Tumor cells promote immune evasion through up regulation of PD-L1 that binds with PD1 on cytotoxic T cells and promote dysfunction. Though therapeutic efficacy of anti PD1 antibody has remarkable effects on different type of cancers it is less effective in breast cancer. Hence more details understanding of PD-L1 mediated immune evasion is necessary. Here we report breast cancer cells secrete extra cellular vesicles in form of exosomes carry PD-L1 and is highly immunosuppressive. TGF-β present in tumor micro-environment orchestrates breast cancer cell secreted exosomal PD-L1 load. Circulating exosomal PD-L1 content is highly correlated with tumor TGF-β level. The later also found to be significantly associated with CD8+CD39+, CD8+PD1+ T cell phenotype. Recombinant TGF-β1 dose dependently induces PD-L1 expression in Texos in vitro and blocking of TGF-β dimmed exosomal PD-L1 level. PD-L1 knocked down exosomes failed to suppress effector activity of activated CD8 T cells like tumor exosomes. While understanding its effect on TCR signalling we found siPD-L1 exosomes failed to block phosphorylation of src family proteins, LAT and PLCγ of CD8 T cells more than PD-L1 exosomes. In-vivo inhibition of exosome release and TGF-β synergistically attenuates tumor burden by promoting Granzyme and IFN-γ release in tumor tissue depicting rejuvenation of exhausted T cells. Thus we establish TGF-β as a promoter of exosomal PD-L1 and unveil a mechanism that tumor cells follow to promote CD8 T cell dysfunction.

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Background Cancer-associated Þbroblasts (CAFs) are known to impact on tumour behaviour but mechanisms controlling this are poorly understood. Methods Breast normal fibroblasts (NFs) or CAFs were isolated from cancers by laser-microdissection or were cultured. Fibroblasts were transfected to manipulate miR-222 or Lamin B Receptor (LBR). Fibroblast conditioned medium was collected and used to treat epithelial BC lines MDA-MB-231 and MDA-MB-157. Migration, invasion, proliferation, or senescence was assessed using transwell, MTT or X-gal assays respectively. Results MiR-222 was up-regulated in CAFs as compared to NFs. Ectopic miR-222 expression in NFs induced CAF-like expression profiles, while miR-222 knock-down in CAFs inhibited CAF phenotypes. LBR was identified as a direct miR-222 target and was functionally relevant since LBR knock-down phenocopied miR-222 over-expression and LBR over-expression phenocopied miR-222 knock-down. MiR-222 over-expression, or LBR knock-down, was sufficient to induce NFs to show CAF characteristics of enhanced migration, invasion and senescence, and furthermore conditioned medium from these fibroblasts induced increased BC cell migration and invasion. The reverse manipulations in CAFs inhibited these behaviours in fibroblasts and inhibited paracrine influences on BC cells. Conclusion MiR-222/LBR have key roles in controlling pro-progression influences of CAFs in BC. This pathway may present therapeutic opportunities to inhibit CAF-induced cancer progression. 3 BACKGROUND Breast cancer (BC) is the leading cause of cancer death worldwide among women 1 , and nearly 2.3 million females are newly diagnosed annually. Although there are initial responses to treatment, many cancers relapse and distant metastases occur in nearly one third of woman; these are typically fatal. The biology behind BC metastases still remains undetermined. Therefore, understanding molecular determinants of metastasis is crucial for finding new therapeutic strategies. The tumour microenvironment consists of immune cells, blood vessels, endothelial cells, fibroblasts and extracellular matrix 4. This microenvironment plays key roles in disease outcome by inducing tumour cell proliferation and aggressiveness 5,6. Cancer-associated Þbroblasts (CAFs), an activated form of tissue-resident fibroblasts present within breast cancers, comprise a major component of the tumour microenvironment 7 , characterised most commonly by expression of-smooth muscle actin 8. CAFs can induce cancer progression 9 and metastasis 10 by secreting various cytokines, chemokines and growth factors (e.g. VEGF, FGF2, TGF , CXCL12, IL6 and IL8) 11,12 and by modulating the extracellular matrix (ECM) that facilitates tumour cell migration and invasion 13,14. CAFs also modulate immune cell function to create an immune suppressive environment during cancer progression 15. The process of transformation of CAFs from resident normal Þbroblasts is achieved by several growth factors 16,17,18 , however mechanisms of transformation have not yet been fully expl...

Ineffective cancer treatment is implicated in metastasis, recurrence, resistance to chemotherapy and radiotherapy, and evasion of immune surveillance. All these failures occur due to the persistence of cancer stem cells (CSCs) even after rigorous therapy, thereby rendering them as essential targets for cancer management. Contrary to the quiescent nature of CSCs, a gene profiler array disclosed that phosphatidylinositol-3-kinase (PI3K), which is known to be crucial for cell proliferation, differentiation, and survival, was significantly upregulated in CSCs. Since PI3K is modulated by cyclic adenosine 3′,5′ monophosphate (cAMP), analyses of cAMP regulation revealed that breast CSCs expressed increased levels of phosphodiesterase 4 (PDE4) in contrast to normal stem cells. In accordance, the effects of rolipram, a PDE4 inhibitor, were evaluated on PI3K regulators and signaling. The efficacy of rolipram was compared with paclitaxel, an anticancer drug that is ineffective in obliterating breast CSCs. Analyses of downstream signaling components revealed a switch between cell survival and death, in response to rolipram, specifically of the CSCs. Rolipram-mediated downregulation of PDE4A levels in breast CSCs led to an increase in cAMP levels and protein kinase A (PKA) expression. Subsequently, PKA-mediated upregulation of phosphatase and tensin homolog antagonized the PI3K/AKT/mTOR pathway and led to cell cycle arrest. Interestingly, direct yet noncanonical activation of mTOR by PKA, circumventing the influence of PI3K and AKT, temporally shifted the fate of CSCs toward apoptosis. Rolipram in combination with paclitaxel indicated synergistic consequences, which effectively obliterated CSCs within a tumor, thereby suggesting combinatorial therapy as a sustainable and effective strategy to abrogate breast CSCs for better patient prognosis.

Our study is probably the first study in India comparing serum resistin levels of CKD patients vis-à-vis control subjects. Further cellular research may be needed to explore this relation.