Oscar Palomares¹ and José Luis Subiza²

¹Department of Biochemistry and Molecular Biology, School of Chemistry, Complutense University, Madrid, Spain ²Inmunotek, Alcalá de Henares, Madrid, Spain

 Nowadays, it is well-accepted the essential role of the immune system in both tumor rejection and tumor progression¹. Effective host antitumor responses are challenged by a plethora of different tumor escape mechanisms^2-4. In this regard, compelling experimental evidence demonstrated that functional regulatory T cells (Tregs), which constitute a heterogeneous group of T cells with potent suppressive capacity, are abnormally expanded in cancer and greatly increased within the tumor tissues, thus playing a key role in tumor development and progression and posing a major barrier to anti-cancer immunity^{3, 5}.

The mechanisms by which Tregs are fueled in hosts bearing malignant tumors and the contribution of the tumor microenvironment (TME) to these processes are not yet fully understood^{1-3, 6}. Dendritic cells (DCs) are professional antigen-presenting cells that are crucial in the orchestration of proper immune responses by linking innate and adaptive immunity⁷. The ability of mature DCs infiltrating tumors to promote highly suppressive Tregs is strongly influenced by exogenous signals encountered in the TME⁷. However, how DCs integrate these signals and the functional resulting outcomes remains elusive.

Malignant transformation is accompanied by changes in tumor cell surface glycoconjugates⁸, including the aberrant or incomplete glycosylation of heavily glycosylated structures such as mucins and proteoglycans⁹. These structures are often overexpressed and shed from tumor cells, having a strong influence on tumor progression by modulating cell adhesion, neo-angiogenesis and metastases¹⁰. In addition, tumor-associated carbohydrates may inhibit anti-cancer immunity through different mechanisms¹¹, including the activation of highly suppressive Tregs. Whether tumor-derived carbohydrate structures encountered by DCs might condition their capacity to polarize functional Tregs, the underlying molecular mechanisms and the potential clinical implications in tumor development, progression and anti-cancer treatments remain incompletely understood.

Murine Ehrlich's tumor (ET) cells, originally derived from a spontaneous mammary carcinoma, lack of H-2 antigens and can grow either in ascitic or solid form in almost any mouse strain across different antigenic backgrounds, thus representing a suitable model to uncover potential novel tumor escape mechanisms ¹². Ca10 is a highly glycosylated ET cell surface macromolecule defined by the binding of monoclonal antibody (mAb) A10^13,14. This antibody specifically recognizes a carbohydrate epitope on the Ca10 macromolecule. Ca10 is spontaneously released from ET cells and can be detected in the serum of ET-bearing mice¹⁴. Although A10 also reacts with glycan moieties located in some human cancer mucins¹⁵, the structural features of the carbohydrate moiety contained in Ca10 and its potential functional implications in cancer remains largely unknown.

In this study, we reveal that Ca10 derived from ET cells is mainly composed of heparan sulfate-related structures that promotes the generation of tolerogenic human DCs, which are able to produce highly suppressive FOXP3⁺ Tregs by mechanisms partially depending on PD-L1, IL-10, IDO, mTOR pathway and glycolysis. In vivo, we observed positive correlations between the serum level of Ca10, the tumor size and the generation of splenic Tregs in mice bearing solid ET. Remarkably, the administration of Ca10 to tumor-free mice also increased the frequency of splenic Tregs, which was fully impaired by the mAb A10. It is worthy to highlighting that in this study we also provide evidence supporting the presence of a circulating human counterpart and show, for the first time, that serum levels of Ca10H are increased in patients suffering from different cancer types compared to healthy individuals. Of note, in prostate cancer patients, these levels are higher in those with bone metastases.

In summary, in this work we demonstrated the involvement of a tumor-associated heparan sulfate structure in the induction of tolerogenic responses in cancer, which might well play an important role in the mechanisms of tumor escape and progression. The discovery of this novel structural-functional relationship might well pave the way for the identification of potential novel tumor biomarkers and for the improvement or development of alternative therapeutic strategies in cancer.

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