Media and methods for growing mammary organoids

Abstract

Disclosed are media, kits and methods for directional formation of breast organoids.Embodiments of the organoid media can be used to bias / enrich the formation of luminal organoids from isolated breast epithelial cells.Embodiments of the modified organoid media can be used to bias / enrich the formation of mixed lineage organoids from isolated breast epithelial cells, or to convert luminal organoids into mixed lineage organoids.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 175,686, filed April 16, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to cell culture applications, more particularly to cell culture applications using cells of the mammary gland, and even more particularly to cell culture applications relating to the growth of multicellular structures composed of specific mammary gland cell types. [Background technology]

[0003] Approximately one in eight women in the United States will be diagnosed with invasive breast cancer during their lifetime. Breast tumors arise in the epithelium of the mammary gland. The breast epithelium is composed of a series of branching ducts that drain the lactogenic acini during lactation. The cells of these ducts and acini are organized as a bistratified epithelium with an inner luminal layer and an outer basal layer. The luminal layer is composed of epithelial cells of two lineages: an estrogen receptor (ER) expressing lineage and a lactogenic lineage. The lactogenic lineage cells in the resting mammary gland are cells that proliferate during pregnancy to produce sac-like structures called acini. The cells lining the acini, or acinar cells, synthesize and secrete milk during lactation. The basal cell layer of the mammary epithelium is composed of basal cells, also called myoepithelial cells, because they have contractile properties (they squeeze milk out of the mammary gland).

[0004] Three lineages of cells in the postnatal gland (basal, ER + , milk) are maintained primarily by their own stem cell populations. +The line expresses high levels of luminal keratins (K) 8 and 18, but not basal K5 and K14. These cells, as the name suggests, express high levels of ER, as well as the progesterone receptor (PR). Basal cells do not express K8 or K18, but do express keratins K5 and K14. The milk line has an intermediate phenotype; in humans, cells of this milk line express both luminal and basal keratins. The milk line in mice has a predominantly luminal phenotype, albeit with low levels of luminal keratin expression, and low but detectable transcript levels of basal keratins. The milk line does not express ER.

[0005] Human breast tumors are divided into three subtypes based on phenotype: ER + , HER2 amplification (HER2 + ), and triple negative (ER - , P.R. - , HER2 - The triple-negative classification can be further divided into basal-like and claudin-like. low The cellular origin of human breast tumors remains incompletely understood, but it is believed that ER tumors are + The tumor is + Derived from HER2 cells + The tumor is + and basal-like tumors are derived from breast lineage cells and express claudin- low There is a hypothesis that the tumor originates from basal cells.

[0006] The in vitro model system will be useful for studying basic mammary biology questions and also for understanding differences in normal mammary cells compared to breast tumor cells. Additionally, the system may be useful for compound screening assays and toxicity testing.

[0007] There remains a need for effective in vitro model systems to study normal and diseased states of the mammary gland. In particular, there remains a need for in vitro model systems to study individual cell types in isolated mammary glands, both in normal and diseased situations. Furthermore, there remains a need for validated culture conditions, including cell culture media, to obtain in vitro model systems that recapitulate the specific structures or cell types being studied, whether in normal or diseased conditions. Summary of the Invention

[0008] The present disclosure relates to media, kits and methods for growing mammary organoids. More specifically, the present disclosure relates to culturing mammary epithelial cells to predictably and reproducibly obtain mammary organoids composed of desired mammary cell types.

[0009] In one broad aspect of the present disclosure, a method is provided for forming breast organoids from isolated breast epithelial cells. Such a method may include contacting breast epithelial cells with an organoid medium that does not contain one or both of an exogenously added WNT signaling agonist and / or an inhibitor of BMP signaling, and culturing the breast epithelial cells in the organoid medium for a sufficient time to form a first population of organoids that are enriched for organoids that are composed of more luminal cells than non-luminal cells (such as basal cells, stromal cells, etc.) when the organoid medium contains one or both of an exogenously added WNT signaling agonist and / or an inhibitor of BMP signaling. In one embodiment, the breast epithelial cells include breast epithelial stem cells or progenitor cells. In one embodiment, the breast epithelial cells are isolated from a primate or rodent. In one embodiment, the non-luminal cells are one or more of basal cells, stromal cells, hematopoietic cells, and endothelial cells.

[0010] In one embodiment, the luminal cells express K8. In one embodiment, the luminal cells co-express K8 and K18. In one embodiment, the basal cells express K5. In one embodiment, the basal cells co-express K5 and K14.

[0011] In one embodiment, organoid medium comprises basal medium and one or both of ERBB1 ligand and / or ERBB4 ligand.In one embodiment, ERBB1 ligand is not EGF or TGFα.In one embodiment, ERBB1 ligand is amphiregulin.In one embodiment, ERBB4 ligand is also the ligand of different ERBB receptor family members.In one embodiment, ERBB4 ligand is heregulin and / or neuregulin3.

[0012] In one embodiment, organoid culture medium comprises the inhibitor of BMP signal transduction but does not include the agonist of WNT signal transduction.In one embodiment, organoid culture medium comprises the agonist of WNT signal transduction but does not include the inhibitor of BMP signal transduction.

[0013] In one embodiment, the WNT signaling agonist (not included in the organoid medium) is R-spondin, a WNT protein, or an engineered mimic of any of the above.

[0014] In one embodiment, the inhibitor of BMP signaling is a protein or small molecule. In one embodiment, the inhibitor of BMP signaling is one or more of noggin, chordin, follistatin, LDN193189 or dorsomorphin.

[0015] In one embodiment, the organoid medium does not contain exogenously added sex hormones. In one embodiment, the sex hormone is progesterone.

[0016] In one embodiment, the organoid is composed of more than 50% luminal cells.

[0017] In one embodiment, the method may further include culturing the first population of organoids in a modified organoid medium to convert the first population of organoids into a second population of organoids.

[0018] In one embodiment, the modified organoid medium is supplemented with EGF. In one embodiment, the modified organoid medium is supplemented with a WNT signaling agonist and / or an inhibitor of BMP signaling.

[0019] In one embodiment, when one or both of the WNT signaling agonist and / or the inhibitor of BMP signaling are not added to the organoid medium, the second population of organoids is composed of more basal cells and fewer luminal cells.

[0020] In one embodiment, the first group of organoids can be passaged in organoid medium 5 times or more.In one embodiment, the second group of organoids can be passaged in modified organoid medium 5 times or more.

[0021] In one embodiment, the method can further comprise contacting the first group or the second group of organoid with the inhibitor of TGF-beta.In one embodiment, the method can further comprise treating with the inhibitor of TGF-beta to obtain the nuclear localization of ER.

[0022] In one embodiment, the first population of organoids is composed of, on average, more than 50% luminal cells and less than 30% basal cells.

[0023] In another broad aspect of the present disclosure, there is provided breast organoid medium formulation.This breast organoid medium comprises basal medium and one or both of the ligand of ERBB1 and the ligand of ERBB4, and lacks one or both of the exogenously added WNT signaling agonist and the inhibitor of BMP signaling.

[0024] In one embodiment, organoid culture medium comprises the inhibitor of BMP signal transduction but does not include the agonist of WNT signal transduction.In one embodiment, organoid culture medium comprises the agonist of WNT signal transduction but does not include the inhibitor of BMP signal transduction.

[0025] In one embodiment, the WNT signaling agonist (not included in the organoid medium) is one or more of R-spondin, WNT protein, or an engineered mimic of any of the above.

[0026] In one embodiment, the inhibitor of BMP signal transduction (not included in organoid medium) is protein or small molecule.In one embodiment, the inhibitor of BMP signal transduction (not included in organoid medium) is one or more of Noggin, Chordin, Follistatin, LDN193189 or Dorsomorphin.

[0027] In one embodiment, the ligand of ERBB1 is not EGF or TGFα. In one embodiment, the ligand of ERBB1 is amphiregulin. In one embodiment, the ligand of ERBB4 is also a ligand of a different ERBB receptor family member. In one embodiment, the ligand of ERBB4 is neuregulin 1 and / or neuregulin 3.

[0028] In one embodiment, the organoid medium does not contain exogenously added sex hormones. In one embodiment, the sex hormone is progesterone.

[0029] In one embodiment, organoids composed of more luminal than non-luminal cells are enriched by culturing isolated mammary epithelial cells in organoid medium.

[0030] In one embodiment, when isolated mammary epithelial cells are cultured in organoid medium containing one or both of exogenously added WNT signaling agonist and / or BMP signaling inhibitor, the organoids that are composed of more luminal cells and less basal cells are enriched by culturing isolated mammary epithelial cells in organoid medium.In one embodiment, mammary epithelial cells comprise mammary epithelial stem cells or progenitor cells.In one embodiment, mammary epithelial cells are isolated from primates or rodents.

[0031] In another broad aspect of the present disclosure, a kit is provided (for formulating the organoid medium, modified organoid medium, and ER nuclear localization medium of the present disclosure).In one embodiment, such a kit can include a basal medium and a first luminal cell-promoting supplement that is added to the basal medium.In one embodiment, the first supplement can include one or both of the ligand of ERBB1 and / or the ligand of ERBB4.In one embodiment, the first supplement can also lack one or both of the exogenously added WNT signaling agonist and / or the inhibitor of BMP signaling.

[0032] In one embodiment, the kit of the present disclosure may further comprise a second mixed lineage promoting supplement added to the basal medium or added to the basal medium supplemented with the first supplement. In one embodiment, the second supplement may comprise a second ligand of ERBB1 that is different from the ligand of ERBB1 in the first supplement. In one embodiment, the second supplement may further comprise one or both of an exogenously added WNT signaling agonist and / or an inhibitor of BMP signaling.

[0033] In one embodiment, the kit of the present disclosure may further include a third ER nuclear localization supplement added to the basal medium or to the basal medium supplemented with the first supplement and / or the second supplement. In one embodiment, the third supplement may include an inhibitor of TGFβ signaling. In one embodiment, the inhibitor of TGFβ signaling is SB431542, RepSox, A77-01 or A83-01.

[0034] Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that this detailed description and the specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, and that various changes and modifications which will become apparent to those skilled in the art from this detailed description are within the spirit and scope of the present invention.

[0035] For a better understanding of the various embodiments described herein, and to more clearly show how they may be effectively practiced, reference is made to the accompanying drawings, which illustrate at least one exemplary embodiment, and which will now be described, the drawings being not intended to limit the scope of the teachings described herein. [Brief description of the drawings]

[0036] [Figure 1] Representative images of organoids formed from unsorted mouse mammary epithelial cells are shown. Images in panels A), B) and C) correspond to serial passaging of organoids in organoid medium containing exogenously added WNT agonist and inhibitor of BMP signaling. Images in panel D show images taken by confocal microscopy of passage 2 of organoids generated in the organoid medium used to form the organoids shown in panels A)-C). Organoids were stained for K8, K14 and the nuclear stain DAPI. Scale bars are 500 μm in panels A)-C) and 100 μm in panel D). [Diagram 2] Representative images of organoids formed from sorted populations of mouse mammary epithelial cells are shown. ER+ luminal cells (panels A and D), mammary cells (panels B and E), and basal cells (panels C and F) were cultured in organoid medium without inhibitors of BMP signaling and either in the presence or absence of RSPO-1. Scale bar is 500 μm. [Diagram 3] Representative images of organoids formed from unsorted mouse mammary epithelial cells are shown. The images in panel A) show organoids formed in medium containing the WNT agonists RSPO-1 or RSPO-3 or without exogenously added WNT signaling agonists. Scale bar is 500 μm. Flow cytometry plots in panel B) show the intercellular phenotype formed in organoids in the organoid medium in panel A). [Figure 4]Figure 1 shows representative images of organoids formed from unselected mouse mammary epithelial cells in serial passage.Luminous organoids are grown in the organoid medium of the present disclosure with or without exogenously added WNT signaling agonist.Scale bar is 500 μm.Passage 2 organoids are stained for K8, K14, and nuclear stain DAPI, and imaged by confocal microscopy.Scale bar is 100 μm. [Diagram 5] Representative images of organoids formed from unselected mouse mammary epithelial cells in serial passage are shown.Luminous organoids do not form or grow in the organoid medium of the present disclosure with or without exogenously added inhibitor of BMP signaling.Scale bar is 500 μm.Passage 2 organoids are stained for K8, K14, and nuclear stain DAPI, and imaged by confocal microscope.Scale bar is 100 μm. [Figure 6] Representative flow cytometry plots of organoids formed from unsorted mouse mammary epithelial cells are shown. Panels A) and B) show representative images of organoids after culturing cells for 14 days (P0) in medium with (panel A) or without (panel B) R-spondin. Cells of organoids shown in panels A) and B) were analyzed by flow cytometry for expression of EpCAM and CD49f (panels C) and D), respectively. A summary of the flow cytometry data is shown in panel E. Results were confirmed in n=8 mice. Scale bar is 500 μm. [Figure 7]Representative images of organoids formed from unsorted human mammary epithelial cells are shown. Organoids were formed in organoid medium supplemented with exogenous RSPO-1, EGF and Noggin. The image in panel A) shows a larger focal field capturing multiple organoids that formed. Scale bar is 500 μm. Each image in panels B) and C) shows one organoid imaged by either bright field or confocal microscopy after staining for K8, K14 and the nuclear stain DAPI. Scale bar is 100 μm. [Figure 8] Figure 1 shows the effect of modulating signaling through BMP receptors on human organoids formed from unsorted human breast epithelial cells. Panel A) Flow cytometry plots compare organoid composition following formation in organoid medium containing either BMP signaling agonists (BMP2 or BMP4) or antagonists (Noggin or LDN193189). Panel B) Flow cytometry plots show the effect of inhibiting signaling through BMP receptors on organoid cell lineage balance for two different donors. [Figure 9] Figure 1 shows the effect of modulating signaling through BMP receptors on human organoids formed from unsorted human mammary epithelial cells. The plot in panel A) shows the total number of cells contained within organoids formed in organoid medium containing either BMP signaling agonists (BMP2 or BMP4) or antagonists (Noggin or LDN193189). The bar graph in panel B) summarizes the composition of organoids formed in organoid medium containing different regulators of BMP signaling. The bar graph represents the mean of two experiments + / - standard deviation, normalized to a control that did not contain exogenously added regulators of BMP signaling. In panel C), organoids formed in medium with or without inhibitors of BMP signaling were imaged by confocal microscopy after staining for K8, K14 and the nuclear stain DAPI. The white arrow indicates the selected area staining brightly for K8. Scale bar is 50 μm. [Figure 10] Figure 1 shows images of luminal-biased human organoids taken at serial passages. Organoids were formed in organoid medium containing a combination of two different inhibitors of BMP signaling or in medium containing each BMP signaling inhibitor individually. Scale bar is 500 μm. [Figure 11] Figure 1 shows the effect of Wnt agonism on organoids formed from unsorted human mammary epithelial cells. Organoids in panel A) were formed in organoid medium containing or omitting agonists of WNT signaling, and representative flow cytometry plots of the composition of such organoids are shown in panel B). Scale bar is 500 μm. Bar graphs in panel C) summarize the organoid composition after formation in medium containing various WNT signaling agonists or antagonists. Bar graphs represent the mean of two experiments + / - standard deviation, normalized to a control without exogenously added WNT signaling modulators. [Figure 12] Figure 1 shows the effect of modulating signaling through various ERBB receptor family members on organoids formed from unsorted human breast epithelial cells. Panel A) Flow cytometry plots show a comparison of organoid composition after formation in either organoid medium without modulation of ERBB receptor family members or with the indicated ERBB receptor family ligands. Panel B) Flow cytometry plots show the effect on organoid formation of organoid medium without exogenous modulators of ERBB family members or with EGF signaling inhibitors for two different donors. [Figure 13]Figure 1 shows the effect of modulating signaling through ERBB family members on organoids formed from unsorted human breast epithelial cells. The plot in panel A) shows the total number of cells contained in organoids formed in organoid medium containing either ERBB agonists (AREG, EGF or NRG1) or antagonists (erlotinib + gefitinib). The bar graph in panel B) summarizes the composition of the resulting organoids after formation in organoid medium containing different ERBB receptor modulators. The bar graph represents the mean of two experiments + / - standard deviation, normalized to a control without exogenously added ERBB family signaling modulators. [Figure 14] Figure 1 shows the effect of modulation of TGFβ-mediated signaling on organoids formed from unsorted human mammary epithelial cells. Panel A) Flow cytometry plots show a comparison of organoid cell composition after formation in organoid medium without modulation of TGFβ signaling or with exogenously added TGFβ1. Panel B) Flow cytometry plots show the effect of an exogenously added inhibitor of TGFβ signaling on organoid formation. [Figure 15] Shows the effect of inhibiting TGFβ-mediated signal transduction on ER localization. In panel A) (human) and panel B) (mouse), organoids are formed in each organoid medium of the present disclosure, and then transiently exposed to different inhibitors of TGFβ signal transduction. After staining for K8, K14, ER and nuclear stain DAPI, organoids are imaged by confocal microscopy. Arrows indicate the colocalization of ER staining and DAPI staining. [Figure 16]Representative images of passage 1, day 10 organoids formed from unsorted human mammary epithelial cells are shown. Organoids were formed in organoid medium without exogenously added RSPO-1, Noggin, progesterone or EGF. The image in panel A) shows a wider focal area capturing multiple organoids that formed. Scale bar is 100 μm. Images in panels B)-D) show one formed organoid, alone or merged, imaged by confocal microscopy after staining for K8, K14 and the nuclear stain DAPI. Scale bar is 50 μm. [Figure 17] Representative confocal microscopy images of organoids formed from unsorted human breast epithelial cells are shown. Luminal-restricted organoids were formed in organoid medium containing either 50 ng / ml neuregulin 3 (NRG3) (A) or 100 ng / ml anti-Müllerian hormone (AMH) (B). Basal-restricted organoids were formed in organoid medium containing 50 ng / ml granulocyte-macrophage colony-stimulating factor (GM-CSF) (C). Organoids were stained for K8, K14 and the nuclear stain DAPI. Scale bar is 100 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The present disclosure relates to the medium and method for growing breast organoids.More specifically, the present disclosure relates to engineering the culture of breast epithelial cells to predictably and reproducibly generate breast organoids that are composed of desired breast cell types.In some embodiments, the breast organoids formed in the medium of the present disclosure or by carrying out the method of the present disclosure can be propagated or passaged in the medium.

[0038] As used in this disclosure, the term "breast organoid(s)" refers to a multicellular structure that recapitulates the general organization of breast epithelium or its particular components, such as ductal segments or terminal ductal lobular units. In one embodiment, breast organoids are composed of a particular cell type (e.g., luminal cells) that is greater than any other cell type (e.g., basal, endothelial, stromal, hematopoietic, etc.). In a related embodiment, such breast organoids may be composed primarily or entirely of one type of breast epithelial cells. In one embodiment, breast organoids are composed of a more balanced mixture of various types of breast epithelial cells.

[0039] As an example of the former, "luminal organoid", "luminal restricted organoid" or "luminal biased organoid" is a breast organoid (formed using the organoid medium of the present disclosure) that is composed of about 50%, about 60%, about 70%, about 80%, about 90% or more luminal cells. Thus, luminal organoids can be composed of more luminal cells than non-luminal cells. When formed using the organoid medium of the present disclosure, such luminal organoids can be referred to as the first population of organoids. In one embodiment, luminal organoids (or the first population of organoids) are composed of more luminal cells than may be present when using a medium that is formulated differently from the organoid medium as disclosed herein.

[0040] As an example of the latter, "mixed lineage organoids" or "branched organoids" are breast organoids that are composed of various cell types (e.g., basal cells, luminal cells, stromal cells, etc.). Mixed lineage organoids can be organized in a manner that is substantially similar to that observed in normal breast tissue. When formed using a medium that is formulated differently from the organoid medium of the present disclosure, such mixed lineage organoids can be referred to as a second population of organoids. Thus, the organoids of the second population of organoids are composed of at least more basal cells and less luminal cells after being exposed to the organoid medium of the present disclosure. Mixed lineage organoids can be formed using the modified organoid medium of the present disclosure, regardless of whether they start from isolated breast epithelial cells or from a first population of organoids.

[0041] As used in this disclosure, the term "mammary epithelial cells" refers to cells present in and isolatable from the mammary gland of a mammal. In one embodiment, mammary epithelial cells may include mammary epithelial stem or progenitor cells. For purposes of this disclosure, mammary epithelial cells may be provided as a single cell suspension, a suspension of fragments of mammary epithelial cells, clumps of mammary epithelial cells, or a mixture of any combination of the foregoing. In one embodiment, mammary epithelial cells may be "mammary epithelial-like cells" when derived from pluripotent stem cells, e.g., induced pluripotent stem cells, embryonic stem cells, etc. In one embodiment, the mammary epithelial cells or mammary epithelial-like cells are derived from a primate, such as a human, or a rodent, such as a mouse.

[0042] Mammary epithelial cells are typically derived from basal cells (e.g., K14 + , K5 + , S.M.A. + , CD49f + , and K8 - and luminal cells, wherein the luminal cells are characterized by one or more of the following: + Lineage (e.g., K8 + , E.R. + , K5 - , K14 - , and CD49f -and milk-producing lines (e.g., K8 + , CD49b + , ALDEFLUOR + In one embodiment, the luminal cells are further subdivided into luminal cells characterized by one or more of the following: expression of K8 ("K8 + In one embodiment, luminal cells may be classified based on co-expression of K8 and K18. In one embodiment, basal cells may be classified based on expression of K14 ("K14 + In one embodiment, luminal cells may be classified based on co-expression of K14 and K5. One skilled in the art will appreciate that the signatures of basal or luminal cells are not exhaustive or exclusive. In some cases, different markers may be useful for distinguishing each cell type. Alternatively, in some cases, different cell types may express common markers at roughly equivalent or different levels.

[0043] Basement and luminal cells are likely to be bipotent, for example, because the sorted luminal cell population is cultured under conditions that promote the eventual emergence of basement cells, and vice versa, provided that a suitable culture environment is used, in one embodiment, the suitable culture environment includes any suitable supplemented culture medium (as further described herein).

[0044] Preparations of mammalian epithelial cells can be obtained using known methodologies or modifications of known methodologies. Typically, mammary glands are excised from a subject and physically / mechanically disrupted using a scalpel or other similar means. Enzymatic digestion usually facilitates further dissociation of the mammary gland, including its connective tissue.

[0045] In one embodiment, breast tissue minced with a scalpel can be gently agitated in a solution containing collagenase and hyaluronidase to disintegrate the extracellular matrix and connective tissue. The resulting liquid fraction can then be subjected to one or more rounds of centrifugation.

[0046] When multiple centrifugations are performed, each may be performed at a stepwise higher centrifugal force and / or longer duration, thereby saving pellets at each step. In one embodiment, two to three centrifugations are sufficient to isolate the cells of interest. For example, after a first centrifugation at about 100 x g for about 0.5 to 1 minute, larger debris including breast epithelial cell fragments can be collected in the pellet. After a second centrifugation at about 200 x g for about 3 to 5 minutes, clumps of breast epithelial cells and individual breast epithelial cells can be collected in the pellet. And, after a third centrifugation at about 400 x g for about 5 minutes, individual cells including stromal cells can be collected in the pellet.

[0047] In some embodiments, depending on the appearance of the pelleted cells, a brief treatment with ammonium chloride may be necessary to lyse contaminating red blood cells.

[0048] To generate a single cell suspension of mammary epithelial cells, the cells of the first pellet, and, if applicable, the cells of the second pellet, can be treated sequentially with a trypsin-containing solution, then a DNase solution, optionally followed by further processing by filtration through a 37 μm filter.

[0049] In some embodiments, the cultured mammary epithelial cells may be contained within relatively large tissue fragments, such as those obtained as a residue following digestion and filtration as described above.

[0050] In some embodiments, the cultured mammary epithelial cells may be single cells obtained using the processes described above.

[0051] In some embodiments, the cultured mammary epithelial cells may be a mixture of single cells and tissue fragments, whether smaller (e.g., those that pass through a 37 μm filter) and / or larger (e.g., those that do not pass through a 37 μm filter).

[0052] However, the treated mammary epithelial cells can be seeded and cultured in the medium disclosed herein (e.g., organoid medium) and / or according to the methods disclosed herein.

[0053] Culture medium In one aspect of the present disclosure, a cell culture medium (i.e., organoid medium) is provided for forming or growing breast organoid.In one embodiment, the organoid medium of the present disclosure maintains and / or grows breast organoid over multiple passages.In one embodiment, the organoid medium can be used to grow / form breast organoid from breast epithelial cells and maintain / grow breast organoid over multiple passages.

[0054] As used in this disclosure, the term "organoid medium" refers to a solution that can be used to form and / or grow and / or proliferate breast organoids from isolated preparations of breast epithelial cells. Furthermore, such organoid medium can be used to passage breast organoids formed from isolated preparations of breast epithelial cells. In one embodiment, the organoid medium comprises a basal medium and is appropriately supplemented with additives for the culture of mammalian cells, particularly primary epithelial cells, such as one or more of salt(s); buffer(s); amino acids; energy source(s) (e.g., glucose, pyruvate, etc.); albumin(s) or albumin surrogates; trace elements; and lipids. Furthermore, the organoid medium can comprise one or more suitable small molecules and / or cytokines or growth factors or mimetics thereof to bias the formation of breast organoids (derived from breast epithelial cells) composed of cells of a desired lineage. In one embodiment, such organoid medium can also promote the growth / proliferation of breast organoids. In certain embodiments, organoid medium is intended to form and / or grow and / or grow luminal organoids that can be included in the first group of organoids.In one embodiment, the same organoid medium can be used to form / passage human and mouse luminal organoids.In one embodiment, different organoid medium can be used to form / passage human and mouse luminal organoids.

[0055] Organoid medium can be formulated using any known and / or commercially available basal medium that can support the culture of epithelial cells, such as breast epithelial cells.For example, basal media routinely used in the culture of epithelial cells, such as breast epithelial cells, include DMEM, DMEM / F12, Adv-DMEM, and Adv-DMEM / F12.In one embodiment, the basal medium is any of DMEM, DMEM / F12, Adv-DMEM, or Adv-DMEM / F12.

[0056] On the other hand, if it is desirable to bias cultures of mammary epithelial cells to form luminal organoids that are composed of more luminal cells than would otherwise occur, it may be necessary to optimize the organoid medium formulation for this specific purpose.

[0057] In one embodiment, the organoid medium comprises at least one mitogen. At least one mitogen can be based on the amino acid sequence corresponding to human gene. In one embodiment, the mitogen can be based on the amino acid sequence corresponding to non-human gene, such as rodent species. In one embodiment, it is appropriate to match the origin (in terms of sequence or source) of the mitogen with the species origin of the isolated breast epithelial cells to be cultured. In some embodiments, it is not necessary to match the origin (in terms of sequence or source) of the mitogen with the species origin of the isolated breast epithelial cells to be cultured.

[0058] In one embodiment, the organoid medium (for forming and maintaining luminal organoid) comprises the ligand of ERBB1. In one embodiment, the ligand of ERBB1 is not epidermal growth factor (EGF). In the same or different embodiment, the ligand of ERBB1 is not transforming growth factor alpha (TGFα). Thus, in one embodiment, the ligand of ERBB1 is not EGF or TGFα, functional fragments or mimics of the above.

[0059] In one embodiment, the ligand of ERBB1 is amphiregulin. In such an embodiment, the concentration of amphiregulin may be about 1 μg / mL to 0.1 ng / mL, or about 500 ng / mL to 0.5 ng / mL, or about 250 ng / mL to 1 ng / mL, or about 125 ng / mL to 2 ng / mL, or about 50 ng / mL to 3 ng / mL.

[0060] In one embodiment, the organoid medium (for forming and maintaining luminal organoid) comprises the ligand of ERBB4.In one embodiment, the ligand of ERBB4 is also the ligand of different ERBB receptor family members.In such an embodiment, the ligand of ERBB4 can also be the ligand of ERBB3.

[0061] In one embodiment, the ligand of ERBB4 is heregulin (also known as neuregulin 1, the ligand of dimerized ERBB3 and ERBB4). In such an embodiment, the concentration of heregulin is about 1 μg / mL to 0.1 ng / mL, or about 500 ng / mL to 0.5 ng / mL, or about 250 ng / mL to 1 ng / mL, or about 125 ng / mL to 2 ng / mL, or about 50 ng / mL to 3 ng / mL. In one embodiment, the ligand of ERBB4 is neuregulin 3, which may be included in the organoid medium within the aforementioned concentration ranges.

[0062] In one embodiment, the organoid medium comprises either ERBB1 or ERBB4 ligand.In one embodiment, the organoid medium comprises both ERBB1 and ERBB4 ligands.

[0063] In one embodiment, the ligand for ERBB4 can be betacellulin, epigen, epiregulin, neuregulin 1, neuregulin 2, neuregulin 3, neuregulin 4, or tomoregulin. In some embodiments, organoid media (for promoting and maintaining basal or mixed organoids) includes two or more ERBB4 ligands.

[0064] In one embodiment, the ligand for ERBB4 is neuregulin 3. In such embodiments, the concentration of neuregulin 3 is about 1 μg / mL to 0.1 ng / mL, or about 500 ng / mL to 0.5 ng / mL, or about 250 ng / mL to 1 ng / mL, or about 125 ng / mL to 2 ng / mL, or about 50 ng / mL to 3 ng / mL. In one embodiment, the concentration of neuregulin 3 is about 50 ng / mL.

[0065] Just as it may be important to activate and / or inhibit certain signaling cascades with a protein, peptide or small molecule ligand, it may also be important not to activate and / or inhibit other signaling cascades.

[0066] Thus, in one embodiment, the organoid culture medium (for forming and maintaining luminal organoids) does not contain exogenously added WNT signaling agonists and / or regulators of BMP signaling (e.g., activators or inhibitors).

[0067] In one embodiment, the WNT signaling agonist that is not included in organoid medium is R-spondin, WNT protein, or any of the above engineered mimics.WNT signaling agonist is used in the culture of certain epithelial organoids, but the present inventors have shown that the inclusion of WNT signaling agonist in the organoid medium of the present disclosure may be unnecessary or harmful to the formation, growth promotion and maintenance of luminal organoid.In addition, the inclusion of WNT signaling agonist in organoid medium can also promote / maintain the formation / growth of mixed lineage organoid.

[0068] In one embodiment, the R-spondin that is excluded from organoid medium is one or more of R-spondin1, R-spondin2, R-spondin3 or R-spondin4.In one embodiment, the R-spondin that is excluded from organoid medium is each of R-spondin1, R-spondin2, R-spondin3 or R-spondin4.Therefore, the concentration of R-spondin that is added to organoid medium to promote and maintain luminal organoid is 0ng / mL or substantially 0ng / mL.

[0069] In one embodiment, the WNT protein that is excluded from organoid medium is one or more of WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11 or WNT16.In another embodiment, the WNT protein that is excluded from organoid medium is each of WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11 or WNT16. Thus, to promote and maintain luminal organoids, the concentration of WNT protein added to the organoid medium is 0 ng / mL, or essentially 0 ng / mL.

[0070] Advances in synthetic biology have allowed the in silico design of protein-based, peptide-based or small molecule-based ligand mimics.The advantages of such mimics include but are not limited to increased activity, increased stability, simplified structure, reduced size, etc.Therefore, in some embodiments, the function of WNT or R-spondin (for example, by its mimics) does not exist in the organoid medium of the present disclosure.

[0071] For example, typical recombinant WNT proteins have little or no effect when added to culture media, likely due to their high hydrophobicity and rapid degradation. To address this challenge, a standard FZD-mediated WNT pathway agonist was engineered to be more potent than wild-type Wnt3a while retaining acceptable biological activity when added to culture media (U-Protein Express).

[0072] In one embodiment, the regulator of BMP signaling that is not included in organoid medium is a protein, peptide or small molecule that inhibits signaling via BMP receptor.In one embodiment, the regulator of BMP signaling that is not included in organoid medium is a protein, peptide or small molecule that activates signaling via BMP receptor.Although regulator of BMP signaling is commonly used to culture certain epithelial organoids, the present inventors have shown that the inclusion of certain regulator(s) of BMP signaling in the organoid medium of the present disclosure may be unnecessary or harmful to the promotion and maintenance of luminal organoid.

[0073] In one embodiment, the inhibitor of BMP signaling that is excluded from the organoid medium (for promoting / maintaining luminal organoid) is a protein or peptide. Such excluded protein (or functionally equivalent peptide) may be one or more of Noggin, Chordin, Follistatin, Sclerostin, CTGF / CCN2, Gremlin, Cerberus, DAN, PRDC, Decorin, Alpha-2 Macroglobulin protein, etc. In one embodiment, the BMP signaling inhibitor that is excluded from the organoid medium is each of Noggin, Chordin, Follistatin, Sclerostin, CTGF / CCN2, Gremlin, Cerberus, DAN, PRDC, Decorin, and Alpha-2 Macroglobulin protein. Thus, the effective concentration of the inhibitor of BMP signaling in the organoid medium for promoting and maintaining luminal organoid is 0ng / mL or substantially 0ng / mL.

[0074] In one embodiment, the inhibitor of BMP signaling that is excluded from the organoid medium (for promoting / maintaining luminal organoid) is a small molecule.Such excluded small molecule can be LDN193189 or Dorsomorphin.In one embodiment, the inhibitor of BMP signaling that is excluded from the organoid medium is each of LDN193189 and Dorsomorphin.Therefore, the effective concentration of the BMP signaling inhibitor in the organoid medium for promoting and maintaining luminal organoid is 0ng / mL or substantially 0ng / mL.

[0075] In one embodiment, the activator of BMP signaling can be a protein such as BMP2 or BMP4 or a peptide thereof.

[0076] Many regulators of BMP signaling are known, and it may be important to select specific regulators to include or not include in organoid medium based on the pathways they regulate. In some embodiments of organoid medium, the optimal formation and maintenance of luminal organoids can be achieved by including two or more different regulators of BMP-mediated signaling. In one embodiment, multiple inhibitors of BMP signaling or multiple activators of BMP signaling can be included in organoid medium. In one embodiment, both activators and inhibitors of BMP signaling can be included in organoid medium. In any embodiment that includes BMP signaling inhibitors or BMP signaling activators or a combination of the two, the specific BMP pathway that is regulated may be an important consideration for the formation of luminal organoids.

[0077] In one embodiment, the organoid culture medium for forming / maintaining luminal organoids contains both ERBB1 and ERBB4 ligands and does not contain exogenously added WNT signaling agonists and / or inhibitors of BMP signaling (or mimetics of either of the above).

[0078] In one embodiment, organoid medium comprises the inhibitor of BMP signal transduction, but does not comprise the exogenously added WNT signal transduction agonist.In one embodiment, organoid medium comprises the inhibitor of WNT signal transduction, but does not comprise the exogenously added BMP signal transduction agonist.

[0079] Biasing or promoting the formation of luminal organoids can mean that by culturing breast epithelial cells in organoid medium, the organoids that are composed of more luminal cells than non-luminal cells are enriched (e.g., compared to forming organoids from breast epithelial cells isolated in a medium different from organoid medium).In one embodiment, the luminal organoids formed in organoid medium comprise more than 70% luminal cells and about 20% or less basal cells.In one embodiment, the luminal organoids formed in organoid medium comprise more than 80% luminal cells and about 15% or less basal cells.In one embodiment, the luminal organoids formed in organoid medium comprise more than about 90% luminal cells and about 5% or less basal cells.

[0080] On the other hand, if it is desired to convert the formation of luminal organoid (i.e., the first group of organoids) into, for example, mixed lineage organoid (i.e., the second group of organoids), modified organoid medium can be used for this purpose.In one embodiment, modified organoid medium can comprise one or more of mitogens, agonists of WNT signaling, or regulators of BMP signaling, or two or more of them.

[0081] In one embodiment, the modified organoid medium (for forming and maintaining mixed lineage organoids) comprises one or more ligands for one or more of ERBB1, ERBB2, ERBB3, or ERBB4.

[0082] In one embodiment, the ligand of ERBB1 may be epidermal growth factor (EGF), transforming growth factor alpha (TGFα), amphiregulin, heparin-binding EGF (HB-EGF), betacellulin, epigen, or epiregulin. In some embodiments, the modified organoid medium may contain two or more ERBB1 ligands.

[0083] In one embodiment, the ERBB1 ligand is EGF and / or TGFα and / or amphiregulin. In such embodiments, the concentration of such ERBB1 ligand(s) may be about 1 μg / mL to 0.1 ng / mL, or about 500 ng / mL to 0.5 ng / mL, or about 250 ng / mL to 1 ng / mL, or about 125 ng / mL to 2 ng / mL, or about 50 ng / mL to 3 ng / mL.

[0084] In one embodiment, the ligand for ERBB3 included in the modified organoid medium can be one or more of Neuregulin 1, Neuregulin 2, or Neuroglycan C. In some embodiments, modified organoid medium (for forming and maintaining mixed lineage organoids) can include two or more ERBB3 ligands.

[0085] In one embodiment, the ligand of ERBB3 is neuregulin 1. In such embodiments, the concentration of neuregulin 1 is about 1 μg / mL to 0.1 ng / mL, or about 500 ng / mL to 0.5 ng / mL, or about 250 ng / mL to 1 ng / mL, or about 125 ng / mL to 2 ng / mL, or about 50 ng / mL to 3 ng / mL.

[0086] In one embodiment, the WNT signaling agonist contained in modified organoid medium is R-spondin, WNT protein, or any of the above engineered mimics.By including WNT signaling agonist in modified organoid medium, alone or in combination with other factors, it can suppress the formation / maintenance of luminal organoid, and the culture of breast epithelial cells can be biased toward mixed lineage organoid (for example, the second group of organoid).

[0087] In one embodiment, the R-spondin contained in the modified organoid medium is one or more of R-spondin1, R-spondin2, R-spondin3, or R-spondin4. In one embodiment, the R-spondin contained in the modified organoid medium is R-spondin1, R-spondin2, R-spondin3, or R-spondin4. Thus, the effective concentration of R-spondin in the modified organoid medium for forming and maintaining mixed lineage organoids is 1 μg / mL to 0.1 ng / mL, or about 500 ng / mL to 0.5 ng / mL, or about 250 ng / mL to 1 ng / mL, or about 125 ng / mL to 2 ng / mL, or about 50 ng / mL to 3 ng / mL.

[0088] In one embodiment, the WNT protein contained in the modified organoid medium is one or more of WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, or WNT16. Thus, the effective concentration of WNT protein in the modified organoid medium for forming and maintaining mixed lineage organoids is 1 μg / mL to 0.1 ng / mL, or about 500 ng / mL to 0.5 ng / mL, or about 250 ng / mL to 1 ng / mL, or about 125 ng / mL to 2 ng / mL, or about 50 ng / mL to 3 ng / mL.

[0089] Because Wnt3a may be unstable in cell culture medium or used under cell culture conditions, in some embodiments, Wnt3a conditioned medium can be a suitable substitute for Wnt3a in the modified organoid medium of the present disclosure.WNT conditioned medium can be produced using known approaches, such as by culturing WNT-producing cells.The concentration of WNT in WNT conditioned medium is typically equivalent to the concentration outlined above.

[0090] The progress of synthetic biology allows the in silico design of ligand mimics.The advantages of such mimics include but are not limited to: increased activity, increased stability, simplified structure, reduced size, etc.Therefore, in some embodiments, mimics are present in the organoid medium of the present disclosure through WNT or R-spondin function.

[0091] For example, typical recombinant WNT proteins have little or no effect when added to culture media, likely due to their high hydrophobicity and rapid degradation. To address this challenge, a standard FZD-mediated WNT pathway agonist was engineered to be more potent than wild-type Wnt3a while retaining acceptable biological activity when added to culture media (U-Protein Express).

[0092] In one embodiment, a WNT mimic is included in the modified organoid medium for promoting the formation / maintenance of mixed lineage organoids while suppressing the formation / maintenance of luminal organoids. In one embodiment, a WNT mimic can be included in such medium alone or together with one or more other agonists of WNT signaling. In an embodiment, when a WNT mimic is included in such medium, the WNT mimic can be a WNT surrogate-FC fusion protein. When a WNT mimic is included in the modified organoid medium for promoting the formation / maintenance of basal or mixed organoids while suppressing the formation / maintenance of luminal organoids, the concentration of the WNT mimic can be about 100nM to 0nM, about 50nM to 0.01nM, about 20nM to 0.05nM, or about 10nM to 0.1nM.

[0093] In one embodiment, the BMP signal transduction regulator contained in the modified organoid medium for promoting the formation / maintenance of mixed lineage organoid while suppressing the formation / maintenance of luminal organoid is protein, peptide or small molecule.In one embodiment, such regulator can be the activator or inhibitor of BMP signal transduction.

[0094] In an embodiment in which the modified organoid medium contains an activator of BMP signaling, examples of such activators include BMP2 or BMP4. Thus, the effective concentration of the activator of BMP signaling in the modified organoid medium can be about 10 μg / mL to 0.1 ng / mL, or about 1 μg / mL to 1 ng / mL, or about 300 ng / mL to 3 ng / mL, or about 150 ng / mL to 5 ng / mL, or about 100 ng / mL to 10 ng / mL, or about 50 ng / mL to 10 ng / mL.

[0095] In embodiments in which the modified organoid medium includes an inhibitor of BMP signaling, examples of such protein (or peptide-based) inhibitors include noggin, chordin, follistatin, sclerostin, CTGF / CCN2, gremlin, cerberus, DAN, PRDC, decorin, alpha-2 macroglobulin protein, etc. Thus, the effective concentration of the inhibitor of BMP signaling in the modified organoid medium can be about 10 μg / mL to 0.1 ng / mL, or about 1 μg / mL to 1 ng / mL, or about 300 ng / mL to 3 ng / mL, or about 150 ng / mL to 5 ng / mL, or about 100 ng / mL to 10 ng / mL, or about 50 ng / mL to 10 ng / mL.

[0096] In an embodiment in which the modified organoid medium contains an inhibitor of BMP signaling, examples of such small molecule inhibitors include LDN193189 or Dorsomorphin. Such inhibitors of BMP signaling contained in the modified organoid medium can be about 1 mM to 0.001 nM, about 1 μM to 0.1 nM, or about 100 nM to 1 nM.

[0097] In one embodiment, the organoid medium may contain inhibitors of BMP signaling, either protein / peptide based or small molecules (or a combination of the two), and thus the modified organoid medium may contain activators or agonists of BMP signaling, or vice versa.

[0098] Thus, in one embodiment, a modified organoid culture medium for inhibiting the formation / maintenance of luminal organoids and promoting the formation / maintenance of mixed lineage organoids comprises two or more of at least one ERBB ligand, at least one agonist of WNT signaling, and at least one regulator of BMP signaling.

[0099] In another aspect of the present disclosure, a kit is provided that comprises components for forming breast organoid.In one embodiment, the kit that is used for forming mouse breast organoid is different from that used for forming human breast organoid.The above description of medium composition is incorporated into the following kit description.

[0100] The kit of the present disclosure includes a basal medium and at least one supplement added to the basal medium. In one embodiment, the kit of the present disclosure includes a basal medium and at least two supplements added to the basal medium. In one embodiment, the kit of the present disclosure includes a basal medium and three or more supplements added to the basal medium.

[0101] Any particular supplement may be combined with the basal medium when it is desired to form a particular type of breast organoid. For example, when it is desired to form a first population of organoids (e.g., luminal restricted organoids or luminal biased organoids), the first supplement added to the basal medium may include one or both of ERBB1 and ERBB4 ligands. In some embodiments, the first supplement also does not include one or both of exogenously added WNT signaling agonists and BMP signaling regulators. In such embodiments, the BMP signaling regulators may be activators or inhibitors of BMP signaling. In related embodiments, the first supplement includes one or both of ERBB1 and ERBB4 ligands and one or both of BMP signaling regulators, and does not include WNT signaling agonists. In related embodiments, the first supplement includes one or both of ERBB1 and ERBB4 ligands and WNT signaling agonists, and does not include BMP signaling regulators. In a related embodiment, the first supplement comprises one or both of a ligand for ERBB1 and a ligand for ERBB4, and does not comprise both a WNT signaling agonist and a modulator of BMP signaling. In one embodiment, the ligand for ERBB1 is not EGF. In one embodiment, neither the basal medium nor the first supplement comprises progesterone.

[0102] The kit may further comprise a second supplement, which is added to the basal medium, to convert the first group of organoids formed (e.g., luminal restricted or luminal biased organoid population) into a second group of organoids (e.g., mixed organoids). The basal medium supplemented with the second supplement (and optionally with the first supplement) is referred to herein as modified organoid medium. In an embodiment of the second supplement, it may comprise one or both of a WNT signaling agonist and a BMP signaling regulator. In such an embodiment, the regulator of BMP signaling may be an activator or inhibitor of BMP signaling. In a related embodiment, the BMP signaling regulator contained in the second supplement is an activator of BMP signaling, such as BMP2 or BMP4. In a related embodiment, the second supplement may further comprise a ligand of ERBB1 that is different from the ligand of ERBB1 contained in the first supplement. In one embodiment, the ligand of ERBB1 contained in the second supplement is EGF.

[0103] The kit may further comprise a third supplement added to the basal medium (the first supplement and the second supplement are either present or absent). The third supplement may comprise an inhibitor of TGFβ signal transduction. In one embodiment, the third supplement is added to the basal medium (or the organoid medium or the modified organoid medium) after the breast organoid of interest is formed, to promote the nuclear localization of estrogen receptor. In one embodiment, the inhibitor of TGFβ signal transduction is SB431542. In one embodiment, the inhibitor of TGFβ signal transduction is RepSox.

[0104] Any of the above media (such as the embodiment included in the kit) can be used in the method of forming and / or maintaining breast organoids, and the type of breast organoids that can be formed and / or maintained depends on the composition of the media (e.g., organoid medium vs. modified organoid medium). Since breast epithelial cells and the organoids that result therefrom are sex hormone responsive, in some embodiments, it may be desirable for the media (e.g., basal medium, organoid medium, and modified organoid medium) of the present disclosure and the supplements added thereto to not contain exogenously added sex hormones, even when used in the method or incorporated in the kit. In one embodiment, the sex hormone is estrogen. In one embodiment, the sex hormone is progesterone.

[0105] method In another aspect of the present disclosure, a method is provided for forming or growing breast organoid.In one embodiment, the method of the present disclosure maintains and / or grows breast organoid over multiple passages.The breast organoid formed / grown / passaged according to the disclosed method can be used for downstream assays, such as to better understand the biology of mammary gland in both normal and diseased conditions, drug screening and toxicity testing, or therapeutic applications.

[0106] The method of forming breast organoid, and in particular the first group of organoids, from isolated breast epithelial cells comprises contacting breast epithelial cells with organoid medium that does not contain one or both of exogenously added WNT signaling agonist and / or BMP signaling regulator.In such an embodiment, the BMP signaling regulator can be the inhibitor of BMP signaling or the activator of BMP signaling.

[0107] In one embodiment, the method uses the organoid culture medium that contains the regulator of BMP signal transduction (for example, the inhibitor or activator of BMP signal transduction) but does not contain WNT signal transduction agonist.In one embodiment, the method uses the organoid culture medium that contains the agonist of WNT signal transduction but does not contain the regulator of BMP signal transduction (for example, the inhibitor or activator of BMP signal transduction).In one embodiment, the method uses the organoid culture medium that does not contain the regulator of BMP signal transduction (for example, the inhibitor or activator of BMP signal transduction) or the WNT signal transduction agonist.

[0108] In one embodiment, the mammary epithelial cells comprise mammary epithelial stem or progenitor cells.

[0109] The isolated mammary epithelial cells can be of any mammalian species. In one embodiment, the mammary epithelial cells are either human mammary epithelial cells or mouse mammary epithelial cells. The isolated mammary epithelial cells can be obtained from commercial suppliers, academic collaborators, or by processing mammary tissue. When starting from mammary tissue, such specimens are usually processed using a combination of mechanical / physical and enzymatic means.

[0110] As described herein, single cell suspensions of breast epithelial cells or suspensions of fragments or clumps of breast epithelial cells are typically generated by cutting breast tissue using a scalpel, tissue dissociator, or the like. After or during the cutting operation, the breast tissue is typically incubated in an enzyme-containing solution to degrade the extracellular matrix and connective tissue. The enzyme solution can include any enzyme or combination of enzymes useful for the purpose of processing breast tissue. By way of non-limiting example, enzymes that may be used for this purpose include collagenase, hyaluronidase, dipase, thermolysin, trypsin, DNase, and the like.

[0111] In one embodiment, the enzymatic treatment of the minced breast tissue is applied sequentially. For example, the minced breast tissue may first be incubated in a solution containing one or both of collagenase and hyaluronidase. Larger fragments of the digested breast tissue may be collected by centrifugation at relatively low centrifugal forces (e.g., <200xg) and set aside. The resulting supernatant may be further centrifuged at progressively higher centrifugal forces (e.g., ≧200xg) to isolate smaller material, including clumps of cells or single cells. Both pellets may be combined and further digested in a trypsin-containing solution to obtain a substantially single-cell suspension.

[0112] In some embodiments, the suspension of substantially single cells or fragments, or a mixture of single cells and fragments, may require DNase treatment to degrade DNA from the lysed cells and reduce the viscosity of the cell suspension.

[0113] After generating a suspension of mammary epithelial cells, particularly if single cells are present therein, it may be desirable to separate specific subsets of mammary epithelial cells from the various cell type states in the sample. Specific subsets of mammary epithelial cells may be isolated from the sample using known methods such as fluorescence activated cell sorting or immunomagnetic cell separation, as commercially available from STEMCELL Technologies.

[0114] The cellular composition of the mammary gland includes both epithelial and non-epithelial cells (fibroblasts, endothelial cells, lymphocytes, adipocytes, neurons and muscle cells). The expression of markers between the corresponding cell types may differ between humans and mice. In humans, epithelial cells can be distinguished from non-epithelial cells by the expression of EpCAM. +Within the epithelial compartment, breast epithelium contains two major lineages: the luminal lineage and the myoepithelial (or basal) lineage. Myoepithelial cells are typically characterized by i) high expression of cytokeratin 5 (K5), cytokeratin 14 (K14), P63, smooth muscle actin (SMA), CD49f and CD29; and ii) the absence of cytokeratin 8 (K8), cytokeratin (K18) and CD24. On the other hand, the luminal lineage is characterized by two subpopulations: mammary progenitor cells and hormone-responsive ER cells. + The ER can be divided into + The lineages can be distinguished by i) high expression of K8, K18, estrogen receptor alpha (ERα), progesterone receptor (PR), and ii) absence of K5, K14, CD49f, and SMA. The milk lineages can be distinguished by i) positive ALDEFLUOR staining, indicating ALDH1A3 expression, ii) expression of K8, K18, and CD49f, iii) expression of K5 and K14, and iv) absence of ERα and SMA. In mice, epithelial cells can be distinguished from non-epithelial cells by expression of EpCAM or CD24. EpCAM + Within the epithelial compartment, breast epithelium contains two major lineages: the luminal lineage and the myoepithelial (or basal) lineage. Myoepithelial cells are typically characterized by i) high expression of cytokeratin 5 (K5), cytokeratin 14 (K14), P63, smooth muscle actin (SMA), CD49f, and CD29; and ii) the absence of cytokeratin 8 (K8), cytokeratin (K18). On the other hand, the luminal lineage is characterized by two subpopulations: mammary progenitor cells and hormone-responsive ER cells. + The ER can be divided into +The lineages can be distinguished by i) high expression of K8, K18, estrogen receptor alpha (ERα), progesterone receptor (PR), and ii) absence of K5, K14, CD49f, CD49b, and SMA. The milk lineages can be distinguished by i) positive ALDEFLUOR staining, indicating expression of ALDH1A3; ii) expression of K8, K18, CD49b, and CD49f; iii) low expression of K5, K14, ERα, and SMA. In some cases, the presence or absence of either or both SMA and ER+ can help further distinguish basal and luminal cells.

[0115] In some embodiments, the isolated breast epithelial cells are cultured with an extracellular matrix. In one embodiment, the isolated breast epithelial cells are seeded into a "dome" of extracellular matrix. In one embodiment, the isolated breast epithelial cells are seeded into a "sandwich" of extracellular matrix, where the cells are seeded on a layer of extracellular matrix and then covered with a covering layer of the same or a different extracellular matrix. In one embodiment, the isolated breast epithelial cells are seeded on top of a layer of extracellular matrix.

[0116] Extracellular matrices for culturing breast epithelial cells are known, and any suitable extracellular matrix is ​​contemplated in the present disclosure. In one embodiment, the extracellular matrix is ​​Matrigel™ (Corning). In one embodiment, the extracellular matrix may be a suitable alternative to Matrigel, such as Cultrex® basement membrane matrix (Trevigen).

[0117] In one embodiment, the extracellular matrix may be one natural component of the extracellular matrix or any combination thereof. Examples of natural components of the extracellular matrix include collagen, laminin, entactin, heparin sulfate, proteoglycan, or fibronectin.

[0118] In one embodiment, the extracellular matrix may be a synthetic matrix, such as a hydrogel formulated with any one or more of the natural extracellular components outlined above.

[0119] The density of breast epithelial cells seeded on a layer of extracellular matrix or within a particular volume of extracellular matrix is ​​determined empirically. In embodiments where breast epithelial cells are seeded within a "dome" of extracellular matrix at the bottom of a 24-well plate, any number of cells from about 1,000 to 50,000 can be included within a 30-50 μL dome. If a different plate format is used, the number of cells and the volume of extracellular matrix may need to be adjusted. Nevertheless, it is understood that up to a clonal density of one cell or cells can be seeded on or in the extracellular matrix.

[0120] As described herein above, the nature of organoid medium will depend on the intended output of the method disclosed herein.Therefore, the above description of organoid medium is incorporated into the following disclosure of the method of forming mammary organoid.

[0121] Where the desired output of the method is a first population of organoids (e.g., luminal organoids), the organoid medium may comprise a basal medium and one or both of a ligand for ERBB1 and / or a ligand for ERBB4 (and in addition, does not comprise an exogenously added WNT signaling agonist and / or one or both of a modulator (e.g., an inhibitor or activator) of BMP signaling).

[0122] Potential ligands of ERBB1 and ERBB4 that can be included in organoid culture medium are described herein above. In one embodiment, the ligand of ERBB1 is amphiregulin. In one embodiment, the ligand of ERBB4 is neuregulin 1. In one embodiment of the organoid culture medium for promoting the formation and / or maintenance of luminal organoid, the culture medium comprises both the ligand of ERBB1 and the ligand of ERBB4. In one embodiment, the ligand of ERBB1 is not EGF or TGF alpha. In one embodiment, the ligand of ERBB1 is not EGF or TGF alpha.

[0123] Also, as mentioned above, when formulating organoid medium for promoting the formation and / or maintenance of luminal organoid, an important consideration is to avoid activating or inhibiting specific signal transduction cascade.Therefore, in one embodiment of such organoid medium, this medium does not contain exogenously added WNT signal transduction agonist and / or BMP signal transduction regulator (for example, its inhibitor or activator).

[0124] In one embodiment, the agonist of WNT signaling not included in the organoid medium (to promote the formation and / or maintenance of luminal organoids) is R-spondin, WNT protein or peptide, or any of the above engineered / synthetic mimetics.

[0125] In one embodiment, the inhibitor of BMP signaling that is not included in the organoid culture medium (for promoting the formation and / or maintenance of luminal organoid) is a protein inhibitor, for example, noggin, or a small molecule inhibitor such as LDN193189 or dorsomorphin.In one embodiment, neither the protein inhibitor of BMP signaling nor the small molecule inhibitor of BMP signaling is included in the organoid culture medium (for promoting the formation and / or maintenance of luminal organoid).

[0126] Therefore, the method further comprises culturing breast epithelial cells in organoid medium for a sufficient time to form a first group of organoids enriched in organoids that are composed of more luminal cells than non-luminal cells.In other words, breast epithelial cells can be cultured in organoid medium for a sufficient time to form a first group of organoids, where the organoids formed using a medium different from organoid medium do not produce any organoids at all or are composed of less luminal cells (and more non-luminal cells) compared to when the organoid medium of the present disclosure is used.

[0127] In one embodiment, luminal organoids can be formed about 3 days after contact with organoid medium. In one embodiment, luminal organoids can be formed about 5 days after contact with organoid medium. In one embodiment, luminal organoids can be formed about 7 days after contact with organoid medium. In one embodiment, luminal organoids can be formed about 10 days after contact with organoid medium. In one embodiment, luminal organoids can be formed about 14 days after contact with organoid medium. In one embodiment, the formed luminal organoids can be cultured for up to 30 days or more before they need to be subcultured.

[0128] In one embodiment, the luminal organoids (i.e., the first population of organoids) formed upon contacting mammary epithelial cells with organoid medium may be passaged 3 or more times, or 5 or more times, or 7 or more times, or 10 or more times.

[0129] If the output of the method is a population of breast organoids enriched in mixed lineage organoids (e.g., a second population of organoids), then modified organoid medium (as described above) may be used to contact and culture the breast epithelial cells or the first population or organoids. Thus, such modified organoid medium can convert the formation of the first population of organoids into the second population of organoids.

[0130] The examples of ERBB ligand, agonist of WNT signaling and regulator (activator or inhibitor) of BMP signaling are as specified above.In one embodiment, the modified organoid medium (for suppressing the formation of luminal organoid and promoting the formation / passage of mixed lineage organoid) comprises at least one ERBB ligand; at least one agonist of WNT signaling; and at least one regulator of BMP signaling.

[0131] In one embodiment, the first group of organoids (e.g., luminal organoids) formed by using the organoid medium of the present disclosure or by carrying out the method of the present disclosure can be converted into a second group of organoids (e.g., mixed lineage organoids).The first group of organoids can be converted into the second group of organoids by using the modified organoid medium of the present disclosure.

[0132] In one embodiment, the mixed lineage organoids (i.e., the second population of organoids) formed by contacting the first population of mammary epithelial cells or organoids with the modified organoid medium may be passaged 3 or more times, or 5 or more times, or 7 or more times, or 10 or more times.

[0133] In one embodiment, the method may further comprise contacting the first group of organoids or the second group of organoids with an inhibitor of TGFβ signaling. The inhibitor of TGFβ signaling can be any inhibitor, since inhibitors of TGFβ signaling are known and commercially available. In one embodiment, the inhibitor of TGFβ signaling is SB431542. In one embodiment, the inhibitor of TGFβ signaling is A77-01. In one embodiment, the inhibitor of TGFβ signaling is A83-01. In one embodiment, the inhibitor of TGFβ signaling is RepSox. By contacting the first group of organoids and / or the second group of organoids with an inhibitor of TGFβ signaling, the nuclear localization of estrogen receptor can be promoted. In one embodiment, the contacting the first group of organoids and / or the second group of organoids with an inhibitor of TGFβ signaling is during the culture step (in organoid medium or modified organoid medium). In one embodiment, contacting the first population of organoids and / or the second population of organoids with an inhibitor of TGFβ signaling is performed temporarily, for example, after formation of the first population of organoids and / or the second population of organoids.

[0134] Regardless of the type of breast organoids formed by carrying out the methods disclosed herein, these organoids may be used in any number of downstream assays, methods, or applications. As an example, breast organoids, whether derived from normal or diseased breast epithelial cells, may be used to screen a panel of compounds to determine their efficacy and / or toxicity. As another example, such breast organoids may be used to investigate basic breast biology or the biology of diseased breast epithelium, such as cancer. As another example, such breast organoids may be used to investigate therapeutic interventions that are grafted into subjects, either human patients or animal models.

[0135] The following non-limiting examples illustrate the present disclosure. EXAMPLES

[0136] Example 1: Processing of breast tissue samples All cells used in this disclosure were obtained from tissues derived from human or mouse subjects in accordance with applicable IRB and ethical requirements.

[0137] Fragments of human breast epithelial cells were isolated from tissue samples obtained from academic collaborators as follows: Excised tissue samples were minced in a petri dish using a scalpel to cut in a cross-hatch pattern. The minced tissue samples were placed in a dissociation flask in standard culture medium supplemented with BSA, insulin, collagenase and hyaluronidase. The flask was gently shaken overnight on an orbital shaker in a 37°C tissue incubator. The following morning, the floating layer of fat was removed by pipette and the remaining liquid was transferred to a tube and briefly centrifuged at low rpm (approximately 80g for 30 seconds). The pellet (pellet A) was set aside and the supernatant was centrifuged further for progressively longer times and at progressively higher rpm (approximately 200g for 4 minutes). The pellet (pellet B) was set aside and the supernatant was centrifuged further for progressively longer times and at progressively higher rpm (approximately 450g for 5 minutes).

[0138] Cryopreserved pellets A and B were dissociated into single cell suspensions by treatment with 0.25% trypsin-EDTA for 5 min at 37° C. (with intermittent trituration) and then centrifuged at 300 g for 5 min. The supernatant was discarded and the pellets were resuspended in 100 μg / ml DNase solution at room temperature for 1 min. The resulting cells were passed through a 37 μm strainer to obtain a single cell suspension.

[0139] A single cell suspension of mouse mammary epithelial cells was isolated from mammary glands as follows: Excised mammary glands were minced and placed in tubes containing standard medium containing 50 μg / ml gentamicin, collagenase and hyaluronidase. Mammary glands were incubated in tissue culture at 37° C. for 2 hours. After incubation, the cell dissociation mixture was washed with advanced DMEM and centrifuged at 300 g for 5 minutes. Depending on the appearance of the pellet, in some cases it is advisable to resuspend the pellet in ammonium chloride to lyse contaminating red blood cells. The pellet is then resuspended in 0.25% trypsin-EDTA as above for 5 minutes at 37° C. and then centrifuged (300 g for 5 minutes). The supernatant was discarded and the pellet was resuspended in 100 ug / ml DNase solution at room temperature for 1 minute. The resulting cells were passed through a 37 μm strainer to obtain a single cell suspension.

[0140] Example 2: Seeding and culturing single-cell suspensions of mammary epithelial cells Single cell suspensions obtained as described in Example 1 were reconstituted in 100% Matrigel™ (Corning) and seeded as domes into the wells of a microplate, at approximately 1-2×10 4 of human cells or approximately 5x10 3 Approximately 25-40 μL of mouse cells were seeded into each dome. Each well contained 0.5-1 mL of one of the various culture media described herein.

[0141] Plated single cell suspensions of mammary epithelial cells were formed into organoids that were passaged approximately every 7 days, with complete medium changes 2-3 times during such 7-day period. To passage the organoids, the Matrigel domes were disrupted by pipetting with pre-warmed 0.25% trypsin-EDTA, incubating at 37°C for approximately 5-15 minutes, and pipetting again. The trypsinization reaction was inactivated by washing with an equal volume of Hanks Balanced Salt Solution supplemented with 2% FBS. Depending on the density and size of the organoids, a 1:2-1:5 split was performed and the cells were seeded as domes as above. Alternatively, a single cell suspension was obtained by straining the cells through a 37 μm strainer and seeded into Matrigel domes as above.

[0142] Example 3: Staining of breast organoids Organoids were retrieved from the Matrigel by incubating in Corning cell recovery solution for 1 hour with rocking on ice. Organoids were then fixed in 4% paraformaldehyde (PFA) for 1 hour at room temperature and stored in PBS at 4°C. After PFA fixation, antigen retrieval was performed by boiling the organoids in sodium citrate buffer at 96°C for 20 minutes. Organoids were permeabilized in a 0.5% Triton-X-100 solution in PBS overnight at room temperature. Samples were blocked by incubating overnight in a solution of 5% goat serum.

[0143] After fixation and permeabilization, organoids were sequentially incubated in primary and secondary antibody solutions (summarized in the table below) overnight at room temperature. Samples were counterstained in 2ug / ml DAPI solution for 20 minutes and dehydrated by stepwise incubation in solutions of increasing methanol concentrations (50%, 80%, then 100% methanol). Organoids were embedded in a 2:1 solution of benzyl benzoate-benzyl alcohol (BABB), transferred to Ibidi glass-bottom chamber slides, and imaged using a Leica SP8 confocal laser scanning microscope. Organoids were washed between each step with immunofluorescence (IF) buffer consisting of PBS with 1% BSA, 0.1% cold fish skin gelatin, 0.2% Triton-X-100, and 0.05% Tween 20. [Table 1]

[0144] Example 4: Formation of mouse mammary branched organoids Single cell suspensions of mouse mammary epithelial cells were obtained according to Example 1 and plated and cultured in medium containing a WNT signaling agonist (e.g., RSPO1), an inhibitor of BMP signaling (e.g., Noggin), and EGF essentially as described in Example 2 (Figure 1).

[0145] In three consecutive passages, mouse mammary organoids formed in the above medium consistently reproduce and maintain organoids characterized by high levels of branching, which is characteristic of organoids that have a significant number of basal cell types and other non-luminal cell types (Figure 1A, 1B, and 1C).After the second passage, the formed organoids are imaged using confocal microscope as described in Example 3, and strong K14 expression is observed in highly branched organoids (Figure 1D).

[0146] Example 5: Formation of mouse luminal-biased organoids in the organoid medium of the present disclosure Single cell suspensions of mouse mammary epithelial cells were obtained according to Example 1. ER cell differentiation was assessed based on differential expression of EpCAM, CD49f and CD49b using a BD FACSAria™ Fusion flow cytometer. + Single cell suspensions were sorted into three cell populations corresponding to epithelial, mammary and basal cell lineages. Non-epithelial cells were further excluded during a sorting strategy based on the expression of CD45, Ter119 and CD31. Sorted cells were seeded and cultured in organoid medium (e.g., MammoCult™ Mouse Organoid Growth Medium, STEMCELL Technologies) without exogenously added inhibitors of BMP signaling, with exogenously added RSPO-1 (about 100 ng / mL) or without exogenously added RSPO-1 essentially as described in Example 2 (Figure 2).

[0147] ER in mouse mammary epithelial cells + Both lines formed luminal organoids with evident cystic tissue with roughly the same efficiency in both media formulations tested (Figures 2A and 2D). In contrast, milk lineages of mouse mammary epithelial cells were biased toward cystic tissue in RSPO-1-deficient culture media compared to a more mixed tissue of basal, luminal, and other cells when cultured in RSPO-1-containing media (Figures 2B and 2E). Similar to the milk lineages, basal lineage cells appeared to be biased toward cystic tissue in RSPO-1-deficient media compared to a primarily mixed tissue when cultured in RSPO-1-containing media, although a notable number of mixed organoids remained (Figures 2C and 2F).

[0148] The observation that each of the three sorted lineages of mouse mammary epithelial cells could be biased towards cystic tissue (i.e., luminal organoids) based on media formulation prompted experiments investigating the effect of different media formulations on bulk (i.e., unsorted) mammary epithelial cells.

[0149] Organoid media were formulated to lack either exogenously added RSPO-1 or RSPO-3, and they were tested on single cell suspensions of bulk mammary epithelial cells obtained according to Example 1, seeded and cultured as described in Example 2. In media containing both RSPO-1 and RSPO-3, a significant number of mixed organoids were obtained, whereas luminal-biased (or cystic) organoids were observed in organoid media lacking exogenously added RSPO (or other Wnt agonists) (Figure 3A), as in Figure 2. Flow cytometric analysis of the cellular composition of organoids formed in either RSPO-containing or RSPO-free media confirmed that the number of basal cells was reduced among organoids formed in the absence of WNT agonist action (Figure 3B).

[0150] In follow-up experiments, organoids were formed from single cell suspensions obtained according to Example 1, seeded and cultured as described in Example 2. Organoids were cultured in the presence or absence of RSPO-1 for three consecutive passages, and at each passage, organoids formed in the absence of RSPO-1 consistently reproduced and maintained the unbranched cystic organization characteristic of luminal cell-restricted organoids (Figure 4; P0, P1, and P2). At the end of the second passage, the formed organoids were processed and stained for K8, K14, and DAPI as described in Example 3. Images obtained using confocal microscopy showed strong K8 expression among organoids formed in the absence of WNT agonism and a clear lack of K14 staining, which was in contrast to the confocal microscopy images of organoids formed in the presence of WNT agonism (Figure 4).

[0151] The above experiment was carried out in a medium containing an inhibitor of BMP signaling to examine the effect of BMP signaling inhibition on the formation of mouse mammary organoids. Organoids were formed from single cell suspensions obtained according to Example 1, and seeded and cultured as described in Example 2 in medium with or without exogenously added inhibitor of BMP signaling (0 ng / mL or ~100 ng / mL) (but with WNT signaling agonist RSPO-1). Bright field and confocal microscopy images taken over successive passages in the above medium formulations revealed a significant number of branched organoids containing a significant number of basal cells, regardless of the medium formulation used (Figure 5).

[0152] Next, the effect of the presence or absence of BMP signaling inhibition in the context of organoid medium with or without exogenously added WNT signaling agonist was examined.Organoids were formed from single cell suspension obtained according to Example 1, and seeded and cultured as described in Example 2.Based on eight independent experiments using organoid medium with about 100ng / mL Noggin and about 100ng / mL RSPO-1 (Figure 6A) and Figure 6C)) or without exogenously added Noggin and RSPO-1 (Figure 6B) and Figure 6D))), in the medium formulation without BMP signaling inhibitor and WNT signaling agonist, a significant decrease in basal cells was observed with an increase in luminal cells (Figure 6E).

[0153] Based on the aforementioned data, it appears that the absence of WNT signaling agonists in the organoid medium has a significant impact on the lineage balance among mammary organoids (e.g., increase in luminal cells), and the presence or absence of inhibitors of BMP signaling is not necessarily required for the formation of luminally biased organoids.

[0154] Example 6: Formation of branched human mammary organoids Single cell suspensions of human mammary epithelial cells were obtained according to Example 1 and plated and cultured in medium containing RSPO-1, Noggin and EGF substantially as described in Example 2 (FIG. 7).

[0155] At day 26, human breast organoids formed in the aforementioned medium consistently regenerated and maintained organoids characterized by high levels of branching, a hallmark of organoids containing numerous basal cells (Figures 7A, 7B, and 7C). Confocal microscopy was used to image the formed organoids, and strong K14 expression was observed in the highly branched organoids (Figure 7C). Basal cells at the periphery of the lobes can be seen stacking on top of each other. Instead of cohesive branching, basal cells in a single collection advance away from the organoid, resulting in "degradative organoid branching." The absence of gland-forming units is reminiscent of classical invasive lobular carcinoma.

[0156] Example 7: Formation of human luminal-biased organoids in the organoid medium of the present disclosure Single cell suspensions of human mammary epithelial cells were obtained according to Example 1. Single cell suspensions of cells were seeded and cultured in organoid medium (e.g., MammoCult™ Human Organoid Growth Media, STEMCELL Technologies) to test the effects of different activators and inhibitors of BMP signaling, essentially as described in Example 2 (Figure 8).

[0157] Organoids were formed in medium containing a BMP signaling activator, either BMP2 at about 50 ng / mL or BMP4 at about 50 ng / mL, and a BMP signaling inhibitor, either Noggin at about 25-200 ng / mL or LDN193189 at about 10-100 nM. Just before the first passage, the formed organoids were dissociated and the balance of cell lineages was analyzed by flow cytometry. Activation of signaling through the BMP pathway resulted in a marked increase in basal cells and a decrease in luminal cells compared to organoids formed in medium containing an inhibitor of BMP signaling (Figure 8A). Follow-up experiments with two different donor samples confirmed that inhibiting signaling through BMPs reduced the number of basal cells, while increasing the number of luminal cells compared to organoid medium without BMP regulation (Figure 8B).

[0158] The effect of modulating BMP-mediated signaling was analyzed in multiple experiments with respect to the total cell number among the organoids formed (Figure 9A) and the lineage balance of cells among the organoids formed (Figure 9B). Organoids formed under the aforementioned conditions were analyzed for total cell number, and it was observed that activation of BMP-mediated signaling by either BMP2 or BMP4 resulted in a significant net loss of total cells compared to conditions in which BMP-mediated signaling was not modulated or was inhibited via either Noggin or LDN193189 (Figure 9A). Similar to the results observed in Figure 8, the effect of activation and inhibition of BMP signaling on the balance of cell lineages was confirmed (Figure 9B). K8 + The relative increase in luminal cells was confirmed by confocal microscopy for organoids formed in organoid medium containing inhibitors of BMP signaling (Figure 9C). Images taken by confocal microscopy also showed that K14 in organoids formed in organoid medium containing inhibitors of BMP signaling was significantly increased in the luminal cells compared with the control group (Figure 9C). + Little, if any, staining of the cells was found.

[0159] Finally, we investigated the potential additive effect of dual inhibition of BMP-mediated signaling (Figure 10). Organoids were formed in medium containing both Noggin and LDN193189, or in medium containing either Noggin or LDN193189. Examination of the cultures by brightfield microscopy revealed that organoid medium containing one inhibitor of BMP signaling appears sufficient to bias the formation of luminal organoids from mammary epithelial cells (Figure 10).

[0160] Overall, the absence of one or more inhibitors of BMP signaling in the organoid medium would generate organoids composed of a significant number of luminal cells, whereas the addition of one or more inhibitors of BMP signaling in the organoid medium would increase the number of luminal cells and decrease the number of basal cells. In contrast, activation of BMP-mediated signaling would negatively affect the lineage balance of organoids if the formation of luminal-restricted or luminal-biased mammary organoids is desired.

[0161] Next, considering the important role of modulation of BMP signaling on the lineage balance of human breast organoids, the potential impact of modulation of WNT signaling on breast organoid formation was examined. Single-cell suspensions of human breast epithelial cells were obtained according to Example 1. Single-cell suspensions of cells were seeded and cultured in organoid medium essentially as described in Example 2, and the effects of different activators (about 0.1-1 nM WNT surrogate or about 20-200 ng / mL RSPO-1) and WNT signaling inhibitors (about 50 ng / mL DKK1 or about 200 nM NSC) were tested (Figure 11).

[0162] Luminal-promoting organoid media (e.g., MammoCult™ Human Organoid Growth Medium, STEMCELL Technologies) were formulated to be supplemented with or without exogenously added RSPO-1. Brightfield images of formed organoids did not show a significant increase or decrease in luminal-biased organoids between the two formulations tested (Figure 11A). This observation was confirmed after analyzing cells from organoids formed in either condition by flow cytometry (Figure 11B). Aggregation of flow cytometry data from multiple organoid formation experiments similarly suggested that modulation of WNT signaling had little effect on the lineage balance of cells in the analyzed organoids (Figure 11C). The percentages of luminal and basal cells appeared to be approximately equivalent regardless of whether the organoid media contained a WNT signaling agonist, a WNT signaling antagonist, or no modulation of WNT signaling. Thus, agonism and / or antagonism of WNT signaling does not appear to be critical for the formation of luminal-restricted or luminal-biased human breast organoids from mammary epithelial cells.

[0163] Since the regulation of WNT signaling is not considered to be important for the formation of luminal-restricted or luminal-biased human breast organoids, and the inhibition of BMP signaling is considered to tilt cell lineage balance in favor of luminal cells, next, the effect of regulating signaling through ERBB family members on the formation of human breast organoids is examined.According to Example 1, the single-cell suspension of human breast epithelial cells is obtained.Single-cell suspension of cells is seeded in organoid medium (e.g., MammoCult™ Human Organoid Growth Medium, STEMCELL Technologies), cultured, and tested the ligand of different ERBB receptor family members (Figure 12), as described in Example 2.

[0164] Organoids were formed in medium containing no ligands for ERBB receptor family members or about 10-50 ng / mL amphiregulin (AREG), about 10-50 ng / mL epidermal growth factor (EGF), or about 50-100 ng / mL heregulin / neuregulin 1 (NRG1). Just before the first passage, the formed organoids were dissociated and the balance of cell lineages was analyzed by flow cytometry. AREG and NRG1 appeared to have little or no effect on the balance of cell lineages, whereas EGF appeared to significantly decrease the number of luminal cells while significantly increasing the number of basal cells among the formed organoids (Figure 12A). In follow-up experiments on two donor samples, the effect of inhibiting EGF receptor-mediated signaling was tested by treatment with approximately 100 nM gefitinib and approximately 100 nM erlotinib, which appeared to have negligible or no effect on cell lineage balance (Figure 12B).

[0165] The effect of modulating signaling through ERBB receptor family members was analyzed in multiple experiments with respect to total cell number among the organoids formed (Figure 13A) and lineage balance of cells among the organoids formed (Figure 13B). Organoids formed in the aforementioned conditions were analyzed for total cell number, and it was observed that the inclusion of an activator of signaling through ERBB family members resulted in a net increase in the total cell number of the formed breast organoids compared to the control condition that did not include modulation of ERBB signaling (Figure 13A). The highest mitogenic activity was observed when either EGF or AREG was included in the organoid medium. Similar to the results observed in Figure 12, the effect on lineage balance of cells was confirmed for the various ERBB receptor family ligands tested (Figure 13B). EGF resulted in the greatest decrease in luminal cells and the greatest increase in basal cells, while AREG appears to have the least effect on the proportion of luminal cells but a large effect on the increase in the proportion of basal cells. In contrast, the inclusion of NRG1 in the organoid medium appeared to result in a higher proportion of luminal cells and a decrease in the proportion of basal cells.

[0166] In the last series of experiments, the effect of adjusting TGFβ-mediated signal transduction on human breast organoid formation is examined.The single cell suspension of human breast epithelial cells is obtained according to Example 1.The single cell suspension of cells is seeded and cultured in organoid medium (e.g., MammoCult™ Human Organoid Growth Medium, STEMCELL Technologies) substantially as described in Example 2, and the effect of the ligand of TGFβ receptor or the inhibitor of TGFβ-mediated signal transduction is tested (Figure 14).

[0167] Organoids were formed in medium containing no ligand for the TGFβ receptor or about 50ng / mL of TGFβ1 (or TGFβ3, data not shown, but results consistent with TGFβ1). Just before the first passage, the formed organoids were dissociated and the balance of cell lineages was analyzed by flow cytometry. The presence of TGFβ1 in the organoid medium appeared to have an overall negative effect on the proportion of luminal and basal cells, while at the same time significantly increasing the proportion of stromal cells among the formed breast organoids (Figure 14A).

[0168] Given that the inclusion of TGFβ1 in the organoid medium has a negative impact on the formation of luminal-restricted or luminal-biased human breast organoids, we investigated the potential role of inhibition of signaling through the TGFβ receptor. Inclusion of either SB431542 at approximately 10 μM, A77-01 at approximately 200–500 nM, or RepSox at approximately 5–25 μM in the organoid medium appeared to decrease the proportion of stromal cells while maintaining or improving the proportion of luminal cells (Figure 14B). Based on these results, inclusion of an inhibitor of TGFβ signaling, such as RepSox, in the organoid medium could increase the proportion of luminal cells compared to organoid medium that does not contain a regulator of signaling through the TGFβ receptor. However, a potentially more interesting finding suggested that transient exposure to an inhibitor of TGFβ signaling (after organoids were formed as described above) promoted localization of estrogen receptors in the nucleus rather than in the cytoplasm (Figure 15). These results were confirmed for both SB43154 and RepSox in both human (Figure 15A) and mouse (Figure 15B) mammary organoids.

[0169] In follow-up experiments, organoid medium was formulated to lack both exogenously added RSPO-1 and Noggin. This medium was tested on single cell suspensions obtained according to Example 1, seeded and cultured as described in Example 2. On day 10 of the first passage, organoids formed without exposure to exogenously added RSPO-1 and Noggin showed cystic morphology and lack of branching, which is characteristic of luminal organoids (Figure 16A). Confocal microscopy of mammary organoids formed under these culture conditions confirmed the presence of luminal cell marker expression (K8) and the apparent absence of basal cell marker expression (K5) (Figure 16B, Figure 16C and Figure 16D).

[0170] Example 8: Additional factors that promote the formation of mammary luminal organoids Other candidate factors are tested in organoid medium to examine their effect on the bias of the formation of breast organoids that are restricted to specific lineages of breast epithelial cells.A single cell suspension of human breast epithelial cells is obtained according to Example 1.A single cell suspension of cells is seeded and cultured in organoid medium (e.g., MammoCult™ Human Organoid Growth Medium, STEMCELL Technologies) supplemented with either 50ng / ml neuregulin (NRG3), 100ng / ml anti-Mullerian hormone (AMH) or 50ng / ml granulocyte-macrophage colony-stimulating factor (GM-CSF) substantially as described in Example 2.

[0171] Day 17 organoids were stained for either K14 or K8 and imaged by confocal microscopy (Figure 17). The inclusion of either NRG3 or AMH in organoid medium appeared to bias the culture of mammary epithelial cells to form luminal restricted organoids (Figure 17A and Figure 17B), while the inclusion of GM-CSF in organoid medium appeared to bias the culture of mammary epithelial cells to form basal restricted organoids (Figure 17C).

[0172] While the present disclosure has been described with reference to what are presently considered to be the preferred embodiments, it is to be understood that the present disclosure is not limited to the disclosed embodiments, but on the contrary, the present disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0173] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims

1. 1. A method for forming mammary organoids from isolated mammary epithelial cells, the method comprising: a) contacting said mammary epithelial cells with organoid medium that does not contain an exogenously added WNT signaling agonist and / or an inhibitor of BMP signaling; b) culturing the mammary epithelial cells in the organoid medium for a time sufficient to form a first population of organoids enriched for organoids comprised of more luminal cells than non-luminal cells; Here, the organoid culture medium comprises a basal medium and one or both of an ERBB1 ligand and / or an ERBB4 ligand.

2. 2. The method of claim 1, wherein the organoid medium comprises an inhibitor of the BMP signaling but does not comprise the WNT signaling agonist.

3. 2. The method of claim 1, wherein the organoid medium comprises the WNT signaling agonist but does not comprise an inhibitor of the BMP signaling.

4. The method of any one of claims 1 to 3, wherein the WNT signaling agonist is R-spondin, a WNT protein, or an engineered mimetic of any of the foregoing.

5. The method of claim 1 , wherein the inhibitor of BMP signaling is a protein or a small molecule.

6. 6. The method of claim 5, wherein the inhibitor of BMP signaling is one or more of noggin, chordin, follistatin, LDN193189 or dorsomorphin.

7. The method of claim 1 , wherein the ligand of ERBB1 is not EGF or TGF alpha.

8. The method of claim 1 , wherein the ERBB1 ligand is amphiregulin.

9. The method of claim 1 , wherein the ligand for ERBB4 is also a ligand for a different ERBB receptor family member.

10. The method of claim 1 , wherein the ERBB4 ligand is neuregulin 1 and / or neuregulin 3.

11. 13. The method of claim 1, wherein said organoid medium does not contain exogenously added sex hormones.

12. 12. The method of claim 11, wherein the sex hormone is progesterone.

13. The method of claim 1 , wherein the non-luminal cells are one or more of basement cells, stromal cells, hematopoietic cells, and endothelial cells.

14. 2. The method of claim 1, wherein the organoid is composed of 50% or more luminal cells.

15. 2. The method of claim 1, further comprising culturing the first population of organoids in a modified organoid medium to convert the first population of organoids into a second population of organoids.

16. 16. The method of claim 15, wherein the modified organoid medium is supplemented with EGF.

17. The method of claim 15 or 16, wherein the modified organoid medium is supplemented with a WNT signaling agonist and / or an inhibitor of BMP signaling.

18. The method of claim 15, wherein when one or both of the WNT signaling agonist and / or the BMP signaling inhibitor are not added to the organoid culture medium, the second population of organoids is composed of more basement cells and fewer luminal cells.

19. 2. The method of claim 1, wherein the first population of organoids can be passaged in the organoid medium five or more times.

20. 16. The method of claim 15, wherein the second population of organoids can be passaged in the modified organoid medium five or more times.

21. 2. The method of claim 1, further comprising contacting the first population of organoids with an inhibitor of TGF-beta.

22. 22. The method of claim 21, further comprising obtaining nuclear localization of the ER.

23. A mammary organoid culture medium comprising a basal medium and one or both of a ligand for ERBB1 and a ligand for ERBB4, the medium lacking an exogenously added WNT signaling agonist and one or both of an inhibitor of BMP signaling.

24. 24. The medium of claim 23, wherein said organoid medium comprises an inhibitor of said BMP signaling but does not comprise said WNT signaling agonist.

25. 24. The medium of claim 23, wherein said organoid medium comprises said WNT signaling agonist but does not comprise said inhibitor of BMP signaling.

26. 24. The medium of claim 23, wherein the WNT signaling agonist is R-spondin, a WNT protein, or an engineered mimetic of any of the foregoing.

27. The medium of claim 23 , wherein the inhibitor of BMP signaling is a protein or a small molecule.

28. 28. The medium of claim 27, wherein the inhibitor of BMP signaling is one or more of noggin, chordin, follistatin, LDN193189 or dorsomorphin.

29. The medium of claim 23 , wherein the ligand for ERBB1 is not EGF or TGF alpha.

30. 24. The method of claim 23, wherein the ligand of ERBB1 is amphiregulin.

31. The medium of claim 23 , wherein the ligand for ERBB4 is also a ligand for a different ERBB receptor family member.

32. The medium of claim 23 , wherein the ligand for ERBB4 is neuregulin 1 or neuregulin 3.

33. 24. The medium of claim 23, wherein said organoid medium does not contain exogenously added sex hormones.

34. 34. The medium of claim 33, wherein the sex hormone is progesterone.

35. 24. The medium of claim 23, wherein culturing isolated mammary epithelial cells in said organoid medium enriches for organoids comprised of more luminal than non-luminal cells.

36. A kit for forming mammary organoids from isolated mammary epithelial cells, the kit comprising: A basal medium; a first supplement added to the basal medium, the first supplement comprising one or both of a ligand for ERBB1 and / or a ligand for ERBB4, and not comprising an exogenously added one or both of an agonist of WNT signaling and / or an inhibitor of BMP signaling; Including the kit.

37. The kit of claim 36, further comprising a second supplement added to the basal medium or the basal medium supplemented with the first supplement, the second supplement comprising a second ligand of ERBB1 different from the ligand of ERBB1 in the first supplement, and one or both of an exogenously added WNT signaling agonist and / or an inhibitor of the BMP signaling.

38. 38. The kit of claim 36 or 37, further comprising a third supplement added to the basal medium or to the basal medium supplemented with the first supplement and / or the second supplement, wherein the third supplement comprises a TGFβ signaling inhibitor.

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