Tertiary lymphoid structures (TLSs) are associated with immunotherapy response, yet the mechanisms controlling their formation and maintenance remain unclear. Using spatial transcriptomics and multiplex imaging across human tumors, we found that CCR7+ mature dendritic cells (DCs) accumulate in TLSs. In a mouse non-small cell lung cancer model that forms mature TLSs, we show that early TLS development requires interferon-γ (IFN-γ)-driven type 1 conventional dendritic cell (cDC1) maturation, migration to tumor-draining lymph nodes (tdLNs), and T cell recruitment. As tumors progress, TLSs persist independently of tdLN T cell egress, coinciding with cDC1 accumulation within intratumoral CCL19 stromal hubs. There, cDC1-major histocompatibility complex class 1 (MHC-I) and -MHC-II concomitant antigen presentation, along with CD40 signaling, sustain TLS, T follicular helper (TFH) cell pool, germinal centers, and tumor-specific immunoglobulin G (IgG). These findings highlight local mature cDC1s as key TLS orchestrators and potential targets to enhance antitumor TLS function.

Mechanism of immune surveillance in males with NOTCH1 liver tumors

While immune checkpoint blockade (ICB) has transformed cancer treatment, only a minority of hepatocellular carcinoma (HCC) patients achieve durable responses. Studies have demonstrated the gut microbiome as an important modulator of anti-tumor immunity, yet its influence on tumorigenesis and ICB response in HCC remains incompletely characterized. We analyzed fecal microbiome composition via 16S rRNA sequencing from 17 patients with resectable HCC (stage Ib, II, and IIIb) receiving anti-PD-1 (NCT03916627). Treatment response was radiographically assessed (RECIST 1.1). Gut microbiota samples were collected pre-treatment and during therapy. To investigate the microbiome’s causal link with HCC tumorigenesis, we employed an immunogenic murine HCC model generated via hydrodynamic tail-vein delivery of MYC-lucOS and CTNNB1 plasmids. Ex-germ-free mice received fecal microbiota transplantation (FMT) from either (i) a clinical non-responder with high Bacteroidetes abundance (44.7%), or (ii) a responder with low Bacteroidetes abundance (0.89%). Overall response rate was 26.3% (5 responders, 14 non-responders). Microbiome profiling revealed no significant difference in alpha diversity between responders and non-responders. Longitudinal analysis between pre- and during-treatment stools showed minimal change in alpha diversity. Taxonomic comparisons between responders and non-responders revealed significant Bacteroidetes phylum enrichment in non-responders (p<0.05). In the murine model, FMT from the non-responder donor markedly accelerated tumor progression and reduced median survival compared with responder-derived FMT (p<0.05). Integrating clinical microbiome profiling with a humanized HCC mouse model, we identify Bacteroidetes enrichment as a contributor to HCC tumor progression. FMT from a non-responder stool with high Bacteroidetes accelerated tumorigenesis and reduced survival relative to responder-derived microbiota with low Bacteroidetes. Because mechanistic microbiome studies in liver cancer have long been limited by the absence of models that faithfully reflect patient tumor biology and immune evasion, we developed a genetically engineered, immunogenic HCC model that enabled us to also investigate the microbiome signatures observed in patients. Together, these findings support Bacteroidetes-targeted microbial modulation as a potential therapeutic strategy to abrogate tumor progression and enhance ICB efficacy in HCC. Joan Shang, Marina Barcena-Varela, Illaria Mogno, Anthony Lozano, Ian Liebling, Kai Mead, Zhihua Li, Lauren Grinspan, Katherine Lindblad, Marina Ruiz de Galarreta, Romain Donne, Sacha Gnjatic, Miriam Merad, Tomi Jun, Celina Ang, Thomas Marron, Jeremiah Faith, Amaia Lujambio. Bacteroidetes Enrichment Associated with Immune Checkpoint Blockade Resistance and Promotes Tumorigenesis in Hepatocellular Carcinoma [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C063.

Sex-dependent immune responses in NOTCH1-driven liver tumors

NOTCH1-driven tumors in females lead to impaired CD8+ T cell priming and activation

Enhancing DC-T cell axis in females harboring NOTCH1-driven tumors has therapeutic effects

Abstract Hepatocellular carcinoma presents strong sexual dimorphism, being two to three times more frequent in males than in females; however, the role of sex in response to immunotherapies in HCC remains unknown. We demonstrate that NOTCH1, an understudied oncogene in HCC, elicits sexually dimorphic antitumor immunity and response to FDA-approved immunotherapies. Surprisingly, males harboring NOTCH1-driven tumors displayed enhanced antitumor immune responses, which, in mice, were mediated by dendritic and T cells. Conversely, females harboring NOTCH1-driven tumors presented immune evasion and resistance to immunotherapies through a defect in dendritic cell (DC)–mediated priming and activation of CD8+ T cells in mice, which was restored therapeutically with CD40 agonism. Mechanistically, the sexually dimorphic immunity was mediated by genes in the sex chromosomes but not by sex hormones. Together, our study unravels an unexpected association between NOTCH1 and sex in cancer immunity and highlights the potential of restoring the DC−CD8+ T-cell axis with CD40 agonism to improve outcomes. Significance: Although HCC presents strong sexual dimorphism, the role of sex in response to immunotherapies remains elusive. With a novel HCC mouse model and validation in patients with HCC, we demonstrate that NOTCH1 disrupts antitumor immunity specifically in females through a mechanism mediated by sex chromosome genes, which is reversed with CD40 agonism. See related commentary by Zhu and Koltsova, p. 452

Hepatocellular carcinoma presents strong sexual dimorphism, being two to three times more frequent in males than in females; however, the role of sex in response to immunotherapies in HCC remains unknown. We demonstrate that NOTCH1, an understudied oncogene in HCC, elicits sexually dimorphic antitumor immunity and response to FDA-approved immunotherapies. Surprisingly, males harboring NOTCH1-driven tumors displayed enhanced antitumor immune responses, which, in mice, were mediated by dendritic and T cells. Conversely, females harboring NOTCH1-driven tumors presented immune evasion and resistance to immunotherapies through a defect in dendritic cell (DC)-mediated priming and activation of CD8+ T cells in mice, which was restored therapeutically with CD40 agonism. Mechanistically, the sexually dimorphic immunity was mediated by genes in the sex chromosomes but not by sex hormones. Together, our study unravels an unexpected association between NOTCH1 and sex in cancer immunity and highlights the potential of restoring the DC-CD8+ T-cell axis with CD40 agonism to improve outcomes. Significance: Although HCC presents strong sexual dimorphism, the role of sex in response to immunotherapies remains elusive. With a novel HCC mouse model and validation in patients with HCC, we demonstrate that NOTCH1 disrupts antitumor immunity specifically in females through a mechanism mediated by sex chromosome genes, which is reversed with CD40 agonism. See related commentary by Zhu and Koltsova, p. 452.

Although oncogenic alterations influence tumor metabolism, how they impose distinct metabolic programs within a shared tissue context remains poorly defined. Here, we developed a rapid mitochondrial profiling platform to compare metabolites and proteins in genetic models of primary liver cancer (PLC). Analyses of six genetically distinct PLCs revealed that mitochondrial energy metabolism is largely dictated by oncogene identity. Kras -driven tumors required creatine metabolism to buffer energy demands during early tumorigenesis, whereas c-MYC -driven tumors relied on oxidative phosphorylation. Among c-MYC -driven PLCs, Pten -deficient tumors accumulated mitochondrial phosphoethanolamine, a precursor for phosphatidylethanolamine (PE) synthesis. Inhibition of PE synthesis selectively impaired the growth of Pten -deficient tumors and extended survival, in part through enhanced infiltration of CD8⁺ T cells and sensitization to TNFα-mediated cytotoxicity. Mechanistically, loss of PE elevated surface TNF receptor 2 (TNFR2), promoting TNFα signaling and pro-inflammatory response. These findings uncover genotype-specific mitochondrial metabolic liabilities and establish PE synthesis as a tumor-intrinsic mechanism of immune evasion in PLC.

Background & Aims:Liver cancer, the third leading cause of cancer-related mortality worldwide, has two main subtypes: hepatocellular carcinoma (HCC), accounting the majority of the cases, and cholangiocarcinoma (CAA). Notch pathway primarily regulates the intrahepatic development of bile ducts, which are lined with cholangiocytes, but it can also be upregulated in 1/3 of HCCs. To better understand the role of NOTCH1 in HCC, we developed a novel mouse model driven by activated Notch1 intracellular domain (NICD1) and MYC overexpression in hepatocytes. Methods:Using the hydrodynamic tail-vein injection method for establishing primary liver tumors, we generated a novel murine model of liver cancer harboring MYC overexpression and NOTCH1 activation. We characterized this model histopathologically as well as transcriptomically, utilizing both bulk and single cell RNA-sequencing. We also performed functional experiments using monoclonal antibodies. Results:MYC;NICD1 tumors displayed a combined HCC-CCA phenotype with temporal plasticity. At early time-points, histology was predominantly "cholangiocellular", which then progressed to mainly "hepatocellular". The "hepatocellular" component was enriched in mesenchymal genes and gave rise to lung metastasis. Metastatic cells were enriched in the TGFB and VEGF pathways and their inhibition significantly reduced the metastatic burden. Conclusions:Our novel mouse model uncovered NOTCH1 as a driver of temporal plasticity and metastasis in HCC, the latter of which is, in part, mediated by angiogenesis and TGFß pathways. Impact and Implications:This study develops a novel murine model of NOTCH1-driven liver cancer, an understudied oncogene in HCC. Using this model, we show that NOTCH1 drives plasticity in HCC and metastasis to the lungs that can be therapeutically targeted through inhibition of VEGF and TGFß pathways. Highlights:NOTCH1 activation in combination with MYC overexpression drives combined HCC-CCA.NOTCH1 activation in hepatocytes drives temporal plasticity.NOTCH1 activation drives metastasis of HCC cells to the lungs, but not of CCA cells.Angiogenesis and TGFß pathways mediate NOTCH1-induced lung metastasis. Graphical abstract:

Background and Aims:Growing evidence highlight the critical role of the gut microbiome in tumorigenesis and response to immunotherapies. However, the impact of gut microbes on hepatocellular carcinoma (HCC) progression and response to immune-checkpoint blockade (ICB) remains unclear due to the lack of combined preclinical and clinical studies. Approach & Results:We performed 16S rRNA of cross-cohort stool samples from 10 HCC responders (R) and 40 non-responders (NR) to ICB at baseline and on-treatment time-points. We identified an enrichment of Bacteroidetes in NR. To study the role of the microbiome in the cancer immune response, we generated an immunogenic mouse model of HCC via hydrodynamic tail-vein injection (HDTVI) of DNA plasmids mimicking common HCC alterations and immunogenicity by expressing model antigens (MYC-lucOS;CTNNB1 tumors). We found that antibiotic (ABX)-induced dysbiosis promoted a pro-tumorigenic effect in the MYC-lucOS;CTNNB1 HCC model by the expansion of a specific Bacteroidetes, Parabacteroides distasonis. Colonization of mice carrying MYC-lucOS;CTNNB1 HCCs with Parabacteroides distasonis confirmed its pro-tumorigenic effect in vivo. Furthermore, we explored the effects of colonizing with microbiotas from patients and showed that microbiota from a NR donor enriched in Bacteroidetes promoted faster tumorigenesis than microbiota from a R donor with reduced Bacteroidetes. We isolated 6 Bacteroidetes species from the NR donor, cultured them, and used them as a cocktail to colonize mice; similarly, mice transplanted with this cocktail showed increased tumorigenesis and reduced survival. Conclusions:This study identified Bacteroidetes enrichment as a potential biomarker of ICB resistance in HCC and, by using immunogenic mouse models, established that Bacteroidetes abundance influences tumor development.

Macrophages are known to dampen tumor immunity. However, identifying druggable targets that modulate these cells to improve existing immunotherapies has been limited by a dearth of studies identifying macrophages that associate with pathological response to immune checkpoint blockade. To fulfill this unmet clinical need, we leveraged transcriptional and spatial profiling of specimens collected from a Phase II clinical trial studying neoadjuvant PD-1 blockade in patients with hepatocellular carcinoma (HCC). We determined that the intratumoral abundance of TREM2-expressing macrophages and serological levels of soluble TREM2 are elevated in patients who responded to PD-1 blockade, compared to non-responders. We validated these findings in a second HCC cohort and in the IMbrave150 trial. These highlight the robust potential for TREM2 macrophages to predict therapeutic responses of HCC to immunotherapy. Therefore, our study provides a novel basis for the use of TREM2 macrophages to strategize treatment for patients with HCC to maximize therapeutic benefit. ### Competing Interest Statement M.M. serves on the scientific advisory board and holds stock from Compugen Inc., Dynavax Inc., Morphic Therapeutic Inc., Asher Bio Inc., Dren Bio Inc., Nirogy Inc., Oncoresponse Inc., Owkin Inc. M.M. serves on the scientific advisory board of Innate Pharma Inc., DBV Inc., and Genenta Inc. M.M. receives funding for contracted research from Regeneron Inc. and Boerhinger Ingelheim Inc. T.U.M. has served on Advisory and/or Data Safety Monitoring Boards for Rockefeller University, Regeneron Pharmaceuticals, Abbvie, Bristol-Meyers Squibb, Boehringer Ingelheim, Atara, AstraZeneca, Genentech, Celldex, Chimeric, Glenmark, Simcere, Surface, G1 Therapeutics, NGMbio, DBV Technologies, Arcus and Astellas, and has research grants from Regeneron, Bristol-Myers Squibb, Merck and Boehringer Ingelheim. S.G. reports past consultancy or advisory roles for Merck and OncoMed; research funding from Regeneron Pharmaceuticals related to the current study, and research funding from Boehringer Ingelheim, Bristol Myers Squibb, Celgene, Genentech, EMD Serono, Pfizer and Takeda, unrelated to the current work. S.G. is a named coinventor on an issued patent (US20190120845A1) for multiplex immunohistochemistry to characterize tumors and treatment responses. The technology is filed through Icahn School of Medicine at Mount Sinai (ISMMS) and is currently unlicensed. This technology was used to evaluate tissue in this study and the results could impact the value of this technology. G.T., M.N., Y.W., G.A., N.J. and J.C.L. are employee and shareholder of Regeneron Pharmaceuticals Inc. C.P., N.F. and J.H. are employees and shareholders of Vizgen Inc. The remaining authors declare no competing interests.

Tertiary lymphoid structures (TLS) are organized immune cell aggregates that arise in chronic inflammatory conditions. In cancer, TLS are associated with better prognosis and enhanced response to immunotherapy, making these structures attractive therapeutic targets. However, the mechanisms regulating TLS formation and maintenance in cancer are incompletely understood. Using spatial transcriptomics and multiplex imaging across various human tumors, we found an enrichment of mature dendritic cells (DC) expressing high levels of CCR7 in TLS, prompting us to investigate the role of DC in the formation and maintenance of TLS in solid tumors. To address this, we developed a novel murine model of non-small cell lung cancer (NSCLC) that forms mature TLS, containing B cell follicles with germinal centers and T cell zones with T follicular helper cells (TFH) and TCF1+PD-1+progenitor exhausted CD8+T cells (Tpex). Here we show that, during the early stages of tumor development, TLS formation relies on IFNγ-driven maturation of the conventional DC type 1 (cDC1) subset, their migration to tumor-draining lymph nodes (tdLN), and recruitment of activated T cells to the tumor site. As tumors progress, TLS maintenance becomes independent of T cell egress from tdLN, coinciding with a significant reduction of cDC1 migration to tdLN. Instead, mature cDC1 accumulate within intratumoral CCR7 ligand-enriched stromal hubs. Notably, timed depletion of cDC1 or disruption of their migration to these stromal hubs after TLS are formed alters TLS maintenance. Importantly, we found that cDC1-mediated antigen presentation to both CD4+and CD8+T cells and intact CD40 signaling, is critical for the maintenance of TLS, the preservation of the TFHcell pool, the formation of germinal center and the production of tumor-specific IgG antibodies. These findings underscore the key role of mature cDC1 in establishing and maintaining functional TLS within tumor lesions and highlight the potential for cDC1-targeting therapies as a promising strategy to enhance TLS function and improve anti-tumor immunity in patients with cancer.