Flow analysis of RANK expression on myeloid cells and identification of bone marrow cells expressing IL-32 in multiple myeloma patients

Abstract

Multiple myeloma is the second most common hematological cancer worldwide, characterized by an increased expansion of malignant plasma cells in the bone marrow, organ dysfunction, immunodeficiency and bone erosion. Nearly all patients develop osteolytic lesions that cause not only sever bone pain but also promote tumor growth and poor prognosis. Increased osteoclastogenesis is the major cause of myeloma induced bone disease. However, the origin of osteoclast precursors has not yet been fully revealed. One of the prominent markers for osteoclast precursors is RANK expression on their surface, and in this study we determined the expression of RANK in the bone marrow granulocytes and monocytes in myeloma patients. We determined the RANK expression on granulocytes and monocytes in bone marrow aspirates collected from patients with myeloma by using a multicolor flow cytometric analysis. Our results showed that very few patients expressed RANK in monocytes and there were significantly fewer RANK+ granulocytes compared to RANK+ monocytes. None of the patients with high tumor load and bone lesions had high proportion of RANK+ granulocytes or monocytes. Based on our results, we are able to conclude that none of these myeloma patients expressed RANK on their granulocytes. Several pro-inflammatory cytokines are proposed to play an important role in myeloma progression as well as bone disease. Interleukin-32 (IL-32) is a pro-inflammatory cytokine that was found by my research group to be elevated in Multiple myeloma. In this study we set out to identify the IL-32 expressing cells in the patient’s bone marrow in both, CD138+ and CD138- fractions. Therefore, we performed flow cytometric analysis of patient’s bone marrow and myeloma cell lines. Our flow cytometric analysis showed that IL-32 was highly expressed in two myeloma cell lines whilst very few IL-32 expressing cells could be detected in these myeloma patients’s bone marrow. However, a sub-population of CD138+ cells was found to express IL-32. Immunophenotyping of CD138- fraction suggested that a small proportion of CD14+ monocytes and CD3+ T cells also expressed IL-32.