Q32 - Size-Dependent Cell-Cell Interactions Promote Survival of Cancer Cell Clusters in Stressful Microenvironments

Introduction: : Tumor size serves as a critical clinical indicator of prognosis in cancer patients [1], with larger tumors correlating with worse patient outcomes. Circulating tumor cell (CTC) clusters, which arise from the intravasation of motile tumor aggregates into the bloodstream, have been found to exhibit greater survival than individual circulating tumor cells [5, 6, 7]. When combined with other techniques, they have emerged as promising diagnostic and prognostic biomarkers in solid tumors [2, 3, 4]. While the enhanced survival and chemoresistance of CTC clusters have been well documented [8, 9], the mechanism of how clustering confers survival advantages to cancer cells from physiological stresses (starvation, hypoxia, shear stress, etc.) is still not clear. In this study, we demonstrate that large clusters are associated with improved cancer cell survival in harsh extracellular environments, which is in turn associated with the organization and intercellular interaction of cells within the cluster that scale with its size.

Materials and

Methods: : We precisely controlled cluster size by seeding 4T1 murine breast cancer cells onto micropatterned coverslips coated with fibronectin [10], featuring circular shapes ranging from 11.3 to 225 µm radii (~1 to 500 cells per cluster, respectively). To investigate the impact of cluster shapes on cell survival, cells were additionally seeded on other patterned shapes (triangles, squares, and pentagons) with the same area as the circular pattern. Once cells reached 100% confluency in the patterns, they were either cultured continuously in growth media containing 10% Fetal Bovine Serum (FBS) as a control or in starvation media with 0% FBS for an additional 24 hours. To detect early or late apoptosis, cells were then labeled with FITC annexin-V or propidium iodide, respectively, with a counterstain of Hoechst 33342 for cell nuclei. Labeled cells were fixed and imaged with a Zeiss LSM 900 Confocal Microscope. To quantify apoptosis, an “apoptotic ratio” was calculated by dividing the number of apoptotic cells by the total number of cells in a cluster. In addition to 2D micropatterned clusters, 3D tumor spheroids of varying sizes were generated and embedded in 3.0 mg/mL rat tail type I collagen and subjected to the same treatment. Statistical tests were performed in GraphPad Prism 10.0 and further image processing was performed in Fiji ImageJ.

Results, Conclusions, and Discussions::

Results: The apoptotic rate of control cancer clusters (Fig. 1A-B) did not exhibit a clear dependence on size in 2D (Fig. 1CD). By contrast, the apoptosis of serum-starved 2D clusters decreased significantly with increasing cluster size. Consequently, large serum-starved clusters exhibited comparably low apoptosis to the control, whereas small, starved clusters had significantly higher apoptosis than the control, suggesting that clustering may enhance the overall cell survival in stressful conditions. Similarly, enhanced survival of large 3D serum-starved clusters/spheroids was observed, albeit both control and starved spheroids appeared to have a size-dependent decrease in apoptosis (Fig. 1D, 1E). As larger clusters have a higher percentage of center cells, we hypothesized that center cells are less susceptible to apoptosis than edge cells, leading to the increased overall survival of larger clusters in serum starvation. Supporting this, we found triangular clusters with a larger perimeter and thus more edge cells displayed higher apoptosis than circular clusters with a smaller perimeter but the same total area (Fig. 1F). However, when comparing the distribution of apoptosis within the clusters, more apoptosis was observed in serum starvation than control only in the center regions (Fig. 1G-H), suggesting the opposite—center cells are more susceptible to apoptosis. Considering that cellular traction stress often increases from the center to the edge of clusters, and cell apoptosis mostly occurs in low-traction regions [11], we suspect larger clusters may have a higher percentage of cells in the high-traction regions, which improves their overall survival in starvation. Given that elevated apoptosis in starvation mostly occurred within a 50-µm-radius region from the center, regardless of cluster size (Fig. 1G-H), we further hypothesize this radius may define the low-traction region and will verify this by measuring the traction stress distribution in our clusters.

Conclusions: Together, we observed a robust effect of large cancer cell clusters in enhancing cell survival under stressful environments. While the exact mechanism of this survival is still under investigation, it is likely attributed to the interaction between neighboring cells that scales up with cluster size. Our findings have important implications for potentially disrupting cancer cell clustering when developing future therapies.

Acknowledgements and/or References (Optional):: This work was supported by funding from the National Institutes of Health (R21GM147958, R21EB034930, and P20GM139768-6405) to J.Z.

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