The role of sialylation in stromal cell-mediated immunosuppression in the colorectal tumour microenvironment
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Date
2023-04-19Embargo Date
2025-04-18
Author
Egan, Hannah
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Abstract
Immunosuppressive tumour microenvironments (TME) inhibit the effectiveness of many
immunotherapies. The hypersialylation of cancer cells contributes significantly to this
suppressive environment, aiding in tumour immune evasion. Sialic acids are bound by
siglec (sialic acid–binding, immunoglobulin (Ig)-like lectin) receptors expressed by
immune cells such as macrophages and T cells. This binding initiates a downstream
inhibitory signalling response similar to what is seen with PD-1/PD-L1 binding. Cancer
associated fibroblasts, or CAFs, are a highly immunosuppressive cell type found in the
colorectal cancer TME and are associated with tumour progression and a worse
prognosis, however, the mechanisms by which they mediate their immunosuppressive
properties have not yet been fully elucidated.
This PhD aimed to investigated if the sialylation profile of CAFs and tumour-conditioned
stromal cells contribute to their immunosuppressive properties; and if this could provide
a novel target, overcoming immunotherapy resistance commonly associated with
approved immunotherapies.
Colorectal cancer cell line secretome, +/- an inflammatory TNF-α stimulus, was
generated and used to condition primary BALB/c bone marrow mesenchymal stromal
cells (BM-MSCs). BM-MSCs recruited into the colorectal cancer TME are known to be
precursors to CAFs, and our model reflects this recruitment process. Intestinal stromal
cells were isolated from primary colorectal cancer patient resections and were termed
cancer associated fibroblasts (CAFs) or normal associated fibroblasts (NAFs) based on
their location relative to the primary tumour. The sialylation profile of these cells were
assessed through the use of lectins and Fc chimeras staining on flow cytometry and an
elevated level of α2,6 and α2,3 linked sialic acid was confirmed in both tumour conditioned mouse stromal cells and CAFs compared to controls. BALB/c BM-MSCs
conditioned with tumour secretome had elevated expression of both α 2,6 linked sialic
acids and siglec-E ligands. When these same cells were cultured with inflammatory
tumour secretome, there was a significant increase in α2,3 linked sialic acid. In primary intestinal stromal cells, CAFs had significantly higher levels of α2,6 linked sialic acids and
siglec-9 ligands compared to patient matched NAFs. Following this, the use of a
sialyltransferase inhibitor, P-3FAX-Neu5Ac, was shown to significantly reduce cell
surface sialic acid expression on stromal cells +/- tumour conditioning, without affecting
cell size or viability. Interestingly, the use of the sialyltransferase inhibitor could
significantly increase the detection of PD-L1 by both flow cytometry and western
blotting, suggesting the PD-L1 may be sialylated.
Stromal cells +/-tumour secretome conditioning were cultured with healthy splenocytes
(mouse) or PBMCs (human) and their immunosuppressive properties were assessed by
flow cytometry. CAFs significantly induced a more exhausted and immunomodulatory T
cell phenotype, highlighted by increased expression of exhaustion markers, PD-1, TIM-3
and LAG-3, and immunomodulatory receptors Siglec-7 and -9 when compared with
NAFs. To elucidate the role of sialylation on CAF-mediated immunosuppression, NAFs
and CAFs were treated with the sialyltransferase inhibitor (SI) P-3FAX-Neu5Ac prior to
co-culture. Reduction of sialic acid expression on NAFs/CAFs was confirmed by flow
cytometry and the SI-treated NAFs/CAFs were then co-cultured with allogeneic T cells
to assess the functional consequences of reduced NAF/CAF sialylation. SI-treated CAFs
induced significantly less CD4+TIM-3
+ and both CD4+
LAG-3
+ and CD8+
LAG-3
+ T cells
compared to their untreated counterparts. Interestingly, SI-treated CAFs also induced
significantly less Siglec-7 and -9 receptor-expressing CD8+ T cells.
An in vivo mouse model of CT26 colorectal cancer was designed to assess the role of
highly sialylated stromal cells. Mice were subcutaneously injected with CT26 cells and
conditioned stromal cells, where the stromal cells had undergone prior desialylation in
the appropriate groups. Mice with highly stromal dense tumours had significantly
supressed T cells, with supressed CD25 and granzyme B expression, both
intratumourally and distally in the spleen and draining lymph node. This suppression
was sialylation dependent, with complete loss of suppression in tumours with
desialylated stromal cells. Macrophages and NK cells in the tumour, draining lymph
nodes and spleens expressed significantly less siglec-G in mice which received co-
injection of CT26 cells and tumour-conditioned MSCs, which was dependent on the
stromal cells’ sialylation profile.
These results demonstrate that stromal cells in the tumour-microenvironment can
regulate immune cells in a sialylation dependent manner and that their
immunosuppressive effects may be reversed through the targeting of sialylation on the
stromal cell surface.