Use of non-small cell lung cancer multicellular tumor spheroids to study the impact of chemotherapy.

BACKGROUND: Lung cancers represent the main cause of cancer related-death worldwide. Recently, immunotherapy alone or in combination with chemotherapy has deeply impacted the therapeutic care leading to an improved overall survival. However, relapse will finally occur, with no efficient second line treatment so far. New therapies development based on the comprehension of resistance mechanisms is necessary. However, the difficulties to obtain tumor samples before and after first line treatment hamper to clearly understand the consequence of these molecules on tumor cells and also to identify adapted second line therapies. METHODS: To overcome this difficulty, we developed multicellular tumor spheroids (MCTS) using characterized Non-Small Cell Lung Cancer (NSCLC) cell lines, monocytes from healthy donors and fibroblasts. MCTS were treated with carboplatin-paclitaxel or -gemcitabine combinations according to clinical administration schedules. The treatments impact was studied using cell viability assay, histological analyses, 3'RNA sequencing, real-time PCR, flow cytometry and confocal microscopy. RESULTS: We showed that treatments induced a decrease in cell viability and strong modifications in the transcriptomic profile notably at the level of pathways involved in DNA damage repair and cell cycle. Interestingly, we also observed a modification of genes expression considered as hallmarks of response to immune check point inhibitors and immunogenicity, particularly an increase in CD274 gene expression, coding for PD-L1. This result was validated at the protein level and shown to be restricted to tumor cells on MCTS containing fibroblasts and macrophages. This increase was also observed in an additional cell line, expressing low basal CD274 level. CONCLUSIONS: This study shows that MCTS are interesting models to study the impact of first line therapies using conditions close to clinical practice and also to identify more adapted second line or concomitant therapies for lung cancer treatment.

Oncolytic attenuated measles virus encoding NY-ESO-1 induces HLA I and II presentation of this tumor antigen by melanoma and dendritic cells.

Antitumor virotherapy stimulates the antitumor immune response during tumor cell lysis induced by oncolytic viruses (OVs). OV can be modified to express additional transgenes that enhance their therapeutic potential. In this study, we armed the spontaneously oncolytic Schwarz strain of measles viruses (MVs) with the gene encoding the cancer/testis antigen NY-ESO-1 to obtain MVny. We compared MV and MVny oncolytic activity and ability to induce NY-ESO-1 expression in six human melanoma cell lines. After MVny infection, we measured the capacity of melanoma cells to present NY-ESO-1 peptides to CD4 + and CD8 + T cell clones specific for this antigen. We assessed the ability of MVny to induce NY-ESO-1 expression and presentation in monocyte-derived dendritic cells (DCs). Our results show that MVny and MV oncolytic activity are similar with a faster cell lysis induced by MVny. We also observed that melanoma cell lines and DC expressed the NY-ESO-1 protein after MVny infection. In addition, MVny-infected melanoma cells and DCs were able to stimulate NY-ESO-1-specific CD4 + and CD8 + T cells. Finally, MVny was able to induce DC maturation. Altogether, these results show that MVny could be an interesting candidate to stimulate NY-ESO-1-specific T cells in melanoma patients with NY-ESO-1-expressing tumor cells.

TP53 mutations correlate with the non-coding RNA content of small extracellular vesicles in melanoma.

Non-coding RNAs (ncRNAs) are important regulators of gene expression. They are expressed not only in cells, but also in cell-derived extracellular vesicles (EVs). The mechanisms controlling their loading and sorting remain poorly understood. Here, we investigated the impact of TP53 mutations on the non-coding RNA content of small melanoma EVs. After purification of small EVs from six different patient-derived melanoma cell lines, we characterized them by small RNA sequencing and lncRNA microarray analysis. We found that TP53 mutations are associated with a specific micro and long non-coding RNA content in small EVs. Then, we showed that long and small non-coding RNAs enriched in TP53 mutant small EVs share a common sequence motif, highly similar to the RNA-binding motif of Sam68, a protein interacting with hnRNP proteins. This protein thus may be an interesting partner of p53, involved in the expression and loading of the ncRNAs. To conclude, our data support the existence of cellular mechanisms associate with TP53 mutations which control the ncRNA content of small EVs in melanoma.

Viral Mimicry Response Is Associated With Clinical Outcome in Pleural Mesothelioma.

Introduction: The aim of this study was to investigate endogenous retrovirus (ERV) expression and type I interferon (IFN) activation in human pleural mesothelioma (PM) and their association with clinical outcome. Methods: The expression of ERV was determined from PM cohorts and mesothelial precursor RNA sequencing data. The expression of ERV was confirmed by quantitative polymerase chain reaction (qPCR). Methylation of genomic DNA was assessed by quantitative methylation-specific PCR. DNA demethylation was induced in cells by demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-CdR) treatment. To block type I IFN signaling, the cells were treated with ruxolitinib or MAVS silencing. The expression of IFN-stimulated genes (ISGs) was determined by qPCR and Western blot. Circulating ERVs were detected by qPCR. Results: Long terminal repeats (LTRs) represent the most abundant transposable elements up-regulated in PM. Within the LTR, ERVmap_1248 and LTR7Y, which are specifically enriched in PM, were further analyzed. The 5-Aza-CdR treatment increased the levels of ERVmap_1248 expression and induced ERVmap_1248 promoter demethylation in mesothelial cells. In addition, ERVmap_1248 promoter was more demethylated in the mesothelioma tissue compared with nontumor tissue. The 5-Aza-CdR treatment of the mesothelial cells also increased the levels of ISGs. Basal ISG expression was higher in the mesothelioma cells compared with the mesothelial cells, and it was significantly decreased by ruxolitinib treatment or MAVS silencing. Furthermore, ISG expression was higher in the tumor tissue with high expression levels of ERVmap_1248. High expression of ERVmap_1248 was associated with longer overall survival and BAP1 mutations. ERVmap_1248 and LTR7Y can be detected in the PM plasma. Conclusions: We provide clues for patient stratification especially for immunotherapy where best clinical responses are associated with an activated basal immune response.

Heterogeneous RNA editing and influence of ADAR2 on mesothelioma chemoresistance and the tumor microenvironment.

We previously observed increased levels of adenosine-deaminase-acting-on-dsRNA (Adar)-dependent RNA editing during mesothelioma development in mice exposed to asbestos. The aim of this study was to characterize and assess the role of ADAR-dependent RNA editing in mesothelioma. We found that tumors and mesothelioma primary cultures have higher ADAR-mediated RNA editing compared to mesothelial cells. Unsupervised clustering of editing in different genomic regions revealed heterogeneity between tumor samples as well as mesothelioma primary cultures. ADAR2 expression levels are higher in BRCA1-associated protein 1 wild-type tumors, with corresponding changes in RNA editing in transcripts and 3'UTR. ADAR2 knockdown and rescue models indicated a role in cell proliferation, altered cell cycle, increased sensitivity to antifolate treatment, and type-1 interferon signaling upregulation, leading to changes in the microenvironment in vivo. Our data indicate that RNA editing contributes to mesothelioma heterogeneity and highlights an important role of ADAR2 not only in growth regulation in mesothelioma but also in chemotherapy response, in addition to regulating inflammatory response downstream of sensing nucleic acid structures.

The DCMU Herbicide Shapes T-cell Functions By Modulating Micro-RNA Expression Profiles.

DCMU [N-(3,4-dichlorophenyl)-N-dimethylurea] or diuron is a widely used herbicide, which can cause adverse effects on human, especially on immune cells, due to their intrinsic properties and wide distribution. These cells are important for fighting not only against virus or bacteria but also against neoplastic cell development. We developed an approach that combines functional studies and miRNA and RNA sequencing data to evaluate the effects of DCMU on the human immune response against cancer, particularly the one carried out by CD8+ T cells. We found that DCMU modulates the expression of miRNA in a dose-dependent manner, leading to a specific pattern of gene expression and consequently to a diminished cytokine and granzyme B secretions. Using mimics or anti-miRs, we identified several miRNA, such as hsa-miR-3135b and hsa-miR-21-5p, that regulate these secretions. All these changes reduce the CD8+ T cells' cytotoxic activity directed against cancer cells, in vitro and in vivo in a zebrafish model. To conclude, our study suggests that DCMU reduces T-cell abilities, participating thus to the establishment of an environment conducive to cancer development.

Human MuStem cells repress T-cell proliferation and cytotoxicity through both paracrine and contact-dependent pathways.

BACKGROUND: Muscular dystrophies (MDs) are inherited diseases in which a dysregulation of the immune response exacerbates disease severity and are characterized by infiltration of various immune cell types leading to muscle inflammation, fiber necrosis and fibrosis. Immunosuppressive properties have been attributed to mesenchymal stem cells (MSCs) that regulate the phenotype and function of different immune cells. However, such properties were poorly considered until now for adult stem cells with myogenic potential and advanced as possible therapeutic candidates for MDs. In the present study, we investigated the immunoregulatory potential of human MuStem (hMuStem) cells, for which we previously demonstrated that they can survive in injured muscle and robustly counteract adverse tissue remodeling. METHODS: The impact of hMuStem cells or their secretome on the proliferative and phenotypic properties of T-cells was explored by co-culture experiments with either peripheral blood mononucleated cells or CD3-sorted T-cells. A comparative study was produced with the bone marrow (BM)-MSCs. The expression profile of immune cell-related markers on hMuStem cells was determined by flow cytometry while their secretory profile was examined by ELISA assays. Finally, the paracrine and cell contact-dependent effects of hMuStem cells on the T-cell-mediated cytotoxic response were analyzed through IFN-γ expression and lysis activity. RESULTS: Here, we show that hMuStem cells have an immunosuppressive phenotype and can inhibit the proliferation and the cytotoxic response of T-cells as well as promote the generation of regulatory T-cells through direct contact and via soluble factors. These effects are associated, in part, with the production of mediators including heme-oxygenase-1, leukemia inhibitory factor and intracellular cell adhesion molecule-1, all of which are produced at significantly higher levels by hMuStem cells than BM-MSCs. While the production of prostaglandin E2 is involved in the suppression of T-cell proliferation by both hMuStem cells and BM-MSCs, the participation of inducible nitric oxide synthase activity appears to be specific to hMuStem cell-mediated one. CONCLUSIONS: Together, our findings demonstrate that hMuStem cells are potent immunoregulatory cells. Combined with their myogenic potential, the attribution of these properties reinforces the positioning of hMuStem cells as candidate therapeutic agents for the treatment of MDs.

Homozygous Co-Deletion of Type I Interferons and CDKN2A Genes in Thoracic Cancers: Potential Consequences for Therapy.

Homozygous deletion (HD) of the tumor suppressor gene CDKN2A is the most frequent genetic alteration in malignant pleural mesothelioma and is also frequent in non-small cell lung cancers. This HD is often accompanied by the HD of the type I interferons (IFN I) genes that are located closed to the CDKN2A gene on the p21.3 region of chromosome 9. IFN I genes encode sixteen cytokines (IFN-α, IFN-߅) that are implicated in cellular antiviral and antitumor defense and in the induction of the immune response. In this review, we discuss the potential influence of IFN I genes HD on thoracic cancers therapy and speak in favor of better taking these HD into account in patients monitoring.

Endogenous retrovirus expression activates type-I interferon signaling in an experimental mouse model of mesothelioma development.

Early events in an experimental model of mesothelioma development include increased levels of editing in double-stranded RNA (dsRNA). We hypothesised that expression of endogenous retroviruses (ERV) contributes to dsRNA formation and type-I interferon signaling. ERV and interferon stimulated genes (ISGs) expression were significantly higher in tumor compared to non-tumor samples. 12 tumor specific ERV ("MesoERV1-12") were identified and verified by qPCR in mouse tissues. "MesoERV1-12" expression was lower in mouse embryonic fibroblasts (MEF) compared to mesothelioma cells. "MesoERV1-12" levels were significantly increased by demethylating agent 5-Aza-2'-deoxycytidine treatment and were accompanied by increased levels of dsRNA and ISGs. Basal ISGs expression was higher in mesothelioma cells compared to MEF and was significantly decreased by JAK inhibitor Ruxolitinib, by blocking Ifnar1 and by silencing Mavs. "MesoERV7" promoter was demethylated in asbestos-exposed compared to sham mice tissue as well as in mesothelioma cells and MEF upon 5-Aza-CdR treatment. These observations uncover novel aspects of asbestos-induced mesothelioma whereby ERV expression increases due to promoter demethylation and is paralleled by increased levels of dsRNA and activation of type-I IFN signaling. These features are important for early diagnosis and therapy.

High Oncolytic Activity of a Double-Deleted Vaccinia Virus Copenhagen Strain against Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is a cancer of the pleura that lacks efficient treatment. Oncolytic immunotherapy using oncolytic vaccinia virus (VV) may represent an alternative therapeutic approach for the treatment of this malignancy. Here, we studied the oncolytic activity of VV thymidine kinase (TK)-ribonucleotide reductase (RR)-/green fluorescent protein (GFP) against MPM. This virus is a VV from the Copenhagen strain that is deleted of two genes encoding the TK (J2R) and the RR (I4L) and that express the GFP. First, we show in vitro that VVTK-RR-/GFP efficiently infects and kills the twenty-two human MPM cell lines used in this study. We also show that the virus replicates in all eight tested MPM cell lines, however, with approximately a 10-fold difference in the amplification level from one cell line to another. Then, we studied the therapeutic efficiency of VVTK-RR-/GFP in non-obese diabetic (NOD) severe combined immunodeficient (SCID) mice that bear peritoneal human MPM tumors. One intraperitoneal infection of VVTK-RR-/GFP reduces the tumor burden and significantly increases mice survival compared to untreated animals. Thus, VVTK-RR - may be a promising oncolytic virus (OV) for the oncolytic immunotherapy of MPM.

Involvement of the M-CSF/IL-34/CSF-1R pathway in malignant pleural mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a rare and aggressive cancer related to asbestos exposure. The tumor microenvironment content, particularly the presence of macrophages, was described as crucial for the development of the disease. This work aimed at studying the involvement of the M-CSF (CSF-1)/IL-34/CSF-1R pathway in the formation of macrophages in MPM, using samples from patients. METHODS: Pleural effusions (PEs), frozen tumors, primary MPM cells and MPM cell lines used in this study belong to biocollections associated with clinical databases. Cytokine expressions were studied using real-time PCR and ELISA. The Cancer Genome Atlas database was used to confirm our results on an independent cohort. An original three-dimensional (3D) coculture model including MPM cells, monocytes from healthy donors and a tumor antigen-specific cytotoxic CD8 T cell clone was used. RESULTS: We observed that high interleukin (IL)-34 levels in PE were significantly associated with a shorter survival of patients. In tumors, expression of CSF1 was correlated with 'M2-like macrophages' markers, whereas this was not the case with IL34 expression, suggesting two distinct modes of action of these cytokines. Expression of IL34 was higher in MPM cells compared with primary mesothelial cells. Particularly, high expression of IL34 was observed in MPM cells with an alteration of CDKN2A. Finally, using 3D coculture model, we demonstrated the direct involvement of MPM cells in the formation of immunosuppressive macrophages, through activation of the colony stimulating factor-1 receptor (CSF1-R) pathway, causing the inhibition of cytotoxicity of tumor antigen-specific CD8+ T cells. CONCLUSIONS: The M-CSF/IL-34/CSF-1R pathway seems strongly implicated in MPM and could constitute a therapeutic target to act on immunosuppression and to support immunotherapeutic strategies.

Frequent Homozygous Deletions of Type I Interferon Genes in Pleural Mesothelioma Confer Sensitivity to Oncolytic Measles Virus.

INTRODUCTION: Oncolytic immunotherapy is based on the use of nonpathogenic replicative oncolytic viruses that infect and kill tumor cells exclusively. Recently, we found that the spontaneous oncolytic activity of the Schwarz strain of measles virus (MV) against human malignant pleural mesothelioma (MPM) depends on defects in the antiviral type I interferon (IFN-I) response in tumor cells. METHODS: In this study, we studied three independent human MPM bio-collections to identify the defects in the IFN-I responses in tumor cells. RESULTS: We show that the most frequent defect is the homozygous deletions (HDs) of all the 14 IFN-I genes (IFN-α and IFN-ß) that we found in more than half of MV-sensitive MPM cell lines. These HDs occur together with the HDs of the tumor suppressor gene CDKN2A also located in the 9p21.3 chromosome region. Therefore, the IFN-I-/- MPM cell lines develop a partial and weak IFN-I response when they are exposed to the virus compared with that of normal cells and MV-resistant MPM cell lines. This response consists of the expression of a restricted number of IFN-stimulated genes that do not depend on the presence of IFN-I. In addition, the IFN-I-/- MPM cell lines infected by MV also develop a pro-inflammatory response associated with stress of the endoplasmic reticulum. CONCLUSION: Our study emphasizes the link between HDs of IFN-I encoding genes and the CDKN2A gene in MPM and sensitivity to MV oncolytic immunotherapy.

Cisplatin unleashes Toll-like receptor 3-mediated apoptosis through the downregulation of c-FLIP in malignant mesothelioma.

Toll-like receptor 3 (TLR3) is an immune receptor that behaves like a death receptor in tumor cells, thereby providing an original target for cancer therapy. The therapeutic potential of TLR3 targeting in malignant mesothelioma, an aggressive and incurable neoplasia of the pleura and peritoneum, has so far not been addressed. We investigated TLR3 expression and sensitivity of human mesothelioma cell lines to the synthetic dsRNA Poly(I:C), alone or in combination with cisplatin, the gold standard chemotherapy in mesothelioma. Activation of TLR3 by Poly(I:C) induced apoptosis of 4/8 TLR3-positive cell lines but not of TLR3-negative cell lines. The combined cisplatin/Poly(I:C) treatment enhanced apoptosis of 3/4 Poly(I:C)-sensitive cell lines and overcame resistance to Poly(I:C) or cisplatin alone in 2/4 cell lines. Efficacy of the combined treatment relied on cisplatin-induced downregulation of c-FLIP, the main regulator of the extrinsic apoptotic pathway, leading to an enhanced caspase-8-mediated pathway. Of note, 6/6 primary cell samples isolated from patients with peritoneal mesothelioma expressed TLR3. Patient-derived cells were sensitive to Poly(I:C) alone while the combined cisplatin/Poly(I:C) treatment induced dramatic cell death. Our findings demonstrate that TLR3 targeting in combination with cisplatin presents an innovative therapeutic strategy in mesothelioma.

MicroRNAs in Tumor Exosomes Drive Immune Escape in Melanoma.

MicroRNAs (miRNA), small noncoding RNAs that regulate gene expression, exist not only in cells but also in a variety of body fluids. These circulating miRNAs could enable intercellular communication. miRNAs are packaged in membrane-encapsulated vesicles, such as exosomes, or protected by RNA-binding proteins. Here, we report that miRNAs included in human melanoma exosomes regulate the tumor immune response. Using microscopy and flow cytometry, we demonstrate that CD8+ T cells internalize exosomes from different tumor types even if these cells do not internalize vesicles as readily as other immune cells. We explored the function of melanoma-derived exosomes in CD8+ T cells and showed that these exosomes downregulate T-cell responses through decreased T-cell receptor (TCR) signaling and diminished cytokine and granzyme B secretions. The result reduces the cells' cytotoxic activity. Using mimics, we found that miRNAs enriched in exosomes-such as Homo sapiens (hsa)-miR-3187-3p, hsa-miR-498, hsa-miR-122, hsa-miR149, and hsa-miR-181a/b-regulate TCR signaling and TNFα secretion. Our observations suggest that miRNAs in melanoma-derived exosomes aid tumor immune evasion and could be a therapeutic target.

A Functional Assay to Determine the Capacity of Oncolytic Viruses to Induce Immunogenic Tumor Cell Death.

Oncolytic immunotherapy efficacy relies partially on the induction of immunogenic tumor cell death following infection with oncolytic viruses (OV) to induce an antitumor immune response. Here, we describe a method to determine if an OV is able to induce such an immunogenic tumor cell death. This method consists in testing whether tumor cells lysed by an OV are able to induce the maturation of human monocyte-derived immature dendritic cells (Mo-iDC).

p53 regulates CD46 expression and measles virus infection in myeloma cells.

In this study, we assessed the sensitivity of myeloma cells to the oncolytic measles virus (MV) in relation to p53 using 37 cell lines and 23 primary samples. We showed that infection and cell death were correlated with CD46 expression, which was associated with TP53 status; TP53 abn cell lines highly expressed CD46 and were preferentially infected by MV when compared with the TP53 wt cell lines (P = .046 and P = .045, respectively). Infection of myeloma cells was fully dependent on CD46 expression in both cell lines and primary cells. In the TP53 wt cell lines, but not the TP53 abn cell lines, activation of the p53 pathway with nutlin3a inhibited both CD46 expression and MV infection, while TP53 silencing reciprocally increased CD46 expression and MV infection. We showed using a p53 chromatin immunoprecipitation assay and microRNA assessment that CD46 gene expression was directly and indirectly regulated by p53. Primary myeloma cells overexpressed CD46 as compared with normal cells and were highly infected and killed by MV. CD46 expression and MV infection were inhibited by nutlin3a in primary p53-competent myeloma cells, but not in p53-deficient myeloma cells, and the latter were highly sensitive to MV infection. In summary, myeloma cells were highly sensitive to MV and infection inhibition by the p53 pathway was abrogated in p53-deficient myeloma cells. These results argue for an MV-based clinical trial for patients with p53 deficiency.

Dysfunction of HPV16-specific CD8+ T cells derived from oropharyngeal tumors is related to the expression of Tim-3 but not PD-1.

BACKGROUND: Human papillomavirus (HPV) type 16 infection is one of the most important etiological agents of oropharyngeal squamous cell carcinoma. Patients with HPV-associated carcinomas of the head and neck were reported to have a better clinical outcome than patients with HPV-negative tumors. Because HPV16 E6 and E7 oncoproteins are highly immunogenic and constitutively expressed, HPV-specific T cell immunity may play the key role in improving the prognosis of these patients. METHODS: Tumor-derived T cells were expanded in high levels of IL-2 and stimulated with HPV16 E6/E7 peptides in the presence or absence of anti-PD-1 monoclonal antibody nivolumab and soluble Tim-3. RESULTS: HPV16-specific tumor-infiltrating T cells were present in 73.1% of HPV-associated oropharyngeal tumors. HPV16 specific CD8+ TILs were able to produce IFNγ upon specific stimulation and predominantly expressed PD-1 but not Tim-3. Specific IFNγ production was further enhanced after a blockade of both PD-1 and Tim-3 pathways but not after a PD-1 blockade alone. Additionally, the specific stimulation of anti-HPV16 CD8+ T cells suppressed Tim-3 upregulation after the PD-1 blockade. CONCLUSION: Our data provide the rationale for combination cancer immunotherapy approaches, including the dual blockade of PD-1 and Tim-3 and, potentially, the use of HPV16-directed therapeutic vaccines.

Oncolytic viruses sensitize human tumor cells for NY-ESO-1 tumor antigen recognition by CD4+ effector T cells.

Oncolytic immunotherapy using oncolytic viruses (OV) has been shown to stimulate the antitumor immune response by inducing the release of tumor-associated antigens (TAA) and danger signals from the dying infected tumor cells. In this study, we sought to determine if the lysis of tumor cells induced by different OV: measles virus, vaccinia virus, vesicular stomatitis virus, herpes simplex type I virus, adenovirus or enterovirus, has consequences on the capacity of tumor cells to present TAA, such as NY-ESO-1. We show that the co-culture of NY-ESO-1neg/HLA-DP4pos melanoma cells with NY-ESO-1pos/HLA-DP4neg melanoma cells infected and killed by different OV induces an intercellular transfer of NY-ESO-1 that allows the recognition of NY-ESO-1neg/HLA-DP4pos tumor cells by an HLA-DP4/NY-ESO-1(157-170)-specific CD4+ cytotoxic T cell clone, NY67. We then confirmed this result in a second model with an HLA-DP4+ melanoma cell line that expresses a low amount of NY-ESO-1. Recognition of this cell line by the NY67 clone is largely increased in the presence of OV productive infection. Altogether, our results show for the first time another mechanism of stimulation of the anti-tumor immune response by OV, via the loading of tumor cells with TAA that sensitizes them for direct recognition by specific effector CD4+ T cells, supporting the use of OV for cancer immunotherapy.