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Biomechanics

Biomechanics - Poster Session E

Poster P11 - Diabetic Hyperglycemia Alters Cancer-Associated Fibroblast Transcriptome to Support Breast Cancer Progression

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Diabetes Mellitus is a metabolic disorder resulting in resistance to, or lack of insulin, leading to hyperglycemia. Diabetics who develop cancer experience worse prognosis, however the mechanism is unclear. Interestingly, a retrospective analysis of 265 patients with breast cancer demonstrated diabetes alone was not indicative of poor outcome (Herrera LA. Exp. Dia. Res. 2012). However, uncontrolled glucose levels (>130 mg/dL) were associated with decreased overall survival in both diabetic and nondiabetic patients relative to normoglycemic patients (Herrera LA. Exp. Dia. Res. 2012). Hyperglycemia increases excess circulating glucose which reacts with proteins in extracellular matrix to crosslink and stiffen them in a nonenzymatic glycation reaction to form Advanced Glycation End-products (AGEs). Independent of diabetes, stiffness is a poor prognostic indicator for cancer. In an MMTV-PyMT model of diabetic breast cancer, we recently demonstrated that hyperglycemia worsened breast cancer prognosis as a result of AGE crosslink accumulation. In addition to increased tumor stiffness and burden, matrix composition was altered in hyperglycemic tumors. Cancer-associated fibroblasts (CAFs) are mechanosensitive, contractile cells in tumors which respond to changes in mechanics to modulate matrix architecture and composition. CAF contractility is maintained through YAP signaling, where increased tissue mechanics enhance CAF contractility and matrix mechanics in a feed-forward manner via YAP. Therefore, we hypothesize that hyperglycemia results in stiffened collagen matrix which reprograms CAFs to further increase matrix mechanics to support cancer progression through YAP-regulated mechanosensing.

Materials and

Methods:  RNA Sequencing Data Analysis

Raw RNA sequencing data (GSE202922) were obtained from the Gene Expression Omnibus (GEO) and preprocessed. Expression data from 66 patients were normalized and converted to log2 counts per million (CPM) for downstream analysis. Single-sample Gene Set Enrichment Analysis (ssGSEA) was performed using the GSVA package in R to transform expression data using a rank transformation, and ssGSEA scores were calculated for each patient. Tumor microenvironment scores, including stromal, immune, and ESTIMATE scores, were calculated using the ESTIMATE package in R.

ScRNAseq of Hyperglycemic Murine Model

Diabetes was induced via streptozotocin (STZ) treatment of MMTV-PyMT mice as previously described (Reinhart-King CA. Sci. Adv. 2022). Briefly, hyperglycemia was induced in mice via serial intraperitoneal STZ injection to kill beta islet cells. 3-4 tumors were isolated from mice at 12 weeks of age. Tumors were minced and enzymatically digested using the Human Tumor Dissociation Kit from Milytenyi Biotec (130-095-929) into a single cell suspension for RNA-sequencing. Stromal cell enrichment was performed by negative selection using EPCAM+ via magnetic-assisted cytometry (MACs). Cells were sequenced using the 10x genomics platform.

Results, Conclusions, and Discussions: Diabetes increases mechanosensing and alters tumor composition

Since hyperglycemia is known to increase matrix stiffness, we hypothesized the transcriptome of diabetic tumors will show enrichment of genes related to mechanosensing. A Single Sample Gene Set Enrichment Analysis (ssGSEA) for 14 gene transcripts downstream of YAP activation (YAP signature) showed diabetic patients have increased enrichment in YAP signature, while ssGSEA of a set of randomly curated genes were not enriched, suggesting diabetic tumors are selectively enriched for mechanosensing genes (Fig.1A).

Next, we asked whether cellular composition is influenced by diabetes. We applied Estimation of STromal and Immune cells in MAlignant Tumors using Expression data (ESTIMATE) to infer the fraction of stromal and immune cells in tumors. ESTIMATE analysis showed a significant increase in stromal scores while the immune score showed no change, suggesting diabetic tumors have an increase in stromal cells. (Fig.1B). Taken together these results indicate diabetics have tumors with altered tumor cell composition and mechanosensing.

CAF transcriptome is altered towards matrix modulation in diabetes

Bulk RNA sequencing data from patients points to an increase in stromal activity in diabetic tumors. Therefore, we sought to understand how diabetes alters CAF transcriptome. To do this we performed single cell RNA sequencing (scRNAseq) on nondiabetic (Ctrl) and diabetic (STZ) mouse tumors. To enrich for CAF transcriptome changes, tumors were de-enriched for cancer cells prior to sequencing. Based on canonical biomarkers, we identified 4 overall cell populations in tumors (Fig. 2A). In agreement with patient data, CAF transcripts were increased while immune populations were not altered (Fig. 2B). Gene Ontology (GO) SLIM biological process analysis of the top differentially enriched CAF genes indicates CAF transcriptome has increased transcription of matrix modulation genes (Fig. 2C). Taken together this data suggest CAFs are transcriptionally altered by diabetes to organize matrix.

Our results are the first to our knowledge to demonstrate a connection between diabetes, changes in tumor composition, and enhanced YAP mechanosensing in breast cancer. Understanding these mechanisms may provide new therapeutic strategies for diabetic breast cancer patients, potentially improving their prognosis.

Acknowledgements (Optional):