Shunta Hori

CXCL1-Mediated Interaction of Cancer Cells with Tumor-Associated Macrophages and Cancer-Associated Fibroblasts Promotes Tumor Progression in Human Bladder Cancer

Tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) are reported to be associated with poor prognosis, depending on their pro-tumoral roles. Current knowledge of TAMs and CAFs in the tumor microenvironment of urothelial cancer of the bladder (UCB) is limited. Therefore, we investigated the paracrine effect induced by TAMs and CAFs in the tumor microenvironment of human UCB. For this, we first carried out immunohistochemical analysis for CXCL1, CD204 (TAM marker), αSMA (CAF marker), E-cadherin, and MMP2 using 155 UBC tissue samples. Next, CXCL1-overexpressing clones of THP-1-derived TAMs and NIH3T3-derived CAFs were developed by lentiviral vector infection. The immunohistochemical study showed high CXCL1 levels in UCB cells to be associated with enhanced recruitment of TAMs/CAFs, higher metastatic potential, and poor prognosis. Three-dimensional (3D) co-culture of UCB cells and TAMs/CAFs suggested that CXCL1 production in TAMs/CAFs play an important role in cell-to-cell adhesion and interaction among cancer cells and these stromal cells. CXCL1-expressing TAMs/CAFs enhanced tumor growth of subcutaneous UCB tumors in nude mice when injected together. In addition, an experiment using the orthotopic bladder cancer model revealed that CXCL1 production in TAMs/CAFs supported tumor implantation into the murine bladder wall and UCB growth when injected together, which was confirmed by clinical data of patients with bladder cancer. Thus, CXCL1 signaling in the tumor microenvironment is highly responsible for repeated intravesical recurrence, disease progression, and drug resistance through enhanced invasion ability. In conclusion, disrupting CXCL1 signaling to dysregulate this chemokine is a promising therapeutic approach for human UCB.

Neutrophil-to-lymphocyte ratio as a detection marker of tumor recurrence in patients with muscle-invasive bladder cancer after radical cystectomy.

PURPOSE High-neutrophil to lymphocyte ratio (NLR) values have been shown to be associated with a poor prognosis in many human malignant tumors. We evaluated the correlation of the NLR with other variables in patients with muscle-invasive bladder cancer after radical cystectomy (RC); in particular, we evaluated chronological changes in the postoperative NLR. METHODS We included the data from a total of 110 patients who underwent RC for muscle-invasive bladder cancer. The NLR was calculated using complete blood counts determined before RC. Kaplan-Meier and Cox proportional regression analyses of recurrence-free survival (RFS), cancer-specific survival (CSS), and overall survival (OS) were performed to identify significant prognostic variables. RESULTS The median patient age was 72 years (41-91 years). In univariate analysis, the pretreatment NLR (≥2.6 vs.<2.6) was associated with RFS (hazard ratio [HR] = 2.41, P = 0.008), CSS (HR = 2.89, P = 0.006), and OS (HR = 2.73, P = 0.002). In multivariate analysis, an NLR≥2.6 and an infiltrative growth pattern at the tumor invasion front were significantly associated with RFS (HR = 2.61, P = 0.023), CSS (HR = 2.58, P = 0.08), and OS (HR = 2.77, P = 0.004). Postoperative chronological analysis revealed that the NLR of 68 patients without recurrence remained low during follow-up, whereas the NLR of the remaining 42 patients with recurrence increased significantly in the last visit before recurrence was detected radiographically (P< 0.01). CONCLUSIONS The NLR and tumor growth pattern were strong predictors of prognosis for patients undergoing RC. Our results suggest that an increase in the NLR during follow-up after RC is a potential marker for the early detection of recurrence.

Collagen type IV alpha 1 (COL4A1) and collagen type XIII alpha 1 (COL13A1) produced in cancer cells promote tumor budding at the invasion front in human urothelial carcinoma of the bladder

Current knowledge of the molecular mechanism driving tumor budding is limited. Here, we focused on elucidating the detailed mechanism underlying tumor budding in urothelial cancer of the bladder. Invasive urothelial cancer was pathologically classified into three groups as follows: nodular, trabecular, and infiltrative (tumor budding). Pathohistological analysis of the orthotopic tumor model revealed that human urothelial cancer cell lines MGH-U3, UM-UC-14, and UM-UC-3 displayed typical nodular, trabecular, and infiltrative patterns, respectively. Based on the results of comprehensive gene expression analysis using microarray (25 K Human Oligo chip), we identified two collagens, COL4A1 and COL13A1, which may contribute to the formation of the infiltrative pattern. Visualization of protein interaction networks revealed that proteins associated with connective tissue disorders, epithelial-mesenchymal transition, growth hormone, and estrogen were pivotal factors in tumor cells. To evaluate the invasion pattern of tumor cells in vitro, 3-D collective cell invasion assay using Matrigel was performed. Invadopodial formation was evaluated using Gelatin Invadopodia Assay. Knockdown of collagens with siRNA led to dramatic changes in invasion patterns and a decrease in invasion capability through decreased invadopodia. The in vivo orthotopic experimental model of bladder tumors showed that intravesical treatment with siRNA targeting COL4A1 and COL13A1 inhibited the formation of the infiltrative pattern. COL4A1 and COL13A1 production by cancer cells plays a pivotal role in tumor invasion through the induction of tumor budding. Blocking of these collagens may be an attractive therapeutic approach for treatment of human urothelial cancer of the bladder.

Integrative Assessment of Pretreatment Inflammation-, Nutrition-, and Muscle-Based Prognostic Markers in Patients with Muscle-Invasive Bladder Cancer Undergoing Radical Cystectomy

Objective: The present study evaluated the clinical relevance of an integrative preoperative assessment of inflammation-, nutrition-, and muscle-based markers for patients with muscle-invasive bladder cancer (MIBC) undergoing curative radical cystectomy (RC). Methods: The analysis enrolled 117 patients and the variables included age, body mass index (BMI), neutrophil-to-lymphocyte ratio, monocyte-to-lymphocyte ratio, platelet-to-lymphocyte ratio, modified Glasgow Prognostic Score (mGPS), prognostic nutritional index (PNI), Controlling Nutritional Status score, psoas muscle index (PMI), and peak expiratory flow (PEF). The correlations among the variables were evaluated and their prognostic values after RC were tested. Results: Three inflammation markers (ratios of blood cell counts) were positively correlated (p < 0.0001). The PNI and the BMI were positively correlated (p = 0.04), although they were inversely correlated with the three inflammation markers (p < 0.0001). Age was not significantly correlated with the inflammation markers and PMI, although older age was associated with lower PNI and lower PEF. The disease-specific survival was independently predicted by T4 tumor, positive N status, and decreased PNI. Overall survival was independently predicted by T4 tumor, mGPS, and pretreatment sarcopenia status. Conclusions: The inflammation-, nutrition-, and muscle-based markers would be useful risk assessment tools for MIBC.

Regulatory T Cells and Tumor-Associated Macrophages in the Tumor Microenvironment in Non-Muscle Invasive Bladder Cancer Treated with Intravesical Bacille Calmette-Guérin: A Long-Term Follow-Up Study of a Japanese Cohort

The clinical significance of regulatory T cells (Treg) and tumor-associated macrophages (TAM) in the tumor microenvironment of human bladder cancer remains unclear. The aim of this study is to explore their relevance to oncological features in non-muscle invasive bladder cancer (NMIBC). We carried out immunohistochemical analysis of forkhead box P3 (FOXP3, Treg maker), CD204 (TAM marker), and interleukin-6 (IL6) using surgical specimens obtained from 154 NMIBC patients. The Treg and TAM counts surrounding the cancer lesion and IL6-positive cancer cell counts were evaluated against clinicopathological variables. We focused on the ability of the Treg and TAM counts around the cancer lesion to predict outcomes after adjuvant intravesical Bacille Calmette–Guérin (BCG) treatment. High Treg counts were associated with female patients, older age, T1 category, and high tumor grade. TAM count was significantly correlated with Treg count and with IL6-positive cancer cell count. In our analysis of 71 patients treated with BCG, high counts of Treg and TAM were associated with shorter recurrence-free survival, and the former was an independent predictor of recurrence. Poor response to intravesical BCG was associated with Treg and TAM in the tumor microenvironment. Disrupting the immune network can be a supplementary therapeutic approach for NMIBC patients receiving intravesical BCG.

Topical and systemic immunoreaction triggered by intravesical chemotherapy in an N-butyl-N-(4-hydroxybutyl) nitorosamine induced bladder cancer mouse model

Intravesical bacillus Calmette-Guerin (BCG) treatment is the most common therapy to prevent progression and recurrence of non-muscle invasive bladder cancer (NMIBC). Although the immunoreaction elicited by BCG treatment is well documented, those induced by intravesical treatment with chemotherapeutic agents are much less known. We investigated the immunological profiles caused by mitomycin C, gemcitabine, adriamycin and docetaxel in the N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced orthotopic bladder cancer mouse model. Ninety mice bearing orthotopic bladder cancer induced by BBN were randomly divided into six groups and treated with chemotherapeutic agents once a week for four weeks. After last treatment, bladder and serum samples were analyzed for cell surface and immunological markers (CD4, CD8, CD56, CD204, Foxp3, and PD-L1) using immunohistochemistry staining. Serum and urine cytokine levels were evaluated by ELISA. All chemotherapeutic agents presented anti-tumor properties similar to those of BCG. These included changes in immune cells that resulted in fewer M2 macrophages and regulatory T cells around tumors. This result was compatible with those in human samples. Intravesical chemotherapy also induced systemic changes in cytokines, especially urinary interleukin (IL)-17A and granulocyte colony stimulating factor (G-CSF), as well as in the distribution of blood neutrophils, lymphocytes, and monocytes. Our findings suggest that intravesical treatment with mitomycin C and adriamycin suppresses protumoral immunity while enhancing anti-tumor immunity, possibly through the action of specific cytokines. A better understanding of the immunoreaction induced by chemotherapeutic agents can lead to improved outcomes and fewer side effects in intravesical chemotherapy against NMIBC.

Aquaporin-2 plays an important role in water transportation through the bladder wall in rats

We investigated the role of the bladder wall in permeating water, focusing on aquaporins.

Tadalafil, a phosphodiesterase type 5 inhibitor, improves bladder blood supply and restores the initial phase of lower urinary tract dysfunction in diabetic rats

To investigate the effect of tadalafil on bladder blood flow and lower urinary tract function in a rat model of diabetes.

Diagnostic and prognostic role of urinary collagens in primary human bladder cancer

Collagen type 4 alpha 1 (COL4A1) and collagen type 13 alpha 1 (COL13A1) produced by urothelial cancer cells support the vital oncogenic property of tumor invasion. We investigated the diagnostic and prognostic capability of COL4A1 and COL13A1 in voided urine and compared the observed values with those of fragments of cytokeratin‐19 (CYFRA21‐1), nuclear matrix protein 22 (NMP‐22), and voided urine cytology in bladder cancer (BCa). We collected voided urine samples from 154 patients newly diagnosed with BCa, before surgery and from 61 control subjects. Protein levels of COL4A1, COL13A1, CYFRA21‐1, and NMP‐22 in urine supernatants were measured using enzyme‐linked immunosorbent assays. Diagnostic performance and optimal cut‐off values were determined by receiver operating characteristic analysis. Urine levels of COL4A1, COL13A1, the combined values of COL4A1 and COL13A1 (COL4A1 + COL13A1), and CYFRA21‐1 were significantly elevated in urine from patients with BCa compared to the controls. Among these biomarkers, the optimal cut‐off value of COL4A1 + COL13A1 at 1.33 ng/mL resulted in 57.4%, 83.7%, 56.1%, 80.7%, and 91.7% sensitivity for low‐grade tumors, high‐grade tumors, Ta, T1, and muscle invasive disease, respectively. We evaluated the prognostic value of preoperative urine levels in 130 non‐muscle invasive BCa samples after the initial transurethral surgery. A high urinary COL4A1 + COL13A1 was found to be an independent risk factor for intravesical recurrence. Although these data need to be externally validated, urinary COL4A1 and COL13A1 could be a potential diagnostic and prognostic biomarker for BCa. This easy‐to‐use urinary signature identifies a subgroup of patients with a high probability of recurrence and progression in non‐muscle invasive and muscle invasive BCa.

The impact of the definition of biochemical recurrence following salvage radiotherapy on outcomes and prognostication in patients with recurrent prostate cancer after radical prostatectomy: a comparative study of three definitions

Purpose The clinical management and follow-up of patients with recurrent prostate cancer after salvage radiotherapy (SRT) has not yet been established, and no standardized definition of biochemical recurrence (BCR) after SRT exists. We compared the impact of applying three different definitions of BCR following SRT on patient outcomes and prognostication. Subjects Patients who received salvage androgen-deprivation therapy before the completion of SRT were excluded. The data of 118 men who had undergone salvage radiation as monotherapy for BCR after radical prostatectomy were reviewed. In all patients, SRT comprised irradiation to the prostatic bed (70 Gy) using three-dimensional conformal radiotherapy techniques. Treatment outcomes, including BCR-free survival and prognostic factors, were analyzed and compared among three definitions: The Nara, Radiation Therapy Oncology Group (RTOG) 9601, and GETUG-AFU 16 definitions. Results The BCR rate differed significantly among the applied definitions. Multivariate analyses identified the same four independent prognostic factors, including primary Gleason pattern 4 or 5, negative resection margin, prostate-specific antigen (PSA) level before SRT 0.5 or more, and PSA doubling time before SRT <6 months, using the RTOG 9601 and GETUG-AFU 16 definitions, whereas only two of the four factors were identified using the Nara definition. Although the results obtained using the RTOG 9601 and GETUG-AFU 16 definitions were similar, the prognostic value of the four factors differed. According to the RTOG 9601 definition of BCR, a negative resection margin on prostatectomy specimens and short PSA doubling time before SRT were associated with no subsequent response in PSA level. Conclusions The applied definition of BCR after SRT can influence the reported BCR-free rate and the potential prognostic factors. Establishment of the standardized definition is needed for the optimal management of patients with recurrent prostate cancer undergoing SRT.