Fig 1: TUDCA induces survival benefit in HCC mouse model after iRFA. Scheme showing animal experimental set-up. An orthotopic HCC model received iRFA was generated and treated with 100 mg/kg TUDCA or vehicles by oral gavage for 14 days. (B) Bile acid composition in plasma in TUDCA-treated mice and control groups (n=12). (C) UDCA% in plasma bile acids. (D) Total bile acid levels in plasma. (E) UDCA levels in plasma. (F) Ratio of primary to secondary bile acids. (G) Ratio of conjugated to unconjugated bile acids. (H) Final photography of orthotopic tumors from each group. (I) Tumor volume. (J) Survival curve. Specifically, at the end of the PBS curve, 8 out of 11 mice died, and at the end of the TUDCA curve, 3 out of 11 mice died. (K) Representative flow cytometry plots of TCRβ+ α-GalCer-CD1d+iNKT cells. (L) Calculation of iNKT cells infiltration in residual tumor tissues. (M) Infiltration of macrophages. (N) Calculation of CD86 on the surface of macrophages. (O) Calculation of CD206 on the surface of macrophages. (P) The ratio of M1 to M2 macrophages in residual tumor tissues. Data are presented as mean±SD. P values represent *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by Student’s t-test. BAs, bile acids; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; HCC, hepatocellular carcinoma; HDCA, hyodeoxycholic acid; iNKT cells, invariant natural killer T cells; iRFA, incomplete radiofrequency ablation; LCA, lithocholic acid; MCA, muricholic acid; PBS, phosphate-buffered saline; TCR, T-cell ratio; TUDCA, tauro-conjugated ursodeoxycholic acid; UDCA, ursodeoxycholic acid.
Fig 2: Reduced infiltration of iNKT cells and M2 macrophage polarization in residual HCC tissues following iRFA. A bilateral subcutaneous HCC-iRFA model was established. One tumor was excised for flow cytometry prior to ablation, while residual neoplastic tissue from the contralateral tumor was harvested for flow cytometric analysis 7 days after incomplete ablation (n=9–12 mice/group). (A) Representative flow cytometry plots of TCRβ+ α-GalCer-CD1d+iNKT cells. (B) Calculation of iNKT cells infiltration. (C) Calculation of infiltration of macrophages. (D) Representative flow cytometry plots of F4/80+CD206+ macrophages. (E) Calculation of CD206 on the surface of macrophages. (F) Representative flow cytometry plots of F4/80+CD86+ macrophages. (G) Calculation of CD86 on the surface of macrophages. (H) The ratio of M1/M2 macrophages. An orthotopic HCC model received iRFA was generated. (I) Representative H&E staining of liver tumor tissues from pre-iRFA and post-iRFA mice (original magnification: 10×). (J) Representative images of immunofluorescence staining of CD86+ and CD206+ macrophages in pre-iRFA (n=4) and post-iRFA (n=4) groups. (K) Calculation of CD86+macrophages in the tumor tissue. (L) Calculation of CD206+macrophages in the tumor tissue. Bar represents 20 µm. Data are presented as mean±SD. P values represent *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by pair t-test. HCC, hepatocellular carcinoma; iNKT cells, invariant natural killer T cells; iRFA, incomplete radiofrequency ablation; TCR, T-cell receptor.
Fig 3: LCA promotes tumor recurrence and inhibits iNKT cell infiltration after iRFA in HCC. (A) Scheme showing animal experimental set-up. An orthotopic HCC model received iRFA was generated and treated with 20 mg/kg LCA or vehicles by oral gavage for 7 days. (B) Bile acid composition in plasma in LCA-treated mice and control groups (n=9–11). (C) LCA% in plasma bile acids. (D) Total bile acid levels in plasma. (E) LCA levels in plasma. (F) Ratio of primary to secondary bile acids. (G) Ratio of conjugated to unconjugated bile acids. (H) Final photography of orthotopic tumors from each group. (I) Tumor volume. (J) Survival curve. Specifically, at the end of the CTR curve, 0 out of 11 mice died, and at the end of the LCA curve, 4 out of 10 mice died. (K) Representative flow cytometry plots of TCRβ+ α-GalCer-CD1d+iNKT cells. (L) Calculation of iNKT cells infiltration in residual tumor tissues. (M) Infiltration of macrophages. (N) Calculation of CD86 on the surface of macrophages. (O) Calculation of CD206 on the surface of macrophages. (P) The ratio of M1 to M2 macrophages in residual tumor tissues. Data are presented as mean±SD. P values represent *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by Student’s t-test. BAs, bile acids; CA, cholic acid; CDCA, chenodeoxycholic acid; CTR, control group; DCA, deoxycholic acid; HCC, hepatocellular carcinoma; HDCA, hyodeoxycholic acid; iNKT cells, invariant natural killer T cells; iRFA, incomplete radiofrequency ablation; LCA, lithocholic acid; MCA, muricholic acid; TCR, T-cell receptor; UDCA, ursodeoxycholic acid.
Supplier Page from BioLegend for PE anti-mouse α-GalCer:CD1d complex