Immune checkpoint reprogramming via sequential nucleic acid delivery strategy optimizes systemic immune responses for gastrointestinal cancer immunotherapy.

Monoclonal antibodies targeting immune checkpoints have been widely applied in gastrointestinal cancer immunotherapy. However, systemic administration of various monoclonal antibodies does not often result in sustained effects in reversing the immunosuppressive tumor microenvironment (TME), which may be due to the spatiotemporal dynamic changes of immune checkpoints. Herein, we reported a novel immune checkpoint reprogramming strategy for gastrointestinal cancer immunotherapy. It was achieved by the sequential delivery of siPD-L1 (siRNA for programmed cell death ligand 1) and pOX40L (plasmid for OX40 ligand), which were complexed with two cationic polymer brush-grafted carbon nanotubes (dense short (DS) and dense long (DL)) designed based on the structural characteristics of nucleic acids and brush architectures. Upon administrating DL/pOX40L for the first three dosages, then followed by DS/siPD-L1 for the next three dosages to the TME, it upregulated the stimulatory checkpoint OX40L on dendritic cells (DCs) and downregulated inhibitory checkpoint PD-L1 on tumor cells and DCs in a sequential reprogramming manner. Compared with other combination treatments, this sequential strategy drastically boosted the DCs maturation, and CD8+ cytotoxic T lymphocytes infiltration in tumor site. Furthermore, it could augment the local antitumor response and improve the T cell infiltration in tumor-draining lymph nodes to reverse the peripheral immunosuppression. Our study demonstrated that sequential nucleic acid delivery strategy via personalized nanoplatforms effectively reversed the immunosuppression status in both tumor microenvironment and peripheral immune landscape, which significantly enhanced the systemic antitumor immune responses and established an optimal immunotherapy strategy against gastrointestinal cancer.

A nomogram predicting distant metastasis risk for gastric cancer patients with preoperative anemia: a multicenter retrospective study.

BACKGROUND: Anemia represents a well-established risk factor for patients diagnosed with gastric cancer, and is often associated with an unfavorable prognosis. In this context, the timely prediction of distant metastasis risk in patients with anemic gastric cancer assumes paramount importance. METHODS: Information of gastric cancer patients complicated with preoperative anemia in the First Affiliated Hospital of Sun Yat-sen University was collected. The cohort from the First Affiliated Hospital of Guangxi Medical University was used as an external validation set. A Nomogram was established based on the risk factors screened by univariate and multivariate logistic regression analyses. RESULTS: A total of 848 gastric cancer patients with preoperative anemia were enrolled. Pyloric obstruction, carcinoma antigen 125, T stage, N stage, tumor size, and preoperative weight loss were independent predictors of distant metastasis in gastric cancer patients with anemia (p < 0.05), based on which a nomogram was constructed. The accuracy, reliability and clinical value of the nomogram were evaluated by concordance index, receiver operating characteristic curve, decision curve analysis, calibration curve and showed good stability and clinical predictive value. CONCLUSIONS: Preoperative anemic gastric cancer patients, complicated with pyloric obstruction, elevated CA125, advanced T and N stage, larger tumor size, and preoperative weight loss, should be paid more attention to distant metastasis.

NAT10 Overexpression Promotes Tumorigenesis and Epithelial-Mesenchymal Transition Through AKT Pathway in Gastric Cancer.

BACKGROUND: N-acetyltransferase 10 (NAT10), the only RNA cytosine acetyltransferase known in humans, contributes to cancer tumorigenesis and progression. This study aims to investigate the effect of NAT10 on the malignant biological properties of gastric cancer (GC) and its underlying mechanism. METHODS: The expression and prognostic significance of NAT10 in GC were analyzed using The Cancer Genome Atlas (TCGA) and Sun Yat-sen University (SYSU) cohorts. The influence of NAT10 on the malignant biological behaviors of GC was detected by Cell Counting Kit-8 (CCK-8) assay, plate colony formation assay, 5-ethynyl-2'-deoxyuridine (EdU), Transwell migration and invasion assays, scratch wound assay, flow cytometric analysis, and animal studies. The overall level of N4 acetylcytidine (ac4C) in GC was detected by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The downstream signal pathways of NAT10 were analyzed by Gene Set Enrichment Analysis (GSEA) and verified by Western blot (WB) and immunofluorescence (IF). RESULTS: The significant upregulation of NAT10 expression in GC was associated with a poor prognosis. The knockdown of NAT10 markedly suppressed GC cell proliferation, migration, invasion, and cell cycle progression. Downregulating NAT10 reduced ac4C levels and inhibited AKT phosphorylation and epithelial-mesenchymal transition (EMT) in GC. CONCLUSIONS: NAT10 functions as an oncogene and may provide a new therapeutic target in GC.

NBS1 lactylation is required for efficient DNA repair and chemotherapy resistance.

The Warburg effect is a hallmark of cancer that refers to the preference of cancer cells to metabolize glucose anaerobically rather than aerobically1,2. This results in substantial accumulation of lacate, the end product of anaerobic glycolysis, in cancer cells3. However, how cancer metabolism affects chemotherapy response and DNA repair in general remains incompletely understood. Here we report that lactate-driven lactylation of NBS1 promotes homologous recombination (HR)-mediated DNA repair. Lactylation of NBS1 at lysine 388 (K388) is essential for MRE11-RAD50-NBS1 (MRN) complex formation and the accumulation of HR repair proteins at the sites of DNA double-strand breaks. Furthermore, we identify TIP60 as the NBS1 lysine lactyltransferase and the 'writer' of NBS1 K388 lactylation, and HDAC3 as the NBS1 de-lactylase. High levels of NBS1 K388 lactylation predict poor patient outcome of neoadjuvant chemotherapy, and lactate reduction using either genetic depletion of lactate dehydrogenase A (LDHA) or stiripentol, a lactate dehydrogenase A inhibitor used clinically for anti-epileptic treatment, inhibited NBS1 K388 lactylation, decreased DNA repair efficacy and overcame resistance to chemotherapy. In summary, our work identifies NBS1 lactylation as a critical mechanism for genome stability that contributes to chemotherapy resistance and identifies inhibition of lactate production as a promising therapeutic cancer strategy.

OLFM4 promotes the progression of intestinal metaplasia through activation of the MYH9/GSK3ß/ß-catenin pathway.

BACKGROUND: Intestinal metaplasia (IM) is classified into complete intestinal metaplasia (CIM) and incomplete intestinal metaplasia (IIM). Patients diagnosed with IIM face an elevated susceptibility to the development of gastric cancer, underscoring the critical need for early screening measures. In addition to the complexities associated with diagnosis, the exact mechanisms driving the progression of gastric cancer in IIM patients remain poorly understood. OLFM4 is overexpressed in several types of tumors, including colorectal, gastric, pancreatic, and ovarian cancers, and its expression has been associated with tumor progression. METHODS: In this study, we used pathological sections from two clinical centers, biopsies of IM tissues, precancerous lesions of gastric cancer (PLGC) cell models, animal models, and organoids to explore the role of OLFM4 in IIM. RESULTS: Our results show that OLFM4 expression is highly increased in IIM, with superior diagnostic accuracy of IIM when compared to CDX2 and MUC2. OLFM4, along with MYH9, was overexpressed in IM organoids and PLGC animal models. Furthermore, OLFM4, in combination with Myosin heavy chain 9 (MYH9), accelerated the ubiquitination of GSK3ß and resulted in increased ß-catenin levels through the Wnt signaling pathway, promoting the proliferation and invasion abilities of PLGC cells. CONCLUSIONS: OLFM4 represents a novel biomarker for IIM and could be utilized as an important auxiliary means to delimit the key population for early gastric cancer screening. Finally, our study identifies cell signaling pathways involved in the progression of IM.

MEX3A promotes colorectal cancer migration, invasion and EMT via regulating the Wnt/ß-catenin signaling pathway.

BACKGROUND: Mex-3 RNA binding family members are well-established to be important in cancer development and progression. However, the functions of Mex-3 RNA binding family member A (MEX3A) in colorectal cancer (CRC) metastasis remain poorly understood. In this study, we aim to reveal the function and the mechanism of MEX3A in promoting CRC metastasis. METHODS: We used multiple databases including TCGA database, UALCAN, LinkedOmics, CancerSEA, GeneMANIA and STRING database to investigate the expression, the functions and underlying molecular mechanism of MEX3A in CRC. Multiple experimental methods were adapted to determine the study, including real-time PCR (qPCR), immunohistochemistry (IHC), western blot (WB), transfection, transwell migration and invasion assays, immunofluorescence (IF). RESULTS: We found that MEX3A was significantly upregulated and correlated to tumor stage and lymph nodal metastasis in CRC through bioinformatics analysis and tissue immunohistochemistry (IHC). The higher expression of MEX3A in CRC correlated with poor recurrence-free survival (RFS) and overall survival (OS). In vitro studies showed that knockdown of MEX3A suppressed EMT transition, invasion and metastasis of CRC cells. Mechanistically, we revealed that MEX3A promotes epithelial-mesenchymal transition (EMT), invasion and metastasis of CRC cells by upregulating the Wnt/ß-catenin signaling pathway. CONCLUSION: In conclusion, our study reveals that MEX3A promotes CRC migration, invasion and EMT via regulating the Wnt/ß-catenin signaling pathway and could be a novel therapeutic target for this patient population.

3D bioprinting platform development for high-throughput cancer organoid models construction and drug evaluation.

The evaluation of anti-tumor drugs is critical for their development and clinical guidance. Tumor organoid models are gaining increased attention due to their ability to better mimic real tumor tissues, as well as lower time and economic costs, which makes up for the shortcomings of cell lines and xenograft models. However, current tumor organoid cultures based on the Matrigel have limitations in matching with high-throughput engineering methods due to slow gelation and low mechanical strength. Here, we present a novel composite bioink for culturing colorectal cancer organoids that provides an environment close to real tissue growth conditions and exhibits excellent photocrosslinking properties for rapid gel formation. Most importantly, the tumor organoids viability in the composite bioink after printing was as high as 97%, which also kept multicellular polar structures consistent with traditional culture methods in the Matrigel. Using 3D bioprinting with this composite bioink loaded with organoids, we demonstrated the feasibility of this drug evaluation model by validating it with clinically used colorectal cancer treatment drugs. Our results suggested that the composite bioink could effectively cultivate tumor organoids using 3D bioprinting, which had the potential to replace less reliable manual operations in promoting the application of tumor organoids in drug development and clinical guidance.

NIFK, a Potential Prognostic and Immunological Biomarker in Pan-- Cancer Analysis, Significantly Regulates Proliferation and Metastasis of Colorectal Cancer.

BACKGROUND: As a binding protein of Ki67, NIFK plays an important role in the mitosis of cells and is closely related to the progression of specific types of tumors. However, there is still a lack of systematic analysis of NIFK in pan-cancer and insufficient research to explore its role in human tumors. METHODS: We systematically evaluated the pan-cancer expression and mutation of NIFK in human cancers using data from The Cancer Genome Atlas (TCGA) through large-scale bioinformatics analysis. In addition, we explored the pan-cancer immunological characteristics of NIFK, especially in colorectal adenocarcinoma (COAD). Furthermore, we used single-cell sequencing to analyze the expression of NIFK in different cells of COAD tissues and performed GO, KEGG, and gene set enrichment analysis of NIFK in COAD. Lastly, we evaluated the effects of NIFK knockdown on the colorectal cancer cell lines in in vitro experiment. RESULTS: We found that NIFK was overexpressed in almost all types of tumors and showed significant prognostic efficacy. Additionally, correlations between NIFK and specific immune features, such as immune cell infiltration, immune checkpoint genes, TMB, and MSI, suggest that NIFK may be used to guide immunotherapy. Subsequently, it was found that the expression of NIFK was significantly upregulated in tumor cells through single-cell sequencing analysis, and the NIFK gene was closely associated with tumor progression and immune therapy response. Finally, we further elucidated the role of NIFK in colorectal cancer and found that downregulation of NIFK expression could inhibit the proliferation, migration, and invasion ability of colorectal cancer cells. CONCLUSION: The results of this study demonstrated that NIFK, as a member of the pan-cancer genes, will serve as a biomarker and a potential therapeutic target for a range of cancer types, providing new insight into precision medicine.

Comparative Study on the H-Abstraction Reactions of Isopropyl Acetate and Propyl Acetate by HO2 and OH Radicals.

Isopropyl acetate (IPA) and propyl acetate (PA) are recognized as promising biofuels suitable for applications as fuel additives and biodiesel models. The H-abstraction reactions with radicals stand out as the fundamental initiating reactions in the combustion kinetic models for IPA and PA. In the present work, the kinetic calculations of IPA and PA plus HO2 and OH radicals were investigated at M06-2X/cc-pVTZ//G4, M08-HX/maug-cc-pVTZ, and CCSD(T)/jul-cc-pVTZ levels. The thermodynamic calculations were obtained based on the G4 and CBS-APNO methods. Rate coefficients were calculated using both transition state theory and canonical variational transition state theory with tunneling correction at the temperature range of 250-2000 K. The total rate constants for the IPA + OH system were fitted as follows: k = 0.4674 × T3.927 exp(2128/T) (cm3 mol-1 s-1), and for the PA + OH system, the total rate constants were determined using the following equation: k = 0.0161 × T4.373 exp(2220/T) (cm3 mol-1 s-1). The rate coefficients of IPA + OH reactions determined based on the M08-HX/maug-cc-pVTZ level effectively replicate the experimental data, while H-abstraction rate coefficients of PA + OH by the CCSD(T)/jul-cc-pVTZ method accurately reproduce the experimental data. Refining the H-abstraction rate coefficients in the kinetic mechanism of PA, as proposed by Dayma et al. [Proc. Combust. Inst. 37 (2019) 429-436], has been achieved through incorporating the present calculated data, leading to the development of a revised mechanism. The validation of the updated mechanism against jet-stirred reactor data is presented, showcasing its effective performance in predicting JSR data.

Exosome circATP8A1 induces macrophage M2 polarization by regulating the miR-1-3p/STAT6 axis to promote gastric cancer progression.

Circular RNAs (circRNAs) play important roles in gastric cancer progression but the regulatory role of circRNAs in controlling macrophage function remains elusive. Exosomes serve as cargo for circRNAs and play a crucial role as mediators in facilitating communication between cancer cells and the tumor microenvironment. In this study, we found that circATP8A1, a previously unreported circular RNA, is highly expressed in both gastric cancer tissues and exosomes derived from plasma. Increased circATP8A1 was associated with advanced TNM stage and worse prognosis in patients with gastric cancer. We showed that  the circATP8A1 knockdown significantly inhibited gastric cancer proliferation and invasion in vitro and in vivo. Functionally, exosome circATP8A1 induced the M2 polarization of macrophages through the STAT6 pathway instead of the STAT3 pathway. Mechanistically, circATP8A1 was shown to activate the STAT6 pathway through competitive binding to miR-1-3p, as confirmed by Fluorescence In Situ Hybridization (FISH), RNA immunoprecipitation, RNA pulldown, and Luciferase reporter assays. The reversal of circATP8A1-induced STAT6 pathway activation and macrophage polarization was observed upon blocking miR-1-3p. Macrophages treated with exosomes from gastric cancer cells overexpressing circATP8A1 were able to promote gastric cancer migration, while knockdown of circATP8A1 reversed these effects in vivo. In summary, exosome-derived circATP8A1 from gastric cancer cells induce macrophages M2 polarization via the circATP8A1/miR-1-3p/STAT6 axis, and tumor progression. Our results highlight circATP8A1 as a potential prognostic biomarker and therapeutic target in gastric cancer.

RNA methylation-related genes INHBB and SOWAHA are associated with MSI status in colorectal cancer patients and may serve as prognostic markers for predicting immunotherapy efficacy.

The role of RNA methylation is vital in the advancement and spread of tumors. However, its exact role in microsatellite instability in colorectal cancer (CRC) is still not fully understood. To address this gap in knowledge, this study investigated the impact of genes associated with RNA methylation on the prognosis and response to immunotherapy in individuals diagnosed with low microsatellite instability (MSI-L) or microsatellite stable (MSS) CRC. The differentially expressed genes (DEGs) in two groups of patients: those with high microsatellite instability (MSI-H) and those with MSI-L/MSS was thoroughly investigated and compared with aims of exploring the association between them and the 60 RNA methylation regulators. We employed these genes and developed an MSI-RMscore to establish a risk signature capable of forecasting patient outcomes. Furthermore, an investigation of the immunophenotypic traits was conducted encompassing patients categorized as high-risk and low-risk. By combining the MSI-RMscore and clinicopathological features, a predictive nomogram was developed, which was subsequently validated using the GEO database. Furthermore, immunohistochemistry was employed to establish the correlation between INHBB and SOWAHA and the MSI status, as well as patient prognosis. Our findings indicated that the high-risk subgroup exhibited unfavorable overall survival rates, reduced responsiveness to immune checkpoint blockers, elevated estimate scores, and increased infiltration of macrophages and fibroblasts. We also confirmed that INHBB and SOWAHA were associated with CRC patient prognosis and MSI status, as well as immunotherapy response. These findings suggest that targeting INHBB and SOWAHA could be a promising strategy to enhance patient responsiveness to immunotherapy.

Combined Hepatocellular-Cholangiocarcinoma: Biology, Diagnosis, and Management.

Background: Combined hepatocellular-cholangiocarcinoma (cHCC-iCCA) is a rare type of primary liver cancer displaying characteristics of both hepatocytic and cholangiocytic differentiation. Summary: Because of its aggressive nature, patients with cHCC-iCCA exhibit a poorer prognosis than those with HCC. Surgical resection and liver transplantation may be considered curative treatment approaches; however, only a minority of patients are eligible at the time of diagnosis, and postoperative recurrence rates are high. For cases that are not eligible for surgery, locoregional and systemic therapy are often administered based on treatment protocols applied for HCC or iCCA. Owing to the rarity of this cancer, there are still no established standard treatment protocols; therefore, the choice of therapy is often personalized and guided by the suspected predominant component. Further, the genomic and molecular heterogeneity of cHCC-iCCA can severely compromise the efficacy of the available therapies. Key Messages: In the present review, we summarize the latest advances in cHCC-iCCA and attempt to clarify its terminology and molecular biology. We provide an overview of the etiology of cHCC-iCCA and present new insights into the molecular pathology of this disease that could contribute to further studies aiming to improve the patient outcomes through new systemic therapies.

M1 polarization of macrophages promotes stress-induced hair loss via interleukin-18 and interleukin-1ß.

Stress-induced hair loss is a prevalent health concern, with mechanisms that remain unclear, and effective treatment options are not yet available. In this study, we investigated whether stress-induced hair loss was related to an imbalanced immune microenvironment. Screening the skin-infiltrated immune cells in a stressed mouse model, we discovered a significant increase in macrophages upon stress induction. Clearance of macrophages rescues mice from stress-induced hair shedding and depletion of hair follicle stem cells (HFSCs) in the skin, demonstrating the role of macrophages in triggering hair loss in response to stress. Further flow cytometry analysis revealed a significant increase in M1 phenotype macrophages in mice under stressed conditions. In searching for humoral factors mediating stress-induced macrophage polarization, we found that the hormone Norepinephrine (NE) was elevated in the blood of stressed mice. In addition, in-vivo and in-vitro studies confirm that NE can induce macrophage polarization toward M1 through the ß-adrenergic receptor, Adrb2. Transcriptome, enzyme-linked immunosorbent assay (ELISA), and western blot analyses reveal that the NLRP3/caspase-1 inflammasome signaling and its downstream effector interleukin 18 (IL-18) and interleukin 1 beta (IL-1ß) were significantly upregulated in the NE-treated macrophages. However, inhibition of the NE receptor Adrb2 with ICI118551 reversed the upregulation of NLRP3/caspase-1, IL-18, and IL-1ß. Indeed, IL-18 and IL-1ß treatments lead to apoptosis of HFSCs. More importantly, blocking IL-18 and IL-1ß signals reversed HFSCs depletion in skin organoid models and attenuated stress-induced hair shedding in mice. Taken together, this study demonstrates the role of the neural (stress)-endocrine (NE)-immune (M1 macrophages) axis in stress-induced hair shedding and suggestes that IL-18 or IL-1ß may be promising therapeutic targets.

Pumilio1 regulates NPM3/NPM1 axis to promote PD-L1-mediated immune escape in gastric cancer.

Abnormal regulation of RNA binding proteins (RBPs) plays an essential role in tumorigenesis and progression, but their functions and mechanisms remain largely elusive. Previously, we reported that Pumilio 1 (PUM1), a RBP, could regulate glycolysis metabolism and promote the progression of gastric cancer (GC). However, the role of PUM1 in tumor immune regulation remains largely elusive. In this study, we report that PUM1 induces immune escape through posttranscriptional regulation of PD-L1 in GC. We used multiplexed immunohistochemistry to analyze the correlation between PUM1 expression and immune microenvironment in GC. The effect of PUM1 deficiency on tumor killing of T cells was examined in vitro and in vivo. The molecular mechanism of PUM1 was evaluated via RNA immunoprecipitation, chromatin immunoprecipitation, Western blot, co-immunoprecipitation, and RNA stability assays. Clinically, elevated PUM1 expression is associated with high-expression of PD-L1, lack of CD8+ T cell infiltration and poor prognosis in GC patients. PUM1 positively regulates PD-L1 expression and PUM1 reduction enhances T cell killing of tumors. Mechanistically, PUM1 directly binds to nucleophosmin/nucleoplasmin 3 (NPM3) mRNA and stabilizes NPM3. NPM3 interacts with NPM1 to promote NPM1 translocation into the nucleus and increase the transcription of PD-L1. PUM1 inhibits the anti-tumor activity of T cells through the PUM1/NPM3/PD-L1 axis. In summary, this study reveals the critical post-transcriptional effect of PUM1 in the modulation of PD-L1-dependent GC immune escape, thus provides a novel indicator and potential therapeutic target for cancer immunotherapy.