(252c) Age-Dependent Stiffening of the Extracellular Matrix Increases the Rate of Cellular Senescence and Cancer Metastasis

Background: Melanoma is the fifth leading cause of cancer related death in the United States. It is often associated with years of excess sun damage and as such disproportionately affects older generations. While the 5-year survival rate for localized melanoma is exceptionally high at 99%, the metastatic disease is much more devastating with survival dropping to ~30%[1]. Like many age-associated pathologies, melanoma likely arises from a combination of intrinsic (e.g., genetic, molecular, extracellular) and extrinsic (e.g., environmental) factors. While significant body of work focuses on targeting genetic aberrations associated with melanoma. The mechano-structural changes of skin with age, and the corresponding impact on age-related phenotypes such as cellular senescence have been largely overlooked.

Cellular senescence reflects an irreversible state of cell cycle arrest that is often associated with normal aging. Senescent cell populations increase with age, and the presence of senescent cells, particularly senescent fibroblasts, within the tissue microenvironment can stimulate pro-growth effects on cancer cells through the secretion of pro-inflammatory factors (i.e., SASP). A key SASP factor, interlukin-6, can promote proliferation in a paracrine manner through activation of STAT3[2].

During aging, skin tissues exhibit increased collagen bundling and, as a result, increased ECM stiffness[3]. In homeostasis, this crosslinking of collagen with other ECM proteins such as fibrillin and elastin enhances the structural stability of the skin[4]–[6]. In solid tumors, the current consensus is that this age-associated stiffening of the matrix drives tumor progression through increased proliferation and migration of cancer cells. Here, we hypothesize that age-dependent matrix stiffening promotes accelerated senescence and associated increased Interlukin-6-containing SASP that drive melanoma cell migration and metastasis.

Methods: Using a gender-balanced cohort of primary dermal fibroblast cell lines from the Baltimore Longitudinal Study of Aging (BLSA), we assess the rate at which fibroblasts enter a state of cellular senescence in the presence of pro-senescence inducing agents. Dermal fibroblasts from four individuals aged 25, 35, 65, and 75 are plated on poly-acrylamide substrates of varying mechanical stiffness between 0.5 kPa and 25 kPa, corresponding to low stiffness of normal/young skin and high stiffness of solid melanoma tumors. The extent of senescence is assessed based on proliferation arrest, hallmark biomarkers of senescence (e.g. SA-βgal, p16, p21, and HMGB1), abundance of secreted factors (e.g., IL-6 and IL-8) in cell culture media, and morphological assessment. Metastatic potential of melanoma cells is characterized by functional assessments of single cell motility in a 3D extracellular matrix.

Results & Conclusion: Our results suggest that dermal fibroblasts cultured on stiff substrates are more susceptible to enter cellular senescence compared to soft. These results suggest 1) that senescence may be regulated via mechanotranduction, and 2) that there is a feedback loop by which increasing matrix stiffness contributes to the onset of cellular senescence, which in turn promotes further matrix stiffening and cancer metastasis.