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Triple-negative breast cancer (TNBC), an aggressive breast cancer subtype lacking effective targeted therapies, is considered to feature a unique cellular microenvironment with high infiltration of tumor-associated macrophages (TAM), which contribute to worsening breast cancer patient outcomes. Previous studies have shown the antitumoral actions of the dietary omega-3 docosahexaenoic acid (DHA) in both tumor epithelial and stromal components of the breast cancer microenvironment. Particularly in breast cancer cells, DHA can be converted into its conjugate with ethanolamine, DHEA, leading to a more effective anti-oncogenic activity of the parent compound in estrogen receptor-positive breast cancer cells. Here, we investigated the ability of DHEA to attenuate the malignant phenotype of MDA-MB-231 and MDA-MB-436 TNBC cell lines, which in turn influenced TAM behaviors. Our findings revealed that DHEA reduced the viability of TNBC cells in a concentration-dependent manner and compromised cell migration and invasion. Interestingly, DHEA inhibited oxygen consumption and extracellular acidification rates, reducing respiration and the glycolytic reserve in both cell lines. In a co-culture system, TNBC cells exposed to DHEA suppressed recruitment of human THP-1 cells, reduced their viability, and the expression of genes associated with TAM phenotype. Interestingly, we unraveled that the effects of DHEA in TNCB cells were mediated by reduced C-C motif chemokine ligand 5 (CCL5) expression and secretion affecting macrophage recruitment. Overall, our data, shedding new light on the antitumoral effects of DHA ethanolamine-conjugated, address this compound as a promising option in the treatment of TNBC patients.

Introduction: Our previous findings evidenced that BCCs engulf MSCs in clinical samples of breast cancer metastasis. Among the BCC-engulfing MSCs, we observed that some of them retain some markers of MSCs, resulting in the generation of hybrid cancer cell population. However, the mechanisms of hybrid cancer cell formation and the phenotypic features of hybrid cancer cells are still unclear. Here, we tested the hypothesis that hybrid cancer cells may acquire in vivo a dormant phenotype, with an increased survival advantage and chemoresistant features. Method: MSC labeled with DsRED (DsRED-MSC) were cultured with MDA-MB-231 labeled with GFP (GFP-231) to generate a hybrid cell-enriched co-culture. Live imaging microscopy, flow cytometry, cytokine array and western blot were used to characterize the hybrid cancer cells. GFP-231 were incubated with phRodo labeled MSC and subjected to phagocytosis assay. In the co-culture of DsRED-MSC with GFP-231 treated with Doxorubicin (Doxo) or untreated, we analyzed the percentage of DsRED+/GFP+ hybrid population. Hybrid-enriched co-culture or GFP-231 in single culture labeled with firefly luciferase were intracardially injected in NOD/SCID mice and monitored for metastases by bioluminescence imaging (BLI), upon Doxo treatment. After collecting the tissues at necropsy, metastases were identified by GFP fluorescence microscopy. Results: In co-cultures, DsRED+/GFP+ hybrid cells had a higher percentage of Ki-67 low in G1 and polyploidy compared to GFP-231+ cells. Hybrid cancer cells have a distinct cytokine profile than control BCCs with increased levels of senescence-associated secretory phenotype (SASP) factors. Hybrid cancer cell formation occurs through a phagocytosis-like mechanism, which involves WNT5A and MSR1. Doxo treatment increased the percentage of DsRED+/GFP+ hybrid cells, whereas reduced the percentage of GFP-231+ cells. In animal study, we observed a lower qualitative BLI intensity in mice injected with hybrid cell-enriched co-cultures compared to control. Doxo treatment increased the metastatic burden in mice inoculated with hybrid cell-enriched co-cultures compared to untreated Conclusions: MSC engulfment by BCCs results in a hybrid multinucleated cell population. Hybrid cells acquire a dormant phenotype characterized by a higher percentage of Ki-67low cells in G1 and a senescent phenotype compared to controls. Hybrid-cell-enriched co-cultures established less metastasis compared to control in vivo, but became resistant and acquire the ability to form metastasis upon doxo treatment. Citation Format: Giuseppina Augimeri, Maria E. Gonzalez, Daniela Bonofiglio, Sebastiano Andò, Celina G. Kleer. Hybrid cells generated by Mesenchymal Stem/Stromal Cell Engulfment enhance breast cancer metastasis upon Doxorubicin treatment in mouse model [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 P5-06-06.

Exosomes, small membrane vesicles secreted by both normal and malignant cells upon fusion of endosomal multivesicular bodies (MVBs) with the plasma membrane,play an important role in cell-to-cell communication. Several reports have highlighted the involvement of exosomes in many aspects of breast cancer (BC) development and progression, thus mounting interest in the potential exploitation of these vesicles and their cargoes as cancer biomarkers, drug delivery systems, and for the development of novel therapies. It has been extensively demonstrated the involvement of the obesity hormone leptin in all steps of breast tumorigenesis, but up to now its role in modulating breast cancer exosome generation has not been investigated. Here, we studied the effect of leptin on exosome biogenesis and secretion in estrogen receptor a (ERa)-positive MCF-7 and triple negative MDA-MB-231 BC cells. First, we revealed by Transmission Electron Microscopy (TEM) that the number of MVBs in the cytoplasm of leptin-treated MCF-7 and MDA-MB-231 BC cells was significantly increased compared to control untreated cells. Next, we characterized size distribution, particle number and protein cargo of exosomes, isolated by ultracentrifugation method, from conditioned media (CM) of cells treated or not with leptin. Nanoparticle Tracking Analysis revealed that the concentration of exosomes in the leptin treated MCF-7- and MDA-MB-231-CM was significantly higher compared to untreated samples. Furthermore, exosomes quantification by Acetylcholinesterase activity showed that the full leptin receptor antagonist, peptide LDFI, abrogated leptin-induced exosome secretion. Exosomes from leptin treated cells showedan increased expression of the leptin target gene Heat shock protein 90 (Hsp90) and its client protein HER2, along with activated leptin signaling effectors (pJAK2, pSTAT3, and pMAPK42/44) compared to exosomes from untreated cells. Mechanistically, our results demonstrated that, among proteins involved in exosome biogenesis, leptin significantly increased protein expression of the well-known exosomal luminal marker Tumor susceptibility gene 101 (Tsg101), without affecting its mRNA levels. Thus, we aimed to analyse specific Tsg101 protein-protein interaction as a possible mechanism able to modulate its stability or half-life within the cells. To answer to this question, we performed, in MCF-7 BC cells, co-immunoprecipitation studies combined with mass spectrometry. Analysis of the spectra identified Hsp90 as a specific Tsg101 interacting protein, and concomitantly immunoblotting assays revealed a specific interaction between HSP90 and Tsg101 in basal condition that was further increased upon leptin exposure. Accordingly, leptin-induced Tsg101 protein levels were completely abrogated in the presence of specific Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamicin. In conclusion, our results demonstrate for the first time that leptin was able to increase exosomes release in BC cells, through an up-regulation of Tsg101 expression at posttranslational level. These findings, providing additional insights into the molecular mechanism governing exosome generation in BC cells, might open new avenues for therapeutic intervention in BC. Citation Format: Gelsomino L, Giordano C, Barone I, Panza S, Augimeri G, Bonofiglio D, Catalano S, Andò S. Leptin modulates exosome biogenesis in breast cancer cells through an enhanced Hsp90/Tsg101 interaction [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-05-04.

Aberrant leptin (Ob) signaling, a hallmark of obesity, has been recognized to influence breast cancer (BC) biology within the tumor microenvironment (TME). Here, we evaluated the impact of leptin receptor (ObR) knockdown in affecting BC phenotype and in mediating the interaction between tumor cells and macrophages, the most abundant immune cells within the TME. The stable knockdown of ObR (ObR sh) in ER&alpha, positive and ER&alpha, negative BC cells turned the tumor phenotype into a less aggressive one, as evidenced by in vitro and in vivo models. In xenograft tumors and in co-culture experiments between circulating monocytes and BC cells, the absence of ObR reduced the recruitment of macrophages, and also affected their cytokine mRNA expression profile. This was associated with a decreased expression and secretion of monocyte chemoattractant protein-1 in ObR sh clones. The loss of Ob/ObR signaling modulated the immunosuppressive TME, as shown by a reduced expression of programmed death ligand 1/programmed cell death protein 1/arginase 1. In addition, we observed increased phagocytic activity of macrophages compared to control Sh clones in the presence of ObR sh-derived conditioned medium. Our findings, addressing an innovative role of ObR in modulating immune TME, may open new avenues to improve BC patient health care.

The overexpression of phosphodiesterase (PDE) 5 is frequently found in various human cancers, such as those of the breast. However, PDE5&rsquo, s role in the tumor microenvironment is still unknown. As PDE5 represents a high-value therapeutic target, we investigated whether the expression and function of PDE5 in breast cancer-associated fibroblasts (CAFs) may be clinically relevant to malignant progression. PDE5 expression was increased in human breast cancer stroma compared with normal stroma and was correlated to a shorter overall survival. Treatment of CAFs, isolated from breast tumor biopsies, with selective PDE5 inhibitors inhibited their proliferation, motility, and invasiveness, and negatively controlled tumor&ndash, stroma interactions in both &lsquo, in vitro&rsquo, and &lsquo, in vivo&rsquo, models. PDE5 stable overexpression transformed immortalized mouse embryonic fibroblasts (MEFs) towards an activated fibroblast phenotype, impacting their intrinsic characteristics and paracrine effects on breast cancer cell growth and migration through an enhanced production of the C-X-C motif chemokine 16 (CXCL16). On the other hand, CAF exposure to PDE5 inhibitors was associated with reduced CXCL16 expression and secretion. Importantly, CXCL16 levels in breast cancer stroma showed a strong correlation with PDE5 levels and poor patient outcomes. In conclusion, PDE5 is overexpressed in breast cancer stroma, enhances the tumor-stimulatory activities of fibroblasts, and impacts clinical outcomes, thus, we propose this enzyme as an attractive candidate for prognosis and a potential target for treatments in breast cancer patients.

Background: Breast tumorigenesis results from the sequential acquisition of multiple genetic alterations in the epithelial cells and is also influenced by reciprocal interactions between the tumor cells and the surrounding stromal microenvironment. In this regard, an important mediator in driving mammary tumorigenesis is leptin, mainly secreted by adipocytes, but also by other cells in the tumor microenvironment. Several studies support the crucial involvement of leptin/leptin receptor (ObR) in the biology of breast cancer; however it still remains to be elucidated how ObR signaling may modulate tumor cell cytokine secretion favoring a permissive microenvironment required for tumor progression. Thus, we evaluated the impact of ObR lack in affecting breast cancer phenotype and in mediating epithelial/stromal tumor reciprocal interaction by using both in vitro and in vivo experimental approaches. Methods: As experimental models we established stable ERα-positive MCF-7 with an empty lentiviral vector (MCF-7 EV) and a lentiviral vector to silence human ObR (MCF-7 shObR), triple-negative MDA-MB-231 and human monocyte cell line derived from an acute monocyte leukemia patient, THP-1. We ran cytokine array to study cytokine secretion pattern in both MCF-7 EV and MCF-7 shObR cells. qRT-PCR, immunofluorescence and ELISA were performed to detect MCP-1 expression and secretion. Co-culture experiments and Boyden Chamber Transmigration assays were assessed to study the recruitment of THP1. Tumor associated macrophage (TAM) markers were detected after co-colture experiments between MCF-7 stable clones, MDA-MB-231 cells and THP1. Orthotopic mammary fat pad implantation of MCF-7 stable clones was performed to test tumor growth and macrophage infiltration. Results: Firstly we demonstrated a reduced activation of leptin signaling, and a decreased growth and motility in MCF-7 shObR cells compared to MCF-7 EV. Cytokine array revealed that the lack of ObR abrogated the release of monocyte chemoattractant protein (MCP1), and dramatically down-regulated interleukin 8 (IL-8) and intercellular adhesion molecule 1 (ICAM-1) secretion. Moreover, in MCF-7 shObR cells we found a diminution of MCP-1 in terms of mRNA and protein expression. As expected the suppression of MCP-1 secretion in these cells blocked the recruitment of monocytes and inhibited TAM development. The specific role of ObR/Ob signaling in regulating MCP-1 expression and secretion was further demonstrated in the presence of the ObR selective antagonist, the LDFI. LDFI treatment markedly down-regulated MCP-1 expression thus blocking macrophage recruitment and polarization in MDA-MB-231 cells. In vivo experiments revealed a decrease of tumor growth in MCF-7 shObR cells compared to MCF-7 EV wherein a dramatic reduction of MCP-1 expression was well reproduced in MCF-7 shObR clones. Conclusions: Our results, for the first time, showed that Ob/ObR signaling pathway may directly control MCP-1 function and tumor cell-polarized macrophages. Thus these data suggest the use of leptin receptor specific antagonist able to block macrophage-mediated effects of tumor microenvironment in sustaining breast cancer growth and progression. Citation Format: Luca Gelsomino, Salvatore Panza, Giuseppina Augimeri, Giuseppina Daniela Naimo, Rocco Malivindi, Giusi La Camera, Alessandro Paolì, Cinzia Giordano, Ines Barone, Loredana Mauro, Daniela Bonofiglio, Stefania Catalano, Sebastiano Andò. The inhibition of leptin receptor impairs macrophage recruitment in the tumor microenvironment blocking breast cancer growth and progression [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-06-11.