Feasibility of intraoperative pathologic examination for sentinel lymph nodes during sentinel node navigation surgery in early gastric cancer: results of pathologic protocol for SENORITA trial.

BACKGROUND: During sentinel node navigation surgery in patients with gastric cancer, intraoperative pathologic examination of sentinel nodes is crucial in determining the extent of surgery. In this study, we evaluated the feasibility and accuracy of intraoperative pathologic protocols using data from a prospective, multicenter, randomized trial. METHODS: A retrospective analysis was conducted using data from the SEntinel Node ORIented Tailored Approach trials from 2013 to 2016. All sentinel lymph nodes were evaluated during surgery with hematoxylin-eosin (HE) staining using a representative section at the largest plane for lymph nodes. For permanent histologic evaluation, sentinel basin nodes were stained with HE and cytokeratin immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) sections and examined with HE for three deeper-step sections at 200-µm intervals. The failure rate of identification by frozen section and the metastasis rate in non-sentinel basins were investigated. RESULTS: Of the 237 patients who underwent sentinel node basin dissection, 30 had lymph node metastases on permanent pathology. Thirteen patients had macrometastasis confirmed in frozen sections as well as FFPE sections (failure rate: 0%). Patients with negative sentinel nodes in frozen sections but micrometastasis in FFPE sections had no lymph node recurrence during the follow-up period (0%, 0/6). However, in cases with tumor-positive nodes in frozen sections, metastases in non-sentinel basins were detected in the paraffin blocks (8.3%, 2/24). CONCLUSIONS: The single-section HE staining method is sufficient for detecting macrometastasis via intraoperative pathological examination. If a negative frozen-section result is confirmed, sentinel basin dissection can be performed safely. Otherwise, standard surgery is required.

NAMPT-Driven M2 Polarization of Tumor-Associated Macrophages Leads to an Immunosuppressive Microenvironment in Colorectal Cancer.

Nicotinamide phosphoribosyltransferase (NAMPT) is a metabolic enzyme with key roles in inflammation. Previous studies have examined the consequences of its upregulated expression in cancer cells themselves, but studies are limited with respect to its role in the other cells within the tumor microenvironment (TME) during colorectal cancer (CRC) progression. Using single-cell RNA sequencing (scRNA-seq) data, it is founded that NAMPT is highly expressed in SPP1+ tumor-associated macrophages (TAMs), a unique subset of TAMs associated with immunosuppressive activity. A NAMPThigh gene signature in SPP1+ TAMs correlated with worse prognostic outcomes in CRC patients. The effect of Nampt deletion in the myeloid compartment of mice during CRC development is explored. NAMPT deficiency in macrophages resulted in HIF-1α destabilization, leading to reduction in M2-like TAM polarization. NAMPT deficiency caused significant decreases in the efferocytosis activity of macrophages, which enhanced STING signaling and the induction of type I IFN-response genes. Expression of these genes contributed to anti-tumoral immunity via potentiation of cytotoxic T cell activity in the TME. Overall, these findings suggest that NAMPT-initiated TAM-specific genes can be useful in predicting poor CRC patient outcomes; strategies aimed at targeting NAMPT may provide a promising therapeutic approach for building an immunostimulatory TME in CRC progression.

Intercellular cross-talk through lineage-specific gap junction of cancer-associated fibroblasts related to stromal fibrosis and prognosis.

Stromal fibrosis in cancer is usually associated with poor prognosis and chemotherapy resistance. It is thought to be caused by fibroblasts; however, the exact mechanism is not yet well understood. The study aimed to identify lineage-specific cancer-associated fibroblast (CAF) subgroup and their associations with extracellular matrix remodeling and clinical significances in various tumor types using single-cell and bulk RNA sequencing data. Through unsupervised clustering, six subclusters of CAFs were identified, including a cluster with exclusively high gap junction protein beta-2 (GJB2) expression. This cluster was named GJB2-positive CAF. It was found to be a unique subgroup of terminally differentiated CAFs associated with collagen gene expression and extracellular matrix remodeling. GJB2-positive CAFs showed higher communication frequency with vascular endothelial cells and cancer cells than GJB2-negative CAFs. Moreover, GJB2 was poorly expressed in normal tissues, indicating that its expression is dependent on interaction with other cells, including vascular endothelial cells and cancer cells. Finally, the study investigated the clinical significance of GJB2 signature score for GJB2-positive CAFs in cancer and found a correlation with poor prognosis. These results suggest that GJB2-positive CAF is a unique fibroblast subtype involved in extracellular matrix remodeling, with significant clinical implications in cancer.

Simultaneous establishment of pancreatic cancer organoid and cancer-associated fibroblast using a single-pass endoscopic ultrasound-guided fine-needle biopsy specimen.

Considering the critical roles of cancer-associated fibroblasts (CAFs) in pancreatic cancer, recent studies have attempted to incorporate stromal elements into organoid models to recapitulate the tumor microenvironment. This study aimed to evaluate the feasibility of patient-derived organoid (PDO) and CAF cultures by using single-pass endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) samples from prospectively enrolled pancreatic cancer patients. The obtained samples were split into two portions for PDO and CAF cultures. PDOs and CAFs were cultured successfully in 54.4% (31/57) and 47.4% (27/57) of the cases, respectively. Both components were established in 21 cases (36.8%). Various clinicopathologic factors, including the tumor size, tumor location, clinical stage, histologic subtype, and tumor differentiation, did not influence the PDO establishment. Instead, the presence of necrosis in tumor samples was associated with initial PDO generation but no further propagation beyond passage 5 (P = 0.024). The "poorly cohesive cell carcinoma pattern" also negatively influenced the PDO establishment (P = 0.018). Higher stromal proportion in tumor samples was a decisive factor for successful CAF culture (P = 0.005). Our study demonstrated that the coestablishment of PDOs and CAFs is feasible even with a single-pass EUS-FNB sample, implying an expanding role of endoscopists in future precision medicine.

Estrogen-responsive cancer-associated fibroblasts promote invasive property of gastric cancer in a paracrine manner via CD147 production.

Estrogen signaling has been extensively studied, especially in cancers that express estrogen receptor alpha (ERα). However, little is known regarding the effect of estrogen on cancer-associated fibroblasts (CAFs). Here, we explored the role of estrogen signaling of CAFs in gastric cancer (GC) progression. We investigated the phenotypic changes in CAFs upon 17ß-estradiol (E2) treatment using ERα-negative/positive CAFs, and the conditioned media (CM) collected from these were compared with regard to cancer cell proliferation, migration, and invasion. A paracrine factor was found using a cytokine array and was confirmed using qRT-PCR, western blotting, and enzyme-linked immunosorbent assays. ERα-CD147-matrix metalloproteinase (MMP) axis was confirmed by knockdown experiments using specific siRNAs. We found that a subset of CAFs expressed ERα. ERα-positive CAFs were responsive to E2, inducing ERα expression in a dose-dependent manner. Although E2 did not induce the proliferation of ERα-positive CAFs, the CM from E2-bound ERα-positive CAFs significantly promoted cancer cell migration and invasion. Cytokine array revealed that CD147 was induced in ERα-positive CAFs upon E2 treatment; this was mediated via ERα. Increased CD147 upregulated MMP2 and MMP9 in CAFs, and also influenced cancer cells in a paracrine manner to increase MMPs and CD147 in cancer cells. High CD147 expression in tumor tissue was associated with a worse prognosis in GC patients. Our data suggest that estrogen signaling activation in CAFs and the byproduct CD147 are among the critical mediators between the interplay of CAFs and cancer cells to facilitate cancer progression.

LCK-Mediated RIPK3 Activation Controls Double-Positive Thymocyte Proliferation and Restrains Thymic Lymphoma by Regulating the PP2A-ERK Axis.

Receptor-interacting protein kinase 3 (RIPK3) is the primary regulator of necroptotic cell death. RIPK3 expression is often silenced in various cancer cells, which suggests that it may have tumor suppressor properties. However, the exact mechanism by which RIPK3 negatively regulates cancer development and progression remains unclear. This report indicates that RIPK3 acts as a potent regulator of the homeostatic proliferation of CD4+ CD8+ double-positive (DP) thymocytes. Abnormal proliferation of RIPK3-deficient DP thymocytes occurs independently of the well-known role for RIPK3 in necroptosis (upstream of MLKL activation), and is associated with an incidental thymic mass, likely thymic hyperplasia. In addition, Ripk3-null mice develop increased thymic tumor formation accompanied by reduced host survival in the context of an N-ethyl-N-nitrosourea (ENU)-induced tumor model. Moreover, RIPK3 deficiency in p53-null mice promotes thymic lymphoma development via upregulated extracellular signal-regulated kinase (ERK) signaling, which correlates with markedly reduced survival rates. Mechanistically, lymphocyte-specific protein tyrosine kinase (LCK) activates RIPK3, which in turn leads to increases in the phosphatase activity of protein phosphatase 2 (PP2A), thereby suppressing hyper-activation of ERK in DP thymocytes. Overall, these findings suggest that a RIPK3-PP2A-ERK signaling axis regulates DP thymocyte homeostasis and may provide a potential therapeutic target to improve thymic lymphoma therapies.

Modeling Pancreatic Cancer with Patient-Derived Organoids Integrating Cancer-Associated Fibroblasts.

Pancreatic cancer is a devastating disease and is highly resistant to anticancer drugs because of its complex microenvironment. Cancer-associated fibroblasts (CAFs) are an important source of extracellular matrix (ECM) components, which alter the physical and chemical properties of pancreatic tissue, thus impairing effective intratumoral drug delivery and resulting in resistance to conventional chemotherapy. The objective of this study was to develop a new cancer organoid model, including a fibrous tumor microenvironment (TME) using CAFs. The CAF-integrated pancreatic cancer organoid (CIPCO) model developed in this study histologically mimicked human pancreatic cancer and included ECM production by CAFs. The cancer cell-CAF interaction in the CIPCO promoted epithelial-mesenchymal transition of cancer cells, which was reversed by CAF inhibition using all-trans retinoic acid. Deposition of newly synthesized collagen I in the CIPCO disturbed the delivery of gemcitabine to cancer cells, and treatment with collagenase increased the cytotoxic effect of gemcitabine. This model may lead to the development of next-generation cancer organoid models recapitulating the fibrous TME.

miR-4742-5p promotes invasiveness of gastric cancer via targeting Rab43: An in vitro study.

miRNA (miR)-4742-5p is a recently identified microRNA regarding progression and metastasis in gastric cancer (GC). However, the biological function of this novel miRNA is largely unknown. We identified that the miR-4742-5p expression level was variably increased in GC cell lines. Suppression of miR-4742-5p using miR-inhibitor reduced the proliferation, migration, and invasion of GC cells with high miR-4742-5p expression, whereas overexpression of miR-4742-5p-mimic enhanced the aforementioned properties in GC cells with low miR-4742-5p expression. miR-4742-5p expression induced the decreases of Zo-1 and E-cadherin expression as well as the increases of vimentin and N-cadherin expression, leading to epithelial-mesenchymal transition (EMT) of cancer cells. RNA sequencing results indicated Ras-related GTP-binding protein 43 (Rab43) as a potential target gene. We identified that the expression of Rab43 is associated with activation of AKT and nuclear factor-kappa B (NF-κB) which are key oncogenic pathways in cancer cells. Our results demonstrate a new component in GC progression, promising a potential therapeutic strategy.

NAMPT mitigates colitis severity by supporting redox-sensitive activation of phagocytosis in inflammatory macrophages.

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the nicotinamide adenine dinucleotide (NAD+) salvage pathway and plays a crucial role in the maintenance of the NAD+ pool during inflammation. Considering that macrophages are essential for tissue homeostasis and inflammation, we sought to examine the functional impact of NAMPT in inflammatory macrophages, particularly in the context of inflammatory bowel disease (IBD). In this study, we show that mice with NAMPT deletion within the myeloid compartment (Namptf/fLysMCre+/-, Nampt mKO) have more pronounced colitis with lower survival rates, as well as numerous uncleared apoptotic corpses within the mucosal layer. Nampt-deficient macrophages exhibit reduced phagocytic activity due to insufficient NAD+ abundance, which is required to produce NADPH for the oxidative burst. Nicotinamide mononucleotide (NMN) treatment rescues NADPH levels in Nampt mKO macrophages and sustains superoxide generation via NADPH oxidase. Consequently, Nampt mKO mice fail to clear dead cells during tissue repair, leading to substantially prolonged chronic colitis. Moreover, systemic administration of NMN, to supply NAD+, effectively suppresses the disease severity of DSS-induced colitis. Collectively, our findings suggest that activation of the NAMPT-dependent NAD+ biosynthetic pathway, via NMN administration, is a potential therapeutic strategy for managing inflammatory diseases.

pSlugS158 immunohistochemistry is a novel promising mitotic marker for FFPE samples: a pilot study.

Slug is a transcription factor belonging to the slug/snail superfamily. The protein is involved in embryonic development and epithelial-mesenchymal transition of tumors. Slug is also under temporal regulation during cell cycle. Here, we examined relationship between pSlugS158 (site-specific phosphorylation) and the cell cycle, and checked whether its phosphorylation level reflects mitotic activity in tissue specimens. Cell cycle analysis was performed after cell synchronization. To evaluate pSlugS158 identifying mitotic figures, we performed immunohistochemistry (IHC) for pSlugS158 in various formalin-fixed paraffin-embedded tissues; in addition, mitotic counts were compared with those in sections stained with hematoxylin and eosin (HE) and IHC for PHH3, a mitotic marker. We found that the level of pSlugS158 protein increased specifically at M phase and decreased at the G1/S phases in vitro. In almost all tested tissues, nuclear stain of pSlugS158 was identified in the cell with mitotic figures. There was no significant difference in mitotic counts between HE- and pSlugS158-stained sections. In conclusion, pSlugS158 may be a novel and practical immunohistochemical marker for detecting mitotic figures in human tissues.

RIPK3 activation induces TRIM28 derepression in cancer cells and enhances the anti-tumor microenvironment.

BACKGROUND: Necroptosis is emerging as a new target for cancer immunotherapy as it is now recognized as a form of cell death that increases tumor immunogenicity, which would be especially helpful in treating immune-desert tumors. De novo synthesis of inflammatory proteins during necroptosis appears especially important in facilitating increased anti-tumor immune responses. While late-stage transcription mediated by NF-κB during cell death is believed to play a role in this process, it is otherwise unclear what cell signaling events initiate this transactivation of inflammatory genes. METHODS: We employed tandem-affinity purification linked to mass spectrometry (TAP-MS), in combination with the analysis of RNA-sequencing (RNA-Seq) datasets to identify the Tripartite Motif Protein 28 (TRIM28) as a candidate co-repressor. Comprehensive biochemical and molecular biology techniques were used to characterize the role of TRIM28 in RIPK3 activation-induced transcriptional and immunomodulatory events. The cell composition estimation module was used to evaluate the correlation between RIPK3/TRIM28 levels and CD8+ T cells or dendritic cells (DC) in all TCGA tumors. RESULTS: We identified TRIM28 as a co-repressor that regulates transcriptional activity during necroptosis. Activated RIPK3 phosphorylates TRIM28 on serine 473, inhibiting its chromatin binding activity, thereby contributing to the transactivation of NF-κB and other transcription factors, such as SOX9. This leads to elevated cytokine expression, which then potentiates immunoregulatory processes, such as DC maturation. The expression of RIPK3 has a significant positive association with the tumor-infiltrating immune cells populations in various tumor type, thereby activating anti-cancer responses. CONCLUSION: Our data suggest that RIPK3 activation-dependent derepression of TRIM28 in cancer cells leads to increased immunostimulatory cytokine production in the tumor microenvironment, which then contributes to robust cytotoxic anti-tumor immunity.

Factors affecting the diagnostic yield of endoscopic transpapillary forceps biopsy in patients with malignant biliary strictures.

BACKGROUND AND AIM: Transpapillary biliary forceps biopsy (TBFB) is a common method to obtain histological evidence for the differential diagnosis of biliary stricture. This study aimed to evaluate the factors associated with a positive cancer diagnosis from TBFB and the number of tissue samples required to increase the diagnostic yield in patients with malignant biliary strictures. METHODS: A total of 376 patients who underwent TBFB for investigation of biliary stricture were included. Factors affecting the diagnostic yield of TBFB were determined using univariate analysis and multivariate logistic regression analyses. RESULTS: Bile duct cancer (odds ratio [OR] = 3.50, P = 0.002), intraductal growing type (OR = 9.01, P = 0.001), and number of tissue samples (n < 5 vs 5 ≤ n < 10, OR = 4.13, P = 0.01; n < 5 vs n ≥ 10, OR = 12.25, P < 0.001; 5 ≤ n < 10 vs n ≥ 10, OR = 2.97, P = 0.046) were significant factors associated with positive results for malignancy. In patients with periductal infiltrating-type bile duct cancer, the number of tissue samples was a significant factor for diagnostic sensitivity (54.3% in the n < 5 group, 83.3% in the 5 ≤ n < 10 group and 98.2% in the n ≥ 10 group) (P < 0.001). CONCLUSIONS: Bile duct cancer, intraductal growing type, and five or more tissue samples were significant predictors of positive TBFB results in patients with malignant biliary stricture. Increasing the number of tissue samples by five or more led to higher sensitivity in bile duct cancer patients with the periductal infiltrating type.

Quantitation of ligand is critical for ligand-dependent MET signalling activation and determines MET-targeted therapeutic response in gastric cancer.

BACKGROUND: Despite the promising preclinical antitumor activity of MET-targeting therapies, most clinical trials have failed. We introduced a new concept of quantitation of stroma-induced hepatocyte growth factor (HGF) to assess the actual MET signalling activity in gastric cancer (GC). METHODS: We treated serially diluted HGF and conditioned media (CM) from cancer-associated fibroblasts (CAFs) on low MET-expressing cancer cells and investigated the phenotypical and signalling changes. Stromal proportion and MET expression in GC samples were assessed, and gene set enrichment analysis (GSEA) from the public database was performed. The antitumor effect of anti-MET treatment was examined, especially when cancer cells were activated in a ligand-dependent manner. RESULTS: Relatively high doses of HGF or high-concentrated CM fully activated MET signalling cascades and promoted cell proliferation/invasion. High stromal proportion denoted worse patient survival in MET-positive GCs than in MET-negative ones. GSEA showed that the gene sets regarding proliferation, migration, and CAF as well as MET pathway signature were enriched in simultaneously MET- and HGF-positive samples. Sufficient ligand-dependent MET signalling activation increased the sensitivity to crizotinib. CONCLUSIONS: We conclude that patients whose tumours have a high stromal proportion and at least low MET expression may benefit more from MET-targeted therapies.

MicroRNA signatures associated with lymph node metastasis in intramucosal gastric cancer.

Although a certain proportion of intramucosal carcinomas (IMCs) of the stomach does metastasize, the majority of patients are currently treated with endoscopic resection without lymph node dissection, and this potentially veils any existing metastasis and may put some patients in danger. In this regard, biological markers from the resected IMC that can predict metastasis are warranted. Here, we discovered unique miRNA expression profiles that consist of 21 distinct miRNAs that are specifically upregulated (miR-628-5p, miR-1587, miR-3175, miR-3620-5p, miR-4459, miR-4505, miR-4507, miR-4720-5p, miR-4742-5p, and miR-6779-5p) or downregulated (miR-106b-3p, miR-125a-5p, miR-151b, miR-181d-5p, miR-486-5p, miR-500a-3p, miR-502-3p, miR-1231, miR-3609, and miR-6831-5p) in metastatic (M)-IMC compared to nonmetastatic (N)-IMC, or nonneoplastic gastric mucosa. Intriguingly, most of these selected miRNAs showed stepwise increased or decreased expression from nonneoplastic tissue to N-IMC to M-IMC. This suggests that common oncogenic mechanisms are gradually intensified during the metastatic process. Using a machine-learning algorithm, we demonstrated that such miRNA signatures could distinguish M-IMC from N-IMC. Gene ontology and pathway analysis revealed that TGF-ß signaling was enriched from upregulated miRNAs, whereas E2F targets, apoptosis-related, hypoxia-related, and PI3K/AKT/mTOR signaling pathways, were enriched from downregulated miRNAs. Immunohistochemical staining of samples from multiple institutions indicated that PI3K/AKT/mTOR pathway components, MAPK1, phospho-p44/42 MAPK, and pS6 were highly expressed and the expression of SMAD7, a TGF-ß pathway component, was decreased in M-IMC, which could aid in distinguishing M-IMC from N-IMC. The miRNA signature discovered in this study is a valuable biological marker for identifying metastatic potential of IMCs, and provides novel insights regarding the metastatic progression of IMC.

Amplification of transglutaminase 2 enhances tumor-promoting inflammation in gastric cancers.

Tumor-promoting inflammation is a hallmark of cancer and is highly associated with tumor progression, angiogenesis, and metastasis. Tumor-associated macrophages (TAMs) are major drivers of tumor-promoting inflammation, but due to the complexity of the tumor microenvironment, the detailed regulatory mechanisms are still under investigation. Here, we investigated a novel role for transglutaminase 2 (TGM2) in the development of tumor-promoting inflammation and recruitment of TAMs to gastric cancer (GC) tissues. When estimated by array comparative genomic hybridization and droplet digital PCR, the copy numbers of the TGM2 gene were amplified in 13.6% (14/103) of GC patients and positively associated with TGM2 expression. Gene set enrichment analysis of expression microarray data for GC samples with high or low TGM2 expression showed that increased TGM2 expression was associated with tumor-promoting inflammation in GC. In addition, the expression of TGM2 was correlated with the expression of markers for macrophages, neutrophils, blood vessels, and lymphatic vessels. Overexpression of TGM2 in GC cells augmented the IL-1ß-induced secretion of macrophage-recruiting chemokines and NF-κB activation. TGM2 protein levels were associated with the expression levels of the macrophage marker CD163 in human GC tissue samples. Moreover, GC patients with high expression of TGM2 had a worse prognosis than those with low expression of TGM2. These results suggest TGM2 as a novel regulator of the tumor microenvironment of GC and provide a promising target for constraining tumor-promoting inflammation.