Most circulating tumor cells (CTC) are detected as single cells, whereas a small proportion of CTCs in multicellular clusters with stemness properties possess 20- to 100-times higher metastatic propensity than the single cells. Here we report that CTC dynamics in both singles and clusters in response to therapies predict overall survival for breast cancer. Chemotherapy-evasive CTC clusters are relatively quiescent with a specific loss of ST6GAL1-catalyzed α2,6-sialylation in glycoproteins. Dynamic hyposialylation in CTCs or deficiency of ST6GAL1 promotes cluster formation for metastatic seeding and enables cellular quiescence to evade paclitaxel treatment in breast cancer. Glycoproteomic analysis reveals newly identified protein substrates of ST6GAL1, such as adhesion or stemness markers PODXL, ICAM1, ECE1, ALCAM1, CD97, and CD44, contributing to CTC clustering (aggregation) and metastatic seeding. As a proof of concept, neutralizing antibodies against one newly identified contributor, PODXL, inhibit CTC cluster formation and lung metastasis associated with paclitaxel treatment for triple-negative breast cancer.
Significance: This study discovers that dynamic loss of terminal sialylation in glycoproteins of CTC clusters contributes to the fate of cellular dormancy, advantageous evasion to chemotherapy, and enhanced metastatic seeding. It identifies PODXL as a glycoprotein substrate of ST6GAL1 and a candidate target to counter chemoevasion-associated metastasis of quiescent tumor cells. This article is featured in Selected Articles from This Issue, p. 1949.
The IgG antibody class forms an important basis of the humoral immune response, conferring reciprocal protection from both pathogens and autoimmunity. IgG function is determined by the IgG subclass, as defined by the heavy chain, as well as the glycan composition at N297, the conserved site of N-glycosylation within the Fc domain. For example, lack of core fucose promotes increased antibody-dependent cellular cytotoxicity, whereas α2,6-linked sialylation by the enzyme ST6Gal1 helps to drive immune quiescence. Despite the immunological significance of these carbohydrates, little is known about how IgG glycan composition is regulated. We previously reported that mice with ST6Gal1-deficient B cells have unaltered IgG sialylation. Likewise, ST6Gal1 released into the plasma by hepatocytes does not significantly impact overall IgG sialylation. Since IgG and ST6Gal1 have independently been shown to exist in platelet granules, it was possible that platelet granules could serve as a B cell-extrinsic site for IgG sialylation. To address this hypothesis, we used a platelet factor 4 (Pf4)-Cre mouse to delete ST6Gal1 in megakaryocytes and platelets alone or in combination with an albumin-Cre mouse to also remove it from hepatocytes and the plasma. The resulting mouse strains were viable and had no overt pathological phenotype. We also found that despite targeted ablation of ST6Gal1, no change in IgG sialylation was apparent. Together with our prior findings, we can conclude that in mice, neither B cells, the plasma, nor platelets have a substantial role in homeostatic IgG sialylation.
The intestinal mucosa is continually exposed to diverse microbial and dietary Ags, requiring coordinated efforts by specialized populations of regulatory T cells (Tregs) to maintain homeostasis. Suppressive mechanisms used by intestinal Tregs include the secretion of anti-inflammatory cytokines such as IL-10 and TGF-β. Defects in IL-10 signaling are associated with severe infantile enterocolitis in humans, and mice deficient in IL-10 or its receptors develop spontaneous colitis. To determine the requirement of Foxp3+ Treg-specific IL-10 for protection against colitis, we generated Foxp3-specific IL-10 knockout (KO) mice (IL-10 conditional KO [cKO] mice). Colonic Foxp3+ Tregs isolated from IL-10cKO mice showed impaired ex vivo suppressive function, although IL-10cKO mice maintained normal body weights and developed only mild inflammation over 30 wk of age (in contrast to severe colitis in global IL-10KO mice). Protection from colitis in IL-10cKO mice was associated with an expanded population of IL-10-producing type 1 Tregs (Tr1, CD4+Foxp3-) in the colonic lamina propria that produced more IL-10 on a per-cell basis compared with wild-type intestinal Tr1 cells. Collectively, our findings reveal a role for Tr1 cells in the gut, as they expand to fill a tolerogenic niche in conditions of suboptimal Foxp3+ Treg-mediated suppression and provide functional protection against experimental colitis.
IgG is a key mediator of immune responses throughout the human body, and the structure of the conserved glycan on the Fc region has been identified as a key inflammatory switch regulating its downstream effects. In particular, the absence of terminal sialic acid has been shown to increase the affinity of IgG for activating Fc receptors, cascading the inflammatory response in a variety of diseases and conditions. Previously, we have shown that IgG sialylation is mediated by B cell-extrinsic processes. Here, we show that the FcRn-mediated recycling pathway within endothelial cells is a critical modulator of IgG sialylation. Building a deeper understanding of how IgG sialylation is regulated will drive the development of novel therapeutics which dynamically tune IgG functionality in vivo.
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