The OVAREX study: Establishment of ex vivo ovarian cancer models to validate innovative therapies and to identify predictive biomarkers.

BACKGROUND: Ovarian cancer is the first cause of death from gynecological malignancies mainly due to development of chemoresistance. Despite the emergence of PARP inhibitors, which have revolutionized the therapeutic management of some of these ovarian cancers, the 5-year overall survival rate remains around 45%. Therefore, it is crucial to develop new therapeutic strategies, to identify predictive biomarkers and to predict the response to treatments. In this context, functional assays based on patient-derived tumor models could constitute helpful and relevant tools for identifying efficient therapies or to guide clinical decision making. METHOD: The OVAREX study is a single-center non-interventional study which aims at investigating the feasibility of establishing in vivo and ex vivo models and testing ex vivo models to predict clinical response of ovarian cancer patients. Patient-Derived Xenografts (PDX) will be established from tumor fragments engrafted subcutaneously into immunocompromised mice. Explants will be generated by slicing tumor tissues and Ascites-Derived Spheroids (ADS) will be isolated following filtration of ascites. Patient-derived tumor organoids (PDTO) will be established after dissociation of tumor tissues or ADS, cell embedding into extracellular matrix and culture in specific medium. Molecular and histological characterizations will be performed to compare tumor of origin and paired models. Response of ex vivo tumor-derived models to conventional chemotherapy and PARP inhibitors will be assessed and compared to results of companion diagnostic test and/or to the patient's response to evaluate their predictive value. DISCUSSION: This clinical study aims at generating PDX and ex vivo models (PDTO, ADS, and explants) from tumors or ascites of ovarian cancer patients who will undergo surgical procedure or paracentesis. We aim at demonstrating the predictive value of ex vivo models for their potential use in routine clinical practice as part of precision medicine, as well as establishing a collection of relevant ovarian cancer models that will be useful for the evaluation of future innovative therapies. TRIAL REGISTRATION: The clinical trial has been validated by local research ethic committee on January 25th 2019 and registered at ClinicalTrials.gov with the identifier NCT03831230 on January 28th 2019, last amendment v4 accepted on July 18, 2023.

The TRIPLEX study: use of patient-derived tumor organoids as an innovative tool for precision medicine in triple-negative breast cancer.

BACKGROUND: Triple negative breast cancers (TNBC) account for approximately 15% of all breast cancers and are associated with a shorter median survival mainly due to locally advanced tumor and high risk of metastasis. The current neoadjuvant treatment for TNBC consists of a regimen of immune checkpoint blocker and chemotherapy (chemo-ICB). Despite the frequent use of this combination for TNBC treatment, moderate results are observed and its clinical benefit in TNBC remains difficult to predict. Patient-derived tumor organoids (PDTO) are 3D in vitro cellular structures obtained from patient's tumor samples. More and more evidence suggest that these models could predict the response of the tumor from which they are derived. PDTO may thus be used as a tool to predict chemo-ICB efficacy in TNBC patients. METHOD: The TRIPLEX study is a single-center observational study conducted to investigate the feasibility of generating PDTO from TNBC and to evaluate their ability to predict clinical response. PDTO will be obtained after the dissociation of biopsies and embedding into extra cellular matrix. PDTO will be cultured in a medium supplemented with growth factors and signal pathway inhibitors. Molecular and histological analyses will be performed on established PDTO lines to validate their phenotypic proximity with the original tumor. Response of PDTO to chemo-ICB will be assessed using co-cultures with autologous immune cells collected from patient blood samples. PDTO response will finally be compared with the response of the patient to evaluate the predictive potential of the model. DISCUSSION: This study will allow to assess the feasibility of using PDTO as predictive tools for the evaluation of the response of TNBC patients to treatments. In the event that PDTO could faithfully predict patient response in clinically relevant time frames, a prospective clinical trial could be designed to use PDTO to guide clinical decision. This study will also permit the establishment of a living biobank of TNBC PDTO usable for future innovative strategies evaluation. TRIAL REGISTRATION: The clinical trial (version 1.2) has been validated by local research ethic committee on December 30th 2021 and registered at ClinicalTrials.gov with the identifier NCT05404321 on June 3rd 2022, version 1.2.

ORGAVADS: establishment of tumor organoids from head and neck squamous cell carcinoma to assess their response to innovative therapies.

BACKGROUND: Radiotherapy is one of the cornerstones of the treatment of Head and Neck Squamous Cell Carcinomas (HNSCC). However, radioresistance is associated with a high risk of recurrence. To propose strategies (such as combinations with drugs) that could over intrinsic radioresistance, it is crucial to predict the response to treatment. Patient-Derived Tumor Organoids (PDTO) are in vitro tridimensional microtumors obtained from patient' own cancer samples. They have been shown to serve as reliable surrogates of the tumor response in patients. METHODS: The ORGAVADS study is a multicenter observational trial conducted to investigate the feasibility of generating and testing PDTO derived from HNSCC for the evaluation of sensitivity to treatments. PDTO are obtained after dissociation of resected tumors remaining from tissues necessary for the diagnosis. Embedding of tumor cells is then performed in extracellular matrix and culture in medium supplemented with growth factors and inhibitors. Histological and immunohistochemical characterizations are performed to validate the resemblance between PDTO and their original tumor. Response of PDTO to chemotherapy, radiotherapy and innovating combinations are assessed, as well as response to immunotherapy using co-cultures of PDTO with autologous immune cells collected from patient blood samples. Transcriptomic and genetic analyses of PDTO allow validation of the models compared to patients' own tumor and identification of potential predictive biomarkers. DISCUSSION: This study is designed to develop PDTO models from HNSCC. It will allow comparing the response of PDTO to treatment and the clinical response of the patients from whom they are derived. Our aim is to study the PDTO ability to predict the clinical response to treatment for each patient in view of a personalized medicine as well as to establish a collection of HNSCC models that will be useful for future innovative strategies evaluation. TRIAL REGISTRATION: NCT04261192, registered February 7, 2020, last amendment v4 accepted on June, 2021.

Deletion of intestinal epithelial AMP-activated protein kinase alters distal colon permeability but not glucose homeostasis.

OBJECTIVE: The intestinal epithelial barrier (IEB) restricts the passage of microbes and potentially harmful substances from the lumen through the paracellular space, and rupture of its integrity is associated with a variety of gastrointestinal disorders and extra-digestive diseases. Increased IEB permeability has been linked to disruption of metabolic homeostasis leading to obesity and type 2 diabetes. Interestingly, recent studies have uncovered compelling evidence that the AMP-activated protein kinase (AMPK) signaling pathway plays an important role in maintaining epithelial cell barrier function. However, our understanding of the function of intestinal AMPK in regulating IEB and glucose homeostasis remains sparse. METHODS: We generated mice lacking the two α1 and α2 AMPK catalytic subunits specifically in intestinal epithelial cells (IEC AMPK KO) and determined the physiological consequences of intestinal-specific deletion of AMPK in response to high-fat diet (HFD)-induced obesity. We combined histological, functional, and integrative analyses to ascertain the effects of gut AMPK loss on intestinal permeability in vivo and ex vivo and on the development of obesity and metabolic dysfunction. We also determined the impact of intestinal AMPK deletion in an inducible mouse model (i-IEC AMPK KO) by measuring IEB function, glucose homeostasis, and the composition of gut microbiota via fecal 16S rRNA sequencing. RESULTS: While there were no differences in in vivo intestinal permeability in WT and IEC AMPK KO mice, ex vivo transcellular and paracellular permeability measured in Ussing chambers was significantly increased in the distal colon of IEC AMPK KO mice. This was associated with a reduction in pSer425 GIV phosphorylation, a marker of leaky gut barrier. However, the expression of tight junction proteins in intestinal epithelial cells and pro-inflammatory cytokines in the lamina propria were not different between genotypes. Although the HFD-fed AMPK KO mice displayed suppression of the stress polarity signaling pathway and a concomitant increase in colon permeability, loss of intestinal AMPK did not exacerbate body weight gain or adiposity. Deletion of AMPK was also not sufficient to alter glucose homeostasis or the acute glucose-lowering action of metformin in control diet (CD)- or HFD-fed mice. CD-fed i-IEC AMPK KO mice also presented higher permeability in the distal colon under homeostatic conditions but, surprisingly, this was not detected upon HFD feeding. Alteration in epithelial barrier function in the i-IEC AMPK KO mice was associated with a shift in the gut microbiota composition with higher levels of Clostridiales and Desulfovibrionales. CONCLUSIONS: Altogether, our results revealed a significant role of intestinal AMPK in maintaining IEB integrity in the distal colon but not in regulating glucose homeostasis. Our data also highlight the complex interaction between gut microbiota and host AMPK.

Regulatory T Cell Stability and Migration Are Dependent on mTOR.

CD4+ Foxp3+ regulatory T cells (Treg) are essential to maintain immune tolerance, as their loss leads to a fatal autoimmune syndrome in mice and humans. Conflicting findings have been reported concerning their metabolism. Some reports found that Treg have low mechanistic target of rapamycin (mTOR) activity and would be less dependent on this kinase compared with conventional T cells, whereas other reports suggest quite the opposite. In this study, we revisited this question by using mice that have a specific deletion of mTOR in Treg. These mice spontaneously develop a severe and systemic inflammation. We show that mTOR expression by Treg is critical for their differentiation into effector Treg and their migration into nonlymphoid tissues. We also reveal that mTOR-deficient Treg have reduced stability. This loss of Foxp3 expression is associated with partial Foxp3 DNA remethylation, which may be due to an increased activity of the glutaminolysis pathway. Thus, our work shows that mTOR is crucial for Treg differentiation, migration, and identity and that drugs targeting this metabolism pathway will impact on their biology.

Tumor necrosis factor receptor family costimulation increases regulatory T-cell activation and function via NF-κB.

Several drugs targeting members of the TNF superfamily or TNF receptor superfamily (TNFRSF) are widely used in medicine or are currently being tested in therapeutic trials. However, their mechanism of action remains poorly understood. Here, we explored the effects of TNFRSF co-stimulation on murine Foxp3+ regulatory T cell (Treg) biology, as they are pivotal modulators of immune responses. We show that engagement of TNFR2, 4-1BB, GITR, and DR3, but not OX40, increases Treg proliferation and survival. Triggering these TNFRSF in Tregs induces similar changes in gene expression patterns, suggesting that they engage common signal transduction pathways. Among them, we identified a major role of canonical NF-κB. Importantly, TNFRSF co-stimulation improves the ability of Tregs to suppress colitis. Our data demonstrate that stimulation of discrete TNFRSF members enhances Treg activation and function through a shared mechanism. Consequently, therapeutic effects of drugs targeting TNFRSF or their ligands may be mediated by their effect on Tregs.

The NF-κB RelA Transcription Factor Is Critical for Regulatory T Cell Activation and Stability.

Regulatory T cells (Tregs) play a major role in immune homeostasis and in the prevention of autoimmune diseases. It has been shown that c-Rel is critical in Treg thymic differentiation, but little is known on the role of NF-κB on mature Treg biology. We thus generated mice with a specific knockout of RelA, a key member of NF-κB, in Tregs. These mice developed a severe autoimmune syndrome with multi-organ immune infiltration and high activation of lymphoid and myeloid cells. Phenotypic and transcriptomic analyses showed that RelA is critical in the acquisition of the effector Treg state independently of surrounding inflammatory environment. Unexpectedly, RelA-deficient Tregs also displayed reduced stability and cells that had lost Foxp3 produced inflammatory cytokines. Overall, we show that RelA is critical for Treg biology as it promotes both the generation of their effector phenotype and the maintenance of their identity.