Tenascin-C expression in the lymph node pre-metastatic niche in muscle-invasive bladder cancer.

BACKGROUND: Markers of stromal activation at future metastatic sites may have prognostic value and may allow clinicians to identify and abolish the pre-metastatic niche to prevent metastasis. In this study, we evaluate tenascin-C as a marker of pre-metastatic niche formation in bladder cancer patient lymph nodes. METHODS: Tenascin-C expression in benign lymph nodes was compared between metastatic (n = 20) and non-metastatic (n = 27) patients with muscle-invasive bladder cancer. Urinary extracellular vesicle (EV) cytokine levels were measured with an antibody array to examine potential correlation with lymph node inflammation. The ability of bladder cancer EVs to activate primary bladder fibroblasts was assessed in vitro. RESULTS: Lymph node tenascin-C expression was elevated in metastatic patients vs. non-metastatic patients, and high expression was associated with worse survival. Urinary EVs contained four cytokines that were positively correlated with lymph node tenascin-C expression. Bladder cancer EVs induced tenascin-C expression in fibroblasts in an NF-κB-dependent manner. CONCLUSIONS: Tenascin-C expression in regional lymph nodes may be a good predictor of bladder cancer metastasis and an appropriate imaging target. It may be possible to interrupt pre-metastatic niche formation by targeting EV-borne tumour cytokines or by targeting tenascin-C directly.

Enhanced metastatic potential in the MB49 urothelial carcinoma model.

Recent data suggest that patients with a basal/stem-like bladder cancer (BC) subtype tend to have metastatic disease, but this is unconfirmed. Here we report the identification of murine MB49 cell line sub-clones with stem-like characteristics in culture. Subcutaneous implantation of S2 and S4 MB49 sub-clones into immunocompetent mice resulted in lung metastases in 50% and 80% of mice respectively, whereas none of the mice implanted with the parental cells developed metastasis. Gene profiling of cells cultured from S2 and S4 primary and metastatic tumors revealed that a panel of genes with basal/stem-like/EMT properties is amplified during metastatic progression. Among them, ITGB1, TWIST1 and KRT6B are consistently up-regulated in metastatic tumors of both MB49 sub-clones. To evaluate clinical relevance, we examined these genes in a human public dataset and found that ITGB1 and KRT6B expression in BC patient tumor samples are positively correlated with tumor grade. Likewise, the expression levels of these three genes are correlated with worse clinical outcomes. This MB49 BC metastatic pre-clinical model provides a unique opportunity to validate and recapitulate results discovered in patient studies and to pursue future mechanistic therapeutic interventions for BC metastasis.

Bladder cancer extracellular vesicles drive tumorigenesis by inducing the unfolded protein response in endoplasmic reticulum of nonmalignant cells.

The field cancerization effect has been proposed to explain bladder cancer's multifocal and recurrent nature, yet the mechanisms of this effect remain unknown. In this work, using cell biology, flow cytometry, and qPCR analyses, along with a xenograft mouse tumor model, we show that chronic exposure to tumor-derived extracellular vesicles (TEVs) results in the neoplastic transformation of nonmalignant human SV-HUC urothelial cells. Inhibition of EV uptake prevented this transformation. Transformed cells not only possessed several oncogenic properties, such as increased genome instability, loss of cell-cell contact inhibition, and invasiveness, but also displayed altered morphology and cell structures, such as an enlarged cytoplasm with disrupted endoplasmic reticulum (ER) alignment and the accumulation of smaller mitochondria. Exposure of SV-HUC cells to TEVs provoked the unfolded protein response in the endoplasmic reticulum (UPRER). Prolonged induction of UPRER signaling activated the survival branch of the UPRER pathway, in which cells had elevated expression of inositol-requiring enzyme 1 (IRE1), NF-κB, and the inflammatory cytokine leptin, and incurred loss of the pro-apoptotic protein C/EBP homologous protein (CHOP). More importantly, inhibition of ER stress by docosahexaenoic acid prevented TEV-induced transformation. We propose that TEVs promote malignant transformation of predisposed cells by inhibiting pro-apoptotic signals and activating tumor-promoting ER stress-induced unfolded protein response and inflammation. This study provides detailed insight into the mechanisms underlying the bladder cancer field effect and tumor recurrence.

Characterization of urinary extracellular vesicle proteins in muscle-invasive bladder cancer.

The mechanisms of bladder cancer progression are unknown, and new treatments and biomarkers are needed. Patient urinary extracellular vesicles (EVs) derive in part from bladder cancer cells and contain a specific protein cargo which may provide information about the disease. We conducted a proteomics study comparing EVs from the muscle-invasive bladder cancer (MIBC) cell line TCCSUP to EVs from normal urothelial line SVHUC. GO term analysis showed that TCCSUP EVs are enriched in proteins associated with the cell membrane, extracellular matrix, and inflammation and angiogenesis signaling pathways. Proteins characteristic of cancer EVs were further screened at the mRNA level in bladder cancer cell lines. In Western blots, three of six proteins examined showed greater than fifteenfold enrichment in patient urinary EVs compared to healthy volunteers (n = 6). Finally, we performed immunohistochemical staining of bladder tissue microarrays for three proteins of interest. One of them, transaldolase (TALDO1), is a nearly ubiquitous enzyme and normally thought to reside in the cytoplasm. To our surprise, nuclei were stained for transaldolase in 94% of MIBC tissue samples (n = 51). While cytoplasmic transaldolase was found in 89-90% of both normal urothelium (n = 79) and non-muscle-invasive samples (n = 71), the rate falls to 39% in MIBC samples (P < 0.001), and negative cytoplasmic staining was correlated with worse cancer-specific survival in MIBC patients (P = 0.008). The differential EV proteomics strategy reported here successfully identified a number of proteins associated with bladder cancer and points the way to future investigation.

Identification of extracellular vesicle-borne periostin as a feature of muscle-invasive bladder cancer.

Muscle-invasive bladder cancer (MIBC) is an aggressive malignancy with high mortality, and heterogeneity in MIBC results in variable clinical outcomes, posing challenges for clinical management. Extracellular vesicles (EVs) derived from MIBC have been shown to promote cancer progression. EVs derived from bladder cell lines were subjected to proteomic analysis, and periostin was chosen for further characterization due to its stage-specific gene expression profile. Knockdown of periostin by RNA interference reduces invasiveness in vitro and produces a rounder morphology. Importantly, treating low grade BC cells with periostin-rich EVs promotes cell aggressiveness and activates ERK oncogenic signals, and periostin suppression reverses these effects. These data suggest that MIBC might transfer periostin in an EV-mediated paracrine manner to promote the disease. To determine the potential of periostin as a bladder cancer indicator, patient urinary EVs were examined and found to have markedly higher levels of periostin than controls. In addition, immunohistochemical staining of a bladder cancer tissue microarray revealed that the presence of periostin in MIBC cells is correlated with worse prognosis. In conclusion, periostin is a component of bladder cancer cells associated with poor clinical outcome, and EVs can transfer oncogenic molecules such as periostin to affect the tumor environment and promote cancer progression.

Near infrared fluorescence imaging after intravenous indocyanine green: initial clinical experience with open partial nephrectomy for renal cortical tumors.

OBJECTIVE: To evaluate the safety of near infrared fluorescence (NIRF) of intravenously injected indocyanine green (ICG) during open partial nephrectomy, and to demonstrate the feasibility of this technology to identify the renal vasculature and distinguish renal cortical tumors from normal parenchyma. METHODS: Patients undergoing open partial nephrectomy provided written informed consent for inclusion in this institutional review board-approved study. Perirenal fat was removed to allow visualization of the renal parenchyma and lesions to be excised. The patients received intravenous injections of ICG, and NIRF imaging was performed using the SPY system. Intraoperative NIRF video images were evaluated for differentiation of tumor from normal parenchyma and for renal vasculature identification. RESULTS: A total of 15 patients underwent 16 open partial nephrectomies. The mean cold ischemia time was 26.6 minutes (range 20-33). All 14 malignant lesions were afluorescent or hypofluorescent compared with the surrounding normal renal parenchyma. NIRF imaging of intravenously injected ICG clearly identified the renal hilar vessels and guided selective arterial clamping in 3 patients. No adverse reactions to ICG were noted, and all surgical margins were negative on final pathologic examination. CONCLUSION: The intravenous use of ICG combined with NIRF is safe during open renal surgery. This technology allows the surgeon to distinguish renal cortical tumors from normal tissue and highlights the renal vasculature, with the potential to maximize oncologic control and nephron sparing during open partial nephrectomy. Additional study is needed to determine whether this imaging technique will help improve the outcomes during open partial nephrectomy.

A novel in vitro assay of tumor-initiating cells in xenograft prostate tumors.

BACKGROUND: The field of prostate cancer has been stymied by the difficulty of cultivating patient-derived samples in the laboratory. In order to help circumvent this challenge, we sought to develop an in vitro assay of human prostate cancer initiation employing a prostate-associated mesenchymal feeder layer. METHODS: Rat seminal vesicle mesenchyme (rSVM) harvested from male neonatal rats was plated in 12-well plates and then irradiated with 30 Gy after approximately 75% confluence. Single-cell suspensions of two human non-adherent prostate cancer xenograft lines (TRPC and LAPC9) were then plated on irradiated rSVM. At 3-4 weeks, three-dimensional solid structures, termed glandoids, were harvested and analyzed or transplanted singly into the renal capsule of immunodeficient mice. Animals were assessed for tumor formation 8-12 weeks after engraftment. Finally, clonality assays were performed to determine whether glandoids usually arise from a single cell and are therefore clonal in origin. RESULTS: Glandoids form with reliable frequency (1/ approximately 300 plated cells), are constituted by relevant cell types (CK8+, CK5-, PSA+) and after implantation into immunocompromised mice, give rise to tumors that recapitulate original xenograft histology and cell composition; defining a glandoid as a tumor-initiating unit. In addition, assessment of red fluorescent protein (RFP)-labeled glandoids revealed either all red or non-red structures, with few areas of fusion, suggesting glandoids are clonal in origin. CONCLUSIONS: The above assay describes an adjunct technique to readily cultivate cells from prostate cancer xenografts in vitro and as such provides a platform on which tumor-initiating cell studies and high-throughput drug discovery may be performed.

Selective expression of CD44, a putative prostate cancer stem cell marker, in neuroendocrine tumor cells of human prostate cancer.

BACKGROUND: Hormonal therapy is effective for advanced prostate cancer (PC) but the disease often recurs and becomes hormone-refractory. It is hypothesized that a subpopulation of cancer cells, that is, cancer stem cells (CSCs), survives hormonal therapy and leads to tumor recurrence. CD44 expression was shown to identify tumor cells with CSC features. PC contains secretory type epithelial cells and a minor population of neuroendocrine cells. Neuroendocrine cells do not express androgen receptor and are quiescent, features associated with CSCs. The purpose of the study was to determine the expression of CD44 in human PC and its relationship to neuroendocrine tumor cells. METHODS: Immunohistochemistry and immunofluorescence were performed to study CD44 expression in PC cell lines, single cells from fresh PC tissue and archival tissue sections of PC. We then determined if CD44+ cells represent neuroendocrine tumor cells. RESULTS: In human PC cell lines, expression of CD44 is associated with cells of NE phenotype. In human PC tissues, NE tumor cells are virtually all positive for CD44 and CD44+ cells, excluding lymphocytes, are all NE tumor cells. CONCLUSIONS: Selective expression of the stem cell-associated marker CD44 in NE tumor cells of PC, in combination with their other known features, further supports the significance of such cells in therapy resistance and tumor recurrence.

Uroflow in murine urethritis.

OBJECTIVES: To develop a noninvasive method to measure urinary flow rate in the mouse. This could be useful for the study of bladder outlet obstruction, as well as processes affecting detrusor function in the awake animal. Genetically engineered mice can improve our understanding of a variety of human bladder diseases. METHODS: A metabolic cage without a fecal separation screen was placed above a precision balance that reported the mass of the excreta pan every 100 ms. A computational algorithm identified voids suitable for assessment of uroflow from other excretory events. These algorithms were verified by comparison with a series of images obtained automatically before and during the excretory events. Intraurethral acetic acid was used to induce urethritis and to verify the sensitivity of the measurement technique. RESULTS: Automatic categorization and characterization of uroflow was successful. Brief exposures of the urethra of the female C57BL6/J mouse to 2% acetic acid decreased uroflow and increased the void duration without a change in the voided volume. CONCLUSIONS: This method will enable studies of urologic function in mice of differing age, sex, strain, and genetic constitution. Murine urethritis can be differentiated from cystitis, known to be associated with a decrease in voided volume. The observed changes were consistent with urethral obstruction induced by local swelling and inflammation.