Genotoxic chemotherapies, including TOP1 inhibitors, have been shown to upregulate tumor PD-L1 expression and confer clinical benefits when combined with ICIs [
41,
42]. Therefore, we investigated whether TLC388 can directly increase tumor PD-L1 expression. As shown in Fig.
4A and B, TLC388 treatment exhibited significantly increased
CD274 (PD-L1) mRNA expression in these cell lines with dose-dependent and time-dependent manner. Furthermore, the total and surface PD-L1 levels were also upregulated in dose-dependent and time-dependent manner (Fig.
4C, D and E, and
4F). Combined treatment with RT and TLC388 led to greater PD-L1 upregulation (Fig.
4G), suggesting that TLC388 may increase susceptibility to ICIs in an ICI-unresponsive MSS-CRC model. Therefore, we treated CT26-bearing BALB/c mice (MSS-CRC) with low-dose TLC388, local radiotherapy, and anti-mouse PD-1 antibodies (Fig.
4H). As shown in Fig.
4I, we found that the response to ICIs alone was unsatisfactory, with a decrease in tumor volume of approximately 30%. In combination with TLC388, there was a significant decreased in tumor volume (2.27±0.49 cm
3 vs. 0.56±0.02 cm
3,
p < 0.001; Fig.
4I and J). Moreover, local radiotherapy also increased the response to ICIs (2.27±0.49 cm
3 vs. 0.64±0.03 cm
3,
p < 0.001; Fig.
4I). Compared with RT alone, RT plus ICIs also led to a 33.3% complete response (2/6) (16.7%, 1/6; Fig.
4I). Furthermore, triple treatment resulted in greater extents of tumor regression and complete response (50%, 3/6; Fig.
4I and J).
Additionally, compared with those in the other groups, the mRNA expression of the proinflammatory cytokines
Ifnα2, Ifnβ1, and
Cxcl10 in the resected tumors was significantly increased in the triple-treatment group, compared to other groups (Fig.
5A and B, and
5C). The recruitment of tumor-infiltrating CD11c
+ DCs and cytotoxic GzmB
+ immune cells was also elevated in the triple-treatment group (Fig.
5D and E, and
5F). These results demonstrated that TLC388 can enhance STING signaling to reshape the tumor microenvironment, leading to a greater antitumor immune response. To comprehensively evaluate the immune cell profiles within the tumor microenvironment, we examined the proportions of dendritic cells (MHC
HiCD11c
+ DCs, CD86
HiCD11c
+ DCs and PD-L1
HiCD11c
+ DCs), T cells (CD4
+ T cells and CD8
+ T cells), effector/memory T cells (CD44
+CD62L
−CD8
+ T
EM cells), cytotoxic T/NK cells (IFNγ
+CD8
+ T cells, IFNγ
+CD49b
+ NK cells) and regulatory T cells (Foxp3
+CD25
+CD4
+ Treg cells). The gating strategies are shown in Fig.
S2. We observed greater infiltration of MHC
HiCD11c
+ DCs and CD86
HiCD11c
+ DCs in the triple-treatment group than in the other groups (Fig.
6A,
S3A,
6B, and
S3B). The density of PD-L1
HiCD11c
+ DCs was increased by RT plus TLC388, implying that PD-L1 expressed on DCs may diminish T cell–mediated cytotoxicity by attenuating T cell activation via an impaired antigen-presenting ability [
43‐
46] (Fig.
6C and
S3C). However, there was no significant increase in the triple-treatment group (Fig.
6C). The density of total tumor-infiltrating CD4
+ and CD8
+ T cells was also increased in the triple-treatment group (Fig.
6D and E, and
S4A). But the increase in expression was not statistically significant compared to that in the other dual treatment groups (Fig.
6D and E, and
S4A). Notably, compared with those in the other groups, the densities of effector/memory CD44
+CD62L
−CD8
+ T
EM cells, cytotoxic IFNγ
+CD8
+ T cells, and cytotoxic IFNγ
+CD49b
+ NK cells were markedly increased in the triple-treatment group, compared to other groups (Fig.
6F,
S4B,
6G, and
6H). The percentage of immunosuppressive Foxp3
+CD25
+CD4
+ Treg cells did not increase in the triple-treatment group (Fig.
6I). Taken together, these results demonstrated that TLC388 significantly reinvigorated cancer immunogenicity, enhancing the recruitment of DCs and functional cytotoxic T cells, thereby improving therapeutic efficacy in poorly immunogenic MSS-CRC.