Abstract P5-06-05: Palmitate induces a senescent-like phenotype in fibroblasts resulting in altered phenotypes in cells of the breast tumor microenvironment

Abstract

Abstract Background: Obese breast cancer patients face a worse prognosis, including an increased risk of recurrence and mortality. While the causative mechanisms have yet to be fully uncovered, emerging evidence implicates palmitate, increased in the obese state, in development of cellular senescence, an inflammatory state associated with proliferation, metastasis, and tumor-associated neutrophil (TAN) polarization, among other measures of carcinogenesis. However, studies have yet to investigate the impact of palmitate on induction of senescence in breast fibroblasts, and no studies have assessed the effect of senescent fibroblasts on neutrophil polarization in the breast tumor microenvironment. This said, we hypothesize that palmitate alters breast cancer cell gene expression and neutrophil phenotype via induction of a senescent-like phenotype in fibroblasts. Methods: HCA2, IMR-90, and human mammary fibroblasts were exposed to palmitate or vehicle in media supplemented with 2% charcoal-stripped fetal bovine serum, after which the cells were measured through qPCR for expression of IL-1a, IL-6 and IL-8, some of the most prominent members of the senescence-associated secretory phenotype. Palmitate-exposed fibroblasts were also subjected to chromogenic staining for senescence-associated beta-galactosidase and immunoenzymatic BrdU analysis, two well-established measures of senescence. Next, in order to study the influence of these fibroblasts on other cells of the breast tumor microenvironment, we assessed their impact on polarization of DMSO-differentiated HL-60 neutrophils by using flow cytometry to measure neutrophil expression of CD54 and CD95, differentially expressed on N1 and N2 neutrophils. Finally, we employed PCR arrays to assess the impact of palmitate-exposed fibroblasts on the expression of 84 genes in MCF-7 and 231 breast cancer cells, measuring activation of pathways related to apoptosis, cell cycle, DNA damage, senescence, telomere maintenance, metabolism, angiogenesis, and the endothelial-to-mesenchymal transition. Results and Conclusions: Palmitate induced pro-inflammatory gene expression and SA-beta-gal activity and decreased BrdU incorporation in fibroblasts. Palmitate also exhibited non-cell-autonomous effects, as palmitate-exposed fibroblasts induced phenotypic changes in both neutrophils and breast cancer cells. These findings are among the first to implicate palmitate-induced fibroblast senescence in the stimulation of non-cell-autonomous changes in the breast tumor microenvironment and will ultimately inform our understanding of the mechanistic connection between the obesity-associated factor palmitate and breast tumorigenesis. Citation Format: Brittany Susanne Harlow, Albert Davalos, Bryan McClellan, Andrew Brenner, Christopher Jolly, Stefano Tiziani, Steve Hursting, Linda deGraffenried. Palmitate induces a senescent-like phenotype in fibroblasts resulting in altered phenotypes in cells of the breast tumor microenvironment [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-05.