Involvement of CX3CR1+ cells appearing in the abdominal cavity in the immunosuppressive environment immediately after gastric cancer surgery.

BACKGROUND: Gastric cancer is primarily treated by surgery; however, little is known about the changes in the intraperitoneal immune environment and the prognostic impact of surgery. Surgical stress and cancer-associated inflammation cause immune cells to mobilize into the abdominal cavity via numerous cytokines. One such cytokine, CX3CR1, has various immune-related functions that remain to be fully explained. We characterized the intraperitoneal immune environment by investigating CX3CR1+ cells in intraperitoneal lavage fluid during gastric cancer surgery. METHODS: Lavage fluid samples were obtained from a total of 41 patients who underwent gastrectomy. The relative expression of various genes was analyzed using quantitative real-time PCR. The association of each gene expression with clinicopathological features and surgical outcomes was examined. The fraction of CX3CR1+ cells was analyzed by flow cytometry. Cytokine profiles in lavage fluid samples were investigated using a cytometric beads array. RESULTS: CX3CR1high patients exhibited higher levels of perioperative inflammation in blood tests and more recurrences than CX3CR1low patients. CX3CR1high patients tended to exhibit higher pathological T and N stage than CX3CR1low patients. CX3CR1 was primarily expressed on myeloid-derived suppressor cells and tumor-associated macrophages. In particular, polymorphonuclear myeloid-derived suppressor cells were associated with perioperative inflammation, pathological N, and recurrences. These immunosuppressive cells were associated with a trend toward unfavorable prognosis. Moreover, CX3CR1 expression was correlated with programmed death-1 expression. CONCLUSIONS: Our results suggest that CX3CR1+ cells are associated with an acute inflammatory response, tumor-promotion, and recurrence. CX3CR1 expression could be taken advantage of as a beneficial therapeutic target for improving immunosuppressive state in the future. In addition, analysis of intra-abdominal CX3CR1+ cells could be useful for characterizing the immune environment after gastric cancer surgery.

Prognostic relevance of tumor-resident memory T cells in metastatic lymph nodes of esophageal squamous cell carcinoma.

Tumor-resident memory T (TRM ) cells in primary tumors are reportedly associated with a favorable prognosis in several malignancies. However, the behaviors and functions of TRM cells in regional lymph nodes (LNs) of esophageal cancer remain poorly understood. The aim of this study was to elucidate the effects of TRM cells in regional LNs of esophageal cancer on clinicopathological findings and prognosis. Specimens of esophageal cancer and primary metastatic LNs (recurrent nerve LNs) were obtained from 84 patients who underwent radical esophagectomy between 2011 and 2017. We performed immunohistochemistry to enumerate and analyze TRM cells, and used flow cytometry to investigate the function of TRM cells. TRM cells were observed in both metastatic LNs and primary tumors. TRM cell-rich specimens exhibited reduced lymphatic invasion and LN metastasis and prolonged survival compared with TRM cell-poor specimens. TRM cells in metastatic LNs were more significantly associated with enhanced survival than TRM cells in primary tumors. TRM cells expressed high levels of granzyme B as a cytotoxicity marker. Our results suggested that high TRM cell infiltration in metastatic LNs improves survival even though LN metastasis is commonly associated with poor prognosis. TRM cells possibly contribute to antitumor immunity in regional LNs.

Evaluation of a simple activity measurement method in rats.

[Purpose] Behavioral analysis is widely used in animal research. However, such analysis requires specialized equipment and can be difficult to perform. Therefore, this study aimed to explore and validate a simple behavioral analysis method. [Participants and Methods] For behavioral assessments, Wistar rats were placed in a rearing cage and videotaped from two directions: overhead and side view. The filmed videos were analyzed using ImageJ software to calculate the distance traveled and activity and inactivity times of the rats. Intraclass correlation coefficients 1 and 2 were calculated to examine the reliability of the behavioral analysis method. [Results] Intraclass correlation coefficients 1 and 2 for distance traveled and activity and inactivity times determined using the behavioral analysis method showed high reliability. [Conclusion] The behavioral analysis method validated in this study used inexpensive and easily accessible equipment and devices. The results show high correlation coefficients for the measurement of distance traveled and activity time performed by experimental animals, demonstrating the reliability of this simple method.

5-Aminolevulinic acid overcomes hypoxia-induced radiation resistance by enhancing mitochondrial reactive oxygen species production in prostate cancer cells.

BACKGROUND: The naturally occurring amino acid 5-aminolevulinic acid (5-ALA) is a precursor of protoporphyrin IX (PpIX) biosynthesised in the mitochondria. When accumulated PpIX is excited by light (wavelength of 625-635 nm), reactive oxygen species (ROS) are generated. Here, we investigated whether 5-ALA may increase the sensitisation of prostate cancer (PCA) cells to radiotherapy through the generation of ROS via its metabolite, PpIX. METHODS: Effect of 5-ALA on PC-3 and DU-145 PCA cell lines treated with ionising radiation (IR) was examined in vitro and in vivo with assessment by clonogenic assay, mitochondrial function and ROS production under normoxia or hypoxia condition. RESULTS: 5-ALA enhanced intra-mitochondrial ROS production immediately after exposure to IR and decreased mitochondrial membrane potential via increase of intra-cellular PpIX. IR with 5-ALA induced mitochondrial dysfunction and increased ATP production, switching energy metabolism to the quiescence. Under hypoxic condition, ROS burst and mitochondrial dysfunction were induced by IR with 5-ALA resulting reducing cancer stemness and radiation resistance. CONCLUSION: These results suggest that combined therapy with 5-ALA and radiation therapy is a novel strategy to improve the anti-cancer effects of radiation therapy for PCA.

Clinical relevance of tertiary lymphoid structures in esophageal squamous cell carcinoma.

BACKGROUND: Tertiary lymphoid structures (TLSs) have been reported to be involved in immune responses in many carcinomas. This study investigated the significance of TLSs in esophageal squamous cell carcinoma, focusing on TLS maturation.  METHODS: The relationships of TLSs with clinicopathological features of 236 patients who underwent curative surgery for stage 0-IV esophageal squamous cell carcinoma were investigated. Mature TLSs, in which the germinal center formation was rich in CD23+ cells, were classified as TLSs containing a germinal center (GC-TLSs). GC-TLS densities were measured, and CD8+ cells were counted. The prognostic impact of GC-TLSs was assessed by Kaplan-Meier plots using the log-rank test for the relapse-free survival. A comparative study of GC-TLSs was performed using the Wilcoxon rank sum test. The relationship between GC-TLSs and CD8+ cells was examined by Spearman's rank correlation coefficient test. RESULTS: TLSs were located mainly at the invasive margin of the tumor in cases with esophageal squamous cell carcinoma. Among the patients treated with neoadjuvant chemotherapy, those with advanced disease had a better prognosis in the GC-TLS high-density group than did those in the GC-TLS low-density group. Patients in whom neoadjuvant chemotherapy was effective had more GC-TLSs than those in whom it was less effective. The density of GC-TLSs and the number of tumor-infiltrating CD8+ cells were higher in patients treated with neoadjuvant chemotherapy than in those without chemotherapy, and a weak correlation between the density of GC-TLSs and the number of tumor-infiltrating CD8+ cells was observed. Moreover, co-culturing of PBMCs with an anticancer drug-treated esophageal squamous cell carcinoma cell line increased the CD20 and CD23 expression in PBMCs in vitro. CONCLUSION: TLS maturation may be important for evaluating the local tumor immune response in patients treated with neoadjuvant chemotherapy for esophageal squamous cell carcinoma. The present results suggest that TLS maturation may be a useful target for predicting the efficacy of immunotherapy, including immune checkpoint inhibitor treatment for esophageal squamous cell carcinoma.

An Axis between the Long Non-Coding RNA HOXA11-AS and NQOs Enhances Metastatic Ability in Oral Squamous Cell Carcinoma.

Long non-coding RNAs (lncRNAs) play critical roles in human cancers. HOXA11 anti-sense RNA (HOXA11-AS) is an lncRNA belonging to the homeobox (HOX) gene cluster that promotes liver metastasis in human colon cancer. However, its role and mechanism of action in human oral squamous cell carcinoma (OSCC) are unclear. In this study, we investigated HOXA11-AS expression and function in human OSCC tissues and cell lines, as well as a mouse model of OSCC. Our analyses showed that HOXA11-AS expression in human OSCC cases correlates with lymph node metastasis, nicotinamide adenine dinucleotide (NAD)(P)H: quinone oxidoreductase 1 (NQO1) upregulation, and dihydronicotinamide riboside (NRH): quinone oxidoreductase 2 (NQO2) downregulation. Using the human OSCC cell lines HSC3 and HSC4, we demonstrate that HOXA11-AS promotes NQO1 expression by sponging microRNA-494. In contrast, HOXA11-AS recruits zeste homolog 2 (EZH2) to the NQO2 promoter to suppress its expression via the trimethylation of H3K27. The upregulation of NQO1 enzymatic activity by HOXA11-AS results in the consumption of flavin adenine dinucleotide (FAD), which reduces FAD-requiring glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity and suppresses glycolysis. However, our analyses show that lactic acid fermentation levels are preserved by glutaminolysis due to increased malic enzyme-1 expression, promoting enhanced proliferation, invasion, survival, and drug resistance. In contrast, suppression of NQO2 expression reduces the consumption of NRH via NQO2 enzymatic activity and increases NAD levels, which promotes enhanced stemness and metastatic potential. In mouse tumor models, knockdown of HOXA11-AS markedly suppressed tumor growth and lung metastasis. From these findings, targeting HOXA11-AS may strongly suppress high-grade OSCC by regulating both NQO1 and NQO2.

Hypomethylation of CLDN4 Gene Promoter Is Associated with Malignant Phenotype in Urinary Bladder Cancer.

The tight junction (TJ) protein claudin-4 (CLDN4) is overexpressed in bladder urothelial carcinoma (BUC) and correlates with cancer progression. However, the mechanism of CLDN4 upregulation and promotion of malignant phenotype is not clear. Here, we analyzed 157 cases of BUC and investigated the hypomethylation of CpG island in the CLDN4 promoter DNA and its correlation with cancer progression. In hypomethylated cases, CLDN4 expression, cell proliferation, stemness, and epithelial-mesenchymal transition were increased. Treatment of three human BUC cell lines with the demethylating agent aza-2'-deoxycytidine (AZA) led to excessive CLDN4 expression, and, specifically, to an increase in CLDN4 monomer that is not integrated into the TJ. The TJ-unintegrated CLDN4 was found to bind integrin ß1 and increase stemness, drug resistance, and metastatic ability of the cells as well as show an anti-apoptosis effect likely via FAK phosphorylation, which reduces upon knockdown of CLDN4. Thus, CLDN4 is overexpressed in BUC by an epigenetic mechanism and the high expression enhances the malignant phenotype of BUC via increased levels of TJ-unintegrated CLDN4. CLDN4 promoter DNA methylation is expected to be a novel indicator of BUC malignant phenotype and a new therapeutic target.

Cranio-caudal puncture with computed tomography-guided drainage of a deep pelvic abscess.

Purpose: Computed tomography (CT)-guided percutaneous drainage has been used to address pelvic abscesses because it is safe and minimally invasive. However, CT-guided drainage has the limitation that the puncture route should be on the same axial slice. A technique for puncturing in the cranio-caudal direction under CT fluoroscopy is needed. Case report: An 82-year-old man with an abscess due to rectal cancer was scheduled for CT-guided drainage to improve his general condition before radical surgery. Drainage was performed via a perineal approach to localize the drainage tract in the resection area to avoid dissemination of cancer cells. To perform a puncture in the cranio-caudal direction we controlled the needle like a joystick and advanced it under CT fluoroscopy while moving the CT gantry cranially to follow the needle tip throughout the puncture. Our unique technique yielded successful CT-guided puncture in the cranio-caudal direction. Conclusions: Our unique technique overcomes the limitations of CT-guided cranio-caudal puncture and may allow the drainage of abscesses whose treatment was heretofore difficult.

Tertiary lymphoid structures show infiltration of effective tumor-resident T cells in gastric cancer.

Several studies have reported that tissue-resident memory T cells (TRM cells) or tertiary lymphoid structures (TLSs) are associated with a good prognosis. The aim of this study was to clarify the association of TRM cells and TLSs in the tumor immune microenvironment in gastric cancer (GC). We performed immunohistochemical and immunofluorescence staining to detect the presence of CD103+ T cells and to assess the association between CD103+ T cells and TLSs. CD103+ T cells were observed in the tumor epithelium accompanied by CD8+ T cells and were associated with a better prognosis in GC. Furthermore, CD103+ T cells were located around TLSs, and patients with CD103high had more rich TLSs. Patients who had both CD103high cells and who were TLS-rich had a better prognosis than patients with CD103low cells and who were TLS-poor. Moreover, for patients who received PD-1 blockade therapy, CD103high and TLS-rich predicted a good response. Flow cytometry was performed to confirm the characteristics of CD103+ CD8+ T cells and showed that CD103+ CD8+ T cells in GC expressed higher levels of PD-1, granzyme B, and interferon-γ than CD103- CD8+ T cells. Our results suggested that CD103+ CD8+ cells in GC are correlated with TLSs, resulting in enhanced antitumor immunity in GC.

Analysis of hand environment factors contributing to the hand surface infection barrier imparted by lactic acid.

BACKGROUND: Organic acids on the surface of human hands contribute to the barrier against transient pathogens. This is the first study to explore the synergistic contribution of lactic acid and other hand environment-related features on the antibacterial properties of the hand surface. MATERIALS AND METHODS: We estimated the contribution of fingerprint depth, skin pH, stratum corneum water content, skin temperature, and sweat rate of the hands to the infection barrier using an observational survey of 105 subjects. The relationship between each factor and the antibacterial activity of the hands was analyzed using Pearson's correlation coefficient. We performed molecular dynamics simulations to study the interaction between lactic acid and bacterial membranes. RESULTS: The amount of lactic acid on the hands and skin temperature contributed positively to the antimicrobial activity (r = 0.437 and P = 3.18 × 10-6 , r = 0.500 and P = 5.66 × 10-8 , respectively), while the skin pH contributed negatively (r = -0.471, P = 3.99 × 10-7 ). The predicted value of the combined antimicrobial effect of these parameters was [antimicrobial activity] = 0.21 × [lactic acid] - 0.25 × [skin pH] + 0.26 × [skin temperature] + 0.98. The coefficient of determination (R2 ) was 0.50. CONCLUSION: The increase in the amount of non-ionic lactic acid due to lower pH and improvement in the fluidity of the cell membrane due to higher temperatures enable the efficient transport of lactic acid into cells and subsequent antimicrobial activity. The proposed mechanism could help to develop an effective hand infection barrier technology.

BRAF Mutation Is Associated with Hyperplastic Polyp-Associated Gastric Cancer.

Gastric hyperplastic polyps (GHP) are frequently found to be benign polyps and have been considered to have a low carcinogenic potential. The characteristics of the hyperplastic polyp-associated gastric cancer (HPAGC) remain unclear. Therefore, we analyzed samples from 102 GHP patients and identified 20 low-grade atypical GHPs (19.6%), 7 high-grade atypical GHPs (6.9%), and 5 intramucosal cancer samples (4.9%). GHP atypia was more common in the elderly and increased with increasing polyp size. In particular, polyps larger than 1 cm were associated with a higher grade and cancer. Furthermore, mucus production decreased with increasing atypia. Although no correlation was found between atypia and Helicobacter pylori infection or intestinal metaplasia, enhanced proliferative ability (Ki-67) did correlate with atypia, as did nuclear 8-hydroxy-2'-deoxyguanosine levels. Interestingly, 4-hydroxynonenal levels in granulation tissue and the area ratio of granulation tissue within polyps also correlated with GHP atypia. In five cases of HPAGC, three cases exhibited caudal type homeobox transcription factor (CDX2)-positive cells and a mixed mucin phenotype, which is considered to be related to H. pylori infection. By contrast, two cases were CDX2 negative, with a gastric mucin phenotype, and H. pylori infection was not observed in the tumor or the surrounding mucosa. In these cases, a v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation (V600E) was detected. All cancer samples showed high stemness and p53 protein accumulation, but no KRAS mutations. The molecular and phenotypic characteristics of the cases characterized by BRAF mutations may represent a novel subtype of HPAGC, reflecting a conserved pathway to oncogenesis that does not involve H. pylori infection. These findings are worthy of further investigation in a large-scale study with a substantial cohort of HPAGC patients to establish their clinical significance.

Combined administration of lauric acid and glucose improved cancer-derived cardiac atrophy in a mouse cachexia model.

Cancer-derived myocardial damage is an important cause of death in cancer patients. However, the development of dietary interventions for treating such damage has not been advanced. Here, we investigated the effect of dietary intervention with lauric acid (LAA) and glucose, which was effective against skeletal muscle sarcopenia in a mouse cachexia model, on myocardial damage. Treatment of H9c2 rat cardiomyoblasts with lauric acid promoted mitochondrial respiration and increased ATP production by Seahorse flux analysis, but did not increase oxidative stress. Glycolysis was also promoted by LAA. In contrast, mitochondrial respiration and ATP production were suppressed, and oxidative stress was increased in an in vitro cachexia model in which cardiomyoblasts were treated with mouse cachexia ascites. Ascites-treated H9c2 cells with concurrent treatment with LAA and high glucose showed that mitochondrial respiration and glycolysis were promoted more than that of the control, and ATP was restored to the level of the control. Oxidative stress was also reduced by the combined treatment. In the mouse cachexia model, myocardiac atrophy and decreased levels of a marker of muscle maturity, SDS-soluble MYL1, were observed. When LAA in CE-2 diet was orally administered alone, no significant rescue was observed in the cancer-derived myocardial disorder. In contrast, combined oral administration of LAA and glucose recovered myocardial atrophy and MYL1 to levels observed in the control without increase in the cancer weight. Therefore, it is suggested that dietary intervention using a combination of LAA and glucose for cancer cachexia might improve cancer-derived myocardial damage.

Targeting claudin-4 enhances chemosensitivity in breast cancer.

Triple negative breast cancer (TNBC) is characterized by highly aggressive phenotype, limited treatment options and a poor prognosis. In the present study, we examined the therapeutic effect of anti-claudin (CLDN)-4 extracellular domain antibody, 4D3, on TNBC. When the expression of CLDN4 and CLDN1 in invasive ductal carcinoma (IDC) was examined in 114 IDC (78 cases from 2004 to 2009 in a single center and 36 cases of tissues array), CLDN1 had lower expression than CLDN4 and was correlated with histological grade. In contrast, expression of CLDN4 was correlated with histological grade, receptor subtype, and stage. CLDN4 expression in human IDC cell lines MCF-7 (luminal subtype) and MDA-468 (TNBC) was at the same level. In both cells, paclitaxel (PTX)-induced growth suppression was enhanced by 4D3. Furthermore, 4D3 increased both intracellular PTX concentration (in both cells) and apoptosis. In the mouse model, 4D3 promoted the antitumor effect of PTX on subcutaneous tumors and reduced lung metastasis. The combination of PTX and 4D3 reduced M2 macrophages and mesenchymal stem cells in the tumor. 4D3 also reduced stemness of the tumors and increased the intratumoral pH. Moreover, concurrent treatment with 4D3, PTX and tamoxifen, or with PTX and tamoxifen in MDA-468 also showed the same level of antitumor activity and survival as MCF-7. Furthermore, in a bone metastasis model, combination of PTX and bisphosphonate with 4D3 promoted tumor growth in both cells. Thus, CLDN4 targeting of the antibody facilitated existing therapeutic effects.

Malic Enzyme 1 Is Associated with Tumor Budding in Oral Squamous Cell Carcinomas.

Budding at the tumor invasive front has been correlated with the malignant properties of many cancers. Malic enzyme 1 (ME1) promotes the Warburg effect in cancer cells and induces epithelial-mesenchymal transition (EMT) in oral squamous cell carcinoma (OSCC). Therefore, we investigated the role of ME1 in tumor budding in OSCC. Tumor budding was measured in 96 human OSCCs by immunostaining for an epithelial marker (AE1/AE3), and its expression was compared with that of ME1. A significant correlation was observed between tumor budding and ME1 expression. The correlation increased with the progression of cancer. In human OSCC cells, lactate secretion decreased when lactate fermentation was suppressed by knockdown of ME1 and lactate dehydrogenase A or inhibition of pyruvate dehydrogenase (PDH) kinase. Furthermore, the extracellular pH increased, and the EMT phenotype was suppressed. In contrast, when oxidative phosphorylation was suppressed by PDH knockdown, lactate secretion increased, extracellular pH decreased, and the EMT phenotype was promoted. Induction of chemical hypoxia in OSCC cells by CoCl2 treatment resulted in increased ME1 expression along with HIF1α expression and promotion of the EMT phenotype. Hypoxic conditions also increased matrix metalloproteinases expression and decreased mitochondrial membrane potential, mitochondrial oxidative stress, and extracellular pH. Furthermore, the hypoxic treatment resulted in the activation of Yes-associated protein (YAP), which was abolished by ME1 knockdown. These findings suggest that cancer cells at the tumor front in hypoxic environments increase their lactate secretion by switching their energy metabolism from oxidative phosphorylation to glycolysis owing to ME1 overexpression, decrease in extracellular pH, and YAP activation. These alterations enhance EMT and the subsequent tumor budding. Tumor budding and ME1 expression are thus considered useful markers of OSCC malignancy, and ME1 is expected to be a relevant target for molecular therapy.

Role of Clostridium perfringens Enterotoxin on YAP Activation in Colonic Sessile Serrated Adenoma/ Polyps with Dysplasia.

Sessile serrated adenoma/polyp with dysplasia (SSA/P-D) is an SSA/P with cellular dysplasia and has a higher risk of progressing to colon carcinogenesis. Previously, we reported that tight junction impairment by Clostridium perfringens enterotoxin (CPE) leads to activation of the transcriptional co-activator yes-associated protein (YAP) in oral squamous cell carcinoma. Here, we investigated whether CPE activates YAP to promote the malignant progression of SSA/P. E-cadherin expression was lower in the 12 cases with SSA/P-D examined than that in normal mucosa, SSA/P, or tubular adenoma (TA). Furthermore, intracellular translocation of claudin-4 (CLDN4) and nuclear translocation of YAP were observed. The CPE gene was detected in DNA extracted from SSA/P-D lesions, but not in SSA/P or TA. Treatment of the rat intestinal epithelial cell line IEC6 with low-dose CPE resulted in intracellular translocation of CLDN4 to the cytoplasmic membrane. Cytoplasmic CLDN4 showed co-precipitation with transcriptional co-activator with PDZ-binding motif, zonula occludens (ZO)-1, large tumor suppressor, and mammalian Ste20-like. Additionally, YAP co-precipitated with ZO-2 under CPE treatment led to decreased YAP phosphorylation and nuclear translocation. YAP activation promoted increase in nuclear TEA domain family member level, expression of cyclin D1, snail, vimentin, CD44, NS and decrease in E-cadherin levels, thereby inducing stemness and epithelial-mesenchymal-transition (EMT). The Hippo complex with the incorporation of CLDN4 increased stability. Upon low-dose CPE treatment, HT29 cells with BRAFV600E gene mutation showed increased growth, enhanced invasive potential, stemness, and induced EMT phenotype, whereas HCT116 cells, which carry KRASG13D gene mutation, did not show such changes. In an examination of 10 colorectal cancers, an increase in EMT and stemness was observed in CPE (+) and BRAF mutation (+) cases. These findings suggest that C. perfringens might enhance the malignant transformation of SSA/P-D via YAP activation. Our findings further highlight the importance of controlling intestinal flora using probiotics or antibiotics.

Role of Nuclear Claudin-4 in Renal Cell Carcinoma.

Claudin-4 (CLDN4) is a tight junction protein to maintain the cancer microenvironment. We recently reported the role of the CLDN4 not forming tight junction in the induction of epithelial-mesenchymal transition (EMT). Herein, we investigated the role of CLDN4 in renal cell carcinoma (RCC), focusing on CLDN4. CLDN4 expression in 202 RCCs was examined by immunostaining. CLDN4 phosphorylation and subcellular localization were examined using high metastatic human RCC SN12L1 and low metastatic SN12C cell lines. In 202 RCC cases, the CLDN4 expression decreased in the cell membrane and had no correlation with clinicopathological factors. However, CLDN4 was localized in the nucleus in 5 cases (2%), all of which were pT3. Contrastingly, only 6 of 198 nuclear CLDN4-negative cases were pT3. CLDN4 was found in the nuclear fraction of a highly metastatic human RCC cell line, SN12L1, but not in the low metastatic SN12C cells. In SN12L1 cells, phosphorylation of tyrosine and serine residues was observed in cytoplasmic CLDN4, but not in membranous CLDN4. In contrast, phosphorylation of serine residues was observed in nuclear CLDN4. In SN12L1 cells, CLDN4 tyrosine phosphorylation by EphA2/Ephrin A1 resulted in the release of CLDN4 from tight junction and cytoplasmic translocation. Furthermore, protein kinase C (PKC)-ε phosphorylated the CLDN4 serine residue, resulting in nuclear import. Contrarily, in SN12C cells that showed decreased expression of EphA2/Ephrin A1 and PKCε, the activation of EphA2/EphrinA1 and PKCε induced cytoplasmic and nuclear translocation of CLDN4, respectively. Furthermore, the nuclear translocation of CLDN4 promoted the nuclear translocation of Yes-associated protein (YAP) bound to CLDN4, which induced the EMT phenotype. These findings suggest that the release of CLDN4 by impaired tight junction might be a mechanism underlying the malignant properties of RCC. These findings suggest that the release of CLDN4 by impaired tight junction might be one of the mechanisms of malignant properties of RCC.

[Biomarkers for Immunotherapy Based on the Local Microenvironment of Gastric Cancer].

Currently, the immunotherapy approved for gastric cancer is immune checkpoint blockade( ICB) therapy. The effects of ICB depend on the T cell-mediated immune response elicited at the cancer site. Based on the results of previous clinical trials, it is clear that an enhanced immune response to cancer improves prognosis. Thus, the development of biomarkers to predict local immune responses may increase the significance of future immunotherapy for gastric cancer. Biomarker research has clearly progressed with the rapid development of genetic analysis technologies, enabling the analysis of data from clinical trials. Not only the molecular biomarkers known to date for ICB biomarkers, but immune cells that influence ICB therapy are also reviewed in this article.

Giving combined medium-chain fatty acids and glucose protects against cancer-associated skeletal muscle atrophy.

Skeletal muscle volume is associated with prognosis of cancer patients. Maintenance of skeletal muscle is an essential concern in cancer treatment. In nutritional intervention, it is important to focus on differences in metabolism between tumor and skeletal muscle. We examined the influence of oral intake of glucose (0%, 10%, 50%) and 2% medium-chain fatty acid (lauric acid, LAA, C12:0) on tumor growth and skeletal muscle atrophy in mouse peritoneal metastasis models using CT26 mouse colon cancer cells and HT29 human colon cancer cells. After 2 weeks of experimental breeding, skeletal muscle and tumor were removed and analyzed. Glucose intake contributed to prevention of skeletal muscle atrophy in a sugar concentration-dependent way and also promoted tumor growth. LAA ingestion elevated the level of skeletal muscle protein and suppressed tumor growth by inducing tumor-selective oxidative stress production. When a combination of glucose and LAA was ingested, skeletal muscle mass increased and tumor growth was suppressed. Our results confirmed that although glucose is an important nutrient for the prevention of skeletal muscle atrophy, it may also foster tumor growth. However, the ingestion of LAA inhibited tumor growth, and its combination with glucose promoted skeletal muscle integrity and function, without stimulating tumor growth. These findings suggest novel strategies for the prevention of skeletal muscle atrophy.

Evaluation of Parameters for Cancer-Induced Sarcopenia in Patients Autopsied after Death from Colorectal Cancer.

Cachexia frequently occurs in cancer patients and is correlated with reduced therapeutic responsiveness and poor prognosis. Although skeletal muscle atrophy is an important factor related to cachexia, biomarkers for its early diagnosis are not yet definitive. In this study, weight loss, body mass index, skeletal muscle index (SMI), serum carcinoembryonic antigen, serum tumor necrosis factor (TNF)-α, serum interleukin (IL)-6, serum high mobility group box (HMGB)-1, and SDS-soluble myosin light chain 1 (SDS-MYL1) of the psoas muscle were examined in 8 autopsied cases of death from colorectal cancer (CRC) as biomarkers of cachexia. SDS-MYL1 was positively correlated to SMI and TNF-α was negatively correlated, but the other factors did not show any correlations with SMI. Multivariate analysis showed that of the 3 cytokines, TNF-α and HMGB1 were correlated with SMI. Furthermore, when the biochemical skeletal muscle maturation marker, SDS-MYL1, was compared with serum cytokines, TNF-α and HMGB1 were negatively correlated but IL-6 was not. In multivariate analysis, only TNF-α was associated with SDS-MYL1. A positive correlation was found between TNF-α and HMGB1. These findings suggest that since TNF-α was inversely correlated with SMI and SDS-MYL1, TNF-α is a serum marker of skeletal muscle atrophy in CRC. Moreover, SDS-MYL1 might be established as a biomarker linked to clinical sarcopenia in experiments in vitro and in vivo.

Magnetic Hyperthermia Using Self-Controlled Heating Elements Consisting of Fe-Al Milling Alloy Induces Cancer Cell Apoptosis while Preserving Skeletal Muscle.

Necrosis-inducing anticancer drugs enhance high-mobility group box 1 (HMGB1) release during cell necrosis, and HMGB1-induced autophagy in skeletal muscle induces muscle atrophy. We evaluated the efficacy of magnetic hyperthermia therapy (MHT) using a low-energy magnetic field and self-controlled heating elements in tumor treatment. MHT-induced apoptosis by heating mouse subcutaneous tumors at 43°C using a heat-controlling iron-aluminum (Fe-Al) milling alloy. In contrast, MHT using Fe line-induced necrosis by heating to approximately 100°C. Furthermore, MHT with Fe-Al milling alloy reduced stemness. In hyperthermia using age line or Fe-Al milling alloy, both of them provided histological degeneration in skeletal muscle; however, qualitative differences were observed. MHT using Fe-line induced pronounced autophagy, decrease of myosin heavy chain content, and increase in serum HMGB1. In contrast, MHT using Fe-Al milling alloy induced heat shock protein 90 but no autophagy and decreased serum HMGB1. Therefore, MHT using Fe-Al milling alloy might be a good method for local treatment of tumors to reduce skeletal muscle atrophy.