Effect of ECM mechanical properties on activation of Cancer-Associated Fibroblasts

Introduction:: The tumor microenvironment (TME) plays a critical role in cancer progression, and within this complex landscape, cancer-associated fibroblasts (CAFs) are one of the prominent cells of TME and have emerged as key regulators [1] Extracellular Matrix (ECM) architecture influences the growth and phenotypical changes of fibroblasts within the tumor microenvironment (TME) [2]. In epithelial cancers, chronic alterations of dynamic reciprocity between CAF–ECM interactions result in regulating tumor-restrictive stroma evolving into a 'new' tumor-permissive environment [3]. This altered stroma, known as desmoplasia, is initiated and maintained by cancer-associated fibroblasts (CAFs) and is characterized by stiff and altered ECM density [3]. Understanding how the physical properties of the ECM influence CAF behavior is crucial for understanding CAFs and their role in constructing desmoplasia. In this study, our objective was to investigate the mechanistic insights under how the physical properties of the ECM, specifically 3D fiber density and stiffness, impact CAF regulation.

We made a comprehensive analysis by utilizing morphometric changes for ECM density and differential phenotypic changes at a 3D volumetric space for ECM stiffness as biophysical cues that probe for differential response of CAF phenotypes. In this study, we investigated the relationship between the physical properties of the ECM, specifically 3D fiber density [4], and stiffness as a variable in modulating CAF regulation. We made a comprehensive analysis by utilizing morphometric cues for ECM density and differential phenotypic changes at a 3D volumetric space for ECM stiffness as biophysical cues in regulating CAF behavior

Materials and Methods:: Matrix network density: Electrospun gelatin matrix fibers were fabricated and cross-linked by a glutaraldehyde vapor mechanism. Normal Human Dermal Fibroblast (NHDF) cells were used as the source for CAF differentiation. Conditioned media (CM_MCF-7) from MCF-7 (Breast cancer cell line) was used to induce the essential paracrine interaction of TME. CAF profiles were evaluated using qPCR to access common CAF markers, including FAP, FSP1, and PDGFRβ. Morphometric analysis revealed density-dependent differential CAF activation patterns and dynamic characteristics..

Matrix stiffness: Gelatin methacryloyl (GelMA) hydrogel with a continuous stiffness gradient ranging from 3 to 38 kPa was developed to recapitulate physiological stiffness conditions [5]. Normal human dermal fibroblasts were encapsulated in this hydrogel, and their morphological characteristics were captured at 3D volumetric space to related volume adaptation as a function of matrix stiffness


Results, Conclusions, and Discussions:: 3D gelatin matrices were prepared at two different network densities: highly dense (HD) and less dense (LD) matrices with an average pore size of 14.20 μm² for HD and 743.65 μm². We compared the CAF activation by CM_MCF-7 on 2D vs. 3D dense substrates. NHDFs on the 3D matrix fibers showed comparatively low gene expression levels of CAF markers than those cultured on the 2D substrate indicating the substrate confinement-dependent constraints. We studied the effect of ECM architecture on CAF activation by comparing two different network densities, HD and LD (Fig. 1 a). Interestingly, we found that the LD network facilitated CAF activation more than the HD network, accompanied by higher cell spreading and upregulation of CAF markers (Fig. 1 b, c). Further investigation of the Akt/mTOR pathway revealed that the LD matrix promoted CAF activation by inducing higher tensional integrity in fibroblasts resulting in upregulation of the mTOR signaling cascade.

Conclusions:

In this study, we developed a 3D matrix model based on gelatin with tunable network density to mimic the ECM matrix of TME. Fibers with a less dense (LD) network positively influence CAF activation in TME. Our findings indicate that the mTOR pathway regulates the activation of CAFs on the LD matrix. When we inhibited mTOR, we observed a significant reduction in the expression of CAF markers on the LD matrix. Based on these results, we propose that targeting mTOR could be a potential therapeutic approach to prevent CAF activation.

In accordance with matrix network density, we presumed that matrix stiffness modulates CAF plasticity. Previous studies have indicated that volume adaptation plays a crucial role in the response of cells to varying stiffness gradients in 3D hydrogels. Cells can contract and expand volumetrically in soft environments, whereas in stiff environments, cells have limited deformability and maintain a low volume [5]. Building upon this understanding, we hypothesized that volumetric adaptation also modulates CAFs and contributes to their morphological response based on stiffness [Fig 2]. We propose that considering matrix stiffness at a 3D scale could be a more effective approach for manipulating CAF subtypes and their tumorigenic functions.


Acknowledgements (Optional): : We acknowledge the financial support provided by the National Research Foundation of Korea (NRF – 2021R1A2C3008408). We also acknowledge members of Soft Nano-Biomaterials Lab, from Mechanobiology Institute -National University of Singapore, for providing us with training and sharing the protocols for preparing gradient stiffness 3D hydrogels.

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