VISTA/CTLA4/PD1 coexpression on tumor cells confers a favorable immune microenvironment and better prognosis in high-grade serous ovarian carcinoma

IntroductionImmunotherapy by blocking immune checkpoints programmed death/ligand (PD1/PDL1) and cytotoxic T-lymphocyte-associated protein 4(CTLA4) has emerged as new therapeutic targets in cancer. However, their efficacy has been limited due to resistance. A new- checkpoint V-domain Ig-containing suppressor of T cell activation (VISTA) has appeared, but the use of its inhibition effect in combination with antibodies targeting PDL1/PD1and CTLA4 has not been reported in ovarian cancer.MethodsIn this study, we investigated the expressions of VISTA, CTLA4, and PDL1 using immunohistochemistry (IHC)on 135 Formalin-Fixed Paraffin-Embedded (FFPE)tissue samples of High-grade serous carcinoma (HGSOC). VISTA, CTLA4, PDL1, PD1, CD8, CD4, and FOXP3 mRNA extracted from 429 patients with ovarian cancer in the Cancer Genome Atlas (TCGA) database was included as a validation cohort. Correlations between these checkpoints, tumor-infiltrating- lymphocytes (TILs), and survival were analyzed.Results and discussionCTLA4 was detectable in 87.3% of samples, VISTA in 64.7%, PD1 in 56.7%, and PDL1 in 48.1%. PDL1 was the only tested protein associated with an advanced stage (p=0.05). VISTA was associated with PDL1, PD1, and CTLA4 expressions (p=0.005, p=0.001, p=0.008, respectively), consistent with mRNA level analysis from the TCGA database. Univariate analyses showed only VISTA expression (p=0.04) correlated with overall survival (OS). Multivariate analyses showed that VISTA expression (p=0.01) and the coexpression of VISTA+/CTLA4+/PD1+ (p=0.05) were associated with better OS independently of the clinicopathological features. Kaplan-Meier analysis showed that the coexpression of the VISTA+/CTLA4+/PDL1+ and VISTA+/CTLA4+/PD1+ checkpoints on tumor cells (TCs)were associated with OS (p=0.02 and p<0.001; respectively). VISTA+/CTLA4+/PD1+ in TCs and CD4+/CD8+TILswere associated with better 2-yer OS. This correlation may refer to the role of VISTA as a receptor in the TCs and not in the immune cells. Thus, targeting combination therapy blocking VISTA, CTLA4, and PD1 could be a novel and attractive strategy for HGSOC treatment, considering the ambivalent role of VISTA in the HGSOC tumor cells

Bidimensional lamellar assembly by coordination of peptidic homopolymers to platinum nanoparticles

A key challenge for designing hybrid materials is the development of chemical tools to control the organization of inorganic nanoobjects at low scales, from mesoscopic (~µm) to nanometric (~nm). So far, the most efficient strategy to align assemblies of nanoparticles consists in a bottom-up approach by decorating block copolymer lamellae with nanoobjects. This well accomplished procedure is nonetheless limited by the thermodynamic constraints that govern copolymer assembly, the entropy of mixing as described by the Flory–Huggins solution theory supplemented by the critical influence of the volume fraction of the block components. Here we show that a completely different approach can lead to tunable 2D lamellar organization of nanoparticles with homopolymers only, on condition that few elementary rules are respected: 1) the polymer spontaneously allows a structural preorganization, 2) the polymer owns functional groups that interact with the nanoparticle surface, 3) the nanoparticles show a surface accessible for coordination

Self-assembly of metallic nanoparticles oriented by peptide polymers

Les nanomatériaux hybrides organique-inorganique (nanocomposites) connaissent un intérêt croissant notamment grâce au développement de nouvelles méthodes permettant leur synthèse. Afin de moduler leurs propriétés, il est important de contrôler la nature chimique des constituants ainsi que leurs interactions. Dans le cadre de ce travail de thèse, nous avons mis en évidence une nouvelle approche de croissance et de structuration de nanoparticules métalliques (Pt, Ir, Au, etc.) en utilisant des polymères peptidiques comme ligands macromoléculaires. A l'instar des protéines, ces derniers sont constitués d'acides aminés et adoptent certaines structurations biomimétiques (hélice α ou feuillet β), un élément peu étudié pour promouvoir des nanocomposites.Ce travail commence par un travail de synthèse consistant à préparer une librairie de polymères peptidiques incorporant des points d'ancrage chimiques pour moduler leur association à des particules métalliques. Nous avons pour cela développé des réactions de greffage sur du poly(γ-benzyl-L-glutamate) (PBLG) avec différentes amines fonctionnalisées pour promouvoir une chimie de coordination spécifique avec les métaux de transitions.La deuxième partie de ce manuscrit décrit ensuite l'utilisation de ces polymères peptidiques comme matrice permettant la croissance contrôlée de nanoparticules métalliques (synthèse in situ). Nous avons ainsi mis en évidence que les polypeptides donnent accès à des nanoparticules hyperbranchées, résultant de la coalescence de nanocristaux ultra-petits, dont la morphologie peut être contrôlée en jouant sur les propriétés du polymère (degré de polymérisation, stœchiométrie des chaines latérales).Dans une dernière partie, les polymères peptidiques ont finalement été utilisés pour guider l'autoassemblage de nanoparticules métalliques préformées. Nous avons ainsi obtenu des structures lamellaires bidimensionnelles, dont la stabilité est assurée par des liaisons de coordination spécifiques entre les groupements fonctionnels du polymère et la surface des nanoparticules. En modulant la taille des polymères, il a été possible de contrôler l’espace entre les lamelles, une modification structurale clé qui permet de modifier les propriétés électriques des nanocomposites.Hybrid organic-inorganic nanomaterials (nanocomposites) are of growing interest due to the development of new synthesis. In order to modulate their properties, it is important to control the nature of the constituents and their interactions. Within the framework of this PhD thesis, we developed a new approach to prepare nanocomposites from metallic nanoparticles (Pt, Ir, Au, etc.) using peptidic polymers (polypeptides). Polypeptides are made up of amino acids and adopt biomimetic structures (α-helix or β-sheets) as do proteins. As macromolecular ligands, they have been understudied in nanocomposite design.This research work first describes how we have prepared a library of polypeptides incorporating tailored side chain moieties modulating the binding with metallic nanoparticles. Indeed, we have fully developed an efficient 2 steps grafting process to introduce onto poly(γ-benzyl-L-glutamate) (PBLG) backbones amines promoting specific coordination binding with transition metals.The second part of the PhD manuscript describes how these polypeptides can be used to promote the in-situ synthesis of metallic nanoparticles. A comprehensive study of this approach have shown that polypeptides give access to hyperbranched nanoparticles, resulting from the coalescence of ultra-small nanocrystals, whose morphology can be controlled by modulating the properties of the polymer (polymerization degree, side chain stoechiometry).Finaly, a third part of the PhD presents how peptidic polymers can direct the self-assembly of preformed metallic nanoparticles. Using specific formulations, we obtained two-dimensional lamellar structures, whose stability is ensured by specific coordination bonding between the functional groups of the polymer and the surface of the nanoparticles. By varying the molar mass of the polymers, it was possible to control the space between the lamellae, a key structural feature that allows the electrical properties of the nanocomposites to be changed

Auto-assemblage des nanoparticules métalliques orienté par des polymères peptidiques

Hybrid organic-inorganic nanomaterials (nanocomposites) are of growing interest due to the development of new synthesis. In order to modulate their properties, it is important to control the nature of the constituents and their interactions. Within the framework of this PhD thesis, we developed a new approach to prepare nanocomposites from metallic nanoparticles (Pt, Ir, Au, etc.) using peptidic polymers (polypeptides). Polypeptides are made up of amino acids and adopt biomimetic structures (α-helix or β-sheets) as do proteins. As macromolecular ligands, they have been understudied in nanocomposite design.This research work first describes how we have prepared a library of polypeptides incorporating tailored side chain moieties modulating the binding with metallic nanoparticles. Indeed, we have fully developed an efficient 2 steps grafting process to introduce onto poly(γ-benzyl-L-glutamate) (PBLG) backbones amines promoting specific coordination binding with transition metals.The second part of the PhD manuscript describes how these polypeptides can be used to promote the in-situ synthesis of metallic nanoparticles. A comprehensive study of this approach have shown that polypeptides give access to hyperbranched nanoparticles, resulting from the coalescence of ultra-small nanocrystals, whose morphology can be controlled by modulating the properties of the polymer (polymerization degree, side chain stoechiometry).Finaly, a third part of the PhD presents how peptidic polymers can direct the self-assembly of preformed metallic nanoparticles. Using specific formulations, we obtained two-dimensional lamellar structures, whose stability is ensured by specific coordination bonding between the functional groups of the polymer and the surface of the nanoparticles. By varying the molar mass of the polymers, it was possible to control the space between the lamellae, a key structural feature that allows the electrical properties of the nanocomposites to be changed.Les nanomatériaux hybrides organique-inorganique (nanocomposites) connaissent un intérêt croissant notamment grâce au développement de nouvelles méthodes permettant leur synthèse. Afin de moduler leurs propriétés, il est important de contrôler la nature chimique des constituants ainsi que leurs interactions. Dans le cadre de ce travail de thèse, nous avons mis en évidence une nouvelle approche de croissance et de structuration de nanoparticules métalliques (Pt, Ir, Au, etc.) en utilisant des polymères peptidiques comme ligands macromoléculaires. A l'instar des protéines, ces derniers sont constitués d'acides aminés et adoptent certaines structurations biomimétiques (hélice α ou feuillet β), un élément peu étudié pour promouvoir des nanocomposites.Ce travail commence par un travail de synthèse consistant à préparer une librairie de polymères peptidiques incorporant des points d'ancrage chimiques pour moduler leur association à des particules métalliques. Nous avons pour cela développé des réactions de greffage sur du poly(γ-benzyl-L-glutamate) (PBLG) avec différentes amines fonctionnalisées pour promouvoir une chimie de coordination spécifique avec les métaux de transitions.La deuxième partie de ce manuscrit décrit ensuite l'utilisation de ces polymères peptidiques comme matrice permettant la croissance contrôlée de nanoparticules métalliques (synthèse in situ). Nous avons ainsi mis en évidence que les polypeptides donnent accès à des nanoparticules hyperbranchées, résultant de la coalescence de nanocristaux ultra-petits, dont la morphologie peut être contrôlée en jouant sur les propriétés du polymère (degré de polymérisation, stœchiométrie des chaines latérales).Dans une dernière partie, les polymères peptidiques ont finalement été utilisés pour guider l'autoassemblage de nanoparticules métalliques préformées. Nous avons ainsi obtenu des structures lamellaires bidimensionnelles, dont la stabilité est assurée par des liaisons de coordination spécifiques entre les groupements fonctionnels du polymère et la surface des nanoparticules. En modulant la taille des polymères, il a été possible de contrôler l’espace entre les lamelles, une modification structurale clé qui permet de modifier les propriétés électriques des nanocomposites

Treatment of anionic dye aqueous solution using Ti, HDTMA and Al/Fe pillared bentonite. Essay to regenerate the adsorbent

In this study, the adsorption removal of an anionic dye (Congo red) by a local bentonite before and after modification was studied. The modification of the bentonite was made by organophilisation using surfactant (HDTMA) and by pillaring process to obtain a bentonite with Ti pillars and with mixed pillars of Fe/Al. The various synthesized materials are characterized by different techniques such as DRX, MET, N2 adsorption-desorption, Zeta potential measurement. Results show the development of the texture and the structure of the bentonite after modification. The various adsorbents synthesized show an increase in the adsorption capacity of Congo Red compared to the initial bentonite. Adsorption isotherms are described by the Langmuir model in all cases except that for Ti pillared bentonite, the Freundlich model is more suitable. Pseudo-second order is better for describing the adsorption process. Also, regeneration of the adsorbent is approached in this study by photochemical way and the results show a total regeneration of the adsorbent. Keywords: Bentonite, Pillaring, Anionic dye, Adsorption, Regeneratio

Enhanced Dielectric Relaxation in Self-Organized Layers of Polypeptides Coupled to Platinum Nanoparticles: Temperature Dependence and Effect of Bias Voltage

International audienceUsing alternative current impedance spectroscopy, we investigate the dynamical conductivity of hybrid nanomaterials composed of helical polypeptide layers containing platinum nanoparticles (PtNP). The electrical characteristics of the self-organized poly(γ-benzyl-l-glutamate) (PBGL) in bidimensional lamellar assembly in the presence of Pt nanoparticles are well modeled and described by a single equivalent circuit of parallel resistance and capacitance. The latter are determined using a comparison between the measured and calculated Nyquist plots, which allows extracting the characteristic relaxation time and frequency of the dipolar relaxation process. We found that the relaxation frequency in the PBLG–PtNP hybrid materials is enhanced by 4 orders of magnitudes compared to pure PBLG, which indicates a much faster dielectric relaxation in PBLG–PtNP due to dipole orientation and dipole–dipole interactions. The temperature dependence of the relaxation time is analyzed using Arrhenius plots, from which the activation energy of the relaxation process is found to be around 0.1 eV. Such a value close to the peptide vibration energy of the PBLG indicates a vibration-assisted relaxation process and a polaronic charge transport mechanism. An advantage of the PBLG–PtNP nanocomposite material is that the activation energy can be finely tuned by the PBLG degree of polymerization. Finally, an important outcome of this work is the investigation of the dielectric relaxation process in PBLG–PtNP under applied DC bias. We found that the activation energy decreases with increasing bias voltage for all degrees of polymerization of the PBLG molecule. This effect is interpreted in terms of electric field-induced alignment of the dipoles and of increased mobility of the polaronic charge carriers. The presence of piezoelectricity in the hybrid material gives the possibility to use the DC bias as a simple mean of monitoring the dynamical conductivity involving polaronic states

DataSheet_3_VISTA/CTLA4/PD1 coexpression on tumor cells confers a favorable immune microenvironment and better prognosis in high-grade serous ovarian carcinoma.docx

IntroductionImmunotherapy by blocking immune checkpoints programmed death/ligand (PD1/PDL1) and cytotoxic T-lymphocyte-associated protein 4(CTLA4) has emerged as new therapeutic targets in cancer. However, their efficacy has been limited due to resistance. A new- checkpoint V-domain Ig-containing suppressor of T cell activation (VISTA) has appeared, but the use of its inhibition effect in combination with antibodies targeting PDL1/PD1and CTLA4 has not been reported in ovarian cancer.MethodsIn this study, we investigated the expressions of VISTA, CTLA4, and PDL1 using immunohistochemistry (IHC)on 135 Formalin-Fixed Paraffin-Embedded (FFPE)tissue samples of High-grade serous carcinoma (HGSOC). VISTA, CTLA4, PDL1, PD1, CD8, CD4, and FOXP3 mRNA extracted from 429 patients with ovarian cancer in the Cancer Genome Atlas (TCGA) database was included as a validation cohort. Correlations between these checkpoints, tumor-infiltrating- lymphocytes (TILs), and survival were analyzed.Results and discussionCTLA4 was detectable in 87.3% of samples, VISTA in 64.7%, PD1 in 56.7%, and PDL1 in 48.1%. PDL1 was the only tested protein associated with an advanced stage (p=0.05). VISTA was associated with PDL1, PD1, and CTLA4 expressions (p=0.005, p=0.001, p=0.008, respectively), consistent with mRNA level analysis from the TCGA database. Univariate analyses showed only VISTA expression (p=0.04) correlated with overall survival (OS). Multivariate analyses showed that VISTA expression (p=0.01) and the coexpression of VISTA+/CTLA4+/PD1+ (p=0.05) were associated with better OS independently of the clinicopathological features. Kaplan-Meier analysis showed that the coexpression of the VISTA+/CTLA4+/PDL1+ and VISTA+/CTLA4+/PD1+ checkpoints on tumor cells (TCs)were associated with OS (p=0.02 and p+/CTLA4+/PD1+ in TCs and CD4+/CD8+TILswere associated with better 2-yer OS. This correlation may refer to the role of VISTA as a receptor in the TCs and not in the immune cells. Thus, targeting combination therapy blocking VISTA, CTLA4, and PD1 could be a novel and attractive strategy for HGSOC treatment, considering the ambivalent role of VISTA in the HGSOC tumor cells.</p

DataSheet_2_VISTA/CTLA4/PD1 coexpression on tumor cells confers a favorable immune microenvironment and better prognosis in high-grade serous ovarian carcinoma.xls

IntroductionImmunotherapy by blocking immune checkpoints programmed death/ligand (PD1/PDL1) and cytotoxic T-lymphocyte-associated protein 4(CTLA4) has emerged as new therapeutic targets in cancer. However, their efficacy has been limited due to resistance. A new- checkpoint V-domain Ig-containing suppressor of T cell activation (VISTA) has appeared, but the use of its inhibition effect in combination with antibodies targeting PDL1/PD1and CTLA4 has not been reported in ovarian cancer.MethodsIn this study, we investigated the expressions of VISTA, CTLA4, and PDL1 using immunohistochemistry (IHC)on 135 Formalin-Fixed Paraffin-Embedded (FFPE)tissue samples of High-grade serous carcinoma (HGSOC). VISTA, CTLA4, PDL1, PD1, CD8, CD4, and FOXP3 mRNA extracted from 429 patients with ovarian cancer in the Cancer Genome Atlas (TCGA) database was included as a validation cohort. Correlations between these checkpoints, tumor-infiltrating- lymphocytes (TILs), and survival were analyzed.Results and discussionCTLA4 was detectable in 87.3% of samples, VISTA in 64.7%, PD1 in 56.7%, and PDL1 in 48.1%. PDL1 was the only tested protein associated with an advanced stage (p=0.05). VISTA was associated with PDL1, PD1, and CTLA4 expressions (p=0.005, p=0.001, p=0.008, respectively), consistent with mRNA level analysis from the TCGA database. Univariate analyses showed only VISTA expression (p=0.04) correlated with overall survival (OS). Multivariate analyses showed that VISTA expression (p=0.01) and the coexpression of VISTA+/CTLA4+/PD1+ (p=0.05) were associated with better OS independently of the clinicopathological features. Kaplan-Meier analysis showed that the coexpression of the VISTA+/CTLA4+/PDL1+ and VISTA+/CTLA4+/PD1+ checkpoints on tumor cells (TCs)were associated with OS (p=0.02 and p+/CTLA4+/PD1+ in TCs and CD4+/CD8+TILswere associated with better 2-yer OS. This correlation may refer to the role of VISTA as a receptor in the TCs and not in the immune cells. Thus, targeting combination therapy blocking VISTA, CTLA4, and PD1 could be a novel and attractive strategy for HGSOC treatment, considering the ambivalent role of VISTA in the HGSOC tumor cells.</p

DataSheet_1_VISTA/CTLA4/PD1 coexpression on tumor cells confers a favorable immune microenvironment and better prognosis in high-grade serous ovarian carcinoma.docx

IntroductionImmunotherapy by blocking immune checkpoints programmed death/ligand (PD1/PDL1) and cytotoxic T-lymphocyte-associated protein 4(CTLA4) has emerged as new therapeutic targets in cancer. However, their efficacy has been limited due to resistance. A new- checkpoint V-domain Ig-containing suppressor of T cell activation (VISTA) has appeared, but the use of its inhibition effect in combination with antibodies targeting PDL1/PD1and CTLA4 has not been reported in ovarian cancer.MethodsIn this study, we investigated the expressions of VISTA, CTLA4, and PDL1 using immunohistochemistry (IHC)on 135 Formalin-Fixed Paraffin-Embedded (FFPE)tissue samples of High-grade serous carcinoma (HGSOC). VISTA, CTLA4, PDL1, PD1, CD8, CD4, and FOXP3 mRNA extracted from 429 patients with ovarian cancer in the Cancer Genome Atlas (TCGA) database was included as a validation cohort. Correlations between these checkpoints, tumor-infiltrating- lymphocytes (TILs), and survival were analyzed.Results and discussionCTLA4 was detectable in 87.3% of samples, VISTA in 64.7%, PD1 in 56.7%, and PDL1 in 48.1%. PDL1 was the only tested protein associated with an advanced stage (p=0.05). VISTA was associated with PDL1, PD1, and CTLA4 expressions (p=0.005, p=0.001, p=0.008, respectively), consistent with mRNA level analysis from the TCGA database. Univariate analyses showed only VISTA expression (p=0.04) correlated with overall survival (OS). Multivariate analyses showed that VISTA expression (p=0.01) and the coexpression of VISTA+/CTLA4+/PD1+ (p=0.05) were associated with better OS independently of the clinicopathological features. Kaplan-Meier analysis showed that the coexpression of the VISTA+/CTLA4+/PDL1+ and VISTA+/CTLA4+/PD1+ checkpoints on tumor cells (TCs)were associated with OS (p=0.02 and p+/CTLA4+/PD1+ in TCs and CD4+/CD8+TILswere associated with better 2-yer OS. This correlation may refer to the role of VISTA as a receptor in the TCs and not in the immune cells. Thus, targeting combination therapy blocking VISTA, CTLA4, and PD1 could be a novel and attractive strategy for HGSOC treatment, considering the ambivalent role of VISTA in the HGSOC tumor cells.</p

VISTA+/CD8+ status correlates with favorable prognosis in Epithelial ovarian cancer

Immunotherapy by blocking immune checkpoint regulators has emerged as a new targeted therapy for some cancers. Among them V-domain Ig suppressor of Tcell activation (VISTA) which is identified as a novel checkpoint regulator in ovarian cancer. This study aimed to investigate the VISTA role in Epithelial ovarian cancer (EOC), and its relationship with tumor-infiltrating lymphocytes (TILs) markers and its prognostic value. The expression of VISTA, CD3, CD8, CD4, FOXP3, and CD56 was assessed in 168 EOC tissue microarrays (TMA) by immunohistochemistry (IHC). In addition, associations between VISTA, TILs, clinicopathological variables, and overall survival (OS) were analyzed. VISTA expression in IGRov1 cells, as well as in PBMC of EOC patient, was evaluated by western blot. VISTA expression was detected in 64,28% of tissues, among which 42.3% were positive for tumor cells (TCs), and 47,9% were positive for immune cells (ICs). In univariate analysis, VISTA expression was significantly associated with a high density of TILs:CD3+ (p = 0,001), CD4+ (p = 0,002) and CD8+ (p≤0,001), in ICs but not in TCs. In terms of OS, multivariate analysis showed a significant association between the high density of CD8+ TILs and VISTA positive staining in ICs (p = 0,044), but not in TCs (p = 0,108). Kaplan-Meier curves demonstrated no correlation between VISTA expression and prolonged OS in both ICs (p = 0,841) and TCs (p = 0,090). Classification of EOC tumor microenvironment based on VISTA and CD8+TILs expression, demonstrated four immune subtypes: VISTA+/CD8+, VISTA+/CD8-, VISTA-/CD8+ and VISTA-/CD8-. The dual positive VISTA+/CD8+ subtype was significantly associated with prolonged OS in both TCs and ICs (p = 0,012 and p≤0,01, respectively), whereas patients with VISTA+/CD8- had the worst OS. Our results showed that VISTA is highly expressed in the IGRov1 cell line and LT-CD8 from a patient with EOC. Our results highlighted the association of VISTA expression and CD8+ TILs in EOC, with prolonged OS in patients with VISTA+/CD8+ and proposed VISTA as a potential immunotherapeutic target in EOC