filtration barrier; MAP1LC3A/B/LC3A/B), microtubule-associated protein 1 light chain 3 a/b; MTOR, mechanistic target of rapamycin; Nphs2, nephrosis 2, podocin; SQSTM1, sequestosome 1; STZ, streptozotocin; TEM, transmission electron microscopy; TUBA, tubulin; a, WT1, Wilms tumor 1.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 endothelialspecific 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.

OBJECTIVEClass 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 METHODSWe 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.RESULTSWe 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.CONCLUSIONSWe 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.

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 g (PPARg) may prevent podocyte injury, but the function of glomerular PPARg in acute, severe inflammatory GN is unknown. Here, we observed marked loss of PPARg abundance and transcriptional activity in glomerular podocytes in experimental RPGN. Blunted expression of PPARg in podocyte nuclei was also found in kidneys from patients diagnosed with crescentic GN. Podocyte-specific Pparg gene targeting accentuated glomerular damage, with increased urinary loss of albumin and severe kidney failure. Furthermore, a PPARg gain-of-function approach achieved by systemic administration of thiazolidinedione (TZD) failed to prevent severe RPGN in mice with podocyte-specific Pparg gene deficiency. In nuclear factor erythroid 2-related factor 2 (NRF2)-deficient mice, loss of podocyte PPARg 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-PPARg 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-PPARg pathway may be a therapeutic target for RPGN. 27: 172-188, 201627: 172-188, . doi: 10.1681 Necrotizing and crescentic rapidly progressive glomerulonephritis (RPGN) is a heterogeneous condition characterized by rapidly declining kidney function. Left untreated, patients with RPGN often require longterm RRT. J Am Soc NephrolCharacteristic histologic features of RPGN include an irreversible loss of podocyte quiescence, aggravated endothelial injury, and the development of cellular crescents, all of which eventually lead to glomerular obsolescence. During crescent formation in mouse

Aims/hypothesisPancreatic cell development is a tightly controlled process. Although information is available regarding the mesodermal signals that control pancreatic development, little is known about the role of environmental factors such as nutrients, including glucose, on pancreatic development. We previously showed that glucose and its metabolism through the hexosamine biosynthesis pathway (HBP) promote pancreatic endocrine cell differentiation. Here, we analysed the role of the transcription factor carbohydrate-responsive element-binding protein (ChREBP) in this process. This transcription factor is activated by glucose, and has been recently described as a target of the HBP.MethodsWe used an in vitro bioassay in which pancreatic endocrine and exocrine cells develop from rat embryonic pancreas in a way that mimics in vivo pancreatic development. Using this model, gain-of-function and loss-of-function experiments were undertaken.ResultsChREBP was produced in the endocrine lineage during pancreatic development, its abundance increasing with differentiation. When rat embryonic pancreases were cultured in the presence of glucose or xylitol, the production of ChREBP targets was induced. Concomitantly, beta cell differentiation was enhanced. On the other hand, when embryonic pancreases were cultured with inhibitors decreasing ChREBP activity or an adenovirus producing a dominant-negative ChREBP, beta cell differentiation was reduced, indicating that ChREBP activity was necessary for proper beta cell differentiation. Interestingly, adenovirus producing a dominant-negative ChREBP also reduced the positive effect of N-acetylglucosamine, a substrate of the HBP acting on beta cell differentiation.Conclusions/interpretationOur work supports the idea that glucose, through the transcription factor ChREBP, controls beta cell differentiation from pancreatic progenitors.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-012-2623-0) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

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.

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 tumoral tissue. We found that the size of hepatoblastoma cells correlates with the size of their nucleus, cytoplasm and mitochondrial mass. We also discovered that the blood capillary controls 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 new investigations in an emerging field that we call onconanotomy.

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 considerable progress in understanding the biology and genetics of cancer, the development of effective therapies is hampered by the lack of sufficient experimental models that recapitulate the genetic diversity of this disease. The recourse to patient-derived xenograft (PDX) for the evaluation of new candidate anticancer drugs is becoming the gold standard in preclinical oncology. The faithful reproduction of patients’ cancer features, and the possibility to generate a large number of models that recapitulate patient population genetic heterogeneity, confer PDXs a critical added value in the evaluation of new candidate drugs. These improved models will hopefully contribute to decrease the attrition rate observed in clinical trials, thus far unacceptably high. Over the last 15 years, we have generated and characterized a collection of 200+ PDXs from different solid tumors that accurately reproduce the histological and molecular heterogeneity of the tumors of origin. This panel has allowed for the preclinical validation of several anticancer drugs that are now used in the clinic. Although being an indispensable tool to complete preclinical studies, the use of PDX in vivo systems for large-scale screening during early drug discovery is hampered by ethical, economical and throughput burdens limiting the number of test articles being tested. To address this problem, we developed a panel of PDX-derived cell lines (PDXDCs) that we propose as a time and cost-effective medium-throughput screening tool to profile the anti-cancer activity of early test compounds. To date, 50+ PDXDCs from various indications such as breast, lung, prostate and many others have been generated and tested for their response in vitro towards standards of care and targeted anti-cancer agents matching patient clinical management. Differently from standard cell line establishment, which is obtained by expansion of a cell clone that survives in vitro plating, our cell line development technology allows for maintenance of tumor cell population heterogeneity. PDXDCs RNA and exome sequencing data faithfully match the parental PDX features, and by modulating experimental parameters, such as 2D or 3D growth conditions, drug exposure duration and endpoint read-outs, we could phenocopy in vitro the corresponding PDXs’ sensitivities to chemotherapies. These results show our PDXDCs panel is a valuable in vitro platform for drug screening to help selecting drug candidates for further validation in parental PDX models in vivo. Citation Format: Olivier Déas, Léa Sinayen, Emilie Indersie, Kathleen Flosseau, Sophie Banis, Enora Le Ven, Jean-Gabriel Judde, Stefano Cairo. PDX-derived cell line platform for pharmacological screening and functional studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1637.