(D-133) High Glucose Increases Matrix Contraction by Cancer-Associated Fibroblasts

Introduction::

Cancer-associated fibroblasts (CAFs) are highly contractile cells that are major constituents of tumor stroma. CAFs can act to restrain tumor growth or inhibit immune cell infiltration through cytokine secretion and by modulation matrix density, and stiffness. Additionally, CAFs utilize contractility to alter matrix architecture. Specifically, CAF derived matrix is more aligned relative to normal fibroblast derived matrix. Matrix alignment is known to be correlated with poor prognosis as studies indicate cells are able to utilize aligned collagen as tracks to escape the primary site. CAF contractility is regulated by YAP nuclear localization, and glucose is known to increase YAP nuclear localization in multiple cell types including fibroblasts, yet this effect has not yet been examined in CAFs. Additionally, CAF are metabolically reprogrammed towards aerobic glycolysis in tumors, yet the direct effect of glucose availability on CAF phenotype and tumor matrix architecture is currently understudied. Therefore, we hypothesize that increased glucose availability will increase YAP nuclear localization and matrix contraction by CAFs leading to increased matrix alignment. To examine this, we utilize a mouse model of hyperglycemic breast cancer and cell-seeded collagen gels cultured in varying glucose concentrations to investigate the effect of glucose on matrix organization by CAFs. Our in vitro results indicate CAFs increase matrix contraction in response to glucose, and our in vivo data indicate that hyperglycemia results in increased collagen alignment in mouse tumors.

Materials and Methods::

Collagen Contraction Assay: Human breast cancer-derived (CAFs) were mixed with rat tail type I collagen to obtain a 2 mg/mL mixture with 2x10⁵ cells/mL. The collagen/cell mixture was seeded into a 24-well plate and allowed to polymerize at 37°C for 30 mins. After polymerization, cell-seeded collagen gels were detached from well sides and cultured for 48 hours in basic media (DMEM) with either 0 or 30 mM glucose concentration. Cell contractility was measured by calculating the percent change in area of the construct.

Collagen Organization Assay: Fixed tumor sections collected from PyMT mice were hydrated and stained using a Picrosirius red stain kit according to the manufacturer’s instructions. After dehydration and mounting, quantitative polarization microscopy was performed on the sections using an inverted Axiovert microscope equipped with a rotatable linear polarizer and a circular polarizer with a Zeiss Axiocam 506 color camera. Orientation psuedocolor images and dispersion angle were generated using the OrientationJ, and directionality Fiji plugins, respectively.

Immunofluorescence Imaging: CAFs were fixed after 48 hours exposure to glucose, blocked in 3% BSA, permeabilized and stained for YAP protein. Images were taken using a Zeiss LSM800 confocal microscope.

Statistical analysis: All statistical analyses were carried  out  using  PRISM 10. Unpaired student’s t tests were used to compare two groups, or One way ANOVA was utilized to compare 3 or more groups. All data are expressed as mean ± SD. All the statistical tests were treated as two-sided and calculated at a level of significance of alpha = 0.05.

Results, Conclusions, and Discussions::

Hyperglycemic glucose levels increase matrix contraction by CAFs: CAFs are hypercontractile cells that metabolically reprogrammed in tumors by cancer cells towards a glycolytic phenotype. CAFs are known to utilize their contractility to align matrix in tumors which can serve to worsen prognosis by aiding tumor cell invasion. Therefore, we asked whether CAFs increase matrix contraction in response to glucose. To do this we employed a collagen contractility assay which allowed CAFs to contract a free-floating collagen gel in response to no glucose, normoglycemic (5mM) or hyperglycemic (30mM) concentrations. Using this assay, we found that CAFs significantly increase matrix contraction in response to hyperglycemic glucose levels. (Fig. 1A) 

Glucose increases YAP nuclear localization in CAFs: CAF contractility is regulated by YAP nuclear localization to induce f-actin rearrangement. Further, glucose is known to increase YAP nuclear localization in multiple cell types. Therefore, to examine how glucose may induce CAF contractility, we looked to YAP nuclear localization. As expected, we confirm through immunofluorescence imaging, that YAP nuclear localization is increased by glucose in CAFs. (Fig.1B) 

Hyperglycemia results in tumors with increased matrix organization: Motivated by our in vitro data, we asked whether prolonged exposure to hyperglycemic glucose levels induced changes in architecture in tumors. To do this, we employed our previously established hyperglycemic mouse model of breast cancer. In brief, we utilize a MMTV-PyMT mouse model and streptozotocin treatment, which eliminates beta islet cells, to induce hyperglycemia. To examine matrix organization as a result of hyperglycemia, we conducted orientation analysis of picrosirius red stained tumor sections from normoglycemic or hyperglycemic mice utilizing the directionality plugin in Fiji. Our analysis shows hyperglycemia results in tumor matrix that is more aligned than normoglycemic mice. Taken together, these results indicate CAFs may respond to high glucose by increasing contraction of the surrounding matrix to influence architecture. (Fig1.C).
Future work will utilize in vitro models to examine CAF-induced matrix organization in response to hyperglycemic glucose levels. Completion of our work provide insight into nutritional regulation of CAF mechanobiology and provide a potential mechanism to inhibit tumor progression.

Acknowledgements (Optional): :

References (Optional): :