(D-151) Investigating Crosstalk of Mechanical and Soluble Cues in Perivascular Matrix

The contractility of stromal cells influences the mechanoregulation of the surrounding perivascular matrix, thereby impacting vascular networks [1]. This includes the mechanical forces generated by stromal cells, their interactions with neighboring cells within the perivascular matrix, and the subsequent remodeling of the matrix through stromal cell paracrine factor secretion and ECM reorganization [2]. However, the mechanisms through which biomechanical behaviors of stromal cells modulate cell-cell signaling and matrix remodeling in the context of vasculature growth and maintenance need to be better understood. To investigate, we analyzed the contractility and cytokine profiles of multiple stromal cell types typically used to create vascularized 3D tissue models to gain insights into their mechanical phenotypes. These studies aim to understand the mechanisms through which stromal cells regulate mechanosignaling in the vasculature, potentially providing insights into novel therapeutic targets for vascular disorders, including cardiac pathologies, wound healing, or cancer-related angiogenesis.

Seven stromal cell lines were subjected to a bead displacement assay to track cellular movement and matrix distortions within a 3D microtissue model made of 10mg/mL fibrin. These cells included breast cancer-associated (CAF), normal breast (NBF), normal breast with empty vector (NBF-EV), normal breast with constitutively active Rho (NBF-caRho), cardiac (NHCF), dermal (NHDF) and lung (NHLF) fibroblasts. The beads were tracked over thirty-minute intervals for three hours, and the bead displacements were measured in a 3D ROI. For stromal cell profiling, CAFs, NBFs, NBF-EVs, NBF-caRhos, NHCFs, NHDFs, and NHLFs were cultured in 3D microtissue models for a duration of 48 hours. Subsequently, the culture media was collected and subjected to cytokine and growth factor profiling analysis at Eve Technologies. These included EGF (Epidermal Growth Factor), FGF-2 (Fibroblast Growth Factor-2), G-CSF (Granulocyte Colony-Stimulating Factor), GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor), IL-1α (Interleukin-1 alpha), IL-1β (Interleukin-1 beta), IL-8 (Interleukin-8), PDGF-AA (Platelet-Derived Growth Factor-AA), PDGF-AB/BB (Platelet-Derived Growth Factor-AB/BB), and VEGF-A (Vascular Endothelial Growth Factor-A).

The bead displacement study revealed significantly greater mechanical activity in CAFs, NHCFs, and NHDFs compared to the lower mechanical activity in the lung and normal breast fibroblasts. This is represented as higher average displacements measured in the bead tracking protocol (Figure 1). These results also highlight the heterogeneous nature of cell contractility within a single cell line, with CAFs, NBFs, NHCFs, and NHDFs having the widest ranges of matrix displacements generated. To further understand mechanical phenotype, we chose to pursue the analysis of cytokines and growth factors to link soluble signaling patterns to mechanical behaviors.  The cytokine and growth factor panel show that FGF-2 and IL-8 concentrations are higher in various stromal cells than other secreted factors (Figure 2). Notably, these secreted factors are known to be involved in angiogenesis through different mechanisms [3]. Concurrent with the bead tracing study, CAFs and NHCFs showed a greater significance in releasing secreted factors into the media, suggesting their potential role in modulating the angiogenic environment. These results emphasize the variance in the concentration of secreted factors across the various cell lines.

These findings highlight the complex interplay between mechanical behaviors and soluble signaling pathways, providing valuable insights into stromal regulation of angiogenesis and revealing a possible mechanism by which stromal cells influence cell-cell interactions within the perivascular matrix and contribute to regulating vascular networks. Based on these studies, it is possible to gain valuable insight into the regulation of vascular networks by stromal mechanical forces and cytokine profiles. Future studies will apply these results to understand cadherin signaling in angiogenesis and vessel maintenance. Previous work has established how VEGF-A regulates vascular endothelial (VE) cadherin signaling [4]; FGF-2 downregulates epithelial (E) cadherin and promotes tumor migration by working synergistically with neural (N) cadherin; and EGF secretion is downstream of E-cadherin adhesion [5]. The observed differences in mechanical activity among various stromal cell lines further highlight their distinct functional characteristics, which could be instrumental in controlling blood vessel function and growth related to cancer, wound healing, cardiac pathologies, or tissue engineering.

References:

1.         Miller, B. and M.K. Sewell-Loftin, Mechanoregulation of Vascular Endothelial Growth Factor Receptor 2 in Angiogenesis. Front Cardiovasc Med, 2021. 8: p. 804934.

2.         Winkler, J., et al., Concepts of extracellular matrix remodelling in tumour progression and metastasis. Nat Commun, 2020. 11(1): p. 5120.

3.         Ucuzian, A.A., et al., Molecular mediators of angiogenesis. J Burn Care Res, 2010. 31(1): p. 158-75.

4.         Vestweber, D., VE-cadherin: the major endothelial adhesion molecule controlling cellular junctions and blood vessel formation. Arterioscler Thromb Vasc Biol, 2008. 28(2): p. 223-32.

5.         Loh, C.Y., et al., The E-Cadherin and N-Cadherin Switch in Epithelial-to-Mesenchymal Transition: Signaling, Therapeutic Implications, and Challenges. Cells, 2019. 8(10).