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Many patients with hepatocellular carcinoma (HCC) do not respond to the first-line immune checkpoint inhibitor treatment. Immunization with effective cancer vaccines is an attractive alternative approach to immunotherapy. However, its efficacy remains insufficiently evaluated in preclinical studies. Here, we investigated HCC-associated self/tumor antigen, α-fetoprotein-based (AFP-based) vaccine immunization for treating AFP (+) HCC mouse models. We found that AFP immunization effectively induced AFP-specific CD8+ T cells in vivo. However, these CD8+ T cells expressed exhaustion markers, including PD1, LAG3, and Tim3. Furthermore, the AFP vaccine effectively prevented c-MYC/Mcl1 HCC initiation when administered before tumor formation, while it was ineffective against full-blown c-MYC/Mcl1 tumors. Similarly, anti-PD1 and anti-PD-L1 monotherapy showed no efficacy in this murine HCC model. In striking contrast, AFP immunization combined with anti-PD-L1 treatment triggered significant inhibition of HCC progression in most liver tumor nodules, while in combination with anti-PD1, it induced slower tumor progression. Mechanistically, we demonstrated that HCC-intrinsic PD-L1 expression was the primary target of anti-PD-L1 in this combination therapy. Notably, the combination therapy had a similar therapeutic effect in the cMet/β-catenin mouse HCC model. These findings suggest that combining the AFP vaccine and immune checkpoint inhibitors may be effective for AFP (+) HCC treatment.

TBX3 behaves as a tumor suppressor or oncoprotein across cancer. However, TBX3 function remains undetermined in intrahepatic cholangiocarcinoma (iCCA), a deadly primary liver malignancy with few systemic treatment options. This study sought to investigate the impact of TBX3 on iCCA. We found that overexpression of TBX3 strongly inhibited human iCCA cell growth. In the Akt/FBXW7ΔF mouse iCCA model, overexpression of Tbx3 reduced cholangiocarcinogenesis in vivo, while inducible genetic knockout of Tbx3 accelerated iCCA growth. RNA-seq identified MAD2L1 as a downregulated gene in TBX3-overexpressing cells, and ChIP confirmed that TBX3 binds to the MAD2L1 promoter. CRISPR-mediated knockdown of Mad2l1 significantly reduced the growth of two iCCA models in vivo. Finally, we found that TBX3 expression is upregulated in ~20% of human iCCA samples, and its high expression is associated with less proliferation and better survival. MAD2L1 expression is upregulated in most human iCCA samples and negatively correlated with TBX3 expression. Altogether, our findings suggest that overexpression of TBX3 suppresses CCA progression via repressing MAD2L1 expression.

Murine Kupffer cells (KCs) comprise CD11bhi and F4/80hi subsets. Tissue-resident macrophages are known to express the tyrosine kinase receptors colony-stimulating factor 1 receptor (Csf1r) and Mer. However, the expression of Csf1r and Mer on KC subsets and the importance of these tyrosine kinases during liver regeneration (LR) are unknown. KCs from wild-type and Csf1r-GFP mice were characterized by flow cytometry. Partial hepatectomy (PH) was performed in mice treated with clodronate liposomes, a Csf1r small molecule inhibitor or depleting antibody, or a small molecule Mer inhibitor. Sera and livers were analyzed. The function of sorted KC subsets was tested in vitro. Mer was specifically expressed on tissue-resident F4/80hi KCs, 55% of which also expressed Csf1r. Mer+Csf1r+ and Mer+Csf1r- KCs had distinct expression of macrophage markers. Csf1r inhibition in mice reduced F4/80hi KCs by approximately 50%, but did not affect CD11bhi KCs. Clodronate liposomes depleted F4/80hi KCs, but also altered levels of other intrahepatic leukocytes. Csf1r inhibition delayed LR, as demonstrated by a 20% reduction in liver-to-body weight ratios 7 days after PH. At 36h after PH, Csf1r inhibition increased serum ALT and histological liver injury, and decreased liver cell proliferation. A small molecule inhibitor of Mer did not alter the percentage of KCs or their proliferation and just modestly delayed LR. In vitro, Csf1r or Mer inhibition did not decrease KC viability, but did attenuate their cytokine response to stimulation. F4/80hi KCs are Mer+ and can be subdivided based on Csf1r expression. Csf1r or Mer inhibition each reduces KC cytokine production and delays LR.

Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare variant of HCC that most frequently affects young adults. Because of its rarity and an absence of preclinical models, our understanding of FL-HCC is limited. Our objective was to analyze chromosomal alterations and dysregulated gene expression in tumor specimens collected at a single center during two decades of experience with FL-HCC. We analyzed 38 specimens from 26 patients by array comparative genomic hybridiziation (aCGH) and 35 specimens from 15 patients by transcriptome sequencing (RNA-seq). All tumor specimens exhibited genomic instability, with a higher frequency of genomic amplifications or deletions in metastatic tumors. The regions encoding 71 microRNAs (miRs) were deleted in at least 25% of tumor specimens. Five of these recurrently deleted miRs targeted the insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) gene product, and a correlating 100-fold upregulation of IGF2BP1 mRNA was seen in tumor specimens. Transcriptome analysis demonstrated intrapatient tumor similarity, independent of recurrence site or time. The p53 tumor suppressor pathway was downregulated as demonstrated by both aCGH and RNA-seq analysis. Notch, EGFR, NRAS, and RB1 pathways were also significantly dysregulated in tumors compared with normal liver tissue. The findings illuminate the genomic and transcriptomic landscape of this rare disease and provide insight into dysregulated oncogenic pathways and potential therapeutic targets in FL-HCC.

ObjectiveLiver metastases are often resistant to immune checkpoint inhibitor therapy (ICI) and portend a worse prognosis compared with metastases to other locations. Regulatory T cells (Tregs) are one of several immunosuppressive cells implicated in ICI resistance of liver tumours, but the role played by Tregs residing within the liver surrounding a tumour is unknown.DesignFlow cytometry and single-cell RNA sequencing were used to characterise hepatic Tregs before and after ICI therapy.ResultsWe found that the murine liver houses a Treg population that, unlike those found in other organs, is both highly proliferative and apoptotic at baseline. On administration of αPD-1, αPD-L1 or αCTLA4, the liver Treg population doubled regardless of the presence of an intrahepatic tumour. Remarkably, this change was not due to the preferential expansion of the subpopulation of Tregs that express PD-1. Instead, a subpopulation of CD29+ (Itgb1, integrin β1) Tregs, that were highly proliferative at baseline, doubled its size in response to αPD-1. Partial and full depletion of Tregs identified CD29+ Tregs as the prominent niche-filling subpopulation in the liver, and CD29+ Tregs demonstrated enhanced suppression in vitro when derived from the liver but not the spleen. We identified IL2 as a critical modulator of both CD29+ and CD29− hepatic Tregs, but expansion of the liver Treg population with αPD-1 driven by CD29+ Tregs was in part IL2-independent.ConclusionWe propose that CD29+ Tregs constitute a unique subpopulation of hepatic Tregs that are primed to respond to ICI agents and mediate resistance.

Introduction There are no approved locoregional therapies for peritoneal carcinomatosis from gastric adenocarcinoma (GA). Cytoreductive surgery with hyperthermic intraperitoneal chemotherapy (CRS–HIPEC) represents a potential treatment for advanced GA with isolated peritoneal metastasis. Patients and Methods Two separate single-institution phase II, single-arm studies evaluating CRS–HIPEC using cisplatin with mitomycin C (NIH: NCT03092518, MDACC: NCT02891447) in patients with GA and confirmed peritoneal metastasis were analyzed. The primary endpoint of each trial was overall survival (OS). Clinical, pathologic, and treatment variables were analyzed for association with outcomes. Results Over 4 years, 41 patients with peritoneal carcinomatosis from GA underwent CRS–HIPEC. All patients had synchronous peritoneal metastasis and received systemic chemotherapy as front-line therapy. A total of 23 patients also received laparoscopic HIPEC prior to open CRS–HIPEC. The majority (63%, n = 26) were male, and median PCI score at CRS–HIPEC was 2. Median OS was 24.9 months from diagnosis and 14.4 months from CRS–HIPEC. Three-year OS was 25% from diagnosis and 22% from CRS–HIPEC. Median RFS was 7.4 months. The rate of 30-day Clavien–Dindo grade ≥ 3 complications was 32%; specifically, the rate of anastomotic leak was 22%. Multivariable analysis identified the number of pathologically positive lymph nodes as an independent predictor of postoperative OS. Conclusions In patients with gastric adenocarcinoma and isolated peritoneal metastasis treated with CRS–HIPEC, 3-year OS was 22% from CRS–HIPEC, and complications were common. The number of pathologic lymph node metastases was inversely correlated with overall survival. Further investigation of CRS–HIPEC for GA should include patient selection based on response to systemic chemotherapy or incorporate novel intraperitoneal treatment strategies.

BackgroundLiver cancer remains the third leading cause of worldwide cancer-related deaths.1 Despite clinical advances, established immunotherapies largely fail patients due to poor immune responses.1 2 Peripheral nerves influence tumors, but brain-liver interactions remain largely unstudied.3 A recent study reported a decreased risk of liver cancers in patients that underwent truncal vagotomy (vagus snip) compared to simple suture procedure.4 Here, we identify a vagal-immune arc regulating hepatic cancer.MethodsC57BL/6 or BALB/c mice (9–12 weeks old) underwent a surgical hepatic vagotomy (HV) or sham procedure (SV). Following established protocols,5 HV and SV mice received orthotopic tumors via intrahepatic injection (2.5x105 RIL-175 or B16-F10 cells) or tail vein/flank injection (1.0x106 A20, RIL-175 cells) to model primary and metastatic liver cancer. Growth of luciferase-labeled cells was measured via in vivo imaging and immune profiling was conducted via flow cytometry and scRNASEQ. Open field test, Y maze, and phenotyper cages assessed mouse behavior. Highly-multiplexed immunofluorescence (CODEX platform) revealed peripheral nerves in clinical resection samples.ResultsPrecise liver denervation reduced tumor growth in three models of primary (RIL-175) and metastatic (B16-F10, A20) tumors (figure 1A). Outcomes remained organ specific as HV mice exhibited reduced tumor burden of intrahepatic, but not subcutaneous, models. HV livers exhibited decreased levels of vagal neurotransmitter acetylcholine (ACh). As immunofluorescent analyses revealed colocalization of peripheral nerves and lymphocytes in clinical liver cancer, we profiled HV immunity. HV livers exhibited broad anti-tumor immunity, notably increased CD8+ T cells and higher expression of intracellular cytokines (IFNγ, TNFα) (figure 1B). We then examined whether immune alterations were a cause or consequence of HV tumor burden. ACh exposure reduced intracellular cytokine levels in ex vivo CD8+ T cells following anti-CD3/CD8 activation. Treatment with bethanechol (ACh receptor agonist, 400 μg/mL drinking water) increased tumor burden and reduced CD8+TNFα+ subsets. Bethanechol failed to promote tumor growth in Rag1KO mice lacking mature B and T cells, and targeted depletion of CD8+ T cells abrogated the effects of vagotomy (figure 1C). Finally, as the vagus nerve is largely comprised of afferent fibers, we assessed murine behavior and ambulation. Tumor-bearing HV mice displayed decreased anxiety-life features and fatigue compared to sham controls.ConclusionsOur findings highlight a vagal-CD8+ T cell axis modulating hepatic tumor burden and behavior. This work furthers the emerging field of cancer neuroscience and identifies ACh signaling targets to alter hepatic immunosuppression and cancer outcomes.ReferencesKim E, Viatour P. Hepatocellular carcinoma: old friends and new tricks. Exp Mol Med. 2020;52:1898–1907.Greten T, et al. Society for Immunotherapy of Cancer (SITC) clinical practice guideline on immunotherapy for the treatment of hepatocellular carcinoma. 2021. JITC. 2021;9:e002794.Monje M, et al. Roadmap for the Emerging Field of Cancer Neuroscience. Cell. 2020;181:219–222.Wu S, et al. Decreased risk of liver and intrahepatic cancer in non-H. pylori-infected perforated peptic ulcer patients with truncal vagotomy: a nationwide study. Sci Rep. 2021;11:e15594.Brown Z, Heinrich B, Greten T. Establishment of orthotopic liver tumors by surgical intrahepatic tumor injection in mice with underlying non-alcoholic fatty liver disease. Methods protoc, 2018;2:e21.Ethics ApprovalThis research was approved by the NCI Division of Intramural Research Animal Care and Use Committee, proposal numbers: MOB-028 and TGOB-015. Patients provided informed consent for clinical tissue acquisition: Institutional Review Board protocol #2017–0365.Abstract 988 Figure 1(A) RIL-175 tumor weight 21 days after intrahepatic injection (2.5x105 cells/mouse). (B) Median fluorescence intensity (MFI) of ex vivo CD4+ and CD8+ T cells following PMA/ionomycin stimulation, proinflammatory IFNg and TNFa displayed. (C) CD8+ T cell depletion RIL-175 model at 21 days (200 µg anti CD8 or lgG control/mouse 1X per week). Bar graphs indicate mean and SEM, data assessed by unpaired t-test, significance determined as p<0.05.

BackgroundEpidemiological studies suggest that bacterial infections promote SLE disease in predisposed individuals, but the underlying mechanisms remain unknown. We have found that a subset of SLE patients has asymptomatic bacteriuria associated with markers of inflammation and flares, suggesting that chronic exposures to microbial products may trigger flares in lupus. Our labs have shown that the bacterial amyloid curli, expressed in multicellular communities (biofilms) by many bacteria including E. coli, plays a major role in triggering lupus autoimmunity during infection. Curli amyloid/DNA complexes strongly activate dendritic cells and macrophages. When given systemically, curli/DNA complexes and infections with curli-expressing E. coli trigger production of anti-dsDNA and anti-chromatin autoantibodies in lupus prone mice and in wild type mice. This stimulation is diminished in TLR2 or TLR9 deficient mice, suggesting a TLR-mediated activation of innate immunity. We have now focused on the effects of curli/DNA complexes on B cells. MethodsYoung wild type C57BL/6 mice, lupus prone Sle1,2,3 mice, 3H9 mice and CD40L-/- mice were injected with curli/DNA complexes from biofilms or infected with amyloid for short and long-term studies. Splenic B cells were stained by flow cytometry ex vivo. For in vitro experiments, B cells were sorted by positive selection with CD45R (B220), supplemented with anti-CD43Ab-Biotin. B cell purity (>98%), proliferation, activation markers and signaling molecules were measured by Flow cytometry, Western Blot and qRT-PCR. Autoantibodies were measured by ELISA.Results In vitro, curli/DNA complexes could induce class switch to IgG, in the absence of T cell help, in wildtype B cells, and even more in Sle1,2,3 and 3H9 B cells, which recognize DNA, suggesting an antigen-specific activation of B cells by curli/DNA. Curli/DNA induced non-canonical NFκB activation and transcription of aicda, the master regulator of class switch recombination. In vivo, exposure to curli/DNA broke tolerance to DNA in 3H9 mice. Moreover, it induced autoantibodies in CD40L-/- mice, though at lower levels than in WT mice.ConclusionsThe induction of non-canonical NFκB activation, aicda, and class switch recombination, in the absence of T cells help in vitro, suggests that the fibrillar structure of curli/DNA complexes can cross-link BCRs, some recognizing DNA, and can also trigger a second pathway which substitutes T cell help to induce isotype switching. The lower levels of autoantibodies elicited by curli/DNA in mice deficient of T cell help suggests that curli/DNA complexes break tolerance to DNA with T cell-independent and T cell-dependent modalities.AcknowledgmentsThis work was supported by the NIH, NIAID grant R21-AI119947 and the Lupus Research Institute, now Lupus Research Alliance, Innovative Grant (SG); NIH NIAMS grant R01-AR061569, R56-AR072115, Lupus Research Alliance, TIL (RC); NIH, NIAID R21-AI125429, R21-AI132996, R21-AR072115 (CT).