Impact of chemotherapy on lung cancer cells and their microenvironment, evaluated in tridimensional multicellular tumor spheroids

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Assessment of new HDAC inhibitors for immunotherapy of malignant pleural mesothelioma

International audienceBackground: Malignant pleural mesothelioma (MPM) is a very rare and highly aggressive cancer of the pleura associated in most cases with asbestos exposure. To date, no really efficient treatments are available for this pathology. Recently, it has been shown that epigenetic drugs, particularly DNA methylation or histone acetylation modulating agents, could be very efficient in terms of cytotoxicity for several types of cancer cells. We previously showed that a hypomethylating agent (decitabine) and a histone deacetylase inhibitor (HDACi) (valproic acid (VPA)) combination was immunogenic and led to the induction of an anti-tumor immune response in a mice model of mesothelioma. However, VPA is not very specific, is active at millimolar concentrations and is responsible for side effects in clinic. To improve this approach, we studied four newly synthetized HDACi, two hydroxamates (ODH and NODH) and two benzamides (ODB and NODB), in comparison with VPA and SAHA. We evaluated their toxicity on immune cells and their immunogenicity on MPM cells in combination with decitabine.Results: All the tested HDACi were toxic for immune cells at high concentrations. Combination with decitabine increased toxicity of HDACi only towards T-cell clone. A decrease in the proportion of regulatory T cells and natural killer cells was observed in particular with VPA and ODH. In MPM cells, all HDACi combinations induced NY-ESO-1 cancer testis antigen (CTA) expression and the recognition of the treated cells by a NY-ESO-1 specific T-CD8 clone. However, for MAGE-A1, MAGE-A3 and XAGE-1b mRNA expression, the results obtained depended on the HDACi used and on the CTA studied. Depending on the MPM cell line studied, molecules alone increased moderately PDL1 expression. When combined, a higher stimulation of this immune check point inhibitor expression was observed. Decitabine-induced anti-viral response seemed to be inhibited in the presence of HDACi.Conclusions: This work shows that the combination of decitabine and HDACi could be of interest for MPM immunotherapy. However, this combination induced PD-L1 expression which suggests that an association with anti-PD-L1 therapy should be performed to induce an efficient anti-tumor immune response

Targeting cell-bound MUC1 on myelomonocytic, monocytic leukemias and phenotypically defined leukemic stem cells with anti-SEA module antibodies

International audienceCell surface molecules aberrantly expressed or overexpressed by myeloid leukemic cells represent potential disease-specific therapeutic targets for antibodies. MUC1 is a polymor-phic glycoprotein, the cleavage of which yields two unequal chains: a large extracellular a subunit containing a tandem repeat array bound in a strong noncovalent interaction to a smaller b subunit containing the transmembrane and cytoplasmic domains. Because the a-chain can be released from the cell-bound domains of MUC1, agents directed against the a-chain will not effectively target MUC1 + cells. The MUC1 SEA (a highly conserved protein module so called from its initial identification in a sea urchin sperm protein, in enterokinase, and in agrin) domain formed by the binding of the a and b chains represents a stable structure fixed to the cell surface at all times. DMB-5F3, a partially human-ized murine anti-MUC1 SEA domain monoclonal antibody, was used to examine MUC1 expression in acute myeloid leukemia (AML) and was found to bind acute myelomonocytic and monocytic leukemia (AML-M4 and AML-M5) cell lines. We also examined monocytic neoplasms freshly obtained from patients including chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia, which were found to uniformly express MUC1. CD34 + / lin − /CD38 − or CD38 + presumed leukemic stem cell populations from CD34 + AML and CD34 − CD38 − or CD38 + populations from CD34 − AML were also found to express MUC1, although at low percentages. Based on these studies, we generated an anti-MUC1 immuno-toxin to directly gauge the cytotoxic efficacy of targeting AML-bound MUC1. Using single-chain DMB-5F3 fused to recombinant gelonin toxin, the degree of AML cytotoxicity was found to correlate with MUC1 expression. Our data support the use of an anti−MUC1 SEA module−drug conjugates to selectively target and inhibit MUC1-expressing myelomo-nocytic leukemic cells

IL ‐7 is expressed in malignant mesothelioma and has a prognostic value

International audienceMalignant pleural mesothelioma (MPM) is an aggressive cancer mainly related to asbestos exposure. Despite recent therapeutic advances, notably immunotherapies, the benefit remains limited and restricted to a small percentage of patients. Thus, a better understanding of the disease is needed to identify new therapeutic strategies. Recently, interleukin 7 receptor (IL-7R) has been described as being expressed by MPM cells and associated with poorer patient survival. Thus, the aim of this work was to study the IL-7R/IL-7 pathway in MPM using patient samples. We found that, although more than 40% of MPM cells expressed IL-7R, IL-7 had no effect on their intracellular signaling. Accordingly, the addition of IL-7 to the culture medium did not affect MPM cell growth. Using The Cancer Genome Atlas (TCGA) database, we showed that high IL7 gene expression in MPM tumors was associated with a higher overall patient survival and an induction of genes involved in the immune response. In pleural effusions (PEs), we found that IL-7 concentration was not a good diagnostic biomarker. However, we observed that high IL-7 levels in PEs were associated with shorter survival of MPM patients, but not of lung cancer patients. The prognostic value of IL-7 was also conserved when only patients with epithelioid mesothelioma, the most common histological type of MPM, were analyzed. Taken together, our study suggests that, although the IL-7R/IL-7 signaling pathway is not functional in MPM cells, IL-7 expression in PEs may have prognostic value in MPM patients

Additional file 5: of Assessment of new HDAC inhibitors for immunotherapy of malignant pleural mesothelioma

Figure S2. Effect of decitabine and HDACi, in combination or not, on MPM cell growth. MPM cells were treated with: VPA 5 mM, SAHA 2.5 μM, ODB 7.5 μM, NODB 2.5 μM, ODH 2.5 μM, and NODH 25 nM (48 h) in combination or not with decitabine (5-aza) 500 nM (72 h pretreatment). Viability was measured using Cell Titer Glo kit (Promega). *p < 0.05, **p < 0.01 and ***p < 0.001. (PDF 179 kb

Additional file 9: of Assessment of new HDAC inhibitors for immunotherapy of malignant pleural mesothelioma

Figure S6. HDACi increase decitabine-induced CTA expression in Meso45 cells. Meso45 cells were treated with: VPA 5 mM, SAHA 2.5 μM, ODB 7.5 μM, NODB 2.5 μM, ODH 2.5 μM, and NODH 25 nM (48 h) in combination or not with decitabine (5-aza) 500 nM (72 h pretreatment). NY-ESO-1, MAGE-A1, MAGE-A3, XAGE-1b and PD-L1 mRNA were measured using real time PCR. (PDF 197 kb

Additional file 6: of Assessment of new HDAC inhibitors for immunotherapy of malignant pleural mesothelioma

Figure S3. HDACi increase decitabine-induced CTA expression in Meso96 cells. Meso96 cells were treated with: VPA 5 mM, SAHA 2.5 μM, ODB 7.5 μM, NODB 2.5 μM, ODH 2.5 μM, and NODH 25 nM (48 h) in combination or not with decitabine (5-aza) 500 nM (72 h pretreatment). NY-ESO-1, MAGE-A1, MAGE-A3, XAGE-1b and PD-L1 mRNA were measured using real time PCR. (PDF 199 kb

Additional file 2: of Assessment of new HDAC inhibitors for immunotherapy of malignant pleural mesothelioma

Table S2. Detailed information about the antibodies used in the experiments. (DOCX 16 kb

Additional file 4: of Assessment of new HDAC inhibitors for immunotherapy of malignant pleural mesothelioma

Figure S1. Effect of HDAC inhibitors on naïve and memory T-cells. Lymphocytes obtained by elutriation were treated with HDACi at the following concentrations for 48 h: VPA 5 mM, SAHA 1 μM, ODB 32 μM, NODB 8 μM, ODH 4 μM, and NODH 50 nM. Graphics represent the effect of the compounds on CD4 (a) and CD8 (b) naïve T-cells and on CD4 (c) and CD8 (d) memory T-cells. Results are expressed as the means ± S.E.M of three independent experiments. (PDF 108 kb

Additional file 7: of Assessment of new HDAC inhibitors for immunotherapy of malignant pleural mesothelioma

Figure S4. Expression of HLA ABC in Meso96 treated with decitabine/HDACi combinations. Meso96 were treated with: VPA 5 mM, SAHA 2.5 μM, ODB 7.5 μM, NODB 2.5 μM, ODH 2.5 μM, and NODH 25 nM (48 h) in combination or not with decitabine (5-aza) 500 nM (72 h pretreatment). Then, HLA ABC expression was measured using flow cytometry. Results are expressed as the means ± S.E.M of three independent experiments. (PDF 91 kb