Repeated systemic administrations of both aminobisphosphonates and human Vγ9Vδ2 T cells efficiently control tumor development in vivo.

Peripheral Vγ9Vδ2 T lymphocytes compose a major γδ T cell subset in primates with broad reactivity against tumor cells. Vγ9Vδ2 T cells are specifically activated by phosphorylated isoprenoid pathway metabolites called "phosphoagonists." Accordingly, pharmacologic inhibitors of the mevalonate pathway, such as aminobisphosphonates (NBP) that upregulate the intracellular production of phosphoagonists, increase antitumor Vγ9Vδ2 T cell responses. Immunotherapeutic protocols exploiting GMP-grade agonist molecules targeting human Vγ9Vδ2 T lymphocytes have yielded promising, yet limited, signs of antitumor efficacy and therefore need to be improved for next-generation immunotherapies. In this study, we used a model of s.c. human tumor xenografts in severely immunodeficient mice to assess the antitumor efficacy of systemic NBP treatments when combined with the adoptive transfer of human Vγ9Vδ2 T cells. We show that infusion of Vγ9Vδ2 T cells, 24 h after systemic NBP treatment, efficiently delays tumor growth in mice. Importantly, our results indicate efficient but transient in vivo NBP-induced sensitization of tumor cells to human Vγ9Vδ2-T cell recognition. Accordingly, repeated and combined administrations of both NBP and γδ T cells yielded improved antitumor responses in vivo. Because Vγ9Vδ2 T cells show similar responsiveness toward both autologous and allogeneic tumors and are devoid of alloreactivity, these results provide preclinical proof of concept for optimized antitumor immunotherapies combining NBP treatment and adoptive transfer of allogeneic human γδ T cells.

Autologous fat grafting: A comparative study of four current commercial protocols.

BACKGROUND: Autologous fat grafting is a widely used technique that gives natural results when treating soft tissue deficiencies. However, there is no consensus on which is the best procedure to use, leading to unpredictable results because of fat graft resorption. OBJECTIVES: This study compared four commercial lipotransfer devices by analyzing the behavior of the processed adipose tissue and outcome of the adipose graft in an in vivo model. METHODS: Four different protocols that used manual, power-assisted or water-assisted lipoaspiration and then decantation, centrifugation, or filtration were used on each of eight patients to process lipoaspirate. Harvested adipose tissue was assessed in vitro for tissue resorption, oil formation, and cytokine secretion. Graft resorption rate was calculated and histological analyses were performed after subcutaneously injecting the harvested adipose tissue in a murine model. RESULTS: All protocols resulted in very low oil formation and histologically healthy grafts. The tissue volume was significantly greater after 2 days in culture when using manual lipoaspiration and soft centrifugations/washing steps (Microfill®/Macrofill®) compared to Water-Assisted Lipoaspiration/Decantation (BodyJet®) and Power-Assisted Lipoaspiration/Filtration (PAL® + PureGraft®). These results were confirmed in mice 1 month after subcutaneous injection, with greater efficiency obtained with protocols that used (A) manual aspiration, (B) soft centrifugations, and (C) washing steps. CONCLUSIONS: We confirmed that the choice of technique used to process adipose tissue during lipotransfer surgery can highly influence fat grafting efficacy. In our study, the use of manual aspiration combined with soft centrifugations led to the best results in the selected models.

Evaluation of intracavitary administration of curcumin for the treatment of sarcomatoid mesothelioma.

A rat model of sarcomatoid mesothelioma, mimicking some of the worst clinical conditions encountered, was established to evaluate the therapeutic potential of intracavitary curcumin administration. The M5-T1 cell line, selected from a collection established from F344 rats induced with asbestos, produces tumors within three weeks, with extended metastasis in normal tissues, after intraperitoneal inoculation in syngeneic rats. The optimal concentration/time conditions for killing M5-T1 cells with curcumin were first determined in vitro. Secondly, the potential of intraperitoneal curcumin administration to kill tumor cells in vivo was evaluated in tumor-bearing rats, in comparison with a reference epigenetic drug, SAHA. Both agents administered at days 21 and 26 after tumor challenge produced necrosis within the solid tumors at day 28. However, tumor tissue necrosis induced with curcumin was much more extensive than with SAHA, and was characterized by infiltration with mononuclear phagocytic cells. In contrast, tumor tissue treated with SAHA contained foci of resistant cells and was infiltrated by many isolated CD8+ cells. The treatment of tumor-bearing rats with 1.5 mg/kg curcumin on days 7, 9, 11 and 14 after tumor challenge dramatically reduced the mean total tumor mass at day 16. Clusters of CD8+ T lymphocytes were observed at the periphery of small residual tumor masses in the peritoneal cavity, which presented a significant reduction in mitotic index, IL6 and vimentin expression compared with tumors in untreated rats. These data open up interesting new prospects for the therapy of sarcomatoid mesothelioma with curcumin and its derivatives.

Stereotaxic administrations of allogeneic human Vγ9Vδ2 T cells efficiently control the development of human glioblastoma brain tumors.

Glioblastoma multiforme (GBM) represents the most frequent and deadliest primary brain tumor. Aggressive treatment still fails to eliminate deep brain infiltrative and highly resistant tumor cells. Human Vγ9Vδ2 T cells, the major peripheral blood γδ T cell subset, react against a wide array of tumor cells and represent attractive immune effector T cells for the design of antitumor therapies. This study aims at providing a preclinical rationale for immunotherapies in GBM based on stereotaxic administration of allogeneic human Vγ9Vδ2 T cells. The feasibility and the antitumor efficacy of stereotaxic Vγ9Vδ2 T cell injections have been investigated in orthotopic GBM mice model using selected heterogeneous and invasive primary human GBM cells. Allogeneic human Vγ9Vδ2 T cells survive and patrol for several days within the brain parenchyma following adoptive transfer and can successfully eliminate infiltrative GBM primary cells. These striking observations pave the way for optimized stereotaxic antitumor immunotherapies targeting human allogeneic Vγ9Vδ2 T cells in GBM patients.

Characterization of preneoplastic and neoplastic rat mesothelial cell lines: the involvement of TETs, DNMTs, and 5-hydroxymethylcytosine.

Malignant mesothelioma (MM) is one of the worst cancers in terms of clinical outcome, urging the need to establish and characterize new preclinical tools for investigation of the tumorigenic process, improvement of early diagnosis and evaluation of new therapeutic strategies. For these purposes, we characterized a collection of 27 cell lines established from F344 rats, after 136 to 415 days of induction with crocidolite asbestos administered intraperitoneally. Four mesotheliomas were distinguished from 23 preneoplastic mesothelial cell lines (PN) according to their propensity to generate tumors after orthotopic transplantation into syngeneic rats, their growth pattern, and the expression profile of three genes. PN cell lines were further discriminated into groups / subgroups according to morphology in culture and the expression profiles of 14 additional genes. This approach was completed by analysis of positive and negative immunohistochemical MM markers in the four tumors, of karyotype alterations in the most aggressive MM cell line in comparison with a PN epithelioid cell line, and of human normal mesothelial and mesothelioma cells and a tissue array. Our results showed that both the rat and human MM cell lines shared in common a dramatic decrease in the relative expression of Cdkn2a and of epigenetic regulators, in comparison with PN and normal human mesothelial cells, respectively. In particular, we identified the involvement of the relative expression of the Ten-Eleven Translocation (TET) family of dioxygenases and Dnmt3a in relation to the 5-hydroxymethylcytosine level in malignant transformation and the acquisition of metastatic potential.

Highly cohesive dual nanoassemblies for complementary multiscale bioimaging.

Innovative nanostructures made of a high payload of fluorophores and superparamagnetic nanoparticles (NPs) have simply been fabricated upon self-assembling in a two-step process. The resulting hybrid supraparticles displayed a dense shell of iron oxide nanoparticles tightly attached through an appropriate polyelectrolyte to a highly emissive non-doped nanocore made of more than 105 small organic molecules. Cooperative magnetic dipole interactions arose due to the closely packed magnetic NPs at the nanoarchitecture surface, causing enhanced NMR transverse relaxivity. Large in vivo MRI T2 contrast was thus obtained with unusually diluted solutions after intravenous injection in small rodents. Two-photon excited fluorescence imaging could be performed, achieving unprecedented location resolution for agents combining both magnetic nanoparticles and fluorescence properties. Finally, TEM imaging of the sectioned mouse tissue succeeded in isolating the core-shell structures, which represents the first image of intact complex magnetic and fluorescent nanoassemblies upon in vivo injection. Such highly cohesive dual nanoarchitectures should open great horizons toward the assessment with high spatial resolution of the drug or labeled stem cell biodistribution.

Vaccination with epigenetically treated mesothelioma cells induces immunisation and blocks tumour growth.

Malignant mesothelioma (MM) is an aggressive tumour associated with poor outcome in patients. Current treatments for MM are of limited efficacy. Our recent findings suggest that epigenetic drugs may induce both cytotoxicity and an immune response against MM cells. Thus, we used a mouse model of MM (AK7) to analyse how epigenetic drugs could modulate MM development in vivo. The treatment of tumour-bearing mice with an epigenetic drug already tested in clinical MM treatments (SAHA/Vorinostat) reduced the tumour mass and induced a moderate lymphocytic infiltration. However, the treatment did not stop tumour development. In order to show the potential effect of this epigenetic drug on tumour immunogenicity, in addition to cell cytotoxicity, we immunised mice either with AK7 cells pre-treated with SAHA, or with one of two cytotoxic drugs (curcumin or selenite), prior to transplantation of live AK7 cells. A specific immune response was observed only in mice immunised with AK7 cells pre-treated with the epigenetic drug (SAHA) and the tumour growth was arrested. An increase in the proportion of CD3+ CD8+ lymphocytes occurred in the peritoneal cavity. We also observed large conglomerates of immune cells in the omentum with clusters of CD8+ T cells, together with lymphocytes directed against residual AK7 cells in the interlobular connective tissue of the pancreas. Our data demonstrate that epigenetic drugs, such as SAHA, can stimulate tumour immunogenicity and improve the recognition of aggressive MM cells by the immune system in vivo.

Expression of NOS1 and soluble guanylyl cyclase by human kidney epithelial cells: morphological evidence for an autocrine/paracrine action of nitric oxide.

BACKGROUND: Nitric oxide plays an important role in the kidney through effects on both renal hemodynamics and tubular functions. Tubular epithelial cells are thus a target for nitric oxide. However, as to whether tubular epithelial cells endogeneously produce nitric oxide under physiologic conditions in human kidney is currently unknown. The aim of the present study was to characterize and localize in situ the nitric oxide synthase (NOS) isoforms (NOS1, NOS2, and NOS3) expressed in human normal kidney, and soluble guanylyl cyclase, the well-known target for nitric oxide. METHODS: Five complementary experimental approaches were used: (1) detection of NOS reductase activity by nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, (2) immunolocalization of the NOS isoforms (NOS1, NOS2, NOS3), (3) immunoblot analysis, (4) quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of NOS mRNA, and (5) measurement of NOS activity as the conversion rate of l-[14C]-arginine to l-[14C]-citrulline. In addition, in situ detection of soluble guanylyl cyclase was assessed by immunohistochemistry. RESULTS: All these techniques led to consistent results showing that epithelial cells of most tubules along the human nephron exhibit functional NOS1, with a corticomedullary gradient observed both at the protein and mRNA levels. Moreover, epithelial cells expressing NOS1 also express soluble guanylyl cyclase, indicating that these cells possess the machinery for autocrine/paracrine effect of nitric oxide. CONCLUSION: The present study demonstrates that NOS1 is strongly expressed in most tubules of the human nephron and therefore invites to consider epithelial cells as one of the major source of nitric oxide in the human kidney under physiologic conditions.