Gastrointestinal stromal tumors (GIST) are rare malignancies originating from mesenchymal tissue, with imatinib as the standard postoperative adjuvant therapy, especially for high-risk patients. However, recurrence after discontinuation remains an important clinical problem. This study aimed to identify factors influencing recurrence after imatinib discontinuation in intermediate- and high-risk GIST patients to guide individualized treatment strategies. Patients who underwent surgical resection and imatinib adjuvant therapy in Nanjing Drum Tower Hospital from 2011 to 2021 were retrospectively analyzed. In this study, end-point of tumor treatment ( EOOT ) recurrence-free survival ( RFS ) was used as the primary endpoint, which was defined as the time from the end of imatinib adjuvant therapy to the recurrence or metastasis of GIST. Univariate and multivariate Cox regression analyses, along with Kaplan-Meier curves, were used to assess the impact of clinicopathological factors. Among 82 patients who discontinued imatinib, 19 experienced recurrence. Tumor site, tumor size, mitotic index, Ki-67 index, risk classification, and mutation type were significantly associated with recurrence (P < 0.05). Kaplan-Meier analysis showed that non-gastric tumors, larger size, high Ki-67, and mitotic index were linked to poorer EOOT RFS. In addition, while the EOOT RFS of patients who had been on the drug for more than 3 years was not statistically significantly different from those who had been on the drug for a shorter period of time, overall the RFS after discontinuation was longer in patients who had been on the drug for a longer period of time. Tumor size, mitotic index, Ki-67 index, and mutation type are key factors influencing recurrence after imatinib discontinuation. Prolonging the time of adjuvant therapy has an important prognostic improvement effect on high-risk patients.

286 Background: MATTERHORN study has demonstrated that immunotherapy (IO) plus triple CT as neoadjuvant treatment significantly improves pCR and prolongs EFS in GC/GEJC. In the previous single-arm SHARED study, we found that IO plus CRT showed promising efficacy and manageable safety as perioperative treatment for GC/GEJC. This study aims to evaluate the efficacy and safety of tislelizumab (TIS) plus CRT, as compared to CRT or CT alone, in the neoadjuvant treatment of pts with GC/GEJC. Methods: Pts with histologically confirmed locally advanced GC/GEJC, stage III-IVa and no prior anticancer therapy were enrolled and randomized (2:2:1) to receive TIS (200 mg) plus CRT (45Gy, SOX or S1+nab-PTX) (arm A), CRT (arm B), or CT alone (SOX or S1+nab-PTX) (arm C) for 3 cycles as neoadjuvant treatment. After surgery, all pts would receive 3 cycles of CT followed by 3 cycles of S-1 as maintenance therapy, with arm A continuing TIS maintenance therapy for 12 months. The primary endpoint was pCR rate assessed in intent to treatment (ITT) set. Secondary endpoints included pCR rate assessed in pts underwent surgery, major pathological response (MPR) assessed in ITT and pts underwent surgery, 2-year EFS/OS rate and safety. Results: Herein, we reported the preliminary results. 87 pts were randomized (arm A, 35; arm B, 33; arm C, 19) with 58.6% T4a/b and 85.1% N2-3. Baseline characteristics were balanced between groups. 60 pts underwent surgery (arm A, 23; arm B, 24; arm C, 13). Efficacy was assessed in pts underwent surgery. The pCR rate was better in arm A versus arm C (34.8% vs. 0%) and arm A versus arm B (34.8% vs. 25%). The MPR rate was higher in arm A versus C (65.2% vs. 15.4%) and was similar between arm A and arm B (65.2% vs. 62.5%). Grade ≥3 TRAE occurred in 14.3% of arm A, 9.1% of arm B and 10.5% of arm C. Any grade irAE was reported in 9 (25.0%) pts of arm A. Conclusions: The preliminary results showed that TIS plus CRT had higher pCR rate over CRT or CT alone for pts with locally advanced GC/GEJC, further confirm the improved efficacy of IO as neoadjuvant therapy for GC/GEJC. Clinical trial information: NCT05687357 .

Background: Neoadjuvant immunochemotherapy (NIT) has significantly improved the prognosis of patients with locally advanced gastric cancer (GC). However, more than half of patients with microsatellite stable disease still exhibit poor treatment response, with immune exclusion being one of the core underlying mechanisms Methods: We integrated multi-omics approaches using samples from a total of 544 patients, and performed validation in an independent cohort of 210 patients. Functional experiments, including genetic ablation and pharmacological inhibition of squalene epoxidase (SQLE), were conducted in vitro and in vivo to evaluate its effects on tumor progression, CD8⁺ T-cell function, and response to anti-PD-1 therapy. Finding: Four distinct GC immunotypes were identified, among which the immune- desert subtype was strongly associated with poor response to NIT. This subtype exhibited enhanced metabolic and proliferative programs, particularly cholesterol homeostasis, and was characterized by elevated SQLE expression. Spatial analyses demonstrated that SQLE-high malignant niches accumulated cholesterol esters and spatially coincided with reduced CD8⁺ T-cell infiltration. A distinct subset of CD8⁺ STMN1⁺ T cells, functionally different from precursor exhausted T cells, was identified as a key mediator of immunotherapy response in immune-desert GC. Mechanistically, SQLE promoted GC cell proliferation and metastasis through activation of the Akt/mTOR/S6K signaling pathway, while simultaneously impairing CD8⁺ STMN1⁺ T- cell effector function by reducing mitochondrial spare respiratory capacity and metabolic flexibility. Importantly, SQLE inhibition or genetic deletion restored CD8⁺ T-cell infiltration and synergized with anti-PD-1 therapy in vivo. Interpretation: Targeting cholesterol metabolism represents a promising strategy to enhance immunotherapy responsiveness in immune desert GC.

Levornidazole is an ornidazole derivative effective against anaerobic pathogens. Levornidazole disodium phosphate, the disodium phosphate salt of levornidazole, exhibits higher water solubility compared to levornidazole. This study aimed to evaluate the efficacy and safety of levornidazole disodium phosphate in patients with intra-abdominal infections (IAIs) caused by anaerobic pathogens. This non-inferiority trial enrolled patients with IAIs in China, randomized 1:1 to either 1 g levornidazole disodium phosphate intravenously once daily (levornidazole group) or 0.5 g ornidazole and sodium chloride intravenously twice daily (control group) for 4–7 days. The primary endpoint was the clinical cure rate at the test-of-cure (TOC) visit. The non-inferiority margin was −8

Postoperative ileus (POI) is characterized by dysregulated inflammation within the intestinal muscular layer, which significantly disrupts gastrointestinal motility and presents a major challenge to postoperative recovery. Although macrophages are known to contribute to inflammation through glycolytic bursts that support rapid energy production, the role of the stimulator of interferon genes (STING) in orchestrating macrophage glycolysis and modulating phenotypic polarization remains poorly defined. To address this gap, we examined the regulatory relationship between STING and macrophage metabolism. Here, we demonstrate that lipopolysaccharide (LPS)-stimulated RAW 264.7 cells display a pronounced enhancement of glycolysis, an effect that was markedly attenuated in STING knockout (STING KO) cells. Further analysis revealed that STING deletion reduces histone lactylation, consequently restricting chromatin accessibility at the hexokinase 2 (HK2) gene loci. Through CUT&Tag sequencing, we identified IRF3 as a transcription factor that directly binds to the promoter regions of HK2 and enhances its expression. Our results delineate a STING-regulated glycolytic feedback loop in macrophages: STING stabilizes hypoxia-inducible factor 1-alpha (HIF1α), thereby amplifying glycolysis and promoting histone lactylation at HK2 loci. This epigenetic modification facilitates IRF3 binding to the HK2 promoter, further boosting HK2 expression and sustaining glycolytic flux. Together, these findings elucidate a molecular mechanism through which STING modulates macrophage polarization via metabolic reprogramming, highlighting the therapeutic potential of targeting STING to regulate macrophage metabolism, alleviate inflammation, and improve outcomes in POI.

Immunosenescence is characterized by immune decline and chronic inflammation. With advancing age, the incidence of tumors increases significantly. Understanding how immunosenescence influences the initiation and progression of tumors, as well as its implications for tumor immunotherapy, has become a matter of urgent importance. This review begins with an analysis of the phenotypic changes and underlying mechanisms associated with immune system and immune cell aging, and further explores the interplay between immunosenescence and tumorigenesis. Evidence indicates that cytokines, cell interactions and other mediators serve as critical links connecting aging and cancer, exerting complex anti-tumor and pro-tumor effects. However, in the context of immunosenescence, these factors collectively contribute to the formation of an immunosuppressive tumor microenvironment (TME) that facilitates tumor immune evasion and proliferation. Clinical data reveal that immunotherapy in older adults is often challenged by variable treatment efficacy and reduced tolerance. This review systematically summarizes the data related to elderly patients in immunotherapy for different types of cancers, and discusses potential immunotherapy sensitization strategies tailored for elderly patients and the mechanisms and immunomodulatory effects of senescence-modulating drugs, with the aim of enhancing therapeutic response rates and improving safety profiles.

Astragaloside IV (AS-IV), a key active component derived from the traditional Chinese medicinal plant Astragali, has been reported to exhibit various biological activities, including antioxidative, anti-inflammatory, immunoregulatory, and antineoplastic properties. This study aimed to elucidate the role of AS-IV in inhibiting gastric cancer (GC) growth, focusing on its impact on cell ferroptosis and the underlying molecular mechanisms. Proliferation and migration of GC cells upon AS-IV treatment were examined using CCK-8, colony formation, and Transwell assays. Ferroptosis induction was analyzed via ELISA, flow cytometry, and transmission electron microscopy. Ferroptosis suppressor protein 1 (FSP1) mRNA stability was assessed by the ActD assay, while RNA immunoprecipitation (RIP) was employed to confirm the interaction between FSP1 mRNA, Fat mass and obesity-associated protein (FTO) demethylase, and YTH N6-methyladenosine RNA-binding protein F2 (YTHDF2). The dual-luciferase reporter assay was used to explore FTO binding to N6-methyladenosine (m6A)-modified sites on FSP1 mRNA. Furthermore, AS-IV’s anti-tumor effects (20 mg/kg) were validated in vivo using gastric cancer xenograft and lung metastasis mouse models. AS-IV significantly suppressed the proliferation and migration of GC cells by inducing ferroptosis. Mechanistically, AS-IV down-regulated FTO, thus impairing its interaction with FSP1 mRNA and leading to increased m6A modification on FSP1 mRNA. This modification facilitated m6A recognition protein YTHDF2-mediated recognition and subsequent degradation of FSP1 mRNA. The reduction of FSP1 triggered ferroptosis, while the overexpression of FSP1 or inhibition of ferroptosis by ferrostatin-1 partially reversed AS-IV’s effects on cell viability and migration. In vivo, AS-IV effectively inhibited tumor growth and metastasis. This study highlights potent anti-GC effects of AS-IV, mediated by the suppression of FSP1 via the FTO/YTHDF2/m6A axis. The treatment of AS-IV inhibits the expression of demethylase FTO in GC cells, subsequently disrupting the binding between FTO protein and FSP1 mRNA. This disruption leads to a increase in the level of m6A modification on FSP1 mRNA, thereby enhancing the recognition and binding of m6A-recognizing protein YTHDF2, promoting the decay of FSP1 mRNA. Ultimately, the downregulation of FSP1 enhances ferroptosis in GC cells. Collectively. AS-IV holds great promise as a novel therapeutic strategy for gastric cancer treatment. This image was created using the Biorender website (https://www.biorender.com/).

ObjectiveTo investigate the influence of lymph node dissection on the prognosis of unresectable locally advanced gastric cancer (LAGC) after conversion immunochemotherapy (CICT).MethodsA total of 287 patients, including 227 from Nanjing Drum Tower Hospital and an external validation cohort of 60 patients (15 from Peking Union Medical College Hospital, 20 from Beijing Friendship Hospital, and 25 from PLA General Hospital) with pathologically diagnosed unresectable primary LAGC (cT3~4N+) underwent radical gastrectomy after multidisciplinary evaluation conversion immunochemotherapy (CICT) were enrolled, and their clinical data were collected. According to the pathological data of patients’ baseline data set, a clinical prediction model of patients’ overall survival (OS) and progression-free survival (PFS) was established, and based on COX regression model, the critical value of the number of lymph node dissection was determined by restricted cubic spline (RCS) analysis.ResultsUnivariate and multivariate COX regression analysis showed that lymph node metastasis (ypN+) was an independent risk factor for OS (P = 0.030) and PFS (P = 0.002). According to the results of COX regression analysis, the prediction model was established. RCS curve showed that the critical number of lymph node dissection was 21 and 28. Patients with 21–28 lymph node dissection had the best OS and PFS. Compared with the patients with 21–28 lymph nodes dissection, the patients with less than 21 lymph nodes dissection had worse OS (P<0.001) and PFS (P = 0.002). Those with more than 28 lymph nodes isolated had worse OS(P = 0.043) and PFS (P = 0.027). The PFS of patients with more than 28 lymph nodes dissection was better than the patients with less than 21lymph nodes dissection (P = 0.031). Although the difference was not statistically significant, the total OS of patients with more than 28 lymph nodes dissection was still higher than that of patients with less than 21 lymph nodes dissection (P = 0.077).ConclusionAmong patients after CICT, lymph node metastasis (ypN+) is an independent risk factor for the prognosis of patients. The survival benefit of patients with less than 21 lymph node dissection is significantly reduced, while those with more than 28 lymph node dissection are less than 21–28 lymph node dissection.

Postoperative complications of gastrectomy severely impact patient prognosis and impose a substantial economic burden. This study aims to assess the effect of adding omega-3 fish oil emulsion to parenteral nutrition on clinical outcomes and cost-effectiveness in gastric cancer populations after gastrectomy. This retrospective cohort study included 150 gastric cancer patients after gastrectomy at Nanjing Drum Tower Hospital between September 2022 and September 2023. Patients were assigned to an omega-3 group and a control group based on whether they received postoperative omega-3 fish oil fat emulsion injection, with 53 matched pairs after propensity score matching (PSM). The primary outcome was the incidence of Grade II or higher complications. An economic evaluation was conducted from a hospital perspective, considering only direct medical costs over a 90-day horizon without discounting, using the incremental cost-effectiveness ratio (ICER). To evaluate the robustness of the primary findings, sensitivity analyses were performed using both one-way and probabilistic methods. After PSM, the omega-3 group demonstrated markedly lower incidences of both Grade II or higher complications (1/53 [1.9

Anoikis is a type of programmed cell death induced by detachment from the extracellular matrix. In cancer cells, anoikis resistance is essential for cancer cell survival in blood circulation and distant metastasis. However, the mechanisms behind anoikis resistance of gastric cancer remain largely unknown. Herein, we demonstrate that NADPH oxidase 4 (NOX4) expression and reactive oxygen species (ROS) generation are upregulated in suspension gastric cell cultures compared with adherent cultures. Silencing of NOX4 decreases ROS generation and downregulates EGFR, sensitizing cells to anoikis. NOX4 overexpression upregulates ROS and EGFR levels and promotes anoikis resistance. NOX4 depletion inhibits gastric cancer survival in blood circulation and attenuates distant metastasis. NOX4 expression is correlated with EGFR expression in patients. In conclusion, induction of NOX4 expression by detachment promotes anoikis resistance of gastric cancer through ROS generation and downstream upregulation of EGFR, which is critical for the metastatic progression of gastric cancer.

Metabolism plays an important role in the occurrence and development of gastric cancer, including in neoadjuvant immunochemotherapy. However, whether nutrition-related indicators can predict the efficacy of neoadjuvant immunochemotherapy and the prognosis of gastric cancer patients has not been addressed. This study systematically screens various nutritional indicators to explore their efficacy in predicting responses to neoadjuvant immunochemotherapy and patients’ prognosis in locally advanced gastric cancer (LAGC). We retrospectively analyzed 134 LAGC patients who underwent radical resection after neoadjuvant immunochemotherapy. According to postoperative tumor regression grade (TRG), these patients were divided into good responder group (TRG1-0) and poor responder group (TRG3-2) (AJCC/CAP guidelines). Inflammatory and/or nutritional markers were compared for their efficacy on predicting patients’ pathological tumor regression response. The univariate and multivariate logistic regression were carried out to identify the independent factors for predicting pathological tumor regression response, and a predictive nomogram model was further established. Among the total 134 LAGC patients, tumor specimens from 71 and 63 patients had TRG1-0 and TRG3-2 tumor responses, respectively. Multivariate analysis showed that controlling nutritional status (CONUT) score and nutrition risk screening 2002 (NRS2002) score were independent predictors of pathological tumor regression response (OR, 0.68; 95

Gastric cancer remains a global health concern with high incidence and mortality rates. Accurate preoperative prediction of lymph node (LN) metastasis is crucial for staging, treatment planning, and prognosis. This study introduces a novel 3D end-to-end lymph node metastasis multi-task learning network (LMML-net) designed to predict LN metastasis across multiple nodal stations in gastric cancer. We analyzed a cohort of 293 patients who underwent gastrectomy with LN dissection. Preoperative CT scans, conducted within two weeks before surgery, were utilized. The LMML-net integrates tumor segmentation and LN metastasis prediction, employing a 3D attention-unet for tumor segmentation and a multi-task learning approach to address metastasis at different nodal stations. LMML-net demonstrated robust predictive performance, achieving AUCs of 0.813, 0.820, and 0.805 for total LN metastasis in training, testing, and validating cohorts, respectively. Notably, the model effectively addressed challenges posed by early gastric cancer and exhibited satisfactory results across various nodal stations. Visualization through GradCam highlighted significant contributions of both tumor and connective tissue areas to the predictions, enhancing the model’s interpretability. The LMML-net exhibits strong predictive capabilities for LN metastasis across multiple stations in gastric cancer, including cases of early-stage disease. This innovative approach holds promise for guiding personalized preoperative treatments and surgical planning, potentially improving patient outcomes in gastric cancer management. Code and models will be available at: https://github.com/yangzhi028/LMML-net.

Conversion immunochemotherapy may enable curative surgery in patients with initially unresectable locally advanced gastric cancer, but its response rate remains low. Little evidence has shown how to easily predict therapeutic efficacy from hematological biomarkers, apart from tissue biomarkers. This study aimed to identify predictive factors for treatment response from hematological biomarkers and propose strategies for non-responsive patients. Clinical and laboratory data from 153 PD-L1 negative and microsatellite stable advanced gastric cancer patients treated with conversion immunochemotherapy followed by radical gastrectomy were retrospectively analyzed. Correlations between key indicators and treatment outcomes were assessed, and a nomogram was developed to predict tumor regression grade (TRG). TRG was strongly associated with the prognosis of advanced gastric cancer treated with conversion immunochemotherapy. Factors significantly related to TRG included hypertension, pre-treatment serum albumin levels, tumor size, post-treatment serum IL-10, IFN-γ, TNF-α, and changes in tumor-associated carbohydrate antigens before and after treatment. Hypertension, pre-treatment serum albumin levels, post-treatment IL-10 levels, and restoration in tumor-associated carbohydrate antigens were identified as independent predictors of TRG. Dynamic monitoring of serum albumin, IL-10, and tumor-associated carbohydrate antigens can help assess treatment response and identify patients sensitive to conversion immunochemotherapy. For non-responsive patients, strategies such as the associations observed require validation in prospective interventional trials before specific interventions can be recommended.

INTRODUCTION:Gastric neuroendocrine neoplasms (g-NENs) are a rare type of stomach tumor. However, limited data exist about the expression and clinical significance of B7 family ligands/receptors in patients with g-NENs. Thus, we conducted this study to address this issue in a cohort of 112 patients with g-NENs. METHODS:Using immunohistochemistry, we mapped and quantified the expression of the B7 family ligands/receptors in 112 g-NEN samples: programmed cell death ligand 1 and 2 (PD-L1 and PD-L2), B7-H3, B7-H4, recombinant human galectin-9 (LGALS9), and CD155. Associations between the marker levels, clinicopathological variables, and survival were evaluated. RESULTS:The percentages of high expression of PD-L1, PD-L2, B7-H3, B7-H4, LGALS9, and CD155 in the cohort of 112 g-NEN cases were 37.5%, 55.4%, 46.4%, 37.5%, 46.4%, and 51.8%, respectively. Elevated expression of PD-L1, PD-L2, B7-H3, B7-H4, LGALS9, and CD155 was significantly associated with several clinicopathological characteristics. K-M analysis indicated that high expression levels of CD155, B7-H3, PD-L2, and LGALS9 were correlated with poor overall survival (OS) ( P < 0.0001, P = 0.0002, P = 0.0319 and P = 0.0120, respectively). Multivariate Cox regression analysis indicated that high CD155 expression, vasculature invasion, and worse World Health Organization pathological grade were independent prognostic factors for OS ( P = 0.007, P = 0.030, and P = 0.019, respectively). DISCUSSION:We detected variable expression of the PD-L1, PD-L2, B7-H3, B7-H4, LGALS9, and CD155 proteins in g-NENs. These results suggest that the expression level of CD155 may be a vital indicator of OS in patients with g-NENs. B7 family ligands/receptors could be potential immunotherapeutic targets for g-NENs.

Acute severe autoimmune hepatitis (AS-AIH) is characterized by rapid progression and poor prognosis, with a current lack of effective targeted treatments. Stem cell therapy has demonstrated significant therapeutic promise across various autoimmune diseases. However, the intricate pathogenesis of AS-AIH has hindered the widespread utilization of mesenchymal stem cells (MSCs) in this domain. Herein, it is demonstrated that necroptosis, as the primary mode of cell death in AIH, is crucial in causing AS-AIH. Inflammatory macrophages are the primary cell population involved in necroptosis. Inhibition of the specificity protein 1/sphingosine kinase 1/sphingosine-1-phosphate (SP1/SK1/S1P) axis is responsible for this phenomenon, leading to excessive activation of the intrahepatic immune system and aggravating liver damage. Furthermore, the S1P/S1PR2/YAP axis is the key pathway in initiating liver regeneration during AS-AIH. S1P synthesized by hepatocytes is the primary source, and this process is also regulated by the SP1/SK1 axis. MSCs promote S1P synthesis by macrophages through the delivery of SP1, which inhibits necroptosis and synergistically enhances liver regeneration. In addition, MSCs also promote S1P synthesis in hepatocytes through the same mechanism, further aiding liver regeneration. These findings unveil the core pathogenesis of AS-AIH and provide a theoretical foundation for using MSCs as a potential targeted therapeutic modality.

Background:Papillary thyroid carcinoma (PTC) often metastasizes to lymph nodes, increasing recurrence risk and reducing survival. This study identifies predictors for contralateral central lymph node metastasis (Cont-CLNM) in unilateral PTC patients with ipsilateral lateral cervical lymph node metastasis (Ipsi-LLNM). Methods:We retrospectively analyzed data, preoperative ultrasound features, and thyroglobulin (Tg) levels in unilateral PTC patients with Ipsi-LLNM treated at the Thyroid Surgery Department of Nanjing Drum Tower Hospital from August 2017 to August 2024. Least absolute shrinkage and selection operator (LASSO) regression was used for variable selection, with independent t-tests and Chi-squared tests assessing differences. Logistic regression analyses identified risk factors for Cont-CLNM, and a nomogram was validated using 1,000 bootstrap resamples. Decision curve analysis (DCA) evaluated clinical impact. Results:Of 105 PTC patients, 56 (53.3%) had Cont-CLNM. LASSO regression identified three predictors: male sex, lymph node metastasis posterior to the recurrent laryngeal nerve (LN-prRLN), and elevated Tg levels. Multivariate regression confirmed these variables' association with Cont-CLNM. Internal validation yielded an area under the curve of 0.771 [95% confidence interval (CI): 0.684-0.857]. A nomogram was developed and validated through DCA. Conclusions:Our findings indicate that combining male gender, LN-prRLN, and Tg levels effectively predicts Cont-CLNM, providing a basis for risk assessment in unilateral PTC.

The microenvironment tends to be immunosuppressive during tumor growth and proliferation. Immunotherapy has attracted much attention because of its ability to activate tumor-specific immune responses for tumor killing. The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is an innate immune pathway that activates antitumor immunity by producing type I interferons. Cyclic dinucleotides (CDNs), produced by cGAS sensing cytoplasmic abnormal DNA, are major intermediate activating molecules in the STING pathway. Nowadays, CDNs and their derivatives have widely worked as powerful STING agonists in tumor immunotherapy. However, their clinical translation is hindered by the negative electrical properties, sensitivity to hydrolytic enzymes, and systemic toxicity. Recently, various CDN delivery systems have made significant progress in addressing these issues, either through monotherapy or in combination with other treatment modalities. This review details recent advances in CDNs-based pharmaceutical development or delivery strategies for enriching CDNs at tumor sites and activating the STING pathway.

Immunotherapy has emerged as a promising therapeutic strategy for cancer. The activation of the stimulator of interferon genes (STING) pathway promotes the polarization of tumor-associated macrophages (TAMs) towards M1 phenotype, with 2', 3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) serving as an inherent activator, which significantly accumulates in the extracellular space of the tumor sites following radiotherapy (RT). However, the electronegativity and hydrophilicity of cGAMP prevent it from crossing the cell membrane into TAMs, hampering subsequent immunotherapy efficacy. Here, positively charged protamine-modified Salmonella (VNP20009), called VNP-protamine (VNP-PRM), were prepared to enrich cGAMP and form a composite bacteria-drug delivery system with the capacity to enter TAMs freely. Via electrostatic interactions between the guanidine groups of protamine and the phosphate groups of cGAMP, VNP-PRM stably enriched cGAMP on their surface and neutralized the electronegativity of cGAMP. The uptake efficiency of cGAMP by TAMs was then significantly enhanced by the active delivery of VNP-PRM, whose inherent motility and cellular invasiveness endowed them with increased potential to bump against and enter the macrophages. Subsequently, the intracellular cGAMP synergized with the immunogenicity of the bacteria to activate the STING pathway and drive M1 polarization, thereby boosting tumor eradication. Therefore, antitumor immunotherapy can be optimized through in situ enrichment and delivery of post-RT cGAMP to TAMs by the engineered bacteria.

Abstract Aim Postoperative ileus (POI) is one of the most common complications after abdominal surgery, especially subsequent to intra-abdominal infection. The underlying mechanism remains unclear. This study is to investigate the role of HIF1α in the progression of POI. Methods Bioinformatics analysis was utilized to analyze the association between HIF1α and POI. A murine model with POI was generated by surgical intestinal manipulation (IM). Gastrointestinal transit (GIT), leukocyte infiltration and inflammatory mediators were measured. Targeting HIF1α with specific inhibitor (2ME2) was performed in both in vivo and in vitro experiment. Human samples were collected for further validation. Results Public RNA sequencing data demonstrated upregulation of HIF1α in murine intestinal muscularis after POI generation. This was confirmed by our murine models as well as human samples, which was accompanied by impaired GIT, higher inflammatory cytokines, inducible nitric oxide synthetase (iNOS) expression and nitric oxide (NO) generation. HIF1α-specific inhibitor 2ME2 reversed inflammatory response in macrophages, suppressed NO production, and eventually ameliorated GI motility. Conclusions HIF1α controls inflammatory response and NO generation in intestinal muscularis, Targeting HIF1α effectively improves GI motility and ameliorates POI development.Fig. 1Gastrointestinal motility injury and inflammatory cell infiltration in the intestinal muscularis after IM-induced POIa Gross pictures of the representational photo in the Sham and IM groups. b FITC-dextran distribution in the intestinal tract of mice 24 h after IM and the genomic center (GC) of FITC-dextran distribution in each experiment group. c Typical HE staining images in each group (×200, ×400). d Typical immunohistochemical staining images with MPO (×200, ×400). Fig. 2 Identification of differentially expressed genes (DEGs) in the GEO dataset GSE134942 a Volcano plot of DEGs. Log2|Foldchange| >1 and P < 0.05 were set as the cutoff values. The red plots represent upregulated DEGs, while the blue plots represent downregulated DEGs. b The top 20 GSEA pathways based on the adjusted P-values. (c–f) Representative results of GSEA analysis of the IM24h group genes. Fig. 3The upregulation of HIF1α was validated in animal models of postoperative ileusa HE staining of the whole layer of the small intestine and the small intestinal muscle layer after the elimination of mucosa. b Representative Western blotting images probed for HIF1α and iNOS extracted from the small intestinal muscularis. c Quantitative RT-PCR analysis of HIF1α, iNOS, IL1β, and COX-2 mRNA levels in murine intestinal muscularis. d The immunofluorescence images of the sham-operated and IM mice intestine. Fig. 4 Validation of HIF1α upregulation in the clinical specimens of patients with intestinal obstruction a Representative Western blotting images probed for HIF1α and iNOS extracted from muscularis in surgically resected intestinal segments. The control group represents the normal intestinal segments of gastric cancer patients that require gastrojejunostomy and small bowel resection. The ileus group represents ischemia intestinal segments from intestinal obstruction patients who underwent bowel anastomosis after the resection of the necrotic bowel. b Quantitative RT-PCR analysis of the HIF1α, iNOS, IL1β, IL6, and COX-2 mRNA levels in human intestinal muscularis. c The immunofluorescence images of the control and ileus clinical intestinal muscularis specimens. Fig. 5Intraperitoneal injection of HIF1α-specific inhibitor 2ME2 relieved gastrointestinal motility injury in vivoa A schematic diagram of the experiment in vivo. b, c FITC-dextran distribution in the intestinal tract of mice 1 day after IM and genomic center (GC) of FITC-dextran distribution in each experiment group. d Representative Western blotting images probed for HIF1α, iNOS, and COX2 extracted from the small intestinal muscularis. e Total NO extracted from the intestinal muscularis homogenate. Fig. 6 2ME2-induced alleviation of the inflammatory cytokines’ expression and NO generation in macrophages a Representative Western blotting images probed for HIF1α, iNOS, and COX2 extracted from 2ME2 treated-RAW264.7 cells subjected to LPS stimulation or hypoxia culture. b Total NO in the cell culture supernatant.