumor cell type heterogeneity. FIG. 17B: Major cell types preserved primary tumor molecular features revealed by module activity analysis using meta modules assembled from normal human development data. Characterization of tumor cells based on the activity of gene modules representing normal human brain development. Gene modules were identified by applying a novel strategy of iterative, hierarchical clustering to a meta-atlas of seven previously published transcriptomic profiles of the developing human cortex (Nano et al., 2023) (top). This pipeline produced 225 modules, which were assigned biological annotations through both extensive literature review of module genes and term enrichment analysis of gene ontology sets, WikiPathway and KEGG pathway databases, and ChEA, ENCODE and TRRUST transcriptional regulatory collections. A module activity score based on average module gene expression was calculated for each tumor cell. Heat map (bottom) shows the average module activity in primary cells or in transplanted tumor cells across three samples grown in the indicated culture conditions. When compared to theircorresponding primary tumors, most HOTT tumors maintained comparable module activity, except for immune function, cell junction, and synapse functions.

[0067] FIG. 18A-18B. Non-neoplastic cells derived from both HOTT organoids and published GBM dataset exhibited the greatest similarity to corresponding cell types found in the developing human brain. (Associated with Figure 12). To investigate the interaction of tumor cells and their surrounding inferCNV-identified non-neoplastic cells, similar annotation strategy was used to define non-neoplastic cell types, where three different reference databases were used with their corresponding UMAPs displayed in the left column, including normal developing brain meta-atlas (A and Bl), adult brains (B2), and normal aging brains (B3). FIG. 18A: High similarity between non-neoplastic organoid cells and the developing human brain cells demonstrated by high predicted. id. scores in both projected normal developing brain atlas UMAP space and its histogram. As expected, the inventors observed the highest predicted scores of HOTT non-neoplastic organoid cells when projected to the developing brain meta- atlas, as demonstrated by their UMAP in the reference UMAP space annotated by cell types (predicted. id), the similarity (predicted. id. score) and histogram of mean predicted scores, respectively. FIG. 18B: The similarity comparison of non-neoplastic cells from published GBM datasets to three reference dataset including the developing brain meta atlas (Bl), normal adult brain cells (B2) and normal senior brain cells (B3). When annotating the non-neoplastic cells from published GBM datasets, the highest predicted scores were also observed when projected to the developing brain meta-atlas (Bl), but not to normal adult brain cells (B2) and normal aging brain cells (B3). The results suggest the applicability of using the HOTT system to study the tumor microenvironment.

[0068] FIG. 19A-19D Additional CellChat analysis on three HOTT samples across culture conditions yielded consistent results, highlighting the role of PTN-PTPRZ1 signaling in mediating interactions between the tumor and its microenvironment. FIG. 19A: Relative contribution of PTN receptors. FIG. 19B: PTN pathway network. FIG. 19C: PTN pathway expression pattern. FIG. 19D: Cell cell interaction signaling pathways ranking.

[0069] FIG. 20A-20D. Additional CellChat analysis on published adult normal brain and GBM datasets with non-neoplastic cells confirmed PTN / PTPRZ1 signaling as the top pathway mediating the interactions in tumor microenvironment but not in normal brain environment. FIG. 20A: Relative contribution of PTN receptors. FIG. 20B: PTN pathway network. FIG. 20C: PTN pathway expression pattern. FIG. 20D: Cell cell interaction signaling pathways ranking.

[0070] FIG. 21A-21E The tumor microenvironment is essential to PTPRZ1 -mediated cell type changes. FIG. 21A: Gliomasphere transplants HOPX and VEGFA immunostaining. Effects of tumor or environmental PTPRZ1 KD on cell fate were assessed with immunostaining of radial glial marker HOPX and mesenchymal marker VEGFA. Tumor KD of PTPRZ1 significantly increases the expression of tumor HOPX and organoid HOPX, suggesting that intrinsic and environmental PTPRZ1 both play a role in suppressing radial glia fate in the tumor. FIG. 21B: Quantification of HOPX and VEGFA expression. Violin plots depicting the quantified tumor and organoid expression of HOPX and VEGFA. Fold change of each experimental condition was calculated based on the average of all the data points in TumorScrOrgScr. Tumor and organoid expression were based on the ratio of GFP+ / Signal of interest+ over total GFP+ cells and GFP- / Signal of interest+ over total GFP- cells, respectively. P-value determined by two-tailed Mann-Whitney test, *p < 0.05. Experiments were conducted in two patient derived gliomasphere lines (GS005 and GS025) with a minimum of 3 but up to 6 replicates per line. FIG. 21C: Gliomaspheres only control knockdown immunostaining. Immunostaining of astrocyte marker GFAP, neuronal markers CTIP2 and MEF2C, radial glia marker HOPX, and mesenchymal markers HM0X1 and VEGFA of gliomaspheres only under scramble or PTPRZ1 KD conditions (n=3). Modulation in cell fate has largely disappeared upon PTPRZ1 KD, highlighting that the presence of a tumor microenvironment is essential for the observed cell type changes. FIG. 21D: Quantification of gliomaspheres only immunostaining. Violin plots depicting the quantified immunostaining in D. P-value determined by two-tailed Mann-Whitney test, *p < 0.05. Experiments were conducted in three patient derived gliomasphere lines (GS005, GS025 and HK408) with 3 replicates per line. FIG. 21E: Immunostaining quantification of NAZ2329-treated gliomasphere transplants. Violin plots depicting the quantified expression of HM0X1, MEF2C, HOPX, and VEGFA of GS transplanted organoids under vehicle and 25 pM NAZ2329 treatment. No significant cell type changes were observed, indicating that cell fate specification was not driven by PTPRZ1 catalytic activity. Experiments were conducted in three GS lines (GS005, GS025 and HK408) with 4 replicates per line. P-value determined by two-tailed Mann-Whitney test.

[0071] FIG. 22A-22I. Generation, characterization, and benchmarking of a synapse- optimized human organoid tumor transplantation (so-HOTT) model. FIG. 22A: Workflow of synapse-optimized human organoid tumor transplantation (so-HOTT) modeling and characterization. Tumors were obtained from freshly resected surgical sampling, dissociated, and labeled with a GFP lentivirus prior to hanging drop transplantation into human cortical organoids. Tumor-transplanted organoids were then maintained in Sasai 4 media with so-HOTT supplements (S4+). These supplements include soluble NLGN3, BDNF, GSK2879552, EPZ-5676, Bay K 8644, and NMDA, which collectively promote neuronal maturation and synapse formation through various modes of action. so-HOTT organoids were characterized by staining for pre- and post-synaptic markers followed by co-localization analysis, as well as single-cell RNA sequencing of GFP+ and GFP- compartments corresponding to tumor cells and organoid cells, respectively. Standard HOTT organoids grown in Sasai 3 were profiled in the same manner and served as baseline for comparative analysis. FIG. 22B: Immunostainings and machine learning-based segmentation showing co-localization of the pre-synaptic marker SYN1 and the post-synaptic marker PSD95 with GFP+ tumor cells. Tumor cells were either obtained from the tumor core or periphery, and were grown in S4+ or S3 media following hanging drop transplantation. FIG. 22C: Quantification of triple-channel colocalization showing increased co-localization of the abovementioned synaptic markers with GFP+ core and peripheral tumor cells grown in S4+ vs. S3. FIG. 22D: UMAP plot showing cell types present in so-HOTT (S4+) and standard HOTT (S3) tumor cells and organoid cells. FIG. 22E: Dot plot showing enriched Synaptic Gene Ontologies (SynGO) terms in so-HOTT versus standard HOTT organoids. Among the genes significantly upregulated in so-HOTT (log2-fold change > 0.25, adjusted p-value < 0.05) include those involved in postsynaptic assembly and organization, neurotransmitter (NT) receptor colocalization to postsynaptic compartments, synaptic adhesion and neurotransmission. FIG. 22F: Feature plots showing relative expression of presynaptic and postsynaptic SynGO modules in tumor cells from the GBM meta-atlas (left) and from so-HOTT (right). FIG. 22G: Violin plots showing expression levels of presynaptic and postsynaptic SynGO modules in tumor cells from the GBM meta-atlas (left) and from so- HOTT (right). FIG. 22H: Dot plots showing relative expression of neurotransmitter receptor and synaptic adhesion gene modules across different tumor cell types from the GBM meta- atlas (top) and so-HOTT (right). Dot sizes correspond to percentage of cells expressing a given module, while the color scale specifies the average expression of each module in a given cell type. FIG. 221: Box plots showing the relative expression of ionotropic glutamatergic receptor submodules (AMP A, NMDA, delta, and kainate) in the GBM meta-atlas (left) and in so-HOTT (right).

[0072] FIG. 23A-23F. Cell-cell communication analysis of so-HOTT using CellChat. FIG. 23A: Bar plots showing the number of inferred interactions and interaction strengths across so-HOTT models derived from core and peripheral tumor cells and grown in either S4+ or S3. FIG. 23B: Relative information flow for various signaling pathways in core (left) and peripheral (right) tumor cells grown in either S4+ or S3. FIG. 23C: Dot plots showing ligand-receptor interactions that are enriched between non-malignant organoid-derived neurons and various tumor cell types when grown in so-HOTT (i.e., S4+) conditions. FIG. 23D: Heatmap showing outgoing (left) and incoming (right) signaling patterns across various non-malignant (N) and tumor (T) cell types in so-HOTT. Pathways are ranked according to relative strength. FIG. 23E: Bar plot showing the relative contribution of ligand-receptor (LR) pairs from the NRXN pathway, the top interaction pathway identified to be activated in so-HOTT. FIG. 23F: Chord diagrams showing cell types that participate in CADM1 interactions in so-HOTT and standard HOTT models derived from the tumor core and periphery.

[0073] FIG. 24A-24C. Anterograde trans-synaptic tracing in so-HOTT. FIG. 24A: Schematic diagram showing the process of monosynaptic spread of AAVl-Cre viruses from a presynaptic neuron to its postsynaptic partner. Upon viral transfer into the postsynaptic cell, Cre recombinase expression occurs, which leads to inversion and expression of a doublefl oxed, inverted open reading frame (DIO)-EGFP reporter. FIG. 24B: Live imaging of so- HOTT organoids with (left) or without AAVl-Cre introduced in the organoid side. GFP+ cells are present in when AAVl-Cre is introduced, indicating the presence of tumor cells synaptically connected to organoid-derived neurons. FIG. 24C: Immunostaining of Nestin+ GBM cells (red) transplanted into cortical organoids with (top) or without (bottom) AAVl-Cre (magenta). Inset shows DIO-EGFP+ cells (green) that are synaptically connected to Cre+ neurons in AAVl-Cre organoids.DETAILED DESCRIPTION

[0074] In some aspects, the present disclosure relates to methods of providing an organoid. The method can comprise incubating a brain organoid and primary cancer cells in a hanging liquid droplet, wherein the primary cancer cells become attached to and / or incorporated into the brain organoid to form a brain cancer organoid (i.e. a brain organoid containing cancer cells). The incubation in the hanging liquid droplet can provide an efficient and cost-effective means to introduce cancer cells (e.g. primary cancer cells) directly into the brain organoid. Also provided are organoids produced by any of the methods provided herein. The brain organoids can be used to monitor, study, and / or analyze the cancer. The brain organoids can be used to screen candidate agents in order to identify agents with anti-cancer activity, such as agents that reduce metastasis, proliferation, growth, invasion, and / or migration, of cancer cells. Thus, the methods provided herein provide superior methods for producing brain cancer organoids and studying cancer cells in vitro, in comparison to certain existing methods. Also provided herein are methods of analyzing migration of target cells, such as cancer cells, in a fused organoidcomprising a first and second organoid. The methods can provide advantageous conditions for analyzing migration of the target cells and for identifying conditions and agents that can affect the migration.I. Exemplary Definitions

[0075] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described.

[0076] In various aspects, the subject of the herein disclosed methods can be a vertebrate, e.g., a mammal. Thus, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. In some aspects, the subject is a human. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. A patient can refer to a subject afflicted with a disease or disorder. The term “patient” can include human and veterinary subjects. The term “subject” as used herein means a vertebrate animal and includes mammals which includes human beings. In some preferred aspects, the subject is a human.

[0077] The terms “optional” or “optionally” as used herein mean that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.

[0078] The terms “about” or “approximately” are used according to their plain and ordinary meaning to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.. The terms may be removed from the associated disclosed value and the exact value may be used instead.

[0079] Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and / or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated aspect that should be considered disclosed unless the context specifically indicates otherwise. It will be furtherunderstood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these aspects are explicitly disclosed.

[0080] When the lower limit value of a given percentage range does not include the % symbol and / or the percentage type (e.g., w / w, v / v, etc.), then the percentage type for the lower limit value is the same as for the upper limit value of the given percentage range. For example, the percentage range of “0.01 to 0.5% w / w” means “0.01% w / w to 0.5% w / w.”

[0081] The terms “wt.%”, “w / w”, “vol.%”, “v / v”, “w / v”, or “mol.%” refers to a weight percentage of a component, a volume percentage of a component, or molar percentage of a component, respectively, based on the total weight, the total volume of material, or total moles, which includes the component. In non-limiting examples, 10 grams of component in 100 grams of a material is 10 wt.% or 10% w / w of component, 10 mL of component in 100 mL of a material is 10 vol.% or 10% v / v of component, and 10 grams of component in 100 mL of a material is 10 w / v of component.

[0082] The use of the word “a” or “an” when used in conjunction with the terms “comprising”, “having”, “including”, or “containing” (or any variations of these words) may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0083] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0084] For purposes of this application, a number value with one or more decimal places can be rounded to the nearest whole number using standard rounding guidelines, i.e., round up if the number being rounded is 5, 6, 7, 8, or 9; and round down if the number being rounded is 0, 1, 2, 3, or 4. For example, 0.42 can be rounded to 0.4.

[0085] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) as used herein are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0086] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.

[0087] The terms “comprise” (and any form of comprise, such as “comprises” and “comprising”), “have” (and any form of have, such as “has” and “having”), “include” (and any form of include, such as “includes” and “including”) and “contain” (and any form of contain, such as “contains” and “containing”) are open-ended linking verbs. As a result, the methods and systems of the present invention that “comprises,” “has,” “includes” or “contains” one or more elements possesses those one or more elements, but is not limited to possessing only those one or more elements. Likewise, an element of a method or system of the present invention that “comprises,” “has,” “includes” or “contains” one or more features possesses those one or more features, but is not limited to possessing only those one or more features, and moreover, any one or more of these features may be excluded.

[0088] The terms “implantation” and “transplantation” as used herein can refer to procedures that facilitate the incorporation of cells (such as target cells and / or cancer cells as described herein) into organoids (such as brain organoids). Thus, in some aspects, “implantation” and “transplantation” may be used interchangeably herein.II. Brain cancer organoids and related methods

[0089] In some aspects, provided herein is a method for providing an organoid, the method comprising: contacting a brain organoid with cancer cells in suspension to form a liquid droplet containing the brain organoid and cancer cells; and incubating the liquid droplet on a solid surface for a first period of time during which the liquid droplet is a hanging liquid droplet that adheres to and hangs beneath the solid surface; wherein the cancer cells become attached to and / or incorporated into the brain organoid to form a brain cancer organoid. The methods provided herein can provide an efficient means to incorporate cancer cells into brain organoids, thereby forming brain cancer organoids. Incubation of the brain organoid with cancer cells in a hanging liquid droplet can enhance and / or accelerate the incorporation of cancer cells into the brain organoid, for example in comparison to methods that do not utilize hanging liquid droplet incubation. The efficient production of brain cancer organoids has utility for research and / or therapeutic application, such as for analyzing cancer cells. The cancer cells can be primary cancer cells from a subject, such as a human subject. Thus, the methods can facilitatean efficient method to analyze cancer cells from a cancer patient, which can facilitate the identification of effective therapies for the patient. The methods can also be compatible with and facilitate the analysis of various kinds of cancer cells in general.

[0090] In some aspects, an organoid is a miniaturized and simplified version of an organ produced in vitro in three dimensions that physiologically mimics the micro-anatomy of the organ. A brain organoid can be produced from stem cells under conditions that facilitate production of brain cells from the stem cells. Methods for producing organoids, such as brain organoids and cortical organoids have been described elsewhere and would be readily adapted to the methods described herein by one having ordinary skill in the art. Details of producing organoids are described, for example, in: Takebe et al. (2019); Science, Vol. 364, Issue 6444, pp. 956-959; Koo et al. (2019), Mol Cells.; 42(9): 617-627; and US20230204565A1, each of which is incorporated by reference herein in its entirety.

[0091] The organoids described herein can be produced by any suitable method. Culturing an organoid suitable for transplanting cells into may comprise culture in one or more media conditions. For example, during maturation of an organoid, the organoid may be cultured in a first media and subsequently transferred to a second media, and the first and second media can be different. Any number of media conditions can be used throughout the maturation of an organoid. The media used to culture the organoid can be any media described herein. For example, during maturation, the organoid can be cultured in Media #1, Media #2, Media #3, or Media #4, as described herein. The organoid can be cultured sequentially in different medias, for example during development, in Media #1, Media #2, Media #3, and Media #4, respectively. In some aspects, culture in different medias can have different desired effects. For example, organoid maturation can be limited by culturing in Media #3. The organoid can be cultured in Media #3 until transplantation (e.g. of cancer cells or target cells), and then the transferred to a different media (e.g. Media #4).

[0092] When producing an organoid, any suitable analysis and / or quality control steps can be performed. For example, a plurality of organoids can be produced simultaneously, and some of the organoids can be analyzed for quality control purposes. Analysis and quality control may include immunostaining for SOX2 protein, identifying rosettes labeled by SOX2, identification of CTIP2 cells, presence of TBR2 positive cells. These and other quality control and analysis measures have been described elsewhere and can be readily performed by one having ordinary skill in the art.

[0093] In some aspects, provided herein are methods that allow for the efficient incorporation of cancer cells (e.g. primary cancer cells such as glioblastoma cells) intoorganoids, such as brain organoids. In some aspects, provided herein is a method of incorporating primary glioblastoma cells into brain organoids. The method can allow for the efficient incorporation of primary cancer cells directly into brain organoids. Thus, in some aspects, the cancer cells can be directly incorporated into the organoid while minimizing the time and / or steps (such as culturing, plating, or passaging) in between obtaining the cells from the subject and incorporation the cells into the organoid. This is advantageous because it can reduce changes in characteristics of the cancer cells in the organoid that might be acquired by additional steps, such as culturing. It also provides a readily accessible and highly relevant model for studying primary cancer cells from a subject. In addition, primary cancer cells, for example when obtained from a patient biopsy, can be limited. The methods to directly incorporate (e.g. implant) cancer cells into brain organoids described herein can facilitate the rapid and efficient generation of a large number of brain cancer organoids from a limited number of primary cancer cells, for example in comparison to other methods that do not utilize the hanging liquid droplet technique described herein. This can provide increased opportunity to study, understand, analyze, and / or identify potential effective anti-cancer agents and / or treatments for the patient that target the cancer.

[0094] In some aspects, the method involves contacting the brain organoid with primary cancer cells in suspension to form a liquid droplet, and incubating the liquid droplet on a solid surface as a hanging liquid droplet for a first period of time. In some aspects, the inventors discovered that culturing the liquid droplet as a hanging liquid droplet greatly increases the efficiency with which primary cancer cells are incorporated into the brain organoid (for example in comparison to similar methods in which a hanging liquid droplet incubation step is not performed). This unexpected result reveals a convenient and cost-efficient method for the efficient generation of brain organoids containing primary cancer cells (e.g. brain cancer organoids, as described herein).

[0095] After the first period of time, the brain cancer organoid can be transferred to a culture condition, such as wherein the brain cancer organoid is subsequently submerged in media. In the culture condition, the primary cancer cells and the brain cancer organoid can be monitored and analyzed over a period of weeks (e.g. at least 1, 2, 3, 4, or more weeks). The monitoring and analyzing of the brain cancer organoid can reveal aspects of the cancer that may be beneficial for treatment. In some embodiments, the brain cancer organoid can be contacted with one or more candidate agents, and the activity of the agents in inhibiting the cancer (e.g. reducing proliferation, growth, invasion, and / or migration) can be assessed. In some embodiments, the methods can be used to identify candidate agents that can be used in amethod of treating a cancer, such as in the subject from which the primary cancer cells are obtained.

[0096] In some aspects, provided herein is a method of culturing cancer cells. The cancer cells can be primary cancer cells obtained from a subject, such as a human subject having cancer. The method can comprise contacting the cancer cells with a brain organoid such that the cancer cells are incorporated into the brain organoid to form an organoid containing the cancer cells (i.e. a brain cancer organoid as described herein). The cancer cells can be cultured in the brain cancer organoid, and monitored and / or analyzed over time. In some aspects, monitoring and analyzing the cancer cells in the organoid can provide a valuable tool for understanding the cancer and discovering potential treatments for the cancer. For example, the brain cancer organoid can be monitored and analyzed to investigate properties of the cancer, including proliferation, migration, invasion, and / or survival, in the context of cells that recapitulate aspects of the natural environment of the cancer. In addition, the brain cancer organoid can be used to identify therapeutic agents that may be useful for treating the cancer.

[0097] In some aspects, provided herein is a method for providing an organoid, a method of making an organoid, a method for culturing primary cancer cells, a method for culturing primary cancer cells obtained from a subject, a method for providing a brain cancer organoid, and / or a method for implanting primary cancer cells from a subject into a brain organoid. The method can comprise contacting a brain organoid with primary cancer cells in suspension to form a liquid droplet containing the brain organoid and primary cancer cells. The method can comprise providing a liquid droplet on a solid surface, the liquid droplet containing primary cancer cells in suspension and a brain organoid. The method can further comprise incubating the liquid droplet on a solid surface for a first period of time during which the liquid droplet is a hanging liquid droplet that adheres to and hangs beneath the solid surface. The primary cancer cells can become attached to and / or incorporated into the brain organoid. The primary cancer cells can become attached to and / or incorporated into the brain organoid, thereby forming a brain cancer organoid, organoid.

[0098] FIG. 1 illustrates an exemplary workflow of a method for producing a brain cancer organoid. In the figure, a brain organoid (shown as being spheroid in shape in this example) is contacted with cancer cells in suspension to form a liquid droplet containing the brain organoid on a solid surface. The surface is inverted to form a hanging liquid droplet. As shown in the figure, the hanging liquid droplet adheres to and hangs beneath the solid surface. The hanging liquid droplet is incubated for a first period of time, during which the cancer cells become attached to and / or incorporated into the brain organoid to form a brain cancer organoid. Oncethe brain cancer organoid is formed, the brain cancer organoid can be transferred to a culture condition, such as one not comprising a hanging droplet. For example, the culture condition can be in a culture well or a dish, for example on a low adhesion substrate. The brain cancer organoid can then be maintained, monitored and analyzed for any suitable period of time, and then harvested for further analysis.

[0099] In some aspects, the methods involving use of a hanging liquid droplet for providing an organoid described herein provide an efficient means to incorporate cells into organoids in general. Thus, in some aspects, the method can comprise contacting an organoid with cells in suspension that are not cancer cells. For example, instead of cancer cells, the cells in suspension can be any other suitable cell that a user desires to incorporate into an organoid. In addition, the organoid does not need to be a brain organoid, and can be any other suitable organoid. Various cells in suspension and organoids suitable for use in accordance with the methods described herein could be readily selected by one having ordinary skill in the art.

[0100] In some aspects, provided herein is a method for providing an organoid. The method can comprise contacting a brain organoid with cancer cells in suspension to form a liquid droplet containing the brain organoid and cancer cells. The method can further comprise incubating the liquid droplet on a solid surface for a first period of time during which the liquid droplet is a hanging liquid droplet that adheres to and hangs beneath the solid surface. The cancer cells can become attached to and / or incorporated into the brain organoid to form a brain cancer organoid.

[0101] The liquid droplet can be any suitable volume that allows formation of a hanging liquid droplet containing the brain organoid and cancer cells in suspension. For example, the liquid droplet can be between 1-10 microliters (pL), 10-20 pL, 20-30 pL, 30-40 pL, 40-50 pL, 50-60 pL, 60-70 pL, 70-80 pL, 80-90 pL, 90-100 pL, or more than 100 pL in volume. In some embodiments, the liquid droplet is 10-50 pL in volume. In some aspects, the exact volume of the hanging liquid droplet is not determined. For example, in some aspects, the hanging liquid droplet is formed by contacting a brain organoid on a solid surface in a minimal amount of media with a volume of cells in suspension, such as 10-15 pL of cells in suspension, to form the liquid droplet (e.g. as described in Example 1).

[0102] In some aspects, the liquid droplet is incubated as a hanging liquid droplet for a first period of time. The first period of time can be of any suitable duration and can be selected by a user in accordance with the specific cancer cells and brain organoids being used to allow for a suitable level of incorporation of cancer cells. The duration of the first period of time can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, orlonger, or can be any value derivable therein. For example, the duration of the first period of time can be less than 1 hour, 1-2 hours, 2-3 hours, 3-4 hours, 4-5 hours, 5-6 hours, 6-7 hours, 7-8 hours, 8-9 hours, 9-10 hours, 10-11 hours, 11-12 hours, 12-13 hours, 13-14 hours, 14-15 hours, 15-16 hours, 16-17 hours, 17-18 hours, 18-19 hours, 19-20 hours, 20-21 hours, 21-22 hours, 22-23 hours, 23-24 hours, or more than 24 hours. In some embodiments, the duration of the first period of time can be 8-12 hours.

[0103] In some aspects, the incorporation of cancer cells into the brain organoid can be assessed based on the percentage of cells in the organoid or a region of the organoid that are cancer cells after the first period of time. The cancer cells can comprise at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or more, of the cells in the brain cancer organoid, for example at the end of, or after the end of, the first period of time. In some aspects, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or more, of the cells in the brain cancer organoid or cancer cells or are derived from the cancer cells, such as after the first period of time.

[0104] In some aspects, a greater number and / or proportion of the cancer cells become attached to and / or incorporated into the brain organoid in comparison to a control method, such as a comparable method in which the brain organoid and cancer cells are not incubated in a hanging liquid droplet for the first period of time. The control method can be a method wherein, during the first period of time, the liquid droplet rests on top of the solid surface. The control method can be a method wherein, during the first period of time, the solid surface is a dish or a well, such as a flat-bottom, u-bottom, or v-bottom culture well.

[0105] Following formation of the brain cancer organoid, the method can further comprise transferring the brain cancer organoid to a culture condition and culturing the brain cancer organoid for a second period of time. In the culture condition, the brain cancer organoid does not need to be cultured in a hanging liquid droplet, and the brain cancer organoid can be cultured according to any suitable means for culturing the organoid. For example, the brain cancer organoid can be submerged in a suitable culture media on a low adhesion substrate. Once the brain cancer organoid is formed and transferred to the culture condition, the organoid can be monitored and analyzed, for example according to any of the methods provided herein, and for any suitable period of time. The cancer cells and / or brain cancer organoid can remain viable for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or more after formation of the brain cancer organoid. The cancer cells and / or brain cancer organoid can remain viable for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2months, at least 3 months, at least 6 months, or more, after formation of the brain cancer organoid.

[0106] The method can comprise or exclude analyzing one or more characteristics of cells in the brain cancer organoid after formation of the brain cancer organoid. For example, the cells in the brain cancer organoid can be analyzed at least one week after formation of the brain cancer organoid. The method can comprise analyzing one or more characteristics of the cancer cells in the brain cancer organoid. The one or more characteristics can comprise or exclude growth, proliferation, survival, gene expression, invasion, and / or migration.

[0107] In some aspects, the terms migration or invasion, with respect to a cancer, can refer to dissemination of cancer cells into surrounding healthy tissue that causes spread of the cancer into the healthy tissue. Cancer cells, such as within a cancer of a nervous system, can spread outside the tumor into the surrounding healthy tissue. This spreading can occur, in some instances, via a process similar to “mitotic somal translocation.” Mitotic somal translocation is displayed by outer radial glial cells and is a migratory behavior where the soma translocates towards the cortical plate before cytokinesis. Mitotic somal translocation is believed to be involved during brain development for germinal zone expansion.

[0108] The method can comprise or exclude determining growth of the cancer cells in the brain cancer organoid at least one week after formation of the brain cancer organoid. In an exemplary method to determine growth, the number of cancer cells in the brain cancer organoid can be assessed immediately after formation of the brain cancer organoid, and then at a later time point (e.g. 1 week or more after formation of the brain cancer organoid), and the cell numbers can be compared to determine if growth (i.e. proliferation) of the cancer cells occurred, and to what degree.

[0109] Similarly, the method can comprise or exclude determining the invasiveness of the cancer cells in the brain cancer organoid, by analyzing the location of cancer cells in the brain cancer organoid at different time points. For example, the cancer cells in the brain organoid can be imaged immediately after formation of the brain cancer organoid, and then at a later time point. In general, the cancer cells will be located peripherally within the brain cancer organoid immediately after formation. At a later time point, the cancer cells may invade new regions, such as deeper regions of the brain cancer organoid, thereby displaying invasiveness.

[0110] The methods provided herein can comprise or exclude assessing and / or detecting a marker specific for the cancer cells. The marker specific for the cancer cells can be a gene or gene product. The gene or gene product can be endogenous or heterologous. For example, the marker can be a fluorescent protein. Alternatively, the marker can be a gene or gene productthat is present in the cancer cells but not the non-cancerous surrounding cells of the brain organoid. One skilled in the art would readily be able to select a suitable method for tracking and analyzing specific cells, such as the cancer cells, within the brain cancer organoid.[OHl] In some aspects, the efficient methods of providing brain cancer organoids provided herein can enable using a plurality of the brain cancer organoids to screen agents that can affect the cancer cells. For example, a plurality of brain cancer organoids can be provided, and each brain cancer organoid can then be cultured in the presence (or absence) of various candidate agents. The cancer cells can be assessed for various characteristics, such as growth, survival, proliferation, migration, and / or invasion, and the characteristics can be compared among different conditions in which different candidate agents were used. In this way, agents can be identified that have a desired effect on the cancer cells, such as reducing growth, survival proliferation, migration, and / or invasion. Such agents can be identified as anti-cancer agents, which can be effective in treatments for cancer, in particular for the cancer from which the cancer cells of the brain cancer organoid were derived. Thus, it can be seen that the methods provided herein can be used in connection with identifying effective anti-cancer agents and therapies for patients having cancer. The method can comprise or exclude screening a candidate agent for activity in reducing cancer cell growth, proliferation, survival, and / or invasion. For example, the method can comprise culturing the brain cancer organoid in the presence of the candidate agent. In some aspects, reduced cancer cell growth, proliferation, survival, and / or invasion as compared to the cancer cell growth, survival, proliferation, and / or invasion, respectively, in the absence of the candidate agent identifies the candidate agent as having activity in reducing cancer cell growth, proliferation, survival, and / or invasion. The activity can comprise or exclude activity in reducing cell growth, proliferation, survival, and / or invasion of the cancer cells. The activity can comprise or exclude activity in reducing cell growth, proliferation, survival, and / or invasion of cancer in a subject. The candidate agent can comprise or exclude a small molecule, oligonucleotide, antibody, peptide, or protein. Such candidate agents and examples thereof are described in detail elsewhere and could be readily selected for testing in accordance with the described methods by one having ordinary skill in the art.

[0112] An antibody can be a polypeptide that specifically binds to an antigen. Typically, an antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains connected by disulfide bonds. Each heavy chain comprises a heavy chain variable region and a heavy chain constant region. Each light chain comprises a light chain variable region and a light chain constant region. Complementarity determining regions (CDR) in the variable heavyand / or light chains determine specificity of an antibody towards its antigen. Antibodies can be whole antibodies or antigen binding fragments thereof or derived therefrom. An antibody can be a monoclonal antibody, polyclonal antibody, human antibody, humanized antibody, chimeric antibody, and further engineered antibody. Examples of antibodies and fragments thereof include a variable domain fragment ("Fv", consisting of the VH and VL domains of a single arm of an antibody), Fab fragment (monovalent fragment consisting of the VH, VL, CHI and CL domains), Fab2 fragment (bivalent), Fab3 fragment (trivalent), Fab’ fragment (Fab with hinge region), F(ab’)2 fragment (bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region), Fd fragment (consisting of the VH and CHI domains), rlgG (reduced IgG or half-lgG), diabodies, triabodies, tetrabodies, minibodies, monovalent antibodies, divalent or multivalent antibodies comprising a fragment of more than one antibody, single chain variable fragment (ScFv), bis-scFv (bispecific), and derivatives of antibodies such as disulfide stabilized Fv fragments, CDR-comprising peptides, as well as epitope-binding fragments of any of the above.

[0113] The candidate agent can also be a chemotherapeutic agent, such as one suitable for inducing killing of a cancer cell, particularly, a cancer cell of the nervous system. Non-limiting examples of chemotherapeutic agents include temozolomide, carmustine, and lomustine. Additional such chemotherapeutic drugs are known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.

[0114] In certain aspects, the culturing methods disclosed herein may be used for screening a candidate agent for activity in reducing cancer cell growth and / or invasion, the method comprising: culturing the primary cancer cells in the brain organoid in the presence of the candidate agent and culturing the primary cancer cells in the brain organoid. Reduced cancer cell growth of and / or invasion as compared to the cancer cell growth and / or invasion, respectively, in absence of the candidate agent identifies the candidate agent as having activity in reducing cancer cell growth and / or invasion. The reduction in cancer cell growth may be a 5% reduction or more (e.g., a 10%, 20%, 30%, 40%, 50%, or more) compared to the growth in absence of the candidate agent. In certain aspects, growth may be measured by tumor volume. The reduction in cancer cell invasion may be determined by measuring spread of cancer cells outside the initial implantation site.

[0115] The cancer cells can be from any suitable source, and a person skilled in the art would be able to readily select cancer cells for use in the methods provided herein. The cancer cells can be from a subject. The cancer cells can be obtained from a biological sample from a subject that has cancer. The biological sample can comprise or exclude a resected tumor, abiopsy, a needle aspirate, a section, and / or a fluid, or a component of any of the foregoing. The subject can be a human subject. The cancer cells can comprise or exclude primary cancer cells. The cancer cells can comprise or exclude glioblastoma cells. The cancer cells can be from a tumor. The subject can have a cancer of the nervous system. The cancer cells can be from a cancer of the nervous system. Such cancers can affect the brain, spinal cord, a part of the neuroendocrine system, such as a neuroendocrine gland, and can include brain stem glioma, pineal astrocytic tumor, pilocytic astrocytomas, diffuse astrocytomas, anaplastic astrocytoma, glioblastoma, oligodendroglial tumor, mixed glioma, ependymal tumor, medulloblastomas, pineal parenchymal tumor, meningeal tumors, germ cell tumors, and craniopharyngiomas. The cancer of the nervous system can comprise or exclude brain stem glioma, pineal astrocytic tumor, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglial tumor, mixed glioma, ependymal tumor, medulloblastoma, pineal parenchymal tumor, meningeal tumor, germ cell tumor, or craniopharyngioma. The cancer of the nervous system can be glioblastoma. The glioblastoma can comprise or exclude neural, proneural, classical, or mesenchymal glioblastoma. The cancer of the nervous system can be astrocytoma.

[0116] In some aspects, the methods provided herein allow for direct implantation of cancer cells into brain organoids with minimal need for handling and / or processing of the cancer cells. The cancer cells can be obtained by resecting a tumor and dissociating the tumor to provide dissociated cancer cells in suspension. In some embodiments, prior to being contacted with the brain organoid, the cancer cells have not been: cultured; plated; trypsinized; passaged; expanded; and / or incubated in suspension or in two-dimensional culture under conditions for proliferation and / or expansion. In some embodiments, the cancer cells have not been frozen prior to being contacted with the brain organoid. However, any of the aforementioned steps, such as culturing, expanding, or freezing, can be performed prior to contacting the brain organoid with the cancer cells, if desired.

[0117] Any suitable brain organoid can be used in connection with the methods provided herein. A variety of methods for producing brain organoids are described and would be readily adapted for use in the methods provided herein by those having skill in the art. The brain organoid can be generated from stem cells, such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). The brain organoid can be generated from human embryonic stem cells (ESCs). The brain organoid can be generated from human induced pluripotent stem cells (iPSCs). The cells of the brain organoid can be genetically modified or not genetically modified. The cells of the brain organoid and / or the cancer cells can be human cells. The brainorganoid cells and cancer cells can be from the same subject or from different subjects. The brain organoid can be a cortical organoid. In some aspects, provided herein is an organoid (e.g. a brain cancer organoid) generated according to any of the methods described herein.

[0118] The brain organoid can be a neurosphere, neural aggregate, neural rossette, cortical spheroid, cortical organoid, cerebral organoid, or whole-brain organoid. The brain organoid may be a cortical organoid. A cortical organoid can be generated, for example, by culturing untransformed hESCs or iPSCs in a medium comprising a Rho kinase inhibitor, a Wnt signal inhibitor and a TGF-beta signal inhibitor on a low adhesion substrate to generate aggregates. Culturing can be carried out for a period of time sufficient to generate the aggregates. The method for generating cortical organoid can further comprise or exclude culturing the aggregates in a medium comprising a Wnt signal inhibitor and a TGF-beta signal inhibitor and lacking a Rho kinase inhibitor, on a low adhesion substrate to generate the cortical organoid. Culturing can be carried out for a period of time sufficient to generate the cortical organoid. In certain aspects, the cortical organoid can express or not express one or more of the telencephalon markers Foxgl and Six 3. The Wnt signal inhibitor may be IWR-1. The TGF- beta signal inhibitor may be SB431542. The Rho kinase inhibitor may be Y-27632. The aforementioned methods are meant to illustrate non-limiting examples of how organoids can be produced for use in the methods herein. Any other suitable methods for producing brain organoids and / or cortical organoids may be used in connection with the methods provided herein.

[0119] To address the issues presented by the conventional methods of culturing cancer cells in genetically modified organoids, certain embodiments of the invention provide a brain organoid generated from an ESC or an iPSC, the organoid further comprising implanted therein a cancer cell derived from a cancer of a nervous system, wherein the ESC or iPSC is not genetically modified to render it oncogenic. The ESC or iPSC can be a human ESC or human iPSC.

[0120] The cancer cell can be a primary cancer cell. The phrase “primary cancer cell” as used herein can refer to a cancer cell that is isolated from a cancer of a subject, for example, a tumor sample from a subject, and is cultured in a manner that maintains the cell’s viability and may allow the cell to grow but does not allow the cell to divide to a significant extent. Accordingly, when a cell is implanted in a brain organoid, it has not significantly divided since being removed from the subject. For example, the cancer cell may not have been cultured for a significant period of time. In certain aspects, the cancer cell may be isolated by dissociating a tumor sample in a suitable culture medium and implanting the dissociated cells. The tumorsample may be fresh or previously frozen. The dissociated cells may be implanted immediately or may be subjected to a selection step for isolating cells positive for a marker, such as any suitable marker. Exemplary markers can include or exclude PTPRZ1 or PTN. After implantation into the brain organoid, the cancer cell can grow and divide. When implanted into a brain organoid, a primary cancer cell can remain viable for at least one week (e.g., at least two weeks, at least three weeks, at least four weeks, or at least 60 days). Such cultures can find use in screening of agents that inhibit growth of cancers and / or inhibit expression of proteins that are required for rendering cancer cells invasive. For example, such cultures may be used for screening for agents that decrease expression of PTPRZ1 or PTN. Agents of interest that may be used for such screens may include or exclude small molecules, nucleic acids, peptides, and proteins, such as antibodies.

[0121] The primary cancer cell can be isolated from a cancer selected from a brain stem glioma, pineal astrocytic tumor, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglial tumor, mixed glioma, ependymal tumor, medulloblastoma, pineal parenchymal tumor, meningeal tumor, germ cell tumor, and craniopharyngioma. In certain aspects, the primary cancer cell may be a PTPRZ1 -positive cancer cell. PTPRZ1 is a member of the PTPR family. PTPRZ1 has two cytoplasmic tyrosine phosphatase domains, an alpha-carbonic anhydrase domain, chondroitin sulfate proteoglycans and a fibronectin type-ill domain. A secreted growth factor pleiotrophin (PTN) interacts with PTPRZ1 and inactivates the phosphatase activity of PTPRZ1. Such inactivation increases tyrosine phosphorylation status of other signaling molecules such as b-catenin, Fyn and RhoGAP.III. Fused brain organoids and related methods

[0122] In some aspects, provided herein are methods for analyzing cell migration. In some aspects, the method comprises analyzing the migration of target cells, such as cancer cells, from a first organoid to a second organoid that are fused together to form a fused organoid. In some aspects, the target cells are initially present in the first organoid and not present in the second organoid, and following fusion of the organoids together, migration of the target cells can be assessed based on the presence of the target cells in the second organoid after a period of time during which the fused organoid is incubated.

[0123] In some aspects, provided herein is a method for analyzing cell migration, the method comprising: a) contacting a first organoid containing target cells with a second organoid to form a fused organoid comprising the first organoid and the second organoid; b)incubating the fused organoid for a period of time; and c) determining the location of one or more of the target cells within the fused organoid.

[0124] An exemplary workflow of a method for analyzing cell migration using a fused organoid is shown in FIG. 4A. In the top panel of FIG. 4A, the figure shows a first organoid containing target cells (such as cancer cells), and a second organoid that does not contain the target cells. The first and / or second organoid can be brain organoids. The first organoid can be a brain cancer organoid, such as a brain cancer organoid formed by any of the methods provided herein, such as a method utilizing hanging droplet incubation to form a brain cancer organoid. Thus, the target cells can be the cancer cells of the brain cancer organoid. The figure shows that the first organoid and second organoid are co-cultured to form a fused organoid. In some embodiments, the first organoid is contacted with the second organoid, and the organoids form a fused organoid, as shown in the middle panel. The organoids can be contacted with one another by any suitable means. In some embodiments, the organoids are placed together in a partition, such as a dish or a well. In some embodiments, the partition does not comprise any additional organoids, such as a third organoid. In some embodiments, the organoids are placed in a tilted dish such that they are pulled by gravity towards the same location in order to contact one another. The organoids can become fused as a result of the contacting.

[0125] As shown in the middle panel of FIG. 4A, the fused organoid can resemble two visually distinguishable spheroids (corresponding to the first and second organoids) fused to one another at an interface, which appears as a furrow or narrowed region between the two organoids of the fused organoid. Thus, even after fusion, the first and second organoids can retain morphologically distinct domains that can be separated at a later timepoint, if necessary. The fused organoid can then be incubated for a period of time to allow migration of the target cells.

[0126] As shown in the bottom panel of FIG. 4A, following the incubation, the location of one or more of the target cells can be determined in order to assess migration. Because target cells are only present in the first organoid prior to the fusion, target cells present in the second organoid following incubation are indicative of migration of the target cells. The location of the target cells can be determined by any suitable means. For example, immunohistochemistry, immunofluorescence, fluorescence microscopy, and / or flow cytometry can be used to reveal the location of the target cells in the fused organoid. One skilled in the art would readily understand how to identify the location of the target cells within the fused organoid by various standard methods. The target cells can be identified based on genotype, gene expression, or expression of any suitable marker, such as a fluorescent protein. Similarly, the identity of thefirst and second organoid can be identified based on genotype, gene expression, or expression of any suitable marker, such as a fluorescent protein. For example, in some embodiments, the first organoid, second organoid, and / or target cells express (or do not express) markers that allow them to be distinguished (e.g. the first organoid can express GFP and / or the second organoid can express RFP). The location of the target cells can be identified within the fused organoid while the fused organoid is intact, for example using fluorescence microscopy. Alternatively, the location of the target cells can be identified after the first and second organoid of the fused organoid are separated, for example by cleaving the fused organoid at the interface. Once separated, the number of target cells present in the separated first organoid and second organoid can then be assessed by any suitable means, including those listed above. In some embodiments, after the separation, the number of target cells present in the separated organoids (e.g. the second organoid) can be assessed by flow cytometry, for example by determining the density and / or number of cells in the organoid expressing a marker specific for the target cells, such as a fluorescent protein (e.g. GFP).

[0127] In some aspects, the methods provided herein are compatible with methods for analyzing various characteristics of cells implanted in organoids, such as of cancer cells in brain organoids. In some aspects, provided herein is a method for analyzing cell migration. The method can comprise: a) contacting a first organoid containing target cells with a second organoid to form a fused organoid comprising the first organoid and the second organoid. The method can further comprise: b) incubating the fused organoid for a period of time. The method can further comprise: c) determining the location of one or more of the target cells within the fused organoid. In some aspects, by determining the location of the one or more target cells within the fused organoid after the incubating, aspects of the migration of the target cells can be determined. For example, if target cells provided in the first organoid are present in the second organoid after the period of time, then migration can be determined to have occurred (i.e. migration of the target cells from the first organoid to the second organoid).

[0128] The target cells can be any suitable cells for migration analysis. The target cells can comprise or exclude cancer cells, such as any suitable cancer cell or any cancer cell described herein, such as a cancer cell of the nervous system. The first and second organoid can be any suitable organoids. For example, the first and second organoids can be brain organoids. The first organoid can be a brain cancer organoid, such as a brain cancer organoid formed by any of the methods described herein, for example in Section II. Thus, the first organoid can be a brain cancer organoid, with the target cells being the cancer cells of the brain cancer organoid. The first organoid and / or the second organoid can be brain organoids.

[0129] The contacting in step a) can comprise or exclude incubating the first organoid and second organoid together in a partition. The partition can be a cell-culture well or dish. The bottom of the partition can be slanted and the first organoid and second organoid can contact one another when pulled downwards by gravity.

[0130] In some embodiments, the partition does not contain a third organoid, or any organoid other than the first organoid and second organoid. The contacting in step a) can comprise providing the first organoid and second organoid together in a composition, and wherein the composition does not comprise a third organoid. In some embodiments, the fused organoid does not comprise a third organoid. Thus, in some aspects, the method comprises forming a fused organoid by providing two isolated organoids together in the absence of other organoids.

[0131] In some aspects, while the fused organoid comprises a single overall structure of cells, the first organoid and second organoid of the fused organoid can still be distinguished. Thus, after formation of the fused organoid, the shape and morphology of the fused organoid can still reveal the presence of two organoids which remain morphologically distinct, which may be readily apparent upon visual inspection. For example, in some embodiments, the fused organoid does not simply resemble a single spheroid-shaped organoid after the fusion. Instead, the fused organoid resembles two organoids joined at a discrete interface. For example, the fused organoid can resemble two roughly ball-shaped groups of cells (corresponding to two spheroid-shaped organoids) joined at an interface. The first organoid and / or second organoid can be spheroid in shape (e.g. roughly in the shape of a sphere, which may be somewhat distorted, in accordance with the plain and ordinary meaning of spheroid, in particular in accordance with the use of spheroid in relation to spheroids in cell culture). The first and second organoids can be spheroid in shape, and fused at an interface between the first organoid and second organoid. The location of the interface can be at a furrow between the two spheroid organoids. For example, the furrow can be a narrowing at the midway point between the two spheroid organoids, that is smaller in diameter than the maximum diameter of the first organoid and second organoid. Thus, the maximum diameter of the interface can be smaller than the maximum diameter of the first organoid and smaller than the maximum diameter of the second organoid. The first organoid and the second organoid can also be distinguished from one another based on morphology, genotype, and / or expression of a marker. Thus, even if the two organoids cannot be distinguished from one another based on basic visual inspection, additional analysis can allow them to be distinguished (e.g. the first or second organoid can express GFP).One skilled in the art would readily be able to devise a scheme for distinguishing the two organoids using any number of methods.

[0132] In some aspects, determining the location of the one or more target cells within the fused organoid comprises detecting the presence of one or more target cells in the first organoid and / or the second organoid. Determining the location of the one or more target cells within the fused organoid can comprise quantifying the number or density of target cells in the first organoid and / or the second organoid. Determining the location of the one or more target cells within the fused organoid can comprise quantifying the number or density of target cells in the second organoid. Determining the location of the one or more target cells within the fused organoid can comprise detecting a marker specific for the target cells.

[0133] The marker specific for the target cells can be any suitable marker, such as an endogenous marker (e.g. a gene or gene expression product) or a heterologous marker (e.g. a transgene or expression product thereof, such as a fluorescent protein). The marker specific for the target cells can comprise or exclude a fluorescent protein. Determining the location of the one or more target cells can comprise or exclude any suitable method, such as fluorescence microscopy, immunohistochemistry, immunofluorescence, and / or flow cytometry. The method can comprise or exclude separating the first organoid from the second organoid prior to determining the location of the one or more target cells. The first organoid and second organoid can be separated, such as cleaved, at the interface, to separate the two organoids of the fused organoid. The method does not need to comprise separating the first organoid from the second organoid prior to determining the location of the one or more target cells, and the location of the target cells can be determined in the context of the intact fused organoid.

[0134] In some embodiments, the second organoid does not contain target cells prior to being contacted with the first organoid (and prior to fusion). Thus, the presence of target cells in the second organoid after the incubating in step b) can be indicative of migration and / or invasion. For example, the presence of target cells in the second organoid can indicate that the target cells migrated from the first organoid to the second organoid. Thus, simply determining the number of target cells in the second organoid after incubation can provide a measure of migration, without needing to determine the precise location of the target cells within the fused organoid. Increased numbers of target cells in the second organoid can be indicative of increased migration and / or invasion.

[0135] In some aspects, the fused organoids containing target cells and related methods provided herein can be used in connection with screening various conditions and / or agents that may affect migration or invasion. For example, a plurality of the fused organoids can be formedin accordance with the described methods, and incubated in the presence (or absence) of various different candidate agents. Migration (e.g. invasion) from the first organoid to the second organoid can be assessed and compared between the various conditions, and agents or conditions can be identified that result in altered (e.g. decreased) migration. Thus, in some aspects, the methods provided herein can provide means to identify agents that may be effective in altering cellular activity, such as decreasing migration and / or invasion of cancer cells (e.g. wherein the target cells are cancer cells). In some aspects, the method comprises or excludes perturbing the fused organoid and / or the target cells and determining the effect of the perturbation on target cell migration. The effect of the perturbation on target cell migration can be determined in comparison to a control fused organoid in which the perturbation is not performed. The perturbation can comprise or exclude providing target cells with a different genetic background than the target cells in the control fused organoid. The perturbation can comprise or exclude altering gene expression in the target cells and / or fused organoid. The perturbation can comprise or exclude incubating the fused organoid and / or the target cells in the presence of a candidate agent. Reduced target cell migration in the presence of the candidate agent can identify the candidate agent as having activity in reducing cell migration and / or invasion.

[0136] The target cells can be any suitable cell type, such as a cell type provided herein, including any of the cell types and cancer cell types described in Section II. For example, the target cells can be cancer cells and thus the reduced cell migration and / or invasion can be reduced cancer cell migration and / or invasion. The candidate agent can be a small molecule, oligonucleotide, antibody, peptide, or protein. The cancer cells can comprise primary cancer cells. The cancer cells can be from a tumor. The cancer cells can be from a cancer of the nervous system. The cancer of the nervous system can comprise or exclude brain stem glioma, pineal astrocytic tumor, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglial tumor, mixed glioma, ependymal tumor, medulloblastoma, pineal parenchymal tumor, meningeal tumor, germ cell tumor, or craniopharyngioma. The cancer of the nervous system can be glioblastoma. The cancer cells can comprise glioblastoma cells. The cancer cells can be from a subject. The cancer cells can be obtained from a biological sample from a subject that has cancer. The subject can be a human subject.IV. Cell Culture and Media

[0137] The methods provided herein employ methods of cell culture, including the culture of organoids, such as brain organoids. Organoids provide a venue to investigate cell behaviorin the context of an environment that mimics physiological conditions present in vivo, such as in the brain, or other organs or microenvironments. Any suitable culture conditions, such as those known in the art, may be used in connection with the methods provided herein to support the described cells and organoids.

[0138] The culture conditions and media used may be any of those described in the Examples section, but are not limited to the conditions described in the Examples. The culture media may comprise Normal Media, as described in the Examples. Various alterations and substitutions can be made to the components of the Normal Media. The media can comprise or exclude any of the following components: serum (such as fetal bovine serum (FBS)), DMEM / F-12 with Glutamax, growth factors (e.g. epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF) or PDGF subunit A (PDGFAA)), vitamins (e.g. vitamin A), insulin, lipids, and other components. Various components can be included or excluded to direct, influence, or investigate outcomes or behaviors of the cells and / or organoids. Table El in the Examples provides examples of how various factors can be included or excluded to promote expected outcomes.

[0139] The medium can comprise or exclude vitamins. In some embodiments, the medium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of any of the following (and any range derivable therein): biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the medium includes combinations thereof or salts thereof. In some embodiments, the medium comprises biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some embodiments, the vitamins include or consist essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, or combinations or salts thereof. In some embodiments, the medium further comprises proteins. In some embodiments, the proteins comprise albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. In some embodiments, the medium further comprises one or more of the following: corticosterone, D- Galactose, ethanolamine, glutathione, L-camitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, or combinations thereof. In some embodiments, the medium comprises one or more of the following: a B-27® supplement, xeno-free B-27® supplement, GS21™ supplement, or combinations thereof. In some embodiments, the medium comprises or futher comprises amino acids, monosaccharides, inorganic ions. In some embodiments, the aminoacids comprise arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof. In some embodiments, the inorganic ions comprise sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In some embodiments, the medium further comprises one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In certain embodiments, the medium comprises one or more vitamins discussed herein and / or one or more proteins discussed herein, and / or one or more of the following: corticosterone, D-Galactose, ethanolamine, glutathione, L- carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I- thyronine, a B-27® supplement, xeno- free B-27® supplement, GS21™ supplement, an amino acid (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharide, inorganic ion (such as sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus) or salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese.

[0140] The medium in certain aspects can be prepared using a medium used for culturing animal cells as their basal medium, such as any of AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, aMEM, DMEM, Ham, RPMI-1640, and Fischer's media, as well as any combinations thereof, but the medium may not be particularly limited thereto as far as it can be used for culturing animal cells. Particularly, the medium may be xeno-free or chemically defined. The medium can be a serum-containing or serum-free medium, or xeno- free medium. From the aspect of preventing contamination with heterogeneous animal-derived components, serum can be derived from the same animal as that of the cells. Serum-free medium can refer to medium with no unprocessed or unpurified serum and accordingly, can include medium with purified blood-derived components or animal tissue-derived components (such as growth factors).

[0141] The medium may contain or may not contain any alternatives to serum. The alternatives to serum can include materials which appropriately contain albumin (such as lipid- rich albumin, bovine albumin, albumin substitutes such as recombinant albumin or a humanized albumin, plant starch, dextrans and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'- thiolgiycerol, or equivalents thereto. The alternatives to serum can be prepared by the method disclosed in International Publication No. 98 / 30679, for example (incorporated herein in its entirety). Alternatively, any commercially available materials can be used for moreconvenience. The commercially available materials include knockout Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).

[0142] In further embodiments, the medium may be a serum-free medium that is suitable for cell development. For example, the medium may comprise B-27® supplement, xeno-free B-27® supplement (available at world wide web at thermofisher.com / us / en / home / technical- resources / media-formulation.250. html), NS21 supplement (Chen et al., J Neurosci Methods, 2008 Jun 30; 171(2): 239-247, incorporated herein in its entirety), GS21™ supplement (available at world wide web at amsbio.com / B-27.aspx), or a combination thereof at a concentration effective for producing T cells from the 3D cell aggregate.

[0143] In certain embodiments, the medium may comprise one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of the following: Vitamins such as biotin; DL Alpha Tocopherol Acetate; DL Alpha-Tocopherol; Vitamin A (acetate); proteins such as BSA (bovine serum albumin) or human albumin, fatty acid free Fraction V; Catalase; Human Recombinant Insulin; Human Transferrin; Superoxide Dismutase; Other Components such as Corticosterone; D-Galactose; Ethanolamine HC1; Glutathione (reduced); L-Carnitine HC1; Linoleic Acid; Linolenic Acid; Progesterone; Putrescine 2HC1; Sodium Selenite; and / or T3 (triodo-I-thyronine).

[0144] The medium may comprise or exclude externally added ascorbic acid. The medium can also contain or exclude one or more externally added fatty acids or lipids, amino acids (such as non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidant substances, 2-mercaptoethanol, pyruvic acid, buffering agents, and / or inorganic salts.

[0145] One or more of the medium components may be added at a concentration of at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, pg / ml, mg / ml, or any range derivable therein.

[0146] Other culturing conditions can be appropriately defined. For example, the culturing temperature can be about 20 to 40°C, such as at least, at most, or about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40°C (or any range derivable therein), though the temperature may be above or below these values. The CO2 concentration can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (or any range derivable therein), such as about 2% to 10%, for example, about 2 to 5%, or any range derivable therein. The oxygen tension can be at least or about 1, 5, 8, 10, or 20%, or any range derivable therein.

[0147] In additional embodiments, any genetic modification compositions or methods may be used to introduce exogenous nucleic acids into cells or to edit the genomic DNA, such as gene editing, homologous recombination or non-homologous recombination, RNA-mediatedgenetic delivery or any conventional nucleic acid delivery methods. Non-limiting examples of the genetic modification methods may include gene editing methods such as by CRISPR / CAS9, zinc finger nuclease, or TALEN technology.

[0148] Any suitable culture vessels can be used for the methods provided herein, including: flask, flask for tissue culture, dish, petri dish, dish for tissue culture, multi dish, micro plate, micro-well plate, multi plate, multi-well plate, micro slide, chamber slide, tube, tray, culture bag, and roller bottle, as long as it is capable of culturing the cells and / or organoids. The cells and / or organoids may be cultured in a volume of at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range derivable therein, depending on the needs of the culture. The culture vessel can be cellular adhesive or non-adhesive and selected depending on the purpose.EXAMPLES

[0149] The following examples are included to demonstrate aspects of the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.Example 1: Efficient implantation of cancer cells into a brain organoid by hanging liquid droplet incubation

[0150] Brain cancer organoids (brain organoids containing cancer cells) were generated by incubating brain organoids with an exemplary cancer cell type (glioblastoma) in a hanging liquid droplet, in order to implant the cancer cells into the organoid.

[0151] Brain cancer organoids containing glioblastoma cells (GBM) were generated using a hanging liquid droplet method, generally according to the workflow shown in FIG. 1. GFP- labeled glioblastoma (GBM) cells were centrifuged and resuspended in GBM / human cortical organoid culture media to form a GBM single-cell suspension. A 10 mL culture dish was prepared, containing 10 mL of base media. The base media was provided to prevent media evaporation during the subsequent hanging drop co-culture of glioblastoma cells and brain organoids. Week 8 human cortical organoids were carefully transferred to the inside of the lidof the culture dish, using autoclaved 1000 uL pipettes with wide opening, while ensuring that individual organoids remained intact and separate from one another. Excess media was removed from the organoids, and 10-15 uL of the GBM single-cell suspension was added on top of each organoid to form a liquid droplet on the surface of the inside of the culture dish lid. The culture dish lid was inverted such that the liquid droplets were hanging liquid droplets, and the lid was placed on top of the 10 mL culture dish containing the base media. The dish was placed in an incubator at 37°C and 5% CO2 for 8-12 hours to allow attachment and / or incorporation of the GBM cells into the brain organoids, thereby forming brain cancer organoids.

[0152] After hanging liquid droplet incubation, the brain cancer organoids were transferred to ultra-low attachment 6-well or 24-well dishes in culture media, and the brain cancer organoids were imaged by fluorescence microscopy to assess incorporation of GBM cells into the brain organoid. As shown in FIG. 2, a ring of GFP-labeled GBM cells around the organoids was observed immediately after the hanging liquid drop incubation. The result was highly consistent among the observed organoids.

[0153] The brain cancer organoids were maintained in culture for monitoring and analysis for several weeks (for example, survival for 4 weeks or more was observed), during which time GBM cells remained present and viable, and migration and proliferation of GBM cells was observed in the brain cancer organoids.

[0154] The results demonstrate a method for efficiently and consistently implanting cancer cells into brain organoids to produce brain cancer organoids for subsequent monitoring and analysis of the cancer cells in the organoid environment.Example 2: Increased efficiency of glioblastoma implantation using hanging liquid droplet incubation

[0155] Methods for producing a brain cancer organoid using a hanging liquid droplet incubation or not using hanging liquid droplet incubation were compared in terms of efficiency of incorporation of cancer cells.

[0156] Brain cancer organoids were generated by three different methods, each method involving incubating the liquid droplet in a different configuration, as illustrated in FIG. 3A. The three methods were: 1) the hanging liquid droplet method described above in Example 1; 2) the method described in Example 1, with the exception that the liquid droplet was incubated as an upright liquid droplet on a surface above the culture medium for preventing evaporation;and 3) the method describe in Example 1, with the exception that the liquid droplet was incubated in a v-bottom well. Following the liquid droplet incubation, the resulting brain cancer organoids were assessed for incorporation of the GFP+ GBM cells. In this experiment, a region of interest defined with respect to the organoid outer boundary was analyzed for the percentage of cells that were GFP+. Consistent with Example 1, brain cancer organoids formed by the hanging liquid droplet method had a high density of incorporated GBM cells after incubation. In contrast, the alternative methods that did not involve hanging liquid droplet incubation had comparatively much lower incorporation of GBM cells after incubation. Dramatically improved incorporation of GBM cells was visually apparent by fluorescence microscopy (as shown in FIG. 3B), and was statistically significant upon quantification (FIG. 3C).

[0157] The results further support the surprising advantage of the methods involving incubation in a hanging liquid droplet for efficiently forming brain cancer organoids. The high efficiency of incorporation supports that primary cancer cells, can be directly and efficiently incorporated into brain cancer organoids.Example 3: Candidate anti-cancer agent screening in brain cancer organoids

[0158] Brain cancer organoids are made using hanging liquid droplet incubation according to the method described in Example 1 and transferred to culture conditions. In some examples, the cancer is a primary cancer from a human subject, such as from a glioblastoma tumor in the subject. The brain cancer organoids are cultured under normal control conditions, or cultured in the presence of various candidate agents for affecting the implanted cancer cells. Cancer cell growth, proliferation, survival, invasion, and migration, are monitored and analyzed for up to several weeks. Agents are identified that reduce the cancer cell growth, proliferation, survival, invasion, and migration. The identified agents represent potential agents for treatment of cancer, for example in the subject from which the cancer was obtained. In some examples, the identified agents are used to treat the cancer, such as the glioblastoma, in the patent.Example 4: Fused organoid assay for assessing cell migration

[0159] An assay for assessing migration of cancer cells in brain cancer organoids was developed, for example as illustrated in FIG. 4A.

[0160] Gliomasphere (GS) cells expressing a GFP reporter were digested into single-cell suspension with TrypLE Express at room temperature for 5 minutes and resuspended in media (Media #3, described below) at a concentration of 50,000,000 cells / mL. The GS cells weretransplanted onto cortical organoids of age week 6-10 using the hanging drop technique as described, for example, in Example 1.

[0161] The transplanted organoids were transferred to ultra-low attachment 6-well plates 12-16 hours later. After 24 hours of culture in the ultra-low attachment 6-well plates, the transplanted organoids were gently rinsed with warm PBS to remove any loosely engrafted GS cells and transferred to an ultra-low attachment 24-well plate. Each well contained the transplanted organoid and a line- and age-matched untransplanted organoid. The co-culture of the transplanted and untransplanted organoid facilitated fusion to form a single fused organoid. The 24-well plate was left slanted and undisturbed in the 37°C incubator for 5 days before the first media change. The fused organoids were maintained in Media #3 with 3 media changes each week for 3-4 weeks before harvest.

[0162] Fused organoids were assessed, for example by fluorescence imaging to observe migration of the GFP+ GS cells into the untransplanted organoid of the fusion. To harvest, fused organoids were carefully separated at the site of fusion with a feather scalpel and a dissection microscope. The fused or severed organoids were processed and assessed by flow cytometry and immunofluorescence. Migration of GFP+ GS cells into the untransplanted organoid of the fusion was observed.

[0163] The described assay supports an in vitro method of assessing cell migration, such as cancer cell migration, for example using fused organoids formed by the hanging droplet method described above. The assay has utility, for example, in assessing candidate agents for anti-cancer activity, such as the ability to inhibit cancer cell migration.Example 5: Assessment of gliomasphere (GS) cell migration in fused organoids following gene expression knockdown

[0164] Gliomasphere (GS) cell migration was assessed using the fused organoid assay after knockdown of genes in the GS cells and / or the surrounding organoid cells.

[0165] Gliomasphere (GS) cells (GS005 or GS025 cell lines) were infected with virus to deliver short hairpin RNAs (shRNA) targeting (i) a scrambled negative control sequence, (ii) PTPRZ1, or (iii) PTPRZ1 and PTN.

[0166] The infected GS cells were transplanted into brain organoids that 1) expressed mCherry, and 2) had either a scramble shRNA or PTPRZ1 knockdown shRNA background. The transplantation was performed using the hanging droplet technique, for example as described in Example 1.

[0167] Each transplanted organoid was allowed to fuse with a line and age-matched untransplanted organoid (i.e. not containing GS cells) 2 days post-transplantation, for example as described in Example 5. The fused organoids were left undisturbed for 5 days and culture medium was refreshed every 2-3 days for 3-4 weeks. The fused organoids were monitored and assessed. Finally, the fused organoids were severed with a scalpel and analyzed by FACS for the number of GFP+ GS cells in each of the organoids.

[0168] FIG. 4B shows an exemplary immunofluorescence image of GS cells (red) and brain organoid cells (green) in a fused organoid. FIG. 5 shows the percentage of GFP+ GS cells that migrated into the untransplanted organoid under the different experimental conditions. FIG. 6 shows the fold change in migration for each condition using organoids with the PTPRZ1 knockdown background normalized to the scramble shRNA background. The results show that migration of GS cells was increased in organoids with a PTPRZ1 knockdownbackground. FIGS. 7A-F and FIGS. 8A-F show exemplary fluorescence microscopy images, each image showing a GFP+ GS cell-transplanted organoid fused with an untransplanted organoid after allowing time for migration. FIGS. 7A-F show images from GS005 GS cell line, and FIGS. 8A-F show images from the GS025 cell line. Percent migration quantification is shown for each exemplary image.

[0169] The results show that the described assay can be used to assess and detect differences in cellular activity, such as migration, under different conditions, and support the utility of the fused organoid migration assay in assessing cancer cells and identifying anticancer agents.Example 6: Human Organoid Tumor Transplantation Identifies Functional Glioblastoma - Microenvironmental Communication Mediated by PTPRZ1

[0170] Complex cell autonomous and nonautonomous properties modulated by tumor microenvironmental cues drive glioblastoma, an aggressive and deadly form of brain cancer. Here, the inventors leverage a novel human organoid tumor transplantation (HOTT) system to explore how extrinsic cues modulate glioblastoma cell type heterogeneity, migration, and cell fate specification. They show that HOTT recapitulates the core features of major patient tumor cell types and key features of peritumor cell types. Exploration of patient tumor - microenvironment interactions in HOTT highlighted PTPRZ1, a receptor tyrosine phosphatase implicated in tumor migration, as a key player. They performed tumor and microenvironmental knockdown experiments, uniquely enabled by the human microenvironment provided by HOTT, observing that tumor knockdown of PTPRZ1 recapitulated previously described roles in migration and maintaining progenitor identity. Unexpectedly, environmental PTPRZ1 knockdown drove opposite migration and cell fate changes in the tumor, even when the tumor was not manipulated. This is a previously undiscovered mode of tumor-microenvironmental communication, highlighting the need to study human glioblastoma in the context of a human microenvironment such as HOTT.

[0171] Glioblastoma (GBM) is an aggressive form of adult brain cancer, with limited treatment options and average survival of 12 - 18 months post diagnosis (Omuro and DeAngelis, 2013; Wen et al., 2020). Molecular profiling of GBM has long shown inter- and intra-tumoral cell type heterogeneity (Neftel et al., 2019b; Ravi et al., 2022; Tirosh et al., 2016; Venteicher et al., 2017), with this diversity of cell types possibly contributing to difficulty in developing effective treatments (Couturier et al., 2020; Neftel et al., 2019b). Cell typesidentified in GBM include ones reactivated from cortical development such as outer radial glia (Bhaduri et al., 2020b; Wang et al., 2020). Outer radial glia are a subtype of neural stem cells (Hansen et al., 2010; Pollen et al., 2015) abundant in the developing human cortex compared to the rodent where they are exceptionally sparse (Lui et al., 2011). Previous research also suggests that other subtypes in the tumor may also reflect human neurodevelopmental programs (Bhaduri et al., 2020b; Couturier et al., 2020), emphasizing the emerging need to study GBM in human patient relevant model systems.

[0172] As models to study GBM have expanded, so has the knowledge of how they interact with their microenvironment. Recent studies have highlighted that GBM cells interact with normal cell types (neurons, astrocytes etc.) through a variety of mechanisms. For example, brain tumors are increasingly being observed to electrically and synaptically integrate into brain circuits (Krishna et al., 2023; Venkatesh et al., 2019), leveraging properties of neurons to promote invasion through the brain (Venkataramani et al., 2022) and being interconnected into functional networks through electrical activity and microtube connections (Osswald et al., 2015). Together, the study of the tumor microenvironment (TME) has evolved into the new field of cancer neuroscience (Mancusi and Monje, 2023; Winkler et al., 2023), highlighting that unique features of the brain make this TME specifically conducive to GBM biology. However, given that the human cortex is markedly different than the rodent in terms of cell types and structural features (Bakken et al., 2021), many open questions exist regarding how the interactions between the human brain and heterogeneous GBM cell types promote key aspects of brain tumor biology. These questions include understanding how tumor - TME interactions drive tumor growth, but also how these interactions mediate cell fate specification and tumor migration.

[0173] It is well understood that during human cortical development, cell fate specification is a crucial process in generating the diverse cell types that ultimately comprise cortical layers and distinct functional areas (Cadwell et al., 2019). Given a subset of these cell types are reactivated in GBM, then understanding how cell fate is regulated in this context is additionally essential to understanding tumor progression. Indeed, it is known that tumor cell types change over the progression of the disease or across contexts (Neftel et al., 2019b), sometimes in response to treatments such as radiation (Gupta and Burns, 2018; Muthukrishnan et al., 2022; Pajonk et al., 2010) and in other cases as recurrent tumors that emerge with more aggressive cell types (Wang et al., 2022). Normal development is characterized by a tightly regulated set of intrinsic and extrinsic factors that influence this cell fate specification and also behavior ofdividing and migrating cells; the inventors reasoned that it would be important to explore these features in the context of GBM and a human TME.

[0174]

[0142]

[0144] Existing organoid transplantation models have relied on established organoid growth conditions for tumor growth (Bhaduri et al., 2020b; Linkous et al., 2019), but have not been optimized in the same way that two-dimension cell line culture has been developed (Hasselbach et al., 2014). The inventors considered that the status quo might not be the most ideal condition for replicating the heterogeneity and molecular features of patient tumors, and sought to integrate an analysis of how media conditions extrinsically influence tumor cell fate with the goal of establishing and interrogating a model for the investigation of how the tumor and the TME reciprocally interact to promote tumor migration and cell fate specification. Therefore, the inventors decided to explore microenvironmental conditions in a human organoid tumor transplantation model (HOTT), beginning with the characterization of different media conditions. It was found that, not only does HOTT recapitulate patient tumor characteristics, but it also recapitulates important features of the peritumor environment cell types that differ from the healthy adult brain. Thus, HOTT is an ideal model to understand cell - cell relationships between the tumor and its organoid microenvironment. These efforts provided confidence in how the HOTT system recapitulates tumor heterogeneity and cell type fidelity, identifying novel tumor - TME interactions mediated by an outer radial glia marker gene, PTPRZ1, in driving tumor migration and cell fate specification changes. Although it was validated, as previously described, that PTPRZ1 is required for tumor migration and maintenance of sternness within GBM cells, it was also discovered that environmental knockdown of PTPRZ1, without modifying the tumor directly, results in increased tumor migration and sternness. This is a previously undescribed mechanism by which the tumor and the TME communicate, and is a novel description of PTPRZ1 biology that is independent of its catalytic activity. This study highlights the value of the HOTT system as a tool to study and manipulate TME interactions, and further emphasizes the need to contextually study human GBM biology within a TME.A. Results:1. Human Organoid Tumor Transplantation (HOTT) is a valuable tool to study intrinsic and extrinsic regulators of GBM biology

[0175] The inventors use a system they termed the HOTT system to explore both intrinsic and extrinsic features of five GBM patient tumors (FIG. 9A). This approach closely mimicked the direct from patient organoid transplantation system previously described (Bhaduri et al.,2020b). Each of these tumors was dissociated and labeled with a lentivirus expressing green fluorescent protein (GFP), and the input tumor was analyzed with single-cell or single-nucleus sequencing. As a substrate for the GBM cell transplantation, the inventors used human embryonic stem cell (hESC) derived cortical organoids generated using established protocols (Bhaduri et al., 2020a; Kadoshima et al., 2013; Pollen et al., 2019). In order to maximally leverage the cell type heterogeneity that exists in cortical organoids while maintaining the feasibility of a consistent organoid pipeline for tumor transplantations, the inventors chose to perform the transplantations at stages after peak neurogenesis and during the process of gliogenesis. Although organoids are a developmental model, the inventors found here that they mimic important aspects of the TME as well, making them an excellent tool for the study of tumor - TME interactions.

[0176] To optimize tumor engraftment, the inventors tested multiple methods of tumor transplantation and observed that the novel hanging drop method resulted in the highest engraftment efficiency FIG. 15B, see Methods). Using this engraftment strategy, the inventors subjected each tumor implanted in the HOTT system to 5 different media conditions (FIG. 15C). These media conditions were designed based upon commonly utilized gliomasphere growth methods and with the knowledge that certain media components such as FBS are associated with poor growth and differentiation (Lee et al., 2006). Thus, the inventors used the standard organoid media minus FBS, predicting this would drive more progenitor populations. Because gliomasphere cultures are often supplemented with growth factors (Lee et al., 2006), the inventors included a condition where they added fibroblast growth factor (FGF) and epidermal growth factor (EGF) to this FBS depleted media, as well as a parallel condition supplementing platelet derived growth factor (PDGF), a ligand for PDGFRA expressing oligodendrocyte precursor cells (Zhu et al., 2014), hypothesizing that distinct growth conditions would yield unique fractions of progenitor populations in the tumor. The inventors additionally used the standard organoid ‘Media 3’ (M3) (Methods) as a baseline, and a condition where they added B27, hypothesizing that this may generate more tumor derived neuronal populations.

[0177] After 3-4 weeks in culture the inventors isolated the tumor cells using fluorescent activated cell sorting (FIG. 15D) and performed single-cell RNA-sequencing on the tumor cells as well as a subset of the normal organoid cells and paired tumor naive organoids from the same differentiation batch. In parallel, HOTTs were fixed for immunofluorescence and fluorescent in situ hybridization assays (FISH, using RNAScope). The sequenced cells were analyzed for copy number variation (CNV) (Tickle T, 2019) (FIG. 15E), accounting for anyfalse positive or negatively sorted cells. Initial analyses were performed with the tumor cells only, and annotations were performed by projecting the collected data onto a reference metaatlas that is a compendium of published datasets (Bhaduri et al., 2020b; Couturier et al., 2020; Darmanis et al., 2017; Jacob et al., 2020; Neftel et al., 2019a; Yu et al., 2020; Yuan et al., 2018) and were benchmarked against existing annotations commonly used in the literature (FIG. 16A - B). The inventors observed across tumors a strong representation of most GBM cell types, with lower representation of mature oligodendrocyte, mesenchymal and immune related programs (FIG. 9B). Across media conditions and across tumors, the inventors saw few biases in terms of cell type representation and in UMAP space observed similar dimensions occupied by the 5 tumors analyzed (FIG. 9B). These initial analyses suggest that HOTT recapitulates tumor heterogeneity and cell types that have been previously described in human GBM.2. HOTT cell type composition is preserved across environmental conditions

[0178] Although there was excellent representation of most cell types across tumors, the inventors wanted to quantify the cell type composition for each tumor and media condition (FIG. 10A, FIG. 16C). Overall, the inventors saw that most media conditions had relatively consistent distributions across cell types, with the exception that there were more progenitors in the FGF and EGF supplemented condition, consistent with prior literature (Lee et al., 2006) and the inventors’ own expectations. However, across conditions the inventors observed that even in growth conditions with FBS, which is notorious for inducing neural stem cell differentiation and thereby losing progenitor populations that could give rise to cancer stem cells (Janiszewska et al., 2012; Wakimoto et al., 2009), they were able to maintain progenitor identities, such as neural progenitor cells and outer radial glia, both by single-cell analysis and immunofluorescence for sternness markers NES and SOX2 FIG. 10B). This deviation from the literature suggests that the presence of a human microenvironment within HOTT changes the way that GBM responds to media conditions, including FBS (FIG. 10A).

[0179] The inventors validated the cell type distributions observed via single-cell RNA- sequencing with immunofluorescence analysis for cell type marker genes. Notably, across media conditions, most markers including HOPX (outer radial glia), CTIP2 (deep layer neurons), and GFAP (astrocytes) were consistent across media conditions, though the increased progenitor compartment in HEF media conditions was also observed (FIG. 10C, FIG. 16D). However, across conditions, the only media condition in which engraftment was achieved across all replicates was in media conditions including FBS in the media (FIG. 10A, FIG. 16C). Across media conditions, the number of GFP+ cells identified via immunofluorescencematched the number of single-cells retrieved for the analysis (FIG. 16E), with the highest numbers in standard organoid media and the FBS depleted, FGF and EGF supplemented condition. These data suggested that progenitor cells are maintained across media conditions, and that although FBS depleted, FGF and EGF supplemented media promoted the most progenitor cells, standard organoid growth conditions were, in fact, the most reliable.3. HOTT shows fidelity to primary tumor cell types

[0180] Given that tumor heterogeneity could be observed across media conditions in the HOTT system, the inventors sought to explore whether the heterogeneity and cell type fidelity was altered in the HOTT system compared to the primary matched tumors and the meta-atlas of published primary molecular profiles. The inventors first looked for how the cell types across media conditions compared to patient tumor heterogeneity by leveraging a correlative analysis in which they compared clusters from individual HOTT media conditions to their matched patient cell types (FIG. 11 A). The inventors observed that not only were diverse patient cell types represented across media conditions, but also many clusters were highly correlated to the original patient tumor, indicating tumor heterogeneity is well preserved in HOTT. Similar observations were made when comparing to other published annotations (FIG. 17A-B), suggesting that regardless of the granularity of the annotation, HOTT recapitulates patient tumor heterogeneity.

[0181] In instances of neurological disease, transcriptomics programs relating to key features of functional biology can be altered in parallel cell type (Jourdon et al., 2023; Mathys et al., 2023), and the inventors sought to explore if this was the case in GBM in general, and whether individual media conditions impacted this transcriptional identity. To perform this analysis, the inventors leveraged two major strategies. First, they performed single-sample gene set enrichment analysis (ssGSEA) (Barbie et al., 2009; Subramanian et al., 2005), using the transcriptomic signatures of cell types in the meta-atlas as the reference gene sets. The inventors then plotted the enrichment scores across all clusters across media conditions (FIG. 17B), noting that high enrichment was observed in each cell type interrogated, and that the distributions were similar across media conditions, suggesting transcriptional programs of parallel cell types across media conditions were largely unaltered. The inventors additionally explored the predicted annotation scores when comparing the HOTT tumor cell types to the reference meta-atlas. By graphing these scores in a bubble plot, the inventors noted that when comparing to primary tumors, HOTT better recapitulates of certain cell types including cycling, neuronal, astrocyte and radial glial cells (FIG. 11C), while mesenchymal cells are less wellrepresented, though they are comparable to the scores generated by the inventors for the matched primary tumors from these samples, indicating fidelity to their primary counterpart.

[0182] Given the reactivation of developmental programs in GBM, the inventors also performed additional module analysis to explore the expression of recently described neurodevelopmental programs (Nano et al., 2023) across the HOTT media conditions compared to the primary tumor (FIG. 17B). The inventors observed similar module activity for most programs, again corroborating the fidelity of the model to the primary tumor context. Nuanced differences related to cell fate, metabolism and immune function were noted. The inventors interrogated these differences further by performing differential gene expression analysis between the matched primary tumors and those in the HOTT system. The inventors observed that immune function was up in primary tumors (FIG. 1 ID). This expected given that the inventors are unable to propagate immune cells in this system and that this is largely true of patient derived models (Akter et al., 2021). Consistent with the observations in the module analysis, in the HOTT system, the inventors observed an upregulation of programs related to translation, nervous system development and axon guidance (FIG. 1 ID), suggesting that the model promotes the expansion of the neurodevelopmental lineages. This may create a unique opportunity to study questions of cell fate specification in this system. These data together provided a nuanced understanding of which cell types are best recapitulated in the HOTT system, highlighting that although proportions of cells can vary based upon extrinsic environmental cues as modulated through the media, the heterogeneity and fidelity of these cell types was consistent across conditions.

[0183] Compared to other existing organoid models, HOTT uniquely enables the study of the direct interactions between the direct from patient tumor samples and a human TME. The inventors sought to leverage this aspect of HOTT and first asked what cell types were present in the organoid microenvironment. The inventors annotated these non-tumor populations by projecting to cell types from the developing human brain (FIG. 1 IE, FIG. 18 A). As expected, the inventors observed high predicted scores and the annotations showed a variety of developmental cell types, consistent with the organoid as a developmental model. The inventors benchmarked this microenvironment to the patient TME by analyzing published datasets from which non-tumor cells were included in the single-cell analysis, a normal byproduct of resection. The inventors annotated these non-tumor cell types as was done previously for the organoid cells. Given these samples were from the adult human brain, they not only used the dataset from the developing brain, but also a normal adult single-cell dataset (Hodge et al., 2019) as well as a study exploring the aging brain (Yang et al., 2022).Fascinatingly, the normal cells obtained from adult tumor biopsies showed the highest similarity to the developing human brain, as opposed to the adult or aging brain (FIG. 1 IE, FIG. 18B). This highlighted the relevance of the organoid system for modeling the GBM microenvironment which, according to data collected here and the analysis of additional published datasets, deviates from the cell types found in the healthy adult brain (FIG. 18B).4. HOTT uncovers bidirectional tumor - microenvironmental communication mediated by PTPRZ1

[0184] With confidence in not only the tumor cell types in HOTT but also the context in which they are grown, the inventors asked whether anything about the organoids exposed to GBM differed compared to the tumor naive organoids from the same batch. To this end, the inventors sorted out the GFP- cells from the standard M3 condition, and across UMAP space observed segregation between the tumor naive and tumor transplanted organoid cells (FIG. 12A). The inventors noted that transplanted organoids showed a higher proportion of neuronally committed populations including intermediate progenitor cells, newborn neurons, and excitatory cells (FIG. 12A). These changes in the organoid suggested communication occurrs between the tumor and the organoid.

[0185] Thus, the inventors investigated potential key signaling pathways that could act as mediators between tumor and their surrounding normal cells by inferring ligand-receptor interactions with CellChat (Jin et al., 2021) and observed that PTN-PTPRZ1 signaling was the most significant signaling pathway upregulated in the HOTT tumors (FIG. 12B, FIG. 19). This was fascinating to us because this signaling pathway that has been implicated in tumor migration (Qin et al., 2017), including through GBM outer radial glia (Bhaduri et al., 2020b). Because the organoid is a developmental model and both PTN and PTPRZ1 are widely expressed during development (Pollen et al., 2015), the inventors were wary of a model specific artifact influencing these results. Further interrogation of datasets from patients containing nontumor microenvironmental cells also showed higher expression of PTPRZ1 and PTN in the TME compared to the normal adult brain (Krawczyk et al., 2022; Wang et al., 2022), giving us confidence in the system to identify novel tumor - TME interactions.

[0186] In the context of patient tumors (Yu et al., 2020), the inventors again observed that PTN - PTPRZ1 signaling between the tumor and the microenvironment topped the predicted list of interaction pathways (FIG. 12B). Notably, the inventors observed that in tumors PTN and PTPRZ1 were highly expressed in all tumor cell types, with overall lower expression in the normal peritumor cells (FIG. 12B). A similar trend was observed with another published dataset containing primary GBM tumors and normal cells (Bhaduri et al., 2020b; Darmanis etal., 2017) (SFig 6). However, when a similar analysis was performed with the normal adult human brain (Jorstad et al., 2023), this pathway was not a top pathway mediating cell - cell interactions (FIG. 12B), and overall interaction strength was observed to be substantially lower than in the HOTT and GBM patient datasets (FIG. 12B, FIG. 20). This reinforces that the PTPRZ1 communication pathway may be a function of the reactivation of developmental programs that the inventors and others have previously observed in GBM; the TME activation of PTPRZ1 signaling is specific to the tumor and peritumor environment and may be mediating an important axis of tumor - normal cell communication (FIG. 12B). These findings were consistent across samples, datasets, and analyses in additional HOTT samples, published datasets, and additional transcriptional profiles of the adult human brain (-FIGS. 19-20).

[0187] The inventors sought to validate these findings in newly resected patient tumor samples. Patient tumor samples were surgically removed from the tumor core (core region) and from the tumor boundary (peripheral region) using recently described amine chemical exchange saturation transfer echo planar imaging (CEST-EPI) (Patel et al., 2024) to define tumor core and peripheral samples. Because of heterogeneity in the spatial distribution of GBM, both samples included neoplastic and non-neoplastic cells, which is consistent with what has been described in published studies (Wang et al., 2022). Thus, the inventors sought to identify the neoplastic cells in the in situ analysis by post-staining the analyzed samples with Nestin (NES). Neoplastic regions were identified based upon the distribution of this staining as well as differences in cell density. The RNAscope analysis observed that PTPRZ1 and PTN were found on both the neoplastic and non-neoplastic / peritumor areas of the tumor in both the core and peripheral regions (FIG. 12C). When analyzing these data, the inventors noted that almost all cells were double positive for both PTN and PTPRZ1, suggesting a joint role in tumors. Additionally, the fact that PTN and PTPRZ1 are in the peripheral surgical sample supports their role in potentially mediating tumor migration, and these data strongly suggested a role for novel signaling interactions between the tumor and the TME.5. Microenvironmental PTPRZ1 has an unexpected role in mitigating tumor migration

[0188] PTPRZ1 has primarily been interrogated for its cell intrinsic role in promoting tumor sternness and invasion. Notably, PTPRZ1 has long been described as a receptor tyrosine phosphatase (Xia et al., 2019) that maintains glioma sternness and drives proliferation (Fujikawa et al., 2017). Additionally, in vivo work showed that PTN ligand binding to PTPRZ1 resulted in increased tumor invasion through Rho / ROCK signaling (Qin et al., 2017). In previous work, the inventors identified PTPRZ1 as a marker for GBM outer radial glia andshowed that PTPRZ1 was required for the outer radial glia cell jump and divide cell behavior known as mitotic somal translocation (Bhaduri et al., 2020b; Ostrem et al., 2017) which interestingly is also regulated by the Rho / Rock pathway (Ostrem et al., 2014). Previous work has not identified a role for PTPRZ1 in communicating with the TME, though PTN has been described to be secreted by microenvironmental macrophages (Shi et al., 2017) and has also been characterized as a chemoattractant for tumor cells (Qin et al., 2017). The data suggests co-localization of PTN and PTPRZ1 in both the single-cell analysis and in the RNAScope characterization of patient tumor samples.

[0189] This led us to hypothesize that PTPRZ1 may be involved in tumor migration through a cell extrinsic role via tumor - TME communication. To explore how migration might be impacted by both intrinsic and environmental PTPRZ1 expression, the inventors devised a novel migration assay in which the inventors transplanted dissociated patient derived gliomaspheres to organoids using the hanging drop method. The inventors used gliomaspheres for this experiment because they enabled us to query both tumor intrinsic phenotypes in culture and to examine if these phenotypes were the same or different after introduction to HOTT. After 2 days in culture, the inventors generated fusion organoids between transplanted and gliomasphere naive organoids. Each fusion served as a migration experiment whereby tumor cells from the transplanted side had an opportunity to migrate across the fusion boundary. The inventors evaluated the fusion efficacy using both cell sorting and immunostaining (FIG. 13 A). For the sorting experiments, the inventors severed the fusions at the junction point, identifying the original transplanted side as the one with GFP expressing cells fully surrounding the organoid, though migration to the other side could be visualized when looking at the live fusion experiment (FIG. 13B). GFP positive cells could then be sorted from each side of the fusion, enabling a quantitative metric of how much migration was observed .

[0190] For these migration studies, the inventors leveraged the versatility of the HOTT system in enabling us to knockdown environmental PTPRZ1, thus enabling the evaluation of whether TME PTPRZ1 contributes at all to the migration phenotype. The inventors performed tumor and / or organoid knockdown for PTPRZ1 using a small hairpin RNA before transplantation and migration was evaluated (FIG. 13D); across both the organoid and the gliomaspheres they observed moderate PTPRZ1 knockdown (FIG. 13C, FIG. 21). Importantly, knockdown of PTPRZ1 had no impact upon PTN, ensuring that they were studying PTPRZ1, PTN independent, effects (FIG. 13C).

[0191] From the FACS quantification, the inventors validated previous studies highlighting that tumor knockdown of PTPRZ1 decreased overall migration rate (Bhaduri et al., 2020b; Qinet al., 2017; Shi et al., 2017). However, when TME PTPRZ1 was knocked down, both in the context of tumor scrambled hairpin and tumor PTPRZ1 KD, the inventors observed increased migration (FIG. 13E). This was further validated using an orthogonal measurement of migration where immunofluorescence for GFP labeled gliomasphere cells was quantified on either side of the fusion border (FIG. 13F). Again, knockdown of PTPRZ1 in the TME corresponded to greater tumor migration. These data contradict the inventors’ expectations about PTPRZ1; the inventors assumed based upon previous studies that PTPRZ1 KD, even in the TME, would reduce migration. However, the data suggest a distinct role for how peritumoral PTPRZ1 may actually be inhibiting tumor migration, and when removed alters aspects of tumor - TME communication that drives behavioral changes.6. Microenvironmental manipulation of PTPRZ1 alters tumor cell fate through a catalytically independent mechanism

[0192] To explore if this cell - cell communication extended to properties of cell fate specification, the inventors performed immunostaining for the major cell types identified in the single-cell analysis and quantified the observations. Again, the inventors were surprised to see that environmental KD of PTPRZ1 modulated changes in tumor cell types compared to the control conditions, even without manipulation of the tumor itself. This implies a degree of cell nonautonomous tumor - microenvironmental communication. For example, environmental KD of PTPRZ1, without manipulation of the tumor directly, drives reduction of the tumor mesenchymal fraction as marked by HM0X1 and of the tumor neuronal fraction as marked by MEF2C (FIG. 14A-B). Additionally, TME KD of PTPRZ1, again without direct manipulation of the tumor itself, increased tumor astrocyte populations as marked by GFAP (FIG. 14C-D); the reduction of neuronal populations and increase in immature astrocytes suggests a shift towards a more stem-like identity. When targeting the tumor directly, tumor KD of PTPRZ1 increased both tumor and organoid CTIP2, a marker of mature deep layer neurons, including when the microenvironment was not directly manipulated, indicating additional tumor and environmental differentiation which is consistent with previous PTPRZ1 literature. Interestingly, the increase in CTIP2 in the organoid is also spatially located near the transplantation, suggesting a local effect consistent with the hypothesis that these cell fate changes are mediated by cell - cell communication through PTPRZ1 FIG. 14C). In support of this hypothesis, the inventors observed that the cell fate changes that occurred upon transplantation of the gliomaspheres were not the same effects observed when looking at gliomasphere cell type composition on its own (FIG. 21D-E), further emphasizing that the microenvironment can change tumor intrinsic properties of cell fate transition.

[0193] As a receptor tyrosine phosphatase, PTPRZ1 is most well described to function through its catalytic activity by which it removes phosphate groups from downstream targets including Src (Xia et al., 2019). To test whether these changes in cell fate are mediated by PTPRZ1 catalytic activity, the inventors utilized a small molecule inhibitor, NAZ2329, that inhibits the catalytic activity of PTPRZ1 by binding to its active site in the DI domain (Fujikawa et al., 2017). At 25 uM, NAZ2329 inhibition increased the accumulation of phosphoSRC as expected (FIG. 14E). Since the small molecule was added to the culture media, PTPRZ1 inhibition occurred on both GS and organoid cells, and so was most closely resembling the tumor and organoid double KD condition. Notably, the increase in tumor GFAP and CTIP2 population observed in this double KD was lost in the NAZ2329-treated condition (FIG. 14F), suggesting the cell type changes are not mediated by PTPRZ1 catalytic activity.

[0194] Overall, the inventors observed that TME PTPRZ1 KD is increasing tumor immature populations, while tumor KD is increasing mature populations. This contradictory role is further consistent with the observations of migratory phenotypes, again, where the environmental PTPRZ1 is a block on migration. This suggests a push - pull model of tumor intrinsic and extrinsic roles for PTPRZ1 signaling (FIG. 14G), and specifically highlights the need to study GBM signaling pathways and biology in the context of a microenvironment as achieved with the HOTT system.B. Discussion

[0195] Effective models of GBM that recapitulate the heterogeneity of human patient tumors and their complex interactions with the TME are a crucial tool in the field of neurooncology to overcoming existing barriers to designing effective treatments for this devastating cancer.

[0196] Advances in the development of organoid models have opened up vast opportunities in the study of GBM and addressed significant limitations of existing models of 2D cell culture and mouse models. The HOTT system presented here and benchmark against both the patient tumor as well as the patient microenvironment allows for the study of tumornormal crosstalk in a human context. Notably, the inventors observed tumor fidelity in both gene expression and cell type composition to the tumor of origin in the HOTT system. Furthermore, the gene expression of each cell type remained consistent across media conditions, including with the inclusion of FBS, suggesting that the existence of normal brain cells around the tumor could be an essential driver in maintaining cell type identity.

[0197] Here, the inventors leveraged HOTT to explore tumor - TME interactions, inadvertently stumbling upon a crucial role for PTPRZ1 in enabling both the tumor and the TME to reciprocally influence one another. The ability for a tumor to mold the microenvironment is described in other systems (de Visser and Joyce, 2023), and in the context of GBM is largely contextualized in terms of the immunosuppressive forces the tumor exerts on the immune populations in the environment (Pombo Antunes et al., 2020). Recent efforts in the field of cancer neuroscience have begun to highlight mechanisms by which the TME can promote tumor growth and integrate into the electrophysiological networks of the brain. The finding that PTPRZ1, a receptor tyrosine phosphatase, can promote cell - cell communication adds a branch to the ways in which GBM functionally communicates with its TME.

[0198] The HOTT migration assay is a novel tool to explore tumor migration in the context of human TME. Commonly used migratory assays in two-dimensions and in mice either entirely lack a microenvironment or rely on non-human contexts. An advantage of HOTT is that it is molecularly tractable, making knockdown experiments in this system substantially more feasible than generating a knockout mouse when interrogating an individual candidate gene. Using this system, the inventors were also able to identify cell fate shifts mediated by reciprocal knockdown; ie tumor knockdown changed the properties of the TME and TME knockdown altered cell fate specification in the tumor. This clearly suggests a non cell autonomous role for PTPRZ1 signaling, independent of its catalytic activity, that has not been described in the literature.

[0199] The work here shows that PTPRZ 1 plays an unexpectedly significant role in driving tumor - TME communication. Altogether, the HOTT system, the analysis of published patient data, and the RNAScope analysis of GBM margins highlights that PTPRZ 1 is present both in the tumor and the peritumor environment. The inventors and others have previously described a role for PTPRZ 1 in promoting tumor sternness and migration (Bhaduri et al., 2020b; Fujikawa et al., 2017; Mikelis et al., 2009; Shi et al., 2017), whereby PTPRZ1 is required for these pro- tumorigenic properties. This aspect of PTPRZ 1 has actually always been puzzling; in various models it has been shown that not only is PTPRZ 1 required for migration and glioma sternness, but also that this is a function of its catalytic activity (Fujikawa et al., 2016; Fujikawa et al., 2017). However, PTPRZ 1 is a phosphatase, removing phosphate groups from downstream pathways such as Src and beta catenin, where the accumulation of phosphorylation is pro- tumorigenic (Meng et al., 2000). Interestingly, previous work has also suggested that PTPRZ1 is most tumorigenic when bound by its ligand, PTN, which interestingly results in the dimerization of the protein, inactivating its catalytic activity.

[0200] PTPRZ1 is not widely expressed in the adult human brain (Jorstad et al., 2023), but it is upregulated as the inventors and others note across GBM cell types (Xia et al., 2019). Why, then, would glioblastoma cells go to great lengths to upregulate PTPRZ1, just to inactivate it? If PTPRZ1 is playing a substantial role in cell - cell communication as suggested here, this cell extrinsic role explains both why catalytically inactive, but highly expressed, PTPRZ1 could be highly functional for glioblastoma cells. The inventors support this hypothesis by the inhibitor based experiments in which they show these impacts upon cell fate specification are dependent on PTPRZ1 expression but not catalytic activity. Future efforts in the HOTT system and in other models will be required to further elucidate whether common downstream pathways are activated by cell extrinsic PTPRZ1 communication as are invoked in traditional PTPRZ1 signaling, and whether the cell - cell communication roles of PTPRZ1 are modulated by PTN or other ligands that may exist in the tumor milieu.

[0201] Importantly, these findings change the interpretation of PTPRZ 1 as a high value therapeutic target; the inventors’ previous work, work of others, and the data in this study highlight that when tumor cells are investigated alone, PTPRZ 1 knockdown is a valuable strategy to decrease migration and promote tumor differentiation. However, it is unlikely that most drugs would specifically target the tumor only, and the observations of TME KD of PTPRZ 1 show antagonist effects. Though the eventual impact on patient tumors cannot be extrapolated, there is a risk that the apparent inhibitory role on tumor progression that the TME plays by expressing PTPRZ 1 would be lost with therapeutics targeting PTPRZ 1, mitigating the effect of a drug or even worsening overall progression. Thus, any therapeutic approaches seeking to target PTPRZ 1 must ensure impressive tumor specificity, or should use alternative approaches to target this important signaling protein at the nexus of migration and cell fate in GBM.

[0202] A fundamental result of this study is that therapeutic efficacy in GBM will eventually rely on treating the whole brain, and thus, the tumor plus its complex TME. The field is aware of this complexity which underlies numerous drug trials that are less efficacious in patients compared to other models (Bagley et al., 2022). Systems like HOTT enable scalable experimental investigation of the tumor as well as its context, and can be performed directly on patient tumor cells. Eventually, one could imagine that future efforts would directly target the microenvironment, which provides the valuable “soil” in which GBM thrives. However, HOTT too is currently limited in its ability to recapitulate the full microenvironment as it lacks a functional immune system. The inventors have observed that when tumors are transplanted into HOTT, few to no immune cells survive after 3-4 weeks culture, and future efforts shouldfocus on extending this capability of the model. For now, however, HOTT provides an essential additional layer of complexity, modeling important aspects of the human GBM TME, and providing novel insights into the ways GBM and the TME functionally interact.C. Methods:1. Stem Cell Culture and Maintenance

[0203] Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) were cultured consistent with other studies (Bhaduri et al., 2020a; Pollen et al., 2019; Velasco et al., 2019). Multiple hESC / hiPSC lines derived from UCLA and CIRM were utilized in this study. In brief, stem cells were cultured on Matrigel (FisherScientific, 354230) coated 6-well plates in mTeSR plus supplemented with 10% mTeSR plus Supplement (Stem Cell Technology, Cat 100-0276), and IX Penicillin / Streptomycin or Primocin (FisherScientific NC9141851). Media changes were performed every other day, and passaging was conducted when cells reached over 75% confluence. During passaging, ReleSR (Cat 5872) was applied at room temperature for 1 minute, aspirated, and the stem cells were left at 37°C for 5 minutes before fresh stem cell media were added to dissociate them into smaller clusters without reducing them to single cells. These smaller clusters were evenly distributed into new Matrigel- coated plates at a 1 :4 or 1 :6 ratio. For freezing, the same procedure as in stem cell passaging was followed, except in the final step, 1 ml of mFreSR (Cat 5855) per well in a 6-well plate was used to resuspend the stem cells. The resuspended cells were then transferred to cryovials for storage at -80°C for 24-48 hours before being transferred to a liquid nitrogen tank for longterm storage.2. Cortical Organoid Generation

[0204] Cortical organoids were generated using an adapted version of the method described first in Kadoshima et al 2013, similar to other studies (Bhaduri et al., 2020a; Bhaduri et al., 2020b; Kadoshima et al., 2013; Velasco et al., 2019). Briefly, 1ml Accutase (Sigma A6964) was added to each well in a 6-well plate with human embryonic stem cells (hESCs) having over 75% confluence. After a 37°C incubation for 5 minutes, 1 mL of Media 1 (Ml) was added to each well. Ml consists of GMEM (LifeTech 11710-035), 20% KnockOut Serum (LifeTech 10828-028), 0.1 mM P-mercaptoethanol(Sigma 21985023), IX Non-Essential Amino Acids (NEAA), IX Sodium Pyruvate, and IX Penicillin / streptomycin or Primocin (FisherScientific NC9141851). The cells were scraped out and transferred to 15 mL tubes. After centrifugation at 300 x g for 5 minutes, cell pellets were resuspended into a single-cell suspension with 1 mL of Ml supplemented with three small molecules: Rock inhibitor Y27632 (20 pM), TGF-Pinhibitor SB431542 (5 pM), and Wnt signaling inhibitor IWRl-endo (3 pM). One million cells in 10 mL of Ml supplemented with the small molecules were evenly distributed into a 96-well V-shaped low attachment plate (S.Bio MS-9096VZ). Cells were allowed to aggregate undisturbed for 72 hours. On the 3rd day, the first media change was performed by replacing 50 pL with 100 pL of fresh Ml containing the small molecules to avoid disturbing the nascent organoids. Subsequently, 100% media changes were performed every other day until Day 7 when the Rock inhibitor was removed from the recipe. At 18 days, the organoids were transferred to a low attachment 6-well plate (Fisher Scientific 07-200-601). Subsequent media changes were performed every other day. From Day 18 to Day 35, Media 2 (M2) was used, which consists of DMEM / F-12 with Glutamax (Lifetech 10565-018), IX N-2 supplement (Lifetech 17502-048), IX Lipid Concentrate (Lifetech 11905-031), and IX Penicillin / streptomycin or Primocin. Starting from Day 35, Media 3 (M3) was applied, which includes DMEM / F-12 with Glutamax, IX N-2 supplement, IX Lipid Concentrate, IX Penicillin / streptomycin or Primocin, 10% Fetal Bovine Serum (GIBCO Catl0082147), 5 pg / mL Heparin (Sigma H3149), and 0.5% Growth factor reduced Matrigel(Corning 354230). Throughout the culture period, live images were periodically taken to monitor organoid growth, and immunostaining was performed at Week 5 and Week 8 to confirm normal neuronal differentiation.3. Media Conditions Used for Experiments

[0205] Five different culture conditions were used in this study: 1) M3 served as a based media for comparison. 2) M3+B27: M3 media supplemented with 10% B27(ThermoFisher Scientific 12587010). 3) M3noFBS: M3 media without FBS supplemented with 10% of DMEM / F-12 with Glutamax (Lifetech 10565-018). 4)M3noFBS+HEF: M3noFBS supplemented with 20 ug / mL Hu EGF (Life Technologies PHG0313), 8 ug / mL Hu FGF-b (Life Technologies cat. # PHG0263) and 2 mg / mL Heparin (Sigma-Aldrich H3149) as their final concentration. 5) M3noFBS+PDGF: M3noFBS supplemented with 50ng / ml PDGFAA (GIBCO PHG0035) as its final concentration.4. Tumor Dissociation and Viral Transduction

[0206] Primary tumors were obtained from Ronald Reagan Hospital at UCLA with appropriate consent forms for genomic analysis covered by Institutional Review Board IRB numbers 10-000655 and 21-000108. Tumors are cut into smaller pieces with a sterile scalpel blade. The material was then transferred to a 5 mL microcentrifuge tube containing 2.5 mL Papain supplemented with 125 uL DNase (Worthington Cat#LK003150) and incubated for 30-45 minutes in a 37°C incubator. After the initial 10 minutes of incubation, the tube was taken out and shaken vigorously for 10 seconds by hand to aid dissociation. This process was repeated after every 5 minutes of incubation thereafter. The material was further broken down with trituration before it was pelleted at 300g for 5 minutes. The pellet was then resuspended in warm media #3 and filtered with a 40 uM mesh. The cells were then infected with a GFP virus (SIgnaGen Cat#SL 100268) at a concentration of 1 uL / 1 million cells supplemented with 1 : 1000 Polybrene Infection Reagent (Millipore Sigma Cat#TR-1003-G) for 90 minutes on a rotator in the 37°C incubator. The cells were subsequently washed three times with warm media before proceeding to organoid transplantation.5. Tumor Transplantation

[0207] After lentiviral infection, cells were counted after being resuspended in 1ml of warm media 3. Depending on the total tumor recovered and experimental design, the whole single cell suspension was distributed into several Eppendorf tubes, one tube for each culture condition. Following a 5 minute spin at 300g, the cell pellet was resuspended in the final volume of each culture media. 500K-1M tumor cells were transplanted onto one organoid in a 10-15ul droplet of corresponding culture media with the following methods. Hanging drop method: To set up the hanging drop tumor transplantation, 8-12-week-old human cortical organoids were transferred onto the lid of a 10 cm dish using wide-bore lOOOul tips. After removing extra media, 10-15ul tumor single cell suspension was added on top of each organoid. 6 organoids of the same culture condition were placed on one lid. Then the lid was carefully flipped onto the 10 cm dish with 10ml base culture media to prevent the media evaporating during the hanging drop stage. These hanging drop co-cultures were 50 maintained for 12 hours in the 37C incubator, before they are moved back to ultra-low attachment 6 well plate (Fisher Scientific 07-200-601) with corresponding culture media. Tumor cells usually surround the organoids and migrate inside after a few days. Such cultures last for about 4 weeks with 3 times media change each week before being harvested for analysis.

[0208] To optimize GBM transplantation, the inventors employed another two methods, i.e., Insert and V-bottom approaches for a head-to-head comparison. For insert transplantation, 8-12-week-old organoids were positioned at the air-liquid interface on Millicell (Millipore) inserts to restrict movement. A 10-15 pl tumor single cell suspension was gently pipetted onto the semi-dry organoids, allowing integration for 30 minutes at 37°C. Subsequently, organoids were carefully lifted from the inserts by increasing the medium volume by 3 ml. After 2 days, organoids were transferred to an ultra-low attachment 6-well plate without inserts, maintainingappropriate culture media. For V-bottom transplantation, 8-12-week-old organoids were placed into V-bottom low attachment 96-well plates, with one organoid per well and 100 pl of appropriate media. A 10-15 pl tumor single cell suspension was slowly pipetted onto each organoid. After 2 days, organoids were transferred to an ultra-low attachment 6-well plate with suitable culture media.6. FACS Isolation of Tumor Cells

[0209] Tumor-transplanted organoids were transferred to a 1.7 mL Eppendorf tube containing 1 mL Papain supplemented with 50 uL DNase (Worthington Cat#LK003150) and incubated for 30-45 minutes in a 37°C incubator. After the initial 10 minutes of incubation, the tube was taken out and shaken vigorously for 10 seconds by hand to aid dissociation. This process was repeated after every 5 minutes of incubation thereafter. The material was further broken down with trituration before it was pelleted at 300g for 5 minutes. The pellet was then resuspended in a cold FACS buffer (PBS + 2% FBS), filtered with a 40 uM mesh, and stained with DAPI to identify non-viable cells. DAPI- / GFP+ tumor cells and DAPI- / GFP- normal cells were sorted and collected for downstream single-cell capture.

[0210] FACS was performed on either a BioRad S3e or BD Biosciences FACSAria II Cell Sorter. A 100 uM nozzle was used for sorting. All sorts were performed with a purity sort precision at a slow speed. Gates were set to remove debris, eliminate doublets, and DAPI staining was used to exclude dead cells. Gates for GFP positivity were set using control, batch matched organoids without transplantation.7. Single-cell Capture and Sequencing

[0211] Single-cells from FACS or from dissociated tumor cells were captured using the 10X v3.1 3’ capture protocol and the 10X Chromium Machine. 10,000 cells were targeted for capture, or if the retrieved number after sorting was less than 20,000, all cells were used for capture. Capture proceeded as detailed in the user manual and manufacturer guidelines were used for library preparation. Sequencing was performed on an Illumina NovaSeq 6000.8. Single-nuclei Capture

[0212] Single-nuclei sequencing was conducted on all samples to capture the full spectrum of patient tumor cell types. Nuclei extractions were carried out from frozen tissue specimens obtained from the UCLA Brain Tumor Translational Resource (BTTR) or from flash-frozen samples preserved prior to dissociation. Frozen tissue samples were directly transferred to a pre-cooled 7ml douncer containing 3ml of ice-cold Buffer A and gently homogenized with 2 rounds, each consisting of 15 dounces over a 5-minute period. The homogenized solution wasthen filtered using a 37pm reversible strainer (STEMCELL #27215 / 27250) into a pre-cooled 15ml tube containing 3ml Buffer B. Subsequently, the mixture was centrifuged at 1000g for 30 minutes at 4°C in a swing-out rotor after inverting the tube 6 times. Careful removal of the supernatant was followed by resuspension of the pellet in 1 OOOpl Wash Buffer, which was then filtered through a 37pm reversible strainer into a 1.5ml low-binding Axygen tube(MCT-150- L-C). Centrifugation at 500g for 5 minutes at 4°C was performed, and the wash step was repeated once more. The pellet was then resuspended in 0.5ml Buffer C and incubated on ice for 5 minutes to permeabilize nuclei. Subsequently, 0.5ml Buffer D was added and gently mixed, and the nuclei were counted. Concurrently, a final centrifugation at 500g for 5 minutes at 4°C was carried out, followed by resuspension in Buffer E at a targeted concentration of 8M nuclei / ml for loading onto the 10X Genomic chip.Buffer ingredient table9. Single-cell Analysis and Quality Control

[0213] Single-cell RNA sequencing (scRNA-seq) reads obtained from HOTT-derived cells, along with their corresponding primary tumor cells, were aligned to the GRCh38 human reference genome. Cell-by-gene count matrices were generated using the lOXGenomics Cell Ranger pipeline with default parameters. Subsequently, data analysis was conducted using the Seurat R package within R Studio. Cells expressing a minimum of 1000 genes and exhibiting less than 10% mitochondrial gene content were retained. Unique Molecular Identifier (UMI) counts were normalized via logarithmic transformation, employing a scaling factor of 10000. Principal Component Analysis (PCA) was executed on the scaled data, utilizing the top 2000 variable genes. The number of dimensions (dims) was determined as has been previously described (Shekhar et al., 2016). Briefly, the inventors selected significant PCs from the larger value between the square of the standard deviation of PCA scores(Seurat. Obj@reductions$pca@stdevA2) and the square of the square root of the ratio of the number of genes to the number of cells plus one (sqrt(length(row.names(Seurat.Obj)) / length(colnames(Seurat.Obj))) + 1)A2). Cells were clustered in the PCA space utilizing Seurat's FindNeighbors and FindClusters functions, with a resolution parameter set to 2.0. Visualization of cells was performed using Uniform Manifold Approximation and Projection (UMAP), employing the previously defined dimensions. To predict and exclude doublets, the DoubletFinder R package (McGinnis et al., 2019) was employed with default parameters.10. Copy Number Variation Analysis

[0214] To mitigate the risk of false positive or negative identification of tumor cells via FACS, copy number variation analysis was performed Infercnv with default parameters (Tickle T, 2019). Batch-matched naive organoids were applied as reference.11. Single-cell Annotation

[0215] To perform tumor cell annotation, a projection to meta-atlas approach was employed to mitigate technical variations. The GBM meta-atlas was constructed by integrating a total of 69,547 cells from seven previously published primary GBM single-cell RNA sequencing (scRNAseq) datasets, which also included normal cells from adjacent regions proximal to the tumor, identified in the original studies (Bhaduri et al., 2020b; Couturier et al., 2020; Darmanis et al., 2017; Jacob et al., 2020; Neftel et al., 2019b; Yu et al., 2020; Yuan et al., 2018). Data analysis was performed using the Seurat R package with identical parameters as specified, except for the resolution parameter, which was set to 0.5. Subsequently, 23 clusters generated by the pipeline underwent biological annotation based on both comprehensive literature review of top-expressing genes and spatial relationships. A UMAP containing a total of 14 typical GBM cell types was generated as a reference for subsequent neoplastic cell annotation in this study. For HOTT normal cell annotation, normal developmental brain meta-atlas was generated as reference in similar fashion using seven previously published transcriptomic profiles of the developing human cortex. The resulting 225 meta clusters were assigned to biological processes and cell type relevant functions through both extensive literature review of module genes and term enrichment analysis of gene ontology sets.

[0216] Upon generation of Seurat query objects from HOTT datasets and reference object from GBM meta-atlas independently, FindTransferAnchors() was employed to identify a set of anchors between them, followed by MapQuery() to map the query data onto the reference in its UMAP space. Consequently, cell type annotation presented as predicted. id was generatedalongside predicted. id. score to indicate the fidelity of such defined cell types to the typical GBM cell types delineated in the GBM meta-atlas.12. Immunofluorescence staining

[0217] The human cortical organoids were fixed in 4% paraformaldehyde for 45 minutes at room temperature. Subsequently, the cortical organoids were rinsed with PBS and equilibrated in 30% sucrose in PBS overnight at 4C. After equilibration, the cortical organoids were embedded in OCT Embedding Matrix (Fisher Sci Cat.#14-373-65) containing OCT (Tissue-Tek, VWR) and 30% sucrose at a 1 : 1 ratio and frozen on a sheet of dry ice. The frozen blocks were either transferred to -80°C for long-term storage or cryosectioned as 10-16 um- thick sections onto glass slides for immunofluorescence staining. To set up the immunostaining, the sections were first rinsed with PBS for 15 minutes and then treated with a citrate-based antigen unmasking solution (10 mM sodium citrate, pH 6, Vector Labs) that was heated to 95C for 20 minutes. Following antigen unmasking, the sections were permeabilized and blocked with a blocking buffer containing 5% donkey serum, 3% bovine serum albumin, and 0.1% Triton X-100 in PBS for 30 minutes at room temperature. The subsequent primary antibody incubations were performed overnight at 4C using the following primary antibodies in the blocking buffer. Mouse -SOX2 (1 :500, Cat # SC-365823, Santa Cruz), mouse -Nestin (1 :500, Cat #: MAB5326, Millipore Sigma), chicken -GFP (1 :500, Cat #: GFP-1020, Aves), chicken -GFAP (1 :500, Cat #: AB4674, Abeam), rabbit -Ki67 (1 :500, Cat #: AB16667, Abeam), rabbit -MEF2C (1 :500, Cat #: NBP1-89468, Novus), rabbit -HOPX (1 :500, Cat #: 11419-1-AP, Proteintech). The primary antibody incubations were followed by three 10-minute PBS washes and secondary antibody incubations with DAPI in the blocking buffer for 2 hours at room temperature. The following secondary antibodies were used. DAPI (1 : 1000, Cat #: 62248, ThermoScientific), AlexaFluor 488 donkey -chicken (1 :500, Cat #: 703- 545-155, Jackson), AlexaFluor 488 donkey -goat (1 :500, Cat # A32814, Invitrogen), AlexaFluor 568 donkey -mouse (1 :500, Cat #: A32773, Invitrogen), AlexaFluor 568 donkey - rat (1 :500, Cat #: A78946, Invitrogen), AlexaFluor 568 donkey -chicken (1 :500, Cat #: Al 1041, Invitrogen), AlexaFluor 647 donkey -rabbit (1 :500, Cat # A31573, Invitrogen). Finally, the slides were mounted with ProLongTM Gold antifade reagent (Invitrogen) and preserved for imaging at 4C.13. Image Analysis

[0218] The image acquisition parameters (light, exposure, and digital gain) were normalized based on the parameters of the images with the highest fluorescence intensities foreach channel. 9 fields of fluorescent images were captured for each condition using EVOS M5000 digital-inverted benchtop microscope and individual channels were saved as TIFF files. ImageJ was used to merge the individual channels, and Imaris image analysis software (Bitplane) was used to quantify the number of double-positive cells expressing GFP and another marker of interest. These quantitative analyses were performed using the Spots and Surfaces features in Imaris. First, the GFP+ cells were determined based on a standardized shortest distance between DAPI+ Spots and GFP Surfaces. Subsequently, double-positive cells were identified based on another standardized shortest distance between GFP+ cells and either Spots or Surfaces representing a specific marker of interest.14. Tumor Cell Type Heterogeneity Preservation Analysis

[0219] HOTT tumor cells from each culture condition were individually analyzed using the Seurat pipeline with default parameters, followed by marker gene identification based on Seurat-derived clusters. Subsequently, the gene score for each gene was calculated by multiplying its average log2FC by the ratio of pct. l to pct.2, followed by pivoting to generate a gene score matrix (cluster by gene). The gene score matrices from each culture condition within a tumor sample were merged by gene, resulting in the HOTT tumor cluster gene score matrix. Simultaneously, its corresponding primary tumor gene score matrix was generated using the same approach, with the exception that marker genes were identified based on annotated tumor cell types. The correlation between each primary tumor cell type gene score matrix and its corresponding HOTT tumor cluster gene score matrix was performed. The resulting correlation matrices of 3 primary tumor samples was then merged into a correlation matrix comprising 205 HOTT-derived clusters against primary tumor cell types, which was used to generate a heatmap using Morpheus via hierarchical clustering and visualized using a relative color scheme.15. ssGSEA Analysis

[0220] The three human tumor datasets (LB4878, LB5024 and LB5028) were merged and each gene was assigned a gene score across the three samples. The Seurat clusters were separated based on culture conditions and each cluster was compared to fourteen gene lists of different cell types using ssGSEA2 (Barbie et al., 2009; Subramanian et al., 2005). The gene lists were obtained from the GBM meta-atlas. A FDR p-value threshold of less than 0.01 was applied to the clusters and their corresponding enrichment scores were plotted on a ridgeplot.16. Module Preservation Analysis

[0221] The preservation of normal developmental gene programs in primary versus transplanted tumor cells was compared using previously identified set of gene meta-modules representative of normal human brain development (Nano et al., 2023). These 225 meta- modules were defined using iterative, hierarchical clustering of seven previously published transcriptomic profiles of the developing human cortex, and biological processes were assigned to these meta-modules through extensive literature review and term enrichment analysis (Nano et al., 2023). The inventors then characterized the preservation of these normal developmental meta-modules in the datasets of primary and transplanted tumor cells by applying the module activity score. This versatile metric is measured for each cell by calculating the average normalized counts per million (CPM) detected for each meta-module gene.17. Differential Gene Expression Analysis

[0222] Differential Gene Expression analysis was performed using the Seurat package Wilcoxon rank sum test with a p-value threshold of less than 0.05. Gene Ontology analysis was conducted using EnrichR.18. Cell Chat Analysis

[0223] The CellChat package (Jin et al., 2021) was employed to assess cell-to-cell communication according to its standard protocol, as outlined in https: / / github.com / sqjin / CellChat. Each tumor sample, combined from various culture conditions within HOTTs, underwent individual analysis using its corresponding Seurat object as input for CellChat. Samples were stratified based on both neoplastic (denoted by "N" followed by their predicted. id) and non-neoplastic (denoted by "T" followed by their predicted. id) cell types. Visualization of signaling pathway ranking was facilitated through netAnalysis signalingRole heatmap. Additionally, PTN receptor contribution plots, signaling pathway network circle plots, and pathway expression pattern 2D plots were generated using netAnalysis contribution, netVisual aggregate, and netAnalysis signalingRole scatter, respectively, after assigning signaling to "PTN".19. Core versus Periphery Tumor Resection

[0224] Acquisition of glioblastoma specimens was approved under Institutional Review Board #10-000655. Patients undergoing surgery for newly diagnosed glioma were prospectively identified and completed signed informed consent. All patients had standard of care magnetic resonance imaging (MRI) (Kaufmann et al., 2020) sequences with and without gadolinium contrast (Gd-DTPA, 0.1 mmol / kg) and T2 sequences within 4 weeks of surgery.Inclusion criteria included: treatment naive, IDH wild-type glioblastoma, contrast-enhancing bulk of tumor with surrounding non-enhancing T2 hyperintense region on magnetic resonance imaging (MRI), no peripheral involvement of eloquent areas, and medical stability for surgery. As previously described (Patel et al., 2024; Patel et al., 2020), MRI based biopsy targets were selected using intraoperative neuronavigation (BrainLab Curve, BrainLab Surgical Navigation System, Munich, Germany). Contrast-enhancing tumor specimens (Core region) were first removed during standard of care resection followed by acquisition of non-enhancing infiltrating tumor specimens (Periphery region), confirmed by pathologic review with hematoxylin-eosin stains. 2 Specimens were immediately transferred to the research laboratory for further experiments.20. RNAScope and Quantification

[0225] The in situ hybridization assay was conducted using the ACD RNAscope™ HiPlexl2 Reagent Kit on FFPE primary GBM samples, followed by acquisition of RNAscope images using the ZEISS LSM 880 confocal system. The 63x confocal microscopy images were processed and visualized using the 3D view feature in Imaris. The total DAPI+ cell number was then determined using automatic spot detection. Subsequently, images with the negative control probes were taken as a reference to assess with high autofluorescence and false positivity, and PTN and PTPRZ1 positive cells were manually counted. Cells with few fluorescent speckles or dim signals were not counted as positive. Finally, the percentage of positive cells was calculated by taking the ratio of manually counted positive cells over the total cell number. The examiner remained blinded to the expected or hypothesized results.21. Gliomasphere Culture and Maintenance

[0226] Gliomaspheres were maintained in a media containing DMEM / F12 (Gibco), B27 minus Vitamin A (Gibco), Penicillin-Streptomycin (Gibco), GlutaMAX (Gibco), supplemented with heparin (5 pg / mL, Sigma), EGF (20 ng / mL, Sigma), and FGF (20 ng / mL, Sigma). The cells were cultured under 37°C and 5% CO2 and routinely tested negative for mycoplasma contamination.22. Knockdown in Organoids and Gliomaspheres with Validation

[0227] The pLKO. l plasmid used to generate the shRNA was obtained from addgene (Plasmid #10878). The puroR cassette was excised and replaced with either mCherry or EGFP, and the hPGK promoter immediately preceding the EGFP was also replaced with a CMV promoter, both via NEBuilder HiFi DNA Assembly reaction (Cat#: E2621L, New England Biolabs). The scramble (5’ -cctaaggttaagtcgccctcg-3 ’ - SEQ ID NO: 1) and PTPRZ1 -targetingsequence (5’ -gaactcacatctgagcattgt-3 ’ - SEQ ID N0:2) were inserted into the backbone with the manufacturer’s instructions. To generate lentivirus, these plasmids were transfected into HEK293T cells alongside psPAX2 and pMD2.G at a mass ratio of 1 pMD2.G : 3 psPAX2 : 4 pLKO.l using Lipofectamine 2000 (Invitrogen, Cat.# 11668019), and media containing the lentivirus was concentrated with Lenti-X Concentrator (Takara Bio, Cat.# 631232) per the manufacturer’s instructions. The concentrated lentivirus was used to infect UCLA6 hESCs at a concentration of 1 :200 and 1 : 1000 Polybrene Infection Reagent (Millipore Sigma, Cat.# TR- 1003-G) during split. Real-time PCR and western blotting were used to validate PTPRZ1 KD.23. Migration Assay

[0228] Gliomasphere cells were dissociated into single cells with TrypLE (ThermoFisher, Cat.# 12605010) and transplanted onto UCLA6 organoids at 500,000 cells / organoid via the hanging drop approach. After 36 hours, the GS-transplanted organoids were allowed to fuse with a tumor-naive, age and line-matched UCLA6 organoid in an ultra-low attachment 24-well plate, with each well harboring one fusion pair. The fusions were left undisturbed for 5 days before the first media change occurred. The fusions were maintained for 3-4 weeks before being harvested for FACS and immunostaining analyses. Part of the fusions were severed with a scalpel and dissociated into single cells with Papain in preparation for FACS analysis on the Biorad S3e Cell Sorter. The migration percentage was calculated by dividing the number of GFP+ cells on the migrated side with the total number of GFP+ cells in the fusion (n=3). The remaining fusions were fixed with 4% PFA for 45 minutes and rehydrated with 30% sucrose overnight at 4C. The organoids were then embedded in OCT and sectioned for GFP immunostaining. Images were acquired using the ZEISS LSM 880 confocal system. 15 to 36 tile images were captured using a lOx lens to incorporate the entire pair of merging organoids. To determine the tumor cell coverage over the entire fusion, eight bins of identical size were applied to each fusion, with the fusion interface in the middle. The amount of GFP+ tumor cells was quantified in each bin using Imaris.24. Western Blot

[0229] Samples were lysed with RIPA buffer (Millipore Sigma, Cat.# R2078) containing cOmplete™ Protease Inhibitor Cocktail (Roche, Cat.# 04693116001) and PhosSTOP (Roche, Cat.# 4906845001). Protein concentrations were determined using Pierce™ BCA Protein Assay (ThermoFisher, Cat.# 23225). Laemmli sample buffer (BioRad, Cat.# 1610747) was added to the lysate and the mixture was heated at 95C for 10 minutes. 30 ug of protein was loaded in each lane of a 4-15% Mini-PROTEAN® TGX™ Precast Protein Gels (BioRad, Cat.#4561083). After SDS-PAGE, the proteins were transferred onto a nitrocellulose membrane and blocked with 5% milk for 1 hour at room temperature. Primary antibodies were added to a solution containing 2.5% Bovine Serum Albumin and 0.02% sodium azide in TBST; anti- PTPRZ1 (BD Biosciences, Cat.# 610179, 1 :500), anti-P-Actin (Sigma- Aldrich, Cat.# Al 978, 1 : 10000), anti-SRC (Abeam, Cat.# abl09381, 1 : 10000), anti-phospho-SRC (Cell Signaling Tech, Cat. #6943T, 1 : 1000). The membranes were incubated overnight at 4C. Secondary antibodies targeting mouse (Fisher Scientific, Cat.# NC9994806, 1 : 10000) or rabbit (Fisher Scientific, Cat.# NC9734651, 1 : 10000) in 5% milk was added to the membranes for 1 hour at room temperature. The membranes were then developed with Immobilon Forte Western HRP substrate (Millipore Sigma, Cat.# WBLUF).25. Quantitative Real-time PCR

[0230] RNA extraction was performed with RNeasy Plus Mini Kit (Qiagen, Cat.# 74136) according to the manufacturer’s manual and the resulting RNA was used to synthesize cDNA using SuperScript™ IV VILO™ Master Mix (Invitrogen, Cat.# 11756050). Real-time PCR targeting PTPRZ1 (5’-ACTCTGAGAAGCAGAGGAG-3’ (SEQ ID NO:3) and 5’- CTGTTGTCTGTAGTATCCATTAG-3’ (SEQ ID NO:4)) or GAPDH (5’- TCAAGGCTGAGAACGGGAAG-3’ (SEQ ID NO: 5) and 5’-CGCCCCACTTGATTTTGGAG-3’ (SEQ ID NO:6)) was performed with Power SYBR™ Green PCR Master Mix (Applied Biosystems, Cat.# 4367659) in three technical replicates.D. References

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[0301] Zhu, Q., Zhao, X., Zheng, K., Li, H., Huang, H., Zhang, Z., Mastracci, T., Wegner, M., Chen, Y., Sussel, L., et al. (2014). Genetic evidence that Nkx2.2 and Pdgfra are major determinants of the timing of oligodendrocyte differentiation in the developing CNS. Development 141, 548-555.Example 7: A synapse-optimized human organoid tumor transplantation model of glioblastoma

[0302] Glioblastoma (GBM) is a primary brain tumor with extreme intratumoral heterogeneity. A subset of GBM cells express postsynaptic proteins and can form neuron-tumor synapses (NTS) with presynaptic neurons in microenvironment. Cortical organoids, which capture key molecular and cytoarchitectural elements of the human cortex, can provide an informative platform for studying NTS. However, NTS has been minimally demonstrated in cortical organoid models due to the immaturity of neurons in these systems. In this study, the inventors engineered culture conditions to coax NTS formation in tumor-transplanted cortical organoids using rationally selected media supplements. This synapse-optimized human organoid tumor transplantation (so-HOTT) GBM model features increased colocalization of pre- and postsynaptic markers within tumor cells. Single-cell RNA sequencing further reveals that so-HOTT activates genes involved in postsynaptic assembly and organization. Benchmarking to the GBM meta-atlas also indicate that so-HOTT mimics the preferential expression of ionotropic glutamate receptors in primary tumors. Cell-cell communication analysis using CellChat also shows that so-HOTT enriches for intercellular interactions important for synaptic communication, including synaptic adhesion and glutamate signaling pathways. Several of these enriched interactions are higher in transplanted GBM cells derived from the tumor periphery than in tumor core, suggesting that so-HOTT preserves known differences in synaptogenic properties of GBM cells from these two regions. Initial functionalassessment of so-HOTT using trans-synaptic tracing indicate neuron-tumor synaptic cleft formation in tumor cells. Collectively, the profiling studies suggest the existence of bona fide neuron-tumor synapses in so-HOTT, and it is envisioned that this novel model will be a valuable tool for studying neuron-tumor communication in GBM.

[0303] Functional integration of glioblastoma (GBM) cells into neural circuits via excitatory synaptic connections has emerged as a critical mechanism promoting tumor progression (1,2). Studies on rodent models, organotypic brain slice cultures, and neuron-GBM co-culture systems have revealed that these neuron-tumor synapses (NTS) transmit excitatory glutamatergic signals, predominantly via AMPA receptors, leading to increased tumor cell proliferation (1,2). Notably, synaptogenic potential is enriched in tumor cell states with neurodevelopmental signatures (3) . Spatial profiling of GBMs further reveal that both synaptic and neurodevelopmental signatures are enriched at the invasive tumor margin, where glioma cells interface with a relatively intact neuronal microenvironment (4) . These findings suggest that synaptic integration is not only a hallmark of specific tumor cell types but are also critically shaped by a neuron-rich tumor microenvironment.Human cortical organoids have emerged as a powerful tool for modeling brain development and disease, including GBM. Several organoid-based platforms have been developed to study glioma biology, including models based on genetic transformation of cerebral organoids (5), co-culture of patient-derived glioma stem cells (GSCs) with cerebral organoids (6), establishment of glioblastoma organoids from tumor explants (7), or individualized, patientspecific tumor organoid systems (8). While most of these approaches recapitulate key aspects of tumor heterogeneity and growth, none have definitively demonstrated the formation of functional NTS within an organoid context. The root cause of this limitation may differ depending on the system used. In genetically engineered organoids or tumor-transplanted models, it is likely due to the limited neuronal maturation typically observed in the organoid scaffold, which may preclude the formation of mature synaptic structures. Tumor transplants utilizing GSCs are further complicated by the selective pressures these cells have experienced, which can lead to substantial deviations from the tumor's native synaptogenic capacity. Lastly, organoids derived from patient tumor explants may show variability in the preservation of non- malignant cell types during the organoid generation process. In this study, the inventors sought to overcome these limitations by developing a synapse-optimized cortical assembloid model of GBM derived from primary tumors transplanted into cortical organoids subjected to an accelerated maturation process. This novel platform offers a more physiologically relevantmodel for exploring neuron-tumor synapses in GBM, making it a valuable resource for disease modeling and mechanistic studies.A. RESULTS1. Addition of rationally selected media supplements increases pre- and post-synaptic protein co-localization with so-HOTT GBM cells

[0304] To simultaneously enhance cortical organoid maturation and synaptic gene expression in GBM cells, the inventors identified and tested supplements that were previously not present in the standard human organoid tumor transplantation (HOTT) media: 1) neuroligin-3 (NLGN3); 2) brain-derived neurotrophic factor (BDNF); 3) and the GENtoniK cocktail (11), a cocktail of four factors that collectively accelerate neuronal maturation in vitro through two synergistic modes of action, namely GSK2879552- and EPZ-5676-dependent chromatin remodeling in immature neurons and N-methyl-D-asparatate (NMD A)- and Bay K 8644-mediated activation of calcium-dependent transcription (Fig. 1A). In addition to these factors, the inventors also added the widely used serum-free supplement B-27 and increased the Matrigel concentration in the culture media to further support neuronal survival. Hereafter this media is referred to as Sasai 4+ (S4+) to distinguish it from the Sasai 3 (S3) media that is used for standard HOTT cultures (see examples 1-6).

[0305] Using the hanging drop method for transplanting primary tumors into cortical organoids (12) , the inventors generated GBM-transplanted organoids and cultured them in S4+ and S3 for four weeks. To test if the S4+ media condition can promote the formation of putative synapses between organoid neurons and transplanted GBM cells, the inventors compared the co-localization of presynaptic (e.g. SYN1, VGLUT1) and postsynaptic markers (e.g., PSD95, GLUA4) with GFP-labeled transplanted primary GBM cells in S4+ vs S3 media conditions. Additionally, to determine if the region from which the tumor cells were derived from are equally or differentially responsive to S4+, they performed this comparative analysis for both GBM cells obtained from the tumor core and the periphery. Using SynBot (13), a machine learning-based automated workflow for three-channel colocalization and quantification, the inventors confirmed that the addition of the S4+ cocktail increases PSD95 / SYN1 colocalization within tumor cell boundaries (Fig. 1B-C). This trend was true for both core and peripheral tumor cells, suggesting that the S4+ media equally impacts the synaptic protein expression in tumor cells regardless of origin. Notably, the markers used are classical excitatory synaptic markers and their increased co-localization in S4+ mimics the known predominance of excitatory neuron-tumor synapses in GBM. Thus, the inventors have generated a novel GBMassembloid model that recapitulates excitatory synaptic signatures found in GBM, which is referred to herein as the synapse-optimized human organoid tumor transplantation (so-HOTT) model.2. so-HOTT recapitulates the enrichment of postsynaptic ionotropic glutamatergic receptors, cell adhesion, and scaffolding proteins in GBM

[0306] Neuron-tumor synapses in GBM are known to be unidirectional, consisting of a non-malignant presynaptic neuron and a postsynaptic tumor cell (1) . To date, the reverse orientation (i.e., GBM cells being presynaptic) has not been demonstrated, despite efforts to test that possibility. Given that the so-HOTT cocktail could potentially broadly induce pre- and post-synaptic neuronal gene expression in both neurons and transplanted tumor cells, the inventors wanted to test whether the postsynaptic bias that GBM cells are known to exhibit in vivo is preserved in the system. Thus, they performed single-cell RNA sequencing (scRNA- seq) on so-HOTT and standard HOTT models (Fig. ID). Differential gene expression analysis (DEG) between so-HOTT and standard HOTT tumor cells and found enrichment of genes related to postsynapse assembly and organization, postsynaptic density, synapse assembly, and regulation of neurotransmitter receptors to postsynaptic compartments (Fig. IE). Independently, the inventors also took the list of pre- and post-synaptic genes from Synaptic Gene Ontologies (SynGO) (14) and further filtered the raw lists for genes are exclusively presynaptic and postsynaptic; genes that could either be both were discarded. They then scored tumor cells from the GBM meta-atlas (15) and so-HOTT against these refined lists. As expected, primary tumor cells exhibit higher postsynaptic than pre-synaptic gene module expression (Fig. 1F-G). Thus, the postsynaptic bias of GBM cells is recapitulated in so-HOTT, indicating that the model preserves the unidirectional nature of neuron-tumor synapses and does not artificially induce transplanted GBM cells to become presynaptic.

[0307] To further benchmark the so-HOTT model against primary tumor data, the inventors took a more granular look at the postsynaptic proteins present in so-HOTT GBM cells. Previous studies have shown that GBM cells selectively express ionotropic glutamatergic receptors, particularly AMPA receptors (AMPARs), relative to other neurotransmitter receptor families (1). These glutamatergic receptors mediate excitatory postsynaptic currents (EPSCs) that induce tumor proliferation of GBM cells, leading us to examine whether their enrichment is also preserved in so-HOTT. To do so, the inventors scored GBM meta-atlas primary tumors and so-HOTT GBM cells against a curated list of gene modules, each corresponding to a specific neurotransmitter receptor family. This analysis revealed that so-HOTT tumor cellspreserve the preferential expression of ionotropic glutamatergic receptors (Fig. 1H). Consistent with primary tumor data, AMPA receptors was notably elevated in so-HOTT GBM cells relative to other ionotropic glutamatergic receptor subtypes (i.e., NMD A, kainate, and delta) (Fig. II). Additionally, out of all the neurodevelopmental GBM cell types, OPC-like tumor cells showed the highest expression of glutamatergic receptors in both the GBM meta-atlas and in so-HOTT (Fig. 1H). This is consistent with the prevailing view that glioma cells exhibiting synaptic gene signatures are similar to OPCs, a progenitor cell type that has been proposed to function as a postsynaptic cell. Thus, the so-HOTT model maintains the known molecular and cell type signatures of synaptically connected GBM cells, reinforcing its utility as a humanspecific and physiologically relevant platform for studying synaptic communication in GBM.3. so-HOTT tumor cells, particularly those derived from the tumor periphery, are enriched for synaptic intercellular interactions

[0308] To determine what modes of intercellular interactions are present in so-HOTT, cellcell communication analysis using CellChat (16) was performed. Strikingly, there were more inferred interactions and greater interaction strengths in both core and peripheral tumor cells grown in S4+ vs S3, indicating that so-HOTT conditions promote intercellular interactions (Fig. 2A). Modes of communication enriched in so-HOTT include pathways involved in synaptic communication, including those facilitated by various synaptic adhesion proteins (Fig. 2B). These proteins include presynaptic neurexins and postsynaptic neuroligins (NRXN / NLGN), latrophilins (ADGRL), neural cell adhesion molecules (NCAM), and synaptic cell adhesion molecules / SynCAMs (CADM) (Fig. 2B-F). Notably, among non- malignant cells, it was observed that most of the outgoing signals emanate from non-malignant neurons and secondarily from astrocytes; meanwhile, among malignant cells, most of the incoming signals were received by astrocytic- and neuronal-like GBM cells (Fig. 2D). Interestingly, the enrichment of the abovementioned interactions in so-HOTT tend to be specifically pronounced in peripheral than in core tumor cells (Fig. 2A-B, 2F). This observation is consistent with past spatial profiling studies, which showed that GBM cells at the tumor periphery have higher synaptogenic potential than those found in the core, as indicated by higher expression of neurodevelopmental and synaptic genes in the former (3,4) . Collectively, these results underscore the prominence of synapse-associated signaling networks in so-HOTT. The observed spatial bias towards the tumor periphery highlights the integral role of original microenvironmental niche for the eventual enrichment of neuron-like modes of intercellular communi cati on post-transpl antati on .4. Initial functional validation of so-HOTT NTS using trans-synaptic tracing

[0309] To provide evidence for functional neuron-tumor synapses in so-HOTT, the inventors performed anterograde trans-synaptic tracing using an AAVl-Cre tracer introduced into cortical organoids and a double-floxed inverse orientation (DIO) EGFP reporter in transplanted GBM cells (Fig. 3 A). This system relies on the monosynaptic spread of AAVl- Cre viruses from presynaptic organoid-derived neurons across synaptic clefts, eventually reaching their postsynaptic partners (17,18). Assuming the transplanted GBM cells are synaptically connected to the neurons, this should culminate in the expression of Cre recombinase that turns on the Cre-dependent reporter in GBM cells. Indeed, addition of AAV 1 - Cre to the cortical organoid scaffold led to sparse EGFP labeling of transplanted GBM cells as early as 1 week post-transplantation (Fig. 3B), indicating that there is small percentage of the tumor that form synapses with the neurons at this time point. Importantly, EGFP reporter expression was not observed in no AAV1 controls, indicating that the conversion is dependent on the spread of AAVl-Cre viruses from the presynaptic side. The inventors are performing additional tracing experiments on several primary tumors to ascertain what percentage of tumor cells are synaptically connected to the Cre-infected neurons in so-HOTT.B. CONCLUSION

[0310] Understanding how GBM cells interact with the human cortex — particularly through synaptic communication — is critical for uncovering the mechanisms that drive tumor heterogeneity and evolution. These insights hold immense promise for the development of targeted, microenvironment-disrupting therapies. Yet, a major barrier has been the lack of physiologically relevant, human-specific models that recapitulate the complex interplay between GBM and the brain. Here, the inventors developed and described a cortical assembloid model of GBM that features enhanced synaptogenic properties, including higher colocalization of synaptic puncta within tumor cells, upregulated expression of known postsynaptic proteins, enriched synaptic interactions with non-malignant neurons, and evidence of true synaptic cleft formation by trans-synaptic tracing. It is envisioned that this novel platform will be widely used by basic and translational researchers in the cancer neuroscience field, as a tractable model for mechanistic and disease modeling studies focusing on neuron-tumor communication.C. METHODS1. Tumor transplantation

[0311] Primary tumors were cut into smaller pieces and subsequently transferred to a papain solution supplemented with DNase (Worthington). Papain dissociation was performed over the course of 30-45 minutes with vigorous shaking at 5-10 minute intervals at 37°C. Tumor pieces were further broken down with trituration. Dissociated tumor cells were then pelleted by centrifugation at 300 x g for 5 minutes and resuspended in warm Sasai 3 (S3) media after filtration through a 40 pM cell strainer. Tumor cells were then infected with a GFP lentivirus (SignaGen SL 100268) at a concentration of 1 pL per 1 million cells and with polybrene (1 : 1000) (Millipore Sigma TR-1003-G) for 90 minutes on a rotator at 37°C. Cells were subsequently washed three times and resuspended in Sasai 3 media at a concentration of 100,000 cells per pL. A total of 1 million tumor cells in a 10 pL volume were then transplanted into cortical organoids using the hanging drop method. The next day, tumor cells were transferred into Sasai 4 media with supplements (S4+) or S3 media for so-HOTT and standard HOTT models, respectively. S4+ media consisted of IX DMEM / F-12 with GlutaMax (Life Technologies 10565-018) with IX N-2 supplement (Life Technologies 17052-048), IX CD lipid concentrate (Life Technologies 11905-031), 100 pg / mL primocin (Invivogen NC9141851), 10% Fetal Bovine Serum (HyClone SH30071.03), 10 mg / mL heparin (Sigma H3149), 1% vol / vol Matrigel (BD Biosciences 354230), IX B-27 supplement (Life Technologies 17054-044), 100 ng / mL soluble NLGN3 (OriGene Technologies TP307955), 10 ng / mL BDNF (Gibco 450-02), and 1 pM each of the following: GSK2879552 (Selleckchem S7796), EPZ-5676 (Selleckchem S7062), N-methyl-D-aspartate (Selleckchem S7072), and Bay K 8644 (Selleckchem S7924). Spent media was replaced every 2-3 days until the point of harvest for immunostainings or single-cell capture.2. Immunostaining

[0312] Tumor-transplanted cortical organoids were fixed in 4% paraformaldehyde for 45 minutes at room temperature, rinsed with PBS, and equilibrated in 30% sucrose in PBS overnight at 4C. Afterwards, embedding was performed in OCT Embedding Matrix (Fisher Sci 14-373-65) containing OCT (Tissue-Tek, VWR) and 30% sucrose at a 1 : 1 ratio. Embeddings were then frozen on top of dry ice. The frozen blocks were either transferred to -80°C for longterm storage or cryosectioned as 10-12 pm-thick sections onto glass slides for immunofluorescence staining. To stain the slides, sections were first rinsed with PBS for 15 minutes and then treated with a citrate-based antigen unmasking solution (10 mM sodiumcitrate, pH 6, Vector Labs) heated to 95°C for 20 minutes. Following antigen unmasking, the sections were permeabilized and blocked with a blocking buffer containing 5% donkey serum, 3% bovine serum albumin, and 0.1% Triton X-100 in PBS for 30 minutes at room temperature. The subsequent primary antibody incubations were performed overnight at 4°C using the following primary antibodies in the blocking buffer: Chicken anti-GFP (1 :500, Aves Labs GFP-1020), mouse anti-PSD95 (1 :200, Abeam 2723), and rabbit anti-SYNl (1 :200, Cell Signaling 5297). The primary antibody incubations were followed by three 10-minute PBS washes and secondary antibody incubations with DAPI in the blocking buffer for 2 hours at room temperature. The following secondary antibodies were used. DAPI (1 : 1000, ThermoScientific 62248), AlexaFluor 488 donkey anti-chicken (1 :500, Jackson 703-545-155), AlexaFluor 568 donkey anti-mouse (1 :500, Invitrogen A32773), AlexaFluor 647 donkey antirabbit (1 :500, Invitrogen A31573). Slides were mounted with ProLongTM Gold antifade reagent (Invitrogen) and imaged on a Zeiss LSM 880 confocal laser scanning microscope. Images were thresholded and segmented using a machine-learning based workflow in ilastik (version 1.4.1b23), and segmented images were analyzed for 3-channel colocalization analysis using SynBot.3. Fluorescence-activated cell sorting (FACS), single-cell capture, and sequencing

[0313] so-HOTT and standard HOTT models were dissociated in papain using the protocol described above. Dissociated cells were suspended in FACS buffer with DAPI (1 :2000) as a live / dead stain. GFP+ and GFP- negative live cells (i.e., DAPInegative) corresponding to tumor and organoid cells, respectively, were then sorted using the BioRad S3e Cell Sorter and captured and processed for particle-templated instant partition sequencing (PIPseq) using the PIPseq V T2 3’ Single Cell RNA Kit (Fluent Biosciences). Libraries were quantified on an Agilent 2100 Bioanalyzer and sequencing was performed on an Illumina NovaSeq X Plus (UCLA Sequencing Core).4. Single-cell analysis

[0314] Single-cell RNA sequencing (scRNA-seq) reads were aligned to the GRCh38 human reference genome using PIPseeker software version 02.01.04 (Fluent Biosciences), with sensitivity set to 3. All downstream analyses were performed in Rusing Seurat (v5.2.1), dplyr, ggplot2, ggprism, tidyverse, and supporting packages. Briefly, count matrices were converted to Seurat objects using the Read 1 OX function and the corresponding metadata (Tumor ID, region, media, sample type) were added to each object. Cells with >5% mitochondrial genecontent or any mycoplasma reads were excluded. Seurat objects were merged, normalized, scaled, and dimensionality reduced using PCA and UMAP. Clustering was performed with an empirically determined number of significant PCs. Merged Seurat objects were projected onto a pre-integrated GBM metaatlas using Seurat’s anchor-based label transfer. Predicted cell identities and mapping scores were appended as metadata for downstream analysis.

[0315] Gene modules representing synaptic or functional gene sets were imported from a curated list and applied to the merged Seurat dataset. For each cell, average log-normalized expression was computed for each module (metamodule) using custom R scripts, generating module activity scores. These scores were added as metadata to the Seurat object for further analysis. Differentially expressed genes (DEGs) were identified using Wilcoxon rank-sum tests (Seurat FindMarkers) between relevant conditions (e.g., S4+ vs S3,). DEGs were filtered by log fold change and adjusted p-value. Enrichment analysis was performed using the EnrichR API across selected pathway and gene ontology databases, with visualization of top enriched terms based on statistical significance and gene overlap.5. Cell-cell communication analysis using CellChatAll analyses were performed in R using Seurat (version 5.2.1) and CellChat (version 2.1.2). Briefly, processed and annotated Seurat objects from the PIPseq experiment were loaded for analysis. Metadata columns were used to define biological groupings (e.g., Tumor lD, Region, Media, Sample Type, and predicted cell type annotations). Samples were subset by Tumor ID and media condition (S4+, S3), yielding four main analysis groups: Core_S4, Core_S3, Periphery_S4, and Periphery_S3. Cell-cell communication was inferred using the CellChat package. For each sample group, a CellChat object was initialized (createCellChat()) using normalized gene expression data from the RNA assay and the custom "Cell ldentity" metadata as the grouping variable. The CellChat human ligand-receptor interaction database (CellChatDB. human) was used for all analyses. Data were subset to signaling genes, and overexpressed genes and interactions were identified. Communication probabilities between cell groups were calculated (computeCommunProb()) and filtered for robustness. Inferred communication networks and pathway-level results were extracted for downstream analysis. Signaling pathway activity was visualized using chord diagrams, bubble plots, and heatmaps (e.g., netVisual_aggregate(), netVisual_bubble(), netVisual_heatmap()). Comparative analyses between media and regional conditions were performed by merging CellChat objects (mergeCellChat()) and generating comparative rank plots (rankNet()). Centrality analysis and signaling role heatmaps were generated to identify dominant sender and receiver populations.D. REFERENCES

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[0324] 9. Venkatesh, H.S., Tam, L.T., Woo, P.J., Lennon, J., Nagaraja, S., Gillespie, S.M.,Ni, J., Duveau, D.Y., Morris, P.J., Zhao, J. J., et al. (2017). Targeting neuronal activity- regulated neuroligin-3 dependency in high-grade glioma. Nature 549, 533-537.

[0325] 10. Taylor, K.R., Barron, T., Hui, A., Spitzer, A., Yalgin, B., Ivec, A.E., Geraghty,A.C., Hartmann, G.G., Arzt, M., Gillespie, S.M., et al. (2023). Glioma synapses recruit mechanisms of adaptive plasticity. Nature, 1-9. .

[0326] 11. Hergenreder, E., Minotti, A.P., Zorina, Y., Oberst, P., Zhao, Z., Munguba, H.,Calder, E.L., Baggiolini, A., Walsh, R.M., Liston, C., et al. (2024). Combined small-molecule treatment accelerates maturation of human pluripotent stem cell-derived neurons. Nat. Biotechnol., 1-11.

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[0329] 14. Koopmans, F., Nierop, P. van, Andres-Alonso, M., Byrnes, A., Cijsouw, T.,Coba, M.P., Cornelisse, L.N., Farrell, R.J., Goldschmidt, H.L., Howrigan, D.P., et al. (2019). SynGO: An Evidence-Based, Expert-Curated Knowledge Base for the Synapse. Neuron 103, 217-234. e4.

[0330] 15. Fazzari, E., Azizad, D.J., Yu, K., Ge, W., Li, M X., Nano, P R, Kan, R.L., Turn,H.A., Tse, C., Bayley, N.A., et al. (2024). Glioblastoma Neurovascular Progenitor Orchestrates Tumor Cell Type Diversity. bioRxiv, 2024.07.24.604840.

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[0333] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred aspects, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which areboth chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims. All the references cited below and herein are incorporated by reference for all purposes.

Claims

CLAIMS1. A method for providing an organoid, the method comprising: contacting a brain organoid with cancer cells in suspension to form a liquid droplet containing the brain organoid and cancer cells; and incubating the liquid droplet on a solid surface for a first period of time during which the liquid droplet is a hanging liquid droplet that adheres to and hangs beneath the solid surface; wherein the cancer cells become attached to and / or incorporated into the brain organoid to form a brain cancer organoid.

2. The method of claim 1, wherein the liquid droplet is 10-50 microliters (pL) in volume.

3. The method of claim 1 or 3, wherein the suspension comprises a first cell culture medium.

4. The method of any one of claims 1-3, wherein the duration of the first period of time is 8-12 hours.

5. The method of any of claims 1-4, wherein the cancer cells comprise at least 5%, at least 10%, or at least 15% of the cells in the brain cancer organoid.

6. The method of any of claims 1-5, wherein at the end of the first period of time, the cancer cells comprise at least 5%, at least 10%, or at least 15% of the cells in the brain cancer organoid.

7. The method of any of claims 1-6, wherein at least 5%, at least 10%, or at least 15% of the cells in the brain cancer organoid are cancer cells or are derived from the cancer cells.

8. The method of any of claims 1-7, wherein at least 5%, at least 10%, or at least 15% of the cells in the brain cancer organoid are cancer cells or are derived from the cancer cells after the first period of time.

9. The method of any of claims 1-8, wherein a greater number and / or proportion of the cancer cells become attached to and / or incorporated into the brain organoid compared to a method in which the brain organoid and cancer cells are not incubated in a hanging liquid droplet, and wherein the method is the same as the method of claim 1 with the exception that during the first period of time the liquid droplet on the solid surface is not a hanging liquid droplet.

13. The method of any of claims 1-9, wherein the method further comprises transferring the brain cancer organoid to a culture condition and culturing the brain cancer organoid for a second period of time.

14. The method of claim 13, wherein in the culture condition, the brain cancer organoid is not cultured in a hanging liquid droplet.

15. The method of claim 13 or 14, wherein in the culture condition, the brain cancer organoid is submerged in a second cell culture medium on a low adhesion substrate.

16. The method of claim 15, wherein the second cell culture medium facilitates neurontumor synapses.

17. The method of any one of claims 1-16, wherein the first and / or second cell culture medium comprises one or more of neuroligin-3 (NLGN3) nucleic acid or protein, brain-derived neurotrophic factor (BDNF) nucleic acid or protein, GSK2879552, EPZ-5676, Bay K 8644, and N-methyl-d-aspartate (NMD A).

18. The method of claim 17, wherein the first and / or second cell culture medium comprises soluble NLGN3 protein and / or soluble BDNF protein.

19. The method of any one of claims 1-18, wherein the first and / or second cell culture medium comprises one or more of DMEM, F-12, GlutaMax, N-2 supplement, lipids, antimicrobial components, Primocin, serum, heparin, Matrigel, and B-27 supplement.

20. The method of any one of claims 1-19, wherein the first and / or second cell culture medium is serum-free.

21. The method of claim 19 or 20, wherein the first and / or second cell culture medium comprises heparin at a concentration of 4-6 pg / ml.

22. The method of any one of claims 19-21, wherein the first and / or second cell culture medium comprises Matrigel at a concentration of 0.5-2% vol / vol.

23. The method of any one of claims 17-22, wherein the first and / or second cell culture medium comprises GSK2879552, EPZ-5676, NMDA, and / or Bay K 8644 at a concentration of 0.5-3 pM.

24. The method of any one of claims 17-23, wherein the first and / or second cell culture medium comprises BDNF at a concentration of 1-100 ng / mL.

25. The method of any one of claims 17-24, wherein the first and / or second cell culture medium comprises NLGN3 at a concentration of 10-500 ng / mL.

26. The method of any one of claims 1-25, wherein the organoid comprises neuron-tumor synapses.

27. The method of any of claims 1-26, wherein the cancer cells remain viable for at least 1 week after formation of the brain cancer organoid.

28. The method of any of claims 1-27, wherein the method comprises monitoring the brain cancer organoid for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks, or longer, after formation of the brain cancer organoid.

29. The method of any of claims 1-28, wherein the method comprises analyzing one or more characteristics of cells in the brain cancer organoid at least one week after formation of the brain cancer organoid.

30. The method of any of claims 1-29, wherein the method comprises analyzing one or more characteristics of the cancer cells in the brain cancer organoid at least one week after formation of the brain cancer organoid.

31. The method of claim 29 or 30, wherein the one or more characteristics comprise growth, proliferation, survival, gene expression, invasion, and / or migration.

32. The method of any of claims 1-31, wherein the method comprises determining growth of the cancer cells in the brain cancer organoid at least one week after formation of the brain cancer organoid.

33. The method of any of claims 1-32, wherein the method comprises determining the invasiveness of the cancer cells in the brain cancer organoid at least one week after formation of the brain cancer organoid.

34. The method of claim 32 or 33, wherein the determining comprises assessing cells expressing a marker specific for the cancer cells.

35. The method of claim 34, wherein the marker specific for the cancer cells is a fluorescent protein.

36. The method of any of claims 1-35, wherein the method further comprises screening a candidate agent for activity in reducing cancer cell growth, proliferation, survival, and / or invasion, wherein the method comprises culturing the brain cancer organoid in the presence of the candidate agent, and wherein reduced cancer cell growth, proliferation, survival, and / or invasion as compared to the cancer cell growth, survival, proliferation, and / or invasion, respectively, in the absence of the candidate agent identifies the candidate agent as having activity in reducing cancer cell growth, proliferation, survival, and / or invasion.

37. The method of claim 36, wherein the activity is activity in reducing cell growth, proliferation, survival, and / or invasion of the cancer cells.

38. The method of claim 36 or 37, wherein the activity is activity in reducing cell growth, proliferation, survival, and / or invasion of cancer in a subject.

39. The method of any of claims 36-38, wherein the candidate agent is a small molecule, oligonucleotide, antibody, peptide, or protein.

40. The method of any of claims 1-39, wherein the cancer cells are from a subject.

41. The method of any of claims 1-40, wherein the cancer cells are obtained from a biological sample from a subject that has cancer.

42. The method of claim 41, wherein the biological sample comprises a resected tumor, a biopsy, a needle aspirate, a section, and / or a fluid, or a component of any of the foregoing.

43. The method of any of claims 1-42, wherein the subject is a human subject.

44. The method of any one of claims 1-43, wherein the cancer cells comprise primary cancer cells.

45. The method of any of claims 1-44, wherein the cancer cells comprise glioblastoma cells.

46. The method of any of claims 1-45, wherein the cancer cells are from a tumor.

47. The method of any of claims 1-46, wherein the subject has a cancer of the nervous system.

48. The method of any of claims 1-47, wherein the cancer cells are from a cancer of the nervous system.

49. The method of claim 48, wherein the cancer of the nervous system is brain stem glioma, pineal astrocytic tumor, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglial tumor, mixed glioma, ependymal tumor, medulloblastoma, pineal parenchymal tumor, meningeal tumor, germ cell tumor, or craniopharyngioma.

50. The method of claim 48 or 49, wherein the cancer of the nervous system is glioblastoma.

51. The method of claim 50, wherein the glioblastoma is neural, proneural, classical, or mesenchymal glioblastoma.

52. The method of claim 48 or 49, wherein the cancer of the nervous system is astrocytoma.

53. The method of any of claims 1-52, wherein the cancer cells are obtained by resecting a tumor and dissociating the tumor to provide dissociated cancer cells in suspension.

54. The method of any of claims 1-53, wherein prior to being contacted with the brain organoid, the cancer cells have not been: cultured; plated; trypsinized; passaged; expanded; and / or incubated in suspension or in two-dimensional culture under conditions for proliferation and / or expansion.

55. The method of any of claims 1-54, wherein the cancer cells have not been frozen prior to being contacted with the brain organoid.

56. The method of any of claims 1-55, wherein the cancer cells have been frozen prior to being contacted with the brain organoid.

57. The method of any of claims 1-56, wherein the brain organoid is generated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

58. The method of any of claims 1-57, wherein the brain organoid is generated from human embryonic stem cells (ESCs).

59. The method of any of claims 1-57, wherein the brain organoid is generated from human induced pluripotent stem cells (iPSCs).

60. The method of any of claims 1-59, wherein the cells of the brain organoid are not genetically modified.

61. The method of any of claims 1-60, wherein the cells of the brain organoid and / or the cancer cells are human cells.

62. The method of any of claims 1-61, wherein the brain organoid cells and cancer cells are from the same subject.

63. The method of any of claims 1-61, wherein the brain organoid cells and cancer cells are from different subjects.

64. The method of any of claims 1-63, wherein the brain organoid is a cortical organoid.

65. An organoid generated according to the method of any of claims 1-64.

66. A method for analyzing cell migration, the method comprising: a) contacting a first organoid containing target cells with a second organoid to form a fused organoid comprising the first organoid and the second organoid; b) incubating the fused organoid for a period of time; and c) determining the location of one or more of the target cells within the fused organoid.

67. The method of claim 66, wherein the target cells are cancer cells.

68. The method of claim 67, wherein the cancer cells are not immortalized.

69. The method of any one of claims 66-68, wherein the first organoid is a brain cancer organoid formed by the method of any of claims 1-65.

70. The method of any of claims 66-69, wherein the first organoid and / or the second organoid are brain organoids.

71. The method of any of claims 66-70, wherein the contacting in step a) comprises incubating the first organoid and second organoid together in a partition.

72. The method of claim 71, wherein the partition is a cell-culture well or dish.

73. The method of claim 71 or 72, wherein the bottom of the partition is slanted and the first organoid and second organoid contact one another when pulled downwards by gravity.

74. The method of any of claims 71-73, wherein the partition does not contain a third organoid.

75. The method of any of claims 66-74, wherein the contacting in step a) comprises providing the first organoid and second organoid together in a composition comprising cell culture medium.

76. The method of claim 75, wherein the composition does not comprise a third organoid.

77. The method of claim 75 or 76, wherein the cell culture medium comprises one or more of neuroligin-3 (NLGN3) nucleic acid or protein, brain-derived neurotrophic factor (BDNF) nucleic acid or protein, GSK2879552, EPZ-5676, Bay K 8644, and N-methyl-d-aspartate (NMDA).

78. The method of claim 77, wherein the first and / or second cell culture medium comprises soluble NLGN3 protein and / or soluble BDNF protein.

79. The method of any one of claims 75-78, wherein the cell culture medium comprises one or more of DMEM, F-12, GlutaMax, N-2 supplement, lipids, anti-microbial components, Primocin, serum, heparin, Matrigel, and B-27 supplement.

80. The method of any one of claims 75-79, wherein the cell culture medium is serum-free.

81. The method of claim 79 or 80, wherein the cell culture medium comprises heparin at a concentration of 4-6 pg / ml.

82. The method of any one of claims 79-81, wherein the cell culture medium comprises Matrigel at a concentration of 0.5-2% vol / vol.

83. The method of any one of claims 77-82, wherein the cell culture medium comprises GSK2879552, EPZ-5676, NMDA, and / or Bay K 8644 at a concentration of 0.5-3 pM.

84. The method of any one of claims 77-83, wherein the first and / or second cell culture medium comprises BDNF at a concentration of 1-100 ng / mL.

85. The method of any one of claims 77-84, wherein the first and / or second cell culture medium comprises NLGN3 at a concentration of 10-500 ng / mL.

86. The method of any one of claims 66-85, wherein the organoid comprises neuron-tumor synapses.

87. The method of any of claims 66-86, wherein the fused organoid does not comprise a third organoid.

88. The method of any of claims 66-87, wherein the first organoid and second organoid are spheroid in shape.

89. The method of any of claims 66-88, wherein the first and second organoids are spheroid in shape, and fused at an interface between the first organoid and second organoid.

90. The method of claim 89, wherein the location of the interface is at a furrow between the two spheroid organoids.

91. The method of claim 89 or 90, wherein the maximum diameter of the interface is smaller than the maximum diameter of the first organoid and smaller than the maximum diameter of the second organoid.

92. The method of any of claims 66-91, wherein the first organoid and the second organoid are distinguished from one another based on morphology, genotype, and / or expression of a marker.

93. The method of any of claims 66-92, wherein determining the location of the one or more target cells within the fused organoid comprises detecting the presence of one or more target cells in the first organoid and / or the second organoid.

94. The method of any of claims 66-93, wherein determining the location of the one or more target cells within the fused organoid comprises quantifying the number or density of target cells in the first organoid and / or the second organoid.

95. The method of any of claims 66-94, wherein determining the location of the one or more target cells within the fused organoid comprises quantifying the number or density of target cells in the second organoid.

96. The method of any of claims 66-95, wherein determining the location of the one or more target cells within the fused organoid comprises detecting a marker specific for the target cells.

97. The method of claim 96, wherein the marker specific for the target cells is an endogenous marker.

98. The method of claim 96, wherein the marker specific for the target cells is a heterologous marker and / or is expressed from a transgene.

99. The method of claim 96, wherein the marker specific for the target cells is a fluorescent protein.

100. The method of any of claims 66-99, wherein determining the location of the one or more target cells comprises fluorescence microscopy, immunohistochemistry, immunofluorescence, and / or flow cytometry.

101. The method of any of claims 66-100, wherein the method comprises separating the first organoid from the second organoid prior to determining the location of the one or more target cells.

102. The method of claim 101, wherein the first organoid and second organoid are separated at the interface.

103. The method of any of claims 66-100, wherein the method does not comprise separating the first organoid from the second organoid prior to determining the location of the one or more target cells.

104. The method of any of claims 66-103, wherein the second organoid does not contain target cells prior to the contacting.

105. The method of any of claims 66-104, wherein the presence of target cells in the second organoid after the incubating in step b) is indicative of migration and / or invasion.

106. The method of any of claims 66-105, wherein increased numbers of target cells in the second organoid is indicative of increased migration and / or invasion.

107. The method of any of claims 66-106, wherein the method comprises perturbing the fused organoid and / or the target cells and determining the effect of the perturbation on target cell migration.

108. The method of claim 107, wherein the effect of the perturbation on target cell migration is determined in comparison to a control fused organoid in which the perturbation is not performed.

109. The method of claim 108, wherein the perturbation comprises providing target cells with a different genetic background than the target cells in the control fused organoid.

110. The method of any of claims 107-109, wherein the perturbation comprises altering gene expression in the target cells and / or fused organoid.

111. The method of any of claims 107-110, wherein the perturbation comprises incubating the fused organoid and / or the target cells in the presence of a candidate agent.

112. The method of claim 111, wherein reduced target cell migration in the presence of the candidate agent identifies the candidate agent as having activity in reducing cell migration and / or invasion.

113. The method of claim 112, wherein the target cells are cancer cells and the reduced cell migration and / or invasion is reduced cancer cell migration and / or invasion.

114. The method of any of claims 111-113, wherein the candidate agent is a small molecule, oligonucleotide, antibody, peptide, or protein.

115. The method of any of claims 67-114, wherein the cancer cells comprise primary cancer cells.

116. The method of any of claims 67-115, wherein the cancer cells are from a tumor.

117. The method of any of claims 67-116, wherein the cancer cells are from a cancer of the nervous system.

118. The method of claim 117, wherein the cancer of the nervous system is brain stem glioma, pineal astrocytic tumor, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglial tumor, mixed glioma, ependymal tumor, medulloblastoma, pineal parenchymal tumor, meningeal tumor, germ cell tumor, or craniopharyngioma.

119. The method of claim 117 or 118, wherein the cancer of the nervous system is glioblastoma.

120. The method of any of claims 67-119, wherein the cancer cells comprise glioblastoma cells.

121. The method of any of claims 67-120, wherein the cancer cells are from a subject.

122. The method of any of claims 67-121, wherein the cancer cells are obtained from a biological sample from a subject that has cancer.

123. The method of claim 121 or 122, wherein the subject is a human subject.

124. A brain organoid comprising neurons and cancer cells, wherein the organoid comprises neuron-cancer cell synapses.

125. The brain organoid of claim 124, wherein the cancer cells comprise at least 5%, at least 10%, or at least 15% of the cells in the brain organoid.

126. The brain organoid of claim 124 or 125, wherein at least 5%, at least 10%, or at least 15% of the cells in the brain cancer organoid are cancer cells or are derived from the cancer cells.

127. The brain organoid of any of claims 124-126, wherein a greater number and / or proportion of the cancer cells become attached to and / or incorporated into the brain organoid in comparison to a method in which the brain organoid and cancer cells are not incubated in a hanging liquid droplet.

128. The brain organoid of any one of claims 124-127, wherein the brain organoid is in or was cultured in a cell culture medium.

129. The brain organoid of claim 128, wherein the cell culture medium comprises one or more of neuroligin-3 (NLGN3) nucleic acid or protein, brain-derived neurotrophic factor (BDNF) nucleic acid or protein, GSK2879552, EPZ-5676, Bay K 8644, and N-methyl-d- aspartate (NMD A).

130. The brain organoid of claim 128 or 129, wherein the cell culture medium comprises soluble NLGN3 protein and / or soluble BDNF protein.- I l l -131. The brain organoid of any one of claims 128-130, wherein the first and / or second cell culture medium comprises one or more of DMEM, F-12, GlutaMax, N-2 supplement, lipids, anti-microbial components, Primocin, serum, heparin, Matrigel, and B-27 supplement.

132. The brain organoid of any one of claims 128-131, wherein the cell culture medium is serum-free.

133. The brain organoid of any one of claims 131-132, wherein the cell culture medium comprises heparin at a concentration of 4-6 pg / ml.

134. The brain organoid of any one of claims 131-133, wherein the cell culture medium comprises Matrigel at a concentration of 0.5-2% vol / vol.

135. The brain organoid of any one of claims 129-134, wherein the cell culture medium comprises GSK2879552, EPZ-5676, NMD A, and / or Bay K 8644 at a concentration of 0.5-3 pM.

136. The brain organoid of any one of claims 129-135, wherein the cell culture medium comprises BDNF at a concentration of 1-100 ng / mL.

137. The brain organoid of any one of claims 129-136, wherein the cell culture medium comprises NLGN3 at a concentration of 10-500 ng / mL.

138. The brain organoid of any one of claims 124-137, wherein the cancer cells are from a subject.

139. The brain organoid of any one of claims 124-138, wherein the cancer cells are obtained from a biological sample from a subject that has cancer.

140. The brain organoid of claim 139, wherein the biological sample comprises a resected tumor, a biopsy, a needle aspirate, a section, and / or a fluid, or a component of any of the foregoing.

141. The brain organoid of any of claims 138-140, wherein the subject is a human subject.

142. The brain organoid of any one of claims 124-141, wherein the cancer cells comprise primary cancer cells.

143. The brain organoid of any one of claims 124-142, wherein the cancer cells comprise glioblastoma cells.

144. The brain organoid of any one of claims 124-143, wherein the cancer cells are from a tumor.

145. The brain organoid of any one of claims 124-144, wherein the subject has a cancer of the nervous system.

146. The brain organoid of any one of claims 124-145, wherein the cancer cells are from a cancer of the nervous system.

147. The brain organoid of claim 146, wherein the cancer of the nervous system is brain stem glioma, pineal astrocytic tumor, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglial tumor, mixed glioma, ependymal tumor, medulloblastoma, pineal parenchymal tumor, meningeal tumor, germ cell tumor, or craniopharyngioma.

148. The brain organoid of claim 146 or 147, wherein the cancer of the nervous system is glioblastoma.

149. The brain organoid of claim 148, wherein the glioblastoma is neural, proneural, classical, or mesenchymal glioblastoma.

150. The brain organoid of claim 146 or 147, wherein the cancer of the nervous system is astrocytoma.

151. The brain organoid of any of claims 124-150, wherein the cancer cells are obtained by resecting a tumor and dissociating the tumor to provide dissociated cancer cells in suspension.

152. The brain organoid of any of claims 124-151, wherein the cancer cells have not been: cultured; plated; trypsinized; passaged; expanded; and / or incubated in suspension or in two- dimensional culture under conditions for proliferation and / or expansion prior to incorporation into the organoid.

153. The brain organoid of any of claims 124-152, wherein the cancer cells have not been frozen prior to incorporation into the brain organoid.

154. The brain organoid of any of claims 124-153, wherein the cancer cells have been frozen prior to incorporation into the brain organoid.

155. The brain organoid of any of claims 124-154, wherein the brain organoid is generated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

156. The brain organoid of any of claims 124-155, wherein the brain organoid is generated from human embryonic stem cells (ESCs).

157. The brain organoid of any of claims 124-156, wherein the brain organoid is generated from human induced pluripotent stem cells (iPSCs).

158. The brain organoid of any of claims 124-157, wherein the cells of the brain organoid are not genetically modified.

159. The brain organoid of any of claims 124-158, wherein the cells of the brain organoid and / or the cancer cells are human cells.

160. The brain organoid of any of claims 124-159, wherein the brain organoid cells and cancer cells are from the same subject.

161. The brain organoid of any of claims 124-159, wherein the brain organoid cells and cancer cells are from different subjects.

162. The brain organoid of any of claims 124-161, wherein the brain organoid is a cortical organoid.

163. The brain organoid of any one of claims 124-162, wherein the brain organoid comprises co-localization of presynaptic and postsynaptic molecules and / or cells.

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