Anca Reschner

Three-Dimensional Culture of Melanoma Cells Profoundly Affects Gene Expression Profile: A High Density Oligonucleotide Array Study

Growth in three‐dimensional (3D) architectures has been suggested to play an important role in tumor expansion and in the resistance of cancers to treatment with drugs or cytokines or irradiation. To obtain an insight into underlying molecular mechanisms, we addressed gene expression profiles of NA8 melanoma cells cultured in bidimensional monolayers (2D) or in 3D multicellular tumor spheroids (MCTS). MCTS containing 10‐30,000 cells were generated upon overnight culture in poly‐Hydroxyethylmethacrylate (polyHEMA) coated plates. Kinetics of cell proliferation in MCTS was significantly slower than in monolayer cultures. Following long‐term culture (>10 days), however, MCTS showed highly compact and organised cell growth in outer layers, with necrotic cores. 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 ≥threefold differences limited to five genes. In contrast, culture in MCTS resulted in the significant, ≥threefold, upregulation of the expression of >100 transcripts while 73 were ≥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.

New dimensions in tumor immunology: what does 3D culture reveal?

Experimental models indicate that tumor cells in suspension, unlike solid tumor fragments, might be unable to produce life-threatening cancer outgrowth when transferred to animal models, irrespective of the number of cells transferred, although they induce specific immune responses. Human tumor cells cultured in three dimensions display increased pro-angiogenic capacities and resistance to interferons, chemotherapeutic agents or irradiation, as compared with cells cultured in two-dimensional (2D) monolayers. Tumor cells cultured in three dimensions were also shown to be characterized by defective immune recognition by cytotoxic T lymphocytes (CTLs) specific for tumor-associated antigens (TAAs) and by a capacity to inhibit CTL proliferation and dendritic cell (DC) functions. Downregulation of human leukocyte antigen (HLA) or TAA expression and high production of lactic acid might play a role in the elicitation of these effects. Here, we propose that growth in 3D architectures might provide new insights into tumor immunology and could represent an integral missing component in pathophysiological tumor immune escape mechanisms.

Differential effects of the tryptophan metabolite 3-hydroxyanthranilic acid on the proliferation of human CD8 + T cells induced by TCR triggering or homeostatic cytokines

Production of indoleamine 2,3‐dioxygenase (IDO) by tumor cells, leading to tryptophan depletion and production of immunosuppressive metabolites, may facilitate immune tolerance of cancer. IDO gene is also expressed in dendritic cells (DC) upon maturation induced by lipopolysaccarides or IFN. We investigated IDO gene expression in melanoma cell lines and clinical specimens as compared to mature DC (mDC). Furthermore, we explored effects of L‐kynurenine (L‐kyn) and 3‐hydroxyanthranilic acid (3‐HAA) on survival and antigen‐dependent and independent proliferation of CD8+ cells. We observed that IDO gene expression in cultured tumor cells and freshly excised samples is orders of magnitude lower than in mDC, providing highly efficient antigen presentation to CD8+ T cells. Non toxic concentrations of L‐kyn or 3‐HAA did not significantly inhibit antigen‐specific CTL responses. However, 3‐HAA, but not L‐kyn markedly inhibited antigen‐independent proliferation of CD8+ T cells induced by common receptor γ‐chain cytokines IL‐2, ‐7 and ‐15. Our data suggest that CD8+ T cell activation induced by antigenic stimulation, a function exquisitely fulfilled by mDC, is unaffected by tryptophan metabolites. Instead, in the absence of effective T cell receptor triggering, 3‐HAA profoundly affects homeostatic proliferation of CD8+ T cells.

Multiple mechanisms underlie defective recognition of melanoma cells cultured in three-dimensional architectures by antigen-specific cytotoxic T lymphocytes.

Cancer cells’ growth in three-dimensional (3D) architectures promotes resistance to drugs, cytokines, or irradiation. We investigated effects of 3D culture as compared to monolayers (2D) on melanoma cells’ recognition by tumour-associated antigen (TAA)-specific HLA-A*0201-restricted cytotoxic T-lymphocytes (CTL). Culture of HBL, D10 (both HLA-A*0201+, TAA+) and NA8 (HLA-A*0201+, TAA−) melanoma cells on polyHEMA-coated plates, resulted in generation of 3D multicellular tumour spheroids (MCTS). Interferon-gamma (IFN-γ) production by HLA-A*0201-restricted Melan-A/MART-127–35 or gp100280–288-specific CTL clones served as immunorecognition marker. Co-culture with melanoma MCTS, resulted in 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. A multiplicity of mechanisms were potentially involved. First, MCTS per se limit CTL capacity of recognising HLA class I restricted antigens by reducing exposed cell surfaces. Second, expression of melanoma differentiation antigens is downregulated in MCTS. Third, expression of HLA class I molecules can be downregulated in melanoma MCTS, possibly due to decreased interferon-regulating factor-1 gene expression. Fourth, lactic acid production is increased in MCTS, as compared to 2D. These data suggest that melanoma cells growing in 3D, even in the absence of immune selection, feature characteristics capable of dramatically inhibiting TAA recognition by specific CTL.

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-μm 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-127–35-specific CTL clones were used to evaluate tumor cell immunorecognition measured as specific IFN-γ 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-127–35 specific CTL clones produced high amounts of IFN-γ 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 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. 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.

The ester-bonded palmitoyl side chains of Pam3CysSerLys4 lipopeptide account for its powerful adjuvanticity to HLA class I-restricted CD8+ T lymphocytes

Molecularly defined adjuvants are urgently required to implement immunization protocols by which CD8+ T cells induction is envisaged. We show here that the synthetic lipopeptide Pam3CysSerLys4 (P3CSK4) strongly enhances the expansion of antigen‐specific IFN‐γ+CD8+ cells in vitro. These effects critically depend on the presence of two ester‐bonded palmitoylated side chains. In fact, T cell expansion is impaired in the presence of derivatives bearing two non‐palmitoylated fatty acid chains, while derivatives with only one amide‐bonded palmitoylated residue are completely inactive and behave like the non‐lipidated peptide backbone. P3CSK4 is not mitogenic for T lymphocytes and can modulte DC immune biological properties. Indeed, doses as low as 100 ng/ml increase CD86, CD83 and CD40 surface expression on DC, fail to induce CCR7, and trigger a defined pattern of soluble factors associated to immune effector functions. In particular, substantial amounts of TNF‐α, IL‐6, CCL2 and CXCL10, in the absence of IFN‐α, IFN‐γ, IL‐15, IL‐12p70 and CX3CL1, can be measured. Accordingly, antigen‐specific CD8+ T cells expanded in vitro express CCR2 and CXCR3 chemokine receptors. Altogether our data suggest that human DC are able to respond to chemically different synthetic lipopeptide analogs and that optimal adjuvanticity to CD8+ T cell induction is achieved by the palmitoylated structures.

Selective responsiveness to common gamma chain cytokines in peripheral blood-derived cytotoxic T lymphocytes induced by Melan-A/MART-127–35targeted active specific immunotherapy

We have comparatively evaluated the proliferative response of CTL induced in metastatic melanoma patients upon immunization against Melan‐A/MART‐127–35 tumor associated antigen (TAA) to IL‐2, IL‐7 or IL‐15 cytokines, sharing a receptor common γ‐chain (cγ‐c cytokines). Twenty‐eight CTL clones were generated from CD8+ T cells obtained from 3 patients during the contraction phase of immune response following a successful vaccine mediated expansion of specific effectors. All clones were able to kill tumor cell lines expressing HLA‐A0201 and Melan‐A/MART‐1, and displayed phenotypic characteristics of effector/memory (CD45RA−/CCR7−) or CD45RA+/CCR7− effector cells in intermediate to late developmental stage (CD28−/CD276±) CTL. Proliferative responses could be elicited or enhanced by IL‐2 and IL‐15, but not IL‐7, in the absence or in the presence of T‐cell receptor (TCR) triggering, respectively. Accordingly, only IL‐2 and IL‐15 were able to promote the survival of the CTL clones under investigation. While all clones expressed high amounts of receptor cγ‐c (CD132), lower, but detectable, expression of IL‐7 receptor alpha chain was also observed. CD8+ cells from one of the patients treated were obtained 6 months after the last vaccine boost and were cultured in the presence of Melan‐A/MART‐127–35 and each of the 3 cytokines under investigation. Consistent with data from CTL clones, expansion of Melan‐A/MART‐127–35 tetramer positive cells was only observed in the presence of IL‐2 or IL‐15 but not IL‐7. Instead, when CD8+ cells from the same patient were sampled shortly (14 days) after an additional vaccination only IL‐2 was able to promote the expansion of Melan‐A/MART‐127–35 tetramer positive cells. Taken together these data suggest a selective responsiveness of TAA‐specific CTL to different cγ‐c cytokines.

Human cytokines activate JAK–STAT signaling pathway in porcine ocular tissue

The JAK/STAT (Janus Tyrosine Kinase, Signal Transducers and Activators of Transcription) pathway is associated with cytokine or growth factor receptors and it is critical for growth control, developmental regulation and homeostasis. The use of porcine ocular cells as putative xenotransplants appears theoretically possible. The aim of this study was to investigate the response of various porcine ocular cells in vitro to human cytokines in regard to the activation of JAK‐STAT signaling pathways.