Tumor budding is a major risk factor for T1 colorectal cancer. Quality control of the pathological diagnosis of budding is crucial, irrespective of the pathologist's experience. This study examines the interobserver variability according to pathologists' experience and evaluates the influence of cytokeratin (CK) immunostaining in the assessment of budding. Hematoxylin-eosin (HE) and CK-immunostained slides of 40 cases with T1 primary colorectal cancer were examined. Budding grades were individually evaluated by 12 pathologists who we categorized into three groups by their experience (expert, with >10 years of experience (n = 4), senior, with 5-10 years (n = 4), and junior, < 5 years (n = 4)). The results revealed a tendency for the more experienced pathologists to assign higher budding grades compared to the less-experienced pathologists. In the junior group, the interobserver variability obtained with HE slides was poor, but it was markedly improved in the evaluation using CK-immunostained slides. The benefit of CK immunostaining was only slight in the expert group. CK immunostaining would be useful when a pathologist is not experienced enough or does not have enough confidence in the assessment of budding.

Gastric cancer grows under a hypoxic environment. HIF-1α is known to play an important role in controlling the production of reactive oxygen species (ROS) in the mitochondria under hypoxic conditions. We previously established HIF-1α knockdown (KD) cells and control (SC) cells in the 58As9 gastric cancer cell line. In this study, we revealed that KD cells, but not SC cells, induced apoptosis under conditions of hypoxia (1% O2) due to excessive production of ROS. A quantitative RT-PCR analysis demonstrated that the expressions of ten genes, which are involved in the control mechanisms of ROS (including the Warburg effect, mitophagy, electron transport chain [ETC] modification and ROS scavenging), were regulated by HIF-1α. Moreover, the promotion of glucose uptake by glucose plus insulin (GI) treatment enhanced the apoptotic effect, which was accompanied by further ROS production in hypoxic KD cells. A Western blot analysis showed that the membranous expression of GLUT1 in KD cells was elevated by glucose and/or insulin treatments, indicating that the GI-induced glucose uptake is mediated by the increased translocation of GLUT1 on the cell membrane. Finally, the anti-tumor effect of HIF-1α knockdown (KD) plus GI was evaluated using a tumor xenograft model, where a hypoxic environment naturally exists. As a result, the GI treatment strongly inhibited the growth of the KD tumors whereby cell apoptosis was highly induced in comparison to the control treatment. In contrast, the growth of the SC tumors expressing HIF-1α was not affected by the GI treatment. Taken together, the results suggest that HIF-1α inhibition plus GI may be an ideal therapy, because the apoptosis due to the destruction of ROS homeostasis is specifically induced in gastric cancer that grows under a hypoxic environment, but not in the normal tissue under the aerobic conditions.

Adipose tissue, together with the mesothelial layer and microvessels, is a major component of the mesenteric peritoneum, and the mesenterium is a target site for peritoneal fibrosis. Adipose tissue has been speculated to play a role in peritoneal dialysis (PD)-related fibrosis, but the precise cellular kinetics of adipose tissue during this process remain to be determined. To clarify this critical issue, we analyzed the kinetics of adipose tissue using a novel peritoneal reconstruction model in which the effects of mesothelial cells or endothelial cells could be identified. Adipose tissue was co-cultured with mesothelial cells or endothelial cells in a combined organ culture and fluid flow stress culture system. Spindle mesenchymal cells and immature adipocytes derived from adipose tissue were characterized by immunohistochemistry. Adipose tissue fragments cultured in this system yielded many spindle mesenchymal cells in non-co-culture conditions. However, the number of spindle mesenchymal cells emerging from adipose tissue was reduced in co-culture conditions with a covering layer of mesothelial cells. Mesothelial cells co-cultured in the separated condition did not inhibit the emergence of spindle mesenchymal cells from adipose tissue. Interestingly, endothelial cells promoted the emergence of lipid-laden immature adipocytes from adipose tissue under fluid flow stress. We have demonstrated that adipose tissue behavior is not only regulated by mesothelial cells and endothelial cells under fluid flow stress, but is also involved in fibrosis and fat mass production in the peritoneum. Our findings suggest that adipose tissue is a potential source of cells for peritoneal fibrosis caused by PD therapy.

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