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. Author manuscript; available in PMC: 2022 Mar 8.
Published in final edited form as: Cell Rep. 2022 Feb 8;38(6):110349. doi: 10.1016/j.celrep.2022.110349

Figure 6. Targeting PD-L1 suppresses the ICC-promoting effect of HSCs in vitro and in a subcutaneous ICC/HSC co-implantation mouse model.

Figure 6.

(A) Control and PD-L1 knockdown HSCs, without or with TGF-β1 stimulation (5 ng/mL) for 24 h, were subjected to RNA sequencing and the data were analyzed using the Morpheus tool. The heatmaps depict the mRNA levels of genes related to HSC activation (left) and the tumor-promoting effect of HSCs (right). The scale bar represents the minimum expression value (blue) to the maximum value (red).

(B) Cell culture media collected from HSCs, stimulated without or with TGF-β1 (5 ng/mL) for 24 h, were subjected to WB for tumor-promoting factors. TGF-β1 promoted HSCs to produce and release tumor-promoting factors and this effect was abrogated by knockdown of PD-L1 in HSCs. **p < 0.01, ***p < 0.001 by ANOVA, n = 5.

(C) HuCCT1 cells tagged by firefly luciferase were mixed with HSCs and they were co-injected into nude mice subcutaneously. HuCCT1 chemiluminescence was quantitated by in vivo tumor imaging using a Xenogen IVIS 200 machine and Living Image software. Quantitative HuCCT1 chemiluminescence data revealed that control HSCs promoted the implantation of HuCCT1 cells in mice and that this effect of HSCs was reduced by knockdown of PD-L1 of HSCs. *p < 0.05 by ANOVA, n = 12 per group.

(D) Tumor size was measured using a caliper at different days, and tumor growth curves were generated. Control HSCs promoted HuCCT1 growth in mice and this effect of HSCs was reduced by knockdown of PD-L1 of HSCs. *p < 0.05 by ANOVA, n = 7 per group.

(E) The lysates of HuCCT1 tumors were subjected to WB, which revealed that the levels of αSMA and HSC-derived tumor-promoting factors were all reduced in tumors arising from HuCCT1/PD-L1 knockdown HSC co-injections than in tumors arising from control co-injections. *p < 0.05, **p < 0.01 by ANOVA, n = 5, 6.

(F) αSMA IF showed that the myofibroblast densities were lower in tumors arising from HuCCT1/PD-L1 knockdown HSC co-injections than in tumors arising from control co-injections. *p < 0.05 by ANOVA, n = 7, 7. Scale bar, 50 μm. All data are represented as mean ± SEM except those in (A).