Clinical utility of a comprehensive genomic profiling test for patient with advanced biliary tract cancer.

BACKGROUND: Biliary tract cancer (BTC) comprises a heterogeneous group of malignancies with poor prognosis because of the limited treatment options. With the recent advances of next generation sequencing technologies, comprehensive genomic profiling (CGP) tests have been widely introduced into daily clinical practice. PATIENTS AND METHODS: We performed a retrospective, multicenter, observation cohort study. The genomic and clinical data of 85 BTC patients, who underwent CGP testing from August 2021 to September 2023, were analyzed. RESULTS: There were 62 (73%) cases in which treatment recommendations were raised during expert meetings, including 34 intrahepatic cholangiocarcinoma (ICC), 20 extrahepatic cholangiocarcinoma (ECC) and 8 gall bladder carcinoma (GBC). The drug accessibility rate of the BTC patients was 15.3% (13 cases): ten ICCs, two ECCs, and one GBC. Five ICC patients (three male and two female) with the FGFR2 fusion gene were treated with pemigatinib. Those patients who received a genomically matched therapy had significantly longer median overall survival than those patients who not received. (n = 13; not reached [95% CI not reached-not reached] vs n = 72; 8.6 months [95% CI 6.6-10.0]; hazard ratio 0.24 [95% CI 0.12-0.49], p = 0.013). The median observation period of pemigatinib treatment was 15.4 months (range 10.1-27.4). The responses were classified as PR in three patients, SD in one patient and PD in one patient. The median progression free survival is 9.0 months. No patient had grade 3/4 AEs requiring discontinuation of the treatment. CONCLUSION: The drug accessibility rate of ICC is high and pemigatinib is effective and well-tolerated in ICC patients harboring FGFR2 gene fusions.

The expression analysis of SerpinB9 in hepatoblastoma microenvironment.

PURPOSE: In this research, we analyzed the expression of serpinB9 in hepatoblastoma and investigated the factors which enhance its expression. METHOD: SerpinB9 expression in hepatoblastoma cell lines and macrophages co-cultured with each other or stimulated by anticancer agents was examined using RT-qPCR and western blotting. Immunohistochemistry for SerpinB9 in hepatoblastoma specimens was performed. Single-cell RNA-sequence data for hepatoblastoma from an online database were analyzed to investigate which types of cells express SerpinB9. RESULT: HepG2, a hepatoblastoma cell line, exhibited increased expression of SerpinB9 when indirectly co-cultured with macrophages. Immunohistochemistry for the specimens demonstrated that serpinB9 is positive not in hepatoblastoma cells but in macrophages. Single-cell RNA sequence analysis in tissues from hepatoblastoma patients showed that macrophages expressed SerpinB9 more than tumor cells did. Co-culture of macrophages with hepatoblastoma cell lines led to the enhanced expression of SerpinB9 in both macrophages and cell lines. Anticancer agents induced an elevation of SerpinB9 in hepatoblastomas cell lines. CONCLUSION: In hepatoblastoma, SerpinB9 is thought to be more highly expressed in macrophages and enhanced by interaction with hepatoblastoma cell.

Macrophage colony-stimulating factor potentially induces recruitment and maturation of macrophages in recurrent pituitary neuroendocrine tumors.

Although pituitary neuroendocrine tumors (PitNETs) are usually benign, some are highly invasive and recurrent. Recurrent PitNETs are often treatment-resistant and there is currently no effective evidence-based treatment. Tumor-associated macrophages (TAMs) promote tumor growth in many cancers, but the effect of TAMs on PitNETs remains unclear. This study investigated the role of TAMs in the incidence of recurrent PitNETs. Immunohistochemical analysis revealed that the densities of CD163- and CD204-positive TAMs tended to increase in recurrent PitNETs. Compared with TAMs in primary lesions, those in recurrent lesions were enlarged. To clarify the cell-cell interactions between TAMs and PitNETs, in vitro experiments were performed using a mouse PitNET cell line AtT20 and the mouse macrophage cell line J774. Several cytokines related to macrophage chemotaxis and differentiation, such as M-CSF, were elevated significantly by stimulation with macrophage conditioned medium. When M-CSF immunohistochemistry analysis was performed using human PitNET samples, M-CSF expression increased significantly in recurrent lesions compared with primary lesions. Although no M-CSF receptor (M-CSFR) expression was observed in tumor cells of primary and recurrent PitNETs, flow cytometric analysis revealed that the mouse PitNET cell line expressed M-CSFR. Cellular proliferation in mouse PitNETs was inhibited by high concentrations of M-CSFR inhibitors, suggesting that cell-to-cell communication between PitNETs and macrophages induces M-CSF expression, which in turn enhances TAM chemotaxis and maturation in the tumor microenvironment. Blocking the M-CSFR signaling pathway might be a novel therapeutic adjuvant in treating recurrent PitNETs.

Potential involvement of IL-32 in cell-to-cell communication between macrophages and hepatoblastoma.

PURPOSE: This study investigated the expression of interleukin 32 (IL-32) in hepatoblastoma, the most common primary pediatric liver tumor, and its possible roles in tumorigenesis. METHODS: IL-32 expression was investigated in two hepatoblastoma cell lines (Hep G2 and HuH 6) in the steady state and after co-culture with macrophages by RNA-seq analysis and RT-qPCR, and after stimulation with chemotherapy. Cultured macrophages were stimulated by IL-32 isoforms followed by RT-qPCR and western blot analysis. IL-32 immunohistochemical staining (IHC) was performed using specimens from 21 hepatoblastoma patients. Clustering analysis was also performed using scRNA-seq data downloaded from Gene Expression Omnibus. RESULTS: The IL-32 gene is expressed by hepatoblastoma cell lines; expression is upregulated by paracrine cell-cell communication with macrophages, also by carboplatin and etoposide. IL-32 causes protumor activation of macrophages with upregulation of PD-L1, IDO-1, IL-6, and IL-10. In the patient pool, IHC was positive only in 48% of cases. However, in the downloaded dataset, IL-32 gene expression was negative. CONCLUSION: IL-32 was detected in hepatoblastoma cell lines, but not in all hepatoblastoma patients. We hypothesized that stimulation such as chemotherapy might induce expression of IL-32, which might be a critical mediator of chemoresistance in hepatoblastoma through inducing protumor activation in macrophages.

The expression of PD-1 ligand 1 on macrophages and its clinical impacts and mechanisms in lung adenocarcinoma.

Programmed cell death-1 (PD-1) and PD-1 ligand 1 (PD-L1) are target molecules for immunotherapy in non-small cell lung cancer. PD-L1 is expressed not only in cancer cells, but also on macrophages, and has been suggested to contribute to macrophage-mediated immune suppression. We examined the clinical significance of PD-L1 expression on macrophages in human lung adenocarcinoma. The mechanism of PD-L1 overexpression on macrophages was investigated by means of cell culture studies and animal studies. The results showed that high PD-L1 expression on macrophages was correlated with the presence of EGFR mutation, a lower cancer grade, and a shorter cancer-specific overall survival. In an in vitro study using lung cancer cell lines and human monocyte-derived macrophages, the conditioned medium from cancer cells was found to up-regulate PD-L1 expression on macrophages via STAT3 activation, and a cytokine array revealed that granulocyte-macrophage colony-stimulating factor (GM-CSF) was a candidate factor that induced PD-L1 expression. Culture studies using recombinant GM-CSF, neutralizing antibody, and inhibitors indicated that PD-L1 overexpression was induced via STAT3 activation by GM-CSF derived from cancer cells. In a murine Lewis lung carcinoma model, anti-GM-CSF therapy inhibited cancer development via the suppression of macrophage infiltration and the promotion of lymphocyte infiltration into cancer tissue; however, the PD-L1 expression on macrophages remained unchanged. PD-L1 overexpression on macrophages via the GM-CSF/STAT3 pathway was suggested to promote cancer progression in lung adenocarcinoma. Cancer cell-derived GM-CSF might be a promising target for anti-lung cancer therapy.

M-CSFR expression in the embryonal component of hepatoblastoma and cell-to-cell interaction between macrophages and hepatoblastoma.

Tumor-associated macrophages (TAMs) have protumor functions in various cancers. However, their significance in hepatoblastoma, the most common liver tumor in children, remains unclear. The aim of this study was to explore the potential roles of TAMs in hepatoblastoma. Immunohistochemical analysis revealed that the density of CD204-positive TAMs was significantly higher in the embryonal component than in other histological subtypes of hepatoblastoma. An in vitro co-culture study with Huh6 cells and human monocyte-derived macrophages (HMDMs) showed that macrophage-colony-stimulating factor receptor (M-CSFR) was strongly up-regulated in the Huh6 cells that were directly co-cultured with HMDMs. The expressions of M-CSFR ligands (interleukin-34 and M-CSF) were also increased by co-culture with HMDMs. The proliferation of HepG2 cells (another hepatoblastoma cell line expressing M-CSFR) was inhibited by an M-CSFR inhibitor. M-CSFR was found to be highly expressed in the embryonal component and in recurrent lesions. The number of CD204-positive macrophages was also higher in the M-CSFR-positive areas than in the M-CSFR-negative areas. Thus, M-CSFR expression appeared to be induced by cell-cell contact with macrophages in hepatoblastoma cells, and M-CSFR inhibitor is potentially effective against M-CSFR-positive hepatoblastoma, especially recurrent cases.

Bactericidal effect of antimicrobial photodynamic therapy (aPDT) on dentin plate infected with Lactobacillus acidophilus.

This study aimed to examine bactericidal effects of a new antimicrobial photodynamic therapy (aPDT) on dentin plates infected with Lactobacillus acidophilus (L. acidophilus). First, we measured the amount of reactive oxygen species (ROS) produced when new photosensitizer (PS), acid red (AR), and brilliant blue (BB) were irradiated with a semiconductor laser. ROS generated from each PS solution by laser irradiation was calculated as the total light emission amount (Relative Light Unit, RLU) using a chemiluminescence measuring device. Second, we examined bactericidal effects of the aPDT on dentin plates infected with L. acidophilus. The bactericidal effects on each group were evaluated by colony count assay and adenosine triphosphate assay. The experimental groups comprised two laser irradiation groups (650 nm laser, 650laser; and 940 nm laser, 940laser), two PS groups (BB and AR), four aPDT groups (650 nm laser irradiation with BB, 650laser-BB; 650 nm laser irradiation with AR, 650laser-AR; 940 nm laser irradiation with BB, 940laser-BB; 940 nm laser irradiation with AR, 940laser-AR), and a control. The ROS in all aPDT groups was significantly higher than in the control. RLU in all groups applied with laser irradiation was significantly lower than that in the control. However, only 650laser-BB showed significantly lower colony counts than the control. 650laser-BB was the most effective in sterilizing the infected dentin plates.

Clinical impact of TROP2 in non-small lung cancers and its correlation with abnormal p53 nuclear accumulation.

Tumor-associated calcium signal transducer 2 (TROP2) is a cell-surface glycoprotein involved in the high malignant potential of several cancers. Antibody-drug conjugates that target TROP2 represent a promising approach for the treatment of TROP2-expressing cancers including lung cancer and breast cancer. TROP2 expression was tested by immunohistochemistry in lung adenocarcinoma (ADC) and squamous cell carcinoma samples, and its correlation with clinicopathological factors, including survival rate and p53 mutation, was statistically analyzed. We found that increased TROP2 expression was significantly associated with a poor clinical course in patients with ADC, but not in patients with squamous cell carcinoma. A more significant association with poor outcome was seen in ADC cases with a high histological grade as well as those without the epidermal growth factor receptor (EGFR) mutation. A significant correlation between TROP2 expression and abnormal p53 nuclear accumulation/expression was also found in ADC. In the present study, we discovered a significant correlation between TROP2 expression and p53 mutation in ADC, and that TROP2 expression was a prognostic factor in ADC cases with a high histological grade as well as those without the EGFR mutation. Signals mediated by mutated p53 might influence TROP2 expression in ADC.

The duodenal tube test is more specific than hepatobiliary scintigraphy for identifying bile excretion in the differential diagnosis of biliary atresia.

PURPOSE: Confirmation of bile excretion into the gastrointestinal tract is important to exclude biliary atresia (BA). We compared the duodenal tube test (DTT) with hepatobiliary scintigraphy (HS) for their efficiency in detecting bile secretion. METHODS: The subjects of this retrospective study were 47 infants who underwent both DTT and HS to diagnose or exclude BA between January 2000 and March 2018. RESULTS: BA was diagnosed in 32 of the 47 patients, and 7 of the remaining 15 non-BA patients underwent intraoperative cholangiography. Among the various DTT parameters, the total bile acid in duodenal fluid (DF-TBA)/serum (S) gamma-glutamyl transferase (γGTP) ratio was found to be the most specific for BA, with sensitivity and specificity of 98.0-100%, respectively. One BA patient in whom cut off values were not met was a premature infant. The sensitivity and specificity of HS were 100-56.3%, respectively. The diagnostic accuracy of the DF-TBA/S-γGTP parameter was higher than that of HS (98.6% vs. 85.1%, respectively). CONCLUSIONS: The DTT could be more a specific method than HS to detect bile excretion. Thus, the DTT should be incorporated into the multidisciplinary diagnostic approach for the differential diagnosis of BA to prevent unnecessary intraoperative cholangiography in patients who do not have BA.

Hydrogen-rich solution attenuates cold ischemia-reperfusion injury in rat liver transplantation.

BACKGROUND: Liver transplantation (LT) is considered the standard treatment for end-stage liver disease, but ideal donors remain in limited supply, resulting in an unavoidable increase in the need to use grafts from marginal donors. The attenuation of ischemia-reperfusion injury (IRI) in such marginal donors is therefore crucial for reducing the possibility of the primary non-function of grafts and graft loss. Some reports have found that molecular-hydrogen showed antioxidant and anti-inflammatory effects in preventing IRI in some non-hepatic transplant models. Therefore, we investigated whether or not molecular-hydrogen could attenuate IRI in LT model rats. METHODS: We used a hydrogen-rich water bath to dissolve hydrogen into solution and graft tissues and performed isogenic and orthotopic LT in Lewis rats with University of Wisconsin (UW) solution. Blood and tissue samples were collected 6 h after the reperfusion. Hepatic enzymes in serum were measured. Pathological findings including the expressions of cytokines and heme oxygenase (HO)-1 in liver tissues were evaluated. RESULTS: The concentration of hydrogen inside the graft tissues increased depending on the storage time, plateauing after 1 h. Serum liver enzyme levels were significantly lower and the histology score of liver damage markedly attenuated in the group given grafts preserved in hydrogen-rich UW solution than in the control group. The hydrogen-rich UW solution group also showed less oxidative damage and hepatocyte apoptosis than the control group, and the expression of proinflammatory cytokines tended to be lower while the protein levels of HO-1 were significantly increased (n = 3-12 per group, P < 0.05). CONCLUSIONS: Storage of liver grafts in hydrogen-rich UW solution resulted in superior functional and morphologic protection against IRI via the up-regulation of HO-1 expression.

Intravital Imaging of Neutrophil Recruitment Reveals the Efficacy of FPR1 Blockade in Hepatic Ischemia-Reperfusion Injury.

Neutrophils are considered responsible for the pathophysiological changes resulting from hepatic ischemia-reperfusion (I/R) injury, which is a complication of trauma, shock, liver resection, and transplantation. Recently, evidence is accumulating that formyl-peptide receptor (FPR) signaling constitutes an important danger signal that guides neutrophils to sites of inflammation. This study aimed to investigate dynamic neutrophil recruitment using two-photon laser-scanning microscopy (TPLSM) in response to FPR1 blockade during hepatic I/R. LysM-eGFP mice were subjected to partial warm hepatic I/R. They were pretreated with an FPR1 antagonist, cyclosporine H (CsH), or formyl peptide, fMLF. Liver was imaged after hepatic laser irradiation or I/R using the TPLSM technique. CsH treatment alleviated hepatic I/R injury, as evidenced by decreased serum transaminase levels, reduced hepatocyte necrosis/apoptosis, and diminished inflammatory cytokine, chemokine, and oxidative stress. In contrast, systemic administration of fMLF showed few effects. Time-lapse TPLSM showed that FPR1 blockade inhibited the accumulation of neutrophils in the necrotic area induced by laser irradiation in vivo. In the CsH-treated I/R group, the number and crawling velocity of neutrophils in the nonperfused area were lower than those in the control group. Meanwhile, FPR1 blockade did not affect monocyte/macrophage recruitment. Hepatic I/R promoted the retention of neutrophils and their active behavior in the spleen, whereas CsH treatment prevented their changes. Intravital TPLSM revealed that formyl-peptide-FPR1 signaling is responsible for regulating neutrophil chemotaxis to allow migration into the necrotic area in hepatic I/R. Our findings suggest effective approaches for elucidating the mechanisms of immune cell responses in hepatic I/R.

Intravital imaging of neutrophil recruitment in intestinal ischemia-reperfusion injury.

BACKGROUND: Neutrophils are known to be key players in innate immunity. Activated neutrophils induce local inflammation, which results in pathophysiologic changes during intestinal ischemia-reperfusion injury (IRI). However, most studies have been based on static assessments, and few have examined real-time intravital neutrophil recruitment. We herein report a method for imaging and evaluating dynamic changes in the neutrophil recruitment in intestinal IRI using two-photon laser scanning microscopy (TPLSM). METHODS: LysM-eGFP mice were subjected to 45 min of warm intestinal ischemia followed by reperfusion. Mice received an intravenous injection of tetramethylrhodamine isothiocyanate-labeled albumin to visualize the microvasculature. Using a time-lapse TPLSM technique, we directly observed the behavior of neutrophils in intestinal IRI. RESULTS: We were able to image all layers of the intestine without invasive surgical stress. At low-magnification, the number of neutrophils per field of view continued to increase for 4 h after reperfusion. High-magnification images revealed the presence or absence of blood circulation. At 0-2 h after reperfusion, rolling and adhesive neutrophils increased along the vasculature. At 2-4 h after reperfusion, the irregularity of crypt architecture and transmigration of neutrophils were observed in the lamina propria. Furthermore, TPLSM imaging revealed the villus height, the diameters of the crypt, and the number of infiltrating neutrophils in the crypt. In the IRI group, the villus height 4 h after reperfusion was significantly shorter than in the control group. CONCLUSIONS: TPLSM imaging revealed the real-time neutrophil recruitment in intestinal IRI. Z-stack imaging was useful for evaluating pathophysiological changes in the intestinal wall.

Outcomes of treatment with daclatasvir and asunaprevir for recurrent hepatitis C after liver transplantation.

AIM: The development of direct-acting oral agents has dramatically changed the treatment strategy of hepatitis C virus (HCV) infection. Here we aimed to reveal the efficacy and safety of daclatasvir (DCV) and asunaprevir (ASV) for recurrent HCV genotype 1 infection after liver transplantation (LT). METHODS: A retrospective study was undertaken on nine patients who underwent a 24-week DCV/ASV treatment regimen for recurrent HCV genotype 1 infection. Five of the patients were men; four had failed treatment with pegylated interferon (Peg-IFN)/ribavirin, two had failed simeprevir/Peg-IFN/ribavirin, one had the resistance-associated variant Y93H in the NS5A region, and one underwent maintenance dialysis. RESULTS: Median time to treatment initiation following LT was 70 months. Of the nine patients treated with DCV/ASV, eight (88.9%) achieved a sustained viral response 12 weeks after completion of therapy (SVR12). The patient with virologic failure had failed simeprevir/Peg-interferon/ribavirin therapy 4 months before undergoing the DCV/ASV treatment regimen. In addition, a resistance-associated variant D168E in the NS3 region was detected in the patient after discontinuation of the DCV/ASV regimen. The trough level of tacrolimus tended to decrease, and renal function showed no significant changes during treatment. Adverse events occurred in two patients (22.2%), but no severe adverse events occurred during treatment. CONCLUSIONS: The DCV/ASV regimen was well tolerated, resulting in high rates of sustained viral response 12 weeks after completion of therapy for LT patients with recurrent HCV genotype 1 infection.

New insight into reactive ductular cells of biliary atresia provided by pathological assessment of SOX9.

BACKGROUND: Biliary atresia (BA) patients may survive until adolescence after effective Kasai procedure (KP). If liver fibrosis progresses even after successful KP, liver transplantation (LTx) is inevitable. Elucidation of its cause and pathophysiology would open the possibility of treating these patients by non-invasive management. SOX9 is a transcription factor that regulates bile duct development and contributes to liver regeneration and fibrosis. To elucidate the role of SOX9 in BA liver, we investigated the SOX9 expression pattern. METHOD: Immunostaining with anti-SOX9 antibody was done on hepatic specimens obtained at the time of KP or LTx. We analyzed the association of SOX9 expression with clinical data. RESULTS: In BA livers, SOX9 was expressed in reactive ductular cells (RDCs), mostly with a nuclear-dominant pattern. SOX9 was also ectopically expressed in hepatocytes, which was more conspicuous at the timing of KP than LTx. SOX9 expression level was significantly correlated with age (days) at which KP was performed, AST and WBC count. CONCLUSIONS: SOX9 may contribute to RDC formation in BA patients, by affecting both RDCs and hepatocytes. SOX9 could be a key molecule to understand the mechanism of RDC formation, and this understanding would provide a therapeutic strategy for effective treatment of BA.

Effect of Laser Irradiation Modes and Photosensitizer Types on Antimicrobial Photodynamic Therapy (aPDT) for Streptococcus sobrinus in the Crown Dentin of Bovine Teeth: An Experimental In Vitro Study.

This study aimed to assess the impact of different laser irradiation modes and photosensitizer types on the bactericidal efficacy of antimicrobial photodynamic therapy (aPDT). Dentin plates were prepared by sectioning the crown dentin of bovine teeth infected with Streptococcus sobrinus (n = 11). Nine aPDTs involving the combination of three 1% solutions of photosensitizers (brilliant blue, BB; acid red, AR; and methylene blue, MB) and three irradiation modes of semiconductor lasers (50 mW for 120 s, 100 mW for 60 s, and 200 mW for 30 s) were performed for each infected dentin plate, and the control consisted of the specimens not applied with aPDT. The bactericidal effects in 10 groups were evaluated using both assays of the colony count (colony-forming-unit: CFU) and adenosine triphosphate (ATP) (relative-light-unit: RLU). The data obtained were analyzed using the Kruskal-Wallis test (α = 0.05). The most aPDT groups exhibited significantly lower RLU and CFU values compared with the control (p < 0.05). The effect of irradiation modes on RLU and CFU values was significant in the aPDT group using BB (p < 0.05) but not in the aPDT group using AR or MB. The aPDT performed with AR or MB exerted a remarkable bactericidal effect.

Chronic intestinal pseudo-obstruction due to adult-onset acquired isolated hypoganglionosis with muscular atrophy in the small intestine: a case report and review of literature.

BACKGROUND: Chronic intestinal pseudo-obstruction (CIPO) is a rare intestinal disorder characterized by impaired propulsion of the digestive tract and associated with symptoms of intestinal obstruction, despite the absence of obstructive lesions. CIPO includes several diseases. However, definitive diagnosis of its etiology is difficult only with symptoms or imaging findings. CASE PRESENTATION: A 56-year-old man was referred to our hospital due to a 3-year history of continuous abdominal distention. Imaging, including computed tomography of the abdomen, and endoscopy revealed marked dilatation of the entire small intestine without any obstruction point. Therefore, he was diagnosed with CIPO. Since medical therapy didn't improve his symptoms, enterostomy and percutaneous endoscopic gastro-jejunostomy were performed. These procedures improved abdominal symptoms. However, he required home central venous nutrition due to dehydration. The pathological findings of full-thickness biopsies of the small intestine taken during surgery revealed decreased number and degeneration of ganglion cells in the normal plexus. These findings led to a final diagnosis of CIPO due to acquired isolated hypoganglionosis (AIHG). CONCLUSIONS: Here, we report the case of a patient with CIPO secondary to adult-onset AIHG of the small intestine. Since AIHG cannot be solely diagnosed using clinical findings, biopsy is important for its diagnosis.

Conversion surgery for stage IV gastric cancer with multiple liver metastases with a complete pathological response to S-1 plus oxaliplatin therapy.

Although patients with stage IV gastric cancer who respond well to systemic chemotherapy can be treated with gastrectomy, the prognosis of patients with multiple liver metastases is poor. We herein describe a patient with stage IV gastric cancer with multiple liver metastases who underwent conversion surgery after systemic treatment with S-1 plus oxaliplatin. The patient was a 62-year-old man. Upper gastrointestinal endoscopy revealed a 30-mm type 2 tumor in the greater curvature of the stomach at the anterior wall, and biopsy revealed a poorly differentiated adenocarcinoma. Imaging showed three suspected liver metastases in liver segment S8. The patient was judged to have gastric cancer, cStage IV (cT3N1M1(H)), and systemic chemotherapy was administered. He was treated with a total of six courses of chemotherapy. After re-evaluation, the primary tumor had shrunk significantly, and liver metastases could not be detected. Confirming no signs of seeding by laparoscopy, robot-assisted pylorus-preserving gastrectomy with D2 dissection and laparoscopic partial hepatic (S8) resection were performed. The patient was diagnosed with a complete pathological response. Conversion surgery is an option for stage IV gastric cancer when distant metastases are controlled with chemotherapy and when R0 resection is possible.

Intravital imaging of immune responses in intestinal inflammation.

To date, many kinds of immune cells have been identified, but their precise roles in intestinal immunity remain unclear. Understanding the in vivo behavior of these immune cells and their function in gastrointestinal inflammation, including colitis, inflammatory bowel disease, ischemia-reperfusion injury, and neutrophil extracellular traps, is critical for gastrointestinal research to proceed to the next step. Additionally, understanding the immune responses involved in gastrointestinal tumors and tissue repair is becoming increasingly important for the elucidation of disease mechanisms that have been unknown. In recent years, the application of intravital microscopy in gastrointestinal research has provided novel insights into the mechanisms of intestine-specific events including innate and adaptive immunities. In this review, we focus on the emerging role of intravital imaging in gastrointestinal research and describe how to observe the intestines and immune cells using intravital microscopy. Additionally, we outline novel findings obtained by this new technique.

IL-34 in hepatoblastoma cells potentially promote tumor progression via autocrine and paracrine mechanisms.

Hepatoblastoma is the most common pediatric liver tumor, but little research has been done on the role of macrophages in hepatoblastoma. The purpose of this study was to gain insight into potential roles for macrophages in hepatoblastoma. Paraffin-embedded specimens from 56 patients who underwent surgical resection were examined with immunohistochemical staining for the macrophage-specific markers, Iba1 and CD163. Significant differences were seen among histological subtypes. Significantly increased numbers of macrophages were detected in embryonal components compared to fetal components in the mixed epithelial type. In vitro studies using human monocyte-derived macrophages and two hepatoblastoma cell lines (HepG2 and Huh6) were performed. Conditioned medium from these cell lines induced increased CD163 expression in macrophages. Direct co-culture with macrophages induced tumor cell proliferation via induction of protumor cytokine secretion from macrophages. Direct co-culture with macrophages also induced interleukin (IL)-34 overexpression by Huh6 cells via Brd4 signaling. IL-34 overexpression promoted tumor cell proliferation and chemoresistance. High IL-34 and Brd4 expression was detected in embryonal components, which have potentially higher proliferation activity than fetal components. In conclusion, IL-34 expression in embryonal components may induce macrophage chemotaxis in a paracrine manner, and tumor cell proliferation and chemoresistance in an autocrine manner. IL-34 is a potential therapeutic target for hepatoblastoma.

SOX9 contributes to the progression of ductular reaction for the protection from chronic liver injury.

The transcription factor sex-determining region Y-box 9 (SOX9) is a biliary epithelial marker ectopically expressed in hepatocytes (SOX9 + hepatocytes). SOX9 + hepatocytes are believed to function in ductular reaction (DR), recognized as an essential phenomenon related to liver regeneration; however, the functional role of SOX9 and clinical implications of SOX9 + hepatocytes in DR progression are unclear. Human and mouse liver samples were subjected to immunohistochemical and gene functional analyses to investigate the functional role of SOX9 and the clinical significance of SOX9 + hepatocytes. SOX9 + hepatocytes were observed in a bile duct ligation (BDL) mouse model. Forced Sox9 expression in mouse hepatocytes by hydrodynamic injection converted them into cholangiocyte-like cells. DR progression was slower in liver epithelium-specific Sox9-knockout BDL mice than in wild-type BDL mice. SOX9 + hepatocytes were also observed in rare pediatric liver disease biliary atresia (BA). In patients with BA who underwent liver transplantation (LT), the median number of SOX9 + hepatocytes at LT was significantly lower than that at Kasai portoenterostomy (KP) performed prior to LT (P < 0.001). The high SOX9 + hepatocyte group at KP demonstrated significantly better native liver survival rates than the low SOX9 + hepatocyte group at a cut-off of 390 cells/mm2 (P = 0.019, log-rank test). Ectopic expression of SOX9 in hepatocytes of chronically injured livers may exert protective effects in DR progression. To our knowledge, this is the first study showing that SOX9 + hepatocyte count at KP can be a promising biomarker to predict native liver survival after KP in patients with BA.