Back to 2025 Abstracts
TARGETING THE GUT MICROBIOME IMPROVES ANTI-TUMOR CYTOTOXIC CD8 T-CELL RESPONSE AGAINST HEPATOCELLULAR CARCINOMA
Dhanisha s. Suresh, Tejeshwar Jain
*, Utpreksha Vaish, Srikanth Iyer, Vikas Dudeja
surgery, The University of Alabama at Birmingham Heersink School of Medicine, Birmingham, AL
Background: Hepatocellular carcinoma (HCC) remains one of the leading causes of cancer-related mortality in the US. Although immunotherapy is a first-line treatment for advanced HCC, only 50% of patients respond. Recently, gut microbiome has emerged as an important modulator of anti-cancer immune response. We evaluated the role of gut microbiome ablation of anti-tumor immune response against HCC.
Methods: A well established carcinogen-induced model of HCC (DEN-CCl4 model) characterized by a single injection of diethylnitrosamine (DEN) at 2 weeks of age followed by weekly injections of carbon tetrachloride (CCl4) from weeks 8-22 was used to spontaneously induce HCC in male C57BL/6J mice. Mice were then randomized into daily oral gavage with 500uL of saline or broad-spectrum antibiotics cocktail (AC) of poorly absorbed oral antibiotics (Vancomycin, Neomycin, Ampicillin, Metronidazole, and Amphotericin B) for 14 weeks, followed by euthanasia. At endpoint, liver weights and number of tumor nodules were measured, and flow cytometry was performed to evaluate anti-tumor immune response. In a separate experiment, 6 wk old male C57BL/6J mice were randomized into saline or AC treatment for 4 weeks for gut microbiome depletion, followed by subcutaneous (s.c) implantation of an HCC cell line derived from spontaneously formed tumors in DEN-CCL4 mice. 16s rRNA sequencing was performed to evaluate changes in the gut microbiome. At endpoint, tumors were analyzed via qPCR and flow cytometry. CD8+ T-cells were isolated from spleen of saline and AC treated tumor bearing mice and cytotoxicity was evaluated
ex-vivo using co-culture with calceinAM labelled HCC tumor cells.
Results: DEN-CCl4 treatment induced spontaneous HCC formation, as expected. AC treatment led to significantly reduced tumor burden, as evidenced by decreased liver weight (Fig 1) and number of surface tumor nodules. Flow cytometry revealed increased infiltration of CD8+ T-cells in the AC group. These results were recapitulated in the s.c. model, where AC treatment led to significantly reduced tumor burden and flow cytometry revealed increased infiltration of cytotoxic CD8+ T-cells (Tc). qPCR of tumors revealed increased expression of markers associated with Tc response including
Ifng, Gmzb and
Pfn. 16s rRNA analysis revealed depletion of majority of the bacterial colonies upon AC treatment.
Ex-vivo analysis of splenic CD8+ T-cells isolated from AC treated mice revealed increased cytotoxicity on calceinAM assay (Fig 2) and increased expression of cytotoxic markers such as
Ifng and
Gmzb on qPCR
. Conclusion: Gut microbiome targeting leads to decreased HCC tumor growth accompanied by an improved cytotoxic CD8+ T-cell response. Gut microbiome can potentially emerge as a novel therapeutic target to improve immunotherapy efficacy against HCC.
Antibiotics cocktail (abx cocktail) treatment leads to significantly reduced tumor burden in DEN-CCL4 model of HCC. n = 9 per group
Antibiotics cockatil treatment leads to significantly increased ex-vivo cytotoxicity of splenic CD8 T-cells. n = 3 per group. **p = <0.01
Back to 2025 Abstracts
