The hypothesis that microvesicle (MV)-mediated microRNA transfer converts non-cancer stem cells into cancer stem cells (CSCs) leading to therapy resistance remains poorly investigated. Here we provide direct evidence supporting this hypothesis, by demonstrating how MV derived from cancer associated fibroblasts (CAF) transfer miR-221 to promote hormonal therapy resistance (HTR) in models of luminal breast cancer. We determined that CAF-derived MV horizontally transferred miR221 to tumor cells and, in combination with hormone therapy activated an ERlo/Notchhi feed-forward loop responsible for the generation of CD133hi CSC. Importantly, MV from patients with HTR metastatic disease expressed high levels of miR221. We further determined that the IL6-pStat3 pathway in promoted the biogenesis of onco-miR-221hi CAF MV and established stromal CSC niches in experimental and patient-derived breast cancer models. Co-injection of patient-derived CAF from bone metastases led to de novo HTR tumors, which was reversed with IL6R blockade. Finally, we generated PDX models from patient-derived HTR bone metastases and analyzed tumor cells, stroma, and MV. Murine and human CAF were enriched in HTR tumors expressing high levels of CD133hi cells. Depletion of murine CAF from PDX restored sensitivity to HT, with a concurrent reduction of CD133hi CSC. Conversely, in models of CD133neg, HT-sensitive cancer cells, both murine and human CAF promoted de novo HT resistance via the generation of CD133hi CSC that expressed low levels of estrogen receptor alpha (ER). Overall, our results illuminate how MV-mediated horizontal transfer of genetic material from host stromal cells to cancer cells trigger the evolution of therapy-resistant metastases, with potentially broad implications for their control.

Recent literature highlights the importance of pro-inflammatory cytokines in the biology of breast cancer stem cells (CSCs), unraveling differences with respect to their normal counterparts. Expansion of mammospheres (MS) is a valuable tool for the in vitro study of normal and cancer mammary gland stem cells. Here, we expanded MSs from human breast cancer and normal mammary gland tissues, as well from tumorigenic (MCF7) and non-tumorigenic (MCF10) breast cell lines. We observed that agonists for the retinoid X receptor (6-OH-11-O-hydroxyphenanthrene), retinoic acid receptor (all-trans retinoic acid (RA)) and peroxisome proliferator-activated receptor (PPAR)-c (pioglitazone (PGZ)), reduce the survival of MS generated from breast cancer tissues and MCF7 cells, but not from normal mammary gland or MCF10 cells. This phenomenon is paralleled by the hampering of pro-inflammatory Nuclear Factor-jB (NF-jB)/Interleukin-6 (IL6) axis that is hyperactive in breast cancer-derived MS. The hindrance of such pathway associates with the downregulation of MS regulatory genes (SLUG, Notch3, Jagged1) and with the upregulation of the differentiation markers estrogen receptor-a and keratin18. At variance, the PPARa agonist Wy14643 promotes MS formation, upregulating NF-jB/IL6 axis and MS regulatory genes. These data reveal that nuclear receptors agonists (6-OH-11-O-hydroxyphenanthrene, RA, PGZ) reduce the inflammation dependent survival of breast CSCs and that PPARa agonist Wy14643 exerts opposite effects on this phenotype.

Endothelial cell senescence is characterized by acquisition of senescence-associated secretory phenotype (SASP), able to promote inflammaging and cancer progression. Emerging evidence suggest that preventing SASP development could help to slow the rate of aging and the progression of age-related diseases, including cancer. Aim of this study was to evaluate whether and how adalimumab, a monoclonal antibody directed against tumor necrosis factor-α (TNF-α), a major SASP component, can prevent the SASP. A three-pronged approach has been adopted to assess the if adalimumab is able to: i) modulate a panel of classic and novel senescence- and SASP-associated markers (interleukin [IL]-6, senescence associated-β-galactosidase, p16/Ink4a, plasminogen activator inhibitor 1, endothelial nitric oxide synthase, miR-146a-5p/Irak1 and miR-126-3p/Spred1) in human umbilical vein endothelial cells (HUVECs); ii) reduce the paracrine effects of senescent HUVECs' secretome on MCF-7 breast cancer cells, through wound healing and mammosphere assay; and iii) exert significant decrease of miR-146a-5p and increase of miR-126-3p in circulating angiogenic cells (CACs) from psoriasis patients receiving adalimumab in monotherapy.TNF-α blockade associated with adalimumab induced significant reduction in released IL-6 and significant increase in eNOS and miR-126-3p expression levels in long-term HUVEC cultures.A significant reduction in miR-146a-5p expression levels both in long-term HUVEC cultures and in CACs isolated from psoriasis patients was also evident. Interestingly, conditioned medium from senescent HUVECs treated with adalimumab was less consistent than medium from untreated cells in inducing migration- and mammosphere- promoting effects on MCF-7 cells.Our findings suggest that adalimumab can induce epigenetic modifications in cells undergoing senescence, thus contributing to the attenuation of SASP tumor-promoting effects.