Cancer modeling platform and methods of use thereof

Abstract

Described herein are in vitro cell constructs (or "organoids") useful as tumor models, comprising live tumor cells derived from a patient. In some embodiments, the in vitro cell constructs include (a) a core comprised of live tumor cells and (b) a shell surrounding (e.g., encapsulating) the core, the shell comprised of live benign cells (e.g., tissue cells, benign or differentiated tumor cells, etc.). Also described herein are methods for making and using such constructs. Further provided are devices useful for evaluating tumor cells in vitro, comprising: (a) a microfluidic device having a chamber and a channel in fluid communication with the chamber; (b) a live tumor cell construct (e.g., an organoid) in the chamber; (c) a growth medium in the chamber and the channel; (d) a pump operatively coupled to the chamber and the channel and configured to circulate the medium from the chamber, through the channel, and back to the chamber; and (e) a microporous membrane (e.g., a TRANSWELL® microporous membrane) in the channel, positioned to allow the medium to flow through it.

Description

[Technical field]

[0001] Related Application Information This application claims the benefit of U.S. Provisional Patent Application No. 62 / 370,320, filed August 3, 2016, and U.S. Provisional Patent Application No. 62 / 522,313, filed June 20, 2017, the disclosures of each of which are incorporated herein by reference.

[0002] Statement of government support This invention was made with Government support under Grant Nos. 1U54TR001362-01 and 5UL1TR001420-03 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] The present application relates to in vitro cellular constructs (or "organoids") that can be used as tumor models, and methods for making and using same. [Background technology]

[0004] Precision medicine holds great promise for improving the treatment of cancer patients. Currently, genetic profiling can be performed on tumor biopsies to narrow the scope of drug selection based on key important mutations, but selecting the absolute best drug for a particular patient remains difficult. Screening drug selection with tumor models made from the patient's own cells can complement genetic screening to determine the most effective drugs. Such patient-specific cancer models can provide personalized predictive data on the effect of drugs on tumor growth and its metastatic potential. Summary of the Invention

[0005] The first aspect of the present invention is an in vitro cell construct (or "organoid") useful as a tumor model, comprising (a) a core of living tumor cells and (b) a shell surrounding (e.g., encapsulating) the core, the shell being made of living benign cells (e.g., tissue cells, benign or differentiated tumor cells, etc.). The living tumor cells and / or living benign cells can be obtained from a subject, such as from a biopsy (e.g., tumor biopsy and / or tissue biopsy) taken from the subject.

[0006] The second aspect of the present invention is an in vitro cell construct (or "organoid") that is useful as a tumor model, comprising living tumor cells from subject.In some embodiments, the living tumor cells are derived from a biopsy (e.g., tumor biopsy) taken from subject.

[0007] A further aspect of the invention is a device useful for evaluating tumor cells in vitro (e.g., useful for evaluating tumor cell metastasis in vitro, for evaluating tumor cell migration and / or invasion in vitro, for evaluating tumor cell-containing constructs in vitro, and / or for evaluating response to a compound of interest, e.g., a drug or prodrug, etc.), comprising: (a) a microfluidic device having a chamber and a channel in fluid communication with the chamber; (b) a living tumor cell construct (e.g., an organoid) in the chamber; (c) growth medium in the chamber and the channel; (d) a pump operatively coupled to the chamber and the channel and configured to circulate the medium from the chamber, through the channel and back to the chamber; and (e) a microporous membrane (e.g., a TRANSWELL® microporous membrane) in the channel positioned to allow medium to flow therethrough.

[0008] Another aspect of the present invention is a device useful for evaluating tumor cells in vitro (e.g., for evaluating the metastasis of tumor cells in vitro, for evaluating the migration and / or invasion of tumor cells in vitro, for evaluating the growth of constructs containing tumor cells in vitro, and / or for evaluating the response to a compound of interest, such as a drug or prodrug), which comprises two or more chambers with at least one organoid in each chamber. The one or more organoids present in the device can be made from cells obtained from a single subject. In some embodiments, the device comprises live tumor cell organoids in the first chamber and liver organoids in the second chamber. Live tumor cell organoids and liver organoids can be formed from cells obtained from the same subject, which can provide individualized analysis.

[0009] The above and other objects and aspects of the present invention are described in more detail below. It should be understood that the present invention can be embodied in various forms, and should not be construed as being limited to the embodiments set forth herein. Rather, such embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. [Brief description of the drawings]

[0010] [Figure 1]A conceptual diagram showing how modified 3D in vitro models can be used to better inform precision medicine treatment regimens for individual patients. The solid arrows represent the current state of the art precision medicine pipeline, where treatment is determined for a patient based on their tumor genetic profile. However, in practice, even after the identification of key mutations of interest, oncologists are often forced to choose between several possible drugs, resulting in a best guess or guess as to what the optimal treatment will be. The dashed arrows show that the introduction of organoids made with patient cells can complement genetic screening of biopsied tumor cells and ultimately predict the optimal therapy for the patient. [Figure 2A] FIG. 1 is a schematic diagram showing the assembly of the microfluidic device layers: (from top to bottom) tubing, polystyrene (PS) slide with fluidic inlets and outlets, adhesive film with chambers for organoids and corresponding fluidic inlets and outlets, glass slide, and photomask layer. [Figure 2B] Schematic diagram showing the in situ patterning technique. The microfluidic chamber (i) is filled with a hydrogel mixture containing HA hydrogel, photoinitiator and tumor cells (ii) and then exposed to UV illumination through a photomask (iii). The exposed hydrogel is crosslinked (iv) and then the non-crosslinked gel is washed away using a clean buffer (v). Finally, the washed-out buffer is replaced with culture medium for incubation (vi). [Figure 2C] FIG. 1 is a schematic diagram showing the entire measurement apparatus, featuring a low-volume, closed-loop fluidic circuit for each organoid, with flow driven by a computer-controlled peristaltic pump. [Figure 2D] 13A-13D are maximum projection confocal and segmented images of tumor constructs on the device after LIVE / DEAD assay on days 7 and 14, showing percent viability of 93.3% and 86.4%, respectively. [Figure 3A-3F]Panels af(i) are maximum projection confocal images of tumor constructs after LIVE / DEAD assay, and panels af(ii) are segmented images using Imaris software. Medium grey represents dead cells, light grey represents live cells. Panel a) is an image of a control at week 1 with >90% viability. Panel b) is an image of a control at week 2. Panel c) shows constructs that were under the influence of carboplatin and pemetrexed drugs for 7 days. Panel d) shows constructs that were under the influence of carboplatin and pemetrexed drugs for 7 days. Panel e) shows constructs that were under the influence of cisplatin and pemetrexed drugs for 7 days. Panel f) shows constructs that were under the influence of cisplatin and pemetrexed drugs for 7 days. [Figure 3G] Graph showing viability (live / dead cells) measurements from segmented images of control and drug-treated organoids. Drug exposure began on day 7. Significance: *p<0.1, **p<0.05, ***p<0.01. [Figure 4A.4B] Figure 1 shows the biomarker-based therapeutic drug screening in patient-derived tumor constructs. In panel A), genetic analysis of tumor biopsy samples identified two mutations, PBRM1 and BAP1. The BAP1 mutation was identified as a potential target by the EZH2 inhibitor DZNep. In panel B), MTS mitochondrial metabolism assay showed that control organoids were the most proliferative compared to organoids treated with DZNep drug, with a decrease in mitochondrial metabolism occurring in proportion to the decrease in cell number. Conversely, HCT116 organoids do not appear to respond negatively to DZNep. [Fig. 4C-4F]FIG. 1 shows biomarker-based therapeutic drug screening in patient-derived tumor constructs. In panel C), further analysis was performed using Annexin V and Ki67 biomarker images, showing that all drug treatments had a higher ratio of Annexin V to Ki67, indicating increased apoptotic cell mass. Representative images of Annexin V and Ki67 fluorescent biomarkers are shown for DZNep treatments. Red staining (medium grey in black and white images) is Annexin V, and green staining (light grey in black and white images) is Ki67. Scale bar indicates 100 um. In panel D), further analysis was performed using Annexin V and Ki67 biomarker images, showing that all drug treatments had a higher ratio of Annexin V to Ki67, indicating increased apoptotic cell mass. Representative images of Annexin V and Ki67 fluorescent biomarkers are shown for DZNep treatments. The red staining (medium gray in the black and white image) is Annexin V, and the green staining (light gray in the black and white image) is Ki67. The scale bar indicates 100 um. In panel E), hematoxylin and eosin stained organoid sections show that as the DZNep concentration increases in mesothelioma organoids, they lose their normal morphology and nucleus-less ghost cells appear. In panel F), contrary to the mesothelioma organoids in E, HCT116 cells do not show dose-dependent changes like the mesothelioma organoids in E, suggesting that DZNep induces cell death more effectively in mesothelioma organoids than in HCT116 organoids. [Figure 5A] Figure 1 shows viable organoids derived from difficult to culture tumors. Panel A) Low-grade appendix (LGA). [Figure 5B] Figure 1 shows viable organoids derived from difficult to culture tumors.Panel B) Well-differentiated papillary peritoneal mesothelioma. [Figure 5C] Figure 1 shows viable organoids derived from difficult to culture tumors. Panel C) As already suspected in clinical practice, LGA organoids do not respond to therapy, whereas high-grade appendiceal tumor organoids do. [Figure 5D] Figure 1 shows viable organoids derived from difficult to culture tumors. Panel D) Generation of viable tumor organoids from a rare sarcoma. [Figure 6] Figure 6A is a diagram showing the 2D drug testing protocol. Figure 6B is a graph showing percent relative cell viability values ​​of HCT116, HT29, CACO2, and SW480 colorectal cancer cell lines in 2D microenvironments after treatment with various concentrations of anticancer drugs. A=regorafenib (μM) B=sorafenib (μM) C=trametinib (nM) D=5-FU (mM) E=dabrafenib (μM). [Figure 7] Figure 7A is a diagram showing the 3D drug testing protocol. Figure 7B is a graph showing percent relative cell viability values ​​of HCT116, HT29, CACO2, and SW480 colorectal cancer cell lines in 3D microenvironments after treatment with various concentrations of anticancer drugs. A=regorafenib (μM) B=sorafenib (μM) C=trametinib (nM) D=5-FU (mM) E=dabrafenib (μM). [Figure 8] Graph comparing the results of 2D and 3D drug screening with regorafenib, sorafenib, and 5-FU on HCT116 and CACO2 colorectal cell lines. Significant differences are again displayed as determined by p-values ​​(<0.10=* <0.05=** <0.01=***). [Figure 9A] FIG. 1 shows the circular organoid drug testing protocol. [Figure 9B] Microscopic images detailing targeted cell death of HCT116 by regorafenib, and universal cell death of both HCT116 and CACO2 by 5-FU at day 7. Green (light grey in black and white image) fluorescent dye (calcein AM) indicates live cells, whereas red (medium grey in black and white image) fluorescent dye (ethidium homodimer 1) indicates dead cells (scale bar = 200 μm). [Figure 9C] Graph showing the ratios of regorafenib and 5-FU at each concentration versus the control ratio, which is the ratio of no treatment. [Figure 10]FIG. 1 depicts the internal and external components of a microfluidic device that circulates media through the device. [Figure 11] 1 is an image showing LIVE / DEAD results showing that liver organoids are required to metabolize capecitabine into the pharmacologically active form of 5-FU and induce increased cell death in cardiac and pulmonary organoids. [Figure 12] Images showing LIVE / DEAD results in a miniaturized system, showing that in the presence of the liver, capecitabine is metabolized to the toxic drug 5-FU, causing toxicity to the heart and lungs. Without the liver, this metabolism does not occur and heart and lung organoid viability is not compromised. [Figure 13] Graph of initial biomarker analysis in standard size and miniaturized systems. [Figure 14] Diagram of a hybrid microreactor fabrication strategy incorporating tape fluidics and adhesive film and / or double-sided tape (DST), poly(methyl methacrylate) (PMMA), and polydimethylsiloxane (PDMS) components. [Figure 15A] 1A-1D are different components and / or views of an example device having two chambers according to an embodiment of the present invention. [Figure 15B] 1A-1D are different components and / or views of an example device having two chambers according to an embodiment of the present invention. [Figure 15C] 1A-1D are different components and / or views of an example device having two chambers according to an embodiment of the present invention. [Figure 15D] 1A-1D are different components and / or views of an example device having two chambers according to an embodiment of the present invention. [Figure 16] FIG. 1 shows an example six chamber device according to an embodiment of the present invention. [Figure 17] 1 is a schematic diagram of a device structure according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention will now be described in more detail hereinafter with reference to the accompanying drawings, which show embodiments of the invention. However, the present invention may be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, such embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0012] The terms used herein are merely for describing certain embodiments and are not intended to limit the present invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms unless the context clearly indicates otherwise. It is further understood that the terms "comprise" or "comprising" as used herein specify the presence of a stated feature, integer, step, operation, element, component and / or group or combination thereof, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or group or combination thereof.

[0013] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. It is further understood that terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of this specification and claims, and should not be interpreted in an ideal or overly formal sense unless expressly defined as such in this specification. Known functions or structures may not be described in detail for brevity and / or clarity. All publications, patent applications, patents and other references mentioned in this specification are incorporated herein by reference. In case of conflict in terms, the specification shall control.

[0014] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0015] Unless the context dictates otherwise, it is specifically contemplated that the various features of the invention described herein may be used in any combination. Moreover, the invention also contemplates that in some embodiments of the invention, any feature or combination of features described herein may be excluded or omitted. For example, if the specification indicates that a complex comprises components A, B, and C, it is specifically contemplated that any of A, B, or C, or combinations thereof, may be omitted and excluded.

[0016] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) should be interpreted as including the recited materials or steps of the claimed invention "and that do not materially affect the basic and novel characteristics." Thus, as used herein, the term "consisting essentially of" should not be interpreted as the equivalent of "comprising."

[0017] As used herein, the term "about" when referring to a measurable value, such as an amount or concentration, means to refer to a variation of up to ±20% of the particular value, such as, but not limited to, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the particular value, as well as the particular value. For example, "about X" means to include X and a variation of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X, where X is a measurable value. The ranges of measurable values ​​provided herein may include any other ranges and / or respective values ​​within the range.

[0018] A "cell" as used in the present invention is generally an animal cell, particularly a mammalian and primate cell, examples of which include, but are not limited to, human, dog, cat, rabbit, monkey, chimpanzee, cow, pig, or goat. The cells are preferably at least somewhat differentiated into a particular cell or tissue type, such as liver, intestine, pancreas, lymph node, smooth muscle, skeletal muscle, central nervous system, peripheral nervous system, skin, immune system, etc. Some cells may be cancer cells, as discussed further below, in which case some cells, as also discussed further below, optionally but preferably, express a detectable compound (naturally or by recombinant technology).

[0019] As used herein, a "subject" is generally a human subject, although aspects of the invention may be practiced with other animal subjects, particularly veterinary mammalian subjects (e.g., dogs, cats, horses, goats, sheep). Subjects may be male or female and of any age, including infants, juveniles, adolescents, adults, and geriatrics.

[0020] "Organoid" is used interchangeably herein with "cell construct" and "three-dimensional tissue construct" and refers to a composition of living cells, typically in a carrier medium, fabricated into a three-dimensional or multi-layered structure (as opposed to a monolayer). Suitable carrier media include hydrogels, such as cross-linked hydrogels, as described below. Further examples of hydrogels include, but are not limited to, those described in International Application PCT / US2015 / 055699, which is incorporated herein by reference. Organoids can include one differentiated cell type, or two or more differentiated cell types, depending on the particular tissue or organ to be modeled or mimicked. Some organoids can include cancer cells, as discussed further below. When organoids include cancer cells, they can include tissue cells and / or cell-free tissue mimics, such as those that include extracellular matrix (or proteins or polymers derived therefrom), hyaluronic acid, gelatin, collagen, alginate, and others, or combinations thereof. Thus, in some embodiments, cells are mixed with extracellular matrix or cross-linked matrix to form organoid or construct, while in other embodiments, cell aggregates such as spheroid or organoid can be preformed and then mixed with extracellular matrix.In some embodiments, organoid is tumor organoid and / or non-tumor organoid.

[0021] In some embodiments, organoid comprises cells that are human-derived cells, and in some embodiments, organoid comprises cells that are human-derived cells.Organoid of the present invention can express and / or produce one or more (for example, 1, 2, 3, 4 or more) biomarkers that are identical to the biomarkers produced by cells in vivo.For example, liver cells in vivo produce albumin, and organoid of the present invention that comprises liver cells can express albumin.In some embodiments, organoid can express the same amount or the amount of biomarkers that are ±20%, ±10%, or ±5% of the average amount that corresponding cells in vivo produce and / or express. Examples of biomarkers include, but are not limited to, albumin, urea, glutathione S-transferase (GST) (e.g., α-GST), chemokines (e.g., IL-8, IL-1β, etc.), prostacyclin, SB100B, neuron-specific enolase (NSE), myelin basic protein (MBP), hormones (e.g., testosterone, estradiol, progesterone, etc.), inhibin A / B, lactate dehydrogenase (LDH), and / or tumor necrosis factor (TNF).

[0022] In some embodiments, the organoids of the present invention have a diameter of about 200 μm to about 350 μm, for example, about 200, 250, 300, or 350 μm. The organoids can contain about 1,500 or 2,000 to about 3,000 or 3,500 cells in total. The organoids of the present invention can be in any suitable shape, for example, any three-dimensional or multi-layer shape. In some embodiments, the organoids of the present invention are in the form of spheroids. In some embodiments, the organoids of the present invention can be self-organized in suspension or medium (e.g., cross-linked hydrogel).

[0023] As used herein, a "growth medium" may be any natural or artificial growth medium (typically an aqueous liquid) that supports the cells used to carry out the present invention. Examples include, but are not limited to, essential medium or minimal essential medium (MEM), or variations thereof, such as Eagle's minimal essential medium (EMEM) and Dulbecco's modified Eagle's medium (DMEM), as well as those that include blood, serum, plasma, lymph, and other synthetic mimics thereof. In some embodiments, the growth medium includes a pH color indicator (e.g., phenol red).

[0024] As used herein, a "candidate compound" or "compound of interest" may be any compound for which the activity of inhibiting cancer cell spread from a primary site to a second site is determined, or the activity in reducing cancer cell viability and / or antitumor activity is determined. For demonstration, marimastat (N-[2,2-dimethyl-1-(methylcarbamoyl)propyl]-2-[hydroxy-(hydroxycarbamoyl)methyl]-4-methyl-pentanamide) is used as a test compound. However, any compound can be used, and generally organic compounds such as proteins, peptides, nucleic acids, and small organic compounds (aliphatic, aromatic, and mixed aliphatic / aromatic compounds) can be used. Candidate compounds can be generated by any suitable technique, including random generation by combinatorial techniques and / or rational design based on a specific target. See, for example, AMStock et al. Targets for anti-metastatic drug development, Curr.Pharm.Des.19(28):5127-34(2013). In some embodiments, the candidate compound is a prodrug, which can be converted into active drug during the implementation of the test and / or method of the present invention.In some embodiments, the prodrug is converted into active drug by liver organoid.

[0025] As used herein, a "detectable compound" may be a fluorescent protein (e.g., red fluorescent protein, green fluorescent protein, etc.); an antigenic protein or peptide that is specifically bound by an antibody conjugated with an enzyme, fluorescent, or radioactive group, or other label; or any other suitable detectable compound. The detectable compound may be one that occurs naturally in cancer cells (e.g., a cell marker protein that is expressed at higher levels in cancer cells than in non-cancerous cells) or one that is inserted into cancer cells by genetic engineering / recombinant DNA technology (i.e., heterologous).

[0026] The cells used to practice the invention are preferably animal cells (e.g., avian, reptile, amphibian, etc.), and in some embodiments are mammalian cells (e.g., dog, cat, mouse, rat, monkey, ape, human). The cells may be differentiated or undifferentiated cells, but in some embodiments are tissue cells (e.g., liver cells such as hepatocytes, pancreatic cells, cardiac muscle cells, skeletal muscle cells, etc.).

[0027] The choice of cells depends on the particular organoids to be made. For example, liver organoids may use liver parenchymal cells. Peripheral or central nervous organoids may use peripheral nerve cells, central nerve cells, glial cells, or a combination thereof. Bone organoids may use osteoblasts, osteoclasts, or a combination thereof. Lung organoids may use lung airway epithelial cells. Lymph node organoids may use follicular dendritic lymphocytes, fibroblastic reticular lymphocytes, leukocytes, B cells, T cells, or a combination thereof. Smooth or skeletal muscle organoids may use smooth muscle cells, skeletal muscle cells, or a combination thereof. Skin organoids may use skin keratinocytes, skin melanocytes, or a combination thereof. The cells may be differentiated when first incorporated into the composition, or undifferentiated cells that are subsequently differentiated may be used. Additional cells may be added to any of the above compositions, and the following cancer cells may be added to the primary or "first" organoids, as described below.

[0028] The cancer cells used in the present invention may be any type of cancer cell, including but not limited to melanoma, carcinoma, sarcoma, blastoma, glioma, and astrocytoma cells. In some embodiments, the cancer cells used in the present invention express N-cadherin and / or exhibit epithelial-mesenchymal transition in the methods taught herein. The cancer cells may be cancer cells from any primary tissue, including but not limited to intestine (small intestine, large intestine, colon), lung, breast, prostate, skin, bone, brain, liver, pancreas, uterine, cervical, testicular, and ovarian cancer cells.

[0029] In some embodiments, the cells can be obtained from a subject, such as, for example, a subject or patient undergoing cancer treatment, and / or a subject or patient with cancer, and / or a subject with an immune deficiency. In some embodiments, the cells are tumor cells, such as, for example, tumor cells obtained from a biopsy, and organoids made from such cells can be used to screen for potentially effective drugs and / or treatments. Examples of tumor organoids obtained from a biopsy include, but are not limited to, mesothelioma, colorectal, appendix, lung, melanoma, and sarcoma organoids. In some embodiments, the cells include benign cells obtained from a tissue biopsy. The cells can be at least partially differentiated into specific cell or tissue types, such as brain, liver, intestine, pancreas, lymph node, smooth muscle, skeletal muscle, central nervous system, peripheral nervous system, skin, immune system, and the like. Cells obtained from biopsy (e.g., tumor and / or benign) can be used to form and / or generate the organoids of the present invention, and the resulting organoids can be generated and / or used in the methods and / or devices of the present invention within about 1, 2, 3, 4, 5, 6, 7, or 8 days after biopsy. In some embodiments, the cells can be labeled with a detectable compound, such as, but not limited to, a fluorescent compound (e.g., dye, protein, etc.).

[0030] In some embodiments, the organoid of the present invention does not make from and / or does not contain the cell of immortalized cell line.The organoid of the present invention can also make and / or use tumor cell and / or high-functioning cell, such as but not limited to primary cell and / or stem cell (for example, induced pluripotent stem cell and / or differentiated iPS-derived cell).

[0031] The aspects and features of the invention described herein can be carried out according to known materials, methods and techniques, or variations thereof, which are obvious to those skilled in the art to which the invention belongs.See, for example, Skardal A, Devarasetty M, et al.A reductionist metastasis-on-a-chip platform for in vitro tumor progression modeling and drug screening.Biotechnology and Bioengineering 113(9), 2020-32(2016);Skardal A, Devarasetty M, et al.A hydrogel bioink toolkit for mimicking native tissue biochemical and mechanical properties in bioprinted tissue constructs.Acta Biomaterialia 25, 24-34(2015);Bhise N, Manoharan V, et al.A liver-on-a-chip platform with bioprinted hepatic spheroids.Biofabrication 8(1);014010(2016).

[0032] As described above, the present invention provides in vitro cellular constructs (or "organoids") useful as tumor models, comprising (a) a core comprised of living tumor cells and (b) a shell surrounding (e.g., encapsulating) the core, wherein the shell is comprised of living benign cells (e.g., tissue cells, benign or differentiated tumor cells, etc.).

[0033] In some embodiments, the viable tumor cells comprise malignant cells.

[0034] In some embodiments, the live tumor cells comprise or consist of colorectal cancer cells (eg, HCT116 cells, HT29 cells, SW480 cells, Caco2 cells, etc.).

[0035] In some embodiments, the tumor cells and benign cells comprise mammalian cells (e.g., human, mouse, rat, monkey, etc.).

[0036] In some embodiments, the viable benign cells comprise epithelial cells.

[0037] In some embodiments, the live benign cells comprise Caco-2 cells.

[0038] In some embodiments, the tumor cells comprise a detectable compound (eg, a fluorescent compound).

[0039] In some embodiments, the core and / or the shell comprise a hydrogel.

[0040] In some embodiments, the constructs are grown in the culture medium for one or two days, or for one to two weeks, or longer.

[0041] According to some embodiments, an in vitro cell construct (or "organoid") is provided that is useful as a tumor model, comprising live tumor cells from a subject. In some embodiments, the live tumor cells are taken and / or derived from a subject, such as, for example, the tumor of the subject. The live tumor cells can be taken and / or derived from a tumor biopsy. In some embodiments, the live tumor cells form the core of the organoid, and the organoid comprises a shell of live benign cells.

[0042] In some embodiments, the organoid comprises live benign cells from a subject. The benign cells can be taken and / or derived from a subject, such as, for example, the tissue of the subject. In some embodiments, the live benign cells can be taken and / or derived from tissue biopsy and / or can be tissue specific. The organoid comprises live benign cells can be separated (e.g., formed and / or present separately in different chambers of the device) from the organoid comprises live tumor cells. In some embodiments, the cells taken and / or derived from a single subject (e.g., using one or more biopsies from the subject) are used to form at least two (e.g., 2, 3, 4, 5, 6, 7, 8 or more) different organoids, with at least one organoid comprising live tumor cells from tumor biopsy from the subject, and at least one other organoid comprising live liver cells from the subject. In some embodiments, cells harvested and / or derived from a single subject (e.g., using one or more biopsies from the subject) are used to form at least two (e.g., 2, 3, 4, 5, 6, 7, 8, or more) organoids, with at least one organoid comprising viable tumor cells obtained from a tumor biopsy from the subject, and at least one other organoid comprising viable benign cells from the subject that are of the same tissue type as the liver tumor cells.

[0043] The organoid of the present invention can provide a patient-specific model system that can be used to determine the treatment of the patient before the start of therapy and / or treatment.In some embodiments, the compound of interest can be screened for anti-tumor activity, optionally in addition to additional screening methods such as, but not limited to, genetic biomarker evaluation and / or genetic profiling.In some embodiments, the organoid and / or method of the present invention can enable and / or provide cell biomarker recognition, quantification of biomarker expression, and / or real-time testing of chemotherapy drug effectiveness.

[0044] A method for screening a compound of interest for anti-tumor activity in vitro can be carried out by (a) providing at least one construct comprising live tumor cells as described herein; (b) contacting the construct with a compound in vitro; and then (c) determining the proliferation of live tumor cells, the reduction in proliferation of live tumor cells (e.g., lack of tumor cell proliferation, tumor cell death, reduced invasion of the shell by tumor cells, etc.) (e.g., compared to tumor cells of at least one similar construct not contacted with the compound and / or compared to live benign cells present in the shell surrounding the construct and / or present in another organoid) that indicates the anti-tumor activity of the compound of interest. In some embodiments, the tumor cells are obtained from a patient suffering from a tumor. In some embodiments, the compound of interest is a prodrug. In some embodiments, if it is determined that the compound reduces the proliferation of tumor cells in vitro, the method of the present invention can further include administering a therapeutically effective amount of the compound to the patient.

[0045] In some embodiments, the method of the present invention comprises obtaining a tumor biopsy from a subject (e.g., a patient) and using the cells to prepare the organoid of the present invention. The tumor biopsy can be partially or completely genetically sequenced to identify mutations, and any identified mutations can indicate and / or suggest one or more compounds of interest for the treatment of the subject (e.g., antitumor activity). The method of the present invention can include screening one or more compounds of interest identified in genetic sequencing, and contacting one or more of the compounds of interest, respectively, and / or combinations thereof, with the organoid made using cells from the tumor biopsy. The method of the present invention can further comprise administering one or more compounds of interest in a therapeutically effective amount to the subject from whom the tumor biopsy was taken, if it is determined that one or more compounds of interest reduce the proliferation of tumor cells in vitro.

[0046] In some embodiments, the method of the present invention comprises preparing tumor cell organoids from live tumor cells obtained from tumor biopsy from a subject, and preparing liver organoids from live liver cells obtained from the same subject or from a different source. In some embodiments, live tumor cells and live liver cells are obtained from the same subject. Live tumor cell organoids and liver organoids can be made in fluid communication with each other (e.g., in a device, e.g., a device described herein), and live tumor cell organoids and / or liver organoids can be contacted with a compound of interest. Furthermore, the proliferation of live tumor cells in live tumor cell organoids can be determined. In some embodiments, the compound of interest is a prodrug that liver organoids can metabolize to produce an active drug, and the activity of the active drug can be determined (e.g., when the proliferation of live tumor cells and / or liver cells is affected by the prodrug and / or the active drug, this activity can be determined). One or more additional organoids can be used in the method of the present invention. In some embodiments, the compound or compounds may be obtained from the same subject and may be derived from the same or a different tissue of the subject.

[0047] In some embodiments, the methods and / or devices of the present invention may provide for and / or enable the parallel screening of two or more (e.g., 2, 3, 4, 5, 6, 7 or more) compounds of interest. In some embodiments, at least one of the two or more compounds of interest is a prodrug.

[0048] In some embodiments, the methods and / or devices of the present invention can provide and / or enable results (e.g., drug screening results and / or therapeutic analysis) within about one or two weeks (e.g., within about 2, 3, 4, 5, 6, 7, 8, 9, or 10 days) after obtaining a biopsy (e.g., a tumor biopsy) from a subject whose cells were used to generate the organoids of the present invention used in the methods and / or devices of the present invention.

[0049] Also described herein is a device useful for evaluating tumor cells in vitro, comprising: (a) a microfluidic device having a chamber and a channel in fluid communication with the chamber; (b) a living tumor cell construct (e.g., an organoid) in the chamber; (c) a growth medium in the chamber and the channel; (d) a pump operatively coupled to the chamber and the channel and configured to circulate the medium from the chamber through the channel and back to the chamber; and (e) a microporous membrane (e.g., a TRANSWELL® microporous membrane) in the channel, positioned to allow the medium to flow through it. In some embodiments, the device is useful for evaluating tumor cell metastasis in vitro, for evaluating tumor cell migration and / or invasion in vitro, for evaluating the growth of constructs containing tumor cells in vitro, and / or for evaluating response to a compound of interest. In some embodiments, the device may be useful for evaluating construct size in vitro, tumor cell number in vitro, and / or tumor cell death in vitro.

[0050] In some embodiments, the device useful for evaluating tumor cells in vitro comprises two or more chambers with at least one organoid in each chamber and in fluid communication with each other. The device can be useful for evaluating the metastasis of tumor cells in vitro, for evaluating the migration and / or invasion of tumor cells in vitro, for evaluating the growth of constructs containing tumor cells in vitro, and / or for evaluating the response to a compound of interest. In some embodiments, the device can be useful for evaluating the size of constructs in vitro, the number of tumor cells in vitro, and / or the death of tumor cells in vitro. One or more organoids present in the device can be made from cells obtained from a single subject. In some embodiments, the device comprises a living tumor cell organoid in a first chamber and a liver organoid in a second chamber. The living tumor cell organoid and the liver organoid can be formed from cells obtained from the same subject, which can provide individualized analysis.

[0051] In some embodiments, the device can comprise a primary chamber that contains live tumor cell organoids; at least one secondary chamber that contains different organoids; at least one primary channel that connects the primary and secondary chambers and allows fluid communication between them (e.g., flow of growth medium, etc.); and optionally, growth medium in the primary chamber, each secondary chamber, and primary channel. In some embodiments, the device comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) secondary chambers, each of which contains organoids that contain different cells from the others. In some embodiments, at least one secondary chamber contains liver organoids, and optionally, the live tumor cell organoids and liver organoids are made from cells obtained from the same subject. Examples of additional devices include, but are not limited to, those described in International Application PCT / US2016 / 054611, the entire contents of which are incorporated herein.

[0052] An example of a device with two chambers is shown in Figure 15. Another example of a device with five chambers is shown in Figure 16, along with how media may flow through the chambers of the device at different stages. Figure 17 shows a schematic diagram of a device structure that can be used to track cells (e.g., tumor cells) labeled with a detectable compound according to an embodiment of the invention.

[0053] In some embodiments, the tumor cell construct comprises a hydrogel.

[0054] In some embodiments, the tumor cell construct consists of a single cell type.

[0055] In some embodiments, the tumor cell construct comprises or consists of colorectal cancer cells (eg, HCT116 cells, HT29 cells, SW480 cells, Caco2 cells, etc.).

[0056] In some embodiments, at least a portion of the microfluidic device is transparent, and the device further comprises a detector (e.g., a camera) operatively associated with the microporous membrane and configured to detect (e.g., image) tumor cells on the microporous membrane.

[0057] In some embodiments, the device used in the method of the invention can be configured to deliver physiological or supraphysiological fluid to a percentage of tissue volume. For example, one or more of the chambers of the device can have an average volume ranging from about 2 μL to about 10 μL. In some embodiments, one or more of the chambers of the device can have an average volume of about 2, 3, 4, 5, 6, 7, 8, 9, or 10 μL. In some embodiments, the device (e.g., an apparatus with 1, 2, 3, 4, 5, 6, or more chambers) may use an amount of liquid less than about 100 μL and / or have a volume less than about 100 μL, such as, for example, about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40 μL or less. As used herein, the volume of the device refers to the volume that fills the chambers and channels of the device. In some embodiments, the device may use an amount of liquid less than about 50 μL and / or have a volume less than about 50 μL.

[0058] Organoid of the present invention can be viable for at least 1, 2, 3, 4 weeks or more.In some embodiments, organoid in device and / or used in method of the present invention can be viable for at least 1, 2, 3, 4 weeks or more.In some embodiments, organoid of the present invention can be viable and comprise at least about 75% or more (for example, about 80%, 85%, 90%, 95% or more) viable cells based on the average number of cells present in organoid for 1, 2, 3, 4 weeks or more.

[0059] As described herein, cells and / or cell samples can be used in the methods of the present invention to form the organoids of the present invention. In the methods of the present invention, viable organoids can be produced. In some embodiments, the methods of the present invention can achieve at least 50% or more engraftment rate, such as about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more. For example, a 90% engraftment rate means 90% of the times that a viable organoid or a plurality of organoids (e.g., a set of organoids) is obtained and / or produced by the methods of the present invention. In other words, at a 90% engraftment rate, 9 cell samples (e.g., tumor cell samples) out of 10 cell samples will produce a viable organoid or a plurality of organoids when produced according to the methods of the present invention. The organoid or a plurality of organoids can be used in other methods and / or diagnosis of the present invention. In some embodiments, the cells and / or cell samples may be derived from a tumor, such as, for example, a mesothelioma biosample and / or a GI tumor biosample.

[0060] A method for screening tumor cells for metastatic activity in vitro can be carried out by (a) providing a device as described above in this specification, (b) circulating culture medium within the device, and (c) quantitatively or qualitatively detecting tumor cells captured on the microporous membrane, where a higher number of captured tumor cells (e.g., compared to other tumor cells under similar conditions and / or non-metastatic cells under similar conditions) indicates a higher metastatic activity of the tumor cells.

[0061] A method for screening tumor cells in vitro for metastatic activity can be performed by (a) providing a device (e.g., a device having a primary chamber containing living tumor cell organoids and at least one secondary chamber) as described above in this specification, (b) circulating culture medium within the device, and (c) quantitatively or qualitatively detecting tumor cells present in one or more of the secondary chambers (e.g., optionally on and / or in the organoids present in the secondary chambers), where a higher number of tumor cells present in one or more of the secondary chambers indicates a higher metastatic activity of the tumor cells (e.g., compared to other tumor cells under similar conditions and / or non-metastatic cells under similar conditions). A method of screening a compound of interest for anti-metastatic and / or anti-tumor activity in vitro can be carried out by (a) providing a device as described herein above, (b) circulating a medium through the device, (c) administering a compound to the tumor cells (e.g., by adding the compound to the medium), and then (d) quantitatively or qualitatively detecting the tumor cells captured on the microporous membrane, where a lower number of captured tumor cells (e.g., compared to other tumor cells under similar conditions but not administered the compound of interest) indicates a higher anti-metastatic activity of the compound of interest. In some embodiments, the tumor cells are obtained from a patient suffering from a tumor. In some embodiments, the method of the present invention further comprises administering a therapeutically effective amount of the compound to the patient if the compound is determined to reduce tumor cell metastasis in vitro, reduce construct size in vitro, reduce tumor cell number in vitro, and / or induce tumor cell death in vitro.

[0062] A method of screening a compound of interest for anti-metastatic and / or anti-tumor activity in vitro can be carried out by (a) providing a device as described herein above, (b) circulating culture medium through the device, (c) administering a compound to the tumor cells (e.g., by adding the compound to the culture medium), and then (d) quantitatively or qualitatively detecting the tumor cells present in the primary chamber and / or the secondary chamber, where a lower number of tumor cells present in a chamber (e.g., compared to other tumor cells under similar conditions but not administered the compound of interest) indicates a higher anti-metastatic and / or anti-tumor activity of the compound of interest. In some embodiments, the tumor cells are obtained from a patient suffering from a tumor. In some embodiments, the method of the present invention further comprises administering a therapeutically effective amount of the compound to the patient if the compound is determined to reduce tumor cell metastasis in vitro, reduce construct size in vitro, reduce tumor cell number in vitro, and / or induce tumor cell death in vitro.

[0063] In some embodiments, the method of the present invention can include screening a compound of interest for anti-metastatic and / or anti-tumor activity in vitro, the method includes: (a) providing a device as described above in this specification; (b) circulating growth medium from a first chamber containing liver organoids to a second chamber containing live tumor cell organoids; (c) administering a compound of interest to the liver organoids and / or live tumor cell organoids (e.g., by adding the compound to growth medium); and (d) determining the reduction (e.g., change in number or density) of tumor cell presence in the second chamber compared to the number of tumor cells present in live tumor cell organoids when the test compound is not administered.One or more additional organoids, such as, but not limited to, organoids of brain, colon, lung, endothelium, heart, epithelium, etc., optionally containing benign and / or tumor cells, can be present in one or more of the additional chambers of the device, and the chambers are in fluid communication with each other.

[0064] In some embodiments, the method of the present invention can include screening compounds of interest in vitro for anti-metastatic and / or anti-tumor activity, the method including: (a) providing a device (e.g., a device with a primary chamber containing live tumor cell organoids and at least one secondary chamber containing a different organoid (e.g., an organoid containing benign cells) as described herein above; (b) circulating medium through the device; (c) administering a compound to the tumor cells (e.g., by adding the compound to the medium); and (d) determining a decrease in the presence (e.g., a change in number or density) of tumor cells in the primary chamber (e.g., associated with live tumor cell organoids) and / or the secondary chamber compared to the number of tumor cells present in the live tumor cell organoids and / or the secondary chamber in the absence of administration of the test compound. Examples of different organoids in one or more secondary chambers can contain benign and / or tumor cells, including, but not limited to, organoids of the brain, colon, lung, endothelium, heart, epithelium, liver, etc.

[0065] Methods of the invention can include labeling cells (e.g., tumor cells) that contain a detectable compound, such as, but not limited to, a fluorescent compound (e.g., dyes, proteins, etc.).These and other aspects of the invention are further described in the Examples that follow. EXAMPLES

[0066] [Example 1] The first objective of this project was to evaluate a 3D model of a colorectal cancer tumor surrounded by normal host tissue. The modified concentric ring construct consisted of a colorectal tumor core and an outer ring of colonic epithelium to model the tumor in a reductionist, yet physiologically relevant environment. The construct was used to measure tumor core growth, any observed tumor cell invasion into the epithelial layer, and drug efficacy. The construct was made with cells suspended in a hyaluronic acid and gelatin-based hydrogel. One of four colorectal cancer cell types was used to make the tumor core, thus creating a model of four levels of malignancy. HCT116 cells were the most malignant, followed by HT29, SW480, and Caco2 cells. Caco2 cells were also used for the outer ring, as they are most similar to healthy epithelial cells. The tumor cores were fluorescently labeled and fluorescent imaging was used to monitor the tumor cores throughout the course of 7–14 days. After this period, the tumor core was shown to be dense, but little invasion was observed. Circular constructs made with Caco2 outer rings and SW480 cores were also treated with the chemotherapeutic drug regorafenib. MTS assays confirmed a reduction in the amount of viable cells after treatment, and LIVE / DEAD staining showed that regorafenib selectively targeted the inner ring of the constructs over the less tumorigenic Caco2 cells.

[0067] The second objective was to develop a system to quantify the number of cells that metastasize from 3D colorectal cancer tumor constructs. A microfluidic perfusion device was designed to capture circulating metastatic cells on a Transwell membrane sealed in a channel within the device. The device was fabricated using soft lithography of polydimethylsiloxane (PDMS). Tumor constructs consisting of a single tumor cell type suspended in a hydrogel were placed in the appropriate chamber, and the device was sealed and fixed. Media was circulated through the device, and images were acquired every 2-3 days for approximately 2 weeks. After 2 weeks, cells were observed on the Transwell membrane, indicating that the device is capable of assisting in the quantification of tumor cells that have entered the circulation from the tumor construct. Further testing will be performed to confirm whether tumor cells with different levels of malignancy quantitatively exhibit different numbers of migration.

[0068] In summary, two such models, the 3D concentric ring tumor model and the microfluidic circulating tumor cell quantification device, provide novel methods to test tumor cell proliferation, the effect of drugs on tumor cell responses, and the quantification of circulating cells.

[0069] [Example 2] 3D tumor organoids were microengineered directly from fresh tumor biopsies to create a patient-specific model system that allows for treatment optimization prior to the initiation of therapy. Here, we demonstrate the initial deployment of this platform using mesothelioma tumor biospecimens removed from patients. We demonstrate the ability to biologically create and maintain viable 3D tumor constructs within a tumor-on-a-chip microfluidic device. We then demonstrate an on-chip chemotherapy screen that mimics patient responses to the drugs cisplatin and pemetrexed. Finally, we demonstrate the efficacy of candidate compounds identified by genetic biomarker assessment in vitro.

[0070] method Fabrication of thin-film microfluidic devices The microfluidic device fabrication method is based on a method already published elsewhere. A cutting plotter (GraphTec-CE6000-40) was used to form channels in a double-sided adhesive film, and the bottom surface of the patterned layer was glued to a clean microscope glass slide. A polystyrene slide (Tedpella.Inc) featuring drilled holes to act as inlets and outlets for fluid delivery to the channels was glued to the top surface. After device construction, PTFE tubing was connected to each port and fixed using UV-cured polyester resin.

[0071] Patient History The patient is a 50-year-old male presenting with debilitating cachexia, ECOG2 functional status and abdominal distension. Diagnostic laparoscopy was performed for histological diagnosis, which confirmed the diagnosis of epithelioid malignant peritoneal mesothelioma. Precision medicine analysis of his tumor revealed two genomic alterations (BAP1 splice site 1729+1G>A and PBRM1 N258fs×6) for which there are no targeted drugs or clinical trials available. Computed tomography (CT) with iv contrast showed copious malignant ascites without signs of disease outside the peritoneal cavity. He was examined for cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC). Due to the excessive tumor volume, there was no hope of achieving complete CRS at that time. He was therefore recommended advanced systemic chemotherapy with the aim of reducing the tumor volume. He was treated with six cycles of cisplatin-gemcitabine, followed by a single cycle of carboplatin-gemcitabine due to concerns about ototoxicity associated with cisplatin. He showed a good clinical response to cisplatin-based chemotherapy, with almost complete disappearance of his malignant ascites on both repeat staging CT and clinical examination performed prior to carboplatin-based chemotherapy. He was then escorted to the operating room and underwent CRS / cisplatin-based HIPEC, with an unremarkable postoperative course. During surgical exploration and prior to HIPEC, the area of ​​omental disease was excised with scissors, placed on ice, and immediately transferred to the laboratory for construction of tumor organoids.

[0072] Obtaining tumor biospecimens and cell processing Tumors were delivered to the laboratory within 1 hour of removal for cell processing. Upon receipt, tumors were washed for 3 cycles of 5 min in phosphate buffered saline (PBS) containing 2% penicillin-streptomycin, followed by 2 cycles of 5 min in Dulbecco's modified Eagle medium (DMEM) containing 2% penicillin-streptomycin. Tumors were individually minced and placed on a shaker plate at 37°C for 18 hours in DMEM containing 2% penicillin-streptomycin and 10% collagenase / hyaluronidase (10X Collagenaze / hyaluronidase in DMEM, STEMCELL Technologies, Seattle, WA). The digested tumors were then filtered through a 100 μm cell filter and centrifuged to produce a pellet. Plasma and non-cellular material were removed, and the pellet was resuspended in 1 mL of BD PharmLyse containing 9 mL of deionized water for 5 minutes (BD PharmLyse, San Diego, CA). A conical beaker was filled to 50 mL with deionized water and centrifuged, the lysis buffer that lysed the cells was aspirated, and the cell pellet was ready for use.

[0073] Preparation of extracellular matrix hydrogels and basic organoid formation ECM-mimetic HA / gelatin-based hydrogels (HyStem-HP, ESI-BIO, Alameda, CA) were prepared as described above. 15、18、27Briefly, the thiolated HA component (Glycosil) and the thiolated gelatin component (Gelin-S) were dissolved in sterile water containing 0.05% w / v of the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Sigma, St. Louis, MO) to prepare a 1% w / v solution. Polyethylene glycol diacrylate (PEGDA) crosslinker (Extralink, ESI-BIO) was dissolved in the photoinitiator solution to prepare a 2% w / v solution. Glycosil, Gelin-S, and Extralink were then mixed in a volume ratio of 2:2:1, respectively. For tumor construct formation, the prepared solution was thoroughly mixed by using a vortex to resuspend the cells (20 million cells / mL), followed by 1 second of UV exposure (365 nm, 18 Wcm -2 ) to initiate crosslinking and organoid formation.

[0074] Tumor-on-a-chip biofabrication Using photopatterning techniques similar to our previous work, we fabricated biological tumor constructs within microfluidic devices. 15 Here, photomasks featuring 1-3 mm openings were fabricated in aluminum foil by the same cutting plotter technique used for channel formation and attached to the bottom surface of the microfluidic device with a matching patterned adhesive film. A solution of hydrogel precursor was then mixed with cells from a patient's mesothelioma biopsy at a density of 20 million cells / mL. The mixture was introduced into each device channel through its inlet, and the organoid constructs were visualized by exposing them to ultraviolet light (365 nm, 18 W cm) through the attached photomask for 1 s. -2) was clarified. This exposure rapidly initiated the stepwise cross-linking reaction of thiolene, thereby encapsulating the cells in the 3D columns. Finally, the unexposed precursor / cell mixture was washed out of the device with phosphate-buffered saline, leaving the 3D patient-derived mesothelioma constructs in each channel. Each channel of the device with cell constructs was connected to a separate reservoir containing DMEM. Media was flushed from the reservoir through the channels by silastic tubing connected to an MP2 Precision micro-peristaltic pump (Elemental Scientific Inc. Omaha, NE). Flow was initiated and maintained at 10 μL / min.

[0075] Histological evaluation 5 μm thick organoid sections were made from paraffin-embedded constructs and then deparaffinized for staining. IHC was used to visualize the biomarkers cytokeratin 5 / 6 (CK5 / 6), calretinin, and thrombomodulin. Blocking was performed by incubation in the presence of Dako Protein Block for 15 minutes. Primary antibodies CK5 / 6 (Abcam, ab17133, raised in mouse) and calretinin (Abcam, ab702, raised in rabbit) or CK5 / 6 and thrombomodulin (Abcam, ab109189, raised in rabbit) were diluted 1:200 in Dako Antibody Diluent and applied to the sections on slides and incubated at room temperature for 1 hour. Secondary Alexa Fluor 488 or Alexa Fluor 594 antibodies with appropriate species reactivity were then applied to all samples at a dilution of 1:200 in Dako Antibody Diluent and left at room temperature for 1 hour (anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 594, Life Technologies, Carlsbad, CA, A-11070). Sections were then incubated in Dapi for 5 minutes before being coverslipped. For subsequent biomarker experiments, the same protocol was used for Annexin V and Ki67 staining (Abcam, Cambridge, MA, ab14196 and ab16667, respectively). Fluorescent images were acquired using a Leica DM400B compound microscope and overlaid for analysis.

[0076] Cisplatin, Carboplatin, and Pemetrexed Drug Trials At the end of the first week, LIVE / DEAD staining was performed on the two constructs as described below to ensure that the cells were alive before the addition of drugs. Two combinations of drugs were considered and tested on this cancer model: cisplatin + pemetrexed and carboplatin + pemetrexed at two different concentrations, 0.1 μm and 10 μm, mixed in DMEM. The DMEM reservoir was replaced with a volume of 2 mL of this drug mixture.

[0077] LIVE / DEAD staining (LIVE / DEAD Viability / Cytotoxicity Kit for Mammalian Cells, Life Technologies, Grand Island, NY) was performed on day 7 at the end of the drug exposure period. First, the circulating medium was flushed from the device channels with clean PBS, and then 1 mL of a mixture of PBS and DMEM (1:1) containing 2 μM calcein AM and 2 μM ethidium homodimer 1 was introduced. The constructs were incubated for 60 min, after which the channels were flushed again with clean PBS. In situ imaging was performed using an Olympus Fluo View™ FV1000 confocal microscope. 5 μm z-stacks were acquired for each construct using filters appropriate for both red and green fluorescence, and then overlaid.

[0078] Confocal images were processed using Imaris MeasurementPro (Bitplane, Concord, MA). With this software, images from each color channel were analyzed for cell size, shape, and fluorescence intensity, and cell location was labeled. For consistency, an area of ​​450 μm × 900 μm was considered for each image. Cell viability was calculated by quantification of the total number of cells identified in the green channel (LIVE) compared to the total number of cells identified in the red channel (DEAD).

[0079] Biomarker-guided experimental drug testing The patient from whom the tumor biopsy was taken participated in the Precision Medicine Program at the Comprehensive Cancer Center at Wake Forest Baptist Medical Center, and a portion of the biopsy was sent for identification of potentially actionable mutations in the tumor. The de-identified data showed a positive mutation in the BAP1 gene, favoring testing of enhancer of zeste homolog 2 (EZH2) inhibitors as experimental targeted therapy.

[0080] Tumor constructs were once again biologically generated and then maintained for 3 days before drug screening. Patient organoids were then exposed to the drug 3-deazaneplanocin A (DZNep), a histone methyltransferase EZH2 inhibitor, at concentrations of 0.1 μM, 1 μM, and 10 μM in cell culture medium for a 96-hour incubation period. Biologically generated organoids using HCT116 cells, a colorectal cancer cell line known to respond to EZH2 inhibition, were tested in parallel as a positive control. Drug effects were quantified by MTS assay and immunohistochemistry. Mitochondrial metabolism was quantified and relative cell numbers were determined by MTS assay (CellTiter 96 One Solution Reagent Promega, Madison, WI). Absorbance values ​​were measured on a Molecular Devices SpectrumMax M5 (Molecular Devices), a tunable plate reader system with a wavelength of 490 nm.

[0081] Immunohistochemistry staining was performed using the biomarkers Annexin V and Ki67 on 5 μm thick organoid sections. Sections were taken from previously unstained organoids that were embedded in paraffin, sectioned, and then deparaffinized for IHC staining. Primary antibody Annexin V was applied to slides at a concentration of 1:200 in Dako Antibody Diluent and left at room temperature for 60 minutes (Annexin V, Abcam, Cambridge, MA, ab14196). In parallel, primary antibody Ki67 was applied to another sample at a concentration of 1:200 in Dako Antibody Diluent (Ki67, Abcam, ab16667). Secondary Alexa488 antibody was applied to all samples at a concentration of 1:200 in Dako Antibody Diluent and left at room temperature for 1 hour (Alexa 488, Life Technologies, Carlsbad, CA, A-11070). Fluorescence images were acquired using a Leica DM400B compound microscope, and adjacent sections of GFP were imaged for Annexin V and Ki67 and overlaid for analysis. Annexin V images were colored red (medium gray in black and white images) and Ki67 images were colored green (light gray in black and white images).

[0082] result Tumor Organoid Strategy for Precision Medicine Our goal was to develop a platform consisting of patient-specific tumors that can test anti-cancer drugs for efficacy and then inform treatment optimization. The concept of personalized precision medicine works as follows: When a tumor biopsy is taken from a patient, some or all of the biosamples are genetically sequenced in part or in whole to identify mutations that may indicate druggable targets. Then, if an FDA-approved drug is available or a clinical trial is underway that targets one of these biomarkers, the drug or trial therapy the patient receives will be adjusted accordingly (Figure 1 - solid arrow). Under optimal conditions, this strategy results in a more effective therapy than standard chemotherapy. However, in practice, frequently multiple mutations are identified and the lack of a clear best treatment results in a series of possible drug options, necessitating additional means of testing such drugs as well as potential experimental drugs. Our method incorporates patient-derived tumor organoids formed using the same biosample material sent for genetic profiling. In this method (Figure 1 - dashed arrow), a portion of a biological sample is taken to biologically generate a large number of microscale 3D tumor organoids using an extracellular matrix hydrogel biomaterial to support the cells. These organoids are then used in a drug screen designed to test agents identified in the genetic screen, thereby determining which agents will be most effective in that patient.

[0083] Patient-specific tumor-on-a-chip biofabrication Panels A-C of Figure 2 represent the process of biological fabrication of the thin-film microfluidic device, in situ integration of tumor organoids, and general device operation. The device, capable of housing six independent tumor organoids, consists of an adhesive film blocking the fluidic channels and organoid chambers, which is sandwiched between a polystyrene slide with an inlet and an attached glass slide with a photomask (Figure 2, panel A). Tumor organoids were patterned using a hydrogel precursor solution containing cells from the patient's tumor, which were then introduced into each of the six channels and photopolymerized as organoids through the openings in the photomask (Figure 2, panel B). After biological fabrication of the organoids, the device was connected via tubing to a micro peristaltic pump and medium reservoir, and then the flow of organoid medium was initiated (Figure 2, panel C).

[0084] First, each tumor construct was left in the presence of circulating DMEM for 7 or 14 days. To validate the platform's ability to maintain viable patient-derived tumor models, we performed LIVE / DEAD analysis on organoids on days 7 and 14 (Figure 2, Panel D). Results showed high cell viability, greater than 90% on day 7 and greater than 85% on day 14, demonstrating that our device architecture and its supporting hydrogel matrix are capable of supporting patient-derived cells for the expansion of experimental methods.

[0085] Tumor-on-a-chip supports viable patient-derived models and chemotherapy drug testing that correlate with patient drug response On day 7, a subset of tumor constructs was exposed to one of two different doses of chemotherapy drug mixtures, carboplatin / pemetrexed and cisplatin / pemetrexed, or no drug. After 7 days of treatment, LIVE / DEAD analysis was performed on all organoids (Figure 3, panels a(i)-f(i) and a(ii)-f(ii)). Under control (no drug) conditions, tumor constructs were observed to maintain high viability, showing a statistically significant drop to 87% after a total of 14 days in the device. However, organoids exposed to the drug mixture showed a significant drop in viability. In samples treated with carboplatin / pemetrexed, the inventors observed a drop in viability to 52.1% at 0.1 μM circulating concentration and 39.8% at 10 μM. At the high dose, although two orders of magnitude higher than the low dose, the observed changes in viability were only of low significance (p<0.1), indicating a potentially moderate drug effect. In contrast, organoids treated with cisplatin / pemetrexed resulted in a decrease in viability of 39.0% at 0.1 μM and 11.8% at 10 μM. These showed significant decreases compared to both each other (p<0.05) and to control measurements (p<0.05 and p<0.001, respectively). Thus, cisplatin / pemetrexed was significantly more effective than carboplatin / pemetrexed in our device. These results are summarized in Figure 3, panel g.

[0086] These data have two central outcomes. First, the data demonstrate that it is possible to sustain cells from a patient's tumor biopsy long-term in a bioengineered organoid system for extensive testing. To the inventors' knowledge, this invention is the first to report this capability. Second, the data show that it is feasible to drug screen such organoids in vitro, resulting in a drug-dependent decrease in cell viability. This is a promising advantage for therapy design, since drug efficacy can in principle be established in a patient-specific manner before administration of treatment. For example, in such measurements, our results suggest cisplatin / pemetrexed as a superior treatment option over carboplatin / pemetrexed. The nature of personalized medicine does not allow for a systematic comparison of the efficacy of the two agents in patients. However, crucially, patients were treated with cisplatin / pemetrexed with good responses. This suggests that patient-specific drug efficacy can be accurately reproduced in our in vitro system.

[0087] Experimental drug screening by genetic biomarker identification Precision medicine testing identified two known mutations in tumor biopsies: BAP1 splice site 1729+1G>A and PBRM1 N258fs×6. BAP1 (BRCA1-associated protein 1) is a deubiquitinase 28 PBRM1 is a tumor suppressor gene associated with several cancers. 29 At the time of testing, none of the mutations were associated with FDA-approved therapies in mesothelioma or any other tumor types.

[0088] However, in further investigations, the inventors demonstrated that by inhibiting EZH2, an enzyme involved in DNA methylation and transcriptional repression, 30We have confirmed that targeting BAP1 mutations has been successful (Figure 4, Panel A). Therefore, we performed drug screening experiments using the EZH2 inhibitor DZNep. As a control population, we generated organoids using the extensively studied colorectal cancer cell line HCT116. Importantly, HCT116 cells were able to inhibit DZNep 31 Treatment with has been shown to arrest the cell cycle but not necessarily undergo significant levels of cell death, therefore, the inventors considered HCT116 cells to be an appropriate control for this study.

[0089] Our results show that after drug treatment, there was no significant decrease in HCT116 proliferation, but a significant decrease in mesothelioma proliferation (Figure 4, Panel B). While HCT116 and mesothelioma controls were similar, mesothelioma treated with DZNep showed decreased proliferation compared to controls with increasing drug concentrations. As DZNep concentrations increased, cell proliferation decreased. Furthermore, comparisons between HCT116 and mesothelioma show a significant decrease in mesothelioma cell proliferation for all therapeutic drug concentrations. Annexin V and Ki67 immunohistochemistry were performed across all conditions for both cell types. Annexin V appeared to increase in mesothelioma with increasing DZNep concentrations, representing an increase in apoptotic cells (Figure 4, Panel C). However, minimal changes in proliferation were seen across all HCT116 conditions, indicating that DZNep did not significantly affect proliferation (Figure 4, Panel D). Hematoxylin and eosin staining was performed on each organoid, and representative images of mesothelioma show ghost cells and ghost cells without nuclei when treated with DZNep (Figure 4, Panel E). This response was not seen in mesothelioma or HCT116 controls, and was absent in any treatment of HCT116 (Figure 4, Panel F). Only mesothelioma responded, further demonstrating that DZNep measurably reduced proliferation in patient samples, with HCT116 acting as an appropriate positive control with no response.

[0090] Consideration Precision oncology, which involves sequencing tumor DNA to identify actionable genetic alterations, is poised to become standard clinical practice for cancer treatment therapy decisions. However, currently, correlations between any reported genetic alterations and potential drugs cannot be confirmed before treatment begins, and only 11% of patients tested for precision medicine will be followed through with precision medicine-guided treatment. Furthermore, there is a high degree of variability in the biological behavior of cancer based on histology, grade, and tumor volume.

[0091] Studies have used patient-derived xenografts (PDXs) to test tumor progression and drug treatment response in patients. Such models do not require immunocompromised mice in which to place biopsies or tumor samples that would cause mouse-derived cells to infiltrate. The cells also adapt to their new environment and show genetic drift from the initial sample, again making them less than ideal. One further limiting factor in the widespread adoption of PDX technology is that only the most aggressive tumor biopsies are successful in "engrafting" when introduced into immunocompromised mice. The poor engraftment rates, generally found to be in the 25-33% percent success rate range, limit the applicability of PDX technology and its predictive application to the majority of cancer patients. In the early days of the studies described herein, and parallel ongoing trials, the main critique of our method was whether we could improve the engraftment rate of PDXs. Notably, this was surprisingly straightforward in our platform. As shown in FIG. 2, the inventors confirm high cell viability at days 7 and 14 of culture. Furthermore, when the inventors perform similar tests with other mesothelioma and other GI tumor biosamples, they observe engraftment rates of greater than 90%. It is important to mention that the 10% non-engraftment rate may reflect acellular samples obtained from low-grade appendiceal mucinous tumor patients. The above stems from the finding that all high-grade samples at this time grew organoids. This is significant and indicates to us that by using 3D, ECM microenvironment-supported organoid technology, it may be possible to generate models that are adoptable to a larger number of patients than is possible with PDX technology. While very promising, this further evidence of engraftment efficiency requires significant further confirmatory testing.

[0092] In this series of experiments, beyond engraftment and viability assessment, we demonstrate the feasibility of two additional proof-of-concept scenarios: 1) correlation of patients with tumor organoids and 2) biomarker testing of experimental drugs. First, we used the mesothelioma organoid platform to screen two of the common chemotherapy regimens used clinically for this type of cancer. In particular, we tested the response of organoids to a combination of cisplatin and pemetrexed versus carboplatin and pemetrexed (Figure 3). Exposure to the drugs for 7 days by injection into the medium circulating through the microfluidic system provided clear results showing that the cisplatin-pemetrexed treatment was more effective. The ratio of dead cells to live cells was higher in the treatment of 10 μM cisplatin and pemetrexed compared to the relative concentrations of carboplatin and pemetrexed. This result by itself validates the inventors' platform in that the platform allows the inventors to successfully perform drug screening studies. But more importantly, the patient from whom such organoids were derived also responded dramatically to cisplatin-based chemotherapy, as demonstrated by the almost complete disappearance of his copious ascites in CT imaging performed at the end of his cisplatin-based chemotherapy. In fact, this patient would not have been a valid candidate if his tumor had not responded to cisplatin-based treatment. This is important, and these correlative results between patients and the inventors' organoid technology are a critical dataset to continue to develop in order to validate this method for clinical application.

[0093] Second, the inventors confirmed the genetic test results using some of the patient's tumor biosamples. The report showed that two mutations specific to this tumor were identified: BAP1 splice site 1729+1G>A and PBRM1 N258fs×6. As mentioned above, BAP1 is a deubiquitinase. 28 PBRM1 is a tumor suppressor gene associated with several cancers. 29At the time of these studies, none of the mutations were associated with FDA-approved therapies or with any ongoing appropriate clinical trials in mesothelioma or any other tumor types. However, the inventors demonstrated that inhibition of EZH2 significantly improved tumor outcomes in animal models and in vitro studies. 30 , indeed confirmed that it has targeted the mutation of BAP1. Therefore, the inventors carried out a drug screening experiment using the EZH2 inhibitor DZNep as a test scenario to ask whether such biomarkers shown in genetic analysis could indeed be used as actionable targets (Figure 4). Indeed, upon treatment with this compound, the inventors observed a decrease in mitochondrial metabolism, a metric that is generally proportional to cell number. Moreover, the inventors observed a trend toward an increase in the expression of Annexin V, indicating apoptosis, versus a trend toward a decrease in the expression of Ki67, indicating proliferating cells. Furthermore, H&E staining shows that cell morphology and intact cell nuclei are damaged by the compound in a dose-dependent manner. Organoids made in the same way from the widely used HCT116 colorectal cancer cell line did not respond in the same way. Rather, DZNep appeared to have little effect in between, regardless of concentration.

[0094] By implementing such a 3D tumor model in a parallelized microfluidic platform with a circulatory system, the inventors not only have the ability to evaluate multiple drug types and doses on a single device, but also gain advantageous conditions to visualize and track the dynamics of tumor progression, migration, and intravasation into the circulatory system that occurs prior to metastasis to distant sites.

[0095] [Example 3] Multiple sets of patient tumor-derived organoids have been generated with engraftment rates of >90% in vitro (vs. <10% in 2D cell culture), which compares favorably with previously reported engraftment rates of 25-33% in patient-derived xenograft (PDX) models, where only the most aggressive biopsies grow. This poor engraftment rate limits the application of PDX technology to predictive diagnostics for many cancer patients. We continue to use our tumor organoid system for drug screening, demonstrating that, like patients, the tumor organoids maintain selective responses to different drugs. To date, we have generated sets of patient-specific organoids from a diverse group of primary tumors and their metastatic sites, including colorectal cancer, high and low grade appendiceal metastatic sites to omentum, ovary and liver, peritoneal mesothelioma, and extremity sarcoma. Described herein are several examples that demonstrate: 1) correlation of tumor organoid drug responses with drug responses in the patients from which the organoids are derived, 2) the ability to test efficacy of therapies designed based on identification of druggable mutations in patient tumors, and 3) construction of viable organoids from tumor populations that are difficult to culture.

[0096] The inventors have demonstrated the ability to generate viable tumor organoid models from cancer types that are rare and difficult to isolate and culture. For example, the inventors have generated viable patient-specific tumor organoids from low-grade appendix (LGA) tumors (Figure 5, panel A) and well-differentiated papillary mesothelioma, traditionally referred to as "benign" mesothelioma (Figure 5, panel B). Both have slow growth patterns and unpredictable transformation capabilities. To date, it has been essentially impossible to establish cell cultures with low proliferation indices from primary cells. Importantly, compared to high-grade organoids and 2D cultures that respond well to treatment, our LGA organoids do not respond to chemotherapeutic agents (Figure 5, panel C). Such data may be the first useful and definitive evidence that LGA is indeed a chemotherapy-resistant tumor and therefore, treatment of LGA patients with chemotherapy is not beneficial and leaves the treatment with unnecessary toxicity. Furthermore, we recently treated limb sarcoma and generated a set of patient-specific sarcoma organoids in our platform that also showed high survival rates (Figure 5, Panel D). Such sarcoma organoids are currently being employed in drug screening studies.

[0097] [Example 4] The restriction of normal cell types to malignant tumor cells is maintained by altered cell-cell interactions and by physical barriers (Lodish H, Berk A, Zipursky SL, et al. 2000). Anticancer drugs must travel to the tumor via the circulatory system, penetrate extravascular tissues, and successfully target cancer cells present inside the organ (Tannock et al. 2002). The annular organoid model attempts to recapitulate the in vivo process of anticancer drug delivery by creating simple 3D microorgans, or organoids, that contain invasive cancer types surrounded by different cell types, and then targeting the internal invasive cell lines using specific anticancer drugs. Additionally, the experiments described herein were performed to determine whether anticancer drugs would be successful in targeting the internal, more invasive cell lines as opposed to the external cell types when both external and internal cell types are present in a 3D microenvironment similar to that found in vivo (effectiveness determined by 3D drug screening as described above).

[0098] To investigate the effect of the extracellular microenvironment on the efficacy of anticancer drugs, we treated four colorectal cancer cell lines (HCT116, HT29, SW480, and CACO2) with various concentrations of anticancer drugs (regorafenib, sorafenib, trametinib, 5-fluorouracil, and dabrafenib) and embedded them in hyaluronic acid-based hydrogels or seeded directly into wells of well plates. Colorectal cancer cells embedded in hyaluronic acid-based hydrogels mimicked cancer cells in an in vitro 3D microenvironment, whereas cells seeded directly into wells represented cancer cells grown in a conventional 2D microenvironment.

[0099] 2D tumor construct drug screening A 2D drug screen using colorectal cancer cell lines residing in monolayer culture was performed to determine the efficacy of anticancer drugs in a traditional drug screening practice that lacks the cell-ECM interactions typically seen in malignant tumors in vivo.

[0100] Cancer cells seeded directly in the wells were given 4-5 hours to reattach to the well bottom. Anticancer drug solutions were then administered in place of the medium (Figure 6, Panel A) and MTS assays were performed 2 days later to determine cell viability. The MTS absorbance results of the anticancer drugs are shown in comparison with the results of the MTS values ​​of the drug-free control (Figure 6, Panel B). At 100 μM concentrations of regorafenib and sorafenib, and 100 mM concentration of 5-FU, cell viability remained below 20% of the control viability for all four of the colorectal cancer cell lines used. However, screening with dabrafenib and trametinib produced different results than the other three anticancer drugs at higher concentrations. Dabrafenib at a concentration of 100 μM produced relative cell viability values ​​ranging from about 36% for HCT116 cells to about 93% for SW480 cells, and trametinib at a concentration of 100 nM produced relative cell viability values ​​ranging from about 39% for CACO2 cells to about 58% for SW480 cells. When treated with 5-FU at a concentration of 1 mM, the relative cell viability was about 39% for SW480 cells and remained below 10% to 22% for HCT116, HT29, and CACO2 cells. Such values ​​were significantly lower than the relative cell viability values ​​of the lowest concentrations of other anticancer drugs.

[0101] 3D tumor construct drug screening Colorectal cancer cell lines were subjected to 3D drug screening and further embedded in hyaluronic acid-based hydrogel constructs to observe how cell-ECM interactions in the presence of hyaluronic acid affect the efficacy of anticancer drugs.

[0102] Polydimethylsiloxane, or PDMS, was used to coat the well bottom. Cells were embedded in a hyaluronic acid-based hydrogel and given 5 days to acclimate to their 3D environment, at which point they were administered different anticancer drug solutions (Figure 7, Panel A). MTS assays were performed on day 7, and results are shown relative to control values ​​(Figure 7, Panel B). At 100 μM concentrations of regorafenib and sorafenib, cell viability remained below 25% of control viability for all four of the colorectal cancer cell lines used. The only exception to this was when 100 μM regorafenib was administered to CACO2 cells, where the percent relative cell viability increased to approximately 48%. Screening with 100 mM 5-FU produced relative cell viability values ​​between 48% when used with SW480 cells and 81% when administered to HCT116 cells. However, only 5-FU at a concentration of 10 mM produced cell viability values ​​ranging between 9% in SW480 cells and 50% in HT29 cells for all four cell lines. At a concentration of 100 μM, dabrafenib showed high efficacy in targeting HCT116 and SW480 cell lines, with relative cell viability values ​​of 38% and 43%. This was in contrast to the HT29 and CACO2 cell lines, which showed higher cell viability values ​​of 93% and 75%. Trametinib at a concentration of 100 μM produced relative cell viability results that fell within a narrow range for the four colorectal cancer cell lines, with values ​​of 54% in SW480 cells and 74% in HT29 cells.

[0103] Comparing 2D and 3D Drug Screening Significance statistics were performed on the 2D and 3D drug screens to see if colorectal cancer cells responded differently to anticancer drugs when in either environment. Significant differences in cell responses to drugs in such drug screens suggest that cell-ECM interactions between cells and hyaluronic acid are to blame. The responses of HCT116 and CACO2 to regorafenib, sorafenib, and 5-fluorouracil are due to morphological differences and similarities between such cell types. Such cells and drugs will be used in the later parts of this study.

[0104] Even when the same cell lines were given the same concentration of anticancer drug solution, it is clear that the cells responded differently to drug treatment when placed in 2D vs. 3D in vitro microenvironments. Significant differences in cell viability were observed between the results of 2D and 3D drug screens using regorafenib, sorafenib, and 5-fluorouracil in HCT116 and CACO2 (Figure 8). In HCT116 cells, both results were significantly different between the 2D and 3D screens with regorafenib (both <0.05), one was significantly different for sorafenib (<0.10), and two were significantly different for 5-FU (both <0.01). In CACO2 cells, both results were significantly different between 2D and 3D screens with regorafenib (both <0.01), three were significantly different for sorafenib (two <0.01 and one <0.05), and three were significantly different for 5-FU (two <0.01 and one <0.10).

[0105] In HCT116 cells, the relative cell viability values ​​were higher when performing 3D drug screening than when performing 2D drug screening, except for the 10 μM solution of sorafenib and the 10 mM solution of 5-FU. In CACO2 cells, the relative cell viability values ​​were also higher when performing 3D drug screening than when performing 2D drug screening, except for the 10 μM solutions of regorafenib and sorafenib and the 10 mM solution of 5-FU. Comparing the results of 2D and 3D drug screening (Figure 8), it can be seen that regorafenib had a more significant effect on the change in cell viability of HCT116 cells than CACO2 cells, sorafenib had a more significant effect on the change in cell viability of CACO2 cells than HCT116 cells (although HCT116 was already significantly affected in both screens), and 5-FU significantly changed the cell viability of both HCT116 and CACO2 cells. During 3D drug screening, regorafenib and sorafenib at 100 μM concentrations showed lower percent relative cell viability values ​​for HCT116 cells compared to CACO2 cells, 48% vs. 21%, and 25% vs. 15%. When focusing on the results of 10 mM 5-FU for both HCT116 and CACO2 cells in the 3D microenvironment, it is evident that these relative cell viabilities were reduced to similar values ​​of 18% and 22%, as 100 mM 5-FU resulted in much higher viability values ​​during drug screening than 10 mM 5-FU.

[0106] Circular organoid drug screening In the circular organoid study, we tested whether cell-ECM or cell-cell interactions between different cell types in a 3D hyaluronic acid-based hydrogel microenvironment could affect the efficacy and specificity of anticancer drugs in targeting specific cancer cells, as previously observed in 3D drug screens.

[0107] Polydimethylsiloxane, or PDMS, was again used to coat the well bottom. HCT116 cells were embedded in hyaluronic acid hydrogel to create constructs on the PDMS surface. CACO2 cells similarly embedded in hyaluronic acid gel were used to create an outer ring surrounding the HCT116 inner ring construct (Figure 9, Panel A). Because HCT116 cells showed lower relative cell viability values ​​compared to CACO2 cells in the 3D microenvironment when administered regorafenib at a concentration of 100 μM (21% vs. 48%), and because 5-FU showed a similar effect on reducing relative cell viability in the 3D microenvironment, such drugs were administered at equivalent concentrations (1 μM, 10 μM, and 100 μM) to the annular constructs to observe and quantify the efficacy of such drugs for the two cell lines for comparison.

[0108] LIVE / DEAD staining was performed on the circular constructs on day 7 and imaged on an Axiovert microscope (Figure 9, Panel B). The imaging software, MATLAB, was used to derive the % dead:% alive (or % red (medium grey in black and white images) vs. % green (light grey in black and white images)) ratio for the circular constructs. The values ​​were then graphed to show the ratios for each concentration of regorafenib and 5-FU versus the control, which was the ratio of no treatment (Figure 9, Panel C). At 100 μM, both regorafenib and 5-FU showed a % dead:% alive ratio of 0.98. This means that roughly half of the HCT116 and CACO2 cells were alive and half were targeted by the anticancer drug. When analyzing the relative cell viability when regorafenib and 5-FU are administered at concentrations of 1 μM and 10 μM, the % death:% viability is significantly higher for the 5-FU solution than for the regorafenib solution. At 1 μM, the % death:% viability is 0.95 for 5-FU, as opposed to 0.48 for regorafenib, and at 10 μM, the % death:% viability is 0.78 for 5-FU, as opposed to 0.08 for regorafenib. The % death:% viability of the three concentrations of regorafenib and 5-FU was substantially higher than the % death:% viability value of the control, which was only 0.03.

[0109] Focusing on the live-dead images in panel B, the inner HCT116 cells appear to be affected by regorafenib to a greater extent than the CACO2 cells, as the majority of red (medium grey in the black and white image) cells appear in the inner ring (right side of the image) but not the outer ring (left side of the image). In contrast, 5-FU appears to target both cell types to a similar extent, as dead cells that were stained with red (medium grey in the black and white image) ethidium homodimer 1 dye appear in both the inner (HCT116) and outer (CACO2) rings. Furthermore, increasing the concentration of the anticancer drug 10-fold observably increases the drug-induced target cell death.

[0110] A microfluidic device to quantify metastasis We developed a PDMS-based microfluidic device (Figure 10) to determine how the rate of cancer cell metastasis changes when cancer cells are embedded in 3D hyaluronic acid-based constructs and administered anticancer drug solutions flowing through a simplified circulatory system. Furthermore, we aimed to determine how the anticancer drug affects cancer cell viability in the "primary" tumor constructs and in the "secondary" cancer cells trapped on a transwell membrane using the microfluidic device.

[0111] Two microfluidic devices were set up with the intention of running for 7 days to observe the feasibility of the device in capturing cells on the transwell membrane that had entered the media circulation. 3D constructs embedded with HCT116 cells were placed in the "construction chambers" of both devices. Untreated media was added to the fluid reservoir of one device (control) and a 10 μM solution of sorafenib was added to the fluid reservoir of the other device. Images of the constructs and transwell membrane of the control device were taken on days 1 and 7 using a Zeiss Axiovert 200M 487-1 microscope. An increase in cells captured on the transwell membrane of the control device between days 1 and 7 was observed as indicated by the green membrane dye DiO. After disassembling the device, the transwell membrane was imaged on a confocal microscope on day 7 and confirmed that the device successfully captured cells that had migrated out of the 3D construct and entered the circulation. The flow rate of the device was recorded as 0.20 mL / min.

[0112] The device circulating sorafenib 10 μM in drug medium solution showed signs of internal fungal infection on day 3 and was dismantled and discarded following this observation. As a result, any data that could have been collected from the device was lost. A small leak from the device outlet was identified as the source of fungal contamination.

[0113] Consideration Such studies established that the 3D extracellular microenvironment plays a key role in determining the efficacy of anticancer drugs. Four colorectal cancer cell lines showed significant differences in cell viability after embedding and encapsulation in hyaluronic acid-based hydrogels and treatment with five anticancer drugs. The circular organoids allowed for a qualitative comparison of drugs targeting different cancer cells when embedded in a 3D hydrogel environment, and showed that the circular organoids could provide a method to measure cell migration. The developed microfluidic device showed the ability to trap metastatic cancer cells, allowed continuous microscopic observation of the 3D construct and the trapped cells, and suggests its use as a model and platform for personalized chemotherapy drug screening.

[0114] material and method Origin of cancer cell lines The HCT-116, HT-29, SW-480, and CACO-2 colorectal cancer cell lines used were derived from the Cell and Viral Vector Core Laboratory at Wake Forest University School of Medicine. Cells were cultured in 15 cm cell culture plates, and standard DMEM-10 (Dulbecco's modified Eagle's medium) containing DMEM high glucose solution (HyClone, Utah, USA), 10% fetal bovine serum, 1% penicillin / streptomycin, and 1% L-glutamine was used as cell growth medium.

[0115] 2D Drug Screening Sterilization and cell passaging: The medium was aspirated from the cell culture plates, and 10 mL of PBS (25°C) was pipetted into each plate. The cell culture plates were manipulated to cover the entire cells in the plate with PBS, and then the PBS was aspirated out of the plate. 5 mL of 0.05% trypsin (37°C) was pipetted into each cell culture plate. The cell culture plates were then placed in a 37°C incubator for approximately 8 minutes. After this, 5 mL of DMEM-10 medium was added to each plate. 10 mL of cells and medium components were then placed into four clean 15 mL centrifuge tubes.

[0116] Counting and loading of cells on hemocytometer: Cells were resuspended in a 15 mL centrifuge tube with a 1000 μL single channel pipette and 100 μL was transferred from each cell line tube into a new 15 mL centrifuge tube. 400 μL of DMEM-10 medium and 500 μL of Gibco trypan blue stain (0.4%) (Thermo-Fischer Scientific, Massachusetts, USA) dye were then pipetted into each tube. The second tube was diluted 1:10 to facilitate cell counting and trypan blue stain was added to exclude dead cells from the cell count. 10 μL of the 1:10 diluted sample was dispensed with a 2-20 μL single channel pipette and loaded into the hemocytometer chamber of a pre-sterilized hemocytometer. Live cells were counted and calculations were performed to determine the total number of cells present for each cancer cell line.

[0117] Cell seeding: The first 15 mL centrifuge tube containing 10 mL of DMEM-10, cells, and trypsin was placed in a centrifuge. The centrifuge settings were 1500 RPM and maximum acceleration for 5 minutes. The medium and trypsin mixture was then aspirated from the tube, leaving the cell pellet intact. 5 mL of DMEM-10 was then pipetted into each tube, resuspending the cells in the process. The calculated volume of 5 mL of cell medium solution was determined to confirm that 25,000 cells of each of the four colorectal cancer cell lines were seeded directly into each well of a polystyrene clear-bottom 96-well plate. 100 μL of DMEM-10 medium was then pipetted directly into the well. The 96-well plate was incubated at 37°C and 5% CO2 for 5 hours to allow the cells to reattach to the bottom of the wells until the medium was aspirated and anticancer drug solution was added.

[0118] Addition of anticancer drugs for screening: The anti-colorectal cancer drugs used in this screening included 5-FU, regorafenib, sorafenib, trametinib, and dabrafenib (Sigma-Aldrich, Missouri, USA). High-concentration drug stock solutions were prepared using DMSO. Low-concentration drug stock solutions were prepared by mixing anticancer drug stock solutions with DMEM-10 medium. For 5-FU, 1 mM, 10 mM, and 100 mM drug solutions were prepared; for regorafenib, sorafenib, and dabrafenib, 1 μM, 10 μM, and 100 μM drug solutions were prepared; and for trametinib, 1 nM, 10 nM, and 100 nM drug solutions were prepared. The medium was carefully aspirated from the wells, and 200 μL of the various drug-medium solutions were placed into the wells containing the cells. For each anticancer drug concentration, three replicates with each of the different cell types were prepared, and the average values ​​were calculated and statistics were performed on the obtained data.The well plates were then incubated at 37°C and 5% CO2 for 24 hours.

[0119] Cell viability assay: After 48 hours of exposure to the anticancer drug solution, cell viability was determined. Cell viability was examined by CellTiter 96® AQueous One Solution Cell Proliferation (MTS) assay (Promega, Wisconsin, USA). The anticancer drug-medium solution was aspirated from the wells, and 200 μL of MTS reagent was carefully pipetted into the wells. The incubation time of the cells with MTS reagent was approximately 45 min. The cell viability assay was performed on a separate 96-well plate, and the absorbance was quantified by an EnVision Multilabel Plate Reader (PerkinElmer, California, USA).

[0120] 3D Cancer Organoids for Drug Screening Well coating with PDMS and cell isolation: Polydimethylsiloxane, i.e. PDMS (DOW Corning, North Carolina, USA) was used with a 1:10 curing agent ratio to coat the bottom of a 96-well plate, and then the plate was placed in an oven at 80° C. for at least 1 hour. For all four colorectal cancer cell types, cells were passaged in the same manner as described above (A) to place 10 μL of 3D hyaluronic acid-based hydrogel constructs containing 100,000 cells each (10,000,000 / μL) into the wells of a 96-well plate. Depending on the cell number, the desired number of cells was isolated in a new 15 mL centrifuge tube as cell-medium solution and subjected to centrifugation.

[0121] Hydrogel components of single cell type constructs: Hyaluronic acid-based hydrogels (ESI BIO, California, USA) were made using a mixture of three components: Heprasil, Gelin-S, and Extralink PEGDA 2-ARM acrylate crosslinker. These components were mixed in a volume ratio of 2:2:1. Prior to mixing, 0.1% Irgacure photoinitiator (Sigma-Aldrich, Missouri, USA) was added to each of the three components, and the three components were placed in a 37°C incubator for approximately 30 minutes to solubilize.

[0122] Formation of organoid constructs: Once solubilized, the medium was aspirated from the 15 mL centrifuge tube (after centrifugation) and the cells were isolated as a pellet. The hydrogel components were added directly into the centrifuge tube containing the cells, and the cells were carefully resuspended in the mixture. 10 μL of the gel-cell solution was placed in the center of the PDMS-coated well. This was continued for all wells containing cells, and repeated for each cell type. After this point, the gel can be exposed to UV light from a Blue Wave 200 spotlight (Dymax, Connecticut, USA) to solidify the hydrogel. Alternatively, it is possible to wait approximately 30 minutes for the gel to form on its own. DMEM-10 medium was then placed into the wells, and the constructs were incubated for 5 days.

[0123] Anticancer drug screening and cell viability: The same five anticancer drugs were added on day 5, and cell viability assays were performed on day 7.

[0124] Circular organoids for comparing the effectiveness of anti-cancer drugs Hydrogel components of circular organoid constructs: The hyaluronic acid-based hydrogel used to generate circular organoids was a mixture of four components: Heprasil, Gelin-S, and PEGDA 2-ARM acrylate crosslinker (Extralink), and alkyne-PEG-alkyne 2-ARM crosslinker (ESI BIO, California, USA). These components were mixed in a volume ratio of 4:4:1:1. Before mixing, 0.1% Irgacure photoinitiator was added to each of the four components, and the three components were placed in a 37°C incubator for approximately 30 minutes to solubilize them. HCT116 and CACO2 cells were isolated by the passaging method described above, and PDMS was used to coat the bottom of a 96-well plate.

[0125] Formation of circular organoid constructs: The four components of the hydrogel were added to the 15 mL centrifuge tube containing the HCT116 cell pellet and the cells were resuspended in this mixture. 10 μL of HCT116 containing hydrogel was placed in the center of the well and the 96-well plate was covered with aluminum foil and left undisturbed for approximately 35 minutes. During this time, the PEGDA 2-ARM acrylate crosslinks the gel while the UV-sensitive 2-ARM alkyne-PEG-alkyne remains uncrosslinked.

[0126] While waiting for the PEGDA to crosslink, add the four hydrogel components to the 15 mL centrifuge tube containing the isolated CACO2 cells and resuspend the cells in the gel. Remove the aluminum foil covering over the 96-well plate and inspect the constructs to see if they have solidified yet. If so, take 25 μL of the CACO2-embedded hydrogel and carefully place it around the periphery of the HCT116 construct. Use the tip of a pipette to physically move the CACO2 gel around the construct on all four sides. Use UV light from a Blue Wave 200 spotlight to crosslink both gels together, forming a fused ring-shaped construct. Add DMEM-10 medium to fill the wells.

[0127] Anticancer drug screening: Drug solutions of 1 μM, 10 μM, and 100 μM of 5-FU were added to the circular constructs, and drug solutions of 1 μM, 10 μM, and 100 μM of regorafenib were added to the other circular constructs on day 5. Three replicates were made for each drug concentration, and a no-drug control was also maintained.

[0128] Live-Dead staining and microscopy: On day 7, circular constructs were subjected to Live-Dead staining and constructs were imaged on an Axiovert 200M 487-1 microscope (Carl Zeiss AG, Oberkochen, Germany). Circular constructs were exposed to medium containing calcein AM and ethidium homodimer 1 stain (Thermo-Fischer Scientific, Massachusetts, USA) at an equivalent concentration of 1.0 μl / mL. Constructs were washed twice with PBS before and after exposure to the Live-Dead staining solution. Percent red vs. percent green ratios were calculated using MATLAB (Math Works, Massachusetts, USA) software to determine drug efficacy for HCT116-CACO2 circular constructs in the presence of regorafenib and 5-FU. The % red vs. % green ratio represents the % dead:% viable cell ratio.

[0129] Microfluidic Devices Construction: Graphtec Studio software was utilized to create a mold for a novel microfluidic device. The device mold was etched into double-sided tape with aluminum foil attached to one side using a Graphtec ce6000-60 plotter (Graphtec, Tokyo, Japan), and then the mold was physically attached to a cell culture plate. 20 mL of PDMS (1:10 curing agent ratio) was placed into the plate, and the PDMS was then placed in an oven at 80° C. overnight to solidify. The PDMS mold was cut from the plate immediately the next day.

[0130] 8.0 μm Isopore Transwell Membrane Filters (Merck Millipore, Massachusetts, USA) were cut to specifically fit the size of the device compartment with the transwell membrane for cell capture. A Plasma Cleaner PDC-32G (Harrick Plasma Inc., New York, USA) was used to set up the transwell membrane between the two PDMS layers by plasma bonding to the PDMS layer. The PDMS layer of the device was disinfected with 70% ethanol solution in a laboratory hood before its use.

[0131] Execution: HCT116 cells were passaged and treated with DiO membrane dye (Thermo-Fischer Scientific, Massachusetts, USA) in 5 μL / mL of DMEM-10 medium for approximately 20 min. 10 μL of hyaluronic acid-based hydrogel constructs containing 100,000 HCT116 cells (10,000,000 / μL) were placed in the organoid chamber. The PDMS layers were pressed together and fixed in place with four screws using plastic clamps on either side of the device. 3 mL of untreated medium was added to the fluid reservoir. Constructs and transwell membranes were imaged using an Axiovert 200M 487-1 microscope, and on day 7, the transwell membranes were imaged with a confocal microscope (Leica Camera, Wetzlar, Germany).

[0132] [Example 5] Screening of five drugs incorporated into organoids Experimental objective: To evaluate the dependency of compound organoid toxicity on liver organoid metabolic function. As an example, liver metabolic activity can severely affect the outcome and efficacy of some drugs. For example, 5FU, a common first-choice cancer treatment, is cytotoxic to many cells in the body, not just tumors. Therefore, 5FU is usually administered in the form of a prodrug, capecitabine, which is essentially inactive until it passes through the liver and is metabolized to its active form.

[0133] Experimental Design and Results: Five tissue platforms, including liver, heart, lung, testis, and brain organoids, were initiated and maintained for 7 days before the start of drug testing. At this point, the liver module was removed from half of the platforms. Capecitabine (20uM) was administered to all platforms, and viability was then assessed by live / dead staining or heartbeat behavior 7 days after exposure (day 14 of the total study).

[0134] The results demonstrated that in the presence of the liver, capecitabine is metabolized to the toxic drug 5-FU, resulting in toxicity to the heart and lungs (Figure 11). Without the liver, this metabolism does not occur and heart and lung organoid viability is not reduced. Surprisingly, brain organoids were damaged in both groups (Figure 11). The LIVE / DEAD results indicate that liver organoids are required to metabolize capecitabine to the drug active form of 5-FU to induce increased cell death in heart and lung organoids.

[0135] Soluble biomarkers including urea, albumin, α-GST, IL-8, and IL-1β were quantified from aliquots of media on days 1, 3, 5, 7, 9, 11, 13, and 15 (Figure 13). Aliquots of media were sent for mass spectrometry to verify the metabolism of capecitabine to 5-FU.

[0136] [Example 6] Screening of five drugs incorporated into organoids in a miniaturized microfluidic platform We have created a microfluidic platform with a reduced footprint and fluid volume and evaluated whether the same multiplex drug testing can be performed on this platform. Minimizing the fluid volume can increase the signal-to-noise ratio of relevant biomarkers in the system circulation.

[0137] Experimental Design and Results: An adhesive film-based microfluidic platform was fabricated as follows. As with the full-scale platform described in Example 4, five tissue constructs, including liver, heart, lung, testis, and brain organoids, were started and maintained for 7 days before drug testing began. At this point, the liver module was removed from half of the platform. Capecitabine (20 uM) was administered to all platforms, and then viability was assessed by live / dead staining or heartbeat behavior 7 days after exposure (day 14 of the total study).

[0138] In the miniaturized system, the presence of the liver metabolizes capecitabine to the toxic drug 5-FU, causing toxicity to the heart and lungs (Figure 12). Without the liver, this metabolism does not occur and heart and lung organoid viability is not reduced. Importantly, in this platform, brain organoid viability is high (Figure 12). The LIVE / DEAD results show that liver organoids are required to metabolize capecitabine to the drug active form of 5-FU to induce increased cell death in heart and lung organoids. Furthermore, brain organoids are viable, which suggests that perhaps scaling down the volume of the system, while not wishing to be bound by any particular theory, would better prepare the medium and support brain organoid viability. This demonstrates that it is possible to maintain brain organoids in this system, along with other organoid types.

[0139] On days 7 and 15, soluble biomarkers including urea, albumin, α-GST, IL-8, and IL-1β were quantified from aliquots of media (Figure 13). Aliquots of media were sent for mass spectrometry to verify the metabolism of capecitabine to 5-FU.

[0140] Miniaturized System Design This system reduces or eliminates the surface area of ​​PDMS exposed to the medium compared to other systems, such as standard size systems, thereby reducing the opportunity for drug compounds, toxins, soluble proteins, and secreted compounds to be adsorbed or absorbed onto or into the device walls. The device fabrication strategy is based on tape microfluidics, laser cut PMMA, and several PDMS molds, which significantly minimizes the amount of PDMS surface area in contact with the medium on the device. Figure 14 provides an overview of this fabrication method.

[0141] The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. The present invention is defined by the following claims, together with equivalents thereto, which are encompassed by the claims. All publications, patent applications, patents, patent disclosures, sequences and / or SNP accession numbers recognized by GenBank, and other references cited herein are hereby incorporated by reference for their teaching in the sentence and / or paragraph to which the reference is made. The inventions described in each claim of the original patent application were as follows: Claim 1: (a) a core consisting of viable tumor cells; (b) a shell that surrounds (e.g., encapsulates) the core; and wherein the shell is comprised of living benign cells (e.g., tissue cells, benign or differentiated tumor cells, etc.), and is useful as a tumor model in vitro (or "organoid"). Claim 2: The construct of claim 1 , wherein the live tumor cells comprise malignant cells. Claim 3: The construct of claim 1 or 2, wherein the live tumor cells comprise or consist of colorectal cancer cells (e.g., HCT116 cells, HT29 cells, SW480 cells, Caco2 cells, etc.), mesothelioma cancer cells (e.g., well-differentiated papillary mesothelioma cells), appendiceal cancer cells (e.g., low-grade appendiceal (LGA) cells), and / or sarcoma cancer cells. Claim 4: The construct of any one of the preceding claims, wherein the tumor cells and the benign cells comprise mammalian cells (e.g., human, mouse, rat, monkey, etc.). Claim 5: The construct of any one of the preceding claims, wherein the live benign cells comprise epithelial cells. Claim 6: 2. The construct of any one of the preceding claims, wherein the live benign cells comprise Caco-2 cells. Claim 7: The construct of any one of the preceding claims, wherein the tumor cells comprise a detectable compound (e.g. a fluorescent compound). Claim 8: 10. The construct of claim 1 , wherein the core and / or the shell comprises a hydrogel. Claim 9: 2. A construct according to any one of the preceding claims grown in culture for one or two days, or for one to two weeks or more. Claim 10: An in vitro cellular construct (or "organoid") useful as a tumor model, comprising living tumor cells derived from a subject. Claim 11: The construct of claim 10, further comprising a shell surrounding (e.g., encapsulating) the core of live tumor cells, the shell being composed of live benign cells (e.g., tissue cells, benign or differentiated tumor cells, etc.), optionally derived from the subject. Claim 12: The construct of claim 10 or 11, wherein the live tumor cell comprises a malignant cell. Claim 13: The construct of any one of claims 10 to 12, wherein the live tumor cells comprise or consist of colorectal cancer cells (e.g., HCT116 cells, HT29 cells, SW480 cells, Caco2 cells, etc.), mesothelioma cancer cells (e.g., well-differentiated papillary mesothelioma cells), appendiceal cancer cells (e.g., low-grade appendiceal (LGA) cells), and / or sarcoma cancer cells. Claim 14: The construct of any one of claims 10 to 13, wherein the tumor cells and the benign cells, if present, comprise mammalian cells (e.g., human, mouse, rat, monkey, etc.). Claim 15: The construct of claim 14 , wherein the live benign cells comprise epithelial cells. Claim 16: The construct of claim 14, wherein the live benign cells comprise Caco-2 cells. Claim 17: 17. The construct of any one of claims 10 to 16, wherein the tumor cells comprise a detectable compound (e.g., a fluorescent compound). Claim 18: 18. The construct of any one of claims 10 to 17, wherein the core and / or the shell comprises a hydrogel. Claim 19: 19. A construct according to any one of claims 10 to 18, grown in culture for one or two days, or for one to two weeks or more. Claim 20: The construct of any one of the preceding claims, wherein the tumor cells and / or benign cells are prepared using a method having an engraftment rate of at least 50%, 60%, 70%, 80%, or 90%. Claim 21: 2. The construct of any one of the preceding claims, comprising at least 75%, 80%, 85%, or 90% viable cells based on the average number of cells in the construct cultured for 1, 2, 3, or 4 weeks. Claim 22: 1. A method for screening a compound of interest for antitumor activity in vitro, comprising: (a) providing at least one construct according to any one of the preceding claims; (b) contacting said compound with said construct in vitro; and then (c) determining the proliferation of the live tumor cells, the reduction of the live tumor cells (e.g., lack of proliferation of the tumor cells, death of the tumor cells, reduced invasion of the shell by the tumor cells, etc.) (e.g., compared to tumor cells of at least one similar construct not contacted with the compound and / or compared to the live benign cells of the shell) that is indicative of anti-tumor activity of the compound of interest; A method comprising: Claim 23: 23. The method of claim 22, wherein the tumor cells are obtained from a patient afflicted with a tumor. Claim 24: 24. The method of claim 22 or 23, wherein the tumor cells are obtained from a tumor of a patient. Claim 25: The method of any one of claims 22 to 24, further comprising providing at least one additional organoid (e.g., a brain, colon, lung, endothelial, cardiac, epithelial, and / or liver organoid), wherein at least one of the additional organoids comprises cells different from at least one of the constructs. Claim 26: 26. The method of claim 25, wherein at least one of the additional organoids comprises benign cells, and optionally, the benign cells are obtained from the same patient as the tumor cells. Claim 27: The method of claim 25 or 26, wherein the benign cells and / or the tumor cells are obtained from a biopsy from the patient, and the compound is contacted with at least one of the constructs and / or at least one additional organoid within about one week after the biopsy. Claim 28: 28. The method of any one of claims 22 to 27, wherein at least one of said additional organoids is a liver organoid. Claim 29: 29. The method of any one of claims 22 to 28, further comprising the step of labeling the tumor cells with a detectable compound. Claim 30: 30. The method of any one of claims 22 to 29, further comprising the step of administering a therapeutically effective amount of the compound to the patient if the compound is determined to reduce proliferation of the tumor cells in vitro. Claim 31: 31. The method of any one of claims 22 to 30, further comprising identifying the compound of interest based on genetic screening information about the tumor cells. Claim 32: 32. The method of any one of claims 22 to 31, further comprising the step of performing a genetic screening analysis on the tumor cells. Claim 33: 33. The method of any one of claims 22 to 32, wherein the compound is a prodrug. Claim 34: (a) a microfluidic device having a chamber and a channel in fluid communication with the chamber; (b) a living tumor cell construct (e.g., an organoid) in the chamber; and (c) a growth medium in the chamber and the channel; (d) a pump operatively coupled to the chamber and the channel and configured to circulate the medium from the chamber, through the channel and back to the chamber; (e) a device useful for evaluating tumor cells in vitro (e.g., useful for evaluating tumor cell metastasis in vitro, for evaluating tumor cell migration and / or invasion in vitro, for evaluating the growth of constructs containing tumor cells in vitro, and / or for evaluating response to a compound of interest), comprising a microporous membrane in the channel (e.g., a TRANSWELL® microporous membrane) positioned to allow the medium to flow through it. Claim 35: The device of claim 34 , wherein the tumor cell construct comprises a hydrogel. Claim 36: The device of claim 34 or 35, wherein the tumor cell construct consists of a single cell type. Claim 37: The device of any one of claims 34 to 36, wherein the tumor cell construct comprises or consists of colorectal cancer cells (e.g., HCT116 cells, HT29 cells, SW480 cells, Caco2 cells, etc.), mesothelioma cancer cells (e.g., well-differentiated papillary mesothelioma cells), appendiceal cancer cells (e.g., low-grade appendiceal (LGA) cells), and / or sarcoma cancer cells. Claim 38: The device of any one of claims 34 to 37, wherein at least a portion of the microfluidic device is transparent, and the device further comprises a detector (e.g., a camera) operatively associated with the microporous membrane and configured to detect (e.g., image) tumor cells on the microporous membrane. Claim 39: A device according to any one of claims 34 to 38, wherein the live tumor cell construct is a construct according to any one of claims 1 to 21. Claim 40: 1. A method for screening tumor cells for metastatic activity in vitro, comprising: (a) providing a device according to any one of the preceding claims; (b) circulating the medium through the device; (c) detecting tumor cells captured on the microporous membrane; wherein a greater number of captured tumor cells (e.g., compared to other tumor cells under similar conditions and / or compared to non-metastatic cells under similar conditions) indicates a greater metastatic activity of the tumor cells. Claim 41: 1. A method for screening tumor cells for metastatic activity in vitro, comprising: (a) providing a device comprising a primary chamber containing live tumor cell organoids and at least one secondary chamber containing at least one organoid containing cells different from said live tumor cell organoids; (b) circulating the medium through the device; (c) detecting tumor cells in at least one of the secondary chambers; wherein a greater number of tumor cells present in at least one of said secondary chambers indicates a greater metastatic activity of said tumor cells (e.g., compared to other tumor cells under similar conditions and / or compared to non-metastatic cells under similar conditions). Claim 42: 42. The method of claim 41, wherein at least one of said organoids is a liver, colon, brain, lung, endothelial, cardiac, or epithelial organoid. Claim 43: The method of claim 41 or 42, wherein at least one of said organoids comprises a liver organoid and an additional organoid, and said liver organoid and said additional organoid are in separate secondary chambers in fluid communication. Claim 44: 1. A method for screening a compound of interest for anti-metastatic and / or anti-tumor activity in vitro, comprising: (a) providing a device according to any one of the preceding claims; (b) circulating the medium through the device; (c) administering the compound to the tumor cells (e.g., by adding the compound to the culture medium); and then (d) detecting tumor cells captured on the microporous membrane; wherein the fewer tumor cells captured (e.g., compared to other tumor cells under similar conditions but not administered the compound of interest) indicates a greater anti-metastatic and / or anti-tumor activity of the compound of interest. Claim 45: 1. A method for screening a compound of interest for anti-metastatic and / or anti-tumor activity in vitro, comprising: (a) providing a device comprising a first chamber containing a liver organoid and a second chamber containing a live tumor cell organoid; (b) circulating a growth medium from the first chamber to the second chamber; (c) administering a compound of interest to the liver organoids and / or the live tumor cell organoids (e.g., by adding the compound to the growth medium); (d) determining a decrease (e.g., a change in number or density) of tumor cells in the second chamber compared to the number of tumor cells present in the live tumor cell organoid in the absence of administration of the test compound; A method comprising: Claim 46: 46. ​​The method of claim 45, further comprising determining a reduction in the presence (e.g., a change in number or density) of tumor cells in the first chamber compared to the number of tumor cells present in the first chamber in the absence of administration of the test compound. Claim 47: The method of claim 45 or 46, wherein the device further comprises at least one additional chamber containing at least one organoid having cells different from the live tumor cell organoids and liver organoids. Claim 48: 1. A method for screening a compound of interest for anti-metastatic and / or anti-tumor activity in vitro, comprising: (a) providing a device comprising a primary chamber containing live tumor cell organoids and at least one secondary chamber containing an additional organoid containing cells different from said live tumor cell organoids; (b) circulating a medium through the device; (c) administering the compound to the tumor cells (e.g., by adding the compound to the culture medium); (d) determining a decrease (e.g., a change in number or density) in the presence of tumor cells in the live tumor cell organoids and / or at least one of the secondary chambers compared to the number of tumor cells present in the live tumor cell organoids and / or at least one of the secondary chambers in the absence of administration of the test compound; A method comprising: Claim 49: 49. The method of claim 48, wherein the additional organoids are brain, colon, lung, endothelial, cardiac, epithelial, and / or liver organoids. Claim 50: 50. The method of any one of claims 40 to 49, wherein the tumor cells are obtained from a patient afflicted with the tumor. Claim 51: 51. The method of any one of claims 40 to 50, further comprising the step of administering to the patient a therapeutically effective amount of the compound if it is determined that the compound reduces metastasis of the tumor cells in vitro, reduces construct size in vitro, reduces tumor cell number in vitro, and / or induces tumor cell death in vitro.

Claims

1. a core comprising live tumor cells and a crosslinked hydrogel, wherein the live tumor cells include malignant cells, and the live tumor cells are not Caco-2 cells; a shell surrounding the core, the shell comprising viable benign cells and a crosslinked hydrogel, the live benign cells comprising viable epithelial cells; An in vitro cell construct useful as a tumor model, wherein the crosslinked hydrogel contained in the core and the crosslinked hydrogel contained in the shell are each formed from a composition comprising thiolated hyaluronic acid, thiolated gelatin, and at least two polyethylene glycol (PEG) crosslinkers, at least one of the at least two polyethylene glycol (PEG) crosslinkers comprising an alkyne.

2. The construct of claim 1 , wherein the live tumor cells comprise or consist of colorectal cancer cells, mesothelioma cancer cells, appendix cancer cells, and / or sarcoma cancer cells.

3. The construct of claim 1 or 2, wherein the tumor cells and the benign cells comprise mammalian cells.

4. 4. The construct of claim 1, wherein the construct is grown in culture for a period of 1 or 2 days.

5. The construct of any one of claims 1 to 4, wherein the live benign cells comprise Caco-2 cells.

6. The construct of any one of claims 1 to 5, wherein the tumor cells comprise a detectable compound.

7. 4. The construct of claim 1, wherein the construct is grown in culture for one to two weeks or more.

8. a core comprising live tumor cells and a crosslinked hydrogel, wherein the live tumor cells include malignant cells, and the live tumor cells are not Caco-2 cells; a shell surrounding said core, said shell comprising living benign cells and a crosslinked hydrogel, said living benign cells comprising living tissue cells; Including, The crosslinked hydrogel contained in the core and the crosslinked hydrogel contained in the shell are each formed from a composition comprising thiolated hyaluronic acid, thiolated gelatin, and at least two polyethylene glycol (PEG) crosslinkers, at least one of the at least two polyethylene glycol (PEG) crosslinkers comprising an alkyne; An in vitro cell construct useful as a tumor model, wherein said live tumor cells are derived from a subject.

9. The construct of claim 8 , wherein the live, benign cells are derived from the subject.

10. 10. The construct of claim 8 or 9, wherein the live tumor cells comprise or consist of colorectal cancer cells, mesothelioma cancer cells, appendix cancer cells, and / or sarcoma cancer cells.

11. The construct of any one of claims 8 to 10, wherein the tumor cells and the benign cells comprise mammalian cells.

12. The construct of claim 11 , wherein the live, benign cells comprise epithelial cells.

13. The construct of any one of claims 8 to 12, wherein the tumor cells comprise a detectable compound.

14. 14. The construct of any one of claims 8 to 13, wherein the construct has been grown in culture for one or two days, or for one to two weeks, or more.

15. 15. The construct of any one of claims 1 to 14, wherein the tumor cells and / or benign cells are prepared using a method having an engraftment rate of at least 50%, 60%, 70%, 80%, or 90%.

16. 16. The construct of any one of claims 1 to 15, comprising at least 75%, 80%, 85%, or 90% viable cells based on the average number of cells in the construct cultured for 1, 2, 3, or 4 weeks.

17. 1. A method for screening a compound of interest for antitumor activity in vitro, comprising: (a) providing at least one construct according to any one of claims 1 to 16; (b) contacting said compound with said construct in vitro; and then (c) determining the proliferation of said viable tumor cells or the reduction in said viable tumor cells, which indicates an anti-tumor activity of said compound of interest; A method comprising:

18. The method of claim 17, wherein the tumor cells are obtained from a patient suffering from a tumor.

19. 19. The method of claim 17 or 18, wherein the tumor cells are obtained from a tumor of a patient.

20. 20. The method of any one of claims 17 to 19, further comprising the steps of providing at least one additional organoid, wherein at least one of the additional organoids comprises cells different from at least one of the constructs, and wherein at least one of the additional organoids comprises live tumor cells, and determining the amount of the live tumor cells in at least one of the additional organoids.

21. 21. The method of claim 20, wherein at least one of the additional organoids comprises benign cells, optionally obtained from the same patient as the tumor cells.

22. The method of claim 18, 19, or 21, wherein the benign cells and / or the tumor cells are obtained from a biopsy from the patient, and the compound is contacted with at least one of the constructs and / or at least one additional organoid within one week of the biopsy.

23. 23. The method of any one of claims 20 to 22, wherein at least one of the additional organoids is a liver organoid.

24. 24. The method of any one of claims 17 to 23, further comprising the step of labeling the tumor cells with a detectable compound.

25. a chamber having a live tumor cell construct; a channel in fluid communication with the chamber; a growth medium in the chamber and the channel; a microporous membrane in the channel positioned to allow the growth medium to flow through the microporous membrane from the chamber; and a fluid circuit in communication with said chamber and said channel, operatively forming a closed loop fluidic system providing fluid flow from said chamber through said channel and said microporous membrane back to said chamber, said device being useful for evaluating tumor cells in vitro, said device comprising: A device, wherein the living tumor cell construct is a construct described in any one of claims 1 to 16.

26. 1. A method for screening tumor cells for metastatic activity in vitro, comprising: (a) providing a device according to claim 25; (b) circulating the medium through the device; (c) detecting tumor cells captured on the microporous membrane; wherein a higher number of captured tumor cells indicates a higher metastatic activity of said tumor cells.

27. 1. A method for screening tumor cells for metastatic activity in vitro, comprising: (a) providing a device comprising a primary chamber containing a living tumor cell organoid and at least one secondary chamber containing at least one organoid containing cells different from said living tumor cell organoid, said living tumor cell organoid being the construct of claim 1; (b) circulating a medium through the device; (c) detecting tumor cells in at least one of the secondary chambers; wherein a greater number of tumor cells present in at least one of said secondary chambers indicates a greater metastatic activity of said tumor cells.

28. 28. The method of claim 27, wherein at least one of the organoids is a liver, colon, brain, lung, endothelial, cardiac, or epithelial organoid.

29. 29. The method of claim 27 or 28, wherein at least one of the organoids comprises a liver organoid and an additional organoid, and the liver organoid and the additional organoid are in separate secondary chambers in fluid communication.

30. 1. A method for screening a compound of interest for anti-metastatic and / or anti-tumor activity in vitro, comprising: (a) providing a device according to claim 25; (b) circulating the medium through the device; (c) administering said compound of interest to said tumor cells; and then (d) detecting tumor cells captured on the microporous membrane; wherein a lower number of captured tumor cells indicates a higher anti-metastatic and / or anti-tumor activity of said compound of interest.

31. 1. A method for screening a compound of interest for anti-metastatic and / or anti-tumor activity in vitro, comprising: (a) providing a device comprising a first chamber containing a liver organoid and a second chamber containing a living tumor cell organoid, said living tumor cell organoid being a construct according to any one of claims 1 to 16; (b) circulating a growth medium from the first chamber to the second chamber; (c) administering a compound of interest to said liver organoids and / or said live tumor cell organoids; (d) determining a reduction in the presence of tumor cells in the second chamber compared to the number of tumor cells present in the live tumor cell organoid in the absence of administration of the compound of interest; A method comprising:

32. 32. The method of claim 31, further comprising determining a reduction in the presence of tumor cells in the first chamber compared to the number of tumor cells present in the first chamber in the absence of administration of the compound of interest.

33. The method of claim 31 or 32, wherein the device further comprises at least one additional chamber containing at least one organoid having cells different from the live tumor cell organoids and liver organoids.

34. 1. A method for screening a compound of interest for anti-metastatic and / or anti-tumor activity in vitro, comprising: (a) providing a device comprising a primary chamber containing a living tumor cell organoid and at least one secondary chamber containing an additional organoid containing cells different from said living tumor cell organoid, said living tumor cell organoid being a construct according to any one of claims 1 to 16; (b) circulating a medium through the device; (c) administering said compound of interest to said tumor cells; (d) determining a decrease in the presence of tumor cells in said live tumor cell organoids and / or at least one of said secondary chambers compared to the number of tumor cells present in said live tumor cell organoids and / or at least one of said secondary chambers in the absence of administration of said compound of interest; A method comprising:

35. 35. The method of claim 34, wherein the additional organoids are brain, colon, lung, endothelial, cardiac, epithelial, and / or liver organoids.

36. 36. The method of any one of claims 26 to 35, wherein the tumor cells are obtained from a patient afflicted with the tumor.

Images