Tracking human multiple myeloma xenografts in NOD-Rag-1/IL-2 receptor gamma chain-null mice with the novel biomarker AKAP-4

Abstract

Background: Multiple myeloma (MM) is a fatal malignancy ranking second in prevalence among hematological tumors. Continuous efforts are being made to develop innovative and more effective treatments. The preclinical evaluation of new therapies relies on the use of murine models of the disease.

Methods: Here we describe a new MM animal model in NOD-Rag1null IL2rgnull (NRG) mice that supports the engraftment of cell lines and primary MM cells that can be tracked with the tumor antigen, AKAP-4.

Results: Human MM cell lines, U266 and H929, and primary MM cells were successfully engrafted in NRG mice after intravenous administration, and were found in the bone marrow, blood and spleen of tumor-challenged animals. The AKAP-4 expression pattern was similar to that of known MM markers, such as paraproteins, CD38 and CD45.

Conclusions: We developed for the first time a murine model allowing for the growth of both MM cell lines and primary cells in multifocal sites, thus mimicking the disease seen in patients. Additionally, we validated the use of AKAP-4 antigen to track tumor growth in vivo and to specifically identify MM cells in mouse tissues. We expect that our model will significantly improve the pre-clinical evaluation of new anti-myeloma therapies.

Figure 1

Measurement of circulating paraproteins and AKAP-4 levels. Mice were bled once a week as described in the Methods section. The assay was run in triplicate for each time point. Graphs display mean protein levels obtained from each tumor group and error bars indicate SEM. No statistically significant difference was evidenced between IgE or IgG and AKAP-4 levels at any of the analyzed time points as evaluated by two-way ANOVA (Bonferroni's post-test p > 0.05 for all comparisons).

Figure 2

Detection of MM cell lines in tumor-bearing mice. U266 and H929 cell lines, or tissues derived from U266 and H929- injected mice analyzed by flow-cytometry. Histograms show the fluorescence intensity measured with the indicated specific antibody (bold lines) or with the corresponding isotypic control (dotted lines). Graphs are representative of comparable results obtained from 5 U266- and 5 H929-challenged mice.

Figure 3

Detection of primary MM cells in tumor-bearing mice. Ficoll-hypaque isolated cells from primary bone aspirate, or cells derived from primary MM-injected mice were analyzed by flow-cytometry. Histograms show the fluorescence intensity measured with the indicated specific antibody (bold lines) or with the corresponding isotypic control (dotted lines). Histograms are representative of comparable results obtained from 5 primary MM-challenged mice.

Figure 4

Flow-cytometry evaluation of tumor-free mice. Six weeks after irradiation, healthy control mice were euthanized and processed in parallel with tumor-bearing mice. Histograms show the fluorescence intensity measured with the indicated specific antibody (bold lines) or with the corresponding isotypic control (dotted lines). Histograms are representative of comparable results obtained from 5 tumor-free mice.

Figure 5

RT-PCR/Western blot analyses of AKAP-4 mRNA/protein expressions and. RT-PCR analysis of mouse tissues was performed to evaluate AKAP-4 expression. AKAP-4 was detected in bone marrow (BM), blood (BL) and spleen (SP) of mice injected with MM cell lines (U266, H929) and primary tumor cells, but not in tumor-free mice. Positive controls were RNA isolated from exponentially in vitro growing cell lines, or from primary cells after ficoll purification. For the evaluation of healthy mice, RNA isolated from human testis was used as a positive control. Analysis of β-actin transcript served to confirm RNA integrity. Negative controls were PCR performed without template (No T), or with RNA not subjected to the reverse transcription step (No RT). Proteins extracted from MM cells and mouse tissues were analyzed for the presence of AKAP-4 protein by Western blot. AKAP-4 protein was detected in MM cell lines (U266 and H929), primary MM cells, and in the bone marrow, blood and spleen of tumor-challenged mice, but not in healthy controls (tumor-free). Total protein extracts from human testis were used as a positive control for the analysis of tumor-free mice. The pictures are representative of comparable results independently obtained from 5 mice in each group.