Mammary Adipose Tissue Research Articles

Histologic Characterization of Tumor-Adjacent Mammary Adipose Tissue in Normal-Weight and Overweight/Obese Patients with Triple-Negative Breast Cancer

Background: Obesity is a risk factor of several types of cancer, including breast cancer. In this study, we aimed to histologically characterize the adipose tissue of the tumor microenvironment (TME) of triple-negative breast cancer (TNBC) in overweight/obese versus normal-weight patients. Methods: TNBC tissue sections from normal-weight (BMI<25) and overweight/obese patients (BMI≥25) were stained with antibodies against CD68, CD163, CD31, CD34, and vimentin. At the invasive tumor front, positive cells were counted in tumor adjacent adipose tissue (AT) and within cancer tissue (CT). Further, the size of the tumor-adjacent and distant mammary adipocytes was determined in perilipin stained sections. Expression of ANGPTL4, CD36 and FABP4, proteins involved in fatty acid metabolism, was analyzed in marginal tumor cells using an immune reactive score. Results: Overweight/obese TNBC patients had significantly larger adipocytes, higher numbers of CD163+ macrophages (BMI<25: 2.80 vs. BMI≥25: 10.45; p = 0.011) and lower numbers of CD31+ (BMI<25: 4.20 vs. BMI≥25: 2.40; p = 0.018) and CD34+ (BMI<25: 14.60 vs. BMI≥25: 5.20; p = 0.045) cells as markers of angiogenesis in the AT as well as a higher frequency of cancer-associated-fibroblast-like cells in the AT and CT (BMI<25: 7.60 vs. BMI≥25: 25.39 in total; p = 0.001). Moreover, expression of CD36 (BMI<25: 2.15 vs. BMI≥25: 2.60; p = 0.041) and ANGPTL4 (BMI<25: 6.00 vs. BMI≥25: 9.80; p = 0.026) was elevated in the TNBC cells of overweight/obese patients. Conclusions: Our data suggest BMI-related changes in the TME of overweight/obese TNBC patients, including hypertrophied adipocytes, reduced vascularization, more M2-like macrophages and CAF-like cells, and an increase in the expression of fatty acid metabolizing proteins in marginal tumor cells, all contributing to a more tumor-promoting, immunosuppressive environment.

Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations.

Obesity is an established risk factor for breast cancer development and worsened prognosis; however, the mechanisms for this association - and the potential benefits of weight loss - have not been fully explored. The adipose environment surrounding breast tumors, which is inflamed in obesity, has been implicated in tumor progression. An emerging therapeutic target for cancer is TREM2, a transmembrane receptor of the immunoglobulin superfamily that is expressed on macrophages in adipose tissue and tumors. We utilized genetic loss of function (Trem2 +,+ and Trem2 -/-) models and dietary (lean, obese, and weight loss) intervention approaches to examine impacts on postmenopausal breast cancer. Remarkably, Trem2 deficiency ameliorated tumor growth in lean, but not obese or weight loss mice. Single-cell RNA sequencing, in conjunction with VDJ sequencing of tumor and tumor-adjacent mammary adipose tissue (mATTum-adj) immune cells, revealed that tumors of lean Trem2 -/- mice exhibited a shift in clonal CD8+ T cells from an exhausted to an effector memory state, accompanied with increased clonality of CD4+ Th1 cells, that was not observed in any other diet-genotype group. Notably, identical T cell clonotypes were identified in the tumor and mATTum-adj of the same mouse. Finally, an immune checkpoint study demonstrated that αPD-1 therapy restricted tumor growth in lean and weight loss, but not obese mice. We conclude that weight history is relevant when considering potential efficacy of TREM2 inhibition in postmenopausal breast cancer. This work reveals immunological interactions between tumors and surrounding adipose tissue, highlighting significant differences under obese and weight loss conditions.

Cancer cell migration depends on adjacent ASC and adipose spheroids in a 3D bioprinted breast cancer model

Breast cancer develops in close proximity to mammary adipose tissue and interactions with the local adipose environment have been shown to drive tumor progression. The specific role, however, of this complex tumor microenvironment in cancer cell migration still needs to be elucidated. Therefore, in this study, a 3D bioprinted breast cancer model was developed that allows for a comprehensive analysis of individual tumor cell migration parameters in dependence of adjacent adipose stroma. In this co-culture model, a breast cancer compartment with MDA-MB-231 breast cancer cells embedded in collagen is surrounded by an adipose tissue compartment consisting of adipose-derived stromal cell (ASC) or adipose spheroids in a printable bioink based on thiolated hyaluronic acid. Printing parameters were optimized for adipose spheroids to ensure viability and integrity of the fragile lipid-laden cells. Preservation of the adipogenic phenotype after printing was demonstrated by quantification of lipid content, expression of adipogenic marker genes, the presence of a coherent adipo-specific extracellular matrix, and cytokine secretion. The migration of tumor cells as a function of paracrine signaling of the surrounding adipose compartment was then analyzed using live-cell imaging. The presence of ASC or adipose spheroids substantially increased key migration parameters of MDA-MB-231 cells, namely motile fraction, persistence, invasion distance, and speed. These findings shed new light on the role of adipose tissue in cancer cell migration. They highlight the potential of our 3D printed breast cancer-stroma model to elucidate mechanisms of stroma-induced cancer cell migration and to serve as a screening platform for novel anti-cancer drugs targeting cancer cell dissemination.

Specificities of mammary and periprostatic adipose tissues: A perspective from cancer research

In addition to the major subcutaneous and visceral adipose tissues (AT), other adipose depots are dispersed throughout the body and are found in close interaction with proximal organs such as mammary and periprostatic AT (MAT and PPAT respectively). These ATs have an effect on proximal organ function during physiological processes and diseases such as cancer. We highlighted here some of their most distinctive features in terms of tissular organization and responses to external stimuli and discussed how obesity affects them based on our current knowledge.

Abstract PO3-13-08: D17104: Aryl Hydrocarbon Receptor Signaling and Other Biomarkers of Fat Metabolism in Human Mammary Adipose Tissue from Women with Malignant Breast Disease

Abstract Background: Obesity is a recognized risk factor for the development of metabolic disease and breast cancer possibly due to chronic inflammation mediated by cytokines secreted by adipose tissue. Given this, our group examined differential expression of several markers of chronic inflammation in adipose tissue of both benign and malignant breast tissue from patients. The aryl hydrocarbon receptor (AhR) is an important cytosolic, ligand-dependent transcription factor found within hepatocytes and adipocytes that is upregulated in many types of cancer. AhR promotes the initiation, progression, and metastasis of cancer cells allowing certain cancers to evade immune recognition (Xue et al. Front. Immunology 2018). Indoleamine 2,3-dioxygenase 1 (IDO1) is an enzyme that generates kynurenine (KYN) from tryptophan (TRP), which acts as an AhR agonist. We sought to determine expression of IDO1 via immunohistochemistry (IHC) in mammary adipose tissue associated with benign or malignant breast. We hypothesized that adipose tissue adjacent to malignant breast tissue may have heightened expression of IDO1 in addition to Cyp1b1, and SPP1; both of which are also markers of increased aryl-hydrocarbon transcriptional activity, compared to adipose tissue noncontiguous to malignant lesions. KYN affects gene expression for insulin resistance and levels are elevated in plasma of older obese women compared to lean women; however, the relationship to breast malignancy is not known. Methods: IDO1 (Cell Signaling, 1:200), Cyp1b1 (Thermo Fisher, 1:300), and SPP1 (Thermo Fisher, 1:200) IHC was performed according to the manufacturers' protocols. Adipose tissue immediately adjacent to, and >2 mm away from, tumors (three DCIS, 11 invasive carcinomas) was evaluated in 14 patients. Adipose tissue in separate tissue blocks from benign breast tissue (three ipsilateral, 11 contralateral) in the same patients was also evaluated. Human plasma KYN levels were assayed by HPLC in the Dartmouth Clinical Pharmacology Shared Resource as described previously (Rojas et al. Obesity 2021). Results: In this preliminary pilot study, there was no difference in staining pattern for IDO1 in adipose tissue present in malignant and benign samples. Two patients showed increased Cyp1b1 and SPP1 staining immediately adjacent to the invasive carcinoma (Figure 1). Patients with benign breast disease did not show increased Cyp1b1 or SPP1 staining. Results will be confirmed using qPCR for mRNA. Plasma samples from patients with benign disease showed a lower level of KYN expression on average (0.370 ug/mL, N = 8) compared to plasma from patients with malignant disease (0.424, N = 10). Additionally, the ratio of KYN:TRP was lower in the benign group (0.065) compared to the cohort with malignancy (0.080). Univariate linear regression will be used to further assess relationships between continuous variables in the study population (e.g., patient age, BMI, KYN plasma levels, benign vs malignant breast disease). Regression lines and the associated slope estimates (b-value) and p-values for regression models will be provided on scatter plots. Conclusions: While there was no difference in IDO1 staining patterns in adipose tissue of malignant and benign samples in this group, tissue samples from patients with invasive carcinoma demonstrated increased Cyp1b1 and SPP1 staining adjacent to the malignant tumor; while benign tissue samples did not show a positive stain. This suggests increased aryl-hydrocarbon transcriptional activity in invasive breast carcinoma—further linking AhR signaling with adipose tissue immune dysregulation and invasive malignancy. Additionally, KYN plasma levels were higher in patients with malignant disease indicating a possible relationship between insulin resistance and breast malignancy. Additional research may offer new insights relating to this important pathway and ultimately treatments aimed at blunting its nefarious effects. Citation Format: Adam Ephraim, Jonathan Pirruccello, Scott Palisoul, Jonathan Marotti, Mary Chamberlin. D17104: Aryl Hydrocarbon Receptor Signaling and Other Biomarkers of Fat Metabolism in Human Mammary Adipose Tissue from Women with Malignant Breast Disease [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 PO3-13-08.

Molecular Characteristics and Polymorphisms of Buffalo (Bubalus bubalis) ABCG2 Gene and Its Role in Milk Fat Synthesis.

The ATP-binding cassette subfamily G member 2 (ABCG2) serves crucial roles in secreting riboflavin and biotin vitamins into the milk of cattle, mice, and humans, as well as in the transportation of xenotoxic and cytostatic drugs across the plasma membrane. However, the specific role of the ABCG2 gene in water buffaloes (Bubalus bubalis), especially its effect on milk fat synthesis in buffalo mammary epithelial cells (BuMECs), remains inadequately understood. In this study, the full-length CDS of the buffalo ABCG2 gene was isolated and identified from the mammary gland in buffaloes. A bioinformatics analysis showed a high degree of similarity in the transcriptional region, motifs, and conservative domains of the buffalo ABCG2 with those observed in other Bovidae species. The functional role of buffalo ABCG2 was associated with the transportation of solutes across lipid bilayers within cell membranes. Among the 11 buffalo tissues detected, the expression levels of ABCG2 were the highest in the liver and brain, followed by the mammary gland, adipose tissue, heart, and kidney. Notably, its expression in the mammary gland was significantly higher during peak lactation than during non-lactation. The ABCG2 gene was identified with five SNPs in river buffaloes, while it was monomorphic in swamp buffaloes. Functional experiments revealed that ABCG2 increased the triglyceride (TAG) content by affecting the expression of liposynthesis-related genes in BuMECs. The results of this study underscore the pivotal role of the ABCG2 gene in influencing the milk fat synthesis in BuMECs.

Pathomorphological Changes in Mammary Glands of 16 Patients Who Underwent Surgical Aquafilling® Removal: A Single-Center Study.

BACKGROUND Aquafilling® is a soft-tissue filler used in various procedures, including breast plastic surgery. Proponents claim it to be safe and effective without causing serious adverse effects. This study aimed to describe histological changes in breast tissue resulting from potentially harmful effects of Aquafilling®. MATERIAL AND METHODS Tissue samples were collected from 16 patients who underwent surgical removal of Aquafilling®. Histopathological evaluations were performed on hematoxylin and eosin-stained slides, with photographs captured using an Olympus BX 43 light microscope and an XC 30 digital camera at 40×, 100×, and 400× total magnification. RESULTS Inflammatory infiltrates, mainly consisting of macrophages and lymphocytes, were observed in the images. Tissue necrosis was visible in some areas. Fibrosis foci and blood vessels with thickened walls and detached endothelium were identified within mammary adipose tissue. CONCLUSIONS Due to the variety of clinical symptoms and presence of inflammation in all examined women, we recommend histopathological examinations for all cases of Aquafilling® surgical removal. The examination should include information on the extent of inflammation, progression of adipose and muscle tissue damage, and assessment of fibrosis severity. This will help clinicians make informed decisions about Aquafilling® use in patients and improve patient outcomes.

NCoA3 upregulation in breast cancer‑associated adipocytes elicits an inflammatory profile.

Nuclear receptor coactivator 3 (NCoA3) is a transcriptional coactivator of NF‑κB and other factors, which is expressed at relatively low levels in normal cells and is amplified or overexpressed in several types of cancer, including breast tumors. NCoA3 levels have been shown to be decreased during adipogenesis; however, its role in tumor‑surrounding adipose tissue (AT) remains unknown. Therefore, the present study assessed the modulation of NCoA3 in breast cancer‑associated adipocytes and evaluated its association with the expression of inflammatory markers. 3T3‑L1 adipocytes were stimulated with conditioned medium from human breast cancer cell lines and the expression levels of NCoA3 were evaluated by reverse transcription‑quantitative (q)PCR. NF‑κB activation was measured by immunofluorescence, and tumor necrosis factor and monocyte chemoattractant protein 1 levels were analyzed by qPCR and dot blot assays. The results obtained from the invitro model were supported using mammary AT (MAT) from female mice, MAT adjacent to tumors from patients with breast cancer and bioinformatics analysis. The results revealed that adipocytes expressing high levels of NCoA3 were mainly associated with a pro‑inflammatory profile. In 3T3‑L1 adipocytes, NCoA3 downregulation or NF‑κB inhibition reversed the expression of inflammatory molecules. In addition, MAT from patients with a worse prognosis exhibited high levels of this coactivator. Notably, adipocyte NCoA3 levels could be modulated by inflammatory signals from tumors. The modulation of NCoA3 levels in synergy with NF‑κB activity in MAT in a tumor context could be factors required to establish breast cancer‑associated inflammation. As adipocytes are involved in the development and progression of breast cancer, this signaling network deserves to be further investigated to improve future tumor treatments.

Abstract P3-05-28: Impact of increased adiposity on extended aromatase inhibitor treatment in postmenopausal patients with estrogen receptor positive (ER+) breast cancer: a retrospective analysis of the SOLE trial

Abstract Background: The risk of late distant recurrence (≥ 5 years) in post-menopausal patients with ER+ BC remains significant even after standard adjuvant endocrine therapy (ET). Obesity, which has reached high proportions in Western Countries, is one of the modifiable factors that can contribute to BC recurrences. While body mass index (BMI) is an easily available surrogate measure of adiposity, it might not reflect accurately patient adiposity, especially in post-menopausal patients. We therefore hypothesize that mammary adipocyte measurements might better capture the adiposity of the patient. In this study, we retrospectively evaluated the prognostic role of both BMI and adipocyte measurements in the SOLE trial. Patients & Methods: The SOLE trial (NCT00553410) consisted of 4,884 postmenopausal women with hormone receptor positive, lymph node-positive BC treated with 5 years of letrozole after having completed 4-6 years of adjuvant ET. BMI was available for 3,606 patients with ER+/HER2- BC and categorized according to the WHO classification (underweight: < 18.5, normal weight 18.5 – 24.9, overweight 25 – 29.9, obesity ≥ 30 kg/m2). Considering the small sample size, patients with underweight (n=35) were grouped together with patients with normal weight for the subsequent analyses. We evaluated the association between clinico-pathological characteristics and BMI using Fisher exact test and Kruskal-Wallis test. Given the previously reported association of BMI with distant recurrences (PMID: 21115856), we considered distant-relapse free interval (DRFI) as the main study endpoint. Cox regression analyses were adjusted for histology, chemotherapy (yes/no), type of local therapy, in addition to previously reported clinico-pathological variables (PMID: 29158011). Then, we conducted a stratified case-cohort study consisting of 311 patients (97 with distant relapse-cases) with successful digital assessment of adipocytes on normal mammary adipose tissue sample collected at surgery, as previously described (PMID: 33120083). Stratification factors were treatment arm and type of prior ET. Weighted Cox regression models (PMID: 17554753, 18712477) were used to evaluate prognostic association with adipocyte size (area in µm²) categorized in quartile of the 75th percentile (Q4 vs Q1-3), as done previously (PMID: 33753731). Results: 1392 (39%) patients were normal weight, 1315 (36%) were overweight and 899 (25%) were obese. Older age at diagnosis, lymph node involvement (≥4), large tumor size and menopausal status were associated with overweight and obesity. After a median follow-up of 84 months, no associations were found between DRFI and BMI in the univariable and multivariable analysis (adjusted HR overweight vs normal=0.99, 95%CI: 0.76 – 1.29, p=0.977 and HR obese vs normal=1.11, 95%CI: 0.84 - 1.47, p=0.425). In the case-cohort study, patients with larger adipocytes had an increased hazard of distant recurrence in the univariable and multivariable analysis (see table 1). Conclusion: In this study, we did not detect any association between BMI and survival outcomes. We however found that larger mammary adipocytes were independently associated with an increased hazard of distant recurrence. These results suggest that mammary adipocytes, which can be easily evaluated on H&E slides using digital pathology, should be considered as a novel marker to evaluate the risk of late recurrence in patients with breast cancer. Citation Format: Edoardo Isnaldi, François Richard, Giuseppe Marano, Patrizia Boracchi, Marion Maetens, Giuseppe Floris, Guy Jerusalem, Andrea Gombos, Alastair M. Thompson, Erika Hitre, Stefan Aebi, Marco Colleoni, Patrizia Dell’Orto, Roswitha Kammler, Patrick Neven, Giuseppe Viale, Meredith Regan, Elia Biganzoli, Christine Desmedt. Impact of increased adiposity on extended aromatase inhibitor treatment in postmenopausal patients with estrogen receptor positive (ER+) breast cancer: a retrospective analysis of the SOLE trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-28.

Abstract PD12-05: PD12-05 Adipocyte directed vaccination to reduce the risk of breast cancer

Abstract Background: Obesity is an important risk factor for breast cancer and women with metabolic syndrome may be at the highest risk. Infiltration of CD8 T-cells into fat is an early event in obesity. Type I cytokines secreted by CD8 T-cells upregulate costimulatory molecules on enlarged adipocytes. The adipocytes, now antigen presenting cells, further stimulate Type I T-cell activation. The resulting T-cells compete for glucose and fatty acids which leads to metabolic dysfunction in both the adipose tissue and the T-cells themselves. The T-cells are not able to maintain tumor immune surveillance and secretion of adipokines promotes malignant transformation. Immunologic memory prevents inflammation from resolving even if an individual becomes normal weight. Strategies to increase Type II (anti-inflammatory) T-cells in inflamed adipose could have clinical benefit. Methods: We developed a method of CD4 epitope identification that includes functional screening for Th1 or Th2 epitopes. We use a multi-algorithm approach to ensure responsiveness across diverse HLA alleles. We identified Th2 selective epitopes associated with high IL-10 secretion for 6 adipocyte associated antigens that become overexpressed in inflamed adipocytes (IGF-IR, HIF-1a, DUSP1, FABP4, PAI-1 and ATGL). The epitopes were highly homologous between mouse and man (median 100% (range-82-100%). When the epitopes were used to immunize mice, all antigens generated a significant IL-10 response compared to control (p< 0.05). We questioned whether our “adipocyte directed” vaccine (ADVac) could prevent the development of breast cancer in obese mice. Results: First, C57BL/6 mice were fed a high fat high sucrose (HFHS) diet or normal chow. When mice became obese, vaccination with ADVac or adjuvant alone (Alum) was initiated. Four weeks after the final vaccine, visceral adipose tissue showed significantly fewer CD8 T-cells in the obese mice immunized with ADVac as compared to the control, p=0.0011. The decrease in CD8 T-cells was specific for adipose tissue as no change was observed in matched spleen. There was a significant increase in T-regulatory cells in the adipose tissue of mice immunized with ADVac as compared to control, p=0.031. Two weeks after the final vaccine, a glucose tolerance test (GTT) and insulin tolerance (ITT) showed blood glucose concentrations were significantly lower at all time points for the ADVac-immunized obese mice as compared to the control obese mice (p< 0.01 for all). TgMMTV-neu develop aggressive breast cancer when made obese. Ten-week old TgMMTV-neu mice were fed a HFHS diet for 4 weeks, then randomized into 2 cohorts when obese, one cohort receiving the adjuvant only (Alum) and one receiving ADVac. Mice were sacrificed at 31 weeks of age when all controls had developed tumor. ITT showed the glucose levels in the blood were significantly lower in the ADVac group as compared to control (p< 0.0001). Fewer CD8 T-cells were observed in mammary adipose tissue of AdVac immunized mice compared to control (p=0.001). There was significantly less leptin detected in the serum of ADVac vaccinated mice compared to Alum immunized, p=0.024. The median age of tumor development was 25 weeks in controls and 29 weeks in the immunized group (p=0.009). Sixty percent (9/15) of the vaccinated mice were tumor free at study termination, whereas 100% of the control mice had developed tumor. Conclusions: ADVac represents the first vaccine to lower breast cancer risk in obesity. Vaccination corrected metabolic dysfunction as evidenced by reversal of diabetes and prevented breast cancer in the majority of obese ADVac immunized mice. Further studies are ongoing evaluating the systemic distribution of ADVac specific T-cells and the safety of the approach. Citation Format: Mary Disis, Lauren Corulli, Erin R. Rodmaker, Denise Cecil. PD12-05 Adipocyte directed vaccination to reduce the risk of breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD12-05.

Maternal Adipocyte Connexin43 Gap Junctions Affect Breastmilk Lactose Levels and Neonate Growth in Mice.

Simple SummaryBreastfeeding offers many health benefits for both mothers and infants. However, overnutrition and a steady increase in obesity in the U.S. has made it harder for many mothers to produce and express breastmilk. Moreover, the quality of breastmilk from obese mothers is frequently compromised in that it contains fewer nutrients and more inflammatory components. In this study, we used mice to model this phenomenon. We found that short-term high-fat feeding at the start of breeding reduces litter size and pups’ body weight. It also impairs adipocyte remodeling during lactation. Connexin43 is the primary building block for gap junctions in the adipose tissue. It is postulated to play an essential role in adipose tissue remodeling to accommodate mammary gland development and breastmilk production. Using genetically engineered mice without Connexin43 in their adipocytes, we demonstrated that the deletion of adipocyte Connexin43 affects the disappearance of adipocytes during lactation and affects milk composition, which is postulated to impair the pups’ growth. Altogether, this study suggests that increasing or enhancing adipocyte Connexin43 gap junctions may help obese mothers produce better breastmilk to support their neonates.Breastfeeding offers a broad spectrum of health benefits for infants. However, overnutrition and a steady increase in maternal obesity in the U.S. have made it harder for many mothers to produce and express breastmilk, and the quality of milk from obese mothers is also frequently compromised. Adipocytes, the primary cell type in the non-lactating breast, display a drastic morphological and functional change during lactation in mice. Lipid-filled adipocytes undergo lipolysis, and lipid droplets disappear to provide fatty acids and energy for breastmilk production. Once the animal stops lactation, these lipid-depleted adipocytes return as lipid-laden cells. This dynamic remodeling of the tissue is likely the result of active intercellular communications. Connexin43 (Cx43) is the most abundant connexin in the mammary adipose tissue that makes up the gap junctions for direct intercellular communications. Its expression is increased during lactation and reduced in obese mammary adipose tissue, which is resistant to lactation-induced remodeling. However, whether Cx43 is required for adipocyte remodeling and breastmilk production to support neonates’ growth has not been established. In this study, we used doxycycline-inducible adipocyte-specific Cx43-deleted mice and demonstrated that adipocyte Cx43 played a vital role in determining the carbohydrate levels in breastmilk, which may subsequently affect neonates’ growth.

Abstract 6147: Immune population changes in mammary adipose tissue during the development of obesity and their influence on triple negative breast cancer progression

Abstract Obesity is associated with increased risk for many cancer types, including triple-negative breast cancer (TNBC). In addition to obesity’s role in TNBC pathogenesis, it is also recognized as a marker of poor prognosis for survival in pre- and post-menopausal women. However, the mechanism underlying how obesity leads to worsened TNBC progression remains unclear. In this study, we aim to characterize the immune population changes in mammary adipose during the development of obesity and their impact on TNBC progression. First, we utilized a diet-induced obesity mouse model to evaluate tumor growth under obese condition. Four weeks after E0771 (TNBC cell line) mammary fat pad injection, TNBC tumors volume were 1.6-fold larger in obese C57BL/6 mice compared to their lean counterparts. To profile the transcriptome changes induced by obesity, we performed single-cell RNA-Seq on mammary adipose stromal vascular fraction and TNBC allograft from lean and obese C57BL/6 female mice. In TNBC allografts, obesity upregulated genes enriched in the extracellular matrix and epithelial-mesenchymal transition pathways in cancer cells, creating a more aggressive phenotype. Consistent with the heavier TNBC tumor burden, lymphocyte infiltration decreased while myeloid population increased in TNBC allografts from obese mice, which indicated an immunosuppressed tumor microenvironment. Macrophage was the dominant population among Cd45+ cells in tumors, constituting 41.2% in lean and 51.2% in obese mice. An increase of adipose tissue macrophage (ATM) population (2.2-fold increase in obese vs lean adipose; 5.6-fold increase in tumor-bearing vs control adipose) was also observed in the obese or tumor-bearing mammary adipose stroma. Using Gene Set Enrichment Analysis (GSEA), we found that ATM adapted to the obese condition by upregulating fatty acid metabolism and adipogenesis hallmarks while downregulating IFNγ response, allograft rejection, and TNFα signal hallmarks. When encountering TNBC development, ATM downregulated similar proinflammatory pathways, while upregulated the hypoxia and glycolysis pathways. Through integrative analysis, we found that the complement pathway is the primary pathway upregulated in ATM under both obese and tumor growth conditions. Major components of the complement pathway, C1qa and C1qb, were significantly upregulated in ATM by TNBC and obesity. Interestingly, C1qa and C1qb were also upregulated in bone-marrow derived macrophage during M2 polarization and when co-culturing with E0771. Taken together, our data showed that the C1qahi/C1qbhi macrophage expanded in mammary adipose tissue during the development of obesity and TNBC, which created an immunosuppressed microenvironment to facilitate TNBC progression. This suggests that the complement pathway may serve as a potential immune therapy target for obese TNBC patients. Citation Format: Shimeng Liu, Yi Zhang, Myles Brown. Immune population changes in mammary adipose tissue during the development of obesity and their influence on triple negative breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6147.

Abstract 2485: Applications of multimodality ex-vivo tissue imaging to improve breast cancer diagnosis and treatment

Abstract Breast cancer is the most common malignancy affecting women worldwide. Existing models being used to study breast cancer include the Patient-Derived Tumor Organoid (PDTO) Model and the Patient-Derived Tumor Xenograft (PDTX) Model. Both models involve the surgical removal of a tumor from a patient which then undergoes processing to grow an organoid or injected into mice to test different therapies outside the body. However, these models not only take a considerable amount of time to develop properly, but in many cases aren’t representative of the most aggressive tumor cells present within a heterogeneous tissue sample in order to draw the most clinically accurate conclusions of a tumor's response to therapy. In order to advance clinical research, more efficient culture systems that are faster growing and more representative of biological characteristics in-vivo are needed. Previous work from the Karczmar Lab discovered that high-resolution magnetic resonance imaging (MRI) can detect regions of aggressive cancer in biopsy specimens from mouse models, and give additional information on characteristics including cell density, tissue composition, and cancer stage. With this recent discovery, it is hypothesized that ex-vivo imaging could be used to identify aggressive tumor cells in human heterogeneous breast cancer tissue samples to produce higher quality and faster-growing models for testing new therapies and guiding individual patient therapy for breast cancer. The primary aim for this study was to specifically identify image-based markers for regions of aggressive cancer by comparing ex-vivo high-resolution MRI with histology and immunohistochemistry in 3D. Distant normal (DN) and tumor (Tu) tissue samples from breast cancer patients were obtained and imaged using T2-weighted 3D Rapid Imaging with Refocused Echoes (RARE) and stained using hematoxylin and eosin (H&E), commonly used by pathologists to diagnose cancers. Then, the ex-vivo T2-weighted 3D RARE images were correlated to the histopathological H&E staining and confirmed by breast radiologists and pathologists. Preliminary results revealed that high-resolution MR imaging detects mammary glands, ducts, and white adipose tissue in DN tissue samples and regions of invasive cancer, including ductal carcinoma in-situ (DCIS) and necrosis, in Tu tissue. Based on these results, aggressive cancer regions were proven to be identified using image-based guidance and potentially be extracted using core-needle biopsy to improve PDTO and PDTX models for studying breast cancer and advancing individual patient therapy. Citation Format: Corazon Avila, Gregory Karczmar, Devkumar Mustafi. Applications of multimodality ex-vivo tissue imaging to improve breast cancer diagnosis and treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2485.

Abstract 2518: Elucidating the impact of obesity and weight loss on breast cancer tumor progression

Abstract Obesity is an established risk factor for breast cancer and worsened prognosis, however the mechanisms for this link have not been fully elucidated. Additionally, the consequence of weight loss (WL) prior to the diagnosis of cancer has not been fully explored. One possible link is via the immune system. Obesity is associated with metabolic dysfunction as well as inflammation of the adipose tissue. While WL is a great tool to combat metabolic dysfunction, we have shown that following WL the adipose tissue does not properly reprogram its immunophenotype and remains in a heightened inflammatory state. Accompanying the heightened inflammatory state and increased immune infiltration found in obesity, a novel subset of macrophages termed “lipid associated macrophages” (LAMs) with high expression of triggering receptor expressed on myeloid cells 2 (Trem2) have been discovered in adipose tissue of obese mice and humans. Data from our lab shows LAMs are found at low levels in the adipose tissue of lean mice and enhanced in weight gain (WG) and WL mice. While other investigators have shown Trem2-expressing tumor associated macrophages contribute to immunosuppression in the tumor microenvironment, the implications of enhanced Trem2+ LAMs found in obesity and WL have not been explored in the context of cancer. The purpose of this study is to elucidate the impact of obesity and WL on breast cancer tumor progression. Female mice were ovariectomized to model post-menopause and to enable greater weight gain. High fat diet (HFD) and low fat diet (LFD) feeding paradigms were used to produce three diet groups: lean, WG, and WL. Lean mice were maintained on LFD for the entirety of the study. WG mice were fed LFD for 12 weeks followed by HFD for 12 weeks. Mice in the WL group were fed HFD for 12 weeks to establish obesity and then fed LFD for 12 weeks to achieve WL. Four weeks prior to the end of the dietary paradigm, mice were injected with E0771 mammary cancer cells into the mammary fat pad. We confirmed that obesity drives accelerated tumor growth progression resulting in significantly increased tumor mass. We found that WL results in an intermediate phenotype between the lean and obese models, suggesting WL may provide some protection to tumor growth, but does not restore the tumor-bearing host to the same level of protection as a consistently lean model. We also demonstrated significantly increased gene expression of both Adgre1 (gene for F4/80) and Trem2 in obese ovarian and mammary adipose tissue compared to lean adipose tissue. Future studies will focus on understanding the mechanistic links between heightened inflammation in obese and WL adipose tissue and tumor progression. Additionally, we aim to elucidate the contribution of Trem2-expressing immune cells to tumor progression in the obese and WL contexts. Citation Format: Elysa M. Wolf, Barbara Fingleton, Alyssa H. Hasty. Elucidating the impact of obesity and weight loss on breast cancer tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2518.

Myoglobin expression by alternative transcript in different mesenchymal stem cells compartments

BackgroundThe metabolic phenotype of stem cells is increasingly recognized as a hallmark of their pluripotency with mitochondrial and oxygen-related metabolism playing a not completely defined role in this context. In a previous study, we reported the ectopic expression of myoglobin (MB) in bone marrow-derived hematopoietic stem/progenitor cells. Here, we have extended the analysis to mesenchymal stem cells (MSCs) isolated from different tissues.MethodsMSCs were isolated from human placental membrane, mammary adipose tissue and dental pulp and subjected to RT-PCR, Western blotting and mass spectrometry to investigate the expression of MB. A combination of metabolic flux analysis and cyto-imaging was used to profile the metabolic phenotype and the mitochondria dynamics in the different MSCs.ResultsAs for the hematopoietic stem/progenitor cells, the expression of Mb was largely driven by an alternative transcript with the protein occurring both in the monomer and in the dimer forms as confirmed by mass spectrometry analysis. Comparing the metabolic fluxes between neonatal placental membrane-derived and adult mammary adipose tissue-derived MSCs, we showed a significantly more active bioenergetics profile in the former that correlated with a larger co-localization of myoglobin with the mitochondrial compartment. Differences in the structure of the mitochondrial network as well as in the expression of factors controlling the organelle dynamics were also observed between neonatal and adult mesenchymal stem cells. Finally, the expression of myoglobin was found to be strongly reduced following osteogenic differentiation of dental pulp-derived MSCs, while it was upregulated following reprogramming of human fibroblasts to induce pluripotent stem cells.ConclusionsEctopic expression of myoglobin in tissues other than muscle raises the question of understanding its function therein. Properties in addition to the canonical oxygen storage/delivery have been uncovered. Finding of Mb expressed via an alternative gene transcript in the context of different stem cells with metabolic phenotypes, its loss during differentiation and recovery in iPSCs suggest a hitherto unappreciated role of Mb in controlling the balance between aerobic metabolism and pluripotency. Understanding how Mb contributes through modulation of the mitochondrial physiology to the stem cell biology paves the way to novel perspectives in regenerative medicine as well as in cancer stem cell therapy.Graphical abstract

Novel Molecular Insight into the ESR1 Mutant/Adipocytes Crosstalk: a Pivotal Contribute of IGF1‐R

BackgroundObesity is considered a global epidemic and an important public health concerns worldwide. This medical condition is also recognized a risk factor for various diseases, including cancers. Epidemiological studies have reported a higher risk of breast cancer (BC) in obese women compared to normal‐weight ones. In fact, obesity may sustain BC development and progression and also, is considered a heavyweight player driving endocrine resistance. Adipocyte secretome‐factors, insulin resistance, hyper activation of insulin‐like growth factor (IGF‐1/IGF‐1R) pathways, and increased levels of estrogen are the mechanisms through which obesity impacts BC biology but there is a huge knowledge gap on the obesity/acquired endocrine resistance relationship. ESR1 mutations (ESR1m), mainly found in metastatic tumors, contribute to the acquired resistance through documented IGF‐1R signaling pathway altered activation. Thus, we evaluated the effects of adipocytes in influencing the behavior of BC cell expressing the most frequent ESR1m, Y537S, wherein the specific role of IGF‐1/IGF‐1R axis will be investigated.MethodsWe employed CRISPR/Cas9‐engineered human breast cancer cells (MCF‐7) and murine‐derived cells (mMTC2) to express Y537S mutation and 3T3‐L1 as adipocyte‐like cells. As ex vivo model we used human adipocytes (hAdipo) isolated from mammary adipose tissue biopsies of healthy women with varying body mass index. We performed co‐culture experiments testing the effects of adipocytes on BC cell behaviour in the presence or not of a specific IGF1R inhibitor, the GSK1838705A.ResultsConditioned media (CM) derived from mature adipocytes (3T3L1‐A) impacts BC cells behavior by enhancing growth, migration and invasion of MCF‐7 and mMCT2, but in a higher extent in the Y537S mutant clones. The latter effects appear to be amplified upon the treatment with CM derived from ex vivo model of hAdipo. In line with these data, microarray analysis evidenced that mutant BC cells exhibited higher mRNA levels of many essential mediators of tumor‐stroma interplay compared to parental cells. Among them, the most significant up‐regulated gene was IGF‐1R. These data have been confirmed by qRT‐PCR. Remarkably, the treatment with GSK1838705A reversed the enhanced growth, migration and invasion only in mutant cells. These data highlight the pivotal role of IGF‐1R axis in the ESR1m cells/tumor microenvironment interactions.ConclusionsOur data clearly demonstrated that adipocytes, the major compartment of tumor stroma, further sustain the aggressive malignant phenotype of mutant cells, mainly involving IGF‐1R signaling pathway. Prospectively our findings address the use of an IGF‐1R inhibitor to block the mutant/stroma cells interplay. In the era of precision medicine, the use of a specific IGF1R inhibitor may help to improve the outcome of BC patients expressing the ESR1 mutations, particularly in obese setting.

Local Biomarkers Involved in the Interplay between Obesity and Breast Cancer.

Simple SummaryBreast cancer is the second most common cancer in women worldwide. The risk of developing breast cancer depends on various mechanisms, such as age, heredity, reproductive factors, physical inactivity, and obesity. Obesity increases the risk of breast cancer and worsens outcomes for breast cancer patients. The rate of obesity is increasing worldwide, stressing the need for awareness of the association between obesity and breast cancer. In this review, we outline the biomarkers—including cellular and soluble factors—in the breast, associated with obesity, that affect the risk of breast cancer and breast cancer prognosis. Through these biomarkers, we aim to better identify patients with obesity with a higher risk of breast cancer and an inferior prognosis.Obesity is associated with an increased risk of breast cancer, which is the most common cancer in women worldwide (excluding non-melanoma skin cancer). Furthermore, breast cancer patients with obesity have an impaired prognosis. Adipose tissue is abundant in the breast. Therefore, breast cancer develops in an adipose-rich environment. During obesity, changes in the local environment in the breast occur which are associated with breast cancer. A shift towards a pro-inflammatory state is seen, resulting in altered levels of cytokines and immune cells. Levels of adipokines, such as leptin, adiponectin, and resistin, are changed. Aromatase activity rises, resulting in higher levels of potent estrogen in the breast. Lastly, remodeling of the extracellular matrix takes place. In this review, we address the current knowledge on the changes in the breast adipose tissue in obesity associated with breast cancer initiation and progression. We aim to identify obesity-associated biomarkers in the breast involved in the interplay between obesity and breast cancer. Hereby, we can improve identification of women with obesity with an increased risk of breast cancer and an impaired prognosis. Studies investigating mammary adipocytes and breast adipose tissue in women with obesity versus women without obesity are, however, sparse and further research is needed.

A nomogram to predict the value of mammary adipose index (MAI) for prediction of response to neoadjuvant chemotherapy in breast cancer patients.

e12607 Background: Breast cancer (BCa) is the most common malignancy in females on a global scale. Neoadjuvant chemotherapy (NACT) is a promising therapeutic approach for the treatment of locally advanced BCa, which result in primary tumor regression and optimal surgical planning. Cumulative evidence has identified a deleterious crosstalk between cancer cells and the surrounding mammary adipose tissue during tumor progression. Mammary adipose index (MAI) is a novel indicator defined by us, which represents the percentage of adipose tissue in breast. Therefore, this study aims to investigate the correlation between MAI and NACT efficacy of BCa by establishing an intuitive nomogram. Methods: Clinical data of 221 BCa patients who received NACT at the First Affiliated Hospital of Nanjing Medical University from January 1, 2018 to May 31, 2020 were retrospectively collected. Besides, patients were divided into major histological response and non-major histological response group in line with Miller-Payne pathology grading. The level of MAI was collected from magnetic resonance imaging of breast, and was divided into two groups according to the cut-off value. The level of MAI and clinicopathological characteristics were collected to assess the predictive roles of NACT response. Univariate analysis was performed by Chi-square test or Fisher’s exact test. Indicators with P < 0.05 were included in multivariate binary logistic regression analysis. The nomogram was constructed based on results of multivariate logistic analysis. The predictive accuracy and discriminative ability were evaluated by C-index and the calibration curve. Results: Among the 221 patients enrolled, 77 cases (34.84%) were represented by major responses of pathologic histology. In univariate analysis, the level of MAI, tumor size, node status, ER status, PR status HER-2 status and Ki-67 status were correlated with NACT. Multivariate Logistic regression analysis showed that tumor size, HER-2 status and the level of MAI were independent influencing factors for the response to NACT in BCa patients. The patients with a high level of MAI (≥0.6916) were more likely to have a worse NACT response. MAI was an independent predictor for NACT efficacy, and area under the curve of ROC, specificity and sensitivity of the predictive model were 0.805, 0.632 and 0.774 respectively. The nomogram established based on these factors showed its discriminatory ability, and the C-index for prediction was 0.881. The calibration curve indicated that the predictive ability of the nomogram was a good fit to actual observation. The difference was statistically significant (P < 0.05). Conclusions: The nomogram constructed in the present study indicated that the level of MAI could predict the efficacy of NACT in BCa patients, which might provide clinical guidance for the selection of appropriate treatment decisions.

ScRNA-seq Reveals a Role of Mammary Luminal Epithelium in Adipocyte Adaptations

Almost four decades of research suggest a dynamic role of ductal epithelial cells in adipocyte adaptation in mammary gland white adipose tissue (mgWAT), but factors that mediate such communication are not known. Here, we identify a complex intercellular crosstalk in mgWAT revealed by single-cell RNA-seq (scRNA-seq) and comprehensive data analysis suggest that epithelial luminal cells during cold exposure undergo major transcriptomic changes that lead to the expression of an array of genes that encode for secreted factors involved in adipose metabolism such as Adropin (Enho), neuregulin 4 (Nrg4), angiopoietin-like 4 (Angptl4), lipocalin 2 (Lcn2), milk fat globule-EGF factor 8 (Mfge8), Insulin-like growth factor-binding protein 1 (Igfbp1), and haptoglobin (Hp). To define the mammary epithelial secretome, we coin the phrase “mammokines”. We validated our cluster annotations and cluster-specific transcriptomics using eight different adipose scRNA-seq datasets including Tabula Muris and Tabula Muris Senis. In situ mRNA hybridization and ex vivo isolated mgWAT luminal cells show highly localized expression of mammokines in mammary ducts. Trajectory inference demonstrates that cold-exposed luminal cells have similar transcriptional profiles to lactation post-involution (PI), a phase defined by reappearance and maintenance of adipocytes in the mammary gland. Concomitantly, we found that under cold exposure female mgWAT maintains more adipogenic and less thermogenic potential than male scWAT and ex vivo removal of luminal epithelial cells from mgWAT markedly potentiates beige adipocyte differentiation. Conditioned media from isolated mammary epithelial cells treated with isoproterenol suppressed thermogenesis in differentiated beige/brown adipocytes and treatment of beige/brown differentiated adipocyte with mammokine LCN2 suppresses thermogenesis and increases adipogenesis. Finally, we find that mice lacking LCN2 show markedly higher cold-dependent thermogenesis in mgWAT than controls, and reconstitution of LCN2 in the mgWAT of LCN2 knockout mice promotes inhibition of thermogenesis. These results show a previously unknown role of mammary epithelium in adipocyte metabolism and suggest a potentially redundant evolutionary role of mammokines in maintaining mgWAT adiposity during cold exposure. Our data highlight mammary gland epithelium as a highly active metabolic cell type and mammokines could have broader implications in mammary gland physiology and lipid metabolism.

Adipocyte‐derived Exosomes: Novel Insight in the Link between Obesity and Breast Cancer

Introduction Obesity has an intricate connection to both risk and clinical behaviour of breast cancer. Adipose tissue dysfunction in obesity results in a release of many adipokines that it is well known to promote breast tumor biology. In addition to soluble factors, exosomes (exo), small membrane-derived vesicles secreted by both normal and malignant cells, have been recently suggested to act as additional mediators of cell-to-cell communication. However, a clear understanding in the role of adipocyte-derived exo in mediating the adverse effects of obesity on BC is still lacking. Herein, we evaluated the involvement of adipocyte-derived exo in sustaining BC development and progression. Methods Exo were isolated and fully characterized from conditioned media (CM) of differentiated 3T3-L1 adipocytes (3T3-L1 A), a well-recognized ‘in vitro’ model of white adipocytes. We tested the effects of 3T3-L1 A exo in modulating the proliferation (MTT/soft agar assays), motility/invasion (wound healing/Boyden Chamber migration/invasion assays), and stemness (mammosphere forming efficiency/Limiting Dilution assays) of estrogen receptor α (ERα)-positive MCF-7 and triple-negative MDA-MB-231 BC cells. We also assessed co-culture experiments between BC cells and CM derived from adipocytes isolated from human mammary adipose tissues (MAT) of adult normal weight and obese women. Results The treatment with 3T3-L1 A exo significantly increased the number of colonies of MCF-7 and MDA-MB-231 cells in anchorage-independent soft agar growth assay. Also, we found that 3T3-L1 A exo induced a more aggressive phenotype in both BC cells in terms of migration, invasion and stemness. In accordance with these data, we found that 3T3-L1 A exo significantly induced the expression of the mRNA levels of genes encoding several proteins involved in Epithelial to Mesenchymal Transition (EMT) and associated with stem cell phenotype. To gain insight into the molecular mechanism we focused our attention on the expression and activity of HIF-1α, a well-known regulator of hypoxic response, involved in invasion, migration, EMT and metastasis in BC. Interestingly, 3T3-L1 A exo increased the expression of HIF-1α and its target genes, and the use of a HIF-1α translation inhibitor reduced migration and invasion capabilities induced by exo. Finally, we found that CM derived from MAT increased the migratory abilities of both employed cells in a significantly higher extent with MAT obese-CM and these effects were completely reversed in the presence of a specific exo uptake inhibitor. Conclusions Our data suggest that adipocyte exo contribute to a more aggressive BC phenotype. This may open new avenues for identifying novel specific biomarkers and innovative therapeutic strategies (i.e. HIF-1α inhibitors) in BC treatment, especially in obese patients.