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Despite considerable progress in understanding the biology and genetics of breast cancer progression, the development of effective therapies need physiological and predictive preclinical models. In this context, breast cancer patient-derived xenograft (PDX) models has become a standard tool as they reproduce accurately the behavior of tumor of origin, in term of histological and molecular phenotype and response to chemotherapy. Although PDXs in vivo models are indispensable for preclinical studies, they suffer from some limitations due to study costs related to tumor maintenance on mice, variable engraftment rate, growth delay and limited throughput for large-scale drug screening.To address this problem and propose a time and cost effective preclinical screening tool, we developed a panel of breast cancer PDX-derived low-passage 2D cell lines as a convenient in vitro pre-screening platform to profile compound activity.30 different breast cancer PDX models including TNBC, HER2+ and ER+ were tested for their capacity to generate cell lines maintaining the characteristics of the parental PDX tumor and usable for in vitro assays.Today, we succeeded with a series of 14 PDX models.Tumor cells isolated from PDX tumor tissue were cultured under different media and matrix conditions, allowing at least 5 passages in culture. A Short Tandem Repeat (STR) comparison profile was done with the parental PDX before performing a master bank. We succeeded in establishing a panel of 14 PDX-derived cellular models (14/30 = 46% success rate).We performed short term 2D cytotoxicity assays and long term colony assays to compare cell lines in vitro drug sensitivity with their parental PDX in vivo drug response and overall, the results show that this panel reproduced the drug response profile of the original PDXs with chemotherapies, PARP inhibitors, an ADC (T-DM1) therapies.Moreover, cellular models engrafted back onto mice showed in vivo response to chemotherapies similar to that of the parental PDX confirming the identical behavior of cell line / PDX couples.As the use of cellular models is still considered as a standard for early preclinical test to evaluate drug response before moving to in vivo assays, our breast cancer PDX-derived cell line platform appeared to be a robust and relevant tool. Furthermore, since the main concern when using in vitro models is the representativeness of the results obtained when transposed to in vivo models, the similarities between cell lines and parental PDX should maximize success of further in vivo preclinical drug development. Citation Format: Stefano Cairo, Olivier Deas, Sophie Banis, Kathleen Flosseau, Enora Le Ven, Jean-Gabriel Judde. A preclinical platform of breast cancer PDX-derived cell lines as a tool for pharmacological screening and functional studies [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-52.

Despite recent progress in the characterization of tumour components, the tri-dimensional (3D) organization of this pathological tissue and the parameters determining its internal architecture remain elusive. Here, we analysed the spatial organization of patient-derived xenograft tissues generated from hepatoblastoma, the most frequent childhood liver tumour, by serial block-face scanning electron microscopy using an integrated workflow combining 3D imaging, manual and machine learning-based semi-automatic segmentations, mathematics and infographics. By digitally reconstituting an entire hepatoblastoma sample with a blood capillary, a bile canaliculus-like structure, hundreds of tumour cells and their main organelles (e.g. cytoplasm, nucleus, mitochondria), we report unique 3D ultrastructural data about the organization of tumour tissue. We found that the size of hepatoblastoma cells correlates with the size of their nucleus, cytoplasm and mitochondrial mass. We also found anatomical connections between the blood capillary and the planar alignment and size of tumour cells in their 3D milieu. Finally, a set of tumour cells polarized in the direction of a hot spot corresponding to a bile canaliculus-like structure. In conclusion, this pilot study allowed the identification of bioarchitectural parameters that shape the internal and spatial organization of tumours, thus paving the way for future investigations in the emerging onconanotomy field., de Senneville et al. demonstrate an integrated workflow combining 3D imaging, manual and machine learning-based semi-automatic segmentation, mathematics and infographics to study the spatial organization of patient-derived hepatoblastoma xenograft tissues. Their approach potentially assists investigations of this childhood liver tumour and other types of tumour tissues.

Despite considerable progress in understanding the biology and genetics of cancer progression, the development of effective therapies against cancer need physiological and predictive preclinical models. In this context, patient-derived xenograft (PDX) models has become a standard tool as they reproduce accurately the behavior of tumor of origin, in term of histological and molecular phenotype and response to chemotherapy. Although PDXs in vivo models are indispensable for preclinical studies, they suffer from some limitations due to study costs related to tumor maintenance on mice, variable engraftment rate, growth delay and limited throughput for large-scale drug screening. To address this problem and propose a time a cost effective preclinical screening tool, we developed a panel of PDX-derived low-passage 2D cell lines as a convenient in vitro pre-screening platform to profile compound activity. Different PDX models including breast, lung, colon, melanoma, glioblastoma and hepatoblastoma were tested for their capacity to generate cell lines maintaining the characteristics of the parental PDX tumor and usable for in vitro assays. Today, we succeeded with a series of 50 PDX models with 83% success rate. Tumor cells isolated from PDX tumor tissue were cultured under different media and matrix conditions, allowing at least 5 passages in culture. A Short Tandem Repeat (STR) comparison profile was done with the parental PDX before performing a master bank. We performed short term 2D cytotoxicity assays and long term colony assays to compare cell lines in vitro drug sensitivity with their parental PDX in vivo drug response and overall, the results show that this panel reproduced the drug response profile of the original PDXs with chemotherapies, PARP inhibitors, an ADC (T-DM1) and FGFR-targeting therapies. Moreover, cellular models engrafted back onto mice showed in vivo response to chemotherapies similar to that of the parental PDX confirming the identical behavior of cell line/PDX couples. As the use of cellular models is still considered as a standard for early preclinical test to evaluate drug response before moving to in vivo assays, our PDX-derived cell line platform appeared to be a robust and relevant tool. Furthermore, since the main concern when using in vitro models is the representativeness of the results obtained when transposed to in vivo models, the similarities between cell lines and parental PDX should maximize success of further in vivo preclinical drug development. Citation Format: Olivier Deas, Sophie Banis, Kathleen Flosseau, Lea Sinayen, Enora Le Ven, Jean-Gabriel Judde, Stefano Cairo. A preclinical platform of PDX-derived cell lines as a tool for pharmacological screening and functional studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3014.

Poly (adenosine diphosphate-ribose) polymerase inhibitors (PARPi) have recently emerged as therapeutic options for patients with homologous recombination-deficient (HRD) breast or ovarian cancer, two heterogeneous diseases associated with high mortality rates. As shown in several clinical studies, patient response to PARPi is invariably followed by eventual mid-long term resistance and progression under treatment. The molecular processes contributing to PARPi-resistance are at present under-explored. Therefore, a huge effort is being made to better understand how to overcome resistance and to identify ad-hoc combinations with other targeted therapies to improve tumor response and extend progression-free survival. We have previously published the PARPi-response profile of a panel of 40 breast cancer (BC) patient-derived xenografts (PDXs). The models tested showed heterogeneous response to PARPi, and only partial association with the genomic status of BRCA genes, the only currently acknowledged clinical marker to select patients that can benefit from PARPi administration. Although these models are ideal preclinical tools for the evaluation of drug combinations to improve tumor response to PARPi as single agent, the use of these models for early preclinical evaluation of combination efficacy is not straightforward in reason of the limited throughput and of the ethical issue with respect of the 3Rs. The use of cellular models is still considered as a standard early preclinical test to evaluate drug response before moving to in vivo assays. However, the main concern when using in vitro models is the representativeness of the results obtained when transposed to in vivo models. In this study, we have setup an in vitro assay that recapitulates the response to PARPi observed in vivo in our BC PDXs, with particular focus on the models that show resistance to PARPi. To this aim we generated 9 cellular models from 9 BC PDX models: HBCx-2, HBCx-3, HBCx-6, HBCx-8, HBCx-9, HBCx-17, HBCx-19, HBCx-39 and T174. Two cellular models (HBCx-3, and HBCx-19) were established from ER+ BC PDX and 7 from ER- BC PDXs and two models, HBCx-8 and HBCx-17, harbor BRCA1 and BRCA2 mutation, respectively. Seven out of 9 models are resistant to PARPi in vivo, with HBCx-6 PDX showing partial tumor regression and HBCx-17 PDX showing tumor stabilization upon treatment. Several different 2D-culture experimental conditions, namely different cell growth conditions, drug concentrations, duration of cell exposure to drugs, time points and readouts, have been tested to evaluate response to olaparib. The results showed that a 2D colony assay is the best experimental strategy to faithfully evaluate tumor cell sensitivity, minimizing the false positive results when compared to the in vivo data.. This cell panel will be used to identify combination of PARPi with a library of FDA-approved targeted therapy to identify the treatments to be moved forward in vivo. Citation Format: Olivier Déas, Romain Rousseau, Sophie Banis, Kathleen Flosseau, Enora Le Ven, Jean-Gabriel Judde, stefano cairo. Establishment of an in vitro assay that phenocopies tumor response to PARP inhibitors in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3810.

International audience; Necrotizing and crescentic rapidly progressive GN (RPGN) is a life-threatening syndrome characterized by a rapid loss of renal function. Evidence suggests that podocyte expression of the transcription factor peroxisome proliferator-activated receptor γ (PPARγ) may prevent podocyte injury, but the function of glomerular PPARγ in acute, severe inflammatory GN is unknown. Here, we observed marked loss of PPARγ abundance and transcriptional activity in glomerular podocytes in experimental RPGN. Blunted expression of PPARγ in podocyte nuclei was also found in kidneys from patients diagnosed with crescentic GN. Podocyte-specific Pparγ gene targeting accentuated glomerular damage, with increased urinary loss of albumin and severe kidney failure. Furthermore, a PPARγ gain-of-function approach achieved by systemic administration of thiazolidinedione (TZD) failed to prevent severe RPGN in mice with podocyte-specific Pparγ gene deficiency. In nuclear factor erythroid 2-related factor 2 (NRF2)-deficient mice, loss of podocyte PPARγ was observed at baseline. NRF2 deficiency markedly aggravated the course of RPGN, an effect that was partially prevented by TZD administration. Furthermore, delayed administration of TZD, initiated after the onset of RPGN, still alleviated the severity of experimental RPGN. These findings establish a requirement for the NRF2-PPARγ cascade in podocytes, and we suggest that these transcription factors have a role in augmenting the tolerance of glomeruli to severe immune-complex mediated injury. The NRF2-PPARγ pathway may be a therapeutic target for RPGN.

OBJECTIVE Class IIa histone deacetylases (HDACs) belong to a large family of enzymes involved in protein deacetylation and play a role in regulating gene expression and cell differentiation. Previously, we showed that HDAC inhibitors modify the timing and determination of pancreatic cell fate. The aim of this study was to determine the role of class IIa HDACs in pancreas development. RESEARCH DESIGN AND METHODS We took a genetic approach and analyzed the pancreatic phenotype of mice lacking HDAC4, -5, and -9. We also developed a novel method of lentiviral infection of pancreatic explants and performed gain-of-function experiments. RESULTS We show that class IIa HDAC4, -5, and -9 have an unexpected restricted expression in the endocrine β- and δ-cells of the pancreas. Analyses of the pancreas of class IIa HDAC mutant mice revealed an increased pool of insulin-producing β-cells in Hdac5−/− and Hdac9−/− mice and an increased pool of somatostatin-producing δ-cells in Hdac4−/− and Hdac5−/− mice. Conversely, HDAC4 and HDAC5 overexpression showed a decreased pool of insulin-producing β-cells and somatostatin-producing δ-cells. Finally, treatment of pancreatic explants with the selective class IIa HDAC inhibitor MC1568 enhances expression of Pax4, a key factor required for proper β-and δ-cell differentiation and amplifies endocrine β- and δ-cells. CONCLUSIONS We conclude that HDAC4, -5, and -9 are key regulators to control the pancreatic β/δ-cell lineage. These results highlight the epigenetic mechanisms underlying the regulation of endocrine cell development and suggest new strategies for β-cell differentiation-based therapies.

International audience; The glomerulus is a highly specialized capillary tuft, which under pressure filters large amounts of water and small solutes into the urinary space, while retaining albumin and large proteins. The glomerular filtration barrier (GFB) is a highly specialized filtration interface between blood and urine that is highly permeable to small and midsized solutes in plasma but relatively impermeable to macromolecules such as albumin. The integrity of the GFB is maintained by molecular interplay between its 3 layers: the glomerular endothelium, the glomerular basement membrane and podocytes, which are highly specialized postmitotic pericytes forming the outer part of the GFB. Abnormalities of glomerular ultrafiltration lead to the loss of proteins in urine and progressive renal insufficiency, underlining the importance of the GFB. Indeed, albuminuria is strongly predictive of the course of chronic nephropathies especially that of diabetic nephropathy (DN), a leading cause of renal insufficiency. We found that high glucose concentrations promote autophagy flux in podocyte cultures and that the abundance of LC3B II in podocytes is high in diabetic mice. Deletion of Atg5 specifically in podocytes resulted in accelerated diabetes-induced podocytopathy with a leaky GFB and glomerulosclerosis. Strikingly, genetic alteration of autophagy on the other side of the GFB involving the endothelial-specific deletion of Atg5 also resulted in capillary rarefaction and accelerated DN. Thus autophagy is a key protective mechanism on both cellular layers of the GFB suggesting autophagy as a promising new therapeutic strategy for DN.