Breast Cancer Clusters Generate Greater Matrix Deformation in Aligned Fibers During Collective Migration

Introduction: Organized extracellular matrix (ECM) architecture, in particular fiber alignment, is an important and essential cue for promoting cell and tissue development and disease progression such as cancer. In breast cancer, increased remodeling of the extracellular matrix results in aligned matrix architecture and is indicative of later stage cancer and metastasis. During breast cancer metastasis, tumor cells leave the primary tumor via collective migration, a mode of migration characterized by groups of cells moving in a coordinated manner. How collective units of cells respond to mechanical cues such as matrix fiber alignment is not well understood. Further, there is a need to generate in vitro models whereby we can modulate matrix architecture to answer these questions. Our lab created microfluidic devices capable of mimicking aligned and random matrix fiber orientations to study cell-matrix interactions with high resolution. The objective of this study is to understand how breast tumor clusters respond to aligned matrix organization. Our hypothesis is that in aligned matrix fibers, breast tumors will have increased elongation and matrix deformation. Understanding how collectively migrating tumor clusters respond and interact with their ECM will identify key drivers of breast cancer metastasis.

Materials and

Methods: We created tumor organoids from primary murine mammary gland tumors of a spontaneous breast cancer mouse model (MMTV-PyMT). Mammary gland tumors were isolated from female 12 week old MMTV-PyMT;K14-GFP mice, following approved IACUC protocol (AD10002197). Tumor organoids were loaded into our microfluidic devices (aligned and randomly oriented) encapsulated in 2 mg/ml collagen 1 and cultured for up to 48 hr. At 24 and 48 hours, samples were fixed and stained for phalloidin and DAPI and imaged on a Zeiss LSM 980 (40xW). For the matrix deformation assay, tumor clusters were loaded into the microfluidic devices in collagen 1 (2mg/ml) with 1% fluorescent beads. Media with SIR-actin (Cytoskeleton) and verapamil was added and the samples were incubated overnight before live cell imaging (Nikon Ti2). Live cell images were processed in FIJI to correct drift and adjust brightness/contrast, and then imported into our custom MATLAB code (CellXMD) to calculate the deformation of the extracellular matrix based on the displacement of the beads around the cells (MATLAB 2023a). Briefly, the MATLAB code detects each bead in all z-frames and tracks the displacement over time and averages to output a bulk ECM deformation rate. Phalloidin IF staining images were used for cell morphology quantification in FIJI. The protrusion angles were determined by measuring the angles of each protrusion from the center of each cell cluster centroid. Histogram values of the protrusion angles were determined by calculating the frequency of the protrusion angles in ten-degree intervals.

Results, Conclusions, and Discussions: To understand how tumor organoids respond to matrix fibers in a dynamic environment, we quantified cell cluster morphology and measured matrix deformation of tumor organoids after culturing in our aligned and random matrix fiber microfluidic devices. Our prior published work demonstrates the ability to promote directional cell migration in response to fiber orientation in a static assay (Lichtenberg, PLOS One 2024). After we incorporated primary tumor organoids into the aligned and random devices, we observed cell cluster spreading in 24hr (Figure 1A). We quantified the fiber alignment around the tumor organoids and determined the fibers have higher alignment coefficient in the aligned device compared to the random device (Figure 1B-C). The alignment coefficient increases as the cells are cultured longer, indicating the cells could be pulling on the matrix to generate more aligned fibers (Figure 1C). Organoid morphology characterization demonstrated the aspect ratio is increased and the roundness is decreased in the aligned devices compared to the random devices (Figure 1D-E). The aspect ratio increases after 48hrs compared to 24hr, suggesting the clusters continue to elongate. The average angle of the organoids is 90° in the aligned devices (Figure 1F), corresponding to the dominant fiber angles in the aligned devices (Figure 1B). In the random devices the organoid angle ranges in value, indicating non-directional spreading. The number of protrusions is greater in the random devices compared to the aligned devices at 24hr (Figure 2A). The number of protrusions in both devices decreases after 48hr, suggesting the cells could be sensing a dominant direction for prolonged protrusions. Histograms show the directional protrusions of the organoids in the aligned devices and non-directional protrusions in the random devices (Figure 2B). Finally, we measured matrix deformation of the clusters and we found greater matrix deformation per protrusion in aligned devices compared to random devices (Figure 2C). Altogether, these findings suggest directional protrusions are associated with increased matrix deformation, alluding to an increased mechanical response of tumor clusters due to the biomechanical stimulus of aligned fibers. Increased mechanical activity of cancer cells promotes cancer progression and metastasis.

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