Neuropilin2 in Mesenchymal Stromal Cells as a Potential Novel Therapeutic Target in Myelofibrosis.

Bone marrow fibrosis in myeloproliferative neoplasm (MPN), myelodysplastic syndromes (MDS), MPN/MDS overlap syndromes and acute myeloid leukemia (AML) is associated with poor prognosis and early treatment failure. Myelofibrosis (MF) is accompanied by reprogramming of multipotent bone marrow mesenchymal stromal cells (MSC) into osteoid and fiber-producing stromal cells. We demonstrate NRP2 and osteolineage marker NCAM1 (neural cell adhesion molecule 1) expression within the endosteal niche in normal bone marrow and aberrantly in MPN, MDS MPN/MDS overlap syndromes and AML (n = 99), as assessed by immunohistochemistry. Increased and diffuse expression in mesenchymal stromal cells and osteoblasts correlates with high MF grade in MPN (p < 0.05 for NRP2 and NCAM1). Single cell RNA sequencing (scRNAseq) re-analysis demonstrated NRP2 expression in endothelial cells and partial co-expression of NRP2 and NCAM1 in normal MSC and osteoblasts. Potential ligands included transforming growth factor ß1 (TGFB1) from osteoblasts and megakaryocytes. Murine ThPO and JAK2V617F myelofibrosis models showed co-expression of Nrp2 and Ncam1 in osteolineage cells, while fibrosis-promoting MSC only express Nrp2. In vitro experiments with MC3T3-E1 pre-osteoblasts and analysis of Nrp2-/- mouse femurs suggest that Nrp2 is functionally involved in osteogenesis. In summary, NRP2 represents a potential novel druggable target in patients with myelofibrosis.

Clinical implications of AGR2 in primary prostate cancer: Results from a large-scale study.

Human anterior gradient-2 (AGR2) has been implicated in carcinogenesis of various solid tumours, but the expression data in prostate cancer are contradictory regarding its prognostic value. The objective of this study is to evaluate the expression of AGR2 in a large prostate cancer cohort and to correlate it with clinicopathological data. AGR2 protein expression was analysed immunohistochemically in 1023 well-characterized prostate cancer samples with a validated antibody. AGR2 expression levels in carcinomas were compared with matched tissue samples of adjacent normal glands. AGR2 expression levels were dichotomized and tested for statistical significance. Increased AGR2 expression was found in 93.5% of prostate cancer cases. AGR2 levels were significantly higher in prostate cancer compared with normal prostate tissue. A gradual loss of AGR2 expression was associated with increasing tumour grade (ISUP), and AGR2 expression is inversely related to patient survival, however, multivariable significance is not achieved. AGR2 is clearly upregulated in the majority of prostate cancer cases, yet a true diagnostic value appears unlikely. In spite of the negative correlation of AGR2 expression with increasing tumour grade, no independent prognostic significance was found in this large-scale study.

SARS-CoV-2 mRNA vaccination-induced immunological memory in human nonlymphoid and lymphoid tissues.

Tissue-resident lymphocytes provide organ-adapted protection against invading pathogens. Whereas their biology has been examined in great detail in various infection models, their generation and functionality in response to vaccination have not been comprehensively analyzed in humans. We therefore studied SARS-CoV-2 mRNA vaccine-specific T cells in surgery specimens of kidney, liver, lung, bone marrow, and spleen compared with paired blood samples from largely virus-naive individuals. As opposed to lymphoid tissues, nonlymphoid organs harbored significantly elevated frequencies of spike-specific CD4+ T cells compared with blood showing hallmarks of tissue residency and an expanded memory pool. Organ-derived CD4+ T cells further exhibited increased polyfunctionality over those detected in blood. Single-cell RNA-Seq together with T cell receptor repertoire analysis indicated that the clonotype rather than organ origin is a major determinant of transcriptomic state in vaccine-specific CD4+ T cells. In summary, our data demonstrate that SARS-CoV-2 vaccination entails acquisition of tissue memory and residency features in organs distant from the inoculation site, thereby contributing to our understanding of how local tissue protection might be accomplished.

Changes of aortic hemodynamics after aortic valve replacement-A four dimensional flow cardiovascular magnetic resonance follow up study.

Objectives: Non-invasive assessment of aortic hemodynamics using four dimensional (4D) flow magnetic resonance imaging (MRI) provides new information on blood flow patterns and wall shear stress (WSS). Aortic valve stenosis (AS) and/or bicuspid aortic valves (BAV) are associated with altered aortic flow patterns and elevated WSS. Aim of this study was to investigate changes in aortic hemodynamics over time in patients with AS and/or BAV with or without aortic valve replacement. Methods: We rescheduled 20 patients for a second 4D flow MRI examination, whose first examination was at least 3 years prior. A total of 7 patients received an aortic valve replacement between baseline and follow up examination (=operated group = OP group). Aortic flow patterns (helicity/vorticity) were assessed using a semi-quantitative grading approach from 0 to 3, flow volumes were evaluated in 9 planes, WSS in 18 and peak velocity in 3 areas. Results: While most patients had vortical and/or helical flow formations within the aorta, there was no significant change over time. Ascending aortic forward flow volumes were significantly lower in the OP group than in the NOP group at baseline (NOP 69.3 mL ± 14.2 mL vs. OP 55.3 mL ± 1.9 mL p = 0.029). WSS in the outer ascending aorta was significantly higher in the OP group than in the NOP group at baseline (NOP 0.6 ± 0.2 N/m2 vs. OP 0.8 ± 0.2 N/m2, p = 0.008). Peak velocity decreased from baseline to follow up in the aortic arch only in the OP group (1.6 ± 0.6 m/s vs. 1.2 ± 0.3 m/s, p = 0.018). Conclusion: Aortic valve replacement influences aortic hemodynamics. The parameters improve after surgery.

Androgen Receptor Splice Variants Contribute to the Upregulation of DNA Repair in Prostate Cancer.

BACKGROUND: Canonical androgen receptor (AR) signaling regulates a network of DNA repair genes in prostate cancer (PCA). Experimental and clinical evidence indicates that androgen deprivation not only suppresses DNA repair activity but is often synthetically lethal in combination with PARP inhibition. The present study aimed to elucidate the impact of AR splice variants (AR-Vs), occurring in advanced or late-stage PCA, on DNA repair machinery. METHODS: Two hundred and seventy-three tissue samples were analyzed, including primary hormone-naïve PCA, primary metastases, hormone-sensitive PCA on androgen deprivation therapy (ADT) and castration refractory PCA (CRPC group). The transcript levels of the target genes were profiled using the nCounter platform. Experimental support for the findings was gained in AR/AR-V7-expressing LNCaP cells subjected to ionizing radiation. RESULTS: AR-Vs were present in half of hormone-sensitive PCAs on androgen deprivation therapy (ADT) and two-thirds of CRPC samples. The presence of AR-Vs is highly correlated with increased activity in the AR pathway and DNA repair gene expression. In AR-V-expressing CRPC, the DNA repair score increased by 2.5-fold as compared to AR-V-negative samples. Enhanced DNA repair and the deregulation of DNA repair genes by AR-V7 supported the clinical data in a cell line model. CONCLUSIONS: The expression of AR splice variants such as AR-V7 in PCA patients following ADT might be a reason for reduced or absent therapy effects in patients on additional PARP inhibition due to the modulation of DNA repair gene expression. Consequently, AR-Vs should be further studied as predictive biomarkers for therapy response in this setting.

RNA Sequencing Reveals Alterations and Similarities in Cell Metabolism, Hypoxia and Immune Evasion in Primary Cell Cultures of Clear Cell Renal Cell Carcinoma.

The treatment of advanced renal cell carcinoma remains a challenge. To develop novel therapeutic approaches, primary cell cultures as an in vitro model are considered more representative than commercial cell lines. In this study, we analyzed the gene expression of previously established primary cell cultures of clear cell renal cell carcinoma by bulk (3'm)RNA sequencing and compared it to the tissue of origin. The objectives were the identification of dysregulated pathways under cell culture conditions. Furthermore, we assessed the suitability of primary cell cultures for studying crucial biological pathways, including hypoxia, growth receptor signaling and immune evasion. RNA sequencing of primary cell cultures of renal cell carcinoma and a following Enrichr database analysis revealed multiple dysregulated pathways under cell culture conditions. 444 genes were significantly upregulated and 888 genes downregulated compared to the tissue of origin. The upregulated genes are crucial in DNA repair, cell cycle, hypoxia and metabolic shift towards aerobic glycolysis. A downregulation was observed for genes involved in pathways of immune cell differentiation and cell adhesion. We furthermore observed that 7275 genes have a similar mRNA expression in cell cultures and in tumor tissue, including genes involved in the immune checkpoint signaling or in pathways responsible for tyrosine kinase receptor resistance. Our findings confirm that primary cell cultures are a representative tool for specified experimental approaches. The results presented in this study give further valuable insights into the complex adaptation of patient-derived cells to a new microenvironment, hypoxia and other cell culture conditions, which are often neglected in daily research, and allow new translational and therapeutic approaches.

Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer.

Aberrant transcriptional activity of androgen receptor (AR) is one of the dominant mechanisms for developing of castration-resistant prostate cancer (CRPC). Analyzing AR-transcriptional complex related to CRPC is therefore important towards understanding the mechanism of therapy resistance. While studying its mechanism, we observed that a transmembrane protein called neuropilin-2 (NRP2) plays a contributory role in forming a novel AR-transcriptional complex containing nuclear pore proteins. Using immunogold electron microscopy, high-resolution confocal microscopy, chromatin immunoprecipitation, proteomics, and other biochemical techniques, we delineated the molecular mechanism of how a specific splice variant of NRP2 becomes sumoylated upon ligand stimulation and translocates to the inner nuclear membrane. This splice variant of NRP2 then stabilizes the complex between AR and nuclear pore proteins to promote CRPC specific gene expression. Both full-length and splice variants of AR have been identified in this specific transcriptional complex. In vitro cell line-based assays indicated that depletion of NRP2 not only destabilizes the AR-nuclear pore protein interaction but also inhibits the transcriptional activities of AR. Using an in vivo bone metastasis model, we showed that the inhibition of NRP2 led to the sensitization of CRPC cells toward established anti-AR therapies such as enzalutamide. Overall, our finding emphasize the importance of combinatorial inhibition of NRP2 and AR as an effective therapeutic strategy against treatment refractory prostate cancer.

Neuropilin-2 and Its Transcript Variants Correlate with Clinical Outcome in Bladder Cancer.

Urothelial bladder cancer ranks among the 10 most frequently diagnosed cancers worldwide. In our previous study, the transmembrane protein neuropilin-2 (NRP2) emerged as a predictive marker in patients with bladder cancer. NRP2 consists of several splice variants; the most abundant of these, NRP2a and NRP2b, are reported to have different biological functions in lung cancer progression. For other cancer types, there are no published data on the role of these transcript variants in cancer progression and the clinical outcome. Here, we correlate NRP2 and its two most abundant transcript variants, NRP2A and NRP2B, with the clinical outcome using available genomic data with subsequent validation in our own cohort of patients with muscle-invasive bladder cancer. In addition to NRP2, NRP1 and the NRP ligands PDGFC and PDGFD were studied. Only NRP2A emerged as an independent prognostic marker for shorter cancer-specific survival in muscle-invasive bladder cancer in our cohort of 102 patients who underwent radical cystectomy between 2008 and 2014 with a median follow-up time of 82 months. Additionally, we demonstrate that high messenger expression of NRP2, NRP1, PDGFC and PDGFD associates with a more aggressive disease (i.e., a high T stage, positive lymph node status and reduced survival).

Targeting glycolysis with 2-deoxy-D-glucose sensitizes primary cell cultures of renal cell carcinoma to tyrosine kinase inhibitors.

PURPOSE: To investigate the synergistic effect of glycolysis inhibition on therapy answer to tyrosine kinase inhibitors in renal carcinoma. METHODS: Primary cell cultures from 33 renal tumors including clear cell RCC (ccRCC), papillary RCC and the rare subtype chromophobe RCC as well as two metastases of ccRCC were obtained and cultivated. The patient-derived cells were verified by immunohistochemistry. CcRCC cells were further examined by exon sequencing of the von Hippel-Lindau gene (VHL) and by RNA-sequencing. Next, cell cultures of all subtypes of RCC were exposed to increasing doses of various tyrosine kinase inhibitors (axitinib, cabozantinib and pazopanib) and the glycolysis inhibitor 2-deoxy-D-glucose, alone or combined. CellTiter-Glo® Luminescence assay and Crystal Violet staining were used to assess the inhibition of glycolysis and the viability of the cultured primary cells. RESULTS: The cells expressed characteristic tissue markers and, in case of ccRCC cultures, the VHL status of the tumor they derived from. An upregulation of HK1, PFKP and SLC2A1 was observed, while components of the respiratory chain were downregulated, confirming a metabolic shift towards aerobic glycolysis. The tumors displayed variable individual responses for the therapeutics. All subtypes of RCC were susceptible to cabozantinib treatment indicated by decreased proliferation. Adding 2-deoxy-D-glucose to tyrosine kinase inhibitors decreased ATP production and increased the susceptibility of ccRCC to pazopanib treatment. CONCLUSION: This study presents a valuable tool to cultivate even uncommon and rare renal cancer subtypes and allows testing of targeted therapies as a personalized approach as well as testing new therapies such as glycolysis inhibition in an in vitro model.

Neuropilin-2 is an independent prognostic factor for shorter cancer-specific survival in patients with acinar adenocarcinoma of the prostate.

Neuropilin-2 (NRP2) is a member of the neuropilin receptor family and known to regulate autophagy and mTORC2 signaling in prostate cancer (PCa). Our study investigated the association of immunohistochemical NRP2 expression with clinicopathological data in PCa patients. For this purpose, we generated a tissue microarray with prostate tissue specimens from 400 PCa patients treated by radical prostatectomy. We focused on patients with high-risk factors such as extraprostatic extension (pT ≥ 3), Gleason score ≥8 and/or the presence of regional lymph node metastases (pN1). Protein levels of NRP2, the vascular endothelial growth factor C (VEGFC) and oncogenic v-ets avian erythroblastosis virus E26 oncogene homolog (ERG) gene as an indicator for TMPRSS2-ERG fusion was assessed in relation to the patients' outcome. NRP2 emerged as an independent prognostic factor for cancer-specific survival (CSS) (hazard ratio 2.360, 95% confidence interval = 1.2-4.8; p = 0.016). Moreover, the association between NRP2 expression and shorter CSS was also especially pronounced in patients at high risk for progression (log-rank test: p = 0.010). We evaluated the association between NRP2 and the TMPRSS2-ERG gene fusion status assessed by immunohistochemical nuclear ERG staining. However, ERG staining alone did not show any prognostic significance. NRP2 immunostaining is significantly associated with shorter CSS in ERG-negative tumors (log-rank test: p = 0.012). No prognostic impact of NRP2 expression on CSS was observed in ERG-positive tumors (log-rank test: p = 0.153). Our study identifies NRP2 as an important prognostic marker for a worse clinical outcome especially in patients with a high-risk PCa and in patients with ERG-negative PCa.

Linking NRP2 With EMT and Chemoradioresistance in Bladder Cancer.

Neuropilin-2 (NRP2) is a prognostic indicator for reduced survival in bladder cancer (BCa) patients. Together with its major ligand, vascular endothelial growth factor (VEGF)-C, NRP2 expression is a predictive factor for treatment outcome in response to radiochemotherapy in BCa patients who underwent transurethral resection. Therefore, we investigated the benefit of combining cisplatin-based chemotherapy with irradiation treatment in the BCa cell line RT112 exhibiting or lacking endogenous NRP2 expression in order to evaluate NRP2 as potential therapeutic target. We have identified a high correlation of NRP2 and the glioma-associated oncogene family zinc finger 2 (GLI2) transcripts in the cancer genome atlas (TCGA) cohort of BCa patients and a panel of 15 human BCa cell lines. Furthermore, we used in vitro BCa models to show the transforming growth factor-beta 1 (TGFß1)-dependent regulation of NRP2 and GLI2 expression levels. Since NRP2 was shown to bind TGFß1, associate with TGFß receptors, and enhance TGFß1 signaling, we evaluated downstream signaling pathways using an epithelial-to-mesenchymal transition (EMT)-assay in combination with a PCR profiling array containing 84 genes related to EMT. Subsequent target validation in NRP2 knockout and knockdown models revealed secreted phosphoprotein 1 (SPP1/OPN/Osteopontin) as a downstream target positively regulated by NRP2.

RNA quality in fresh-frozen gastrointestinal tumor specimens-experiences from the tumor and healthy tissue bank TU Dresden.

The term "pre-analytics" summarizes all procedures concerned with specimen collection or processing as well as logistical aspects like transport or storage of tissue specimens. All or these variables as well as tissue-specific characteristics affect sample quality. While certain parameters like warm ischemia or tissue-specific characteristics cannot be changed, other parameters can be assessed and optimized. The aim of this study was to determine RNA quality by assessing the RIN values of specimens from different organs and to assess the influence of vacuum preservation. Samples from the GI tract, in general, appear to have lower RNA quality when compared to samples from other organ sites. This may be due to the digestive enzymes or bacterial colonization. Processing time in pathology does not significantly influence RNA quality. Tissue preservation with a vacuum sealer leads to preserved RNA quality over an extended period of time and offers a feasible alternative to minimize the influence of transport time into pathology.

The cancer-associated FGFR4-G388R polymorphism enhances pancreatic insulin secretion and modifies the risk of diabetes.

The fibroblast growth factor receptor 4 (FGFR4)-R388 single-nucleotide polymorphism has been associated with cancer risk and prognosis. Here we show that the FGFR4-R388 allele yields a receptor variant that preferentially promotes STAT3/5 signaling. This STAT activation transcriptionally induces Grb14 in pancreatic endocrine cells to promote insulin secretion. Knockin mice with the FGFR4 variant allele develop pancreatic islets that secrete more insulin, a feature that is reversed through Grb14 deletion and enhanced with FGF19 administration. We also show in humans that the FGFR4-R388 allele enhances islet function and may protect against type 2 diabetes. These data support a common genetic link underlying cancer and hyperinsulinemia.

Monocytes/macrophages support mammary tumor invasivity by co-secreting lineage-specific EGFR ligands and a STAT3 activator.

BACKGROUND: Tumor-associated macrophages (TAM) promote malignant progression, yet the repertoire of oncogenic factors secreted by TAM has not been clearly defined. We sought to analyze which EGFR- and STAT3-activating factors are secreted by monocytes/macrophages exposed to tumor cell-secreted factors. METHODS: Following exposure of primary human monocytes and macrophages to supernatants of a variety of tumor cell lines, we have analyzed transcript and secreted protein levels of EGFR family ligands and of STAT3 activators. To validate our findings, we have analyzed TAM infiltration levels, systemic and local protein levels as well as clinical data of primary breast cancer patients. RESULTS: Primary human monocytes and macrophages respond to tumor cell-derived factors by secreting EGFR- and STAT3-activating ligands, thus inducing two important oncogenic pathways in carcinoma cells. Tumor cell-secreted factors trigger two stereotype secretory profiles in peripheral blood monocytes and differentiated macrophages: monocytes secrete epiregulin (EREG) and oncostatin-M (OSM), while macrophages secrete heparin-binding EGF-like growth factor (HB-EGF) and OSM. HB-EGF and OSM cooperatively induce tumor cell chemotaxis. HB-EGF and OSM are co-expressed by TAM in breast carcinoma patients, and plasma levels of both ligands correlate strongly. Elevated HB-EGF levels accompany TAM infiltration, tumor growth and dissemination in patients with invasive disease. CONCLUSIONS: Our work identifies systemic markers for TAM involvement in cancer progression, with the potential to be developed into molecular targets in cancer therapy.

The FGFR4-G388R polymorphism promotes mitochondrial STAT3 serine phosphorylation to facilitate pituitary growth hormone cell tumorigenesis.

Pituitary tumors are common intracranial neoplasms, yet few germline abnormalities have been implicated in their pathogenesis. Here we show that a single nucleotide germline polymorphism (SNP) substituting an arginine (R) for glycine (G) in the FGFR4 transmembrane domain can alter pituitary cell growth and hormone production. Compared with FGFR4-G388 mammosomatotroph cells that support prolactin (PRL) production, FGFR4-R388 cells express predominantly growth hormone (GH). Growth promoting effects of FGFR4-R388 as evidenced by enhanced colony formation was ascribed to Src activation and mitochondrial serine phosphorylation of STAT3 (pS-STAT3). In contrast, diminished pY-STAT3 mediated by FGFR4-R388 relieved GH inhibition leading to hormone excess. Using a knock-in mouse model, we demonstrate the ability of FGFR4-R385 to promote GH pituitary tumorigenesis. In patients with acromegaly, pituitary tumor size correlated with hormone excess in the presence of the FGFR4-R388 but not the FGFR4-G388 allele. Our findings establish a new role for the FGFR4-G388R polymorphism in pituitary oncogenesis, providing a rationale for targeting Src and STAT3 in the personalized treatment of associated disorders.

A single nucleotide change in the mouse genome accelerates breast cancer progression.

In the growth factor receptor gene FGFR4 the presence of the common single nucleotide polymorphism Arg388 has been associated with progression of various types of cancer including breast cancer. However, a causative relationship is not readily assigned due to genetic heterogeneity in different patient cohorts. To address this issue, we compared the effects of this allele on malignant progression in the WAP-TGFalpha transgenic mouse model of breast cancer. A knock-in strain was generated to introduce an analogous Arg385 allele into the murine FGFR4 gene. Mouse embryonic fibroblasts derived from this strain displayed accelerated cell transformation, with transformed cells exhibiting greater motility and invasive behavior. In the in vivo context of TGFalpha-induced mammary carcinogenesis, tumor development and progression was significantly advanced in tumor mass, size, and onset of pulmonary metastases. Our findings definitively identify the FGFR4 Arg388 allele as a functional prognostic marker for breast cancer progression.

Neurological and behavioral abnormalities, ventricular dilatation, altered cellular functions, inflammation, and neuronal injury in brains of mice due to common, persistent, parasitic infection.

BACKGROUND: Worldwide, approximately two billion people are chronically infected with Toxoplasma gondii with largely unknown consequences. METHODS: To better understand long-term effects and pathogenesis of this common, persistent brain infection, mice were infected at a time in human years equivalent to early to mid adulthood and studied 5-12 months later. Appearance, behavior, neurologic function and brain MRIs were studied. Additional analyses of pathogenesis included: correlation of brain weight and neurologic findings; histopathology focusing on brain regions; full genome microarrays; immunohistochemistry characterizing inflammatory cells; determination of presence of tachyzoites and bradyzoites; electron microscopy; and study of markers of inflammation in serum. Histopathology in genetically resistant mice and cytokine and NRAMP knockout mice, effects of inoculation of isolated parasites, and treatment with sulfadiazine or alphaPD1 ligand were studied. RESULTS: Twelve months after infection, a time equivalent to middle to early elderly ages, mice had behavioral and neurological deficits, and brain MRIs showed mild to moderate ventricular dilatation. Lower brain weight correlated with greater magnitude of neurologic abnormalities and inflammation. Full genome microarrays of brains reflected inflammation causing neuronal damage (Gfap), effects on host cell protein processing (ubiquitin ligase), synapse remodeling (Complement 1q), and also increased expression of PD-1L (a ligand that allows persistent LCMV brain infection) and CD 36 (a fatty acid translocase and oxidized LDL receptor that mediates innate immune response to beta amyloid which is associated with pro-inflammation in Alzheimer's disease). Immunostaining detected no inflammation around intra-neuronal cysts, practically no free tachyzoites, and only rare bradyzoites. Nonetheless, there were perivascular, leptomeningeal inflammatory cells, particularly contiguous to the aqueduct of Sylvius and hippocampus, CD4+ and CD8+ T cells, and activated microglia in perivascular areas and brain parenchyma. Genetically resistant, chronically infected mice had substantially less inflammation. CONCLUSION: In outbred mice, chronic, adult acquired T. gondii infection causes neurologic and behavioral abnormalities secondary to inflammation and loss of brain parenchyma. Perivascular inflammation is prominent particularly contiguous to the aqueduct of Sylvius and hippocampus. Even resistant mice have perivascular inflammation. This mouse model of chronic T. gondii infection raises questions of whether persistence of this parasite in brain can cause inflammation or neurodegeneration in genetically susceptible hosts.