Abstract PO4-28-10: Single-Cell Transcriptomic Assessment of Stromal and Immune Crosstalk on Breast Cancer Invasion Using 3D Tumor-On-Chip Model
Authors: Ravi Kalpana , Sakala Lydia , Park Jin , LaBaer Joshua , Nikkhah Mehdi 
Published: May 2024

Abstract
Introduction: Our understanding of cancer has evolved over the last decades, with treatments increasingly targeting stromal cells in the tumor microenvironment (TME). The TME comprises transformed cancer cells and various non-cancerous cell types such as fibroblasts, macrophages, 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 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 cells, patient-derived CAFs, and THP-1 derived naïve (M𝟇) macrophages. Sum159 mixed with collagen and Matrigel forms the tumor region, while CAF’s and M𝟇 embedded in collagen form the stromal region. Functional and real-time assessments such as migration, morphometric analysis, and proliferation studies were conducted to understand the stromal influence on cancer cells. Gene expression analysis on an array of macrophage activation genes was performed to identify the polarized state of the macrophages after cellular interactions. Finally, Single Cell-RNA sequencing was carried out to unveil the drivers of stromal-immune crosstalk on invasion. Results and Discussion: Using the TME on-a-chip model, we compared cancer cell invasion across 4 different culture conditions namely monoculture (Sum only), co-culture (Sum+CAF, Sum+ M𝟇) and tri-culture (Sum+CAF+ M𝟇). Cancer cell migration was significantly increased in the triculture condition compared to others and stromal components also enhanced cancer cell proliferation. Hierarchical clustering of migration and morphological changes demonstrated that the co-culture conditions grouped together while CAF+ M𝟇 exerted the highest influence on cell migration and clustered separately. To understand the bi-directional communication between tumor and stroma, we assessed the polarized states of naïve macrophages using qPCR. Gene expression analysis showed that tumor-educated macrophages showed characteristics of M1 and M2 phenotypes and communicated reciprocally with tumor cells. Finally, to investigate the molecular mechanics of complex cellular interactions, we performed sc-RNA sequencing on cells extracted from the device and 2D cells (controls). Unsupervised clustering identified 11 different clusters, categorizing cells as Sum159 (tumor cells), CAFs (Stromal), and M𝟇 (Immune) based on cell-specific markers. Notably, we observed a noticeable shift in the clusters of Sum159 cells between 2D and 3D samples, emphasizing the difference between these two cultures. Conclusion: Using the TME on-a-chip, we confirmed the critical influence of microenvironmental components on tumor progression through functional assessment. Gene-expression analysis revealed bi-directional communication between noncancerous stromal components and tumor cells highlighting the importance of microenvironmental crosstalk. Furthermore, sc-RNA sequencing revealed the differences between 2D and 3D cultures. Ongoing work aims to perform transcriptomic and bioinformatic analysis on the sc-RNA sequencing data to gain a deeper understanding of the multifaceted communication within our platform.
Citation Format: Kalpana Ravi, Lydia Sakala, Jin Park, Joshua LaBaer, Mehdi Nikkhah. Single-Cell Transcriptomic Assessment of Stromal and Immune Crosstalk on Breast Cancer Invasion Using 3D Tumor-On-Chip Model [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-10.