Primary liver cancer is the third leading cause of cancer deaths globally (1, 2), It mainly includes hepatocellular carcinoma, cholangiocarcinoma, etc., especially hepatocellular carcinoma, which is mainly caused by liver cirrhosis or nonalcoholic steatohepatitis (3, 4). Despite the availability of various treatments, including surgical resection and immunotherapy” or “such as surgical resection and immunotherapy, surgical resection being the most prevalent, most patients experience poor five-year survival rates, and prone to recurrence (5). Postoperative combination immunotherapy has emerged as a primary treatment strategy for various cancers in recent years. Nevertheless, its effectiveness frequently remains limited in HCC (6), Therefore, we need to have a deeper understanding of the tumor immune microenvironment of HCC in order to provide better treatment for patients.

Research has identified the presence of TLS within tumor tissues. These organized aggregates of immune cells are primarily formed through the recruitment of B cells, T cells, dendritic cells (DCs), and other immune cells by chemokines or other factors. Among these components, B cells constitute the predominant cellular population within TLS (7). B cells are also a major component of the tumor microenvironment in many types of cancer (8). The maturation markers of tertiary lymphoid structures, CD21 and CD23, are both expressed by B cells. CD21 is primarily expressed on follicular dendritic cells and B lymphocytes. CD23, a differentiation antigen of B lymphocytes, is also expressed by these cells (8). Both markers serve to visualize the follicular dendritic cells network within germinal centers, which plays a crucial role in regulating B lymphocyte activation. The antitumor function of TLS formed within human tumors is primarily dependent on antibody-mediated humoral immune responses driven by B lymphocyte-secreted antibodies (7, 9). Prognostic studies in diverse cancers consistently report that the presence of TLS is associated with superior objective response rates to immunotherapy and better clinical outcomes when compared to TLS-negative tumors (10–12), At present, we know very little about the formation mechanism of TLS.

Recent years have witnessed increasing research interest in bacteria residing within tumors (13), It has been proposed in report that intratumoral bacteria might contribute to the development of tertiary lymphoid structures. Bacterial colonization and persistent growth occur within tumors (14), In cancers such as breast cancer (15) and colorectal cancer (16), these intratumoral bacteria primarily reside intracellularly within cancer cells (17), infection with bacteria leads to the upregulation of pro-oncogenic genes in tumor cells (18), enhancing their proliferative capacity and promoting cancer progression and metastasis (17), For instance, Fusobacterium nucleatum exhibits pro-tumorigenic effects in breast cancer (19). Interestingly, however, intratumoral bacteria (ITB) may also exert a stimulatory effect on immune cell infiltration within tumors, potentially playing a beneficial role in immunotherapy. For instance, in colorectal cancer, studies have found that patients with higher levels of Fusobacterium nucleatum exhibit better treatment responses (20), This bacterium appears to modulate TLS via regulatory T cells (21). Conversely, while TLS have been extensively studied in gastrointestinal cancers (22, 23), including HCC (24–26), their mechanisms of formation in HCC specifically remain poorly understood.

In this study, we collected tumor and paired adjacent non-tumor tissues from 153 HCC patients. Hematoxylin and eosin staining and immunofluorescence (IF) were employed to investigate the relationships between ITB, immune cell infiltration, and TLS within the tumor microenvironment (TME). Additionally, we analyzed the clinical relevance of TLS in HCC.

This study included a cohort of 153 patients diagnosed with HCC who underwent curative resection at the First Affiliated Hospital of Air Force Medical University between January 2014 and December 2023. All patients were treatment-naïve prior to surgery, and HCC diagnosis was histologically confirmed post-resection. Clinical data and pathological parameters were retrospectively collected from medical records. Patients were followed up every 3 months for the first two years after surgery, and subsequently every 6 months until death or the last follow-up date March 2025。Recurrence-free survival (RFS) was defined as the time from surgery to recurrence or death. Overall survival (OS) was defined as the time from surgery to death or the last follow-up. This study was approved by the Ethics Committee of the First Affiliated Hospital of Air Force Medical University (Approval No. KY20232280-X-1) in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants.

In recent years, the intratumoral microbiota, particularly bacteria, is increasingly recognized as a key regulator of tumor progression and responses to immunotherapy. Previous studies have suggested that ITB may influence the tumor microenvironment and immunotherapy efficacy by inducing local necrosis, suppressing T-cell infiltration, or modulating immune checkpoint pathways (18, 27–29). Notably, these microbes may exhibit dual roles: while potentially promoting cancer progression, they can also exert antitumor effects through immune cell recruitment (30). In hepatocellular carcinoma, the presence and functional significance of ITB remain to be fully explored. Our study analyzed 153 retrospectively collected HCC surgical resection samples. Using 16S rRNA fluorescence in situ hybridization (FISH) and immunofluorescence staining for lipopolysaccharide and lipoteichoic acid, we observed bacteria exhibiting a punctate pattern within the perinuclear region of tumor cells. These bacteria were predominantly Gram-negative, although their abundance was lower than in other gastrointestinal tumors. Gram-negative bacteria were primarily localized within tumor cells and stromal cell populations, with CD20+ B cells also positioned near LPS signals. These findings suggest that ITB may exist primarily intracellularly, within both cancer and immune cells. This observation aligns with the findings of Xue et al., who reported distinct bacterial communities between HCC tumor and non-tumor tissues, with some bacteria correlating with metabolites (31). It also resonates with Liu et al.’s observation of bacterial presence within inflammatory areas alongside CD8+ T cells and myeloid-derived suppressor cells in HBV-related HCC (32). Collectively, these findings suggest that ITB in HCC may possess the potential to modulate the local tumor microenvironment, representing a promising therapeutic target worthy of further exploration (33).

Given the importance of immunotherapy in cancer, we focused on the impact of ITB on the tumor microenvironment, particularly tertiary lymphoid structures (TLS). As key immune components within the TME, TLS have been associated with favorable prognoses across multiple cancers, including melanoma (34), colon cancer (35), lung cancer (36), pancreatic cancer (22), and esophageal cancer (37). However, the prognostic significance of TLS in HCC—encompassing both intratumoral TLS and peritumoral TLS—remains controversial. Emerging research indicates that immunotherapy modulates TLS density, and higher intratumoral TLS levels correlate with improved recurrence-free survival (24). To better evaluate the overall relevance of TLS in HCC, we adopted a novel approach: rather than assessing iTLS or pTLS independently (as in prior studies), we analyzed them as an integrated TME immune structure for prognostic evaluation. A key finding of this study is the significant correlation between enrichment of Gram-negative bacteria in HCC tissues and TLS presence, whereas Gram-positive bacteria were relatively scarce. Notably, our immunofluorescence (IF) analysis revealed a spatial association between CD20+ B cells (core components of TLS) and bacterial localization. This suggests ITB, particularly Gram-negative species, may promote TLS formation and maturation by recruiting CD20+ B cells.

This finding is highly innovative, representing the first direct in situ linkage of intratumorally colonizing bacteria to TLS establishment in a solid tumor, specifically HCC. It extends previous observations that gut microbiota may influence HCC TLS (38), underscoring the importance of studying the local tumor microbiota—which may exert more direct effects within the TME compared to gut microbes. Furthermore, while Sun et al. found intratumoral microbes remodel the HCC TME and influence CD68+ macrophage infiltration (39), our study specifically focuses on the TLS niche. We identify CD20+ B cells as potential mediators of bacterial effects, promoting TLS quantity and maturation. This provides a new perspective on how microbes precisely regulate the HCC immune landscape. Nevertheless, a fundamental and clinically significant question remains: How do intratumoral bacteria specifically drive CD20+ B cell recruitment and subsequent TLS formation/maturation?

Based on our findings, we hypothesize that bacterial-derived antigens may activate intratumoral B cells, promoting their expansion and facilitating TLS construction and maintenance. The interaction between B cells and T cells—mediated through mechanisms such as antigen presentation and co-stimulatory molecules—is crucial for TLS maturation and function (8). Our observation that bacteria predominantly localize to the perinuclear region could enhance the accessibility of their products to immune cells. Furthermore, bacterial metabolites, as mentioned by Xue et al., may also contribute to regulating immune cell migration and function (31). B-cell-specific chemokines, such as CXCL13, are key drivers for recruiting B cells to inflammatory sites and initiating TLS formation (23, 40). Therefore, given that LPS is a potent Toll-like receptor 4 agonist, we speculate that ITB might stimulate cells within the TME, via receptor signaling pathways like Toll-like receptor 4, to produce critical chemokines such as CXCL13 (41, 42). This could directly recruit circulating CD20+ B cells to the tumor site, thereby promoting TLS formation. An intriguing parallel observation comes from clear cell renal cell carcinoma, where intratumoral fungi were found to suppress lipid catabolism and induce T cell exhaustion (43). This further highlights the functional diversity of the tumor microbiome. Consequently, a more comprehensive understanding of immune cell-mediated mechanisms shaping microbial distribution within tumors requires consideration of other components of the tumor microbiome, including fungal species. While gut microbes may serve as a source of intratumoral bacteria – primarily translocating via the enterohepatic circulation – studies in pancreatic cancer report no such association (44). These mechanisms represent plausible pathways, future studies employing metagenomic sequencing and strain-specific culture are essential for validation.

This study has several limitations. Our characterization of bacteria was limited; we used LPS as a pan-marker for Gram-negative bacteria and were unable to identify specific bacterial species or strains. Furthermore, LTA staining yielded a low positive rate and weak signals in our study. While this does not preclude a role for Gram-positive bacteria in localized microenvironments, our current data indicate their overall contribution to TLS formation is likely limited. Future validation using metagenomic sequencing and strain-specific culture is essential and represents a primary limitation of this work. Importantly, the immunomodulatory functions of different bacterial species can vary significantly, even exhibiting opposing effects. Consequently, the observed association between intratumoral bacteria and TLS in HCC may be primarily driven by immunogenic Gram-negative species. Future research should employ 16S rRNA gene sequencing and single-bacterium isolation/culture techniques to precisely identify key bacterial species associated with TLS formation in HCC and characterize their functional profiles. Additionally, our retrospective cohort had a limited sample size and was from a single center. HCC etiology, geographic region, and patient lifestyle factors could significantly influence intratumoral microbiota composition and the TME. Larger-scale, multi-center prospective studies with detailed epidemiological data are needed to validate and generalize our findings and explore heterogeneity across different patient subgroups.

In summary, this study reveals a significant correlation between the enrichment of intratumoral Gram-negative bacteria and the presence of TLS in HCC. It is the first to propose a mechanism whereby bacteria, potentially via recruitment of CD20+ B cells, may promote TLS formation and maturation, providing a novel perspective on the regulation of the HCC immune microenvironment. We established an integrated iTLS/pTLS evaluation standard for stratifying HCC patients and predicting prognosis based on TLS. Despite limitations such as the lack of precise bacterial classification, this work provides a foundation for future research to identify key pro-TLS bacterial species and elucidate their mechanisms of action. Such efforts promise not only to deepen our understanding of TLS biology but also to pave the way for novel therapeutic strategies. Combining interventions targeting these pathways with existing immunotherapies, such as immune checkpoint inhibitors (ICIs), holds potential to overcome immune resistance and ultimately improve the long-term clinical outcomes of HCC patients. Continued exploration of the intratumoral microbiota – including bacteria, fungi, and other components – and their interactions with host immunity represents a highly promising new direction in cancer immunotherapy.