Expanding the Utilization of Formalin-Fixed, Paraffin-Embedded Archives : Feasibility of miR-Seq for Disease Exploration and Biomarker Development from Biopsies with Clear Cell Renal Cell Carcinoma

Novel predictive tools for clear cell renal cell carcinoma (ccRCC) are urgently needed. MicroRNAs (miRNAs) have been increasingly investigated for their predictive value, and formalin-fixed paraffin-embedded biopsy archives may potentially be a valuable source of miRNA sequencing material, as they remain an underused resource. Core biopsies of both cancerous and adjacent normal tissues were obtained from patients (n = 12) undergoing nephrectomy. After small RNA-seq, several analyses were performed, including classifier evaluation, obesity-related inquiries, survival analysis using publicly available datasets, comparisons to the current literature and ingenuity pathway analyses. In a comparison of tumour vs. normal, 182 miRNAs were found with significant differential expression; miR-155 was of particular interest as it classified all ccRCC samples correctly and correlated well with tumour size (R-2 = 0.83); miR-155 also predicted poor survival with hazard ratios of 2.58 and 1.81 in two different TCGA (The Cancer Genome Atlas) datasets in a univariate model. However, in a multivariate Cox regression analysis including age, sex, cancer stage and histological grade, miR-155 was not a statistically significant survival predictor. In conclusion, formalin-fixed paraffin-embedded biopsy tissues are a viable source of miRNA-sequencing material. Our results further support a role for miR-155 as a promising cancer classifier and potentially as a therapeutic target in ccRCC that merits further investigation.Peer reviewe

Axl-inhibitor bemcentinib alleviates mitochondrial dysfunction in the unilateral ureter obstruction murine model

Renal fibrosis is a progressive histological manifestation leading to chronic kidney disease (CKD) and associated with mitochondrial dysfunction. In previous work, we showed that Bemcentinib, an Axl receptor tyrosine kinase inhibitor, reduced fibrosis development. In this study, to investigate its effects on mitochondrial dysfunction in renal fibrosis, we analysed genome-wide transcriptomics data from a unilateral ureter obstruction (UUO) murine model in the presence or absence of bemcentinib (n = 6 per group) and SHAM-operated (n = 4) mice. Kidney ligation resulted in dysregulation of mitochondria-related pathways, with a significant reduction in the expression of oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO), citric acid cycle (TCA), response to reactive oxygen species and amino acid metabolism-related genes. Bemcentinib treatment increased the expression of these genes. In contrast, AKT/PI3K signalling pathway genes were up-regulated upon UUO, but bemcentinib largely inhibited their expression. At the functional level, ligation reduced mitochondrial biomass, which was increased upon bemcentinib treatment. Serum metabolomics analysis also showed a normalizing amino acid profile in UUO, compared with SHAM-operated mice following bemcentinib treatment. Our data suggest that mitochondria and mitochondria-related pathways are dramatically affected by UUO surgery and treatment with Axl-inhibitor bemcentinib partially reverses these effects.publishedVersio

AXL targeting reduces fibrosis development in experimental unilateral ureteral obstruction

The AXL receptor tyrosine kinase (RTK) is involved in partial epithelial-tomesenchymal transition (EMT) and inflammation – both main promoters of renal fibrosis development. The study aim was to investigate the role of AXL inhibition in kidney fibrosis due to unilateral ureteral obstruction (UUO). Eight weeks old male C57BL/6 mice underwent UUO and were treated with oral AXL inhibitor bemcentinib (n = 22), Angiotensin-converting enzyme inhibitor (ACEI, n = 10), ACEI and bemcentinib (n = 10) or vehicle alone (n = 22). Mice were sacrificed after 7 or 15 days and kidney tissues were analyzed by immunohistochemistry (IHC), western blot, ELISA, Sirius Red (SR) staining, and hydroxyproline (Hyp) quantification. RNA was extracted from frozen kidney tissues and sequenced on an Illumina HiSeq4000 platform. After 15 days the ligated bemcentinib-treated kidneys showed less fibrosis compared to the ligated vehicle-treated kidneys in SR analyses and Hyp quantification. Reduced IHC staining for Vimentin (VIM) and alpha smooth muscle actin (aSMA), as well as reduced mRNA abundance of key regulators of fibrosis such as transforming growth factor (Tgfb), matrix metalloproteinase 2 (Mmp2), Smad2, Smad4, myofibroblast activation (Aldh1a2, Crlf1), and EMT (Snai1,2, Twist), in ligated bemcentinib-treated kidneys was compatible with reduced (partial) EMT induction. Furthermore, less F4/80 positive cells, less activity of pathways related to the immune system and lower abundance of MCP1, MCP3, MCP5, and TARC in ligated bemcentinib-treated kidneys was compatible with reduction in inflammatory infiltrates by bemcentinib treatment. The AXL RTK pathway represents a promising target for pharmacologic therapy of kidney fibrosis.publishedVersio

Clear Cell Renal Cell Carcinoma is linked to Epithelial‐to‐Mesenchymal Transition and to Fibrosis

Clear cell renal cell carcinoma (ccRCC) represents the most common type of kidney cancer with high mortality in its advanced stages. Our study aim was to explore the correlation between tumor epithelial‐to‐mesenchymal transition (EMT) and patient survival. Renal biopsies of tumorous and adjacent nontumorous tissue were taken with a 16 g needle from our patients (n = 26) undergoing partial or radical nephrectomy due to ccRCC. RNA sequencing libraries were generated using Illumina TruSeq® Access library preparation protocol and TruSeq Small RNA library preparation kit. Next generation sequencing (NGS) was performed on Illumina HiSeq2500. Comparative analysis of matched sample pairs was done using the Bioconductor Limma/voom R‐package. Liquid chromatography‐tandem mass spectrometry and immunohistochemistry were applied to measure and visualize protein abundance. We detected an increased generic EMT transcript score in ccRCC. Gene expression analysis showed augmented abundance of AXL and MMP14, as well as down‐regulated expression of KL (klotho). Moreover, microRNA analyses demonstrated a positive expression correlation of miR‐34a and its targets MMP14 and AXL. Survival analysis based on a subset of genes from our list EMT‐related genes in a publicly available dataset showed that the EMT genes correlated with ccRCC patient survival. Several of these genes also play a known role in fibrosis. Accordingly, recently published classifiers of solid organ fibrosis correctly identified EMT‐affected tumor samples and were correlated with patient survival. EMT in ccRCC linked to fibrosis is associated with worse survival and may represent a target for novel therapeutic interventions.Peer reviewe

AXL targeting reduces fibrosis development in experimental unilateral ureteral obstruction

The AXL receptor tyrosine kinase (RTK) is involved in partial epithelial-to-mesenchymal transition (EMT) and inflammation - both main promoters of renal fibrosis development. The study aim was to investigate the role of AXL inhibition in kidney fibrosis due to unilateral ureteral obstruction (UUO). Eight weeks old male C57BL/6 mice underwent UUO and were treated with oral AXL inhibitor bemcentinib (n = 22), Angiotensin-converting enzyme inhibitor (ACEI, n = 10), ACEI and bemcentinib (n = 10) or vehicle alone (n = 22). Mice were sacrificed after 7 or 15 days and kidney tissues were analyzed by immunohistochemistry (IHC), western blot, ELISA, Sirius Red (SR) staining, and hydroxyproline (Hyp) quantification. RNA was extracted from frozen kidney tissues and sequenced on an Illumina HiSeq4000 platform. After 15 days the ligated bemcentinib-treated kidneys showed less fibrosis compared to the ligated vehicle-treated kidneys in SR analyses and Hyp quantification. Reduced IHC staining for Vimentin (VIM) and alpha smooth muscle actin (alpha SMA), as well as reduced mRNA abundance of key regulators of fibrosis such as transforming growth factor (Tgf beta), matrix metalloproteinase 2 (Mmp2), Smad2, Smad4, myofibroblast activation (Aldh1a2, Crlf1), and EMT (Snai1,2, Twist), in ligated bemcentinib-treated kidneys was compatible with reduced (partial) EMT induction. Furthermore, less F4/80 positive cells, less activity of pathways related to the immune system and lower abundance of MCP1, MCP3, MCP5, and TARC in ligated bemcentinib-treated kidneys was compatible with reduction in inflammatory infiltrates by bemcentinib treatment. The AXL RTK pathway represents a promising target for pharmacologic therapy of kidney fibrosis.Peer reviewe

Galaxy colours in high redshift X-ray selected clusters - I: Blue galaxy fractions in eight clusters

We present initial results from a wide-field, multi-colour imaging project, designed to study galaxy evolution in X-ray selected clusters at intermediate (z~0.25) and high redshifts (z~0.5). In this paper we give blue galaxy fractions from eight X-ray selected clusters, drawn from a combined sample of three X-ray surveys. We find that all the clusters exhibit excess blue galaxy populations over the numbers observed in local systems, though a large scatter is present in the results. We find no significant correlation of blue fraction with redshift at z>0.2 although the large scatter could mask a positive trend. We also find no systematic trend of blue fraction with X-ray luminosity. We show that the blue fraction is a function of (a) radius within a cluster, (b) absolute magnitude and (c) the passbands used to measure the colour. We find that our blue fractions (f_b) from galaxy colours close to restframe (U-B), f_b~0.4, are systematically higher than those from restframe (B-V) colours, f_b~0.2. We conclude this effect is real, may offer a partial explanation of the widely differing levels of blue fraction found in previous studies and may have implications for biases in optical samples selected in different bands. While the increasing blue fraction with radius can be interpreted as evidence of cluster infall of field galaxies, the exact physical processes which these galaxies undergo is unclear. We estimate that, in the cores of the more massive clusters, galaxies should be experiencing ram--pressure stripping of galactic gas by the intra--cluster medium. The fact that our low X-ray luminosity systems show a similar blue fraction as the high luminosity systems, as well as a significant blue fraction gradient with radius, implies other physical effects are also important.Comment: Accepted for publication in MNRA

Epithelial-to-mesenchymal transition in clear cell renal cell cancer and renal fibrosis

Background: Clear cell renal cell cancer (ccRCC) is a common cancer in humans and still has a high mortality. Chronic kidney disease (CKD) is a worldwide public health issue due to its increasing incidence and high morbidity and mortality related to accelerated cardiovascular diseases. Renal fibrosis is the histopathological correlate of CKD. Epithelial-to-mesenchymal transition (EMT) is a biological process where cells acquire mesenchymal characteristics such as loss of cell-cell adhesion, enhanced migratory capacity and production of extracellular matrix. In the case of cancer, EMT is involved in tumor progression, aggressiveness and therapy resistance. In the case of fibrosis, partial EMT drives the activation of myofibroblasts and the accumulation of extracellular matrix. AXL receptor tyrosine kinase is an EMT induced effector and is known to play a role in cancer aggressiveness, therapy resistance and fibrosis development. Renal biopsy archives such as the Kidney Biopsy Registry of the Norwegian Renal Registry are valuable sources for renal tissue analyses. Modern molecular analysis of archival renal biopsies could help to investigate pathomechanisms, and define potential biomarkers and new drug targets of renal diseases. The aims of this work were to: 1) evaluate the best method to extract RNA from archival formalin-fixed paraffin-embedded (FFPE) renal biopsies to enable RNA sequencing; 2) to study EMT markers in ccRCC tissues of our patient cohort; and 3) to detect AXL in unilateral ureteral obstruction (UUO), a murine renal fibrosis model, and to quantify fibrosis after inhibition of AXL by the selective small molecule BGB324 in UUO. Methods: In paper I, we tested seven commercially available RNA extraction kits on rat and human FFPE renal biopsy sections and on laser-capture microdissected (LCM) glomerular cross-sections to enable RNA sequencing from archival FFPE renal tissues. In paper II, FFPE sample pairs of ccRCC and adjacent normal tissues from our ccRCC cohort were used to characterize EMT markers by immunohistochemistry (IHC) and to analyze RNA, miRNA sequencing and proteomic data with respect to EMT and fibrosis. In paper III, we analyzed AXL in kidneys subjected to UUO in C57Bl/6 mice and assessed fibrosis extent by Sirius Red (SR) staining and hydroxyproline content after treatment with BGB324 or its vehicle. These analyses were supplemented by RNA sequencing including pathway analyses and by ELISA. We later analyzed the presence of AXL in diabetic and nephrosclerotic FFPE human biopsy tissues by IHC and RNA sequencing. Results: In paper I, four commercially available RNA extraction kits were suitable to isolate sufficient RNA from one human FFPE renal biopsy section and from around 100 LCM glomerular cross-sections with the Illumina TruSeq RNA Access Library Preparation kit. In paper II we demonstrated that EMT and fibrosis are prominent features in ccRCC samples of our cohort, that EMT related genes such as AXL, vimentin and matrix metalloproteinase 14 (MMP14) are associated to tumor stage in our cohort, and that expression levels of AXL, MMP14 and Klotho correlate with survival in publicly available datasets of ccRCC patient cohorts. In paper III, we found that AXL was distinctly more expressed in ligated compared to non-ligated kidneys in UUO. BGB324 treatment attenuated fibrosis development in ligated kidneys compared to ligated vehicle treated kidneys 15 days after obstruction. Tissue analyses and RNA sequencing data were compatible with reduced (partial) EMT and reduced inflammation in ligated BGB324 treated compared to the vehicle treated control. Conclusion: Paper I: RNA sequencing from human FFPE renal biopsies is feasible from one single biopsy section and from around 100 LCM glomerular cross-sections using the Illumina TruSeq RNA Access Library Preparation kit. Archival renal biopsy tissues are thus suitable for transcriptome sequencing. Paper II: EMT and fibrosis are substantial characteristics of ccRCC in our patient cohort and are associated with prognosis based on publicly available ccRCC datasets. Genes related to EMT including AXL may therefore be further evaluated as potential treatment targets. Paper III: AXL is involved in fibrosis development in UUO and inhibition of AXL by BGB324 reduces fibrosis development of UUO. AXL is present also in fibrotic human renal biopsy tissues and AXL therefore represents a potential therapeutic target for renal fibrosis

Lipoproteins in Staphylococcus aureus mediate inflammation by TLR2 and iron-dependent growth in vivo

Lipoproteins (Lpp) are ligands of TLR2 and signal by the adaptor MyD88. As part of the bacterial cell envelope, Lpp are mainly involved in nutrient acquisition for Staphylococcus aureus. The impact of Lpp on TLR2-MyD88 activation for S. aureus in systemic infection is unknown. S. aureus strain SA113 deficient in the enzyme encoded by the prolipoprotein diacylglyceryl transferase gene (Deltalgt), which attaches the lipid anchor to pro-Lpp, was used to study benefits and costs of Lpp maturation. Lpp in S. aureus induced early and strong cytokines by TLR2-MyD88 signaling in murine peritoneal macrophages. Lpp contributed via TLR2 to pathogenesis of sepsis in C57BL/6 mice with IL-1beta, chemokine-mediated inflammation, and high bacterial numbers. In the absence of MyD88-mediated inflammation, Lpp allowed bacterial clearing from liver devoid of infiltrating cells, but still conferred a strong growth advantage in mice, which was shown to rely on iron uptake and storage in vitro and in vivo. With iron-restricted bacteria, the Lpp-related growth advantage was evident in infection of MyD88(-/-), but not of C57BL/6, mice. On the other hand, iron overload of the host restored the growth deficit of Deltalgt in MyD88(-/-), but not in immunocompetent C57BL/6 mice. These results indicate that iron acquisition is improved by Lpp of S. aureus but is counteracted by inflammation. Thus, lipid anchoring is an evolutionary advantage for S. aureus to retain essential proteins for better survival in infection