Background Hypersialylation of cancer cells induces an immunosuppressive microenvironment. The binding of sialic acid by siglec receptors expressed on immune cells initiates a downstream response via immunoreceptor tyrosine-based inhibitory motif (ITIM) signalling. Cancer associated fibroblasts (CAFs) in the colorectal cancer (CRC) microenvironment are highly immunosuppressive and associated with poor survival. The role of sialyation in stromal cell-mediated immunosuppression, however, is unknown. Here, we investigated if sialylation of CAFs contributed to their potent immunosuppressive properties.
Methods Tumour cell secretome (TCS) from multiple CRC cell lines was used to condition primary human and mouse bone marrow-derived stromal cells, as CAF precursors. Normal and CAFs were isolated from colon tumour resections. Stromal cells were cultured with stimulated splenocytes (mouse) or PBMCs (human) and their immunosuppressive properties were assessed by flow cytometry. An in vivo mouse model of CT26 CRC was used to assess the role of highly sialylated stromal cells in tumour development. Mice were injected subcutaneously with CT26 cells alone, or co-injected with TCS-conditioned stromal cells, either control or de-sialylated. 14 days post induction, tumous, draining lymph nodes and spleen were assessed for frequency and expression of T cell activation markers.
Results Tumour conditioning resulted in significantly higher expression of both α2,6-linked sialic acid and specific Siglec ligands on stromal cells. CAFs were significantly more sialylated than stromal cells isolated from adjacent normal associated tissue (NAFs) (figure 1). Following co-culture, CAFs induced significantly higher levels of CD8+ T cells with an exhausted phenotype as determined by TIM-3 and PD-1 expression. Siglec-7 and -9 receptors were induced by CAFs on CD8 T cells. Furthermore, de-sialylation of CAFs, specifically, prior to co-culture resulted in a significant reduction in exhausted CD8+ T cells and attenuation of their immunosuppressive ability (figure 2).
14d post tumour induction in vivo, mice with TCS-conditioned, stromal-dense tumours had significantly fewer activated CD4+ and CD8+ CD25-expressing T cells, both intra-tumourally and distally in the draining lymph nodes and spleen. They also had low levels of cytotoxic granzyme B-expressing CD8+ T cells. Interestingly, this suppression was sialylation-dependent. De-sialylation of TCS-conditioned stromal cells led to restoration of activated T cell levels in tumours and peripheral lymphoid tissues, as well as a marked increase in cytotoxic T cells (figure 3).
Conclusions These results demonstrate, for the first time, that tumour stromal cells suppress activated T cells through sialic acid dependent interactions. We show that targeting stromal cell sialylation may represent a novel immune checkpoint to reactivate anti-tumour immunity.
Ethics Approval The animal study was approved by the Animals Care Research Ethics Committee of the National University of Ireland, Galway (NUIG) and conducted under individual and project authorisation licenses from the Health Products Regulatory Authority (HPRA) of Ireland (AE19125/P077). The study using human samples was approved by University Hospital Galway Ethics committee under an ethically approved protocol (Clinical Research Ethics Committee, Ref: C.A. 2074).
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