A laboratory scale device for microencapsulation of genetically engineered cells into alginate beads

The microencapsulation of recombinant cells, widely used for in vitro high-density cell culture, is a novel and potentially cost-effective method of in vivo heterologous protein delivery, where the protein producing cells are immunologically protected from tissue rejection. We report here a simple, reliable and inexpensive laboratory method to generate calcium alginate microcapsules containing genetically engineered, interleukin-2 expressing, Chinese hamster ovary (CHO) cells.in vitro high-density cell culture, is a novel and potentially cost-effective method of in vivo heterologous protein delivery, where the protein producing cells are immunologically protected from tissue rejection. We report here a simple, reliable and inexpensive laboratory method to generate calcium alginate microcapsules containing genetically engineered, interleukin-2 expressing, Chinese hamster ovary (CHO) cells.in vivo heterologous protein delivery, where the protein producing cells are immunologically protected from tissue rejection. We report here a simple, reliable and inexpensive laboratory method to generate calcium alginate microcapsules containing genetically engineered, interleukin-2 expressing, Chinese hamster ovary (CHO) cells.Fil: Fiszman, Gabriel Leon. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Karara, Armando Luis. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Finocchiaro, Liliana Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Glikin, Gerardo Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentin

Suicide gene therapy on spontaneous canine melanoma: correlations between in vivo tumors and their derived multicell spheroids in vitro

To validate the use of multicellular spheroids to predict the efficacy of herpes simplex thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene therapy in the respective in vivo tumors, we established and characterized 15 melanoma-derived cell lines from surgically excised melanoma tumors. Three HSVtk-lipofected cell lines were not sensitive to GCV in any culture configuration, other five displayed similar sensitivity as monolayers or spheroids, and only one resulted more sensitive when grown as spheroids. Other six cell lines manifested a relative multicellular resistance (MCR) phenotype growing as spheroids, compared with the same cells growing as monolayers. The reverse correlation between the MCR and the monolayers survival to HSVtk/GCV suggests that one of the main causes of MCR would be the rapid cell repopulation after suicide gene treatment. The high correlation of MCR with the spheroids radial growth and with the mitotic index of the respective originary tumors supported this re-growth involvement. A remarkable finding was the high correlation in HSVtk/GCV sensitivity between in vivo tumor and the corresponding derived cell lines growing as spheroids (R 2 0.85). This strongly encourages the implementation of spheroids as highly realistic experimental model for optimizing and predicting the in vivo response of the respective tumors to therapeutic strategies.Fil: Gil Cardeza, Maria Lourdes. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; ArgentinaFil: Villaverde, Marcela Solange. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fiszman, Gabriel Leon. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Altamirano, Natalia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; ArgentinaFil: Cwirenbaum, R. A.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; ArgentinaFil: Glikin, Gerardo Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; ArgentinaFil: Finocchiaro, Liliana Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; Argentin

Autoantibodies against muscarinic receptors in breast cancer. Its role in tumor angiogenesis.

The presence of autoantibodies in cancer has become relevant in recent years. We demonstrated that autoantibodies purified from the sera of breast cancer patients activate muscarinic acetylcholine receptors in tumor cells. Immunoglobulin G (IgG) from breast cancer patients in T1N0Mx stage (tumor size¡Ü2 cm, without lymph node metastasis) mimics the action of the muscarinic agonist carbachol stimulating MCF-7 cell proliferation, migration and invasion. Angiogenesis is a central step in tumor progression because it promotes tumor invasion and metastatic spread. Vascular endothelial growth factor-A (VEGF-A) is the main angiogenic mediator, and its levels have been correlated with poor prognosis in cancer. The aim of the present work was to investigate the effect of T1N0Mx-IgG on the expression of VEGF-A, and the in vivo neovascular response triggered by MCF-7 cells, via muscarinic receptor activation. We demonstrated that T1N0Mx-IgG (10−8 M) and carbachol (10−9 M) increased the constitutive expression of VEGF-A in tumor cells, effect that was reverted by the muscarinic antagonist atropine. We also observed that T1N0Mx-IgG and carbachol enhanced the neovascular response produced by MCF-7 cells in the skin of NUDE mice. The action of IgG or carbachol was reduced in the presence of atropine. In conclusion, T1N0Mx-IgG and carbachol may promote VEGF-A production and neovascularization induced by breast tumor cells via muscarinic receptors activation. These effects may be accelerating breast tumor progression.Fil: Lombardi, Maria Gabriela. Consejo Nacional de Invest.cientif.y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Centro de Estudios Farmacologicos y Botánicos; Argentina;Fil: Negroni, María Pía.Fil: Pelegrina, Laura Tatiana. Consejo Nacional de Invest.cientif.y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Centro de Estudios Farmacologicos y Botánicos; Argentina;Fil: Castro, Maria Ester. Consejo Nacional de Invest.cientif.y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Centro de Estudios Farmacologicos y Botánicos; Argentina;Fil: Fiszman, Gabriel Leon. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr.Ángel Roffo"; Argentina;Fil: Azar, María Eugenia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr.Ángel Roffo"; Argentina;Fil: Cresta Morgado Carlos. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr.Ángel Roffo"; Argentina;Fil: Sales Maria Elena. Consejo Nacional de Invest.cientif.y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Centro de Estudios Farmacologicos y Botánicos; Argentina

Autoanticuerpos contra receptores muscarínicos en cáncer de mama

Se ha investigado exhaustivamente, la capacidad de células transformadas para inducir una respuesta inmune eficaz en portadores de tumor. En particular, la detección y el papel de anticuerpos (Acs) específicos contra antígenos (Ags) tumorales en los pacientes con cáncer ha dado resultados contrapuestos. Previamente caracterizamos la expresión de receptores colinérgicos muscarínicos (RCM) en células LM3, derivadas de un adenocarcinoma mamario murino espontáneo de la cepa BALB/c. Estas células presentan una mayor expresión de RCM en comparación con células normales de epitelio mamario murino, NMuMG. En este trabajo, investigamos la capacidad de las proteínas RCM sobre-expresadas para inducir una respuesta humoral autóloga en portadores del tumor LM3 y la función de los Acs formados en la progresión tumoral. Detectamos autoAcs contra RCM en la fracción IgG purificada del suero de portadores de tumor pequeño (Tp) y tumor grande (TG) formados 14 días y 28 días después de la inoculación subcutánea de células tumorales, respectivamente. La IgG proveniente del suero de portadores de Tp estimula significativamente la proliferación de células tumorales, mientras que la de portadores de TG la inhibe. Ambos efectos fueron reducidos por el pretratamiento de las células LM3 con el antagonista muscarínico atropina (AT). La IgG purificada de animales normales estimuló la proliferación de células LM3 pero el efecto no fue modificado por AT. La IgG de portadores de tumor desplazó la unión del radioligando muscarínico tritiado, bencilato de quinuclidinilo ([3H]-QNB) a los RCM en células LM3 en forma dependiente de la concentración, lo que evidenció la interacción de los Acs con los receptores expresados en dichas células. Asimismo, la IgG de portadores de tumor reconoció, en ensayos de inmunomarcación de homogenatos de corazón murino, una proteína cuyo peso molecular coincide con la que reconoce un Ac monoclonal específico contra el receptor muscarínico de acetilcolina M2. Resultados análogos se obtuvieron en homogenatos de tumor y lisados de células LM3. Además observamos que la IgG purificada de portadores de TG estimula la respuesta neovascular inducida por células LM3 con participación de los RCM. Concluimos que los autoAcs presentes en el suero de portadores de tumor ejercen efectos protumorales diferentes por vía muscarínica: mientras que en portadores de Tp estimulan la proliferación de células LM3 en portadores de TG, promueven la neovascularización.Fil: Fiszman, Gabriel Leon. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cattaneo, Valentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: de la Torre, Eulalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Colombo, Lucas Luis. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Middonno, Cristina. 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: Sales, María Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaUnidad documental simpl

Interacciones estroma-epitelial al tamoxifeno

Tamoxifen, a selective estrogen receptor modulator, is the standard endocrine treatment for hormone receptor positive breast cancer, both in the initial adjuvant therapy and as treatment of patients with metastatic disease. However, about one third of patients with estrogen receptor (ER)-á positive tumors are refractory to tamoxifen therapy and a high percentage of patients who initially respond to tamoxifen develop resistance. Most breast cancers that acquire endocrine resistance retain ER-á expression, suggesting that loss of ER is not a common mechanism of resistance to endocrine therapy. Diverse signal transduction pathways influence the functional activity of ER, in addition to steroid ligand, and are critical in the responsiveness of tumors to anti-hormonal drugs. In particular, it is now well established that activation of several growth factor signaling cascades can promote resistance. However, although these pathways can be modulated by the tumor microenvironment, no studies to our knowledge have investigated this subject in an ER-á positive cell context. In this article we discuss the development of a new mouse model of estrogen responsive/tamoxifen sensitive breast cancer and propose that microenvironmental factors may be critical in the response to endocrine therapy, and could in the future be a rational target for the treatment of tamoxifen resistant breast cancer.á positive tumors are refractory to tamoxifen therapy and a high percentage of patients who initially respond to tamoxifen develop resistance. Most breast cancers that acquire endocrine resistance retain ER-á expression, suggesting that loss of ER is not a common mechanism of resistance to endocrine therapy. Diverse signal transduction pathways influence the functional activity of ER, in addition to steroid ligand, and are critical in the responsiveness of tumors to anti-hormonal drugs. In particular, it is now well established that activation of several growth factor signaling cascades can promote resistance. However, although these pathways can be modulated by the tumor microenvironment, no studies to our knowledge have investigated this subject in an ER-á positive cell context. In this article we discuss the development of a new mouse model of estrogen responsive/tamoxifen sensitive breast cancer and propose that microenvironmental factors may be critical in the response to endocrine therapy, and could in the future be a rational target for the treatment of tamoxifen resistant breast cancer.á expression, suggesting that loss of ER is not a common mechanism of resistance to endocrine therapy. Diverse signal transduction pathways influence the functional activity of ER, in addition to steroid ligand, and are critical in the responsiveness of tumors to anti-hormonal drugs. In particular, it is now well established that activation of several growth factor signaling cascades can promote resistance. However, although these pathways can be modulated by the tumor microenvironment, no studies to our knowledge have investigated this subject in an ER-á positive cell context. In this article we discuss the development of a new mouse model of estrogen responsive/tamoxifen sensitive breast cancer and propose that microenvironmental factors may be critical in the response to endocrine therapy, and could in the future be a rational target for the treatment of tamoxifen resistant breast cancer.á positive cell context. In this article we discuss the development of a new mouse model of estrogen responsive/tamoxifen sensitive breast cancer and propose that microenvironmental factors may be critical in the response to endocrine therapy, and could in the future be a rational target for the treatment of tamoxifen resistant breast cancer.Fil: Pontiggia, Osvaldo Alejandro. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "dr.angel Roffo". Departamento de Radioterapia y Cancer Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fiszman, Gabriel Leon. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "dr.angel Roffo". Departamento de Radioterapia y Cancer Experimental; ArgentinaFil: Rodriguez, Vanina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "dr.angel Roffo". Departamento de Radioterapia y Cancer Experimental; ArgentinaFil: Bal, Elisa Dora. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "dr.angel Roffo". Departamento de Radioterapia y Cancer Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Simian, Marina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "dr.angel Roffo". Departamento de Radioterapia y Cancer Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Breast cancer stem cells are involved in Trastuzumab resistance through the HER2 modulation in 3D culture

Breast cancer human cells culture as spheroids develop autophagy and apoptosis, which promotes Trastuzumab resistance in HER2 overexpressing cells. Our aim was to study the association of the hostile environment developed in 3D with the breast cancer stem cells population and the HER2 modulation.Human mammary adenocarcinoma cell lines were cultured as spheroids using the hanging drop method. We generated hypoxia conditions by using a hypoxic chamber and CoCl2 treatment. Breast cancer stem cells were measured with mammosphere assays, the analysis of CD44+CD24low population by flow cytometry and the pluripotent gene expression by RT-qPCR. HER2 expression was evaluated by flow cytometry and Western blot. MTS assays were conducted to study cell viability.Hostil environment developed in spheroids, defined by hypoxia and autophagy, modulated the response to Trastuzumab. In HER2+ cells with acquired resistance, we observed an increase in the breast cancer stem cell population. In BT474 spheroids, Trastuzumab induced the acquisition of resistance, along with an increase in breast cancer stem cells. Also, in 3D culture conditions we determined a modulation in the HER2 expression.Moreover, breast cancer stem cells showed enhanced HER2 expression. Finally, cells without HER2 gene amplification cultured as spheroids were sensitive to Trastuzumab, diminishing HER2 expression and cancer stem cells.Our findings show that 3D architecture is able to modulate breast cancer stem cell population and HER2 distribution, modifying the cell response to Trastuzumab.Fil: Rodríguez, Cristina Elisa. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Berardi, Damian Emilio. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Abrigo, Marianela. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Todaro, Laura Beatriz. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Bal, Elisa Dora. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Fiszman, Gabriel Leon. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentin

Herpes simplex virus thymidine kinase/ganciclovir system in multicellular tumor spheroids

We have developed multicellular spheroids (MCS) established from LM05e and LM3 spontaneous Balb/c-murine mammary adenocarcinoma and B16 C57-murine melanoma derived cell lines as an in vitro model to study the efficacy of the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide system. We demonstrated for the first time that HSVtk-expressing cells assembled as MCS manifested a GCV resistance phenotype compared to the same cells grown as sparse monolayers. HSVtk-expressing LM05e, LM3 and B16 spheroids were 16-, three- and nine-fold less sensitive to GCV than their respective monolayers, even though they could express transgenes 10-, eight- and five-fold more efficiently. Mixed populations of HSVtk- and their respective βgal-expressing cells displayed a cell-type specific bystander effect that was higher in monolayers than in MCS. However, HSVtk-expressing cells in two- or three-dimensional cultures were always significantly more sensitive to GCV than the βgal-expressing counterparts, supporting the feasibility of this suicide approach in vivo. We present evidence showing that HSVtk-expressing tumor cells, when transferred from monolayers to MCS, displayed: (i) lower GCV cytotoxic activity and bystander effect; (ii) higher and efficient expression of genes transferred as lipoplexes; (iii) lower cell proliferation rates; and (iv) changes in intracellular Bax/Bcl-xL rheostat of mitochondria-mediated apoptosis.Fil: Finocchiaro, Liliana Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Bumaschny, Viviana Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Karara, Armando Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Fiszman, Gabriel Leon. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Casais, Cecilia C.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Glikin, Gerardo Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentin

Lipofection of early passages of cell cultures derived from murine adenocarcinomas: In vitro and ex vivo testing of the thymidine kinase/ganciclovir system

Early passages of cultured cells derived from four spontaneous Balb/c murine adenocarcinomas were used to explore the feasibility of a nonviral HSVtk-based suicide gene therapy system. After lipofection with pCMVtk, the transiently HSVtk expressing P07 (lung), M3, M05, and M38 (mammary gland) cells were, respectively, about 130-, 30-, 120-, and 170-fold more sensitive to ganciclovir (GCV) in vitro than their respective controls. Eighty percent of Balb/c mice subcutaneously inoculated with ex vivo pCMVtk-lipofected P07 cells, followed by intraperitoneal GCV injection for 7 days, displayed a complete inhibition of tumor growth for over 70 days. Control animals started to display tumors 13 days after inoculation. We present evidence showing that early passages of cultured tumor cells can efficiently express lipofected genes and that they are sensitive to the lipoplex-mediated HSVtk/GCV system.Fil: Karara, Armando Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Bumaschny, Viviana Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Fiszman, Gabriel Leon. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Casais, Cecilia C.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Glikin, Gerardo Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Finocchiaro, Liliana Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentin

Establishment of an in vitro estrogen-dependent mouse mammary tumor model: A new tool to understand estrogen responsiveness and development of tamoxifen resistance in the context of stromal-epithelial interactions

Currently, to our knowledge, there are no continuous cell lines derived from estrogen dependent, tamoxifen sensitive spontaneous mouse mammary carcinomas. We describe here the establishment and characterization of a cell line derived from the M05 mouse mammary tumor, LM05-Mix, composed of both an epithelial and a fibroblastic component. From it the respective epithelial LM05-E and fibroblastic LM05-F cell lines were generated by limiting dilution. Immunofluorescence studies confirmed that the epithelial cells were positive for E-cadherin, cytokeratins and vimentin whereas the fibroblastic cells were negative for the epithelial markers and positive for a-smooth muscle actin and vimentin. Both cell types expressed estrogen and progesterone receptors, although only the epithelial LM05-E cells were stimulated by estradiol and inhibited by tamoxifen. In the bicellular LM05-Mix cell line estradiol proved to stimulate cell proliferation whereas the response to tamoxifen was dependent on confluency and the degree of epithelial-fibroblastic interactions. The presence of membrane estrogen receptors in both cell types was suggested by the achievement of non-genomic responses to short treatments with estradiol, leading to the phosphorylation of ERK1/2. Finally, cytogenetic studies suggest that these two cell types represent independent cell populations within the tumor and would not be the result of an epithelial-mesenchymal transition. This model presents itself as a valuable alternative for the study of estrogen responsiveness and tamoxifen resistance in the context of epithelial-stromal interactions.Fil: Pontiggia, Osvaldo Alejandro. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rodriguez, Vanina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Fabris, Victoria Teresa. 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: Raffo, Diego Alejandro. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bumaschny, Viviana Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Fiszman, Gabriel Leon. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bal, Elisa Dora. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Simian, Marina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Immunoglobulin G from breast cancer patients in stage I stimulates muscarinic acetylcholine receptors in MCF7 cells and induces proliferation: participation of nitric oxide synthase-derived nitric oxide

INTRODUCTION: Muscarinic acetylcholine receptors (mAChR) belong to the G-protein-coupled receptor family and are extensively expressed in most cells in mammals. We had reported the expression of mAChR in murine and human breast tumors. METHODS: The presence of antibodies in the sera of patients with different tumors directed against self-proteins has been recently described. In this work, we investigated the presence of autoantibodies against mAChR in the sera of breast cancer patients in stage I (T1N0Mx-IgG). IgG purification was performed by affinity chromatography in protein G-agarose. We also studied the ability of these antibodies to modulate the proliferation of MCF-7 breast tumor cells by the MTS colorimetric assay. The ability of T1N0Mx-IgG to stimulate muscarinic signaling pathway via nitric oxide synthase was tested by Griess reaction. RESULTS: We demonstrated M(3) and M(4) receptors expression in MCF-7 cells. T1N0Mx-IgG promotes cell proliferation, mimicking the action of the muscarinic agonist carbachol. This effect was preferentially due to M(3) receptor activation in tumor cells via phospholipase C-induced nitric oxide liberation by calcium-dependent nitric oxide synthases. IgG from control patients was unable to produce this effect. DISCUSSION: IgG from patients with breast cancer in early stages could be promoting tumor progression by muscarinic activation, and its presence could be determining the prognosis of this illness.Fil: Negroni, María Pía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Fiszman, Gabriel Leon. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Azar, María Eugenia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Cresta Morgado, Carlos. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Español, Alejandro Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Pelegrina, Laura Tatiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: de la Torre, Eulalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Sales, Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentin