Suppressor Cells In Tumor-bearing Mice Research Articles

A single cell sequencing approach to understand the nature of myeloid-derived suppressor cells in tumor-bearing mice

Abstract Peripheral T Cell Lymphoma (PTCL), a type of non-Hodgkin’s lymphoma, is a heterogeneous group of mature T cells that have undergone transformation into a malignant form. The tumor microenvironment (TME) is very heterogeneous and is comprised of different cell types. Myeloid-derived suppressor cells (MDSC) are one of the vital components of the TME. Further, MDSC are associated with tumor growth, angiogenesis and enhanced metastasis. The aim of this study was to compare MSDCs isolated from TME and splenic MDSCs from tumor-bearing hosts (TBHs) as well as splenic MDSCs from naïve mice by using single cell RNA sequencing (scRNASeq). For this reason, C57BL/6 mice were injected with 1×106 EL4 cancer cells subcutaneously to induce tumor growth and 14 days later, cells were isolated from these three tissues, and flow cytometry and scRNASeq were performed. The flow cytometry results showed an increase in MDSC recruitment in the spleens from TBHs in comparison with spleens from naïve mice. scRNASeq analysis showed most of the genes upregulated in MDSCs from TME and spleen of TBHs were responsible for generation, recruitment and proliferation of MDSCs (Lgals5, CXCL10, Alox5ap, CCL6, IL1b, S100A8/A9/A11, CCL4, CCL5). Furthermore, we found several genes overexpressed in MDSCs promoted the proliferation and migration of cancer cells as well as tumor formation (Lyz2, Plac8, Ifitm3, Vim, S100A6, Crip1). In addition, we found some genes have anti-tumor effects by suppressing T and B cells (Lgals5, Ly6c2). In summary, these studies demonstrated that MDSCs from TME and spleens of TBHs have unique features for inducing immunosuppression, as well as recruitment and expansion of MDSCs in TBHs, which make these cells critical for target for immunotherapy.

Astaxanthin Treatment Induces Maturation and Functional Change of Myeloid-Derived Suppressor Cells in Tumor-Bearing Mice.

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells which accumulate in stress conditions such as infection and tumor. Astaxanthin (ATX) is a well-known antioxidant agent and has a little toxicity. It has been reported that ATX treatment induces antitumor effects via regulation of cell signaling pathways, including nuclear factor erythroid-derived 2-related factor 2 (Nrf2) signaling. In the present study, we hypothesized that treatment with ATX might induce maturation of MDSCs and modulate their immunosuppressive activity. Both in vivo and in vitro treatment with ATX resulted in up-regulation of surface markers such as CD80, MHC class II, and CD11c on both polymorphonuclear (PMN)-MDSCs and mononuclear (Mo)-MDSCs. Expression levels of functional mediators involved in immune suppression were significantly reduced, whereas mRNA levels of Nrf2 target genes were increased in ATX-treated MDSCs. In addition, ATX was found to have antioxidant activity reducing reactive oxygen species level in MDSCs. Finally, ATX-treated MDSCs were immunogenic enough to induce cytotoxic T lymphocyte response and contributed to the inhibition of tumor growth. This demonstrates the role of ATX as a regulator of the immunosuppressive tumor environment through induction of differentiation and functional conversion of MDSCs.

Metformin inhibits the function of granulocytic myeloid-derived suppressor cells in tumor-bearing mice

Metformin, an oral medicine broadly used for the treatment of type 2 diabetes, has been found to significantly improve tumor incidence and survival in large-scale clinical analysis. In recent years, the antitumor effect and mechanism of metformin have received much attention. Myeloid-derived suppressor cells (MDSCs), a major immunosuppressive cell type that accumulates in tumor-bearing hosts, can inhibit T cells and promote tumor immune escape. The mechanism by which metformin exerts its anti-tumor effect by regulating MDSCs remains unclear. Here, we found that metformin could inhibit the accumulation and suppressive capacity of G-MDSCs, delay tumor progression and elicit Th1 and CTL responses in murine colon cancer CT-26 cell-transplanted mice. In additionally, metformin could enhance the phosphorylation of AMPK, reduce STAT3 phosphorylation levels, and down-regulate the inhibitory function of G-MDSCs in vitro. These results suggest that metformin may be a potential clinical benefit for antitumor immunotherapy in tumor-bearing mice.

Long noncoding RNA Pvt1 regulates the immunosuppression activity of granulocytic myeloid-derived suppressor cells in tumor-bearing mice

BackgroundMyeloid-derived suppressor cells (MDSCs) participate in tumor-elicited immunosuppression by dramatically blocking T-cell-induced antitumor responses, thereby influencing the effectiveness of cancer immunotherapies. Treatments that alter the differentiation and function of MDSCs can partially restore antitumor immune responses. The long noncoding RNA plasmacytoma variant translocation 1 (lncRNA Pvt1) is a potential oncogene in a variety of cancer types. However, whether lncRNA Pvt1 is involved in the regulation of MDSCs has not been thoroughly elucidated to date.MethodsMDSCs or granulocytic MDSCs (G-MDSCs) were isolated by microbeads and flow cytometry. Bone marrow derived G-MDSCs were induced by IL-6 and GM-CSF. The expression of lncRNA Pvt1 was measured by qRT-PCR. Specific siRNA was used to knockdown the expression of lncRNA Pvt1 in G-MDSCs.ResultsIn this study, we found that knockdown of lncRNA Pvt1 significantly inhibited the immunosuppressive function of G-MDSCs in vitro. Additionally, lncRNA Pvt1 knockdown reduced the ability of G-MDSCs to delay tumor progression in tumor-bearing mice in vivo. Notably, lncRNA Pvt1 was upregulated by HIF-1α under hypoxia in G-MDSCs.ConclusionsTaken together, our results demonstrate a critical role for lncRNA Pvt1 in regulating the immunosuppression activity of G-MDSCs, and lncRNA Pvt1 might thus be a potential antitumor immunotherapy target.

Correction: CpG Blocks Immunosuppression by Myeloid-Derived Suppressor Cells in Tumor-Bearing Mice.

Correction: CpG Blocks Immunosuppression by Myeloid-Derived Suppressor Cells in Tumor-Bearing Mice.

Baccatin III, a precursor for the semisynthesis of paclitaxel, inhibits the accumulation and suppressive activity of myeloid-derived suppressor cells in tumor-bearing mice.

Myeloid-derived suppressor cells (MDSCs) mediate tumor-associated immune suppression in both cancer patients and tumor-bearing animals. Reduction or elimination of MDSCs reduces the rate of tumor progression and improves cancer therapies that employ mechanisms of immunity. Here we show that baccatin III, which is the precursor for the semisynthesis of paclitaxel, exerts anti-tumor immunomodulatory activity in very low doses (0.05-0.5mg/kg), although it is regarded as an inactive derivative of paclitaxel. Oral administration of baccatin III significantly reduced the growth of tumors induced by engrafting BALB/c mice with either 4 T1 mammary carcinoma or CT26 colon cancer cells. Baccatin III (0.5mg/kg) did not exert anti-tumor activity in athymic nude mice. Baccatin III decreased the accumulation of MDSCs in the spleens of the tumor-bearing mice. Furthermore, MDSCs isolated from baccatin III-treated mice, compared with those isolated from vehicle-treated mice, had a significantly reduced suppressive effect on T cells treated with the anti-CD3 and anti-CD28 monoclonal antibodies. Moreover, these cells produced significantly reduced amounts of reactive oxygen species and nitric oxide. These results suggest that baccatin III reduced tumor progression by inhibiting the accumulation and suppressive function of MDSCs.

Generation of a new therapeutic peptide that depletes myeloid-derived suppressor cells in tumor-bearing mice

Cancer immune evasion is an emerging hallmark of disease progression. Functional studies to understand the role of myeloid-derived suppressor cells (MDSC) in the tumor microenvironment however, are limited by the lack of available specific cell surface markers. We adapted a competitive peptide phage display platform to identify candidate peptides binding MDSC specifically and generated peptide-Fc fusion proteins (peptibody). In multiple tumor models peptibody injection iv completely depleted blood, splenic, and intratumoral MDSC in tumor-bearing mice, without affecting proinflammatory immune cell types, such as dendritic cells. While control Gr-1 antibody depleted primarily granulocytic MDSC, peptibodies depleted both granulocytic and monocytic subsets. Remarkably, peptibody treatment was associated with inhibition of tumor growth in vivo, which was superior to Gr-1. Immunoprecipitation of MDSC membrane proteins identified S100 family proteins as candidate targets. Our strategy may be useful to identify novel diagnostic and therapeutic surface targets on rare cell subtypes, including human MDSC.

651. Oncolytic Reovirus Induces Reduction in Immunosuppressive Activity of Myeloid-Derived Suppressor Cells in Tumor-Bearing Mice

651. Oncolytic Reovirus Induces Reduction in Immunosuppressive Activity of Myeloid-Derived Suppressor Cells in Tumor-Bearing Mice

Anti-Gr-1 antibody depletion fails to eliminate hepatic myeloid-derived suppressor cells in tumor-bearing mice.

Recent studies show that the liver is a preferred organ for the accumulation of MDSC. In this study, we examined the effect of systemic RB6-8C5 treatment on hepatic MDSC in tumor-bearing mice. EL4 tumor-bearing mice were injected i.p. with RB6-8C5, and hepatic, splenic, and blood MDSCs were analyzed by flow cytometry. Unexpectedly, hepatic MDSC remained in the liver, although RB6-8C5 completely eliminated them from the spleen and peripheral blood 24 h after treatment. Secondary antibody staining confirmed the presence of RB6-8C5-bound MDSC in the liver of mice with s.c. tumors. Similar observations were made in two other (colon and melanoma) tumor models. Whereas RB6-8C5 injection induced cell death of hepatic MDSC, as shown by Annexin V/7-AAD staining, these cells were replaced immediately, leading to a constant, increased frequency of hepatic MDSC. Adoptively transferred MDSC migrated preferentially to the liver after RB6-8C5 treatment, suggesting that hepatic MDSCs are reconstituted rapidly after depletion. Finally, hepatic MDSC remained immunosuppressive despite RB6-8C5 injection. Our study demonstrates that RB6-8C5 is not suitable for depletion of hepatic MDSCs and analysis of their function.

IFN-γ producing T cells contribute to the increase of myeloid derived suppressor cells in tumor-bearing mice after cyclophosphamide treatment

It has been reported that treatment with cyclophosphamide (CTX) as a chemotherapeutic drug in cancer patients or tumor-bearing mice can result in an increase in the proportion of myeloid derived suppressor cells (MDSCs) in blood and lymphoid organs. Here we sought to clarify the possible mechanism of this unwanted increase in proportion of MDSCs in tumor-bearing mice after CTX treatment. We found that both CD4+ T cells and CD8+ T cells underwent an expansion and activation before the increase of MDSCs in the early period of CTX treatment in 4T1 breast tumor-bearing mice. The proportion of MDSCs in nude mice lacking T cells after CTX therapy was comparable to that in nude mice without CTX treatment. T cell transfer to 4T1-bearing nude mice enhanced the proportion of MDSCs in tumor-bearing mice after CTX therapy. The co-culture of MDSCs and T cells in vitro also showed that CD4+ T cells and CD8+ T cells could facilitate the expansion and survival of MDSCs, and this effect was mediated by IFN-γ released by T cells. These results gave an explanation of the unwanted consequence resulted from CTX treatment in tumor-bearing mice. It also provided some insights into the strategies for eliminating the bad side of CTX treatment and to make it more effective in cancer therapy.

Characterization of the nature of granulocytic myeloid-derived suppressor cells in tumor-bearing mice

MDSCs are a group of cells with potent immune-suppressive activity. These cells accumulate in many pathologic conditions and play a major role in the regulation of immune responses. The nature of MDSC remains highly debatable. In cancer, most MDSCs are represented by cells with granulocytic phenotype and morphology, G-MDSC. The relationship between G-MDSCs and Neu remains unclear. In this study, we have found that G-MDSCs, from tumor-bearing, and Neu, from tumor-free, mice share a common morphology and phenotype. However, in contrast to Neu, a substantial proportion of G-MDSCs expressed M-CSFR and a CD244 molecule. Neu had significantly higher phagocytic activity, expression of lysosomal proteins, and TNF-α than corresponding G-MDSCs, which had significantly higher activity of arginase, MPO, and ROS. In contrast to G-MDSC, neither rested nor mobilized Neu suppressed T cells. G-MDSC survived 2 days in culture in the presence of GM-CSF and within 24 h, became phenotypic and functionally similar to Neu. Tumor-associated G-MDSC shared most characteristics of splenic G-MDSC, rather then Neu. These data suggest that in cancer, despite morphological and phenotypic similarities, G-MDSCs are functionally distinct from Neu and are comprised of pathologically activated precursors of Neu.

CpG Blocks Immunosuppression by Myeloid-Derived Suppressor Cells in Tumor-Bearing Mice

The Toll-like receptor (TLR) 9 ligand CpG has been used successfully for the immunotherapy of cancer. Chronic CpG application in tumor-free hosts leads, however, to the expansion of myeloid-derived suppressor cells (MDSC), which can cause T-cell suppression and may thus hamper the development of an effective immune response. Here, we investigated the effect of TLR9 activation on the function of MDSC in tumor-bearing mice. We investigated the effect of CpG treatment on the number, phenotype, and function of MDSC in mice bearing subcutaneous C26 tumors and in CEA424-TAg mice bearing autochthonous gastric tumors. CpG treatment blocks the suppressive activity of MDSC on T-cell proliferation in both tumor models. Inhibition of MDSC function by CpG was particularly pronounced for a highly suppressive Ly6G(hi) polymorphonuclear subset of MDSC. We further show that TLR9 activation by CpG promotes maturation and differentiation of MDSC and strongly decreases the proportion of Ly6G(hi) MDSC in both tumor-bearing and tumor-free mice. We demonstrate that IFN-α produced by plasmacytoid dendritic cells upon CpG stimulation is a key effector for the induction of MDSC maturation in vitro and show that treatment of mice with recombinant IFN-α is sufficient to block MDSC suppressivity. We show here for the first time that TLR9 activation inhibits the regulatory function of MDSC in tumor-bearing mice and define a role for the antitumoral cytokine IFN-α in this process.

Subsets of Myeloid-Derived Suppressor Cells in Tumor-Bearing Mice

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of cells that play a critical role in tumor associated immune suppression. In an attempt to identify a specific subset of MDSC primarily responsible for immunosuppressive features of these cells, 10 different tumor models were investigated. All models showed variable but significant increase in the population of MDSC. Variability of MDSC expansion in vivo matched closely the effect of tumor cell condition medium in vitro. MDSC consists of two major subsets of Ly6G(+)Ly6C(low) granulocytic and Ly6G(-)Ly6C(high) monocytic cells. Granulocytic MDSC have increased level of reactive oxygen species and undetectable level of NO whereas monocytic MDSC had increased level of NO but undetectable levels of reactive oxygen species. However, their suppressive activity per cell basis was comparable. Almost all tumor models demonstrated a preferential expansion of granulocytic subset of MDSC. We performed a phenotypical and functional analysis of several surface molecules previously suggested to be involved in MDSC-mediated suppression of T cells: CD115, CD124, CD80, PD-L1, and PD-L2. Although substantial proportion of MDSC expressed those molecules no differences in the level of their expression or the proportion, positive cells were found between MDSC and cells from tumor-free mice that lack immune suppressive activity. The level of MDSC-mediated T cell suppression did not depend on the expression of these molecules. These data indicate that suppressive features of MDSC is caused not by expansion of a specific subset but more likely represent a functional state of these cells.

Chemokine-mediated rapid turnover of myeloid-derived suppressor cells in tumor-bearing mice

AbstractTumor growth is associated with aberrant myelopoiesis, including the accumulation of CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) that have the potential to promote tumor growth. However, the identity, growth, and migration of tumor-associated MDSCs remain undefined. We demonstrate herein that MDSCs at tumor site were composed primarily of bone marrow-derived CD11b+Gr-1hiLy-6Cint neutrophils and CD11b+Gr-1int/dullLy-6Chi macrophages. Unexpectedly, in vivo bromodeoxyuridine (BrdU) labeling and parabiosis experiments revealed that tumor-infiltrating macrophages were replenished more rapidly than neutrophils. CCR2 deficiency caused striking conversion of infiltrating cellular dominance from macrophages to neutrophils in the tumor with the excessive production of CXCR2 ligands and granulocyte-colony stimulating factor in the tumor without affecting tumor growth. Overall, our data established the identity and dynamics of MDSCs in a tumor-bearing host mediated by chemokines and elucidated unexpected effects of the paucity of macrophages on tumor development.

Biphasic development of splenic suppressor cells in tumor-bearing mice

The splenic lymphocytes of A/J mice bearing anaplastic carcinoma (15091 A) exhibit a biphasic response to Concanavalin A-stimulated DNA synthesis during the progressive growth of tumors. In this paper we attribute the changing lymphocyte responses to Con A to the biphasic development of splenic suppressor cells in tumor-bearing mice. Anaplastic carcinoma in syngeneic A/J mice offers an ideal tumor model to study the functional relationship of suppressor cells to other parameters of immune response such as T-cell cytotoxicity.