Calreticulin Fragment 39-272 Promotes B16 Melanoma Malignancy through Myeloid-Derived Suppressor Cells In Vivo

Abstract

Calreticulin (CRT), a multifunctional Ca2+-binding glycoprotein mainly located in the endoplasmic reticulum, is a tumor-associated antigen that has been shown to play protective roles in angiogenesis suppression and anti-tumor immunity. We previously reported that soluble CRT (sCRT) was functionally similar to heat shock proteins or damage-associated molecular patterns in terms of ability to activate myeloid cells and elicit strong inflammatory cytokine production. In the present study, B16 melanoma cell lines expressing recombinant CRT fragment 39-272 (sCRT/39-272) in secreted form (B16-CRT), or recombinant enhanced green fluorescence protein (rEGFP) (B16-EGFP), were constructed for investigation on the roles of sCRT in tumor development. When s.c. inoculated into C57BL/6 mice, the B16-CRT cells were significantly more aggressive (in terms of solid tumor growth rate) than B16-EGFP controls in a TLR4- and myeloid-derived suppressor cells (MDSC)-dependent manner. The B16-CRT-bearing mice showed increased Gr1+ MDSC infiltration in tumor tissues, accelerated proliferation of CD11b+Ly6G+Ly6Clow (G-MDSC) precursors in bone marrow, and higher percentages of G-MDSCs in spleen and blood, which was mirrored by decreased percentage of dendritic cells (DC) in periphery. In in vitro studies, recombinant sCRT/39-272 was able to promote migration and survival of tumor-derived MDSCs via interaction with TLR4, inhibit MDSC differentiation into DC, and also elicit expression of inflammatory proteins S100A8 and S100A9 which are essential for functional maturation and chemotactic migration of MDSCs. Our data provide solid evidence for CRT as a double-edged sword in tumor development.

Immunosuppression by Mutated Calreticulin Released from Malignant Cells

“…Based on the combined results obtained by these experiments, we conclude that extracellular CALR is indeed immunosuppressive. This immunosuppression may also involve shifts in the composition of immune subpopulations (such an increase in MDSC) when CALR becomes elevated at the systemic level, as previously reported (He et al, 2017). At the conceptual level, the aforementioned results support the general notion that oncogenesis must be facilitated by the simultaneous occurrence of two processes, namely (1) the activation of cell-autonomous oncogenic drivers (and the inactivation of oncosuppressors) to create malignant cells and (2) the escape from immunosurveillance by ''hiding'' from the immune system or by active immunosuppression.…”

“…CALR can be digested by proteases such as neutrophil elastase to generate a factor called vasostatin that inhibits angiogenesis (Mans et al, 2012). The systemic injection of CALR fragments into mice can also stimulate the expansion of myeloid-derived suppressor cells (MDSC) (He et al, 2017) or that of immunosuppressive B cells (Hong et al, 2013).…”

Linking cellular stress responses to systemic homeostasis

“…The clinical relevance of this process is supported by the fact that CALR exposure on cancer cells correlates not only with eIF2α phosphorylation but also with prognostically favourable anticancer immune responses 65,66 . Of note, dying cancer cells can also release soluble CALR, which limits the ability of macrophages to present phagocytosed antigens (as a consequence of MHC class II downregulation) 67 and favours the accumulation of myeloidderived suppressor cells (MDSCs) 68 , hence potentially promoting tumour progression. Thus, the net microenvironmental effect of UPR ER -driven CALR signalling may depend on the balance between the surface-bound and soluble variants of the molecule.…”

Dual roles of myeloid‑derived suppressor cells induced by Toll‑like receptor signaling in cancer (Review)

“…MPL induces the accumulation of MDSCs both in vitro and in vivo by inhibiting DC development from myeloid cells ( 57 ). In addition, in a melanoma mouse model, soluble calreticulin (sCRT39-272) was demonstrated to promote the migration and survival of tumor-derived MDSCs via interactions with TLR4 ( 52 ). Notably, Li et al ( 58 ) demonstrated that exogenous S100A4 upregulates TLR4 receptor expression on MSC2 cells and protects MDSCs from apoptosis via the TLR4/extracellular regulated protein kinases (ERK)1/2 signaling axis, both in vitro and in vivo .…”

“…De Veirman et al ( 54 ) demonstrated that S100A9 acts as a chemokine for multiple myeloma (MM) cells and induces MDSCs to express and secrete inflammatory and promyeloma cytokines, including TNF-α, IL-6 and IL-10. In addition, He et al ( 52 ) demonstrated that TLR4 signaling inhibits MDSC differentiation into DCs and promotes their functional maturation, and the chemotactic migration of MDSCs by initiating the expression of S100A8 and S100A9. Recent studies on colorectal cancer ( 53 ) and MM ( 54 ) have demonstrated that S100A9 promotes the expression levels of Arg1, iNOS and IL-10, and ROS production in MDSCs via TLR4-NF-κB signaling cascades, thereby inhibiting CD8 + T cell activity and promoting tumor progression ( 53 ).…”