Role of tumor architecture in elicitation of effector functions of human cytotoxic T-lymphocytes recognizing melanoma associated antigens

Abstract

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. 3D architectures might also play a role in impairing immunorecognition of cancer cells by cytotoxic T lymphocytes (CTLs) specific for tumor-associated antigens. Culture of HBL, D10 (both HLA-A*0201+, TAA+) and NA8 (HLA-A*0201+, TAA-) melanoma cell lines on poly-Hydroxyethylmethacrylate-coated plates, resulted in generation of 3D multicellular tumor spheroids (MCTS). Kinetics of cell proliferation in MCTS was significantly slower than in monolayer cultures. Following long-term culture (>10-15 days) MCTS showed highly compact and organised cell growth in outer layers, with necrotic cores. To obtain an insight into the role played by tumor architecture in the elicitation of specific gene expression patterns, we addressed gene expression profiles of NA8 melanoma cells cultured in two-dimensional monolayers (2D) or in 3D (MCTS). Oligonucleotide microarray analysis of the expression of over 20,000 genes was performed on cells cultured in standard 2D, in the presence of collagen as model of extracellular matrix (ECM), or in MCTS. Gene expression profiles of cells cultured in 2D in the presence or absence of ECM were highly similar, with more than threefold differences limited to five genes. In contrast, culture in MCTS resulted in the significant, more than threefold, upregulation of the expression of >100 transcripts, while 73 transcripts were more than threefold downregulated. In particular, genes encoding CXCL1, 2, and 3 (GRO-α, -β, and γ), IL-8, CCL20 (MIP-3α), and Angiopoietin-like 4 were significantly upregulated, whereas basic-FGF and CD49d encoding genes were significantly downregulated. Oligonucleotide chip data were validated at the gene and protein level by quantitative real-time PCR, ELISA, and cell surface staining assays. Taken together, our data indicate that structural modifications of the architecture of tumor cell cultures result in a significant upregulation of the expression of a number of genes previously shown to play a role in melanoma progression and metastatic process. Then we investigated the effects of 3D culture on the recognition of melanoma cells by antigenspecific HLA class I-restricted Cytotoxic T-Lymphocytes (CTL). IFN-γ production can be used as a surrogate marker for tumor cell immunorecognition. Co-culture of melanoma spheroids with HLA-A0201 restricted Melan-A/MART-127-35-specific CTL clones resulted in significantly defective TAA recognition by CTL as compared to 2D, as witnessed by decreased IFN-γ production and decreased Fas Ligand, perforin and granzyme B gene expression. Indeed, Melan- A/MART-1 expression, at both gene and protein levels, was significantly decreased in 3D as compared with 2D tumor cell cultures. Concomitantly, a parallel decrease of HLA class I 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. We further identified a multiplicity of mechanisms potentially involved. In particular : 1) MCTS per se limit CTL capacity of recognizing HLA class I restricted antigens by reducing exposed cell surfaces. 2) Expression of melanoma differentiation antigens is down-regulated in tumor cell spheroids as compared to 2D unrelated to hypoxia or increased Oncostatin M gene expression but rather to decreased MITF gene expression. 3) Expression of HLA class I molecules is frequently down-regulated in melanoma MCTS, as compared to 2D, possibly due to decreased IRF-1 gene expression. 4) Lactate production by melanoma cells is increased in MCTS, as compared to 2D and lactate significantly inhibits TAA triggered IFN-γ production by CTL. 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 and a constellation of mechanisms are involved in causing this impairment of immunorecognition.