Yanina Langle

Bacillus Calmette Guerin Induces Fibroblast Activation Both Directly and through Macrophages in a Mouse Bladder Cancer Model

Background Bacillus Calmette-Guerin (BCG) is the most effective treatment for non-muscle invasive bladder cancer. However, a failure in the initial response or relapse within the first five years of treatment has been observed in 20% of patients. We have previously observed that in vivo administration of an inhibitor of nitric oxide improved the response to BCG of bladder tumor bearing mice. It was described that this effect was due to a replacement of tumor tissue by collagen depots. The aim of the present work was to clarify the mechanism involved in this process. Methodology/Principal Findings We demonstrated that BCG induces NIH-3T3 fibroblast proliferation by activating the MAPK and PI3K signaling pathways and also differentiation determined by alpha-smooth muscle actin (alpha-SMA) expression. In vivo, intratumoral inoculation of BCG also increased alpha-SMA and collagen expression. Oral administration of L-NAME enhanced the pro-fibrotic effect of BCG. Peritoneal macrophages obtained from MB49 tumor-bearing mice treated in vivo with combined treatment of BCG with L-NAME also enhanced fibroblast proliferation. We observed that FGF-2 is one of the factors released by BCG-activated macrophages that is able to induce fibroblast proliferation. The involvement of FGF-2 was evidenced using an anti-FGF2 antibody. At the same time, this macrophage population improved wound healing rate in normal mice and FGF-2 expression was also increased in these wounds. Conclusions/Significance Our findings suggest that fibroblasts are targeted by BCG both directly and through activated macrophages in an immunotherapy context of a bladder murine model. We also described, for the first time, that FGF-2 is involved in a dialog between fibroblasts and macrophages induced after BCG treatment. The fact that L-NAME administration improves the BCG effect on fibroblasts, NO inhibition, might represent a new approach to add to the conventional BCG therapy.

Inhibition of nitric oxide is a good therapeutic target for bladder tumors that express iNOS.

Bladder cancer is the second cause of death for urological tumors in man. When the tumor is nonmuscle invasive, transurethral resection is curative. On the other hand, radical cystectomy is the treatment chosen for patients with invasive tumors, but still under treatment, these patients have high risk of dying, by the development of metastatic disease within 5 years. It is therefore important to identify a new therapeutic target to avoid tumor recurrences and tumor progression. Nitric oxide (NO) is an important biological messenger known to influence several types of cancers. In bladder cancer, production of NO and expression and activity of inducible NO synthase was associated to recurrence and progression. The objective of this work was to analyze if inhibition of nitric oxide production could be considered a therapeutic target for bladder tumors expressing iNOS. Using a bladder cancer murine model with different invasiveness grade we have demonstrated that NO inhibition was able to inhibit growth of bladder tumors expressing iNOS. Furthermore, invasive properties of MB49-I orthotopic growth was inhibited using NO inhibitors. This paper also shows that levels of NO in urine can be correlated with tumor size. In conclusion, inhibition of NO could be considered as a therapeutic target that prevents tumor growth and progression. Also, urine NO levels may be useful for measuring tumor growth.

Role of Peroxisome Proliferator Activated Receptor-Gamma in Bacillus Calmette-Guérin Bladder Cancer Therapy

PURPOSE We evaluated the effects of combined PPARg agonist with bacillus Calmette-Guérin in bladder cancer growth in vitro and in vivo, focusing on the tissue remodeling mechanisms induced by bacillus Calmette-Guérin. MATERIALS AND METHODS PPARs are a superfamily of nuclear receptors that are transcription factors activated by ligands. Activation of PPARg, the γ subtype, causes proliferation inhibition or differentiation of tumor cells. Previously, we reported that the inhibition of murine bladder tumor growth induced by bacillus Calmette-Guérin, which is the standard treatment for patients with nonmuscle invasive, high grade bladder cancer, increased PPARg expression in vitro and in vivo. In vitro the cell growth inhibition induced by bacillus Calmette-Guérin was enhanced by the PPARg agonist 15-d-PGJ2, raising the possibility that PPARg activation may be a therapeutic modality for this disease. RESULTS In MB49 cells bacillus Calmette-Guérin and 15-d-PGJ2 induced PPARg expression, nuclear translocation and transcriptional activity. In vivo bacillus Calmette-Guérin reduced tumor size, an effect that was partially reversed when bacillus Calmette-Guérin was combined with the PPARg agonist rosiglitazone. The same result was found when we analyzed the effect of the PPARg antagonist BADGE (Fluka Chemical, Buchs, Switzerland) combined with bacillus Calmette-Guérin. Analysis of the activation of macrophages and fibroblasts demonstrated that rosiglitazone inhibited the tissue remodeling mechanisms induced by bacillus Calmette-Guérin. CONCLUSIONS Results suggest that PPARg is involved in the antitumor action of bacillus Calmette-Guérin. However, exogenous PPARg agonists would not be a favorable therapeutic modality because they can inhibit the tissue remodeling needed for an overall satisfactory bacillus Calmette-Guérin response.

Inducible Nitric Oxide Synthase and PPARγ are Involved in Bladder Cancer Progression

PURPOSE We evaluated the role of inducible nitric oxide synthase and PPARγ as prognostic factors for bladder cancer. MATERIALS AND METHODS Inducible nitric oxide synthase and PPARγ were evaluated by Western blot and immunohistochemistry in a mouse bladder cancer model of nonmuscle invasive and invasive MB49-I tumor cells, and in human bladder cancer samples. RESULTS Inducible nitric oxide synthase expression was negative in mouse normal urothelium and higher in invasive than in noninvasive MB49 tumors. In vitro inducible nitric oxide synthase activity, determined as nitrite, was higher in MB49-I than in MB49 cells (p <0.001). In human samples expression was also associated with tumor invasion. Nuclear PPARγ expression was negative in normal mouse urothelium but higher in nonmuscle invasive MB49 than in MB49-I tumors. Similarly in human tumors low PPARγ was associated with poor prognosis factors, such as high histological grade (p = 0.0160) and invasion status (p = 0.0352). A positive correlation was noted between inducible nitric oxide synthase and PPARγ in low histological grade and nonmuscle invasive tumors (Pearson correlation index 0.6368, p = 0.0351, 0.4438 and 0.0168, respectively). As determined by gene reporter assay, PPARγ activity was induced by nitric oxide only in nonmuscle invasive MB49 cells (p <0.001). CONCLUSIONS Results suggest that increased PPARγ controls inducible nitric oxide synthase expression at early tumor stages. However, regulation is lost at advanced tumor stages, when the increase in inducible nitric oxide synthase and the decrease in PPARγ seem to be associated with bladder cancer progression.

FGFR3 Down-Regulation is Involved in bacillus Calmette-Guérin Induced Bladder Tumor Growth Inhibition

PURPOSE Bacillus Calmette-Guérin is the standard treatment for patients with nonmuscle invasive high histological grade bladder cancer. Previously we found that bacillus Calmette-Guérin induces murine bladder cancer MB49 cell death in vitro and in vivo, generating tissue remodeling, which involves the release of fibroblast growth factor (FGF)-2. MATERIALS AND METHODS We studied the effect of bacillus Calmette-Guérin treatment on FGF-2 and FGF receptor (FGFR) expression in bladder cancer. RESULTS In vitro FGF-2 increased MB49 cell proliferation but did not reverse bacillus Calmette-Guérin induced cell death. Increased FGF-2 expression was detected after bacillus Calmette-Guérin treatment. Moreover MB49 cells expressed high FGFR3 levels, which decreased after treatment. Similar results were observed in human T24 bladder cancer cells. In vivo MB49 tumors expressed higher FGFR3 levels than normal urothelium. Tumor FGFR3 decreased after treatment and correlated with tumor growth inhibition in response to bacillus Calmette-Guérin. In a pilot bioassay using 11 human bladder tumors treated ex vivo with bacillus Calmette-Guérin we found a subgroup of 41% of patients in whom FGFR3 was decreased after treatment. CONCLUSIONS Based on bladder cancer murine model results we infer that down-regulation of FGFR3 is a predictive marker of a good response to bacillus Calmette-Guérin therapy. The decrease in FGFR3 in response to bacillus Calmette-Guérin occurred not only in a murine model but also in a human bladder cancer cell line and in some patient samples. More patients and increased followup are needed to establish the predictive role of FGFR3 as a marker in human bladder cancer.

Analysis of tumoral spheres growing in a multichamber microfluidic device

Lab on a Chip (LOC) farming systems have emerged as a powerful tool for single cell studies combined with a non‐adherent cell culture substrate and single cell capture chips for the study of single cell derived tumor spheres. Cancer is characterized by its cellular heterogeneity where only a small population of cancer stem cells (CSCs) are responsible for tumor metastases and recurrences. Thus, the in vitro strategy to the formation of a single cell‐derived sphere is an attractive alternative to identify CSCs. In this study, we test the effectiveness of microdevices for analysis of heterogeneity within CSC populations and its interaction with different components of the extracellular matrix. CSC could be identify using specific markers related to its pluripotency and self‐renewal characteristics such as the transcription factor Oct‐4 or the surface protein CD44. The results confirm the usefulness of LOC as an effective method for quantification of CSC, through the formation of spheres under conditions of low adhesion or growing on components of the extracellular matrix. The device used is also a good alternative for evaluating the individual growth of each sphere and further identification of these CSC markers by immunofluorescence. In conclusion, LOC devices have not only the already known advantages, but they are also a promising tool since they use small amounts of reagents and are under specific culture parameters. LOC devices could be considered as a novel technology to be used as a complement or replacement of traditional studies on culture plates.

Flavonoid silybin improves the response to radiotherapy in invasive bladder cancer

Conservative treatment for invasive bladder cancer (BC) involves a complete transurethral tumor resection combined with chemotherapy (CT) and radiotherapy (RT). The major obstacles of chemo‐radiotherapy are the addition of the toxicities of RT and CT, and the recurrence due to RT and CT resistances. The flavonoid Silybin (Sb) inhibits pathways involved in cell survival and resistance mechanisms, therefore the purpose of this paper was to study in vitro and in vivo, the ability of Sb to improve the response to RT, in two murine BC cell lines, with different levels of invasiveness, placing emphasis on radio‐sensitivity, and pathways involved in radio‐resistance and survival. In vitro, Sb radio‐sensitized murine invasive cells through the inhibition of RT‐induced NF‐κB and PI3K pathways, and the increase of oxidative stress, while non‐invasive cells did not show to be sensitized. In vivo, Sb improved RT‐response and overall survival in invasive murine tumors. As Sb is already being tested in clinical trials for other urological cancers and it improves RT‐response in invasive BC, these results could have translational relevance, supporting further research.