Cancer Letters

Cancer Letters

Volume 371, Issue 1, 1 February 2016, Pages 117-124
Cancer Letters

Original Articles
Bone marrow PMN-MDSCs and neutrophils are functionally similar in protection of multiple myeloma from chemotherapy

https://doi.org/10.1016/j.canlet.2015.10.040Get rights and content

Highlights

  • Myeloid cells play a critical role in regulation of myeloma growth.

  • PMN-MDSCs and neutrophils equally protect myeloma cells from chemotherapy.

  • PMN-MDSCs and neutrophils mediate chemoprotective effect through soluble factors.

Abstract

Multiple myeloma (MM) is an incurable cancer of plasma cells localized preferentially in the bone marrow (BM). Resistance to chemotherapy represents one of the main challenges in MM management. BM microenvironment is known to play a critical role in protection of MM cells from chemotherapeutics; however, mechanisms responsible for this effect are largely unknown. Development of MM is associated with accumulation of myeloid-derived suppressor cells (MDSCs) mostly represented by pathologically activated relatively immature polymorphonuclear neutrophils (PMN-MDSCs). Here, we investigated whether PMN-MDSCs are responsible for BM microenvironment-mediated MM chemoresistance. Using in vivo mouse models allowing manipulation of myeloid cell number, we demonstrated a critical role for myeloid cells in MM growth and chemoresistance. PMN-MDSCs isolated from MM-bearing host are immunosuppressive and thus, functionally distinct from their counterpart in tumor-free host neutrophils. We found, however, that both PMN-MDSCs and neutrophils equally promote MM survival from doxorubicin and melphalan and that this effect is mediated by soluble factors rather than direct cell–cell contact. Our data indicate that targeting PMN-MDSCs would enhance chemotherapy efficacy in MM.

Introduction

Multiple myeloma (MM) is an incurable hematological malignancy characterized by a clonal proliferation of plasma cells that accumulate preferentially in the bone marrow (BM). One of the main challenges in this disease is chemotherapy resistance. The tumor microenvironment is now recognized as one of the leading factors that promotes chemoresistance; however, mechanisms responsible for this effect are still to be identified.

We and others have previously demonstrated that development of MM is associated with accumulation in BM of myeloid-derived suppressor cells (MDSCs) [1], [2], [3], [4], [5]. These cells are morphologically and phenotypically similar to immature neutrophils or monocytes but distinct in functional and biochemical characteristics and in their ability to suppress immune responses [6]. In mice, MDSCs are defined by co-expression of CD11b and Gr1 and lack of expression of markers of mature macrophages (MΦ) and dendritic cells (DC). Two major subsets of MDSCs have been identified: granulocytic or polymorphonuclear (PMN-MDSC) characterized by a CD11b+Ly6G+Ly6Clow phenotype and monocytic (M-MDSC) with a phenotype of CD11b+Ly6GLy6Chigh. Counterparts of PMN-MDSCs and M-MDSCs in control tumor-free mice are neutrophils and monocytes, respectively, that have similar phenotypes.

In cancer patients, MDSCs are defined as immature myeloid cells that are co-purified with the mononuclear cell (MNC) fraction and have a phenotype of CD33+CD14CD11b+, with PMN-MDSCs distinguished from M-MDSCs by expression of CD15 or CD66b. In healthy donors, very few MNCs have the CD33+CD14-−CD11b+ phenotype; these cells are called immature myeloid cells (IMC), since they are not immune suppressive. In MM patients, the vast majority (~95%) of MDSCs in the BM is represented by PMN-MDSCs. Human mature neutrophils are not co-purified with the MNC fraction but are found in the pellet formed following Ficoll-Paque gradient centrifugation. MDSCs together with mature neutrophils comprise the largest cellular population in the BM.

While some cellular populations in the human BM microenvironment, including BM stroma (BMS), MΦ, plasmacytoid DCs (pDCs), conventional DCs (cDCs), and osteoclasts, have been previously implicated in MM chemoresistance in vitro [7], [8], [9], [10], [11], the number of these cells in BM is small. At the same time, the contribution of BM PMN-MDSCs and mature neutrophils in MM chemoresistance has yet to be determined.

Our study for the first time demonstrates that cells of neutrophilic granulocyte lineage isolated from both MM-bearing and tumor-free hosts have similar chemoprotective effects on MM cells. This effect does not require direct contact with tumor cells and is mediated through soluble factors produced by these myeloid cells. Taken together, our data indicate that targeting PMN-MDSCs and neutrophils in patients with MM can improve the efficacy of chemotherapy and, thus, outcome of this disease.

Section snippets

Cell lines

Human MM U266, MM1.S, NCI-H929 and RPMI-8226 cell lines were obtained from ATCC. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic (Invitrogen, Grand Island, NY). Mouse MM DP42 and 38ATLN (ATLN) cell lines were established and kindly provided by Dr. Brian Van Ness (University of Minnesota, Minneapolis, MN) and were cultured in RPMI-1640 medium supplemented with 10% FBS, 5 mM glutamine, 50 µM 2-mercaptoethanol, 1%

Proportion of different myeloid cell populations in MM-bearing mice

We used a DP42 mouse model of MM in which disease is localized to the BM and closely resembles the clinical characteristics of MM [4], [15]. Initially, we determined the proportion of different myeloid cell populations in the BM of tumor-free and MM-bearing mice. In the BM of tumor-free mice, CD11b+Gr1+ cells comprising CD11b+Ly6G+Ly6Clow neutrophils (89%) and CD11b+Ly6GLy6Chigh monocytes (11%) represented the largest population of myeloid cells (Fig. 1A). Their counterpart in MM-bearing mice,

Discussion

Tumor microenvironment (TME) is known to play a critical role in the regulation of tumor cell chemosensitivity. However, cellular mechanisms responsible for this effect and contributions of various TME cell populations in this process are not entirely understood. The majority of the cellular compartment in the BM where MM cells reside is constituted of cells of myeloid lineage. Previous in vitro studies have implicated some types of myeloid cells including MΦ, cDC and pDC in MM chemoprotection

Conflict of interest

None.

Acknowledgments

Support for this work was provided by the Multiple Myeloma Research Foundation (Y.N.) and NIH grant T32CA009171 (S.E.H.). Support for Shared Resources utilized in this study was provided by Cancer Center Support Grant CA010815 to the Wistar Institute and P30-CA76292 to the H. Lee Moffitt Cancer Center and Research Institute. The authors would like to thank Dr. Alexandra Pisklakova, Eileen R. Grigson and Tess Chase for technical assistance and Ashley Durand (H. Lee Moffitt Cancer Center) and

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