Comparative analysis of 10X Chromium vs. BD Rhapsody whole transcriptome single-cell sequencing technologies in complex human tissues.

The development of single-cell omics tools has enabled scientists to study the tumor microenvironment (TME) in unprecedented detail. However, each of the different techniques may have its unique strengths and limitations. Here we directly compared two commercially available high-throughput single-cell RNA sequencing (scRNA-seq) technologies - droplet-based 10X Chromium vs. microwell-based BD Rhapsody - using paired samples from patients with localized prostate cancer (PCa) undergoing a radical prostatectomy. Although high technical consistency was observed in unraveling the whole transcriptome, the relative abundance of cell populations differed. Cells with low mRNA content such as T cells were underrepresented in the droplet-based system, at least partly due to lower RNA capture rates. In contrast, microwell-based scRNA-seq recovered less cells of epithelial origin. Moreover, we discovered platform-dependent variabilities in mRNA quantification and cell-type marker annotation. Overall, our study provides important information for selection of the appropriate scRNA-seq platform and for the interpretation of published results.

Metformin Overcomes the Consequences of NKX3.1 Loss to Suppress Prostate Cancer Progression.

BACKGROUND: The antidiabetic drug metformin has known anticancer effects related to its antioxidant activity; however, its clinical benefit for prostate cancer (PCa) has thus far been inconclusive. Here, we investigate whether the efficacy of metformin in PCa is related to the expression status of NKX3.1, a prostate-specific homeobox gene that functions in mitochondria to protect the prostate from aberrant oxidative stress. OBJECTIVE: To investigate the relationship of NKX3.1 expression and metformin efficacy in PCa. DESIGN, SETTING, AND PARTICIPANTS: Functional studies were performed in vivo and in vitro in genetically engineered mouse models and human LNCaP cells, and organotypic cultures having normal or reduced/absent levels of NKX3.1. Correlative studies were performed using two independent retrospective tissue microarray cohorts of radical prostatectomies and a retrospective cohort of prostate biopsies from patients on active surveillance. INTERVENTION: Metformin was administered before or after the induction of oxidative stress by treatment with paraquat. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Functional endpoints included analyses of histopathology, tumorigenicity, and mitochondrial function. Correlative endpoints include Kaplan-Meier curves and Cox proportional hazard regression models. RESULTS AND LIMITATIONS: Metformin reversed the adverse consequences of NKX3.1 deficiency following oxidative stress in vivo and in vitro, as evident by reduced tumorigenicity and restored mitochondrial function. Patients with low NKX3.1 expression showed a significant clinical benefit from taking metformin. CONCLUSIONS: Metformin can overcome the adverse consequences of NKX3.1 loss for PCa progression by protecting against oxidative stress and promoting normal mitochondrial function. These functional activities and clinical correlates were observed only with low NKX3.1 expression. Thus, the clinical benefit of metformin in PCa may depend on the status of NKX3.1 expression. PATIENT SUMMARY: Prostate cancer patients with low NKX3.1 are likely to benefit most from metformin treatment to delay disease progression in a precision interception paradigm.

Glucocorticoid treatment influences prostate cancer cell growth and the tumor microenvironment via altered glucocorticoid receptor signaling in prostate fibroblasts.

Despite significant therapeutic advances in recent years, treatment of metastatic prostate cancer (PCa) remains palliative, owing to the inevitable occurrence of drug resistance. There is increasing evidence that epithelial glucocorticoid receptor (GR) signaling and changes in the tumor-microenvironment (TME) play important roles in this process. Since glucocorticoids (GCs) are used as concomitant medications in the course of PCa treatment, it is essential to investigate the impact of GCs on stromal GR signaling in the TME. Therefore, general GR mRNA and protein expression was assessed in radical prostatectomy specimens and metastatic lesions. Elevated stromal GR signaling after GC treatment resulted in altered GR-target gene, soluble protein expression, and in a morphology change of immortalized and primary isolated cancer-associated fibroblasts (CAFs). Subsequently, these changes affected proliferation, colony formation, and 3D-spheroid growth of multiple epithelial PCa cell models. Altered expression of extra-cellular matrix (ECM) and adhesion-related proteins led to an ECM remodeling. Notably, androgen receptor pathway inhibitor treatments did not affect CAF viability. Our findings demonstrate that GC-mediated elevated GR signaling has a major impact on the CAF secretome and the ECM architecture. GC-treated fibroblasts significantly influence epithelial tumor cell growth and must be considered in future therapeutic strategies.

STAT3/LKB1 controls metastatic prostate cancer by regulating mTORC1/CREB pathway.

Prostate cancer (PCa) is a common and fatal type of cancer in men. Metastatic PCa (mPCa) is a major factor contributing to its lethality, although the mechanisms remain poorly understood. PTEN is one of the most frequently deleted genes in mPCa. Here we show a frequent genomic co-deletion of PTEN and STAT3 in liquid biopsies of patients with mPCa. Loss of Stat3 in a Pten-null mouse prostate model leads to a reduction of LKB1/pAMPK with simultaneous activation of mTOR/CREB, resulting in metastatic disease. However, constitutive activation of Stat3 led to high LKB1/pAMPK levels and suppressed mTORC1/CREB pathway, preventing mPCa development. Metformin, one of the most widely prescribed therapeutics against type 2 diabetes, inhibits mTORC1 in liver and requires LKB1 to mediate glucose homeostasis. We find that metformin treatment of STAT3/AR-expressing PCa xenografts resulted in significantly reduced tumor growth accompanied by diminished mTORC1/CREB, AR and PSA levels. PCa xenografts with deletion of STAT3/AR nearly completely abrogated mTORC1/CREB inhibition mediated by metformin. Moreover, metformin treatment of PCa patients with high Gleason grade and type 2 diabetes resulted in undetectable mTORC1 levels and upregulated STAT3 expression. Furthermore, PCa patients with high CREB expression have worse clinical outcomes and a significantly increased risk of PCa relapse and metastatic recurrence. In summary, we have shown that STAT3 controls mPCa via LKB1/pAMPK/mTORC1/CREB signaling, which we have identified as a promising novel downstream target for the treatment of lethal mPCa.

The Oncogenic Protein Kinase/ATPase RIOK1 Is Up-Regulated via the c-myc/E2F Transcription Factor Axis in Prostate Cancer.

The atypical protein kinase/ATPase RIO kinase (RIOK)-1 is involved in pre-40S ribosomal subunit production, cell-cycle progression, and protein arginine N-methyltransferase 5 methylosome substrate recruitment. RIOK1 overexpression is a characteristic of several malignancies and is correlated with cancer stage, therapy resistance, poor patient survival, and other prognostic factors. However, its role in prostate cancer (PCa) is unknown. In this study, the expression, regulation, and therapeutic potential of RIOK1 in PCa were examined. RIOK1 mRNA and protein expression were elevated in PCa tissue samples and correlated with proliferative and protein homeostasis-related pathways. RIOK1 was identified as a downstream target gene of the c-myc/E2F transcription factors. Proliferation of PCa cells was significantly reduced with RIOK1 knockdown and overexpression of the dominant-negative RIOK1-D324A mutant. Biochemical inhibition of RIOK1 with toyocamycin led to strong antiproliferative effects in androgen receptor-negative and -positive PCa cell lines with EC50 values of 3.5 to 8.8 nmol/L. Rapid decreases in RIOK1 protein expression and total rRNA content, and a shift in the 28S/18S rRNA ratio, were found with toyocamycin treatment. Apoptosis was induced with toyocamycin treatment at a level similar to that with the chemotherapeutic drug docetaxel used in clinical practice. In summary, the current study indicates that RIOK1 is a part of the MYC oncogene network, and as such, could be considered for future treatment of patients with PCa.

Morphological and Tissue Characterization with 3D Reconstruction of a 350-Year-Old Austrian Ardea purpurea Glacier Mummy.

Glaciers are dwindling archives, releasing animal mummies preserved in the ice for centuries due to climate changes. As preservation varies, residual soft tissues may differently expand the biological information content of such mummies. DNA studies have proven the possibility of extracting and analyzing DNA preserved in skeletal residuals and sediments for hundreds or thousands of years. Paleoradiology is the method of choice as a non-destructive tool for analyzing mummies, including micro-computed tomography (micro-CT) and magnetic resonance imaging (MRI). Together with radiocarbon dating, histo-anatomical analyses, and DNA sequencing, these techniques were employed to identify a 350-year-old Austrian Ardea purpurea glacier mummy from the Ötztal Alps. Combining these techniques proved to be a robust methodological concept for collecting inaccessible information regarding the structural organization of the mummy. The variety of methodological approaches resulted in a distinct picture of the morphological patterns of the glacier animal mummy. The BLAST search in GenBank resulted in a 100% and 98.7% match in the cytb gene sequence with two entries of the species Purple heron (Ardea purpurea; Accession number KJ941160.1 and KJ190948.1) and a 98% match with the same species for the 16 s sequence (KJ190948.1), which was confirmed by the anatomic characteristics deduced from micro-CT and MRI.

Complement and Fungal Dysbiosis as Prognostic Markers and Potential Targets in PDAC Treatment.

Pancreatic ductal adenocarcinoma (PDAC) is still hampered by a dismal prognosis. A better understanding of the tumor microenvironment within the pancreas and of the factors affecting its composition is of utmost importance for developing new diagnostic and treatment tools. In this context, the complement system plays a prominent role. Not only has it been shown to shape a T cell-mediated immune response, but it also directly affects proliferation and apoptosis of the tumor cells, influencing angiogenesis, metastatic spread and therapeutic resistance. This makes complement proteins appealing not only as early biomarkers of PDAC development, but also as therapeutic targets. Fungal dysbiosis is currently the new kid on the block in tumorigenesis with cancer-associated mycobiomes extracted from several cancer types. For PDAC, colonization with the yeast Malassezia seems to promote cancer progression, already in precursor lesions. One responsible mechanism appears to be complement activation via the lectin pathway. In the present article, we review the role of the complement system in tumorigenesis, presenting observations that propose it as the missing link between fungal dysbiosis and PDAC development. We also present the results of a small pilot study supporting the crucial interplay between the complement system and Malassezia colonization in PDAC pathogenesis.

Comprehensive characterization of the prostate tumor microenvironment identifies CXCR4/CXCL12 crosstalk as a novel antiangiogenic therapeutic target in prostate cancer.

BACKGROUND: Crosstalk between neoplastic and stromal cells fosters prostate cancer (PCa) progression and dissemination. Insight in cell-to-cell communication networks provides new therapeutic avenues to mold processes that contribute to PCa tumor microenvironment (TME) alterations. Here we performed a detailed characterization of PCa tumor endothelial cells (TEC) to delineate intercellular crosstalk between TEC and the PCa TME. METHODS: TEC isolated from 67 fresh radical prostatectomy (RP) specimens underwent multi-omic ex vivo characterization as well as orthogonal validation of both TEC functions and key markers by immunohistochemistry (IHC) and immunofluorescence (IF). To identify cell-cell interaction targets in TEC, we performed single-cell RNA sequencing (scRNA-seq) in four PCa patients who underwent a RP to catalogue cellular TME composition. Targets were cross-validated using IHC, publicly available datasets, cell culture expriments as well as a PCa xenograft mouse model. RESULTS: Compared to adjacent normal endothelial cells (NEC) bulk RNA-seq analysis revealed upregulation of genes associated with tumor vasculature, collagen modification and extracellular matrix remodeling in TEC. PTGIR, PLAC9, CXCL12 and VDR were identified as TEC markers and confirmed by IF and IHC in an independent patient cohort. By scRNA-seq we identified 27 cell (sub)types, including endothelial cells (EC) with arterial, venous and immature signatures, as well as angiogenic tip EC. A focused molecular analysis revealed that arterial TEC displayed highest CXCL12 mRNA expression levels when compared to all other TME cell (sub)populations and showed a negative prognostic role. Receptor-ligand interaction analysis predicted interactions between arterial TEC derived CXCL12 and its cognate receptor CXCR4 on angiogenic tip EC. CXCL12 was in vitro and in vivo validated as actionable TEC target by highlighting the vessel number- and density- reducing activity of the CXCR4-inhibitor AMD3100 in murine PCa as well as by inhibition of TEC proliferation and migration in vitro. CONCLUSIONS: Overall, our comprehensive analysis identified novel PCa TEC targets and highlights CXCR4/CXCL12 interaction as a potential novel target to interfere with tumor angiogenesis in PCa.

KDM5D predicts response to docetaxel chemotherapy in metastatic castration resistant prostate cancer patients.

BACKGROUND: The administration of docetaxel chemotherapy is one therapeutic option to delay disease progression and increase overall survival in metastatic castration resistant prostate cancer (mCRPC). However, about 15% of patients are primary resistant to chemotherapy and hence would benefit from an alternative mCRPC treatment. Despite intensive research, there are no robust clinical validated biomarkers to predict mCRPC therapy response. Thus, the aim of the study was to determine KDM5D expression in archival radical prostatectomy specimens of patients medicated with docetaxel at time of mCRPC development in order to correlate KMD5D expression with treatment response. METHODS: We used in situ hybridization (ISH) (RNA scope 2.5 HD) to determine KDM5D expression in tissue samples of 28 prostate cancer patients. KDM5D status was correlated to chemotherapy response (PSA and radiographic response). RESULTS: Data revealed that KDM5D is significantly overexpressed in tumor cells (P<0.0001) but also in benign cells (P<0.02) of those patients who responded to chemotherapy compared to non-responders. CONCLUSIONS: To summarize, KDM5D is a promising novel biomarker predicting response to docetaxel chemotherapy already at the time of localized disease and thus potentially avoiding metastatic biopsies in the mCRPC stage of disease.

Implications of Standardized Uptake Values of Oral Squamous Cell Carcinoma in PET-CT on Prognosis, Tumor Characteristics and Mitochondrial DNA Heteroplasmy.

Under aerobic conditions, some cancers switch to glycolysis to cover their energy requirements. Taking advantage of this process, functional imaging techniques such as PET-CT can be used to detect and assess tumorous tissues. The aim of this study was to investigate standardized uptake values and mitochondrial DNA mutations in oral squamous cell carcinoma. A cohort of 57 patients underwent 18[F]FDG-PET-CT and standardized uptake values were collected. In 15 patients, data on mitochondrial DNA mutations of the tumor were available. Kaplan-Meier curves were calculated, and correlation analyses as well as univariate Cox proportional hazard models were performed. Using ROC analysis to determine a statistical threshold for SUVmax in PET investigations, a cut-off value was determined at 9.765 MB/mL. Survival analysis for SUVmax in these groups showed a Hazard Ratio of 4 (95% CI 1.7-9) in the high SUVmax group with 5-year survival rates of 23.5% (p = 0.00042). For SUVmax and clinicopathological tumor features, significant correlations were found. A tendency towards higher mtDNA heteroplasmy levels in high SUVmax groups could be observed. We were able to confirm the prognostic value of SUVmax in OSCC, showing higher survival rates at lower SUVmax levels. Correlations between SUVmax and distinct tumor characteristics were highly significant, providing evidence that SUVmax may act as a reliable diagnostic parameter. Correlation analysis of mtDNA mutations suggests an influence on metabolic activity in OSCC.

Expression of ADAM Proteases in Bladder Cancer Patients with BCG Failure: A Pilot Study.

Although Bacillus Calmette Guérin (BCG) remains a mainstay of adjuvant treatment in high-risk, non-muscle-invasive bladder cancer, BCG failure occurs in up to 40% of patients, with radical cystectomy (RC) as the inevitable therapeutic consequence. Current data suggest that PD-L1 immunosuppressive signaling is responsible for BCG failure, supporting the therapeutic rationale of combining checkpoint inhibitors with BCG. To address the immune cascade in 19 RC specimens obtained after BCG failure, we applied a small immunohistochemical (IHC) panel consisting of selected markers (PD-L1, GATA-3, a disintegrin and metalloproteinase (ADAM) proteases, IL-10/IL-10R). A modified quick score was used for IHC semi-quantification of these markers in tumor cells (TC) and immune cells (IC) within two different regions: muscle-invasive bladder cancer (MIBC) and primary/concurrent carcinoma in situ (CIS). Contrary to expectation, PD-L1 was consistently low, irrespective of tumor region and cell type. Intriguingly, expression of ADAM17, which has been reported to release membrane-bound PD-L1, was high in both tumor regions and cell types. Moreover, expression of GATA3, IL-10, and IL-10R was also increased, indicative of a generally immunosuppressive tumor microenvironment in BCG failure. ADAM10 expression was associated with advanced tumor disease at RC. Our findings raise the possibility that ADAM proteases may cleave PD-L1 from the surface of bladder TC and possibly also from IC. Therefore, IHC assessment of PD-L1 expression seems to be insufficient and should be supplemented by ADAM10/17 in patients with BCG failure.

Defining a therapeutic range for regeneration of ischemic myocardium via shock waves.

Shockwave therapy (SWT) represents a promising regenerative treatment option for patients with ischemic cardiomyopathy. Although no side-effects have been described upon SWT, potential cellular damage at therapeutic energies has not been addressed so far. In this work, we aimed to define a therapeutic range for shock wave application for myocardial regeneration. We could demonstrate that SWT does not induce cellular damage beneath energy levels of 0.27 mJ/mm2 total flux density. Endothelial cell proliferation, angiogenic gene expression and phosphorylation of AKT and ERK are enhanced in a dose dependent manner until 0.15 mJ/mm2 energy flux density. SWT induces regeneration of ischemic muscle in vivo via expression of angiogenic gene expression, enhanced neovascularization and improved limb perfusion in a dose-dependent manner. Therefore, we provide evidence for a dose-dependent induction of angiogenesis after SWT, as well as the absence of cellular damage upon SWT within the therapeutic range. These data define for the first time a therapeutic range of SWT, a promising regenerative treatment option for ischemic cardiomyopathy.

Profiling of Mitochondrial DNA Heteroplasmy in a Prospective Oral Squamous Cell Carcinoma Study.

While a shift in energy metabolism is essential to cancers, the knowledge about the involvement of the mitochondrial genome in tumorigenesis and progression in oral squamous cell carcinoma (OSCC) is still very limited. In this study, we evaluated 37 OSCC tumors and the corresponding benign mucosa tissue pairs by deep sequencing of the complete mitochondrial DNA (mtDNA). After extensive quality control, we identified 287 variants, 137 in tumor and 150 in benign samples exceeding the 1% threshold. Variant heteroplasmy levels were significantly increased in cancer compared to benign tissues (p = 0.0002). Furthermore, pairwise high heteroplasmy frequency difference variants (∆HF% > 20) with potential functional impact were increased in the cancer tissues (p = 0.024). Fourteen mutations were identified in the protein-coding region, out of which thirteen were detected in cancer and only one in benign tissue. After eight years of follow-up, the risk of mortality was higher for patients who harbored at least one ∆HF% > 20 variant in mtDNA protein-coding regions relative to those with no mutations (HR = 4.6, (95%CI = 1.3-17); p = 0.019 in primary tumor carriers). Haplogroup affiliation showed an impact on survival time, which however needs confirmation in a larger study. In conclusion, we observed a significantly higher accumulation of somatic mutations in the cancer tissues associated with a worse prognosis.

OXPHOS remodeling in high-grade prostate cancer involves mtDNA mutations and increased succinate oxidation.

Rewiring of energy metabolism and adaptation of mitochondria are considered to impact on prostate cancer development and progression. Here, we report on mitochondrial respiration, DNA mutations and gene expression in paired benign/malignant human prostate tissue samples. Results reveal reduced respiratory capacities with NADH-pathway substrates glutamate and malate in malignant tissue and a significant metabolic shift towards higher succinate oxidation, particularly in high-grade tumors. The load of potentially deleterious mitochondrial-DNA mutations is higher in tumors and associated with unfavorable risk factors. High levels of potentially deleterious mutations in mitochondrial Complex I-encoding genes are associated with a 70% reduction in NADH-pathway capacity and compensation by increased succinate-pathway capacity. Structural analyses of these mutations reveal amino acid alterations leading to potentially deleterious effects on Complex I, supporting a causal relationship. A metagene signature extracted from the transcriptome of tumor samples exhibiting a severe mitochondrial phenotype enables identification of tumors with shorter survival times.

Toll-like receptor 3 mediates ischaemia/reperfusion injury after cardiac transplantation.

OBJECTIVES: Ischaemia and subsequent reperfusion during heart transplantation inevitably result in donor organ injury. Toll-like receptor (TLR)-3 is a pattern recognition receptor activated by viral and endogenous RNA released by injured cells. We hypothesized that ischaemia/reperfusion injury (IRI) leads to RNA release with subsequent TLR3 activation in transplanted hearts. METHODS: Human endothelial cells were subjected to IRI and treated with TLR3 agonist polyinosinic-polycytidylic acid or a TLR3/double-stranded RNA complex inhibitor. TLR3 activation was analysed using reporter cells. Gene expression profiles were evaluated via next-generation sequencing. Neutrophil adhesion was assessed in vitro. Syngeneic heart transplantation of wild-type or Tlr3-/- mice was performed following 9 h of cold ischaemia. Hearts were analysed for inflammatory gene expression, cardiac damage, apoptosis and infiltrating leucocytes. RESULTS: IRI resulted in RNA release with subsequent activation of TLR3. Treatment with a TLR3 inhibitor abrogated the inflammatory response upon IRI. In parallel, TLR3 stimulation caused activation of the inflammasome. Endothelial IRI resulted in TLR3-dependent adhesion of neutrophils. Tlr3-/- animals showed reduced intragraft and splenic messenger ribonucleic acid (mRNA) expression of proinflammatory cytokines, resulting in decreased myocardial damage, apoptosis and infiltrating cells. Tlr3 deficiency protected from cardiac damage, apoptosis and leucocyte infiltration after cardiac transplantation. CONCLUSIONS: We uncover the release of RNA by injured cells with subsequent activation of TLR3 as a crucial pathomechanism of IRI. Our data indicate that TLR3 represents a novel target in the prevention of IRI in solid organ transplantation.

The Impact of Cand1 in Prostate Cancer.

Evidence has accumulated asserting the importance of cullin-RING (really interesting new gene) ubiquitin ligases (CRLs) and their regulator Cullin-associated neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8) dissociated protein 1 (Cand1) in various cancer entities. However, the role of Cand1 in prostate cancer (PCa) has not been intensively investigated so far. Thus, in the present study, we aimed to assess the relevance of Cand1 in the clinical and preclinical setting. Immunohistochemical analyses of radical prostatectomy specimens of PCa patients showed that Cand1 protein levels are elevated in PCa compared to benign areas. In addition, high Cand1 levels were associated with higher Gleason Scores, as well as higher tumor recurrence and decreased overall survival. In line with clinical findings, in vitro experiments in different PCa cell lines revealed that knockdown of Cand1 reduced cell viability and proliferation and increased apoptosis, therefore underlining its role in tumor progression. We also found that the cyclin-dependent kinase inhibitor p21 is significantly upregulated upon downregulation of Cand1. Using bioinformatic tools, we detected genes encoding for proteins linked to mRNA turnover, protein polyubiquitination, and proteasomal degradation to be significantly upregulated in Cand1high tumors. Next generation sequencing of PCa cell lines resistant to the anti-androgen enzalutamide revealed that Cand1 is mutated in enzalutamide-resistant cells, however, with little functional and clinically relevant impact in the process of resistance development. To summarize the present study, we found that high Cand1 levels correlate with PCa aggressiveness.

p300 is upregulated by docetaxel and is a target in chemoresistant prostate cancer.

Administration of the microtubule inhibitor docetaxel is a common treatment for metastatic castration-resistant prostate cancer (mCRPC) and results in prolonged patient overall survival. Usually, after a short period of time chemotherapy resistance emerges and there is urgent need to find new therapeutic targets to overcome therapy resistance. The lysine-acetyltransferase p300 has been correlated to prostate cancer (PCa) progression. Here, we aimed to clarify a possible function of p300 in chemotherapy resistance and verify p300 as a target in chemoresistant PCa. Immunohistochemistry staining of tissue samples revealed significantly higher p300 protein expression in patients who received docetaxel as a neoadjuvant therapy compared to control patients. Elevated p300 expression was confirmed by analysis of publicly available patient data, where significantly higher p300 mRNA expression was found in tissue of mCRPC tumors of docetaxel-treated patients. Consistently, docetaxel-resistant PCa cells showed increased p300 protein expression compared to docetaxel-sensitive counterparts. Docetaxel treatment of PCa cells for 72 h resulted in elevated p300 expression. shRNA-mediated p300 knockdown did not alter colony formation efficiency in docetaxel-sensitive cells, but significantly reduced clonogenic potential of docetaxel-resistant cells. Downregulation of p300 in docetaxel-resistant cells also impaired cell migration and invasion. Taken together, we showed that p300 is upregulated by docetaxel, and our findings suggest that p300 is a possible co-target in treatment of chemoresistant PCa.

Amplification of 7p12 Is Associated with Pathologic Nonresponse to Neoadjuvant Chemotherapy in Muscle-Invasive Bladder Cancer.

Pathologic downstaging (pDS) to neoadjuvant chemotherapy (NAC) is one of the most important predictors of survival in muscle-invasive bladder cancer (MIBC). The use of NAC is limited as pDS is only achieved in 30% to 40% of cases and predictive biomarkers are still lacking. We performed a comprehensive immunomolecular biomarker analysis to characterize the role of immune cells and inhibitory checkpoints, genome-wide frequencies of copy number alterations, mutational signatures in whole exome, and tumor mutational burden in predicting NAC response. Our retrospective study included 23 primary MIBC patients who underwent NAC, followed by radical cystectomy. pDS to NAC was a significant prognostic factor for better recurrence-free survival (P < 0.001), with a median time to recurrence of 41.2 versus 5.5 months in nonresponders. DNA damage repair alterations were noticed in 38.1% (n = 8), confirming a positive correlation with high tumor mutational burden (P = 0.007). Chromosomal 7p12 amplification, including the genes HUS1, EGFR, ABCA13, and IKZF1, predicted nonresponse in patients with a sensitivity, a negative predictive value, and a specificity of 71.4%, 87.5%, and 100%, respectively. Total count of CD3+ T cells/mm2 tumor was a significant predictor of NAC response. In conclusion, 7p12 amplification may predict nonresponse to NAC and worse survival in MIBC. Multicenter, prospective trials with sufficient statistical power may further fortify these findings.

Robo 4 - the double-edged sword in prostate cancer: impact on cancer cell aggressiveness and tumor vasculature.

Background: The magic roundabout receptor 4 (Robo 4) is a tumor endothelial marker expressed in the vascular network of various tumor entities. However, the role of Robo 4 in prostate cancer (PCa), the second common cause of cancer death among men in -developed countries, has not been described yet. Thus, the present study investigates for the first time the impact of Robo 4 in PCa both in the clinical setting and in vitro. Methods and Results: Immunohistochemical analyses of benign and malignant prostate tissue samples of 95 PCa patients, who underwent radical prostatectomy (RPE), revealed a significant elevated expression of Robo 4 as well as its ligand Slit 2 protein in cancerous tissue compared to benign. Moreover, increased Robo 4 expression was associated with higher Gleason score and pT stage. In advanced stage we observed a hypothesis-generating trend that high Robo 4 and Slit 2 expression is associated with delayed development of tumor recurrence compared to patients with low Robo 4 and Slit 2 expression, respectively. In contrast to so far described exclusive expression of Robo 4 in the tumor vascular network, our analyses showed that in PCa Robo 4 is not only expressed in the tumor stroma but also in cancer epithelial cells. This finding was also confirmed in vitro as PC3 PCa cells express Robo 4 on mRNA as well as protein level. Overexpression of Robo 4 in PC3 as well as in Robo 4 negative DU145 and LNCaP PCa cells was associated with a significant decrease in cell-proliferation and cell-viability. Conclusion: In summary we observed that Robo 4 plays a considerable role in PCa development as it is expressed in cancer epithelial cells as well as in the surrounding tumor stroma. Moreover, higher histological tumor grade was associated with increased Robo 4 expression; controversially patients with high Robo 4 tend to exert lower biochemical recurrence possibly reflecting a protective role of Robo 4.