Establishment of a novel experimental model for muscle-invasive bladder cancer using a dog bladder cancer organoid culture.

In human and dogs, bladder cancer (BC) is the most common neoplasm affecting the urinary tract. Dog BC resembles human muscle-invasive BC in histopathological characteristics and gene expression profiles, and could be an important research model for this disease. Cancer patient-derived organoid culture can recapitulate organ structures and maintains the gene expression profiles of original tumor tissues. In a previous study, we generated dog prostate cancer organoids using urine samples, however dog BC organoids had never been produced. Therefore we aimed to generate dog BC organoids using urine samples and check their histopathological characteristics, drug sensitivity, and gene expression profiles. Organoids from individual BC dogs were successfully generated, expressed urothelial cell markers (CK7, CK20, and UPK3A) and exhibited tumorigenesis in vivo. In a cell viability assay, the response to combined treatment with a range of anticancer drugs (cisplatin, vinblastine, gemcitabine or piroxicam) was markedly different in each BC organoid. In RNA-sequencing analysis, expression levels of basal cell markers (CK5 and DSG3) and several novel genes (MMP28, CTSE, CNN3, TFPI2, COL17A1, and AGPAT4) were upregulated in BC organoids compared with normal bladder tissues or two-dimensional (2D) BC cell lines. These established dog BC organoids might be a useful tool, not only to determine suitable chemotherapy for BC diseased dogs but also to identify novel biomarkers in human muscle-invasive BC. In the present study, for the 1st time, dog BC organoids were generated and several specifically upregulated organoid genes were identified. Our data suggest that dog BC organoids might become a new tool to provide fresh insights into both dog BC therapy and diagnostic biomarkers.

Inhibition of autophagy with expression of NADPH oxidase subunit p22phox in preneoplastic lesions in a high-fat diet and streptozotocin-related hepatocarcinogenesis rat model.

To study the effects of autophagy inducer carbamazepine (CBZ) in a high-fat diet (HFD)/streptozotocin (STZ)-related early hepatocarcinogenesis model, we determined autophagic flux by immunohistochemical analysis of autophagy marker expression in preneoplastic liver foci and compared that with the expression of the NADPH oxidase subunit. Male F344 rats were fed a basal diet or HFD and subjected to two-stage hepatocarcinogenesis; diabetes mellitus was induced via STZ administration. Several STZ-treated, HFD-fed rats were administered CBZ (a total of five doses every one or two days) at week 7 and 8. STZ-treated, HFD-fed rats decreased ß cells in the islet of Langerhans and increased adipophilin-positive lipid droplets in the liver; moreover, they had a larger area of glutathione S-transferase placental form-immunopositive preneoplastic liver foci, which was associated with inhibition of autophagy and induction of the NADPH oxidase subunit, as demonstrated by increased immunohistochemical expression of an autophagosome receptor marker microtubule-associated protein light chain 3 (LC3)-binding protein p62, and of an NADPH oxidase subunit p22phox in the preneoplastic foci. An increased trend of an autophagy phagophore marker LC3 in preneoplastic foci was also detected. CBZ administration could induce autophagy and impair p22phox expression, as shown by altered expression of autophagy regulators (Atg5, Atg6, Lamp1, Lamp2, and Lc3), NADPH oxidase subunits (P22phox and P67phox), and antioxidant enzymes Gpx1 and Gpx2. These results suggest that inhibition of autophagy and induction of p22phox might contribute to HFD/STZ-related early hepatocarcinogenesis in rats; however, the effects of CBZ administration on the STZ/HFD-increased preneoplastic foci were marginal in this study.

Immunohistochemical expression of autophagosome markers LC3 and p62 in preneoplastic liver foci in high fat diet-fed rats.

Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive deposition of droplets in hepatocytes. Patients with NAFLD can be at risk for nonalcoholic steatohepatitis, which can lead to hepatocellular carcinoma. Autophagy is a cellular pathway that is crucial for survival and homeostasis, and which protects against pathophysiological changes like obesity and cancer. We determined the expression of autophagy markers in preneoplastic hepatic lesions and the effects of an autophagy repressor chloroquine (CQ) or inducer amiodarone (AM) in a steatosis-related hepatocarcinogenesis model. Male F344 rats were fed a control diet or high fat diet (HFD), and subjected to initiation and promotion steps with N-nitrosodiethylamine injection at week 0 and a partial hepatectomy at week 3. Several HFD-fed rats were administered 0.1% CQ and 0.5% AM in their drinking water during week 2 and 8. CQ and AM did not improve HFD-induced obesity. AM, but not CQ, significantly decreased the number of glutathione S-transferase placental form-positive preneoplastic liver foci in the liver. Autophagosome markers LC3 and the LC3-binding protein p62 were heterogeneously expressed in the preneoplastic foci. CQ might inhibit autophagy by significantly increased p62/LC3 ratio, while AM might have a potential of inducing autophagy by showing an increased gene expression of the autophagy regulator, Atg5. These results suggest that preneoplastic lesions express autophagosome markers and that AM might decrease steatosis-related early hepatocarcinogenesis by potentially inducing autophagy in HFD-fed rats, while inhibition of autophagy by CQ did not alter the hepatocarcinogenesis. However, an immunohistochemical trial revealed a technical limitation in detecting autophagosome markers because there were variations in each preneoplastic lesion.

Ameliorating effect of postweaning exposure to antioxidant on disruption of hippocampal neurogenesis induced by developmental hypothyroidism in rats.

Developmental hypothyroidism as a model of autism spectrum disorders disrupts hippocampal neurogenesis through the adult stage. The present study investigated the ameliorating effect of postweaning exposure to antioxidant on the hypothyroidism-induced disruptive neurogenesis. Mated female Sprague-Dawley rats were treated with 0 or 10 ppm 6-propyl-2-thiouracil (PTU) as an anti-thyroid agent in drinking water from gestational day 6 to postnatal day (PND) 21 on weaning. PTU-exposed male offspring were fed either basal diet, diet containing α-glycosyl isoquercitrin (AGIQ) at 5,000 ppm or α-lipoic acid (ALA) at 1,000 ppm as an antioxidant from PND 21 to PND 77. PTU-exposure decreased DCX+ and NeuN+ granule cell lineage subpopulations, synaptic plasticity-related FOS+ granule cells, and hilar PVALB+ and GAD67+ GABAergic interneurons, increased hilar SST+ and CALB2+ interneurons, and upregulated Gria3, Otx2, and antioxidant enzyme genes in the dentate gyrus on PND 77. These results suggest disruption of neurogenesis remained in relation with increase of oxidative stress and compensatory responses to the disruption at the adult stage. AGIQ recovered expression of some antioxidant enzyme genes and was effective for restoration of NeuN+ postmitotic granule cells and PVALB+ and SST+ interneurons. In contrast, ALA was effective for restoration of all interneuron subpopulations, as well as postmitotic granule cells, and upregulated Grin2a that may play a role for the restoration. Both antioxidants recovered expression of Otx2 and AGIQ-alone recovered Gria3, suggesting a reversal of disruptive neurogenesis by compensatory responses. Thus, postweaning antioxidant exposure may be effective for ameliorating developmental hypothyroidism-induced disruptive neurogenesis by restoring the function of regulatory system.