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EP-4736152-A1 - METHODS OF PRODUCING A TUMOR AVATAR

EP4736152A1EP 4736152 A1EP4736152 A1EP 4736152A1EP-4736152-A1

Abstract

Three-dimensional tumor avatars comprising an artificial scaffold, tumor cells, tumor associated endothelial cells and tumor associated fibroblast cells are provided. Kits comprising the 3D tumor avatars, methods of producing the 3D tumor avatars and methods of using the 3D tumor avatars are also provided.

Inventors

  • ROZIC, Gabriela
  • KOMAN, Igor

Assignees

  • Ariel Scientific Innovations Ltd

Dates

Publication Date
20260506
Application Date
20240627

Claims (20)

  1. 1. A three-dimensional (3D) tumor avatar comprising: an artificial scaffold, tumor cells, and tumor microenvironmental (TME) cells comprising tumor associated endothelial cells (TECs) and cancer associated fibroblasts (CAFs).
  2. 2. The 3D tumor avatar of claim 1, wherein said TME cells are derived from a tumor tissue or a surrounding tissue thereof.
  3. 3. The 3D tumor avatar of claim 1 or 2, wherein said TME cells and said tumor cells belong to a single histologic type of said tumor.
  4. 4. The 3D tumor avatar of claim 3, wherein said histologic type is carcinoma or sarcoma.
  5. 5. The 3D tumor avatar of any one of claims 1 to 4, wherein said TME cells and said tumor cells belong to a single primary or metastatic site of said tumor.
  6. 6. The 3D tumor avatar of claim 5, wherein said primary site of said tumor is selected from the group consisting of: gastrointestinal cancer, kidney cancer, liver cancer, breast cancer, bladder cancer, prostate cancer, ovary cancer, uterine cancer, head and neck cancer, thyroid cancer, glioma, neuroblastoma, melanoma, and lung cancer.
  7. 7. The 3D tumor avatar of any one of claims 1 to 6, wherein said 3D tumor avatar is a patient-personalized tumor avatar, and wherein said TECs and said CAFs are present in said patient-personalized tumor avatar at a ratio found in a sample obtained or derived from said patient.
  8. 8. The 3D tumor avatar of any one of claims 1 to 7, wherein said TECs and said CAFs are present in said 3D tumor avatar culture at a ratio ranging from 1 : 10 to 1:1.
  9. 9. The 3D tumor avatar of any one of claims 1 to 8, wherein the ratio between tumor cells and CAFs in said 3D tumor avatar is between 1:10 and 1:1, the ratio between tumor cells and TECs in said 3D tumor avatar is between 1:10 and 1:1, or both.
  10. 10. The 3D tumor avatar of any one of claims 1 to 9, wherein said artificial scaffold comprises Matrigel, vitronectin, fibronectin, or any combination thereof.
  11. 11. The 3D tumor avatar of claim 10, wherein said artificial scaffold is a Matrigel dome.
  12. 12. The 3D tumor avatar of claim 11, wherein said Matrigel dome comprises a Matrigel concentration of at least 20%.
  13. 13. The 3D tumor avatar of any one of claims 10 to 12, wherein said artificial scaffold comprises a concentration gradient of Matrigel, fibronectin, vitronectin or any combination thereof, and wherein said gradient of Matrigel is from 1-100% concentration, said gradient of fibronectin is from 0-50 ng/ul and said gradient of vitronectin is from 0-50 ng/ul.
  14. 14. The 3D tumor avatar of any one of claims 1 to 13, wherein said CAFs comprise inflammatory CAFs (iCAFs) and extracellular matrix-remodeling CAFs (eCAFs).
  15. 15. The 3D tumor avatar of any one of claims 1 to 14, further comprising tumor associated mesenchymal cells (TAMCs).
  16. 16. The 3D tumor avatar of claim 15, wherein said TAMCs are tumor associated mesenchymal stem cells (TA-MSCs).
  17. 17. The 3D tumor avatar of any one of claims 1 to 16, further comprising peripheral blood lymphocytes (PBLs).
  18. 18. The 3D tumor avatar of any one of claims 1 to 17, further comprising tumor associated adipocytes (TAAs), tumor associated immune cells, or both.
  19. 19. The 3D tumor avatar of any one of claims 1 to 18 wherein said tumor cells are primary cells from a subject and said TME cells are also primary cells from said subject.
  20. 20. The 3D tumor avatar of any one of claims 1 to 19, wherein said tumor cells are primary cells from a subject, said CAFs are primary cells from said subject and said TECs are from a TEC cell line.

Description

METHODS OF PRODUCING A TUMOR AVATAR CROSS REFERENCE TO RELATED APPLICATIONS [001] This application claims the benefit of priority of Israeli Patent Application No. 304102, filed June 27th, 2023, the contents of which are all incorporated herein by reference in their entirety. FIELD OF INVENTION [002] The present invention is in the field of cancer diagnostics. BACKGROUND OF THE INVENTION [003] The tumor microenvironment (TME) constitutes a permissive milieu for the induction, selection, and expansion of cancer cells and has been shown to play diverse and critical roles in tumor proliferation, angiogenesis, metastasis, medication resistance and immune evasion. The TME consists of different types of cells which have altered gene expression in cancer, including tumor associated stromal cells (TASCs), tumor endothelial cells (TECs), and immune cells, as well as extracellular matrix components. TASCs include cancer-associated fibroblasts (CAFs), tumor associated mesenchymal stem cells (TA- MSCs), tumor associated adipocytes (TAAs), and myofibroblasts. CAFs differ from normal fibroblasts in biological characteristics and gene expression profile. TECs can acquire cytogenetic abnormalities compared with normal endothelial cells (NECs) from adjacent normal tissue and may be able to acquire resistance to drugs. TAAs exhibit tumor-associated metabolic and transcriptomic phenotype. Tumor associated immune cell population correlate with better chances for survival. TA-MSCs can change their functional and phenotype following recruitment and interaction with cancer cells. The high level of TME inter- and intra- tumor heterogeneity make it an important variable in precision oncology decisions, such as cancer drug screening. [004] Organoids, composed of tumor epithelial cells, are 3D in vitro culture systems that have long term genetic stability in culture, simulate the epithelial architecture of the tumor of origin and represent epithelial tumor cells heterogeneity. However, a culture system that mainly includes the cancer epithelial cells, cannot completely recapitulate the TME due to the absence of CAFs, TECs, immune cells, and non-cellular components. [005] A tumor “avatar”, or “mini-tumor” refers to an in-vitro model composed of tumor cells, TME cells and extracellular matrix components. The incorporation of fibroblasts and endothelial cells in a two-dimensional (2D) or a 3D mini-tumor models, have been previously described (e.g., Teixeira FC et al. “Engineering a Vascularized 3D Hybrid System to Model Tumor-Stroma Interactions in Breast Cancer.” Front Bioeng Biotechnol. 2021; 9:647031, hereby incorporated by reference in its entity). Nonetheless, these cells were not derived from a TME niche, thus did not exhibit the phenotypic and the molecular features that specifically characterize the TME cells, and therefore could not accurately mimic the cross-talk between TME cells and tumor cells. [006] There is a great need for new methods of developing three- dimensional (3D) in vitro models of cancer, that would include TME cells, in addition to cancer cells, for mimicking the TME-tumor interaction. An in vitro model that recapitulates the intra- and inter- phenotypic and/or molecular differences between patients, not only of the tumor cells but also of the tumor niche, would allow to augment the sensitivity of cancer drugs screening, to serve as a research model for better understanding of cancer disease mechanisms, as well as to facilitate highly precise personalized onco-medicine. SUMMARY OF THE INVENTION [007] The present invention provides three-dimensional mini-tumors comprising an artificial scaffold, tumor cells, tumor associated endothelial cells and tumor associated stromal cells. Kits comprising the 3D mini-tumors, methods of producing the 3D minitumors and methods of using the 3D mini-tumors are also provided. [008] According to a first aspect, there is provided a three-dimensional (3D) tumor avatar comprising: an artificial scaffold, tumor cells, and tumor microenvironmental (TME) cells comprising tumor associated endothelial cells (TECs) and cancer associated fibroblasts (CAFs). [009] According to some embodiments, the TME cells are derived from a tumor tissue or a surrounding tissue thereof. [010] According to some embodiments, the TME cells and the tumor cells belong to a single histologic type of the tumor. [Oi l] According to some embodiments, the histologic type is carcinoma or sarcoma. [012] According to some embodiments, the tumor cells are tumor epithelial or tumor cells in epithelial-mesenchymal transition state or tumor mesenchymal cells. [013] According to some embodiments, the TME cells and the tumor cells belong to a single primary or metastatic site of the tumor. [014] According to some embodiments, the primary site of the tumor is selected from the group consisting of: gastrointestinal cancer, kidney cancer, liver cancer, breast cancer, bladder cancer, prostate cancer, ovary cancer, uterine