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CD73 is a cell surface 5'nucleotidase (NT5E) and key node in the catabolic process generating immunosuppressive adenosine in cancer. Using a murine monoclonal antibody surrogate of Oleclumab, we investigated the effect of CD73 inhibition in concert with cytotoxic therapies (chemotherapies as well as fractionated radiotherapy) and PD-L1 blockade. Our results highlight improved survival in syngeneic tumor models of colorectal cancer (CT26 and MC38) and sarcoma (MCA205). This therapeutic outcome was in part driven by cytotoxic CD8 T-cells, as evidenced by the detrimental effect of CD8 depleting antibody treatment of MCA205 tumor bearing mice treated with anti-CD73, anti-PD-L1 and 5-Fluorouracil+Oxaliplatin (5FU+OHP). We hypothesize that the improved responses are tumor microenvironment (TME)-driven, as suggested by the lack of anti-CD73 enhanced cytopathic effects mediated by 5FU+OHP on cell lines in vitro. Pharmacodynamic analysis, using imaging mass cytometry and RNA-sequencing, revealed noteworthy changes in specific cell populations like cytotoxic T cells, B cells and NK cells in the CT26 TME. Transcriptomic analysis highlighted treatment-related modulation of gene profiles associated with an immune response, NK and T-cell activation, T cell receptor signaling and interferon (types 1 & 2) pathways. Inclusion of comparator groups representing the various components of the combination allowed deconvolution of contribution of the individual therapeutic elements; highlighting specific effects mediated by the anti-CD73 antibody with respect to immune-cell representation, chemotaxis and myeloid biology. These pre-clinical data reflect complementarity of adenosine blockade with cytotoxic therapy, and T-cell checkpoint inhibition, and provides new mechanistic insights in support of combination therapy. [ABSTRACT FROM AUTHOR]

Proliferation of interstitial fibroblasts is a hallmark of progressive renal fibrosis commonly resulting in chronic kidney failure. The intermediate-conductance [Ca.sup.2+]-activated [K.sup.+] channel ([K.sub.Ca]3.1) has been proposed to promote mitogenesis in several cell types and contribute to disease states characterized by excessive proliferation. Here, we hypothesized that [K.sub.Ca]3.1 activity is pivotal for renal fibroblast proliferation and that deficiency or pharmacological blockade of [K.sub.Ca]3.1 suppresses development of renal fibrosis. We found that mitogenic stimulation up-regulated [K.sub.Ca]3.1 in murine renal fibroblasts via a MEK-dependent mechanism and that selective blockade of [K.sub.Ca]3.1 functions potently inhibited fibroblast proliferation by [G.sub.0]/[G.sub.1] arrest. Renal fibrosis induced by unilateral ureteral obstruction (UUO) in mice was paralleled by a robust up-regulation of [K.sub.Ca]3.1 in affected kidneys. Mice lacking [K.sub.Ca]3.1 ([K.sub.Ca][3.1.sup.-/-]) showed a significant reduction in fibrotic marker expression, chronic tubulointerstitial damage, collagen deposition and [alpha][SMA.sup.+] cells in kidneys after UUO, whereas functional renal parenchyma was better preserved. Pharmacological treatment with the selective [K.sub.Ca]3.1 blocker TRAM-34 similarly attenuated progression of UUO-induced renal fibrosis in wild-type mice and rats. In conclusion, our data demonstrate that [K.sub.Ca]3.1 is involved in renal fibroblast proliferation and fibrogenesis and suggest that [K.sub.Ca]3.1 may represent a therapeutic target for the treatment of fibrotic kidney disease. ion channels | fibroblasts | kidney | organ fibrosis

Abstract Background The reliability of gene expression profiling-based technologies to detect transcriptional differences representative of the original samples is affected by the quality of the extracted RNA. It strictly depends upon the technique that has been employed. Hence, the present study aimed at systematically comparing silica-gel column (SGC) and guanidine isothiocyanate (GTC) techniques of RNA isolation to answer the question which technique is preferable when frozen, long-term stored or fresh lung tissues have to be evaluated for the downstream molecular analysis. Methods Frozen lungs (n = 3) were prepared by long-term storage (2.5 yrs) in -80°C while fresh lungs (n = 3) were harvested and processed immediately. The purity and quantification of RNA was determined with a spectrophotometer whereas the total amounted copy numbers of target sequences were determined with iCycler detection system for assessment of RNA intactness (28S and 18S) and fragment sizes, i.e. short (GAPDH-3' UTR), medium (GAPDH), and long (PBGD) with 200 bp, 700 bp, and 1400 bp distance to the 3'ends of mRNA motif, respectively. Results Total yield of RNA was higher with GTC than SGC technique in frozen as well as fresh tissues while the purity of RNA remained comparable. The quantitative reverse transcriptase-polymerase chain reaction data revealed that higher mean copy numbers of 28S and a longer fragment (1400 bp) were obtained from RNA isolated with SGC than GTC technique using fresh as well as frozen tissues. Additionally, a high mean copy number of 18S and medium fragment (700 bp) were obtained in RNA isolated with SGC technique from fresh tissues, only. For the shorter fragment, no significant differences between both techniques were noticed. Conclusion Our data demonstrated that although the GTC technique has yielded a higher amount of RNA, the SGC technique was much more superior with respect to the reliable generation of an intact RNA and effectively amplified longer products in fresh as well as in frozen tissues.

Proliferation of interstitial fibroblasts is a hallmark of progressive renal fibrosis commonly resulting in chronic kidney failure. The intermediate-conductance Ca2+-activated K+ channel (KCa3.1) has been proposed to promote mitogenesis in several cell types and contribute to disease states characterized by excessive proliferation. Here, we hypothesized that KCa3.1 activity is pivotal for renal fibroblast proliferation and that deficiency or pharmacological blockade of KCa3.1 suppresses development of renal fibrosis. We found that mitogenic stimulation up-regulated KCa3.1 in murine renal fibroblasts via a MEK-dependent mechanism and that selective blockade of KCa3.1 functions potently inhibited fibroblast proliferation by G0/G1 arrest. Renal fibrosis induced by unilateral ureteral obstruction (UUO) in mice was paralleled by a robust up-regulation of KCa3.1 in affected kidneys. Mice lacking KCa3.1 (KCa3.1-/-) showed a significant reduction in fibrotic marker expression, chronic tubulointerstitial damage, collagen deposition and αSMA+ cells in kidneys after UUO, whereas functional renal parenchyma was better preserved. Pharmacological treatment with the selective KCa3.1 blocker TRAM-34 similarly attenuated progression of UUO-induced renal fibrosis in wild-type mice and rats. In conclusion, our data demonstrate that KCa3.1 is involved in renal fibroblast proliferation and fibrogenesis and suggest that KCa3.1 may represent a therapeutic target for the treatment of fibrotic kidney disease. [ABSTRACT FROM AUTHOR]

Gardos channel, the erythrocyte Ca2+-activated K+ channel (KCa3.1), is considered a major regulator of red blood cell (RBC) volume by mediating efflux of potassium and thus cell dehydration and shrinkage. However, the functional importance of KCa3.1 in RBC in vivo is incompletely understood. Here, we used KCa3.1−/−-mice to investigate the consequences of KCa3.1 deficiency for RBC indices, functions, and sequestration. RBCs of KCa3.1−/−-mice of all ages were mildly macrocytic but their biconcave appearance being preserved. RBC number, total hemoglobin, and hematocrit were unchanged in the adult KCa3.1−/−-mice and increased in the premature KCa3.1−/−-mice. Filterability, Ca2+-dependent volume decrease and osmotic tolerance of RBCs lacking KCa3.1 were noticeably reduced when compared to RBC of wild-type littermates. Deformability to increasing shear stress was unchanged. Strikingly, KCa3.1−/−-mice developed progressive splenomegaly which was considerable (∼200% of controls) in the >6-month-old mice and was paralleled by increased iron deposition in the aged mice presumably as a consequence of enhanced RBC sequestration. Daily injections of the KCa3.1-blocker TRAM-34 (120 mg/kg) also produced mild splenomegaly in wild-type mice. We conclude that genetic deficit of erythroid KCa3.1 causes mild RBC macrocytosis, presumably leading to reduced filterability, and impairs volume regulation. These RBC defects result in mild but progressive splenomegaly. [ABSTRACT FROM AUTHOR]