Poster S15 - Metastatic Cancer Cell Clusters Subjected to Physiological Fluid Shear Stresses – Survival, Proliferation, and Aggression Enhancement in Aggregates

Introduction: During the metastatic cascade, cancer cells travel through the bloodstream as circulating tumor cells (CTC). Less than 0.1% of these cells survive the high shear stress exposure and interactions with normal immune cells within the circulation. Cancer cells, however, are known to travel in clusters in circulation, conferring increased metastatic potential as well as higher shear stress and treatment resistance. Such phenomena leave patients with poorer prognoses as diseases are diagnosed later in the metastatic cascade. Yet the biology of these cell clusters remains poorly understood. The development of in vitro models to study cluster transport and biology will reveal the behavior of clustered versus singular CTCs as well as yield new insights into the interactions between cells in homotypic – cancer cells only – and heterotypic – cancer cell and cancer-assisting immune cell – aggregates. We therefore engineered a tunable superhydrophobic array device (SHArD). The SHArD-C was applied to culture clinically relevant models of CTC clusters, allowing for the characterization and quantification of cell-cell interactive behavior both in static culture and in vivo, in the presence of physiologically relevant lower and higher fluid shear stresses, recreating stresses experienced in peripheral venous pathways as well as near the cardiac corridor, respectively.

Materials and

Methods: The human prostate cancer cell line LNCAP (ATCC, CRL-1740) and human colorectal cancer cell line HCT116 (ATCC, CCL-247) were used throughout the study. HCT116 is a human colorectal carcinoma cell line isolated from the primary tumor of a patient and grow with an epithelial morphology. The LNCaP cell line was isolated from a lymph node lesion of a metastatic prostate cancer patient and is androgen sensitive. For 3D culturing of cancer clusters using the SHArD-C, total cell plating number was determined by calculating the number of cells per mm2 to achieve an initial plating density of about three, five, and seven cells per microwell – for a total of ~12,100 clusters per device – to best model clinically isolated aggregates which range from 2-100 cells per cluster. Clusters were imaged via brightfield microscopy to determine roundness and volume using Feret diameters and areas. Clusters were also imaged via confocal microscopy to visualize E-cadherin expression within cellular aggregates, and nuclei were stained with DAPI. Compactness was calculated from confocal microscopy images. These analyses were also performed on monolayer (cells left to grow into aggregates naturally at the bottom of a 24-well plate) and single-cell controls that were subjected to laminar and turbulent flow conditions using cone-and-plate viscometers and syringe pumps, respectively. This equipment was used to model the shear stresses experienced by cellular aggregates when travelling through the peripheral vascular network, i.e., in vessels of skeletal muscle (2-13 dyn/cm2), and within the cardiac corridor and ventricles of the heart (~3950 dyn/cm2).

Results, Conclusions, and Discussions: The size of cell clusters grown in the SHArD-C was easily controlled by varying the average number of cells loaded into each microwell. Cluster sizes were analyzed by approximating the cluster volume: 12 ± 6, 20 ± 6, and 39 ± 15 µm3 for the small, medium, and large clusters, respectively (Fig. 1A-C). E-cadherin is important for cell-cell adhesion in epithelial cells and can affect cell proliferation, migration, cell polarization, and survival. Additionally, E-cadherin contributes to CTC cluster survival through anoikis prevention and FSS resistance via tightly linking cells to one another creating a shielding layer to defend against high shearing forces. LNCaP clusters cultured using the SHArD-C showed the greatest fluorescent signal at boundaries shared with other cells by 36% compared to monolayer controls (Fig. 1D&E), suggesting that the SHArD-C is more effective at producing tightly bound clusters.

When exposed to sustained lower levels of FSS, LNCaP clusters grown using the monolayer method exhibited a 68.3% decrease in cluster volume (Fig. 1A&F) compared to no significant change in volume in the SHArD-C clusters, demonstrating that these clusters can withstand similar forces as encountered in the circulation. Cells were also sheared using a syringe pump system that simulates forces experienced when passing through the heart. Aggregates were exposed to 4000 dyne/cm2 over 10 pulses, once every 2 min, approximating the time taken to traverse the circulatory system and return to the heart. Compared to the single cell samples and monolayer culture methods, cells cultured into cluster form using the SHArD-C showed resistance to cell death and contained significantly higher percentages of viable cells before and after FSS (Fig. 1G&H). In summary, we have demonstrated that SHArD-C aggregates have higher cell-cell junction protein expression, do not disaggregate under lower, laminar FSS, and conserve cell viability after exposure to high FSS. Current methods for culturing CTC clusters in vitro do not achieve comparable results, making our device an attractive approach, as such physiologically relevant models enable better understanding of the role of collective migration units during the metastatic cascade.

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