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

Cathepsin B is involved in the apoptosis intrinsic pathway induced by Bacillus Calmette-Guérin in transitional cancer cell lines

Bacillus Calmette-Guérin (BCG) is the most effective treatment for superficial and in situ transitional bladder cancer. Although the complete mechanisms for its effect are not fully understood yet, both immunological and direct effects on tumor cells have been proposed. It has been proposed that apoptotic tumor cells could be better inducers of immunity than necrotic ones. Thus, apoptosis of bladder cancer cells could contribute to a global response to BCG. Lysosomal hydrolase cathepsin B (CB) is involved in the apoptotic process and has a key role in breast cancer cell programmed death through the activation of a pro-apoptotic protein BID. Truncated BID participates in the mitochondrial apoptotic pathway that involves the activation of pro-caspase 9. The possibility that CB can be involved in apoptosis of TCC line has not been explored yet. Therefore, we analyzed the participation of CB in BCG-induced apoptosis of human and murine TCC lines. Apoptosis was evaluated by a morphologic assay and CB activity by a substrate-specific colorimetric method. Expression of CB, BID and pro-caspase 9 was determined by Western blotting. BCG induced apoptosis of murine (MBT2, MB49) and human (T24) TCC lines. An increase in both CB activity and protein was also observed. The apoptosis of T24 and MB49 cell lines was mediated by activation of pro-caspase 9 and BID, both proteins are involved in mitochondrial apoptosis. Apoptosis and activation of pro-caspase 9 and BID were inhibited by CA-074Me (CA), a cell permeable CB inhibitor. Thus, CB is involved in BCG-induced apoptosis of TCC lines, using at least in part the mitochondrial pathway.Fil: Sandes, Eduardo Omar. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Lodillinsky, Catalina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cwirenbaum, Ruth Ana. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Argüelles, Claudia Lidia. Dirección Nacional de Instituto de Investigación.Administración Nacional de Laboratorios e Institutos de Salud "Dr. Carlos G. Malbrán"; ArgentinaFil: Casabé, Alberto. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Eijan, Ana Maria. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

S100A9 expression associated with nitric oxide is a good marker of prognosis in patients with bladder cancer: its inhibition as a potential therapeutic target

El óxido nítrico (NO) es producido por las enzimas NO sintasas (NOS). Anteriormente describimos que la isoforma inducible (iNOS) se expresa en el 50% de los cánceres de vejiga (CaV) humanos, asociada a recurrencia e invasión. La producción de NO puede alterar la expresión de proteínas relacionadas con la progresión tumoral. Utilizando el modelo de CaV murino MB49 que genera tumores no músculo invasores (NMI) y expresa iNOS identificamos que el tratamiento con el inhibidor de la producción de NO, L-NAME, redujo la expresión de la proteína S100A9, vinculada a la generación de una respuesta inmune supresora mediante el reclutamiento de células supresoras derivadas del linaje mieloide (MDSC). Objetivos: Evaluar 1) S100A9 como marcador pronóstico en pacientes con CaV. 2) La inhibición de NO como blanco terapéutico utilizando el modelo murino, enfocándonos en la expresión de S100A9, las MDSC y el crecimiento tumoral solo o bajo tratamiento con BCG. Identificamos que S100A9 se expresa en células tumorales y células inmunes que infiltran los tumores (células acompañantes). Detectamos una correlación positiva entre iNOS y S100A9 en las células tumorales, sugiriendo que su expresión estaría vinculada. Los tumores invasores presentan mayor número de células acompañantes positivas para S100A9, en comparación con los tumores NMI (p <0,05). Este infiltrado es a predominio de monocitos/macrófagos (CD14) en los tumores NMI (p <0,01), mientras que en los invasores hay un nivel similar de células CD14 y CD15 (granulocitos). En el modelo MB49, demostramos que L-NAME redujo el crecimiento tumoral ortotópico (46% de los ratones presentaron remisión completa) y la expresión de S100A9 en los tumores. Observamos que el número de MDSC en ganglio y bazo estaba aumentado y que los valores solo se normalizaban bajo tratamiento combinado de BCG+L-NAME. Conclusión: Estos hallazgos respaldan la hipótesis de que la inhibición del NO es un buen blanco terapéutico reduciendo en parte la generación de una respuesta inmune supresora. La expresión de S100A9 en células inmunológicas es un buen marcador de progresión tumoral, asociado a la producción de NO.Fil: Langle, Yanina Verónica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sandes, Eduardo Omar. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Belgorosky, Denise. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Balarino, Natalia Patricia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Prack Mc Cormick, Bárbara Patricia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marino, Lina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Rojas Bilbao, Érica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Malagrino, Héctor. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Pasik, Leonardo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Casabé, Alberto. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Eijan, Ana Maria. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

NO inhibition increases wound healing closure generated by peritoneal MACs.

<p>(A) Peritoneal MACs from tumor bearing mice either treated or not with BCG (MACs-T-BCG and MACs-T respectively), were placed in dorsal skin wound of normal mice. L-NAME (2 mM) or 1400 W (1 uM) was added onto the wound. The wound closure was calculated as the percentage of the initial wound area (day zero), a: p<0.01, b: p<0.001 vs PBS-gli, c: p<0.05 vs MACs-T-BCG control. (B) Peritoneal MACs-T-BCG were placed in dorsal skin wound of mice treated orally with L-NAME (0.2 g/kg mouse), a: p<0.0001 vs PBS-gli, b: p<0.01 vs MAC-T-BCG control.</p

Combined therapy of BCG and L-NAME exerts an antitumoral effect through modulation of MACs and fibroblasts in the tumor microenvironment.

<p>BCG directly induces fibroblast proliferation via MAPK and PI3K signaling pathways as well as alpha-SMA and collagen expression. BCG induces in MACs the production of NO and soluble factors including FGF-2 which induce fibroblast proliferation. BCG therapy increases collagen deposition and expression of alpha-SMA and FGF-2 in bladder tumors The treatment with L-NAME, improves the stimulation of fibroblasts by BCG.</p

BCG induces fibroblast differentiation.

<p>(A) Immunofluoresce staining of NIH-3T3 treated with BCG (3×10⁶ CFU/ml) for 24 h revealed with anti-collagen I and anti-alpha-SMA antibody. Scale: bar = 100 um. (B) Western Blot from fibroblast homogenates treated with BCG (3×10⁶ CFU/ml) at different times to determinate collagen I and alpha-SMA induction. (C) Densitometric units of collagen I or (D) alpha-SMA were determined using analysis software, relativized to beta-actin and referred as a fold change of control a: p<0.05, b: p<0.01.</p

BCG induces fibroblast activation through macrophages.

<p>(A) NO production was determined in the supernatants of both peritoneal MACs from MB49 tumor bearing mice (MACs-T) and RAW 264.7 cells treated in vivo with BCG (6×10⁶ CFU/ml) or in vitro (3×10⁶ CFU/ml) respectively, were evaluated in the supernatant by Griess reagent. a: p<0.0001 vs control. RAW 264.7 cells were treated with BCG (3×10⁶ CFU/ml) for 24 h, and then cells were extensively washed with PBS. Serum-free medium was added and incubation was continued for 24 hours to obtain the CM. (B) fibroblast treated for 48 h with the CM from peritoneal or RAW 264.7 cells previously treated with BCG (3×10⁶ CFU/ml) plus L-NAME (2 mM). Fibroblast viability was evaluated by MTS and referred as a percentage of control (RAW 264.7 or NIH-3T3 untreated cell lines), a: p<0.05 and b: p<0.01 vs control, c: p<0.05 vs MACs-T control. (C) Western Blot to determine collagen I and alpha-SMA from fibroblast homogenates treated for 24 h with CM from RAW 264.7 previously treated for 24 h with BCG plus L-NAME. Relative expression level was normalized to beta-actin and referred as a fold change of control, a: p<0.05, b: p<0.01. (D) Masson Trichome (top panel) and immunohistochemical staining (bottom panel), to determine collagen fibers and alpha-SMA respectively, were performed in MB49 tumors growing subcutaneously in mice treated with BCG, L-NAME or BCG+L-NAME. White arrows show blue stain of collagen fibers. Yellow arrows show brown positive staining for alpha-SMA. Scale: bar = 100 um.</p

FGF-2 secreted by BCG-treated MACs induces NIH-3T3 cells proliferation.

<p>(A) Immunofluorescence staining with anti-FGF-2 antibody of RAW 264.7 MACs treated with BCG (3×10⁶ CFU/ml) for 24 h. (B) Western blot from lysates of RAW 264.7 treated with BCG (3×10⁶ CFU/ml) for 8 and 24 h. 20 ng of purified murine FGF-2 was used as a positive control. Relative expression level was normalized to GAPDH and referred as a fold change of control, a: p<0.05 (C) fibroblasts were treated with CM from RAW 264.7 cells previously treated with BCG (3×10⁶ CFU/ml), L-NAME or BCG plus L-NAME for 24 h. Control was carried out with exhausted culture media (CM 3T3, from the same NIH-3T3 cells). 0.5 ng/ml of FGF-2 in CM 3T3 was used as a positive control. NIH-3T3 proliferation was monitored by ³(H) thymidine incorporation. The CM was preincubated 1 h with 10 µg/ml of the blocking monoclonal anti FGF-2 antibody (DB3), or normal IgG as a control, a: p<0.001 vs CM NIH-3T3, b: p<0.001 vs IgG. (D) Immunohistochemical staining to determinate FGF-2 expression was performed in skin wounds. Wounds were treated with peritoneal MACs from tumor bearing mice treated or not with BCG (MAC-T and MAC-T-BCG respectivesly) alone or locally combined with NO inhibitors (E). Immunohistochemical staining of FGF-2 was performed in s.c. MB49 tumors from mice treated with BCG, L-NAME or BCG plus L-NAME. Scale: bar = 100 um.</p