Marc Grégoire

Utility of osteopontin and serum mesothelin in malignant pleural mesothelioma diagnosis and prognosis assessment.

Purpose: Malignant mesothelioma is a highly aggressive tumor and is often diagnosed too late for a curative treatment. We compared diagnostic and prognostic values of mesothelin and osteopontin in 172 patients suspected of malignant pleural mesothelioma (MPM) and in a control group of 112 asymptomatic asbestos-exposed subjects. Experimental Design: Osteopontin and mesothelin were assayed with commercial ELISA kits in a series of 43 patients with pleural metastases of various carcinomas, 33 patients with benign pleural lesions associated with asbestos exposure, 96 patients with MPMs, and 112 asbestos-exposed healthy subjects. Results were correlated with patients diagnosis and survival. Results: Serum osteopontin level was higher in MPM patients compared with healthy asbestos-exposed subjects and had a good capability to distinguish between these two populations. However, osteopontin was unable to distinguish between MPM and pleural metastatic carcinoma or benign pleural lesions associated with asbestos exposure. Neither plasma nor pleural fluid osteopontin were more powerful in this respect. Serum mesothelin had a good ability for diagnosing MPM but was unable to identify patients with nonepithelioid mesothelioma subtypes. Survival analysis identified tumor histologic subtype along with serum osteopontin and serum mesothelin as independent prognostic factors in mesothelioma patients. Conclusions: Osteopontin has a lower diagnostic accuracy than mesothelin in patients suspected of MPM. Insufficient specificity limits osteopontin utility as diagnostic marker. Both molecules have a potential value as prognostic markers.

The role of fibroblasts in tumor behavior

The prominent desmoplastic or stromal reaction seen in many invasive carcinomas suggests that stromal cells play a role in cancer pathogenesis. Investigations based on cell typing, using antibodies to cytoskeletal constituents, have revealed that most tumors contain various types of fibroblasts. Stromal cells with myofibroblastic differentiation features are the predominant cell type at the periphery of epithelial tumors. These tumoractivated fibroblasts play a major role in tumor development and spread, affecting the proliferation, differentiation, invasion or regression of cancer cells. This review considers the events inducing the different fibroblastic responses and the role of tumor-activated fibroblasts in both tumor development and anti-cancer treatments.

IDO expands human CD4+CD25high regulatory T cells by promoting maturation of LPS-treated dendritic cells

We have previously shown that human monocyte‐derived dendritic cells (DC) express indoleamine 2,3‐dioxygenase (IDO), as well as several other enzymes of the kynurenine pathway at the mRNA level upon maturation. The tolerogenic mechanisms of this pathway remain unclear. Here we show that LPS‐treated DC metabolize tryptophan as far as quinolinate. We found that IDO contributes to LPS and TNF‐α + poly(I:C)‐induced DC maturation since IDO inhibition using two different inhibitors impairs DC maturation. IDO knock‐down using short‐hairpin RNA also led to diminished LPS‐induced maturation. In line with these results, the tryptophan‐derived catabolites 3‐hydroxyanthranilic acid and 3‐hydroxykynurenine increased maturation of LPS‐treated DC. Concerning the molecular mechanisms of this effect, IDO acts as an intermediate pathway in LPS‐induced production of reactive oxygen species and NF‐κB activation, two processes that lead to DC maturation. Finally, we show that mature DC expand CD4+CD25high regulatory T cells in an IDO‐dependent manner. In conclusion, we show that IDO constitutes an intermediate pathway in DC maturation leading to expansion of CD4+CD25high regulatory T cells.

Induction of a Caspase-3-like Activity by Calcium in Normal Cytosolic Extracts Triggers Nuclear Apoptosis in a Cell-free System

Calcium is involved in several steps of the apoptotic process. In nuclei, endonucleases are presumed to be the main targets of calcium; however, little is known about its role during the cytosolic phase of apoptosis. We used a cell-free system to address this question. Our results show that CaCl2 triggered nuclear apoptosis (i.e. typical morphological change and DNA fragmentation) at concentrations of 5 mm. This concentration was lowered 10-fold by the co-incubation with cytosolic extracts from nonapoptotic cells. Apoptotic changes induced by the incubation of nuclei with CaCl2 in the presence of these cytosols were strongly reduced in the presence of an inhibitor of caspase-3 and to a lesser extent by an inhibitor of caspase-1. We also show that calcium-induced apoptosis is affected by protease inhibitors such as N-tosyl-l-phenylalanine chloromethyl ketone, but not by calpain or several lysosomal protease inhibitors. The addition of CaCl2 to the cell-free system increased a caspase-3 activity in nonapoptotic cytosols as shown by specific antibodies and an enzymatic assay. No activation of a caspase-3-like activity by the addition of cytochrome c was observed in these extracts under similar conditions. The enhanced caspase-3 activity induced by calcium was inhibited by protease inhibitors affecting morphological nuclear apoptosis except for those responsible for the degradation of lamin A. These results suggest that CaCl2 could trigger, in normal cells, an apoptotic cascade through the activation of cytosolic caspase-3 activity.

Standardized generation of fully mature p70 IL-12 secreting monocyte-derived dendritic cells for clinical use.

Abstract. Dendritic cells (DC) have been shown to be efficient antigen-presenting cells (APC) and, as such, could be considered ideal candidates for cancer immunotherapy. Immature DC (iDC) efficiently capture surrounding antigens; however, only mature DC (mDC) prime naive T lymphocytes. Clinical trials using DC-based tumor vaccines have achieved encouraging, but limited, success, possibly due to the use of immature or incompletely mature DC. Thus, it was apparent that a method capable of generating large numbers of fully functional iDC, their pulsing with desired form of tumor antigens and the subsequent complete and reproducible maturation of iDC is needed. Therefore, we compared two different methods of producing large numbers of iDC. Both protocols yielded comparable numbers of cells with an iDC phenotype with phagocytic function. We next determined which of the clinically applicable activators could induce the complete and reproducible maturation of DC, in order to define the most suitable combination for future clinical trials. Only a combination of TNFα + Poly (I:C), or a previously described cytokine cocktail of TNFα + IL-1β + IL-6 + prostaglandin E2, induced the complete activation of the whole DC population, as assessed by the cell surface expression of CD83 and costimulatory molecules. The matured DC were functionally superior to iDC in their ability to stimulate the proliferation of allogeneic lymphocytes and autologous keyhole limpet hemocyanin (KLH)-specific T lymphocytes. Furthermore, only the combination of TNFα + Poly (I:C) activated DC to produce large amounts of biologically active p70 IL-12. Thus DC maturation by TNFα + Poly (I:C) could efficiently bias T cell response towards Th1 response. Implementation of our results into clinical protocols used for DC generation could be beneficial for future immunotherapy trials.

Carbon Monoxide Inhibits TLR-Induced Dendritic Cell Immunogenicity

Heme oxygenase-1 (HO-1) exerts its functions via the catabolism of heme into carbon monoxide (CO), Fe2+, and biliverdin, as well as by depletion of free heme. We have recently described that overexpression of HO-1 is associated with the tolerogenic capacity to dendritic cells (DCs) stimulated by LPS. In this study, we demonstrate that treatment of human monocyte-derived DCs with CO blocks TLR3 and 4-induced phenotypic maturation, secretion of proinflammatory cytokines, and alloreactive T cell proliferation, while preserving IL-10 production. Treatment of DCs with biliverdin, bilirubin, and deferoxamine or replenishing intracellular heme stores had no effect on DC maturation. HO-1 and CO inhibited LPS-induced activation of the IFN regulatory factor 3 pathway and their effects were independent of p38, ERK, and JNK MAPK. HO-1 and CO treatment also inhibited mouse DC maturation in vitro and mouse DC immunogenic properties in vivo, as shown by adoptive cell transfer in a transgenic model of induced diabetes. Thus, for the first time, our data show that CO treatment inhibits DC immunogenicity induced by TLR ligands and that blockade of IFN regulatory factor 3 is associated with this effect.

Measles Virus Induces Oncolysis of Mesothelioma Cells and Allows Dendritic Cells to Cross-Prime Tumor-Specific CD8 Response

Despite conventional medical and surgical treatments, malignant pleural mesothelioma (MPM) remains incurable. Oncovirotherapy (i.e., the use of replication-competent virus for cancer treatment) is currently explored in clinical trials. In this study, we investigated the antineoplastic potential of a new oncolytic viral agent, a live-attenuated measles virus (MV) strain derived from the Edmonston vaccine lineage (Schwarz strain). We evaluated both oncolytic activity and immunoadjuvant properties of the MV vaccine strain on mesothelioma tumor cells. Infectivity, syncytium formation, and cytolytic activity of MV were studied on a panel of mesothelioma cells derived from pleural effusions of MPM patients. We observed that MV infected preferentially MPM cell lines in comparison with nontransformed mesothelial cells, leading to an efficient killing of a significant fraction of tumor cells. A cytoreductive activity was also evidenced through formation of multinuclear cellular aggregates (syncytia). The susceptibility of MPM cell lines to measles infection was assessed by the analysis of cell surface expression of the MV vaccine receptor (CD46). We also evaluated whether MV infection of mesothelioma cells could elicit an autologous antitumor immune response. We showed that MV Schwarz strain induced apoptotic cell death of infected mesothelioma cells, which were efficiently phagocytosed by dendritic cells (DC). Loading of DCs with MV-infected MPM cells induced DC spontaneous maturation, as evidenced by the increased expression of MHC and costimulatory molecules along with the production of proinflammatory cytokines. Priming of autologous T cells by DCs loaded with MV-infected MPM cells led to a significant proliferation of tumor-specific CD8 T cells. Altogether, these data strongly support the potential of oncolytic MV as an efficient therapeutic agent for mesothelioma cancer.

Immunomodulatory effects of tumor-associated fibroblasts in colorectal-tumor development

In order to elucidate the role of myofibroblasts in tumor development, we compared fibroblastic reactions and their implications in the immune response in progressive and regressive rat colorectal‐tumor models. Immunohistochemical analyses revealed that T lymphocytes and monocytes/macrophages were found outside progressive tumors that were surrounded by a large sheath of myofibroblasts. In vitro experiments using fibroblast‐ vs. myofibroblast‐containing collagen gels showed that the mechanical properties of these tumor‐activated myofibroblasts prevent penetration of T lymphocytes and macrophages within tumor nodules. These results indicate that tumor‐activated myofibroblasts may prevent physical contact between cancer‐cells and immune cells, an essential phenomenon for effective destruction of cancer cells. Successful immunotherapy against cancer should therefore include complementary treatments against these tumor‐associated fibroblasts. Int. J. Cancer 81:629–636, 1999.

Measles Virus Vaccine–Infected Tumor Cells Induce Tumor Antigen Cross-Presentation by Human Plasmacytoid Dendritic Cells

Purpose: Plasmacytoid dendritic cells (pDC) are antigen-presenting cells specialized in antiviral response. The measles virus vaccine is proposed as an antitumor agent to target and specifically kill tumor cells without infecting healthy cells. Experimental Design: Here, we investigated, in vitro, the effects of measles virus vaccine–infected tumor cells on the phenotype and functions of human pDC. We studied maturation and tumor antigen cross-presentation by pDC, exposed either to the virus alone, or to measles virus vaccine–infected or UV-irradiated tumor cells. Results: We found that only measles virus vaccine–infected cells induced pDC maturation with a strong production of IFN-α, whereas UV-irradiated tumor cells were unable to activate pDC. This IFN-α production was triggered by the interaction of measles virus vaccine single-stranded RNA (ssRNA) with TLR7. We observed that measles virus vaccine–infected tumor cells were phagocytosed by pDC. Interestingly, we showed cross-presentation of the tumor antigen NYESO-1 to a specific CD8+ T-cell clone when pDC were cocultured with measles virus vaccine–infected tumor cells, whereas pDC were unable to cross-present NYESO-1 after coculture with UV-irradiated tumor cells. Conclusions: Altogether, our results suggest that the use of measles virus vaccine in antitumor virotherapy induces immunogenic tumor cell death, allowing pDC to mature, produce high amounts of IFN-α, and cross-present tumor antigen, thus representing a mode of recruiting these antigen-presenting cells in the immune response. Clin Cancer Res; 19(5); 1147–58. ©2012 AACR.

Inducible Hsp70 as target of anticancer immunotherapy: Identification of HLA-A*0201-restricted epitopes

The design of a broad application tumor vaccine requires the identification of tumor antigens expressed in a majority of tumors of various origins. We questioned whether the major stress‐inducible heat shock protein Hsp70 (also known as Hsp72), a protein frequently overexpressed in human tumors of various histological origins, but not in most physiological normal tissues, constitutes a tumor antigen. We selected the p391 and p393 peptides from the sequence of the human inducible Hsp70 that had a high affinity for HLA‐A*0201. These peptides were able to trigger a CTL response in vivo in HLA‐A*0201‐transgenic HHD mice and in vitro in HLA‐A*0201+ healthy donors. p391‐ and p393‐specific human and murine CTL recognized human tumor cells overexpressing Hsp70 in a HLA‐A*0201‐restricted manner. Tetramer analysis of TILs showed that these Hsp70 epitopes are targets of an immune response in many HLA‐A*0201+ breast cancer patients. Hsp70 is a tumor antigen and the Hsp70‐derived peptides p391 and p393 could be used to raise a cytotoxic response against tumors of various origins.