Cancer cells growing in three-dimensional (3D) space encounter unique cell-to-cell interactions, mechanical constraints, and variable access to nutrients, oxygen, and other soluble factors that can regulate essential processes of tumor cell survival, metastasis, and drug resistance. To develop new models that better represent these tumor features without the use of animals, researchers have engineered small scale tissue or organ mimics. “Tissues- and organs-on-chips are engineered 3D microphysiological systems containing human cell units designed to replicate the structure and function of complex tissues and organs,” explained Dr. Chris Miller, a staff scientist at UW and Fred Hutchinson Cancer Center. “They are called chips because the manufacture of these microfluidic devices, which is often with silicon-based materials, resembles the microfabrication of computer chips.” In Dr. Shreeram Akilesh’s lab at UW, Dr. Miller and colleagues engineered a “renal cell carcinoma (RCC)-on-a-chip, a human vascularized 3D system to study renal tumor angiogenesis.” This system can be used to learn more about this complex kidney cancer, an incurable disease for most once it becomes metastatic. Currently, the field lacks effective targeted therapies of these solid tumors and treatments that limit RCC metastasis. Drs. Miller and Akilesh became interested in using the RCC-on-a-chip system to investigate the mechanics of immunotherapy delivery to RCC solid tumors. Leading this endeavor, Dr. Miller “collaborated with Dr. Hootie Warren, a pioneer in T-cell immunotherapy who has a long-standing interest in kidney cancer along with Dr. Scott Tykodi, who leads the RCC clinical program, and Dr. Yuexin Xu, an RCC-focused computational immunologist.” This group of researchers developed an RCC-on-a-chip 2.0 system and insights from this adapted platform revealed that T cells and T cell-based immunotherapy cannot easily migrate through dense matrix representative of the solid tumor. However, intriguing observations of tumor migration and apoptosis could be measured in this system, highlighting key capabilities for its use in drug screening of anti-migratory and anti-cancer drugs for metastatic RCC. These findings were published recently in Neoplasia.
First, the researchers wanted to better understand gene expression differences between the standard 2D and structurally complex 3D RCC cell culture systems. To do this, the researchers analyzed changes in gene expression levels between the two culture conditions for two human RCC cell lines. The 3D spheroid RCC system had decreased enrichment of cell growth factors, but increased enrichment of genes involved in epithelial to mesenchymal transition as compared to the 2D cultured cells. These findings were supported by observations that 2D RCC cells continue to grow in culture while the 3D RCC spheroids had limited growth. Similarly, cytotoxic drugs were more effective in killing RCC spheroids than cells cultured in 2D. Lastly, the epithelial to mesenchymal transition is often correlated with tumor cell migration. Excitingly, the researchers were able to observe migrating spheroids in the RCC-on-a-chip platform from the bottom to the top of the chip. This phenotype was striking and sparked the idea of investigating migration of tumor cells and infiltration of T cells engineered to kill cancer cells.
A clear next step was to determine which anti-migration drugs were able to impede spheroid migration in the RCC-on-a-chip platform. Megan Fung, a bioengineering student and Mary Gates and Levinson Emerging Scholar, developed a robust method for unbiased quantification of cell death in RCC-on-a-chip which was performed alongside treatments with the anti-migration drugs to evaluate cell death and spheroid migration together. Intriguingly, several of these drugs (latrunculin, blebbistatin, and AT13148) restricted migration without killing the cells, demonstrating a separation between pathways that mediate RCC survival and migration. This discovery highlights the potential application of the RCC-on-a-chip platform to dissect the specific mechanisms driving RCC metastasis.
Following characterization of the chip system, the researchers turned to their main objective, the investigation of T cell infiltration into solid RCC tumors in collaboration with Dr. Carla Jaeger-Ruckstuhl. The researchers performed T cell migration assays in the RCC-on-a-chip platform in two settings of normal, low-density matrix or tumor-like, high-density extracellular matrix (ECM). “They found that while T cells robustly infiltrated into chips containing collagen with the stiffness found in normal healthy tissues, this migration was severely reduced in chips containing collagen with the stiffness found in tumors and limited tumor antigen-dependent killing of the spheroids,” stated Dr. Miller. “This system models a key challenge that therapeutic T cells are likely to encounter on their path to kill solid tumors with a dense ECM.” Miller continued to explain that significant efforts on engineering T cells to kill the cancer cells have been made as well as methods to overcome immunosuppression in the tumor microenvironments, but less is known about the physical barriers of T cell infiltration into RCC solid tumors.
Since the ECM surrounding the tumor provides physical resistance to T cell infiltration, it is possible that T cell-based immunotherapies may have reduced efficiency in mediating tumor cell death in the 3D system as compared to the 2D system. Chimeric antigen receptor (CAR) T cells designed to target and kill specific cancer cells could perform this duty in 2D at a 0.5:1 ratio of CAR T cell to tumor cell. To increase the odds of CAR T cell-based tumor cell killing, the researchers added the tumor spheroids, and the CAR T cells to the chip at the same time. Under these conditions with tumor-like matrix, 8X more CAR T cells were needed in the chip platform than the 2D system to perform equal tumor cell killing. Utilizing this platform of RCC-on-a-chip will provide insight into the physical barrier of dense tumor tissue and how to dismantle this barrier. “We are now adding a blood vessel barrier to the chips and have developed methods to isolate the T cells from the chips that can successfully infiltrate into stiff collagen,” shared Dr. Miller. “We plan to identify the properties of T cells that are competent for crossing tumor blood vessels and collagen infiltration to produce a therapeutic T-cell product with superior ability to infiltrate solid tumors.” The researchers hope that this RCC-on-a-chip will be relatively simple for others to use for continued study of RCC in 3D. These initial studies emphasize the importance of studying cancer systems in relevant 3D models to aid in the development of innovative cancer therapies.
The spotlighted research was funded by the Department of Defense Kidney Cancer Research Program, Mary Gates Research Scholarship, generous support of philanthropic donors to the Warren Lab, and the Consortium Cancer Center Support Grant.
Fred Hutch/University of Washington/Seattle Children's Cancer Consortium members Drs. Hootie Warren, Shreeram Akilesh, and Scott Tykodi contributed to this work.
Miller CP, Fung M, Jaeger-Ruckstuhl CA, Xu Y, Warren EH, Akilesh S, Tykodi SS. 2023. Therapeutic targeting of tumor spheroids in a 3D microphysiological renal cell carcinoma-on-a-chip system. Neoplasia. 46:100948. Online ahead of print.