Culture of melanoma cells in 3-dimensional architectures results in impaired immunorecognition by cytotoxic T lymphocytes specific for Melan-A/MART-1 tumor-associated antigen

Objective: To assess the effects of the culture of melanoma cells in 3-dimensional (3D) architectures on their immunorecognition by cytotoxic T lymphocytes (CTLs) specific for tumor-associated antigens. Summary Background Data: Growth in 3D architectures has been shown to promote the resistance of cancers to treatment with drugs, cytokines, or irradiation, thereby potentially playing an important role in tumor expansion. We investigated the effects of 3D culture on the recognition of melanoma cells by antigen-specific HLA class I-restricted CTLs. Methods: Culture of HBL melanoma cells expressing Melan-A/Mart-1 tumor-associated antigen and HLA-A0201 on poly-2-hydroxyethyl methacrylate (polyHEMA)-coated plates resulted in the generation of aggregates of 400- to 500-,um diameters containing on average 30,000 cells and characterized by slower proliferation, as compared with monolayer (2-dimensional) cultures. HLA-A0201 restricted Melan-A/Mart-1(27-35)-specific CTL clones were used to evaluate tumor cell immunorecognition measured as specific IFN-gamma production. Comparative gene and protein expression in 2D and 3D cultures was studied by real-time PCR and flow cytometry, respectively. Overall differences in gene expression profiles between 2D and 3D cultures were evaluated by high-density oligonucleotide array hybridization. Results: HLA-A0201 restricted Melan-A/Mart-1(27-35) specific CTL clones produced high amounts of IFN-gamma upon short-term (4-24 hours) coincubation with HBL cells cultured in 2D but not in 3D, thus suggesting altered antigen recognition. Indeed, Melan-A/Mart-1 expression, at both gene and protein levels, was significantly decreased in 3D as compared with 2D cultures. Concomitantly, a parallel decrease of HLA class 1 molecule expression was also observed. Differential gene profiling studies on HBL cells showed an increased expression of genes encoding molecules involved in intercellular adhesion, such as junctional adhesion molecule 2 and cadherin-like 1 (>20- and 8-fold up-regulated, respectively) in 3D as compared with 2D cultures. Conclusions: Taken together, our data suggest that mere growth of melanoma cells in 3D architectures, in the absence of immunoselective pressure, may result in defective recognition by tumor-associated antigen-specific CTL.