Macrophages, Myeloma, Mouse Models and Methodologies

Abstract

Macrophages are highly plastic phagocytic cells, which function in both innate and adaptive immunity and have been implicated in supporting neoplastic progression, including in the haematological malignancy multiple myeloma (MM). In this thesis, the role of macrophages in MM disease establishment and progression was explored. Additionally, standard techniques and methodologies were evaluated, in order to enable further investigation of bone marrow (BM) macrophages in MM. The initial homing and establishment of MM plasma cells (PC) within the BM is an essential first step in MM disease development. Here we show, for the first time, that depletion of macrophages by administration of clodronate-liposomes (clo-lip) resulted in impaired MM PC homing and retention within the BM, leading to a greater than 95% reduction in MM tumour burden in vivo. This was attributed, in part, to decreased levels of macrophagederived insulin-like growth factor 1 (IGF-1). These studies demonstrate a role for macrophages in MM disease establishment and progression and highlight the potential of targeting macrophages as a therapy for MM patients. Next, we employed conventional flow cytometry to identify specific macrophage subpopulations that may be involved in MM. During our analysis, we discovered that traditional BM cell isolation techniques cause fragmentation of murine macrophages resulting in the acquisition of macrophage-derived remnants on the surface of other BM cells. Specifically, our data demonstrated that cells staining positive for the traditional BM macrophage markers F4/80, CD169 and VCAM1 by conventional flow cytometry, exhibited phenotypic characteristics and a gene expression signature consistent with granulocytes. Furthermore, imaging flow cytometry revealed F4/80-positive macrophage remnants adhering to the cell surface of Cd11b+Ly6G+ granulocytes, with whole macrophages rarely detected. These findings have broad implications for studies investigating BM macrophages, particularly those that rely solely on conventional flow cytometric methods. In order to identify a suitable murine MM model for future studies manipulating BM macrophages through genetic or pharmacological means in immunocompetent mice, an extensive characterisation of the Vk*MYC MM transplant lines Vk*MYC-4929 and Vk14451-GFP was undertaken. Notably, serum paraprotein analysis, which is the standard, and often sole, method of quantifying MM tumour burden in these models, had no correlation with BM tumour burden in either line. Furthermore, BM involvement was significantly reduced following successive splenic passage in vivo. These studies also revealed for the first time the presence of macroscopic liver lesions in tumour-bearing mice. Collectively, these results highlight potential model-specific caveats and emphasise the importance of BM-specific analyses of tumour burden within these models. Overall, these studies illustrate the importance of macrophages in MM, identifying a novel role for macrophages in MM PC BM homing and highlighting the potential for macrophage-directed therapies in the treatment of MM. These studies also identify limitations of well-established methodologies, including conventional flow cytometric analysis of BM macrophages, and the reliance on serum paraprotein for tumour burden analysis in Vk*MYC MM transplant models. Notably, the studies described in this thesis will aid in the development of new experimental strategies to examine macrophages in MM, thereby enabling the investigation of macrophage-targeted therapeutic compounds in MM.