Poster T3 - Exploring Heterogenous Cancer-Immune Cell Dynamics with Tumor-on-Chip Models

Introduction: Breast cancer is the most common cancer and second leading cause of cancer-related deaths in women(1). A particularly aggressive subtype of breast cancer is triple-negative breast cancer (TNBC), which lacks progesterone receptors, estrogen receptors, and HER2 expression. The absence of these receptors limits treatment options, adversely impacting patient outcomes. The challenge of TNBC is coupled with the issue of disease heterogeneity, which is seen on a variety of levels, including between patients, within tumors, and across space and time(2). This intricacy presents a clinical hurdle in determining effective treatments for TNBC. While considerable effort is being devoted to understanding breast cancer heterogeneity, many aspects of it remain poorly understood. One such aspect is macrophage diversity, a dominant immune cell type in the tumor microenvironment(3). In this study, we investigate the interactions between clonal breast cancer cell subpopulations and macrophages with diverse functional phenotypes, applying a reverse-engineering approach to mimic intratumor heterogeneity in TNBC.

Materials and

Methods: Cultures were generated using diverse 4T1 murine TNBC cell clones with endogenous fluorescent proteins and RAW264.7 murine macrophages with varying polarization states. 4T1 clonal cells were cultured in monoculture (4T1-E, 4T1-M), in co-culture, (50:50, 4T1-E:4T1-M), and tri-culture (25:25:50, 4T1-E:4T1-M:RAW264.7) Experiments were performed for cells in 2D culture and cells encapsulated in type I collagen hydrogels. Cultures were subjected to live cell imaging to monitor dynamic cell-cell interactions. We measured cell migration characteristics, spheroid morphology, and tunneling nanotubule formation. Furthermore, we determined changes in macrophage polarization ratios using immunofluorescence staining of CD86 and CD206 for M1 and M2 polarization states, respectively. We coupled our results with published RNA-sequencing data of 4T1 clonal cells and polarized RAW264.7 cells to determine potential pathways for therapeutics.

Results, Conclusions, and Discussions: Mixtures comprising 4T1 clonal cells with noninvasive, epithelial (E) and invasive, mesenchymal (M) phenotypes and/or RAW264.7 macrophages were cultured in varied microenvironments. We find that cells exhibited divergent behaviors in mono-, co-, and tri-cultures. In comparison to flattened cell morphologies observed in standard 2D culture, a subset of cells cultured on collagen coated substrates formed 3D clusters reminiscent of loosely adherent spheroids (Fig. 1A). While all conditions displayed modest spheroid formation, the largest spheroid areas were observed in 50:50 mixtures of E and M clones. Unexpectedly, the addition of macrophages to 50:50 mixtures of E:M clonal cell mixtures reduced spheroid sizes back to the baseline observed for either clone in monoculture (Fig. 1B). We performed live cell imaging to more closely examine how the presence of macrophages may reduce spheroid sizes. Preliminary results revealed the formation of dynamic heterotypic cell-cell bridges between TNBC clonal cells and macrophages, reminiscent of tunneling nanotubes (Fig. 1C). We hypothesize that these structures may directly enhance cell-substrate adhesion or do so indirectly through cell-material exchange that alters cell phenotype. Ongoing work aims to elucidate these complex cancer-immune cell interactions by tracking individual and collective cell behaviors, as well as generating co-cultures with varying ratios of pre-polarized macrophages with M1 and M2 states. Future experiments will extend these studies to an established tumor-on-a-chip model to investigate the effects of macrophage functional phenotypes on tumor growth, invasion, and escape(4). Overall, this research provides insight into how tumor-immune heterogeneity and dynamic, heterotypic cell-cell interactions impact breast cancer progression.
This work emphasizes the importance of evaluating intratumor heterogeneity as seen by changes in cancer spheroid formation in the presence of macrophages. This area of study will provide insight into the role of the epithelial-mesenchymal transition on tumor cell invasion and immune resistance, allowing for future improvement in treatment strategies to reduce recurrence and increase survival in breast cancer patients.

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