Delayed in Vivo Disease Progression Is Associated with High Proportions of CD45⁺ Myeloma Cells in the 5T2MM Murine Model¹

MM³ is a plasma cell malignancy with the BM as the main affected organ (reviewed in Ref. 1). The majority of the MM cells in patients have a CD45⁻ phenotype. The common leukocyte antigen, CD45, is a transmembrane protein tyrosine phosphatase expressed on all of the B cells. During maturation toward plasma cells its expression is gradually lost, and fully matured plasma cells are CD45⁻ (2). Although a heterogeneous CD45 expression pattern on human MM cells in the BM has been recognized by several groups (3, 4, 5), the role of the CD45⁺ and CD45⁻ MM cells in myeloma pathogenesis is unclear. Our group uses the 5T2MM mouse model to investigate the in vivo roles of CD45 subsets in myeloma biology. In analogy to the human situation, the 5T2MM cells have a heterogeneous CD45 expression (6). Compared with the CD45⁻ 5T2MM cells, CD45⁺ 5T2MM cells express higher levels of the insulin-like growth factor I receptor (6), which is involved in their chemotaxis to the BM (7). CD45⁺ 5T2MM cells also express higher levels of proteases involved in their invasion, including matrix metalloproteinase-9 and urokinase type plasminogen activator receptor (8). Moreover, CD45⁺ 5T2MM cells have enhanced in vitro chemotaxis toward the BM microenvironment and have higher in vitro invasive capacities (8). In line with these findings, in vivo experiments indicated a higher homing capacity of CD45⁺ MM cells to the BM (6). Despite these phenotypic differences in the expression of key molecules involved in BM homing and the functional differences in the BM migratory capacities, the impact of CD45 heterogeneity on the outcome of myeloma disease is unknown. In this work we provide in vivo data indicating that mice bearing high proportions of CD45⁺ 5T2MM show distinct clinical features and have a delayed disease progression compared with animals with predominantly CD45⁻ MM cells.

5T2 myeloma cells originate from spontaneously developed myeloma in aged C57BL/KaLwRijHsd mice (9, 10). The model was initiated and is continued by i.v. injection of diseased BM cells into young (6–10 weeks old) syngenic recipients (Harlan, Horst, the Netherlands) as described previously (7). 5T2MM cells have a heterogeneous CD45 expression (6) and are a model for the most common form of human myeloma (11).

BM cells were flushed out from femora and mononuclear cells (BMNC) were isolated by centrifugation of the samples on Lympholyte M (Cedarlane, Hornby, Ontario, Canada). Tumor load was assessed by staining of the BMNC with anti-5T2MM idiotype-specific antibodies (12). Rat antimouse IgG1-PE (Becton Dickinson, San Jose, CA) was used as secondary reagent. CD45 expression on the 5T2MM cells was analyzed by double staining with 5T2MM idiotype and with CD45-FITC (clone AMS4508; Biosource International, Camarillo, CA). Mice were bled before killing, and serum paraprotein levels were quantified by electrophoresis (12).

Student’s t test was used for statistical analysis. P ≤ 0.05 were considered as significant.

The majority (92%) of naïve mice injected with 5T2MM cells are terminally diseased at 10–12 weeks after tumor inoculation. Typically, these animals have paralyzed hind legs as illustrated in Fig. 1,A. Top panels of Fig. 2 illustrate that the BM of these mice contain predominantly CD45⁻ MM cells, in analogy with the common human situation. Myeloma cells isolated from these mice are used for experiments and for the continuation of the model. Thus, the dot plots in the top panel of Fig. 2 are also representative for the phenotype of the 5T2MM cells inoculated in the naïve mice. The remaining, nondiseased mice (∼8% of the animals) are usually euthanasized. However, in this study, these mice were followed until signs of overt disease were observed. The mice had a delayed tumor take and were terminally diseased at 14–24 weeks after tumor injection. These mice had completely different clinical features (Fig. 1,B) than the early (normal) diseased animals (Fig. 1,A). These animals typically had a bowed back and never got paralyzed, in contrast with the normal diseased mice. These clear differences in take time and disease appearance lead us to determine the differentiation status of the MM cells in these mice. Dot plots in the bottom panels of Fig. 2 illustrate that the myeloma cells had a predominant CD45⁺ phenotype. In Table 1 the take time, tumor load, serum paraprotein concentration and percentage of CD45⁺ 5T2MM cells between the two groups are compared. Besides the differences in take time and percentage of CD45 5T2MM cells, mice with a delayed take had higher tumor load and paraprotein levels, most probably attributable to the longer duration of the disease.

Our data are in line with very recent observations of Moreau et al. (13) that high levels of CD45⁻ MM cells in myeloma patients are associated with poor clinical prognosis. As already mentioned, CD45⁺ 5T2MM cells are the predominant in vivo BM homing clone (6). In a recent work we demonstrated that after homing the CD45⁺ 5T2MM cells retain this phenotype in the early disease stages and gradually differentiate into CD45⁻ MM cells during the disease progression (14). In the end stage of the disease the majority of the MM cells have obtained a CD45⁻ phenotype. Apparently this differentiation does not occur in a minority (8%) of the mice. The mechanisms underlying these observations are not understood currently. However, the data underline the prognostic value of CD45 expression and are in keeping with our finding of high in vivo proliferative capacity of CD45⁻ 5TMM cells compared with CD45⁺ MM cells (6).

In conclusion, in previous works we demonstrated that CD45⁺ and CD45⁻ 5T2MM cells have different chemotactic, invasive, proliferative, and BM homing capacities (6, 8). The data in this work strongly indicate that CD45 subsets of the 5T2MM cells are also associated with the final outcome of myeloma disease, suggesting that these subsets may have crucial roles in myeloma biology.