Spontaneous canine melanoma derived spheroids display individual multicellular resistance patterns to suicide gene and chemotherapy

ALTAMIRANO N.A.,; KARARA A.L; VILLAVERDE M.S; FISZMAN G.L; GLIKIN G.C.; FINOCCHIARO L.M.E

Cancer Drug Resistance Research Perspectives. L.S. Torres (Ed.).

Nova Science Publishers, Inc

Lugar: New York, U.S.A; Año: 2007; p. 119 - 137

Spontaneous canine melanoma derived spheroids display individual multicellular resistance patterns to suicide gene and chemotherapy Natalia A. Altamirano, Armando L. Karara, Marcela S. Villaverde, Gabriel L. Fiszman, Gerardo C. Glikin & Liliana M. E. Finocchiaro Unidad de Transferencia Genética, Instituto de Oncología "Ángel H. Roffo", Universidad de Buenos Aires, Av. San Martín 5481, (1417) Buenos Aires, Argentina ABSTRACT We have previously demonstrated that, compared to monolayer, spheroid configuration of in vitro cultured tumor cells induce moderate resistance to the herpes simplex thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene therapy system, without preventing the efficacy of the treatment. To establish the suitability of this approach on spontaneous canine melanoma, we isolated, established and characterized six melanoma derived cell lines from surgically excised hepatic metastasis (Ll) and from oral (Ds, Fk, Kg, Sc) and ocular (Ak) primary tumors. Immunocytochemistry evidenced uniform vimentin staining in every tumor cell line while S100 was in Kg > Ak = Ll = Sc >Ds = Fk. Melan A and gp100 were detected in Ak = Ds > Kg = Sc, being absent in amelanic Fk and Ll. None of the cell lines expressed cytokeratin. The cytotoxicity of increasing concentrations (0.1 to 1000 μg/ml) of the pro-drug GCV was tested on monolayers and spheroids of transiently expressing HSVtk or β-galactosidase (βgal) cells. Although HSVtk did not sensitize Fk and Ll cells to GCV in any culture configuration, HSVtk-lipofected Ak, Ds, Kg and Sc cell lines manifested a relative GCV resistance phenotype when grown as spheroids, compared to the same cells grown as monolayers. HSVtk-expressing Ak, Ds, Kg and Sc spheroids, resulted about 3-, 25-, 980- and 950-fold less sensitive to GCV than the respective monolayers. The multicellular resistance (MCR) phenotype of spheroids was also observed when melanoma cells were treated with the chemotherapeutic drugs (1 to 1000 nM) paclitaxel (PTX) and vincristine (VC). Ak, Ds, Fk, Kg, Ll and Sc spheroids resulted 2, 4, >6, >23, 3 and 7-fold less sensitive to PTX and 6, >580, >19, 6, >135, and >66-fold less sensitive to VC than their respective monolayers. The in vitro behavior of spheroids correlated with the clinical outcome of the HSVtk/GCV treatment observed by canine melanoma patients in vivo. Our results suggest: (i) a correlation between the absence of melan A and gp100 antigens and spheroids resistance to suicide gene and chemotherapy; and (ii) a general underlying MCR mechanism opposing to the different treatments.