Subproject 1 – Cancer and metastasis

Participants: Fátima Aparecida Bottcher, Liliana Andrade, Marianne Pinotti, José Aristodemo Pinotti, Carlos Lenz Cesar, João Ernesto de Carvalho (Coordenador da Divisão de Farmacologia e Toxicologia – Centro Pluridisciplinar de Pesquisas Quimícas, Biológicas e Agrícolas). Mary Ann Foglio (Coordenadora da Divisão de Fitoquímica do CPQBA). This collaboration has initiated and involve one PostDoc (FAPESP) under supervision of Dr. Lenz e Dr. Bottcher.

The objectives of this project is to employ biophotonic microscopical techniques SHG, Multiphoton, optical tweezers and CARS, as tools for the study of cellular mechanisms for tumor development, focusing on cell-cell interactions and the hability to invade and metastasize. The studies will concentrate in mammary and ovary cancer. Mortality by ovary cancer is highest among gimnecological cancers, especially because of the late of diagnosis, when cancer has progressed to advanced stages. The lack of a methodology for diagnosing early ovary lesions is a known fact contributing to the high mortality. The search for new diagnosing methods that could better identify early lesions and consequently lead to a precocious diagnostic is a chalenge and a stimulus for research in this area. In a first approach, we will acquire images from a library of retrospective cases with the diverse biophotonic techniques aiming at comparing them with the classical methodologies and trying to find new information that could improve tumor staging. The following situations will be investigated non-neoplasic tissues, in situ carcinomas, borderline tumors and invasive tumors. Special attention will be given to the collagenous matrix as assessed by SHG. Comparisons with conventional images might show where biophotonics are superior and define which techniques should be used with tumor cells in vitro. The library of ovary cancer cases correspond to the research line Role of cadherin E, N, catenins alfa and beta and galectin-3 in ovary cancer metastasis, and are ready for use. Using immunohistochemistry, we could observe that galectin-3 is a differencial marker of malignant or benign ovary neoplasiasm. The literature suggest strongly that galectin-3 acts as a major angiogenic factor contributing to metastasis, and this will be under focus of the present project. The other part of this project is the study of cells and cell lineages in culture. In this sense, mammary non-neoplasic and tumor cells, obtained from primary cultures and cell lineages will be employed. Cell elasticity will be measured under different situations, especially after the establishment of cell-cell contacts. Samples from (1) non-neoplasic mammary gland (obtained from aesthetical reducing mammoplasty) and MCF-10; (2) mammary tumors and MCF-7 cells (low metastatic potential and ER-positive), biopsies from in situ, invasive and metastatic tumors (3) MDA-MB435 lineage (high metastatic potential. Beside these, the other lineages OVCAR (ovary), NCI ADR (ovário resistent ovary cell), PCO 3 (prostate), HT 29 (colon cancer), NCI 460 (lung), 786 0 (kidney), UACC (melanoma) e k562 (leucemia) will be used. Optical tweezers will be employed to put the tumor cells in contact with different cells in monolayers, and parameters such as elasticity and viscosity will be measured. CARS, multiphoton, SHG, and FLIM will be used to observe changes in cell behavior, as compared to the immortalized cell lineages. Early events of mammary carcinogesis are poorly known and this turns this study particularly important. The collaboration with the clinic EGIMAJAP will be essential for obtaining tissues and providing clinical support for the study. The clinic will fund part of the study. The clinic EGIMAJAP will also contribute to the study “Role of stem cells in triple-negative mammary tumors”, which will use CARS, SHG and multiphoton to investigate new paradigms on mammary gland carcinogenesis. In the last tem years, the Division of Pharmacology and Toxicology (DFT) at CPQBA-Unicamp has searched for new anti-cancer drugs. In this search, the DFT mantains cell cultures from diverse tumor types on which active principles extracted from Brazilian flora are tested, so, and finally, the new photonic microscopical tools made available through this project will allow the study of mechanisms of action of active principles screened at CPQBA-Unicamp, in parallel to the main objectives which are (1) evaluate putative anticancer components from natural, synthetic and semi-synthetic sourcers on cultured human tumor cells (in vitro); (2) select the best candidates through determination of the type of cell death they promote, and (3), evaluate the activity in murine tumor models such as Ehrlich solid tumor and B16F10 melanomas.