(C-107) Mechanistic Assessment of the Synergistic Role of Stromal and Immune Cells on Cancer Invasion using a Tumor-On-Chip Model

Introduction:: Our understanding of cancer has evolved over the last decades, and treatments are more focused on targeting the stromal cells within the tumor microenvironment (TME). The TME is composed of transformed cancer cells and a rich diversity of non-cancerous cell types such as fibroblasts, macrophages, dendritic cells, and endothelial cells. Despite this understanding, disrupting the tumor-stromal interactions are not targeted mainstream, as the stromal cells in the TME are genetically stable and can induce both beneficial and adverse effect on tumorigenesis. Thus, ex vivo tumor models that can faithfully recapitulate these critical tumor-stroma interactions are required to mechanistically understand the multifaceted reaction that is occurring within the TME. In this study, we established an innovative and multi-cellular TME on-a-chip model and assessed the synergistic effect of patient-derived Cancer Associated Fibroblast (CAF’s) and macrophages (M𝟇) on breast cancer progression.


Materials and Methods:: The influence of stromal components on cancer progression was assessed using the microfluidic model that allows spatial organization of various cell types. Tri-culture invasion model was established using Sum159 breast cancer cell, patient derived CAFs and THP-1 derived naïve (M𝟇) macrophages. Sum159 mixed with collagen and Matrigel forms the tumor region (red), while CAF’s and M𝟇 embedded in collagen forms the stromal region (green) (Fig 1A). Functional and real time assessment such as migration, morphometric analysis and proliferation studies were conducted to understand the stromal influence on cancer cell. Gene expression analysis on array of macrophage activation genes was performed to further probe into the polarized state of the macrophages within the 3D model.


Results, Conclusions, and Discussions::
Results and Discussion:




Utilizing the TME on-a-chip model, we compared the invasive behavior of cancer cells across 4 different culture conditions namely monoculture (Sum only), co-culture (Sum+CAF, Sum+ M𝟇) and tri-culture (Sum+CAF+ M𝟇) (Fig 1B). We found a significant increase in migration distance of the cancer cells in the triculture condition as opposed to others (Fig 1C). In parallel, the influence of the stromal cells on proliferative potential of the cancer cells was evaluated. Cancer cells in the presence of CAF+ M𝟇 exhibited more positive expression of Ki-67 marker (Fig 1D). Hierarchical clustering of migration and morphological changes demonstrated that the co-culture conditions grouped together whereas CAF+ M𝟇 exerted highest influence on cell migration and clustered separately (Fig 1E). To couple our understanding of the bi-directional communication between tumor and stroma, we primarily performed qPCR assessment of polarized states of naïve macrophages. Relatively higher expression of M1 and M2 activation marker expression was observed in the tri-culture condition (Fig 1F). This finding confirms that tumor educated macrophages are skewed towards both M1, M2 phenotype and their reciprocal communication with tumor cells.



Conclusion:

The biological analysis using the TME on-a-chip confirmed the critical influence of microenvironmental components on tumor progression. Gene-expression analysis of macrophage activation state further confirmed the communication between tumor and non-cancerous stromal components are bi-directional. To further our understanding of the underlying multifaceted communication occurring within our platform, ongoing work is to assess the transcriptomic profiles of the 3D tissue using single cell RNA-Sequencing.




Acknowledgements (Optional): :
The authors would like to acknowledge (NSF) CBET Award #1914680.




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