ABSTRACT: Single cell RNA sequencing of CD45+ immune cells from tumor and tumor-adjacent mammary adipose tissue in a postmenopausal breast cancer model of lean, obese, and weight loss Trem2+/+ and Trem2-/- mice
Project description:Triggering receptor expressed on myeloid cells 2 (Trem2) has been shown to be immunosuppressive in cancer and increased in obese adipose tissue. We utilized high fat and low fat diet to generate lean, obese, and weight loss mice in Trem2 wild-type (Trem2+/+) and Trem2 deficient (Trem2-/-) conditions. Once obesity and weight loss were established, mice were injected with E0771 breast cancer cells and tumors were allowed to grow for 4 weeks. We wanted to better understand the immune landscape of both the tumor and the surrounding adjacent mammary adipose tissue (mAT). Therefore, the CD45+ immune cells from both the tumor and tumor-adjacent mAT were isolated and sequenced using single-cell RNA sequencing and V(D)J sequencing.
Project description:Adipose tissue stromal cells contribute to the regulation of adipose tissue in lean and obese states. Myeloid cells such as adipose tissue macrophages (ATMs) and dendritic cells (ATDCs) undergo both quantitative and qualitative changes with obesity. Due to similarity in markers the identify of adipose tissue dendritic cells and macrophages has been elusive. We have refined prior protocols to unambiguously discern ATM and ATDC in mice. We used microarrays to compare the profiles of ATMs and ATDC from gonadal adipose tissue from lean, obese, and formerly obese mice. We also isolated preadipocytes (PA) from lean and obese mice for comparison. Male C57Bl/6 mice were fed normal diet (ND) or high fat diet (HFD) for 16 weeks. Weight loss (WL) mice were switched from the HFD to ND for 8 weeks. RNA was purified from FACS sorted cell populations (live cells only) obtained from gonadal/epididymal adipose tissue depots. ATMs were defined as CD11c+ (CD45+CD64+ CD11c+) or CD11c- (CD45+CD64+ CD11c-) ATMs. ATDC were defined as CD64- CD11c+. PA were defined as CD31- CD45- Sca1+ PDGFRA+.
Project description:Background Understanding how obesity impacts human mammary adipose tissue (MAT) biology is crucial for deciphering its role in mammary epithelium during both physiological and pathophysiological processes, including breast cancer. Hypertrophic mammary adipocytes and Crown-like Structures are present in MAT of obese patients but whether these changes initiate a fibro-inflammatory response at the tissue level remains insufficiently explored. Objective We aimed to investigate the markers of adipose tissue dysfunction (immune cell infiltration, secretion pattern and fibrosis) in tumor-free MAT of obese versus lean patients Methods Tumor-free MAT were obtained from 61 lean and obese women who underwent mastectomy for breast cancer risk reduction or treatment. Immune and non-immune cell infiltration was determined using flow cytometry. Bulk transcriptomic was used to characterize the phenotype of CD206+ macrophages whose infiltration is increased in obese. Conditioned-medium were prepared from MAT to characterize their secretome and dose adipokines and cytokines by ELISA assay. The extra-cellular matrix (ECM) deposition was evaluated by Masson trichrome staining on cross-stained sections and 3D imaging of red picrosirius-stained tissues. Results We observed an increase in CD206+/HLA-DR+ macrophages in the stromal vascular fraction of MAT from obese patients compared to lean ones. Other immune cell infiltration and endothelial or adipose progenitor cell numbers were similar between groups. Bulk transcriptomics on CD206+ macrophages revealed a significant decrease in ECM component secretion and processing in obese samples. Additionally, no heightened secretion of pro-inflammatory cytokines or MCP-1 was noted in obese samples. ECM characterization indicated an absence of fibrosis, with obese MAT showing reduced collagen secretion and deposition compared to lean counterparts. Conclusions Obesity does not associate with inflammation or fibrosis in MAT, underscoring its unique behavior.
Project description:Adipocytes isolated from lean and obese postmenopausal women with no significant differences in metabolic syndrome parameters demonstrate changes in multiple inflammatory, metabolic and structural gene families. Purified adipocyte samples were isolated from subcutaneous adipose tissue surgical biopsies of 7 obese (BMI>30) and 7 lean (BMI<25) postmenoposal women and gene expression was quantified with Agilent-014850, 4X44K human whole genome platform arrays (GPL6480)
Project description:Purpose: To profile the transcriptomes of omental adipose tissues from obese and lean humans. Methods: Omental adipose tissues from obese and lean patients were subjected to RNA-Seq. Results: Differential expression analysis identified 206 dysregulated genes (p-value < 0.05 by moderated t-test and fold change M-bM-^IM-% 2 in obesity) that are known to be involved in a multitude of functions, including response to stress, inflammatory response and leukocyte adhesion. Differential splicing analysis uncovered the possible role of TLR4 RNA splicing in obesity. Our findings suggest that, as a person experiences weight gain/obesity, the adipose splicing pattern of TLR4 transcripts changes in favor of activation of TLR4 signaling, which in turn may contribute to the progression of obesity-related inflammation and complications. Conclusion: This study provides a look into the transcriptome of disease-state adipose tissue in obesity, and demonstrates the potential importance of aberrant RNA splicing and expression in obesity-associated immune dysregulation. Study design is of cross-sectional nature. Seven samples (three obese and four lean) were analyzed.
Project description:We did single cell RNA sequencing on three lean and three obese donors. For each donor, we sorted CD45 positive and CD45 negative cells and prepared 10X scRNA-seq libaries. We observed cell type compositon changes and expression changes in the lean and obese donors.
Project description:lean control, obese, and formerly obese C57BL6N mice which underwent weight loss via vertical sleeve gastrectomy or calorie restriction were injected with E0771 cells and tumor growth was monitored
Project description:lean control, obese, and formerly obese C57BL6N mice which underwent weight loss via low-fat diet or verticle sleeve gastrectomy were injected with E0771 cells and tumor growth was monitored
Project description:lean control, obese, and formerly obese C57BL6N mice which underwent weight loss via low-fat diet or verticle sleeve gastrectomy were injected with E0771 cells and tumor growth was monitored
