Differential nitric oxide release and sensitivity to injury in different murine mammary tumor cell lines

The purpose of this study was to determine whether nitric oxide (NO) production by different mammary tumor cell lines correlated with their sensitivity to NO mediated injury. Three mammary tumor cell lines LM2, LM3 and LMM3 syngeneic to BALB/c mice were cultured in vitro with IFNgamma + LPS. Different levels of NO production among the three lines were detected in culture supernatants. The only tumor cell line which did not produce NO (LM2) showed the highest sensitivity to SNP-derived NO cytotoxicity (87%), while LM3 and LMM3 which both produced higher levels of NO than LM2, showed lower cytotoxicity by SNP (39% and 22% respectively). Spleen cells (SC) from M2 tumor bearing mice (TBM) were able to lyse LM2 cells by NO-dependent mechanisms. SC from M3-TBM exerted cytotoxicity against LM3 cells mainly by NO-independent mechanisms. Thus, we postulate an inverse correlation between NO production and NO mediated cytotoxicity in the three mammary tumor cell lines. It is possible that tumor cells producing NO develop mechanisms to resist NO injury.Fil: Eijan, Ana Maria. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Davel, L. E.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Rueda, H. A.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Rozenberg, G.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Sacerdote de Lustig, Eugenia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Jasnis, Maria Adela. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaUnidad documental simpl

Autophagy Protects from Trastuzumab-Induced Cytotoxicity in HER2 Overexpressing Breast Tumor Spheroids.

Multicellular tumor spheroids represent a 3D in vitro model that mimics solid tumor essential properties including assembly and development of extracellular matrix and nutrient, oxygen and proliferation gradients. In the present study, we analyze the impact of 3D spatial organization of HER2-overexpressing breast cancer cells on the response to Trastuzumab. We cultured human mammary adenocarcinoma cell lines as spheroids with the hanging drop method and we observed a gradient of proliferating, quiescent, hypoxic, apoptotic and autophagic cells towards the inner core. This 3D organization decreased Trastuzumab sensitivity of HER2 over-expressing cells compared to monolayer cell cultures. We did not observe apoptosis induced by Trastuzumab but found cell arrest in G0/G1 phase. Moreover, the treatment downregulated the basal apoptosis only found in tumor spheroids, by eliciting protective autophagy. We were able to increase sensitivity to Trastuzumab by autophagy inhibition, thus exposing the interaction between apoptosis and autophagy. We confirmed this result by developing a resistant cell line that was more sensitive to autophagy inhibition than the parental BT474 cells. In summary, the development of Trastuzumab resistance relies on the balance between death and survival mechanisms, characteristic of 3D cell organization. We propose the use of spheroids to further improve the understanding of Trastuzumab antitumor activity and overcome resistance

Differential response of myeloid-derived suppressor cells to the nonsteroidal anti-inflammatory agent indomethacin in tumor-associated and tumor-free microenvironments

Myeloid-derived suppressor cells (MDSCs) are key regulatory cells that control inflammation and promote tumor-immune escape. To date, no specific immunomodulatory drug has proven efficacy in targeting the expansion and/or function of these cells in different pathophysiologic settings. In this study, we identified a context-dependent effect of the nonsteroidal anti-inflammatory drug indomethacin (IND) on MDSCs, depending on whether they were derived from tumor microenvironments (TME) or from tumor-free microenvironments (TFME). Treatment of mice bearing the LP07 lung adenocarcinoma with IND inhibited the suppressive activity of splenic MDSCs, which restrained tumor growth through mechanisms involving CD8(+) T cells. The same effect was observed when MDSCs were treated with IND and conditioned media from LP07 tumor cells in vitro. However, in the absence of a tumor context, IND enhanced the intrinsic suppressive function of MDSCs and amplified their protumoral activity. In a model of autoimmune neuroinflammation, IND-treated MDSCs differentiated in TFME attenuated inflammation, whereas IND-treated MDSCs differentiated in TME aggravated clinical symptoms and delayed resolution of the disease. Mechanistically, IND reduced arginase activity as well as NO and reactive oxygen species production in MDSCs differentiated in TME but not in TFME. Moreover, expression of the C/EBP-â transcription factor isoforms correlated with the suppressive activity of IND-treated MDSCs. Our study unveils the dual and context-dependent action of IND, a drug that serves both as an anti-inflammatory and anticancer agent, which differentially affects MDSC activity whether these cells are derived from TME or TFME. These results have broad clinical implication in cancer, chronic inflammation and autoimmunityFil: Blidner, Ada Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Salatino, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Mascanfroni, Ivan Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Diament, Miriam. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Bal, Elisa Dora. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Maria A. Jasnis. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Slobodanka M. Klein. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentin

Cells growth in 3D.

<p>(a) Kinetics of BT474 and MCF7 spheroids growth, n = 6. Upper insert corresponds to representative photographs taken to the same spheroid along time (40X). (b) Hematoxylin-Eosin staining of BT474 spheroid at 7 and 21 days growth showing the necrotic cores and the peripheral rims of viable cells (200X).</p

Autophagy inhibition.

<p>(a) Viability (MTS assay) of BT474 and resistant BT474-MR cells cultured as monolayers treated for 5 days with increasing concentrations of Tz relative to isotype IgG control; (b) Viability of BT474 and BT474 MR treated for 5 days with increasing concentrations of 3-MA. (c) Viability of BT474 and BT474-MR cells cultured in 2D treated with Tz (1 μg/ml), 3-MA (1 mM), or their combination for 3 days. (d) Diameter of BT474 and BT474-MR spheroids (n = 6) treated with Tz (50 μg/ml), 3-MA (1 mM) or their combination for 6 days. Each experiment was repeated at least three times (*p<0.05).</p

Autophagy in BT474 cells.

<p>(a) Western blot (WB) analysis of LC3 in 2D and 3D cultures after 24 h treatment with Tz. (b) Autophagic flux analyzed by WB in cell monolayers treated with Tz (1μg/ml) and 5 nM Bafilomycin A1 (BAF) (c) Cellular distribution of autophagosomes with LC3 stain. Arrows point to autophagosomes. Images show representative zones of BT474 cell monolayers (200x). (d) Immunofluorescence for LC3 (green) and Propidium iodide (PI, red) in spheroids treated for 15 days with Tz (50 μg/ml) or control IgG. Magnified images correspond to the zones limited by the white squares in the left photographs (600x).</p

Study of spheroid subpopulations.

<p>Spheroids were treated with 50 μg/ml Tz or control IgG for 15 days. (a) H&E (200x); immunofluorescence for pHER2 and HER2, HIF-1α (600x) and cleaved caspase 3 (400x). (b) Immunohistochemistry for Ki67 and quantification of Ki67+ cells (*p<0.05). (c) Confocal analysis of p27 (200x). Filled white arrows correspond to nuclear staining and black filled arrows correspond to cytoplasmatic staining.</p

Apoptosis of cells cultured in 2D and 3D after 6 h treatment with Tz, 3-MA or their combination.

<p>Representative Annexin V/ Propidium iodide graphs are shown. Triplicate cultures were run per group, and the experiment was repeated three times (*p<0.05).</p

Dose-dependent effect of Tz on cells growth.

<p>(a) Western blot analysis for AkT, pAkT and β-actin of BT474 spheroids after 24 h or 5 days of Tz treatment. (b) Viability (MTS assay) of BT474 and MCF7 cells cultured as monolayers treated for 5 days with increasing concentrations of Tz (0.005–50 μg/ml) relative to isotype IgG control. (c) Volume was evaluated in BT474 and MCF7 spheroids chronically treated during 15 days with 50, 10 and 1 μg/ml Tz or human IgG. Each point of the curves expresses the percentage in size change respect to the initial pretreated size (considered 100%), and represents the mean ± SD (n = 6). Inserts (right) correspond to 15 days-treated spheroids (40X). (d) BT474 spheroids chronically treated were analyzed by flow cytometry and the quantification of cell cycle distribution with propidium iodide staining is shown. Triplicate cultures were run per group, and each experiment was repeated twice (*p<0.05). (e) Cells obtained from spheroids chronically treated with Tz (BT474-ETz) or IgG (BT474-EIgG) were cultured as monolayers and treated with increasing concentrations of Tz (0.01–50 μg/ml).</p