Poster V2 - The role of cancer-associated fibroblast-remodeled collagen matrix on monocyte recruitment

Introduction: High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological cancer with a median overall survival of about 32.5 months and a five-year survival rate of less than 30%. This poor prognosis is attributed to the late-stage diagnosis along with the metastasis to secondary sites. Monocytes circulating in the bloodstream sense tumor-secreted factors that recruit these cells to the tumor microenvironment (TME). However, the mechanisms that drive monocyte infiltration in the TME are poorly understood. Cancer-associated fibroblasts (CAFs) represent an abundant cell type in the TME and contribute to tumor desmoplasia by secreting and remodeling the extracellular matrix (ECM). These ECM modifications include alterations in the architectural organization and composition. Collagen type I (Col1) is the most abundant ECM component in metastatic ovarian cancer implants in the omentum. In the clinic, there is a positive correlation between monocytes and CAFs numbers in omental metastasis which has been shown to have an impact on cancer progression and survival rates. Most studies on tumor-monocyte crosstalk have been predominately performed in 2D environments which do not accurately recapitulate the TME. Utilizing 3D and microfluidic in vitro platforms better mimics the in vivo environment in which ovarian cancer spreads. Here, we present a microfluidic platform using decellularized CAF-remodeled collagen matrices to study the relationship between matrix architecture and its role in modulating monocyte recruitment.

Materials and

Methods: To derive CAFs, mesenchymal stem cells (MSC isolated from a tumor-infiltrated omentum) were treated with 50µg/mL ascorbic acid (Sigma Aldrich, 50-179-1245) and 100 ng/ml recombinant human connective tissue growth factor (CTGF) (PeproTech, 120-19) for 4 weeks, with medium change every third day. Collagen matrices (rat tail collagen type I solution at 2mg/mL, Fisher, CB354249) were seeded with these MSC-derived fibroblasts (1*106 cells/mL). Matrices were fixed using 4% paraformaldehyde and endpoint staining was performed using anti-FAP (Biolegend, 621151) and anti-COL1 (Fisher, MA1-26771) antibodies. A Zeiss LSM700 confocal microscope was used to image the CAF-remodeled collagen matrices. Collagen alignment quantification was performed using the TWOMBLI plugin in ImageJ. After generating masks, the metric for fiber alignment analysis was based on how organized the alignment of the collagen fibers from each other was with values index ranging from 0 to 1 (Figure 1: 0 indicating randomly arranged fibers and 1 parallel fibers to each other). Additionally, the average length of migratory tunnels (Figure 1) was measured using ImageJ.

Results, Conclusions, and Discussions: Matrices remodeled by tumor MSC-derived fibroblasts exhibited higher collagen fiber alignment compared to control matrices. Additionally, in these matrices we found that 3D tunnels were formed after 48 hrs of remodeling, while no tunnels were observed in the control condition (Fig. 1B). This is due to the MSC-derived fibroblast degrading and contracting the matrix compared to non-remodeled matrices. Quantification of the tunnel formation length was performed using ImageJ with 84.07 +/- 22.65 µm average length (Fig. 1C) and fiber alignment with a value of 0.268 (Fig. 1D). These results demonstrate our ability to generate conditions of CAF-driven matrix remodeling to study monocyte migration.

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