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US-20260126428-A1 - METHODS AND SYSTEMS TO SIMULTANEOUSLY MONITOR CANCER CELL VIABILITY AND COLLECTIVE MIGRATION IN THREE DIMENSIONS

US20260126428A1US 20260126428 A1US20260126428 A1US 20260126428A1US-20260126428-A1

Abstract

A rapid and economical human microphysiological system (“spheroid-on-a-chip”) to investigate therapies targeting collective cancer cell migration in a 3D collagen extracellular matrix is described. The 3D system can be used to identify compounds that inhibit spheroid collective migration without inducing cell death and can accelerate the development of new drugs that target metastasis.

Inventors

  • Christopher P. Miller
  • Megan Fung
  • Scott S. TYKODI
  • Edus H. WARREN
  • Shreeram Akilesh

Assignees

  • FRED HUTCHINSON CANCER CENTER
  • UNIVERSITY OF WASHINGTON

Dates

Publication Date
20260507
Application Date
20251104

Claims (20)

  1. 1 - 71 . (canceled)
  2. 72 . A method of screening a test compound for anti-metastatic properties, the method comprising: adding a tumor spheroid-collagen solution to a central matrix channel of a microfluidic culture chip; polymerizing biomimetic collagen within the tumor spheroid-collagen solution to form a biomimetic collagen matrix; adding a first amount of a test compound media to an upper injection port of the microfluidic culture chip; adding a second amount of the test compound media to a lower injection port of the microfluidic culture chip wherein the second amount is less than the first amount; staining the microfluidic culture chip with a fluorescence compound, wherein the staining creates labeled tumor spheroids; and performing Z-stack confocal imaging using a laser scanning microscope to form Z-stacks and to assess anti-gravitational migration of the labeled tumor spheroids within the collagen matrix thereby screening the test compound for anti-metastatic properties.
  3. 73 . The method of claim 72 , further comprising adding tumor spheroids into a collagen solution to form the tumor spheroid-collagen solution.
  4. 74 . The method of claim 72 , wherein the tumor spheroids are A498 spheroids or HEPG2 spheroids.
  5. 75 . The method of claim 72 , wherein the tumor spheroids have 100-500 cells/spheroid.
  6. 76 . The method of claim 72 , comprising adding 1 mg/mL or 2.5 mg/mL tumor spheroid-collagen solution to the microfluidic culture chip.
  7. 77 . The method of claim 72 , wherein polymerizing collagen within the tumor spheroid-collagen solution is for 20-40 minutes at 37° C.
  8. 78 . The method of claim 72 , comprising adding 50-100 μL of the test compound media in the upper injection port and 25-75 μL of the test compound media in the lower injection port.
  9. 79 . The method of claim 72 , wherein the test compound is a putative anti-migratory compound.
  10. 80 . The method of claim 72 , further comprising creating a Montage gallery of each z-stack position of labeled tumor spheroids.
  11. 81 . The method of claim 72 , wherein the Z-stacks are obtained from a bottom of the collagen matrix that is marked by fluorescence of one or more posts separating the central matrix channel and parallel media channels.
  12. 82 . The method of claim 72 , wherein the z-stacks span a 4-8 μm step size.
  13. 83 . The method of claim 72 , wherein an unbiased script-based automated system quantifies at least one of a cell invasion, a cell migration, or a cell death.
  14. 84 . A system for screening a compound for anti-metastatic properties against renal cell carcinoma (RCC), the system comprising: a microfluidic culture chip, wherein the microfluidic culture chip comprises at least 2 sites per chip, each site containing injection ports for a central matrix channel and parallel media channels flanking the sites, and posts with gaps in between on each side of the matrix channel separating the central matrix channel and parallel media channels; a biomimetic collagen-RCC tumor spheroid matrix in the central matrix channel of the microfluidic culture chip, wherein collagen within the collagen-RCC tumor spheroid matrix is polymerized and comprises 4-20 tumor spheroids/μL; and an upper injection port and a lower injection port, each comprising a test compound media with a pressure gradient between the upper injection port and the lower injection port.
  15. 85 . The system of claim 84 , wherein tumor spheroids within the collagen-RCC tumor spheroid matrix are A498 spheroids.
  16. 86 . The system of claim 84 , wherein tumor spheroids within the collagen-RCC tumor spheroid matrix have 100-500 cells/spheroid.
  17. 87 . The system of claim 84 , wherein collagen within the collagen-RCC tumor spheroid matrix was polymerized at 37° C. for 20-40 minutes.
  18. 88 . The system of claim 84 , comprising 50-100 μL of the test compound media in the upper injection port and 25-75 μL of the test compound media in the lower injection port.
  19. 89 . The system of claim 84 , wherein a test compound within the test compound media is a putative anti-migratory compound.
  20. 90 . The system of claim 84 , wherein the bottom of the collagen-RCC tumor spheroid matrix is marked by fluorescence of one or more posts separating the central matrix channel and parallel media channels.

Description

CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to U.S. Provisional Patent Application No. 63/716,090 filed on Nov. 4, 2024 the entire contents of which are incorporated by reference herein. STATEMENT OF GOVERNMENT SUPPORT This invention was made with government support under W81XWH-19-1-0789 awarded by the Defense Health Agency, Medical Research and Development Branch. The government has certain rights in the invention. FIELD OF THE DISCLOSURE The present disclosure provides methods and systems to simultaneously monitor cancer cell viability and migration in three dimensions (3D). The methods and systems can use microfluidic tissue culture-chip assays to monitor 3D collective cell migration and cell death. The disclosed platform can be used to simultaneously evaluate tumor spheroid cell death and collective migration and has utility for evaluating anti-migratory compounds. BACKGROUND OF THE DISCLOSURE Cancer is the second leading cause of death in the US (Siegel et al., Cancer J Clin. 2023; 73(1):17-48) and new therapies are urgently needed. Unfortunately, the failure rate for the development of new cancer treatments is estimated to be >96% (Printz, Cancer. 2015; 121(10):1529-30). A key limitation to drug development is inadequate preclinical tumor models. The 2-dimensional (2D) growth of cancer cells in dishes does not faithfully recapitulate human tumor biology in vivo, while human xenograft and genetically engineered mouse models are expensive, time-consuming, and also fail to accurately mimic immune interactions relevant to human tumors. While immunocompetent transgenic mouse models with rapidly progressing, metastatic cancer suitable for therapeutic evaluation are available for some cancer types such as breast and prostate, they are lacking for others such as renal cell carcinoma (RCC), the most common type of kidney cancer (van der Min et al., Sci Rep. 2023; 13(1):8246). The search for more robust in vitro tumor models has stimulated interest in economical human microphysiological systems to better predict efficacy and toxicity (Ewart et al., Commun Med (Lond). 2022; 2(1):154). A key component of these systems is the ability to culture tumor cells as spheroids in a 3-dimensional (3D) extracellular matrix (ECM) which better reflects the properties of tumors in the tumor microenvironment (TME) in vivo. Advantages of 3D culture of spheroids include the ability to reproducibly define their composition and the density, stiffness, and porosity of the ECM. These systems can recapitulate in vivo tumor architecture, cell cycle heterogeneity, tumor cell-to-cell and cell-to-ECM interactions, ECM mechanical forces, central hypoxia, nutrient-, pH-, and soluble factor-gradients, gene expression, and drug resistance (Manduca et al., Front Immunol. 2023; 14:1175503; Tan et al., Adv Drug Deliv Rev. 2021; 176:113852). Importantly, 3D tumor-on-a-chip models allow for isolation and study of the impact of individual variables. In turn, this enables a stepwise increase in complexity of the model system including incorporation of the tumor vasculature, stromal and immune cells, and modulation of flow-based perfusion and soluble factor gradients. SUMMARY OF THE DISCLOSURE The present disclosure provides methods and systems to simultaneously monitor cancer cell viability and migration in three dimensions (3D). The methods and systems can use microfluidic tissue culture-chip assays to monitor 3D collective cell migration and cell death. The disclosed platform can be used to simultaneously evaluate tumor spheroid cell death and collective migration and has utility for evaluating anti-migratory compounds. Spheroids are normally observed collecting at the bottom of a 3D space due to gravitational forces. The methods and systems described herein unexpectedly observed an anti-gravitational migration (i.e., away from the directional pull of gravity) of the spheroids towards the top of the 3D space. With this unexpected anti-gravitational migration, the methods and systems herein can be used to test compounds for their anti-metastatic properties (i.e., compounds with anti-metastatic properties prevent collective anti-gravitational migration of the spheroids). BRIEF DESCRIPTION OF THE FIGURES The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. Applicants consider the color versions of the drawings as part of the original submission and reserve the right to present color images of the drawings in later proceedings. FIGS. 1A-1C. 3D microfluidic culture system for renal cell carcinoma (RCC) spheroids. Schematic of the idenTx 3 chip containing 3 sites per chip, each site containing injection ports for the central matrix channel and parallel media channels flanking the sites (1A, upper panel). Schematic of the structure of the posts w