(D-128) Engineered 3D Tumor Immune Microenvironment On-a-Chip to Study the Interaction between T cells and Macrophages in Breast Cancer

Introduction:: Breast cancer is the second leading cause of death in the US. Evolving knowledge about the tumor microenvironment (TME) is driving innovation in designing novel therapies against hard-to-treat breast cancer. The TME encompasses non-malignant cells, an immune microenvironment, a hypoxic and acidic niche along with mechanical and metabolic cues (Fig 1A). Targeting the immune components of TME has emerged as a promising approach for cancer therapy. Current immunotherapies have made progress in restoring anti-tumor immunity, but it is still a daunting task to discover effective cancer therapies to counteract tumor escape. Hence there is a pressing need to better understand the complex tumor-immune dynamics and crosstalk within the TME. To evaluate the cancer-immune interaction, this study aimed at investigating the crosstalk between naïve macrophages and cytotoxic T cells in driving tumor progression using an organotypic 3D ex vivo tumor on-a-chip model. The presented microfluidic platform consists of two distinct regions namely: The tumor region and the stroma region separated by trapezoidal microposts to ensure interconnectivity between regions thereby incorporating high spatial organization.


Materials and Methods:: The microfluidic platform had an organotypic architecture with distinct, yet integrated tumor and stromal entities. Triple Negative breast cancer cell line- SUM159 encapsulated in Collagen and Matrigel was loaded in the tumor region of the devices. THP1 monocytes were differentiated into naïve macrophages [M0] using Phorbol 12-myristate 13-acetate. TALL-104 T cells were used as the model for cytotoxic T cells. The immune cells were encapsulated in collagen and were loaded into the stroma region. (Fig 1B) represents the triculture platform. Four experimental conditions were tested. 1) Monoculture of SUM159 2) Coculture of SUM159+ M0 3) Coculture of SUM159+ T cells 4) Triculture of SUM159+ M0 + T cells. Upon the formation of the model, invasion, and proliferation assays along with morphometric analysis and flow cytometry were carried out to assess the cancer-immune interaction as well as to evaluate the fate of naïve macrophages.


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


The SUM159 cells migration into the stroma region was monitored for 72 hours where the media was changed every other day. It was found that in the presence of M0 macrophage, there was a significant increase in migration compared to the other conditions as seen in (Fig 1: C (i) &D (i)). The presence of T cells in triculture reduced the migratory behavior of cells. Also, it was observed that there was a significant upregulation in Ki67 expression in SUM159+M0 compared to SUM159, SUM159+T cells, and SUM159+ M0+T cells (Fig 1 D (ii)). No significant difference was observed across conditions for Live/Dead Assay. Further, the morphology of tumor cells was analyzed by staining the cells for F-Actin. (Fig 1C (ii) & D(iii)) represent the data obtained from the morphometric analysis. Cells were analyzed for shape descriptors such as circularity, aspect ratio, and protrusiveness. In the presence of M0 macrophages, cells were less circular with enhanced aspect ratio and protrusiveness compared to the other conditions. The trend was reversed in coculture with T cells where the cells were more rounded with a lesser aspect ratio and protrusiveness.




Conclusion:


In our triculture platform, we have successfully recapitulated the complex Tumor Immune Microenvironment by incorporating multiple immune cell populations within an appropriate 3D matrix. Our invasion and morphometric analyses yielded clear results that highlight the significant changes in the morphology and migratory behavior of tumor cells across different conditions. This study highlights the complexity of TME and underscores the critical role of immune cells in regulating cancer progression. The next stage of our research would be to evaluate the fate of naïve macrophages to better understand their behavior within the TME.


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








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