Proteolytic cleavage of IgG hinge in the tumor microenvironment associated with impairment of antitumor immunity
Introduction
The tumor microenvironment contains various stromal cells, including immune cells, cancer-associated fibroblasts, and endothelial cells. Our previous studies have shown that the single-hinge cleaved antibodies (scIgGs) have impaired Fc and cause cancer immune evasion. However, little is known about how the scIgGs can impact the outcomes of cancer patients in the clinic. In this study, we analyzed the levels of scIgGs in tumor tissues from a large cohort of breast cancer patients (n=547). We demonstrated a significant association between the levels of tumor scIgGs and multiple poor prognostic markers such as tumor grade, lymph node metastasis, and cancer recurrence. Using 4T1 and 4T1/IdeS mouse tumor models, we revealed that scIgGs in tumor tissues (4T1/IdeS) showed more tumor growth than the control 4T1 tumors. The scIgG tumors showed immune suppressive microenvironment as evidenced in reduced neutrophil and mast cell infiltration, and cytokine production by transcriptome analysis using RNAseq analysis.
Methods
In this study, we examined the antibody single-hinge cleavage in human breast tumor tissues as detected by IHC. To determine the roles of elevated scIgGs in tumor progression, we established a high scIgG containing allograft 4T1 tumor model and compared it with the counterpart control tumors. Also, we detected the infiltrated immune cells by flow cytometry. The transcriptomes of 4T1-IdeS tumors and control tumors were analyzed using total RNA-sequencing technology (MD Anderson Core). Digital cytometry was performed from bulk RNA-seq data using CIBERSORTx. We determined levels of cytokine/chemokines in tumor lysates using a reverse-phase protein array (Ray Bio).
Results
IHC in human breast tumors demonstrated that positive scIgG staining in tumor tissues had a significant association with lymph node metastasis, tumor grade, and cancer recurrence (Figure 1A, B, and C). Production of Fc-impaired scIgGs by constitutive expression of IdeS in mouse tumor models has been well documented (1-4). We detected increased tumor growth and tumor weight in scIgG-containing tumors in the syngeneic allograft 4T1-IdeS tumor model (Figure 2A and B), as has been reported for other tumor models (4). In order to determine if the presence of tumor scIgGs impacted the tumor microenvironment, we analyzed immune cell infiltration and inflammatory cytokine production. We found that the tumors with elevated levels of scIgGs in 4T1-IdeS tumors showed significantly decreased in macrophage population in comparison with that in the counterpart control tumors (Figure 3A). RNA-seq analysis indicated that 4T1-IdeS and control tumors displayed differential gene expression (Figure 3B). Gene ontology analysis showed that the genes downregulated in 4T1-IdeS tumors participate in multiple pro-inflammatory pathways (Figure 3C), including TNF signaling and cytokine-cytokine receptor interaction pathways (Figure 3D and E). Deconvolution of RNA-seq data by digital cytometry showed that increased single-hinge cleavage is associated with reduced neutrophil infiltration and of mast cell activation (Figure 4A). Analysis of clinical samples from the TCGA cohort showed that breast cancer patients with a reduced proportion of activated mast cells have shorter overall survival (Figure 4B). Finally, we confirmed the levels of cytokines expression by RPPA. Among the 62 proteins quantified, large portions of cytokine/chemokines were reduced in scIgG-containing tumors when compared with their counterpart controls (Figure 5A). We found significantly reduced levels of IFN-g and IL-1-beta in scIgG-containing tumors (Figure 5 B and C).
Conclusion
Taken together, these findings provided strong evidence for a new mechanism of cancer immune evasion involving Fc-impaired scIgGs and shed light on a potential new immune-therapeutic strategy by activating macrophages, up-regulating cytokine signaling pathways and enhancing cross-talk between humoral and cellular immunity.