Takashi Aoi

Overactivated microglia play a key role in sepsis-associated encephalopathy (SAE), although the involvement of T cells is unclear. γδT cells in the brain and meninges regulate normal fear responses via interleukin (IL)-17 in healthy mice. In our sepsis model, the mice showed exacerbated anxious behavior at 10 days post-induction (dpi). At 8 dpi, IL-17 mRNA was significantly upregulated in the brains of septic mice compared with those of control mice. Simultaneously, the number of γδT cells increased in the brains of septic mice in a severity-dependent manner. Additionally, IL-17-producing γδT cells, expressing both the C-X-C motif receptor (CXCR) 6 and the C-C motif receptor (CCR) 6, increased in mice brains, dependent on the severity of sepsis. The frequency of γδT cells in the meninges fluctuated similarly to that in the brain, peaking at 8 dpi of sepsis. Behavioral tests were performed on septic mice after the continuous administration of anti-γδTCR (α-γδTCR) or anti-IL-17A (α-IL-17A) antibodies to deplete the γδT cells and IL-17A, respectively. Compared with IgG-treated septic mice, α-γδTCR- and α-IL-17A-treated septic mice showed suppressed microglial activation and improvements in anxious behavior. These results suggested that CCR6⁺CXCR6⁺ IL-17-producing γδT cells in the brain and meninges promote the exacerbation of SAE and sepsis-induced psychological disorders in mice.

Background: Our study and several studies have reported that in some cancers, including pancreatic ductal adenocarcinoma (PDAC), the expression of squamous lineage markers, such as esophagus-tissue-specific genes, correlated with a poor prognosis. However, the mechanism by which the acquisition of squamous lineage phenotypes leads to a poor prognosis remains unclear. We previously reported that retinoic acid signaling via retinoic acid receptor γ (RARγ signaling) determines the differentiation lineage into the esophageal squamous epithelium. These findings hypothesized that the activation of RARγ signaling contributed to acquiring squamous lineage phenotypes and malignant behavior in PDAC. Methods: This study utilized public databases and immunostaining of surgical specimens to examine RARγ expression in PDAC. We evaluated the function of RARγ signaling by inhibitors and siRNA knockdown using a PDAC cell line and patient-derived PDAC organoids. The mechanism of the tumor-suppressive effects by blocking RARγ signaling was examined by a cell cycle analysis, apoptosis assays, RNA sequencing and Western blotting. Results: RARγ expression in pancreatic intraepithelial neoplasia (PanIN) and PDAC was higher than that in the normal pancreatic duct. Its expression correlated with a poor patient prognosis in PDAC. In PDAC cell lines, blockade of RARγ signaling suppressed cell proliferation by inducing cell cycle arrest in the G1 phase without causing apoptosis. We demonstrated that blocking RARγ signaling upregulated p21 and p27 and downregulated many cell cycle genes, including cyclin-dependent kinase 2 (CDK2), CDK4 and CDK6. Furthermore, using patient-derived PDAC organoids, we confirmed the tumor-suppressive effect of RARγ inhibition and indicated the synergistic effects of RARγ inhibition with gemcitabine. Conclusions: This study clarified the function of RARγ signaling in PDAC progression and demonstrated the tumor-suppressive effect of selective blockade of RARγ signaling against PDAC. These results suggest that RARγ signaling might be a new therapeutic target for PDAC.

Background: The chorioallantoic membrane (CAM) assay is a well-established technique to evaluate tumor invasion and angiogenesis and may overcome the shortcoming of the patient-derived xenograft (PDX) mouse model. Currently, few reports have described lung cancer invasion and angiogenesis in the CAM assay. We therefore used the CAM assay in the evaluation of lung cancer. Method: Lung cancer cell line-derived organoids or lung cancer cell lines were transplanted into the CAM on embryonic development day (EDD) 10, and an analysis was performed on EDD 15. Microscopic and macroscopic images and movies of the grafts on the CAM were captured and analyzed. The relationships between the graft and chick vessels were evaluated using immunohistochemistry. Results: We transplanted lung cancer cell lines and cell line-derived organoid into a CAM to investigate angiogenesis and invasion. They engrafted on the CAM at a rate of 50–83%. A549-OKS cells showed enhanced cell invasion and angiogenesis on the CAM in comparison to A549-GFP cells as was reported in vitro. Next, we found that A549-TIPARP cells promoted angiogenesis on the CAM. RNA-seq identified 203 genes that were upregulated more than twofold in comparison to A549-GFP cells. A pathway analysis revealed many upregulated pathways related to degradation and synthesis of the extracellular matrix in A549-TIPARP cells. Conclusions: The CAM assay can be used to evaluate and research invasion and angiogenesis in lung cancer. The elevated expression of TIPARP in lung cancer may induce angiogenesis by remodeling the extracellular matrix.