Mapping of the three-dimensional lymphatic microvasculature in bladder tumours using light-sheet microscopy.

BACKGROUND: Cancers are heterogeneous and contain various types of irregular structures that can go undetected when examining them with standard two-dimensional microscopes. Studies of intricate networks of vasculature systems, e.g., the tumour lymphatic microvessels, benefit largely from three-dimensional imaging data analysis. METHODS: The new DIPCO (Diagnosing Immunolabeled Paraffin-Embedded Cleared Organs) imaging platform uses three-dimensional light-sheet microscopy and whole-mount immunolabelling of cleared samples to study proteins and micro-anatomies deep inside of tumours. RESULTS: Here, we uncovered the whole three-dimensional lymphatic microvasculature of formalin-fixed paraffin-embedded (FFPE) tumours from a cohort of 30 patients with bladder cancer. Our results revealed more heterogeneous spatial deviations in more advanced bladder tumours. We also showed that three-dimensional imaging could determine tumour stage and identify vascular or lymphatic system invasion with higher accuracy than standard two-dimensional histological diagnostic methods. There was no association between sample storage times and outcomes, demonstrating that the DIPCO pipeline could be successfully applied on old FFPE samples. CONCLUSIONS: Studying tumour samples with three-dimensional imaging could help us understand the pathological nature of cancers and provide essential information that might improve the accuracy of cancer staging.

Evolution of cystectomy care over an 11-year period in a high-volume tertiary referral centre.

OBJECTIVES: To describe the evolution in radical cystectomy (RC) care over 11 years at a referral centre. PATIENTS AND METHODS: The clinical data of patients undergoing either open RC (ORC) or robot-assisted RC (RARC) for cT1-4aN0M0 bladder cancer (BCa) at our centre between January 2006 and December 2016 were retrospectively evaluated. Crude and propensity score-weighted log-binomial regression analyses were conducted to assess the association between pre- and peri-operative variables and the risk of reoperation, intensive care unit (ICU) admission and death <90 days after RC. RESULTS: A total of 814 patients were considered. The percentage of RARCs performed increased (from 10% to 100%) between 2006 and 2013. Overall, 29% of the patients received neoadjuvant chemotherapy (12-37% from 2006 to 2016). Despite no differences in terms of operating time, pelvic lymph node dissection (PLND) was more commonly attempted during RARC and extended PLND was more frequently performed in the RARC group (72% vs 19%; P < 0.001). Ileal conduit was the preferred urinary diversion in both groups, and more patients in the RARC group underwent neobladder construction (34% vs 14%; P < 0.001). The overall rates of re-intervention, ICU admission and death within 90 days of RC were 8.9%, 5.4% and 2.9%, respectively. On crude analysis, RARC was associated with a reduced risk of ICU admission (relative risk [RR] 0.42, 95% confidence interval [CI] 0.23-0.77; P = 0.005), reintervention (RR 0.58, 95% CI 0.37-0.90; P = 0.015) and death (RR 0.37, 95% CI 0.16-0.85; P = 0.020); however, these risk reductions were not statistically significant on weighted analyses. CONCLUSIONS: The introduction of RARC has coincided with a reduction in the rate of ICU admission, reoperation and death within 90 days of surgery, without compromising operating time, PLND extent or neobladder utilization.

Intracellular calcium release modulates polycystin-2 trafficking.

BACKGROUND: Polycystin-2 (PC2), encoded by the gene that is mutated in autosomal dominant polycystic kidney disease (ADPKD), functions as a calcium (Ca(2+)) permeable ion channel. Considerable controversy remains regarding the subcellular localization and signaling function of PC2 in kidney cells. METHODS: We investigated the subcellular PC2 localization by immunocytochemistry and confocal microscopy in primary cultures of human and rat proximal tubule cells after stimulating cytosolic Ca(2+) signaling. Plasma membrane (PM) Ca(2+) permeability was evaluated by Fura-2 manganese quenching using time-lapse fluorescence microscopy. RESULTS: We demonstrated that PC2 exhibits a dynamic subcellular localization pattern. In unstimulated human or rat proximal tubule cells, PC2 exhibited a cytosolic/reticular distribution. Treatments with agents that in various ways affect the Ca(2+) signaling machinery, those being ATP, bradykinin, ionomycin, CPA or thapsigargin, resulted in increased PC2 immunostaining in the PM. Exposing cells to the steroid hormone ouabain, known to trigger Ca(2+) oscillations in kidney cells, caused increased PC2 in the PM and increased PM Ca(2+) permeability. Intracellular Ca(2+) buffering with BAPTA, inositol 1,4,5-trisphosphate receptor (InsP3R) inhibition with 2-aminoethoxydiphenyl borate (2-APB) or Ca(2+)/Calmodulin-dependent kinase inhibition with KN-93 completely abolished ouabain-stimulated PC2 translocation to the PM. CONCLUSIONS: These novel findings demonstrate intracellular Ca(2+)-dependent PC2 trafficking in human and rat kidney cells, which may provide new insight into cyst formations in ADPKD.

Na,K-ATPase signal transduction triggers CREB activation and dendritic growth.

Dendritic growth is pivotal in the neurogenesis of cortical neurons. The sodium pump, or Na,K-ATPase, is an evolutionarily conserved protein that, in addition to its central role in establishing the electrochemical gradient, has recently been reported to function as a receptor and signaling mediator. Although a large body of evidence points toward a dual function for the Na,K-ATPase, few biological implications of this signaling pathway have been described. Here we report that Na,K-ATPase signal transduction triggers dendritic growth as well as a transcriptional program dependent on cAMP response element binding protein (CREB) and cAMP response element (CRE)-mediated gene expression, primarily regulated via Ca(2+)/calmodulin-dependent protein (CaM) kinases. The signaling cascade mediating dendritic arbor growth also involves intracellular Ca(2+) oscillations and sustained phosphorylation of mitogen-activated protein (MAP) kinases. Thus, our results suggest a novel role for the Na,K-ATPase as a modulator of dendritic growth in developing neurons.

GIT1 protects against breast cancer growth through negative regulation of Notch.

Hyperactive Notch signalling is frequently observed in breast cancer and correlates with poor prognosis. However, relatively few mutations in the core Notch signalling pathway have been identified in breast cancer, suggesting that as yet unknown mechanisms increase Notch activity. Here we show that increased expression levels of GIT1 correlate with high relapse-free survival in oestrogen receptor-negative (ER(-)) breast cancer patients and that GIT1 mediates negative regulation of Notch. GIT1 knockdown in ER(-) breast tumour cells increased signalling downstream of Notch and activity of aldehyde dehydrogenase, a predictor of poor clinical outcome. GIT1 interacts with the Notch intracellular domain (ICD) and influences signalling by inhibiting the cytoplasm-to-nucleus transport of the Notch ICD. In xenograft experiments, overexpression of GIT1 in ER(-) cells prevented or reduced Notch-driven tumour formation. These results identify GIT1 as a modulator of Notch signalling and a guardian against breast cancer growth.

Development and internal validation of a nomogram predicting extracapsular extension in radical prostatectomy specimens.

OBJECTIVES: To present a nomogram predicting the side-specific probability of extracapsular extension (ECE) in radical prostatectomy (RP) specimens. METHODS: Three hundred and fifty-four patients with T1c-T3a prostate cancer undergoing RP were included in the analysis. A receiver operating characteristic (ROC) analysis was carried out to evaluate the predictive values of each clinical and pathological factor, separately and in combination. Based on logistic regression analysis, a nomogram predicting the side-specific probability of ECE was developed. RESULTS: Overall, 146 (40%) of 354 patients and 165 (23%) of 708 lobes had ECE pathologically. The areas under the ROC curve (AUC) of the standard features, such as serum PSA, clinical stage and biopsy Gleason sum on each side, in predicting side-specific probability of ECE were 0.624, 0.627, and 0.747, respectively. When these three features were combined, AUC increased to 0.773 which was not significantly different from 0.791 of maximum percent of cancer alone (P = 0.613) and significantly enhanced by including maximum percent of cancer on each side, 0.799 (P = 0.022). The resulting nomogram was internally validated and had excellent calibration. CONCLUSIONS: The accuracy in predicting ECE is increased by combining standard clinical factors (clinical stage, serum PSA, highest Gleason score) and biopsy features, such as maximum percent of cancer in the cores. The developed nomogram is helpful when deciding whether or not neurovascular bundles can be preserved.

Nomogram to predict seminal vesicle invasion using the status of cancer at the base of the prostate on systematic biopsy.

OBJECTIVE: The aim of this study was to predict seminal vesicle invasion (SVI) by developing a new nomogram based on clinical features including the status of cancer at the base of the prostate on systematic biopsy. METHODS: We studied the 466 patients with T1-3N0M0 prostate cancer who were treated with radical prostatectomy at three institutions. Preoperative clinical variables were correlated with the presence or absence of SVI with an area under the curve (AUC) of receiver-operator characteristics analysis. A nomogram was developed to predict SVI based on logistic regression analysis. RESULTS: A total of 81 patients (17%) had SVI. Cancer was present in a biopsy core from the base of the prostate in 209 patients, of whom 32.5% had SVI, compared with only 5% of the 257 patients without cancer at the base of the prostate (P < 0.005). On multivariate analysis, serum prostate-specific antigen, biopsy Gleason score, clinical T stage, and presence or absence of cancer in a biopsy core at the base of the prostate were significant predictors of SVI (P < 0.005 for all). The AUC of a standard model including clinical stage, Gleason score, and prostate-specific antigen was 0.83, which was significantly enhanced by including the presence of cancer at the base of the prostate (none, unilateral or bilateral lobes) (AUC 0.87, P= 0.023). Based on the logistic analysis, we developed the nomogram to predict SVI. The calibration plots appeared to be excellent. CONCLUSION: The information of presence or absence of cancer at the base from prostate biopsy and the resulting nomogram allow an accurate prediction of SVI in patients undergoing radical prostatectomy for prostate cancer.

[Treatment strategy for elderly patients with prostate cancer].

Because of both the indolent and aggressive nature of prostate cancers, it is not easy to select the best treatment for elderly patients with a high prevalence of comorbidities. Since the growth of prostate cancer is generally slow and all treatments adversely affect the quality of life to some degree, conservative treatment may well be the best option for elderly patients with prostate cancer. In fact, previous studies have indicated that the rate of prostate cancer death was not high in patients with low-intermediate risk of prostate cancer who were treated conservatively. However, it is also true that we often encounter elder patients with a locally advanced cancer who had not been exposed to PSA screening. Previous reports also support aggressive treatment such as hormonal therapy, external radiation and brachytherapy alone or in combination for healthy elderly men with high-risk prostate cancer. Thus, after a careful evaluation of the nature of the cancer and comorbidity, we found that both conservative and aggressive treatments are applicable for elderly patients with prostate cancer to maintain their quality of life.

Three-dimensional single-cell imaging for the analysis of RNA and protein expression in intact tumour biopsies.

Microscopy analysis of tumour samples is commonly performed on fixed, thinly sectioned and protein-labelled tissues. However, these examinations do not reveal the intricate three-dimensional structures of tumours, nor enable the detection of aberrant transcripts. Here, we report a method, which we name DIIFCO (for diagnosing in situ immunofluorescence-labelled cleared oncosamples), for the multimodal volumetric imaging of RNAs and proteins in intact tumour volumes and organoids. We used DIIFCO to spatially profile the expression of diverse coding RNAs and non-coding RNAs at the single-cell resolution in a variety of cancer tissues. Quantitative single-cell analysis revealed spatial niches of cancer stem-like cells, and showed that the niches were present at a higher density in triple-negative breast cancer tissue. The improved molecular phenotyping and histopathological diagnosis of cancers may lead to new insights into the biology of tumours of patients.

BCG-induced cytokine release in bladder cancer cells is regulated by Ca2+ signaling.

Bacillus Calmette-Guérin (BCG) is widely used in the clinic to effectively treat superficial urinary bladder cancer. However, a significant proportion of patients who fail to respond to BCG risk cystectomy or death. Though more than 3 million cancer treatments with BCG occur annually, surprisingly little is known about the initial signaling cascades activated by BCG. Here, we report that BCG induces a rapid intracellular Ca2+ (calcium ion) signal in bladder cancer cells that is essential for activating the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and for synthesizing and secreting proinflammatory cytokines, including interleukin 8 (IL-8). A similar Ca2+ response was observed when cells were exposed to the supernatant of BCG. Studying cellular molecular mechanisms involved in the BCG signaling event, we found pivotal roles for phospholipase C and the Toll-like receptor 4. Further assessment revealed that this signaling pathway induces synthesis of IL-8, whereas exocytosis appeared to be controlled by global Ca2+ signaling. These results shed new light on the molecular mechanisms underlying BCG treatment of bladder cancer, which can help in improving therapeutic efficacy and reducing adverse side effects.

Single-cell RNA-seq analysis reveals the platinum resistance gene COX7B and the surrogate marker CD63.

Cancers acquire resistance to systemic treatment with platinum-based chemotherapy (eg, cisplatin [CDDP]) as a result of a dynamic intratumoral heterogeneity (ITH) and clonal repopulation. However, little is known about the influence of chemotherapy on ITH at the single-cell level. Here, mapping the transcriptome of cancers treated with CDDP by scRNA-seq, we uncovered a novel gene, COX7B, associated with platinum-resistance, and surrogate marker, CD63. Knockdown of COX7B in cancer cells decreased the sensitivity of CDDP whereas overexpression recovered the sensitivity of CDDP. Low COX7B levels correlated with higher mortality rates in patients with various types of cancer and were significantly associated with poor response to chemotherapy in urinary bladder cancer. Tumor samples from patients, who underwent CDDP therapy, showed decreased COX7B protein levels after the treatment. Analyzing scRNA-seq data from platinum-naïve cancer cells demonstrated a low-COX7B subclone that could be sorted out from bulk cancer cells by assaying CD63. This low-COX7B subclone behaved as cells with acquired platinum-resistance when challenged to CDDP. Our results offer a new transcriptome landscape of platinum-resistance that provides valuable insights into chemosensitivity and drug resistance in cancers, and we identify a novel platinum resistance gene, COX7B, and a surrogate marker, CD63.

Publisher Correction: Whole-tissue biopsy phenotyping of three-dimensional tumours reveals patterns of cancer heterogeneity.

In this Article originally published, owing to a technical error, author affiliations were incorrectly assigned in the HTML version; the PDF was correct. These errors have now been corrected.

Na,K-ATPase generates calcium oscillations in hippocampal astrocytes.

Na,K-ATPase maintains not only ionic homeostasis, but also participates in a multiprotein complex mediating intracellular signalling. We show that ouabain, a specific ligand for Na,K-ATPase, evokes calcium oscillations in hippocampal astrocytes in primary cultures. Coimmunoprecipitation studies suggest that the mechanism underlying these calcium oscillations involves a multiprotein complex consisting of ankyrin-B, the inositol 1,4,5-trisphosphate receptor and Na,K-ATPase. The ouabain/Na,K-ATPase multi-protein complex induced calcium-dependent downstream activation of the transcription factor nuclear factor-kappaB. Calcium oscillations and nuclear factor-kappaB activation were blocked following intracellular calcium store depletion. Thus, the specific Na,K-ATPase ligand ouabain induced inositol 1,4,5-trisphosphate receptor-dependent calcium oscillations in hippocampal astrocytes, which mediates nuclear factor-kappaB activation.

Whole-tissue biopsy phenotyping of three-dimensional tumours reveals patterns of cancer heterogeneity.

Intratumoral heterogeneity is a critical factor when diagnosing and treating patients with cancer. Marked differences in the genetic and epigenetic backgrounds of cancer cells have been revealed by advances in genome sequencing, yet little is known about the phenotypic landscape and the spatial distribution of intratumoral heterogeneity within solid tumours. Here, we show that three-dimensional light-sheet microscopy of cleared solid tumours can identify unique patterns of phenotypic heterogeneity, in the epithelial-to-mesenchymal transition and in angiogenesis, at single-cell resolution in whole formalin-fixed paraffin-embedded (FFPE) biopsy samples. We also show that cleared FFPE samples can be re-embedded in paraffin after examination for future use, and that our tumour-phenotyping pipeline can determine tumour stage and stratify patient prognosis from clinical samples with higher accuracy than current diagnostic methods, thus facilitating the design of more efficient cancer therapies.

Production of erythropoietin and multiple cytokines by metanephric adenoma results in erythrocytosis.

This is the first report of direct evidence that metanephric adenoma cells produce erythropoietin and other types of cytokines, which may be the cause of the high incidence of erythrocytosis in patients with this tumor. The purpose of the study was to establish a metanephric adenoma cell line in vitro from nephrectomized tumor tissue in order to investigate the ability of metanephric adenoma cells to produce erythropoietin and other types of cytokines. The tumor tissue was obtained from a 16-year-old boy who had developed metanephric adenoma with erythrocytosis and was served for cell culture. Significantly high concentrations of erythropoietin, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), interleukin-6 (IL-6), and IL-8 were detected in the cell culture supernatant. Southern hybridization showed specific positive signals for GM-CSF, G-CSF, IL-6, IL-8 and erythropoietin. The number of chromosomes was 46-XY without any structural abnormalities in cytogenetic analysis of the cultured cells.