Macrophages are an abundant component of myeloma microenvironment and protect myeloma cells from chemotherapy drug-induced apoptosis

Abstract

Multiple myeloma remains an incurable disease. One of the major problems is that myeloma cells develop drug resistance on interaction with bone marrow stromal cells. In this study, we examined the effects of macrophages (Mvarphis), a type of stromal cells, on myeloma cell survival and response to chemotherapy. We showed that Mvarphi, in particular tumor-associated Mvarphi, is a protector of myeloma cells. The protective effect was dependent on direct contact between Mvarphis and myeloma cells. Mvarphis protected both myeloma cell lines and primary myeloma cells from spontaneous and chemotherapy drug-induced apoptosis by attenuating the activation and cleavage of caspase-dependent apoptotic signaling. These findings are clinically relevant because we found that CD68+ Mvarphis heavily infiltrate the bone marrow of patients with myeloma but not the bone marrow of control patients. Thus, our results indicate that Mvarphis may contribute to myeloma cell survival and resistance to chemotherapeutic treatment in vivo.

Figure 1

Macrophages protect myeloma cells from apoptosis. (A) Dot plots showing apoptotic myeloma (MM.1S) cells in cultures or cocultures with nMφs or TCCM-treated Mφs (tMφ) in the presence or absence of dexamethasone (Dex) or melphalan (Mel). Numbers inside dot plots indicate the percentages of live cells. (B) Apoptotic myeloma cells (percentage of melphalan control, which is 100% of apoptotic cells) in cocultures with either nMφs or tMφs in the presence of melphalan. Four commonly used myeloma cell lines, ARP-1, MM.1S, RPMI-8226, and U266, were tested. (C) Percentages of apoptotic myeloma cells in cocultures without or with tMφs either in direct contact (MM + tMφ) or separated by Transwell inserts (MM + tMφ, Transwell) in the presence of melphalan. Culture of myeloma cells in medium and coculture of myeloma cells with tMφs served as controls. (D) Effects of anti–ICAM-1 antibody on blocking tMφ-mediated myeloma apoptosis protection. Shown is the percentage of apoptotic myeloma ARP-1 cells pretreated with 10 μg/mL anti–ICAM-1 antibody or mouse IgG, in (co)cultures with tMφs in the presence of melphalan. Culture of myeloma cells in medium and coculture of myeloma cells with tMφs served as controls. Similar results were obtained with other myeloma cell lines. (E) Percentages of spontaneous apoptotic primary myeloma cells in culture medium only (primary MM) or in cocultures with tMφs (primary MM + tMφ) at 24 hours and 48 hours after isolation of the myeloma cells. Representative results from experiments with primary myeloma cells from one patient of 4 examined are shown. (F) Percentage of apoptotic myeloma ARP-1 cells in coculture with Mφs generated from MM patients in the presence of melphalan. Representative results from experiments with Mφs from 1 patient of 3 examined using this and other cell lines are shown. *P < .05; **P < .01.

Figure 2

The mechanism of macrophage-mediated antiapoptosis in myeloma cells. (A) Western blot analysis showing the protein expression of cleaved PARP (cPARP), cleaved caspase-3 (cCas-3), Bcl-xL, Bcl-2, Bad, and Bax in ARP-1 myeloma cells cultured alone or cocultured with tMφs in the presence of melphalan (5 μM). The level of β-actin served as loading control. Results from 1 representative experiment of 3 performed with ARP-1 are shown. Similar results were obtained with other myeloma cell lines. (B) Levels of IL-6 in normal medium, TCCM, and in the supernatants of nMφs and tMφ, measured by enzyme-linked immunosorbent assay. (C) Dot plots showing apoptotic myeloma cells in culture medium (Med), in cocultures with tMφ, and in coculture with tMφs and IL-6–neutralizing antibody (αIL-6) in the presence or absence of melphalan (Mel). Numbers inside dot plots indicate the percentages of live cells. (D) Percentages of melphalan (Mel)–induced, annexin V–positive apoptotic myeloma (ARP-1) cells in culture medium only (Med) or in cocultures with untreated tMφs or with cytochalasin D (CD)–pretreated tMφs (CD-tMφ). Infiltration of Mφs in the bone marrow of myeloma patients. (E) Immunochemistry staining by CD68 antibody to identify Mφs in bone marrow biopsies from a control patient without malignancy and from a randomly selected myeloma patient. Representative results from experiments with bone marrow biopsies from 1 of 4 myeloma and 4 control patients examined are shown. (F) Percentages of infiltrated Mφs in bone marrow biopsies of MM and control patients. Data were derived from the numbers (mean ± SD) of CD68⁺ Mφs in a total of 1000 cells counted in bone marrow biopsies of 4 patients with MM and 4 controls. **P < .01.