Abstract PO4-24-07: The Impact of GPER Modulation on Cancer Associated Fibroblasts on Extracellular Matrix Formation in Breast Cancer
Authors: Fertal Shelby , Burkel Brian , Murdoch Fern , O'Leary Kathy , Schuler Linda , Ponik Suzanne 
Published: May 2024

Background: The G-protein coupled estrogen receptor (GPER) is a noncanonical estrogen receptor (ER) that is estimated to be present in roughly 50-60% of all breast cancer (BC) subtypes. Previous studies have suggested that the presence of GPER during primary disease aids in disease progression, metastasis, as well as chemotherapy resistance due to the agonistic activity of traditional chemotherapies for ER+ BC, tamoxifen and fulvestrant. It is also well established that the extracellular matrix (ECM), specifically collagen, contributes to the overall risk and progression of BC in patients. Importantly, fibroblasts are one of the primary cell types responsible for ECM deposition in the tumor microenvironment. We hypothesize that the modulation of cancer-associated fibroblasts (CAFs) via estrogen signaling through GPER regulates the deposition and structure of key ECM proteins, such as collagen, to aid in disease progression.

Methods: CAFs, isolated from human BC surgical resections, were plated at 100% confluency, and incubated with either 10nM β-estradiol (E2), 50nM G1 (a GPER specific agonist) or a vehicle control and 50ug/mL ascorbic acid daily for 14 days to induce matrix deposition. Phenol-free, high glucose DMEM with 10% charcoal stripped FBS was used as media to reduce FBS sourced estrogen. Following the 14-day incubation, cell proliferation was quantified prior to extracting the cells to isolate the CAF-derived matrix (CDM). The organization and composition of ECM was analyzed in CDMs through immunofluorescence (IF) or western blot, respectively. An unpaired t-test was conducted on biological triplicates and between vehicle control and either G1 or E2. Significance was determined as p&lt; 0.05.

Results: Culturing CAFs with E2 resulted in a 10% increase in cell proliferation (n=3, p&lt; 0.05) and a 1.6-fold (n=3, p&lt; 0.05) increase in total ECM deposition by western blot. Key ECM proteins such as collagen, fibronectin, and periostin had a 1.8, 1.3, and 1.3-fold increase, respectively (n=3, p&lt; 0.05), in protein deposition with E2 treatment when normalized to cell number. Collagen fiber analysis of IF stained CDM for collagen resulted in a coefficient of alignment with E2 of 0.76 compared to 0.51 without (n=3 for E2 and n=6 for vehicle, p&lt; 0.05). To confirm E2 was acting through GPER, CDMs were subsequently generated with exposure to 50nM G1, a specific GPER agonist. Collagen fiber analysis of the G1 exposed CDMs resulted in a coefficient of alignment with G1 of 0.71 compared to 0.51 without (n=3 for G1 and n=6 for vehicle, p&lt; 0.05).

Conclusions: This study demonstrates that estrogen signaling through GPER modulates the deposition of fibronectin, periostin, and collagen as well as the organization of collagen architecture. These results highlight the need to further investigate how the interplay between the ECM, GPER activity, and fibroblasts impacts breast cancer progression.

Citation Format: Shelby Fertal, Brian Burkel, Fern Murdoch, Kathy O'Leary, Linda Schuler, Suzanne Ponik. The Impact of GPER Modulation on Cancer Associated Fibroblasts on Extracellular Matrix Formation in Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-24-07.