Lina Marino

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.

Abstract 2881: Cytotoxic effect of trastuzumab on macrophage-infiltrated human mammary tumor spheroids

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The HER2 receptor, overexpressed in 20-25% of breast invasive tumors, is associated with low disease-free survival. Trastuzumab (Tz), monoclonal antibody anti HER2 is used as immunotherapy in HER2+ mammary tumors; however, 60% of patients are unresponsive to this therapy. The aim of our study is the analysis of the effect of Tz on the growth of human mammary tumor cells cultured in 3D (shperoids). The human adenocarcinoma cell line BT474 (HER2+) was used to generate spheroids grown in liquid RPMI1640 medium. One spheroid/well was seeded on a 1.5% agar layer, reaching 2.5 mm diameter at day 62 of culture. Differential expression of HER2, HIF-1, Bcl-2 and Ki67 were detected by immunohistochemistry. Proliferating, quiescent as well as apoptotic cells towards the hypoxic core of the tumor spheroids were identified. The effect of Tz (10 and 50 ug/ml/spheroid, 3 times/week) on the growth of 3D cultures of BT474 cells was evaluated, beginning with a volume of 0.23 ± 0.03 mm3 (day 0). Tz 10 ug/ml arrested spheroids growth since day 7 (3 doses) while Tz 50 ug/ml induced a significant reduction in the volume since the first dose (day 0) until day 27, when spheroids showed 71% of growth inhibition compared to untreated or human IgG controls (Tz 50= 0.12 ± 0.04 mm3 vs control= 0.45 ± 0.03 mm3, p<0.001). Spheroids showed lower sensitivity to the cytostatic effect of Tz 10 ug/ml when compared to the same cells cultured as monolayers. Tz exerted a direct and dose-dependent effect on BT474 tumor spheroids: while it was cytostatic at the lower dose (Tz10), it was cytotoxic at the higher (Tz50). Nitric oxide (NO) levels measured by Griess reaction in tumor spheroid supernatants were significantly higher upon Tz50 treatment compared to untreated controls (p<0.01), suggesting NO is involved in the direct cytotoxic mechanism of Tz. BT474 cells growing in monolayers responded to TZ with higher NO levels than 3D cultures. In order to assess whether immune system and tissue architecture have an impact on the Tz cytotoxic effect, we co-cultured single BT474 tumor spheroids with macrophages from human peripheral blood (ratio1:5). At 10 days co-culture, the macrophages had a type I response (M1) against tumor cells. In fact, we observed that the presence of macrophages in the culture increased the cytotoxicity of Tz. This period of time was not enough to switch macrophages to the M2 phenotype. Conditioned media from Tz50-treated BT474 spheroids decreased MMP9 production by macrophages cultures. We propose this 3D culture model as a useful tool to study susceptibility to trastuzumab therapy in HER2 + tumor cells, as well as to analyze the mechanisms of action and resistance of this treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2881. doi:1538-7445.AM2012-2881

Abstract 226: Trastuzumab effect on cell death and proliferation of HER2+ mammary adenocarcinoma cells using a tridimensional culture

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC HER2 is overexpressed in 20-25% invasive breast tumors, and associated with low free disease survival. Trastuzumab (Tz), monoclonal antibody anti HER2, is used as immunotherapy to treat HER2+ tumors; however more than half of them are resistant or acquire resistance during treatment. Cellular spheroids are a model of cell growth in 3D that mimic the structure of in vivo avascular tumors. We have previously demonstrated that tumor spheroids (TS) present different subpopulations, with a gradient of proliferative, quiescente and apoptotic cells from the outer rim towards the hypoxic core. The aim of this study was to analyze the biological characteristics of the different cell populations that constitute the TS and their response to Tz. TS of HER2+ human mammary tumor cells (cell line BT474) were cultured as cell suspensions on agar (hanging drop method) in 48-well plates (1 spheroid / well). Cell cycle at days 7, 14 and 21 of TS growth was analyzed by flow cytometry. A significant increase only in the G2/M phase (6.6%, 10.3% and 13.3% respectively) was detected. TS were incubated with Tz at a concentration of 50ug/ml; a commercial IgG (50ug/mL) was used as negative control. The effect on cell growth kinetics was evaluated with photographs taken at different times, observing a reduction of the spheroids volume until they reached a decrease by 71% at day 20 (p<0.01 vs controls). Tz induced an increase in the% of cells arrested in G1 (80.8% vs 64.7% control IgG, p<0.001) and a decrease in the proliferative subpopulation (7.4% vs 11.4%, p<0.05). A decrease in the% of apoptotic cells was also detected (Annexin V/IP). When we analyzed cell viability at different time points, we observed that Tz elicited changes in live to dead cells ratio. A reversion of the inhibitory effect of Tz on TS growth was observed after antibody withdrawal. Spheroids were fixed and included in paraffin to analyze the expression of HER2, pHER2, clived caspase 3, HIF-1a, p27, p21 and cyclinD1 by confocal microscopy. HER2 expression was not modulated by Tz; however, we observed lower expression of its activated receptor (pHER2). HIF-1a was expressed only in cells around the central core while clived caspase 3 was expressed in the central core. After Tz incubation, both markers were no longer detected. We can conclude that Tz exerted a potent antiproliferative and direct cytotoxic effect on BT474 tumor cells growing in 3D; these activities might be due to the partial inhibition of HER2 activation. Tz inhibited the formation of the hypoxic core and therefore the apoptotic subpopulation, generating thereby a smaller TS composed only of remaining Tz-resistant living cells. We propose that this model could be useful to study the mechanisms involved in the effect and on the resistance to Tz. Citation Format: Cristina E. Rodriguez, Sara Reidel, Luciana Moverer, Lina Marino, Elisa Bal de Kier Joffe, Maria A. Jasnis, Gabriel L. Fiszman. Trastuzumab effect on cell death and proliferation of HER2+ mammary adenocarcinoma cells using a tridimensional culture. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 226. doi:10.1158/1538-7445.AM2013-226

Abstract 548: Interaction between mammary adenocarcinoma cells and macrophages in a three-dimensional model of tumor spheroids

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Solid tumors contain heterogeneous cellular subpopulation due to a limit supply of oxygen, nutrients and wastes. This microenvironment affects local cell growth, survival and overall therapeutic efficacy. Three-dimensional (3D) cell model reflect this situation and is a good model to study of the interaction with the immune system. Spheroids of the murine LM3 and human MCF-7 mammary adenocarcinoma cell lines were cultured with RAW 264.7 macrophages and human monocytes isolated from peripheral blood from normal volunteers respectively Spheroids were maintained in culture dishes pre-treated with 1.5% agar. In both tumor models, macrophages/monocytes were added after 10-13 days spheroids growth. Control cultures were maintained as monolayers in normoxic and hypoxic conditions (hypoxic chamber 1% O2 saturated during 4-6h). A growth curve of the spheroids were performed, reaching a peak of >300um (+/-) at day 15. In MCF-7 model, cell viability, monocyte distribution inside spheroids and protein expression were determined by hematoxiline-eosine staining and immunohitochemistry with specific antibodies. Results showed that 3D growth followed a concentric organization of proliferant (Ki67+), quiescent (Ki67-/Bcl-2+) and dead cells (CAS+) from periphery towards the hypoxic core (HIF-1+). In LM3 spheroids an enhanced arginase activity (140,64±2,05 vs. 18,17±1,22, p<0,05) and nitric oxide (NO) levels (10,21±1,15 vs 0,55±0,50, p<0,05) were also detected compared to LM3 cells growing as monolayer. It was also detected a decrease NO levels during spheroids growth (4 days: 38,7±8,3 vs. 13 days: 10,21±1,15). On the other hand, arginase activity and NO levels of spheroids cocultured with macrophages were lower than the spheroids alone (96,41±3,63 vs.317,7048±8,82 and 4,1±1,56 vs. 10,22±1,15; p<0,05 respectively). These results show that arginine metabolic pathways, either by NOS or arginase, are both more activated in spheroids than in tumor cell monolayer and coculture spheroids. In MCF7 and LM3 models, HIF-1alpha and iNOS expression was detected by western blot using hypoxic monolayers as a control. Culture system in 3D appears to be an ideal model to analyze the role of hypoxia and the mechanisms that modulate interactions between tumor and immune cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 548.