Tertiary lymphoid structures (TLSs) have emerged as critical prognostic and immunotherapeutic indicators in cancer, with their clinical significance modulated by spatial distribution patterns and density. Here, we performed integrated single-cell and spatial transcriptomic profiling of 30 gastric cancer (GC) specimens stratified by TLS spatial localization patterns. Comparative analysis shows pronounced enrichment of CXCL13⁺ T lymphocytes (TLCs), CXCR5⁺ germinal center B lymphocytes (gc_B cells), LAMP3⁺CD80⁺ activated dendritic cells (DCs), and SELP⁺ACKR1⁺ high endothelial venule (HEV) cells within intratumoral-TLS (iTLS) rich tumors compared to peritumoral-TLS (pTLS) and desert-TLS (dTLS) tumor subtypes. Multimodal cell-cell interaction analysis and functional experiments demonstrate that HEV expressed VCAM1 and ICAM1 recruits and activates CXCL13⁺ TLC through the CXCL13-ACKR1 pathway, which promotes TLS formation via CXCL13-CXCR5-dependent crosstalk with B lymphocytes. We further develop a single-cell/spatial TLS signature that captures the cellular ecosystem of iTLS-containing tumor, demonstrating predictive value for immunotherapy outcomes in GC patients.

Despite significant advancements in identifying novel therapeutic targets and compounds, cancer stem cells (CSCs) remain pivotal in driving therapeutic resistance and tumor progression in gastric cancer (GC). High‐resolution knowledge of the transcriptional programs underlying the role of CSC niche in driving tumor stemness and progression is still lacking. Herein, spatial and single‐cell RNA sequencing of 32 human gastric mucosa tissues at various stages of malignancy, illuminating the phenotypic plasticity of tumor epithelium and transcriptional trajectory from mature gastric chief cells to the CSC state, which is associated with activation of EGFR and WNT signaling pathways, is conducted. Moreover, the CSCs interact with not only the immunosuppressive CXCL13⁺ T cells and CCL18⁺ M2 macrophages to evade immune surveillance, but also the inflammatory cancer‐associated fibroblasts (iCAFs) to promote tumorigenesis and maintain stemness, which construct the CSC niche leading to inferior prognosis. Notably, it is uncovered that amphiregulin (AREG) derived from iCAFs promotes tumor stemness by upregulating the expression of SOX9 in tumor cells, and contributes to drug resistance via the AREG‐ERBB2 axis. This study provides valuable insight into the characteristics of CSC niche in driving tumor stemness and progression, offering novel perspective for designing effective strategies to overcome GC therapy resistance.

A miniature CRISPR-Cas12f has been demonstrated to serve as an effective genome editing tool in gram negative bacteria as well as human cells. Here, we developed an alternative method to edit the genome of Bacillus anthracis based on the AsCas12f1 nuclease from Acidibacillus sulfuroxidans. When the htrA gene on the chromosome and the lef gene on the plasmid pXO1 were selected as targets, the CRISPR-AsCas12f1 system showed very high efficiency (100%). At the same time, a high efficiency was observed for large-fragment deletion. Our results also indicated that the length of the homologous arms of the donor DNA had a close relationship with the editing efficiency. Furthermore, a two-plasmid CRISPR-AsCas12f1 system was also constructed and combined with the endonuclease I-SceI for potential multi-gene modification. This represents a novel tool for mutant strain construction and gene function analyses in B. anthracis and other Bacillus cereus group bacteria.

Helicobacter pylori (H. pylori) is a gram-negative pathogen classified as a class I carcinogen. The H. pylori urease B subunit (UreB) and heat shock protein A (HspA) are two important vaccine candidate antigens. In this study, we evaluated the immunogenicity and immunoprotective effect of the attenuated Shigella vector vaccine SH02 expressing the UreB-HspA fusion protein of H. pylori in a mouse model. Oral SH02 with or without subcutaneous injection of rUreB-HspA induced antigen-specific serum IgG, mucosal sIgA, and T cells immune response. Subcutaneous injection of the candidate antigen rUreB-HspA enhanced the level of serum antigen-specific IgG antibodies (p < 0.0001) and the levels of IgG1/IgG2a/IgG2b subtypes. In addition, injection boost also increased the proportion of spleen antigen-specific CD4+CD154+ T cells (p < 0.001), and the proportion of CD4+CD154+ T cells that secrete IFN-γ and IL-17A. Following the H. pylori challenge, the levels of H. pylori colonization in the two experimental groups (Groups A and B) significantly reduced compared with the control group (p < 0.001), indicating that the candidate vaccine yielded a preventive effect of anti-H.pylori infection. Compared with the non-subcutaneous booster injection group (Group A), the subcutaneous booster injection group (Group B) exhibited less gastric inflammation, but there was no significant difference in the level of colonization (p > 0.05). These results lay a foundation for the development of a vaccine against H. pylori and the optimization of immunization methods and procedures to prevent H. pylori infection.

The Bacillus anthracis S-layer protein, BslA, plays a crucial role in mammalian infection. BslA is required to mediate adherence between host cells and vegetative forms of bacteria and this interaction promotes target organs adherence and blood–brain barrier (BBB) penetration in vivo. This study attempts to identify the potential eukaryotic ligand(s) for B. anthracis BslA protein. Biochemical approaches have indicated that the putative host cell ligand(s) for BslA is a surface protein, which is independent of the sugar components for binding to Bs1A. A ligand screening using blot overlays, far Western blots and mass spectrometry analyses revealed that BslA binds to mammalian laminin. ELISA based solid-phase binding assays and surface plasmon resonance assays demonstrated that there were high affinity interactions between BslA(260–652) and laminin. The SPR results also revealed the dissociation constants values of 3.172 × 10−9M for the binding of BslA(260–652) to laminin. These data demonstrated that laminin is a ligand for BslA.