The role of psychological factors in the holistic regulation of cancer has garnered significant attention. Psychological eustress induced by an enriched environment (EE) inhibits various cancers including melanoma, but underlying mechanisms remain largely unknown. The brain-gut axis may play a key role in modulating psychological factors and cancer progression. This study investigates the role of gut microbiota in EE-induced anti-melanoma effects. C57BL/6 mice were housed in EE or standard environment (SE). Longitudinal fecal samples from EE and SE mice underwent 16S rRNA gene sequencing. SE melanoma-bearing mice received fecal microbiota transplantation (FMT) from EE or SE donors to assess the microbiota’s causal role in EE’s anti-tumor effects. Bacterial species upregulated by EE in tumor-bearing mice were identified;Parabacteroides distasonis (Pd) was selected for therapeutic administration. Immune cells in spleen and tumor were quantified by flow cytometry. Natural killer (NK) cell function was tested using anti-NK1.1 depletion. EE increased the alpha diversity of the gut microbiota and alleviated the dysbiosis caused by melanoma. FMT from EE mice to SE mice inhibited melanoma growth, suggesting that the gut microbiota contributes to EE’s anti-tumor effects. EE upregulated seven bacterial species in tumor-bearing mice, including Pd. Oral administration of Pd inhibited melanoma growth and increased intratumoral NK/NKT cell proportions and NK cell granzyme B expression. NK cell depletion abrogated Pd’s anti-tumor effect. This study underscores the interconnectedness of psychological eustress, gut microbiota, and cancer, providing preclinical insights into holistic cancer treatments. Leveraging the brain-gut-cancer axis, targeting psychological factors and gut microbiota could offer a potential adjunctive strategy for melanoma management.

Background Natural killer (NK) cells are key effectors in antitumor immunity, yet their function is markedly suppressed by transforming growth factor-β (TGF-β) in the tumor microenvironment. SMAD7 is an established intracellular antagonist of TGF-β signaling, but its specific role within NK cells remains poorly defined. Methods The clinical relevance of SMAD7 in tumor-infiltrating NK cells was evaluated via integrative analyses of public single-cell and bulk transcriptomic datasets. Functional studies included loss-of-function experiments using NK cell-conditional Smad7 knockout mice in syngeneic tumor models and gain-of-function experiments using a SMAD7-overexpressing human NK-92MI cell line. NK cell antitumor function was assessed through cytotoxicity assays and by measuring the expression of selected effector and exhaustion markers. SMAD7-mediated transcriptional targets were identified by integrating RNA sequencing, chromatin immunoprecipitation followed by quantitative PCR, and luciferase reporter assays. The potential role of SMAD7 in NK cell-based therapy was evaluated in adoptive transfer tumor models. Results High SMAD7 expression in tumor-infiltrating NK cells was associated with a favorable patient prognosis across multiple cancer types. Conditional deletion of SMAD7 in NK cells markedly impaired their antitumor cytotoxicity, leading to accelerated tumor progression in mouse models of both pancreatic and liver cancers. In contrast, SMAD7 overexpression enhanced NK cell cytotoxicity and alleviated functional exhaustion, partly by counteracting TGF-β-mediated suppression. Notably, we uncovered a previously unrecognized nuclear function of SMAD7 in NK cells. SMAD7 directly binds to the STAT5A promoter and promotes its transcription, thereby strengthening STAT5A signaling in NK cells. Disruption of STAT5A largely abolished the enhanced cytotoxicity conferred by wild-type SMAD7 and completely abrogated the effect of a SMAD7 mutant, which was defective in TGF-β receptor binding. In in vivo therapeutic studies, adoptive transfer of SMAD7-overexpressing NK cells showed superior antitumor efficacy against both pancreatic and liver cancers, and SMAD7 modification also significantly improved tumor control and prolonged survival in a chimeric antigen receptor (CAR)-NK cell therapeutic model for liver cancer. Conclusions Our findings identify SMAD7 as a key enhancer of NK-cell antitumor activity through canonical inhibition of TGF-β signaling and non-canonical activation of STAT5A transcription. Modulating SMAD7 offers a promising approach to improve NK cell-based immunotherapy.

The gut microbiota has been implicated in the development and progression of pancreatic cancer (PC), yet few specific bacterial species have been reported to exert functional effect on PC. This study aimed to identify novel PC-associated gut bacterial species with tumor-regulatory effects and explore the potential molecular mediators of their effect. A bidirectional two-sample mendelian randomization (MR) analysis was performed using summary statistics from the MiBioGen consortium (n = 18,340) for gut microbiota and from the GWAS Catalog (587 PC cases, 455,761 controls) to assess their causal relationship. A two-step MR analysis incorporating blood metabolite data (n = 8,299 from the GWAS Catalog) was conducted to investigate potential mediation via circulating metabolites. Functional validation of identified bacterial taxa was carried out in murine models of PC via oral gavage of representative species. Six bacterial taxa, including four genera, demonstrated reliable evidence of causal associations with PC. Eggerthella and Parasutterella were identified as protective for PC, whereas Sutterella and Coprococcus1 were associated with increased PC risk. Additionally, 26 blood metabolites showed causal associations with PC. After evaluating the causal relationships between PC-associated gut microbiota and blood metabolites, we found that the Eggerthella genus and Sutterella genus exert causal effect on PC by reducing the circulating levels of deoxycarnitine and hypotaurine, respectively. In two distinct orthotopic PC models, oral administration of Eggerthella lenta (E. lenta, the type species of the Eggerthella genus) or Parasutterella excrementihominis (P. excrementihominis, the type species of the Parasutterella genus) consistently exerted significant tumor-suppressive effects on the growth of pancreatic tumors. Moreover, E. lenta administration enhanced the infiltration of CD8⁺ T cells and NKT cells within orthotopic pancreatic tumors, suggesting a potential role of E. lenta in reshaping the tumor immune microenvironment to favor antitumor immunity. This study identifies E. lenta and P. excrementihominis as novel PC-associated gut bacterial species with tumor-suppressive effects, offering potential candidates for probiotic-based interventions in PC. Not applicable.

Caloric restriction (CR) induces tumor resistance in mammals, but its mechanisms remain poorly understood. Here, we found that CR altered the proportions and gene expression profiles of tumor-infiltrating neutrophils (TINs). Depletion of neutrophils largely abrogated CR-induced tumor inhibition across multiple murine cancer models, underscoring their critical role in CR's broad anti-tumor effect. CR-induced gene expression changes in TINs were associated primarily with lipid-related processes, notably downregulating hypoxia-inducible lipid droplet-associated (HILPDA). This downregulation reduced lipid accumulation in TINs, limiting tumor growth and enhancing anti-tumor immunity by decreasing lipid transfer to tumor and immune effector cells. Upstream, CR reduced hypoxia-inducible factor 1 (HIF-1α) mRNA expression in circulating neutrophils by decreasing insulin-like growth factor 1 (IGF-1), thereby limiting HILPDA expression in TINs. Patients with lung cancer who had low baseline neutrophil HIF-1α mRNA exhibited improved responses to combined immunotherapy. These findings identify a novel neutrophil- and lipid-centered mechanism for CR-induced tumor inhibition, suggesting the IGF-1/HIF-1α/HILPDA axis as a therapeutic target.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with limited effective treatment options. Emerging evidence links enriched environment (EE)-induced eustress to PDAC inhibition. However, the underlying mechanisms remain unclear. In this study, we explored the role of gut microbiota in PDAC-suppressive effects of EE. We demonstrated that depletion of gut microbiota with antibiotics abolished EE-induced tumor suppression, while fecal microbiota transplantation (FMT) from EE mice significantly inhibited tumor growth in both subcutaneous and orthotopic PDAC models housed in standard environment. 16S rRNA sequencing revealed that EE enhanced gut microbiota diversity and selectively enriched probiotic Lactobacillus, particularly L. reuteri. Treatment with L. reuteri significantly suppressed PDAC tumor growth and increased natural killer (NK) cell infiltration into the tumor microenvironment. Depletion of NK cells alleviated the anti-tumor effects of L. reuteri, underscoring the essential role of NK cell-mediated immunity in anti-tumor response. Clinical analysis of PDAC patients showed that higher fecal Lactobacillus abundance correlated with improved progression-free and overall survival, further supporting the therapeutic potential of L. reuteri in PDAC. Overall, this study identifies gut microbiota as a systemic regulator of PDAC under psychological stress. Supplementation of psychobiotic Lactobacillus may offer a novel therapeutic strategy for PDAC.

BACKGROUND:Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with few well-established risk factors. Emerging epidemiological evidence suggests a link between hepatitis B virus (HBV) infection and PDAC. However, the underlying mechanisms remain unclear. METHODS:High-throughput sequencing-based approach was employed to identify HBV integrations in tumour and para-tumour tissues of PDAC. The biological functions of KMT2B were evaluated in PDAC cell lines as well as in subcutaneous and orthotopic mouse models of PDAC. Chromatin immunoprecipitation sequencing and RNA sequencing were used to identify the pathway involved in PDAC development. RESULTS:HBV integration was detected in approximately one-third of HBV DNA-positive PDAC and adjacent para-tumour tissues. A total of 425 viral‒host junctions were identified, with the majority located in intergenic regions (51.29%), followed by introns (43.29%) and exons (2.35%) of the human genome. Lysine methyltransferase 2B (KMT2B, also known as MLL4), a gene frequently targeted by HBV integration in hepatocellular carcinoma, was also found to be interrupted by HBV in PDAC. KMT2B was significantly upregulated in PDAC and promoted malignant behaviours both in vitro and in vivo. Mechanistically, KMT2B exerts its oncogenic effects by regulating the downstream target gene FYN through histone H3K4 trimethylation, leading to the activation of the PI3K/Akt signalling pathway. CONCLUSION:HBV integration is a common event in HBV-related PDAC and KMT2B has been identified as a novel PDAC-related gene. KEY POINTS:Hepatitis B virus (HBV) integrates in both tumour and adjacent para-tumour tissues of pancreatic ductal adenocarcinoma (PDAC). KMT2B, a target gene of HBV integration, promotes PDAC proliferation and metastasis in vivo and in vitro experiments. KMT2B exerts its oncogenic effects by regulating the downstream target gene FYN via histone H3K4 trimethylation, activating the PI3K/Akt signalling pathway.

Dear Editor, Hepatitis B virus (HBV) infection has been considered an important role in hepatocellular carcinoma (HCC). However, the evidence between HBV and gastric cancer (GC) is limited.1 In this study, HBV integration is identified, for the first time, from HBV-related GC and induces genomic instability and structural variations (SVs) in GC. HBV-integrated genes may act as potential driver genes in GC carcinogenesis. GC is an aggressive malignancy characterized by a high incidence and mortality globally.2 Growing evidence has shown an association between HBV infection and GC.3 However, the evidence is limited to sero-epidemiological studies. Herein, twelve paired GC and para-tumour tissues from patients with HBV surface antigen (HBsAg)-seropositive who underwent radical resection were collected (Tables S1 and S2). HBV DNA (HBV S/C/P/X gene), especially covalently closed circular DNA (cccDNA), was important for HBV regulation and replication. By nested polymerase chain reaction (PCR),4 HBV S and/or C genes were detected in 7/12 of GC and 8/12 of para-tumour tissues (Figure 1A and Table S2). Moreover, we used digital droplet PCR to determine the presence of cccDNA in the above HBV DNA-positive tissues (Figure 1B). HBV cccDNA was detected in 85.7% of GC and 87.5 % of para-tumour samples. To date, this is the first study to detect cccDNA in GC cells. However, there was no significant difference between the level of cccDNA in tumour and para-tumour samples (Figure S1). We further performed a multiplex immunofluorescence assay of viral proteins in the above HBV DNA-positive tissues (Figure 1C). Compared to that in para-tumour tissues, the expression of HBcAg tended to increase in the GC tissues (Figure 1D). The expression of HBcAg (Figure 1E), rather than HBsAg or HBx expression (Figure S2), was significantly related to the level of cccDNA. The above data provide direct evidence demonstrating the HBV infection in GC. HBV DNA integration into the host genome has been considered an important mechanism for HBV-related carcinogenesis in the liver1 and extrahepatic cancers.4, 5 To date, the role of HBV integration in GC has not yet been studied. Herein, the genomic DNAs of seven GC and eight para-tumour tissues containing HBV S and/or C gene, and one paired sample (8 N and 8 T) that showed no existence of HBV DNA as control, were subjected to high-throughput viral integration detection.6 A total of 434 integration sites (supporting read number ≥ 2) were identified from 71.4% (5/7) of HBV DNA-positive GC and 87.5% (7/8) of para-tumour tissues, including five paired tumour and para-tumour samples (Tables S3 and S4). The discovery that HBV integrates into para-tumour tissues may suggest its early occurrence post-infection, potentially during the precancerous phase. Interestingly, we found that cccDNA levels were obviously related to the number of HBV integration sites (Figure 2A), indicating that the HBV integration events were correlated with viral replication. HBV integration sites occurred throughout the entire viral and human genomes, but some breakpoints were found in localized hotspots (Figure 2B,C). In GC samples, HBV integration was more frequently shown on chromosomes 7, 11 and 21 and occurred less frequently on chromosome 4 (Figure 2D). We also analyzed the integration breakpoint in the HBV genome. The viral-host junctions were more prone to have occurred at the 3′-end of the C gene and X gene both in tumour and para-tumour tissues (Figure 2E,F). Besides, genomic instability-correlated genomic elements around the HBV integration site were analyzed. We found that CpG islands were enriched (Figure S3), suggesting that HBV integration may increase genomic instability in GC. To further investigate the role of viral integration on genomic instability and SVs, we performed a long-read genome sequencing in two GC samples with the highest HBV integration events (2T and 2N, Figure 3A). The genome windows spanning HBV integration breakpoints within 500 kb were focused.7 Inversions were enriched around integration sites in both 2N and 2T samples (Figure 3B). Deletions near HBV integration breakpoints were larger in size than the other SVs in the 2T sample (Figure 3C), suggesting that integration events had a stronger genomic impact on these deletions. Moreover, the distance distribution of SVs to HBV-integrated breakpoints was analyzed to explore the spatial connection between HBV integration and SVs. A raised proportion of deletions and insertions were observed close to the HBV integration sites (Figure 3D). Thus, the genomic instability and SVs induced by HBV integration may have significant functional effects on GC development, which requires further investigation. Genes recurrently interrupted by HBV in HCC usually play a role in liver carcinogenesis.8 Moreover, cancer-related genes with distant viral integrations can also be driven by HBV-induced carcinogenesis.9 Recurrent HBV integration sites, defined as HBV targeting the same gene or inserting into the vicinal intergenic sequences within a 500 kb distance in different tissue samples are summarized in Table S5. Strikingly, sprouty RTK signalling antagonist 3 (SPRY3) was repeatedly inserted by HBV three times. SPRY3 was repeatedly integrated by HBV 102 times in our previous analysis of viral integration in HCC tissues,5 suggesting that SPRY3 may play some common and vital mechanisms in driving cancer progression. Then, we chose the SPRY3 for further functional study. Consistent with the observation that SPRY3 was obviously upregulated in GC tissues from the Cancer Genome Atlas (TCGA) dataset (Figure 4A), three GC cell lines exhibited higher SPRY3 expression than the normal human gastric epithelial cell line at the mRNA level (Table S6 and Figure S4A). Clinicopathological analysis revealed that increased SPRY3 was positively correlated with Lauren's classification and MSI status (Table S7). Patients with higher expression levels of SPRY3 have shorter overall survival (Figure 4B), and SPRY3 expression was an independent predictor of GC aggressiveness (Table S8). We then silenced SPRY3 (Table S9 and Figure S4B) and found that the downregulation of SPRY3 led to a significantly decreased rate of GC cell growth, migration and invasion (Figure 4C–E). Moreover, the silencing of SPRY3 significantly decreased the tumour burden in the subcutaneous model (Figure 4F). Overall, the present study reveals the HBV infection and integration in GC by histological and molecular evidence. These findings provide a new direction for research into the mechanism of HBV-associated GC initiation. Yifu He was responsible for the study concept and design, analysis and interpretation of data, critical revision of the manuscript for important intellectual content and obtaining funding. Hong Tu was responsible for the study design. Mengge Li and Shusheng Wu revised the manuscript. Mengge Li performed experiments, collected and analyzed data and wrote the manuscript. Ying Yan and Wenju Chen performed the literature search and carried out the data inclusion and extraction. Shusheng Wu and Huiqin Luo performed the quality assessment. Lulu Cao and Wenju Chen collected samples and analyzed data. Shusheng Wu and Jiayu Niu reviewed clinical data and performed statistical analyses. All authors read and approved the final manuscript. The authors declare no conflict of interest. This study was funded by grants from the National Natural Science Foundation of China (82203225 to Mengge Li), Fundamental Research Funds for the Central Universities (WK9110000172 to Mengge Li), Health Commission of Anhui Province Scientific Research Project (AHWJ2021b090 to Shusheng Wu and AHWJ2021b105 to Yifu He), Hefei Key Common Technology Research and Major Scientific and Technological Achievement Project (2021YL005 to Yifu He) and Anhui Province Key Research and Development Program Project (202104j07020044 to Yifu He). All human samples used in experiments were approved by the institutional ethics review committee of the First Affiliated Hospital of USTC and conducted according to the principles of the Declaration of Helsinki. Informed consent was obtained from all patients. We confirmed that all studies are conducted following relevant guidelines/regulations. All experiments involving mice were approved by the Animal Care and Use Committees of the First Affiliated Hospital of USTC, and were performed in accordance with the Institutional Animal Welfare Guidelines. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Influence of EE on NK population in the peripheral blood and spleen of tumor-bearing mice.

Supplementary figures 1-8. Supplementary Figure 1. The amino acid sequence of scFv GC33 in the construct of anti-GPC3 CAR. Supplementary Figure 2. The expression of CD64 and CD86 in the artificial antigen-presenting cell aK562-64/86. Supplementary Figure 3. Tumor outgrowth of Huh-7 cells was completely abolished by GPC3-targted CAR T cells in vivo. Supplementary Figure 4. Growth suppression on established s.c. HCC xenografts by GPC3-targeted CAR T cells. Supplementary Figure 5. Mice bearing orthotopic Huh-7 tumors. Supplementary Figure 6. The up-regulation of Bcl-XL protein in αGPC3-28BBZ CAR T cells was stimulated by GPC3-specific antigen. Supplementary Figure 7. The GPC3 expression in the normal kidney and gastric glands. Supplementary Figure 8. The homogeneity of GPC3 expression was analyzed in the human primary HCC tissues (n=75).

Intratumoral microbiome is defined as the microbes that reside within tumor tissues to form the tumor microenvironment. Although substantial advances have been made in the research on virus infection and cancer during the past century, little is known about the existence and the role of intratumoral bacteria or fungi in cancer. In recent years, with the development of high-throughput sequencing technology, evidence has emerged to support the existence and functional activity of intratumoral bacteria. This review summarized the recent research progress in intratumoral microbiome, particularly their contribution to the carcinogenesis and chemoresistance of breast cancer, pancreatic cancer and lung cancer. The future perspectives of intratumoral microbiota in clinical application were also discussed.

Exposure to EE reduces anxiety-like behavior, decreases body weight, and increases food intake in mice.

EE prevents experimental lung metastases of murine melanoma cells.

Primers used in quantitative real-time PCR analysis.

EE increases the expression levels of INF-gamma and granzyme B in NK cells.

EE decreases serum leptin levels in wild-type and Beige mice.

Supplementary table. Primer sequences used for construction of the different CARs using the splicing PCR by overlap extension technique.

EE does not appear to influence the expression levels of the ligands of NKG2D and CCR5 in tumors.

Supplementary Materials and Methods.

BACKGROUND AND AIMS:The liver has the unique ability of regeneration, which is extremely important for restoring homeostasis after liver injury. Although clinical observations have revealed an association between psychological stress and the liver, whether stress has a causal influence on the liver regeneration remains markedly less defined.APPROACH AND RESULTS:Rearing rodents in an enriched environment (EE) can induce eustress or positive psychological stress. Herein, EE-induced eustress was found to significantly enhance the ability of liver regeneration after partial hepatectomy or carbon tetrachloride-induced liver injury based on the more rapid restoration of liver/body weight ratio and the significantly increased number of proliferating hepatocytes in EE mice. Mechanistically, the cytokine array revealed that IL-22 was markedly increased in the regenerating liver in response to EE. Blockade of IL-22 signaling abrogated the enhanced liver regeneration induced by EE. Group 1 innate lymphoid cells (ILCs), including type 1 ILCs (ILC1s), have been identified as the major sources of IL-22 in the regenerating liver. EE housing led to a rapid accumulation of hepatic ILC1s after partial hepatectomy and the EE-induced enhancement of liver regeneration and elevation of IL-22 was nearly eliminated in ILC1-deficient Tbx21-/- mice. Chemical sympathectomy or blockade of β-adrenergic signaling also abolished the effect of EE on ILC1s and attenuated the enhanced liver regeneration of EE-housed mice.CONCLUSION:The study findings support the brain-liver axis and suggest that environment-induced eustress promotes liver regeneration through the sympathetic nerve/ILC1/IL-22 axis.