Immune checkpoint inhibitors have shown clinical benefit in several cancer entities including metastatic microsatellite instable colorectal carcinomas. However, for the majority of metastatic colorectal carcinomas the potential and limitations of immune checkpoint inhibition is not fully understood. In this study, the effects of sole and dual CTLA-4 and PD-L1 blockade were investigated in a microsatellite stable highly aggressive orthotopic mouse model of colon cancer. Dual CTLA-4 and PD-L1 inhibition resulted in tumor growth stagnation and completely blocked liver metastasis. Sole CTLA-4 and PD-L1 inhibition only moderately reduced metastatic spread of the colon cancer cells, though CTLA-4 blockade being superior to PD-L1 inhibition. Dual immune checkpoint blockade and sole CTLA-4 inhibition significantly increased intratumoral CD8+ and CD4+ T cells and reduced FOXP3+/CD4+ Treg cells. This was associated with increased expression levels of the pro-inflammatory Th1/M1-related cytokines IFN-γ, IL-1α, IL-2, and IL-12. Moreover, tumors treated with combined immune checkpoint blockade showed the strongest increase in intratumoral iNOS+ macrophages, reduction of PD-L1+ and Tie2+ macrophages and the lowest expression of M2/Th2-related IL-4, TARC and COX-2. The assessment of further microenvironmental changes by DCE-MRI and immunohistology revealed no alterations in functional tumor vascularization upon combined immune checkpoint blockade, but a significant increase in intratumoral fibroblasts and collagen I deposition. Thus, the synergistic inhibitory effects of dual immune checkpoint inhibition can be explained by anti-tumorigenic T cell responses mediated by CTLA-4 inhibition and M1 macrophage polarization predominantly induced by PD-L1 blockade. This was accompanied by pronounced fibroblast activation highlighting the interconnection between immunogenicity and desmoplasia.

免疫检查点抑制剂已在包括转移性微卫星不稳定结直肠癌在内的多种癌症中显示出临床益处。然而，对于大多数转移性结直肠癌，免疫检查点抑制的潜力和局限性尚未完全了解。在这项研究中，在结肠癌的微卫星稳定高侵袭性原位小鼠模型中研究了单一和双重CTLA-4和PD-L1阻滞的作用。双重抑制CTLA-4和PD-L1导致肿瘤生长停滞并完全阻断肝转移。单独的CTLA-4和PD-L1抑制作用仅适度降低了结肠癌细胞的转移扩散，尽管CTLA-4阻断作用优于PD-L1抑制作用。双重免疫检查点封锁和唯一的CTLA-4抑制作用显着增加了肿瘤内CD8 +和CD4 + T细胞并减少了FOXP3 + / CD4 + Treg细胞。这与促炎性Th1 / M1相关细胞因子IFN-γ，IL-1α，IL-2和IL-12的表达水平升高有关。此外，用联合免疫检查点阻断治疗的肿瘤在肿瘤内iNOS +巨噬细胞的增加最强，PD-L1 +和Tie2 +巨噬细胞的减少最强，M2 / Th2相关的IL-4，TARC和COX-2的最低表达。通过DCE-MRI和免疫组织学对进一步微环境变化的评估显示，在联合免疫检查点封锁后，功能性肿瘤血管化没有改变，但肿瘤内成纤维细胞和胶原蛋白I沉积显着增加。因此，双重免疫检查点抑制的协同抑制作用可以通过CTLA-4抑制和主要由PD-L1阻断引起的M1巨噬细胞极化介导的抗致瘤性T细胞应答来解释。这伴随着明显的成纤维细胞活化，突出了免疫原性和增生之间的相互关系。