Pattern recognition receptors (PRRs) are crucial modulators of the tumor immune microenvironment (TIME); yet, their comparative efficacy remains poorly characterized. Platelet factor 4 (PF4/CXCL4) exerts paradoxical effects in cancer, and its regulation by PRR signaling is unclear. Here, we systematically screened PRR agonists in murine tumor models and identified TLR8 agonists as the most potent inducers of a pro-immunogenic TIME, superior to agonists targeting TLR3/7/9 or NOD1/2. TLR8 activation drove CD45+ leukocyte infiltration, potentiated conventional dendritic cell (cDC) and macrophage phagocytosis and migration, amplified recruitment of CD8+ and conventional CD4+ (cCD4+) T cells, lowered Treg proportions, elicited pro-inflammatory and tumoricidal phenotypes in innate immune cells and CD8+ T cells. Notably, TLR8 agonism suppressed tumor growth in both immunocompetent and T cell-deficient mice, indicating the involvement of both innate and adaptive immunity. Mechanistically, TLR8 agonists upregulated PF4 expression in macrophages, cDC2, and CD8+ T cells via the NF-κB pathway. PF4 in turn recruited cCD4+ T cells via CXCR3, and its local overexpression mimicked the antitumor effect of TLR8 activation. Beyond PF4, TLR8 signaling mediated PF4-independent effects, including Treg suppression via IFN-γ and enhanced macrophage phagocytosis. Combination of a TLR8 agonist with anti-PD-1 therapy markedly and synergistically improved survival of tumor-bearing mice. Thus, TLR8 agonists optimally remodel the TIME through PF4-dependent T cell recruitment and PF4-independent ancillary mechanisms. Our finding that the antitumor activity of locally induced PF4 contrasts with its reported protumor effects when expressed systemically clarifies the context-dependent duality of PF4 in cancer. These results position TLR8 agonists as promising candidates for combination immunotherapy.

Triple-negative breast cancer (TNBC) is a particularly aggressive subtype of breast cancer with a high risk of relapse and metastasis. Due to limited tumor immune infiltration in TNBC, the effectiveness of immunotherapy is constrained. In this study, analysis of human datasets (TCGA-BRCA and Kim cohorts) revealed that tumor necrosis factor superfamily member 9 (TNFSF9) and C-X-C motif chemokine ligand (CXCL9) expressions correlate with improved prognosis and enhanced immune cell infiltration. To exploit this, we engineered human umbilical cord mesenchymal stem cells (hUC-MSCs) to co-express TNFSF9 and CXCL9 (MSC-T9C9). In murine TNBC models, the engineered MSC-T9C9 recruits CD8+ T cells and natural killer (NK) cells to the tumor site, thereby increasing immune infiltration and remodeling the tumor immune microenvironment through activating CD8+ T cells and NK cells. This therapeutic strategy proved both effective and well-tolerated. Moreover, MSC-T9C9 enhanced the prognosis and therapeutic efficacy of anti-PD-1 immunotherapy in vivo. These findings demonstrate that the engineered MSC co-expressing chemokine CXCL9 and costimulatory ligand TNFSF9 effectively suppresses TNBC growth by reprogramming the intra-tumoral immune landscape, which offers a promising and safe immunotherapeutic strategy for TNBC treatment.

BACKGROUND:Immunogenic cell death (ICD) represents a promising strategy to suppress tumor growth and potentiate immunotherapy efficacy, driving the demand for novel and clinically translatable ICD inducers. Here, we investigated the potential of cepharanthine (CEP) for inducing ICD in multiple representative solid tumors. METHODS:The ICD-inducing potential of a series of natural alkaloids was screened by flow cytometry. CEP's activity and mechanism were characterized using flow cytometry, western blot, immunofluorescence, ATP quantification, cell thermal shift assay (CETSA), surface plasmon resonance (SPR), bulk and single-cell RNA sequencing. The translational potential of CEP, either as monotherapy or in combination with other immunotherapies, was evaluated in both syngeneic mouse tumor models and patient-derived organoid systems. Toxicity in mice was assessed through hematological and biochemical parameters. RESULTS:CEP effectively induced ICD in multiple representative solid tumor models (breast cancer, colorectal cancer, esophageal cancer, liver cancer, lung cancer) by promoting damage-associated molecular patterns (DAMPs) release, enhancing phagocytosis by antigen-presenting cells, and activating T cell-mediated immunity. Mechanistically, CEP directly bound to protein kinase C zeta (PKCζ), leading to the inhibition of NF-κB signaling via suppressing p65 nuclear translocation, and downregulation of poly ADP-ribose polymerase 1 (PARP1) transcription. PARP1 downregulation triggers intracellular reactive oxygen species (ROS) accumulation, subsequently inducing DNA damage and endoplasmic reticulum (ER) stress. Furthermore, CEP synergized with PD-1 blockade and OX40 agonism, thereby enhancing antitumor efficacy in vivo. CONCLUSIONS:CEP is a potent and preclinically well-tolerated ICD inducer that acts by targeting the PKCζ/NF-κB/PARP1 axis. Its synergy with immune checkpoint inhibitors and co-stimulatory agonists underscores its translational potential for combination cancer immunotherapy.

BACKGROUND:Major histocompatibility complex class I (MHC)-I loss is a prevalent mechanism for immune evasion and resistance to immunotherapy. However, how MHC-I loss shapes the tumor microenvironment and influences immune cell interactions, ultimately affecting tumor growth, remains largely unknown. METHODS:We established B2m knockout (MHC-I-deficient) tumor cells using CRISPR/Cas9 and evaluated their growth in subcutaneous and orthotopic mouse models. Immune profiling was performed using flow cytometry and single-cell RNA sequencing. Antibody-mediated cell depletion was used to assess the functional contributions of specific immune subsets. Chemokine expression was analyzed by bulk RNA sequencing, quantitative PCR, ELISA and western blotting, and its functional relevance was determined using knockout or overexpression in tumor cells implanted in vivo. Signaling pathways were interrogated using pharmacological inhibition, RNA interference and western blotting. RESULTS:MHC-I loss promoted tumor growth in MC38, AKR, and LLC1 models, but unexpectedly suppressed Hepa1-6 and orthotopic MYC;Trp53 -/- hepatocarcinoma growth. This differential effect correlated with changes in immune infiltrates. CD4+ T cells, natural killer (NK) cells, and macrophages were required for suppression of MHC-I-deficient Hepa1-6 tumors. CD4+ T cells were essential for recruiting NK cells and monocytes/macrophages and for inducing their tumoricidal phenotypes, including iNOS (inducible nitric oxide synthase) expression in macrophages. The differential infiltration of CD4+ T cells was driven by opposite regulation of CXCL16 on B2m knockout: upregulation in Hepa1-6 cells and downregulation in other models. CXCL16 exerted potent antitumor effects by recruiting CD4+ T cells. Mechanistically, B2M loss regulated CXCL16 via suppression of Akt in MC38 and AKR cells, but via activation of NF-κB in Hepa1-6 cells. CONCLUSION:CXCL16-driven CD4+ T cells are central regulators of antitumor immunity against MHC-I-deficient tumors. The context-dependent regulation of CXCL16 by MHC-I loss determines the immune landscape and tumor outcome, highlighting a potential therapeutic avenue for targeting MHC-I-deficient cancers.

The induction of immunogenic cell death (ICD) impedes tumor progression via both tumor cell-intrinsic and -extrinsic mechanisms, representing a robust therapeutic strategy. However, ICD-targeted therapy remains to be explored and optimized. Through kinome-wide CRISPR-Cas9 screen, NUAK family SNF1-like kinase 1 (NUAK1) is identified as a potential target. The ICD-provoking effect of NUAK1 inhibition depends on the production of reactive oxygen species (ROS), consequent to the downregulation of nuclear factor erythroid 2-related factor 2 (NRF2)-mediated antioxidant gene expression. Moreover, the mevalonate pathway/cholesterol biosynthesis, activated by spliced form of X-box binding protein 1 (XBP1s) downstream of ICD-induced endoplasmic reticulum (ER) stress, functions as a negative feedback mechanism. Targeting the mevalonate pathway with CRISPR knockout or the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) inhibitor simvastatin amplifies NUAK1 inhibition-mediated ICD and antitumor activity, while cholesterol dampens ROS and ICD, and therefore also dampens tumor suppression. The combination of NUAK1 inhibitor and statin enhances the efficacy of anti-PD-1 therapy. Collectively, our study unveils the promise of blocking the mevalonate-cholesterol pathway in conjunction with ICD-targeted immunotherapy.

Aerobic exercise has been recommended as a non-pharmacological treatment for asthma. Previous studies have shown that circMETTL9 regulates cellular inflammation, apoptosis, and oxidative stress levels. However, whether aerobic exercise can modulate the expression of circMETTL9 to alleviate chronic allergic airway inflammation remains unclear. In this study, we established a mouse model of chronic allergic lung inflammation with aerobic exercise intervention to assess its effects. Our results demonstrate that aerobic exercise exerts anti-inflammatory, anti-proliferative, anti-apoptotic, and anti-oxidative stress effects by regulating the circMETTL9/EIF4A3/IGFBP3 axis. Mechanistically, we found that circMETTL9 binding to EIF4A3 does not affect EIF4A3 expression. However, EIF4A3 positively regulates both the protein and mRNA levels of IGFBP3. Specifically, circMETTL9 binds to EIF4A3 to inhibit IGFBP3 transcription and translation. This study identifies a novel potential target and research direction for treating chronic allergic lung inflammation.

As one of the major challenges to global health, the innovation of prevention and treatment methods for tumors has consistently been a focal point in medical research. In recent years, bacterial membrane vesicles (MVs), particularly outer membrane vesicles (OMVs) secreted by Gram-negative bacteria, have garnered significant attention due to their unique biological characteristics and potential anti-tumor effects. OMVs are bilayer lipid nanocapsules that are actively released by bacteria during their growth, typically ranging in diameter from 20 to 300 nm. They are rich in various biomolecules, including lipids, proteins, nucleic acids, and other small molecules. These components not only reflect the outer membrane structure of bacteria but also contain numerous pathogen-associated molecular patterns (PAMPs) related to bacterial pathogenicity and immunogenicity. Consequently, OMVs play an important role in bacterial resistance, antimicrobial activity, gene transfer, signal transduction, and immune regulation. Research and application of OMVs in anti-tumor therapy have made significant progress. This paper reviews the classification, characteristics, preparation, safety evaluation, biological functions, and specific research advancements of OMVs as antitumor drugs, immunomodulators, and carriers. Additionally, common methods for the preparation and modification of OMVs, including preliminary extraction, purification, characterization, and drug loading, are discussed. This paper also summarizes the challenges faced by OMVs in anti-tumor research and outlines future development directions, aiming to provide a reference for the further application of OMVs in tumor treatment.

Emerging evidence suggests that aerobic exercise exerts beneficial effects on asthma. Previous studies have demonstrated that cell communication can drive the formation of macrophage extracellular traps (METs). However, the potential of aerobic exercise to mediate communication between airway epithelial cells and macrophages, thereby influencing MET formation, remains unexplored. Our data reveal that the upregulation of circular RNA METTL9 (circMETTL9), derived from methyltransferase-like protein 9 (METTL9) in airway epithelial cells, promotes the formation of METs. Notably, aerobic exercise was found to downregulate the expression of circMETTL9, thereby facilitating communication between airway epithelial cells and macrophages and inhibiting METs formation. Mechanistically, circMETTL9 and insulin-like growth factor binding protein 3 (IGFBP3) compete for binding to the DEXDc domain of eukaryotic translation initiation factor 4A3 (EIF4A3), which regulates METs via the C-X-C motif chemokine ligand 12(CXCL12) -C-X-C chemokine receptor type 4(CXCR4) signaling axis. This study provides robust molecular evidence supporting aerobic exercise as a foundational pulmonary rehabilitation strategy for lung protection in asthma. Targeting circMETTL9, mimicking this exercise-mediated pathway, represents a promising therapeutic approach. These insights offer a direct mechanistic rationale for refining exercise-based rehabilitation protocols and developing novel targeted therapies.

Background Lipid metabolism plays a pivotal role in asthma pathogenesis. However, a comprehensive analysis of the importance of lipid metabolism-related genes (LMRGs) in regulating the immune microenvironment in asthma remains lacking. The transcriptome matrix was downloaded from the Gene Expression Omnibus (GEO) dataset. Differentially expressed analysis and weighted gene coexpression network analysis (WGCNA) were conducted on the GSE74986 dataset to select hub LMRGs, and gene set enrichment analysis (GSEA) was conducted to explore their biological functions. The CIBERSORT algorithm was used to determine immune infiltration in the asthma and control groups, and the correlation of diagnostic biomarkers and immune cells was performed via Spearman correlation analysis. Subsequently, a competitive endogenous RNA (ceRNA) network was constructed to investigate the hidden molecular mechanism of asthma. The expression levels of the hub genes were further validated in the GSE143192 dataset, and RT‒qPCR and immunofluorescence were performed to verify the reliability of the results in the OVA asthma model. Lastly, the ceRNA network was confirmed by qRT-PCR and RNAi experiments in the characteristic cytokine (IL-13)-induced asthma cellular model. Results ASAH1 , ACER3 and SGPP1 were identified as hub LMRGs and were mainly involved in protein secretion, mTORC1 signaling, and fatty acid metabolism. We found more infiltration of CD8 + T cells, activated NK cells, and monocytes and less M0 macrophage infiltration in the asthma group than in the healthy control group. In addition, ASAH1 , ACER3 , and SGPP1 were negatively correlated with CD8 + T cells and activated NK cells, but positively correlated with M0 macrophages. Within the ceRNA network, SNHG9 - hsa-miR-615-3p - ACER3 , hsa-miR-212-5p and hsa-miR-5682 may play crucial roles in asthma pathogenesis. The low expression of ASAH1 and SGPP1 in asthma was also validated in the GSE74075 dataset. After SNHG9 knockdown, miR-615-3p expression was significantly upregulated, while that of ACER3 was significantly downregulated. Conclusion ASAH1 , ACER3 and SGPP1 might be diagnostic biomarkers for asthma, and are associated with increased immune system activation. In addition, SNHG9 - hsa-miR-615-3p - ACER3 may be viewed as effective therapeutic targets for asthma. Our findings might provide a novel perspective for future research on asthma.

Understanding the biological functions and processes of genes, particularly those not yet characterized, is crucial for advancing molecular biology and identifying therapeutic targets. The hypothesis guiding this study is that the 3D proximity of genes correlates with their functional interactions and relevance in prokaryotes. We introduced 3D-GeneNet, an innovative software tool that utilizes high-throughput sequencing data from chromosome conformation capture techniques and integrates topological metrics to construct gene association networks. Through a series of comparative analyses focused on spatial versus linear distances, we explored various dimensions such as topological structure, functional enrichment levels, distribution patterns of linear distances among gene pairs, and the area under the receiver operating characteristic curve by utilizing model organism Escherichia coli K-12. Furthermore, 3D-GeneNet was shown to maintain good accuracy compared to multiple algorithms (neighbourhood, co-occurrence, coexpression, and fusion) across multiple bacteria, including E. coli, Brucella abortus, and Vibrio cholerae. In addition, the accuracy of 3D-GeneNet's prediction of long-distance gene interactions was identified by bacterial two-hybrid assays on E. coli K-12 MG1655, where 3D-GeneNet not only increased the accuracy of linear genomic distance tripled but also achieved 60% accuracy by running alone. Finally, it can be concluded that the applicability of 3D-GeneNet will extend to various bacterial forms, including Gram-negative, Gram-positive, single-, and multi-chromosomal bacteria through Hi-C sequencing and analysis. Such findings highlight the broad applicability and significant promise of this method in the realm of gene association network. 3D-GeneNet is freely accessible at https://github.com/gaoyuanccc/3D-GeneNet.

Genetic sequencing has revolutionized immunotherapy in colorectal cancer (CRC). Recent clinical trials have revealed a positive response to immunotherapy-based systemic therapies in CRC patient subgroups with microsatellite instability (MSI)-High or DNA polymerase epsilon (POLE) mutation. However, the unsatisfactory response rates was the major limitation in real-world practice of the precision immunotherapy in CRC. Adding photodynamic therapy (PDT) to systemic immunotherapy has showed synergetic anti-tumor effect by modulating tumor microenvironment, while the eligible patient’s subgroups which would benefit from this combination remained equivocal. Here we reported a synchronous colorectal cancer patient with MSI-High and POLE mutation who had accelerated response in less than 2 cycles (42 days) of immunotherapy-based systemic therapies after tumor-directed PDT and has remained progression-free by far. This case enlightened the synergetic effect of PDT in immunotherapy-treated CRC patients, with the MSI and POLE-mutation status as predictors of survival benefits.

Tembusu virus (TMUV) is an emerging pathogenic flavivirus associated with acute egg-drop and fatal encephalitis in domestic waterfowl. Since its initial identification in mosquitoes in 1955, TMUV has been confirmed to infect ducks, pigeons, sparrows, geese, and chickens, posing a significant threat to the poultry industry. Here, we sequenced two DTMUV strains isolated in 2019 and systematically investigated the possible origin, genetic relationships, evolutionary dynamics, and transmission patterns of TMUV based on complete virus genome sequences in the public database. We found that TMUV can be divided into four major clusters: TMUV, cluster 1, cluster 2, and cluster 3. Interestingly, we found that cluster 2.2 (within cluster 2) is the most commonly involved in interspecies transmission events, and subcluster 2.1.2 (within cluster 2.1) is currently the most prevalent cluster circulating in Asia. Notably, we also identified three positively selected sites in the E and NS1 proteins, which may be involved in virus replication, immune evasion, and host adaptation. Finally, phylogeographic analysis revealed that cluster dispersal originated in Southeast Asia and that short-distance transmission events have occurred frequently. Altogether, these data provide novel insights into the evolution and dispersal of TMUV, facilitating the development of rapid diagnostics, vaccines, and therapeutics against TMUV infection.

Supplementary data of in vitro cell line experiments, human stomach tumor database/sample analysis and animal experiments.

Supplementary Figure 1 from Repression of Vascular Endothelial Growth Factor Expression by the Runt-Related Transcription Factor 1 in Acute Myeloid Leukemia

Supplementary Figure Legend from Repression of Vascular Endothelial Growth Factor Expression by the Runt-Related Transcription Factor 1 in Acute Myeloid Leukemia

2019年12月新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)在全球蔓延,促使世界卫生组织宣布2019冠状病毒病(COVID-19)流行为国际性突发公共卫生事件.人群普遍易感SARS-CoV-2,而肿瘤患者由于肿瘤生长和抗癌治疗抑制全身免疫系统,成为更易感染并发展为重症SARS-CoV-2症状的脆弱人群.SARS-CoV-2感染后的炎症因子风暴与免疫抑制在SARS-CoV-2感染的肿瘤患者体内产生矛盾,其表现和预后及治疗结果仍未明确.本文基于现有研究文献综述,旨在了解SARS-CoV-2感染对肿瘤患者的生物学及生存预后的影响,以期为疫情期间肿瘤患者的临床治疗提供参考.

新型冠状病毒肺炎(COVID-19)患者的死亡原因主要有代谢性酸中毒,脓毒性休克,肺水肿,多器官衰竭,深静脉血栓等,这些全身表现与炎症风暴引起的肺、肾等重要脏器的严重病理损伤相关.患者疾病的发生、发展、恶化与体内淋巴细胞亚群紊乱,细胞因子风暴产生等免疫失衡机制密切相关.其中细胞免疫发挥了至关重要的作用.重症患者血液中诸多炎症因子表达显著升高,另外,IL-6是造成细胞因子风暴的典型促炎因子,可作为判断病情预后的依据.IL-6阻断可成为阻断细胞因子风暴的有效方法.

Programmed death-1 (PD-1) antibody treatment has been recently approved for patients with advanced gastric cancer. Despite its success in several types of cancer, the efficacy of anti-PD-1 therapy is limited by many factors, such as tumor infiltration of effector lymphocytes and expression level of programmed death ligand-1 (PD-L1) which can be quantified as a combined positive score (CPS). An overexpression of PD-L1/ PD-L2 has been identified in Epstein-Barr virus (EBV)–positive gastric adenocarcinoma, suggesting these patients may benefit from PD-1/PD-L1 blockade.1The Cancer Genome Atlas Research NetworkNature. 2014; 513: 202-209Crossref PubMed Scopus (3739) Google Scholar However, EBV-associated gastric cancer accounts for only 10% of all cases worldwide.2Nishikawa J. et al.Cancers. 2014; 6: 2259-2274Crossref PubMed Scopus (45) Google Scholar Accordingly, a meta-analysis for clinical trials with immune checkpoint inhibitors for advanced gastric cancer or esophago-gastric junction tumors indicates that the objective response rate for anti-PD-1 treatment is 12% and the progression-free survival is 1.61 months.3Chen C. et al.OncoImmunology. 2019; 8e1581547Crossref Scopus (26) Google Scholar The overall benefits from anti-PD-1/PD-L1 therapy for patients with gastric cancer remains unsatisfactory, and exploration of other immunotherapeutic targets is warranted.The majority of clinically approved cancer immunotherapies target T-cell-mediated adaptive immunity. Until recently, various approaches targeting the innate immunity such as antagonist for the phagocytosis checkpoint CD47 have been developed.4Feng M. et al.Nat Rev Cancer. 2019; 19: 568-586Crossref PubMed Scopus (279) Google Scholar CD24 is the most recent “don’t eat me” signal identified in ovarian and breast tumor cells and suppresses phagocytosis via Siglec-10 expressed on macrophages.5Barkal A.A. et al.Nature. 2019; 572: 392-396Crossref PubMed Scopus (399) Google Scholar However, the outcome of systemic inhibition of CD24 has not been fully explored in an immunocompetent mouse model of cancer.As reported, CD24 overexpression is associated with poor survival of patients with gastric cancer, which implies its potential as a therapeutic target.6Wu J.X. et al.PLoS One. 2014; 9e114746Google Scholar To investigate this issue, we first compared CD24 expression in normal and tumor tissues. Human gastric cancer samples exhibited a higher CD24 mRNA level than normal stomach tissues (Figure A1A). What is more, in a clinically relevant gastric cancer mouse model established by treatment with Helicobacter (H.) felis bacteria and chemical carcinogen N-methyl-N-nitrosourea (MNU), a higher percentage of gastric epithelial cells expressed CD24 in the tumor model than the normal control (Figure A1B). Interestingly, H. felis/MNU treatment strongly reduced PD-L1 expression in the mouse stomach (Figure A1C). Consistently, the expression level of PD-L1 in epithelial cells was also negligible (Figure 1B), which means this model can be considered CPS low.It has been pointed out that antigen expression screening in patients for targeted therapies is essential to achieve the maximum therapeutic benefit of the treatment.7Bussing D. et al.AAPS J. 2021; 23: 56Crossref PubMed Scopus (2) Google Scholar Thus, it can be speculated that the expression level of the inhibitory molecules is closely related to the responsiveness of these antibodies. Therefore, we further compared the expression levels of CD24 with the other two immune inhibitory molecules PD-L1 and CD47 in human gastric cancer samples and found that CD24 mRNA level was higher than either PD-L1 or CD47 (Figure 1A). In H. felis/MNU-induced gastric cancer in mice, membrane expression of CD24 protein in gastric epithelial cells was also more prevalent than PD-L1 and CD47 (Figure 1B). Together, CD24 serves as a potential therapeutic target and its abundance may be indicative for the suitableness of anti-CD24 therapy for gastric cancer.To evaluate the therapeutic potential of systemic inhibition of CD24, we applied the CD24 blocking antibody in comparison with one of the most popular immunotherapeutic agents, PD-1 antibody in the H. felis/MNU-induced gastric cancer model (Figure 1C). Anti-CD24 treatment greatly repressed the tumor growth, while anti-PD-1 treatment showed no significant effect (Figure 1D and E). The pathological features including metaplastic and dysplastic grades were significantly reduced by CD24 antibody (Figure 1F). Moreover, anti-CD24 treatment did not change the ratio of CD24+ cells in the stomach, suggesting that the therapeutic effect was not attributed to antibody-mediated depletion of CD24-expressing cells (Figure A2). Consistently, in a recent study using H/K-ATPase-IL-1β or gastrin-deficient mice, anti-PD-1 treatment also failed to inhibit the growth of established stomach tumors.8Kim W. et al.Gastroenterology. 2021; 160: 781-796Abstract Full Text Full Text PDF PubMed Scopus (34) Google ScholarWe further analyzed the immune cells in the stomach tumors by fluorescence activated cell sorting. The ratios of CD45+ cells in total cells were similar among all groups, while both anti-CD24 and anti-PD-1 treatments increased the ratios of CD4+ and CD8+ T cells and decreased the percentage of regulatory T cells in the stomach (Figure 2A–D), even though PD-1 antibody did not shrink the tumor. Then diving deeply into the T-cell population, we found no significant difference in the ratio of CD69+CD4+ T cells while there was a striking bump of CD69+CD8+ T cells after anti-CD24 or anti-PD-1 treatments (Figure A3), suggesting CD24 blockade also boosted the activity of CD8+ T cells. Notably, neutrophils showed a significant upregulation in the stomach upon anti-PD-1 treatment, which might at least partly result in immune suppression and compromised antitumor effects mediated by T cells (Figure 2E) because tumor-associated neutrophils have been shown to foster gastric cancer progression.9Wang T.T. et al.Gut. 2017; 66: 1900-1911Crossref PubMed Scopus (212) Google Scholar Downregulation of PD-L1 expression by H. felis/MNU treatment in the mouse stomach might represent another reason for the limited effectiveness of anti-PD-1 therapy (Figure A1C).Figure 2Analysis of tumor microenvironment upon anti-CD24 and anti-PD-1 therapies in H. felis/MNU-induced gastric cancer model. Fluorescence activated cell sorting analysis of immune cells including CD45+ (A), CD4+ T (B), CD8+ T (C), regulatory T cells (D), and neutrophils (E) in the stomachs of H. felis/MNU mice treated with αCD24, αPD-1, or control antibodies. ∗P < .05, ∗∗P < .01, N.S., not significant.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Given an identified role of CD24 in the inhibition of macrophage-mediated phagocytosis previously, we also investigated whether CD24 inhibition might influence the phenotype of tumor-associated macrophages (TAMs). A trending shift from M2-like to M1-like phenotype was observed in the CD24 antibody-treated H. felis/MNU mice (Figure A4A–E). This indicated that targeting CD24 might also contribute to polarization of the TAMs to M1 phenotype. Because it was observed that CD24 inhibition led to an enhanced phagocytosis of B cells by macrophages in vitro,5Barkal A.A. et al.Nature. 2019; 572: 392-396Crossref PubMed Scopus (399) Google Scholar we also explored whether systemic blockade of CD24 in vivo would jeopardize B cells. Anti-CD24 treatment showed no obvious impact on the B cells from the spleens but significantly increased the ratios of B cells in both blood and stomach (Figure A4F). From the functional side, expression of the activation marker CD69 on B cells was not affected by CD24 antibody either (Figure A4G). Thus, systemic administration of CD24 antibody did not cause unwanted effects on the B cells; on the contrary, it promoted their presence in the stomach.Driven by some unsatisfactory therapeutic results of PD-1 inhibitors in the clinic and the potential of phagocytosis checkpoint CD24 as an immunotherapeutic target,5Barkal A.A. et al.Nature. 2019; 572: 392-396Crossref PubMed Scopus (399) Google Scholar our work went deeper into the therapeutic potential of anti-CD24 treatment in an immunocompetent, clinically relevant model of spontaneous gastric cancer and demonstrated that CD24 was a superior therapeutic target to PD-1 in this mouse model.Given the critical role of innate immunocytes in priming adaptive immune responses, accumulating evidence has highlighted a dual role of CD47, the first identified phagocytosis checkpoint, in eliciting innate and adaptive antitumor immunity.4Feng M. et al.Nat Rev Cancer. 2019; 19: 568-586Crossref PubMed Scopus (279) Google Scholar,10Liu X. et al.Nat Med. 2015; 21: 1209-1215Crossref PubMed Scopus (427) Google Scholar Consistently, our study also showed that systemic CD24 blockade coordinated both adaptive immunity via boosting tumor-infiltrating effector T and B lymphocytes and innate immunity via polarizing TAMs to M1 phenotype. Collectively, anti-CD24 therapy holds great promises for the treatment of gastric cancer by motivating both innate and adaptive immunity and could serve as an alternative or extra option for the nonresponders to anti-PD-1 therapy. Programmed death-1 (PD-1) antibody treatment has been recently approved for patients with advanced gastric cancer. Despite its success in several types of cancer, the efficacy of anti-PD-1 therapy is limited by many factors, such as tumor infiltration of effector lymphocytes and expression level of programmed death ligand-1 (PD-L1) which can be quantified as a combined positive score (CPS). An overexpression of PD-L1/ PD-L2 has been identified in Epstein-Barr virus (EBV)–positive gastric adenocarcinoma, suggesting these patients may benefit from PD-1/PD-L1 blockade.1The Cancer Genome Atlas Research NetworkNature. 2014; 513: 202-209Crossref PubMed Scopus (3739) Google Scholar However, EBV-associated gastric cancer accounts for only 10% of all cases worldwide.2Nishikawa J. et al.Cancers. 2014; 6: 2259-2274Crossref PubMed Scopus (45) Google Scholar Accordingly, a meta-analysis for clinical trials with immune checkpoint inhibitors for advanced gastric cancer or esophago-gastric junction tumors indicates that the objective response rate for anti-PD-1 treatment is 12% and the progression-free survival is 1.61 months.3Chen C. et al.OncoImmunology. 2019; 8e1581547Crossref Scopus (26) Google Scholar The overall benefits from anti-PD-1/PD-L1 therapy for patients with gastric cancer remains unsatisfactory, and exploration of other immunotherapeutic targets is warranted. The majority of clinically approved cancer immunotherapies target T-cell-mediated adaptive immunity. Until recently, various approaches targeting the innate immunity such as antagonist for the phagocytosis checkpoint CD47 have been developed.4Feng M. et al.Nat Rev Cancer. 2019; 19: 568-586Crossref PubMed Scopus (279) Google Scholar CD24 is the most recent “don’t eat me” signal identified in ovarian and breast tumor cells and suppresses phagocytosis via Siglec-10 expressed on macrophages.5Barkal A.A. et al.Nature. 2019; 572: 392-396Crossref PubMed Scopus (399) Google Scholar However, the outcome of systemic inhibition of CD24 has not been fully explored in an immunocompetent mouse model of cancer. As reported, CD24 overexpression is associated with poor survival of patients with gastric cancer, which implies its potential as a therapeutic target.6Wu J.X. et al.PLoS One. 2014; 9e114746Google Scholar To investigate this issue, we first compared CD24 expression in normal and tumor tissues. Human gastric cancer samples exhibited a higher CD24 mRNA level than normal stomach tissues (Figure A1A). What is more, in a clinically relevant gastric cancer mouse model established by treatment with Helicobacter (H.) felis bacteria and chemical carcinogen N-methyl-N-nitrosourea (MNU), a higher percentage of gastric epithelial cells expressed CD24 in the tumor model than the normal control (Figure A1B). Interestingly, H. felis/MNU treatment strongly reduced PD-L1 expression in the mouse stomach (Figure A1C). Consistently, the expression level of PD-L1 in epithelial cells was also negligible (Figure 1B), which means this model can be considered CPS low. It has been pointed out that antigen expression screening in patients for targeted therapies is essential to achieve the maximum therapeutic benefit of the treatment.7Bussing D. et al.AAPS J. 2021; 23: 56Crossref PubMed Scopus (2) Google Scholar Thus, it can be speculated that the expression level of the inhibitory molecules is closely related to the responsiveness of these antibodies. Therefore, we further compared the expression levels of CD24 with the other two immune inhibitory molecules PD-L1 and CD47 in human gastric cancer samples and found that CD24 mRNA level was higher than either PD-L1 or CD47 (Figure 1A). In H. felis/MNU-induced gastric cancer in mice, membrane expression of CD24 protein in gastric epithelial cells was also more prevalent than PD-L1 and CD47 (Figure 1B). Together, CD24 serves as a potential therapeutic target and its abundance may be indicative for the suitableness of anti-CD24 therapy for gastric cancer. To evaluate the therapeutic potential of systemic inhibition of CD24, we applied the CD24 blocking antibody in comparison with one of the most popular immunotherapeutic agents, PD-1 antibody in the H. felis/MNU-induced gastric cancer model (Figure 1C). Anti-CD24 treatment greatly repressed the tumor growth, while anti-PD-1 treatment showed no significant effect (Figure 1D and E). The pathological features including metaplastic and dysplastic grades were significantly reduced by CD24 antibody (Figure 1F). Moreover, anti-CD24 treatment did not change the ratio of CD24+ cells in the stomach, suggesting that the therapeutic effect was not attributed to antibody-mediated depletion of CD24-expressing cells (Figure A2). Consistently, in a recent study using H/K-ATPase-IL-1β or gastrin-deficient mice, anti-PD-1 treatment also failed to inhibit the growth of established stomach tumors.8Kim W. et al.Gastroenterology. 2021; 160: 781-796Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar We further analyzed the immune cells in the stomach tumors by fluorescence activated cell sorting. The ratios of CD45+ cells in total cells were similar among all groups, while both anti-CD24 and anti-PD-1 treatments increased the ratios of CD4+ and CD8+ T cells and decreased the percentage of regulatory T cells in the stomach (Figure 2A–D), even though PD-1 antibody did not shrink the tumor. Then diving deeply into the T-cell population, we found no significant difference in the ratio of CD69+CD4+ T cells while there was a striking bump of CD69+CD8+ T cells after anti-CD24 or anti-PD-1 treatments (Figure A3), suggesting CD24 blockade also boosted the activity of CD8+ T cells. Notably, neutrophils showed a significant upregulation in the stomach upon anti-PD-1 treatment, which might at least partly result in immune suppression and compromised antitumor effects mediated by T cells (Figure 2E) because tumor-associated neutrophils have been shown to foster gastric cancer progression.9Wang T.T. et al.Gut. 2017; 66: 1900-1911Crossref PubMed Scopus (212) Google Scholar Downregulation of PD-L1 expression by H. felis/MNU treatment in the mouse stomach might represent another reason for the limited effectiveness of anti-PD-1 therapy (Figure A1C). Given an identified role of CD24 in the inhibition of macrophage-mediated phagocytosis previously, we also investigated whether CD24 inhibition might influence the phenotype of tumor-associated macrophages (TAMs). A trending shift from M2-like to M1-like phenotype was observed in the CD24 antibody-treated H. felis/MNU mice (Figure A4A–E). This indicated that targeting CD24 might also contribute to polarization of the TAMs to M1 phenotype. Because it was observed that CD24 inhibition led to an enhanced phagocytosis of B cells by macrophages in vitro,5Barkal A.A. et al.Nature. 2019; 572: 392-396Crossref PubMed Scopus (399) Google Scholar we also explored whether systemic blockade of CD24 in vivo would jeopardize B cells. Anti-CD24 treatment showed no obvious impact on the B cells from the spleens but significantly increased the ratios of B cells in both blood and stomach (Figure A4F). From the functional side, expression of the activation marker CD69 on B cells was not affected by CD24 antibody either (Figure A4G). Thus, systemic administration of CD24 antibody did not cause unwanted effects on the B cells; on the contrary, it promoted their presence in the stomach. Driven by some unsatisfactory therapeutic results of PD-1 inhibitors in the clinic and the potential of phagocytosis checkpoint CD24 as an immunotherapeutic target,5Barkal A.A. et al.Nature. 2019; 572: 392-396Crossref PubMed Scopus (399) Google Scholar our work went deeper into the therapeutic potential of anti-CD24 treatment in an immunocompetent, clinically relevant model of spontaneous gastric cancer and demonstrated that CD24 was a superior therapeutic target to PD-1 in this mouse model. Given the critical role of innate immunocytes in priming adaptive immune responses, accumulating evidence has highlighted a dual role of CD47, the first identified phagocytosis checkpoint, in eliciting innate and adaptive antitumor immunity.4Feng M. et al.Nat Rev Cancer. 2019; 19: 568-586Crossref PubMed Scopus (279) Google Scholar,10Liu X. et al.Nat Med. 2015; 21: 1209-1215Crossref PubMed Scopus (427) Google Scholar Consistently, our study also showed that systemic CD24 blockade coordinated both adaptive immunity via boosting tumor-infiltrating effector T and B lymphocytes and innate immunity via polarizing TAMs to M1 phenotype. Collectively, anti-CD24 therapy holds great promises for the treatment of gastric cancer by motivating both innate and adaptive immunity and could serve as an alternative or extra option for the nonresponders to anti-PD-1 therapy. 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