Targeted disruption of bone marrow stromal cell-derived Gremlin1 limits multiple myeloma disease progression in vivo

In most instances, multiple myeloma (MM) plasma cells (PCs) are reliant on factors made by cells of the bone marrow (BM) stroma for their survival and growth. To date, the nature and cellular composition of the BM tumor microenvironment and the critical factors which drive tumor progression remain imprecisely defined. Our studies show that Gremlin1 (Grem1), a highly conserved protein, which is abundantly secreted by a subset of BM mesenchymal stromal cells, plays a critical role in MM disease development. Analysis of human and mouse BM stromal samples by quantitative PCR showed that GREM1/Grem1 expression was significantly higher in the MM tumor-bearing cohorts compared to healthy controls (p < 0.05, Mann-Whitney test). Additionally, BM-stromal cells cultured with 5TGM1 MM PC line expressed significantly higher levels of Grem1, compared to stromal cells alone (p < 0.01, t-test), suggesting that MM PCs promote increased Grem1 expression in stromal cells. Furthermore, the proliferation of 5TGM1 MM PCs was found to be significantly increased when co-cultured with Grem1-overexpressing stromal cells (p < 0.01, t-test). To examine the role of Grem1 in MM disease in vivo, we utilized the 5TGM1/KaLwRij mouse model of MM. Our studies showed that, compared to immunoglobulin G (IgG) control antibody-treated mice, mice treated with an anti-Grem1 neutralizing antibody had a decrease in MM tumor burden of up to 81.2% (p < 0.05, two-way ANOVA). The studies presented here demonstrate, for the first time, a novel positive feedback loop between MM PCs and BM stroma, and that inhibiting this vicious cycle with a neutralizing antibody can dramatically reduce tumor burden in a preclinical mouse model of MM.Kimberley C. Clark, Duncan R. Hewett, Vasilios Panagopoulos, Natalya Plakhova, Khatora S. Opperman, Alanah L. Bradey, Krzysztof M. Mrozik, Kate Vandyke, Siddhartha Mukherjee, Gareth C.G. Davies, Daniel L. Worthley, and Andrew C.W. Zannettin

Macrophages in multiple myeloma: key roles and therapeutic strategies

Published: 06 January 2021Macrophages are a vital component of the tumour microenvironment and crucial mediators of tumour progression. In the last decade, significant strides have been made in understanding the crucial functional roles played by macrophages in the development of the plasma cell (PC) malignancy, multiple myeloma (MM). Whilst the interaction between MM PC and stromal cells within the bone marrow (BM) microenvironment has been extensively studied, we are only just starting to appreciate the multifaceted roles played by macrophages in disease progression. Accumulating evidence demonstrates that macrophage infiltration is associated with poor overall survival in MM. Indeed, macrophages influence numerous pathways critical for the initiation and progression of MM, including homing of malignant cells to BM, tumour cell growth and survival, drug resistance, angiogenesis and immune suppression. As such, therapeutic strategies aimed at targeting macrophages within the BM niche have promise in the clinical setting. This review will discuss the functions elicited by macrophages throughout different stages of MM and provide a comprehensive evaluation of potential macrophage-targeted therapies.Khatora S. Opperman, Kate Vandyke, Peter J. Psaltis, Jacqueline E. Noll and Andrew C. W. Zannettin

Clodronate-liposome mediated macrophage depletion abrogates multiple myeloma tumor establishment in vivo

Multiple myeloma is a fatal plasma cell malignancy that is reliant on the bone marrow microenvironment. The bone marrow is comprised of numerous cells of mesenchymal and hemopoietic origin. Of these, macrophages have been implicated to play a role in myeloma disease progression, angiogenesis, and drug resistance; however, the role of macrophages in myeloma disease establishment remains unknown. In this study, the antimyeloma efficacy of clodronate-liposome treatment, which globally and transiently depletes macrophages, was evaluated in the well-established C57BL/KaLwRijHsd murine model of myeloma. Our studies show, for the first time, that clodronate-liposome pretreatment abrogates myeloma tumor development in vivo. Clodronate-liposome administration resulted in depletion of CD169+ bone marrow-resident macrophages. Flow cytometric analysis revealed that clodronate-liposome pretreatment impaired myeloma plasma cell homing and retention within the bone marrow 24 hours postmyeloma plasma cell inoculation. This was attributed in part to decreased levels of macrophage-derived insulin-like growth factor 1. Moreover, a single dose of clodronate-liposome led to a significant reduction in myeloma tumor burden in KaLwRij mice with established disease. Collectively, these findings support a role for CD169-expressing bone marrow-resident macrophages in myeloma disease establishment and progression and demonstrate the potential of targeting macrophages as a therapy for myeloma patients.Khatora S. Opperman, Kate Vandyke, Kimberley C. Clark, Elizabeth A. Coulter, Duncan R. Hewett, Krzysztof M. Mrozik, Nisha Schwarz, Andreas Evdokiou, Peter I Croucher, Peter J Psaltis, Jacqueline E Noll and Andrew CW Zannettin

Fragmentation of tissue-resident macrophages during isolation confounds analysis of single-cell preparations from mouse hematopoietic tissues

Mouse hematopoietic tissues contain abundant tissue-resident macrophages that support immunity, hematopoiesis, and bone homeostasis. A systematic strategy to characterize macrophage subsets in mouse bone marrow (BM), spleen, and lymph node unexpectedly reveals that macrophage surface marker staining emanates from membrane-bound subcellular remnants associated with unrelated cells. Intact macrophages are not present within these cell preparations. The macrophage remnant binding profile reflects interactions between macrophages and other cell types in vivo. Depletion of CD169+ macrophages in vivo eliminates F4/80+ remnant attachment. Remnant-restricted macrophage-specific membrane markers, cytoplasmic fluorescent reporters, and mRNA are all detected in non-macrophage cells including isolated stem and progenitor cells. Analysis of RNA sequencing (RNA-seq) data, including publicly available datasets, indicates that macrophage fragmentation is a general phenomenon that confounds bulk and single-cell analysis of disaggregated hematopoietic tissues. Hematopoietic tissue macrophage fragmentation undermines the accuracy of macrophage ex vivo molecular profiling and creates opportunity for misattribution of macrophage-expressed genes to non-macrophage cells.Susan M. Millard, Ostyn Heng, Khatora S. Opperman, Anuj Sehgal, Katharine M. Irvine, Simranpreet Kaur, Cheyenne J. Sandrock, Andy C. Wu, Graham W. Magor, Lena Batoon, Andrew C. Perkins, Jacqueline E. Noll, Andrew C.W. Zannettino, David P. Sester, Jean-Pierre Levesque, David A. Hume, Liza J. Raggatt, Kim M. Summers, and Allison R. Petti