Figure 1
Features of PCTs arising in C.IL6/MYC mice. (a) Targeted Tg insertion of Myc in C.iMycΔEμ mice. Shown are schemes of the normal mouse Igh locus (top) and the targeted Igh locus (bottom) harboring the inserted Myc gene (red box, not to scale) in the intervening genomic region between the Ig heavy-chain joining locus, JH (gray rectangle), and the switch region (black dot) of the Cμ locus (labeled open rectangle). The Eμ and Eα enhancers are depicted by blue diamonds that are labeled. Note that Eμ has been deleted during gene targeting so that only Eα is available to drive Myc expression in cis. The ‘iMyc’ transgene insertion site is also the preferred recombination site of chromosomal t(8;14)(q24;q32) IGH-MYC exchanges in human sporadic Burkitt lymphoma and chromosomal T(12;15) Igh-Myc exchanges in a subset of inflammation-dependent peritoneal plasmacytomas in strain C mice. (b) Generation of strain C.IL6Myc mice. The mice were selected from crosses of homozygous Tg (TG/TG) C.iMycΔEμ mice and heterozygous Tg (TG/+) C.H2-Ld-IL6 mice. In accordance with a Mendelian trait, approximately half of offspring exhibited the desired doubly heterozygous Tg genotype. (c) Necropsy photographs of a PCT-bearing C.IL6Myc mouse before (left panel) and after (right panel) opening the abdominal wall. The mouse harbors enlarged, highly vascularized peripheral lymph nodes (LNs) in the CLN, ALN and ILN regions. The grossly enlarged MLN penetrating the gut loops and the moderately enlarged SPL become visible after removal of the abdominal wall. A mouse that developed hind leg paralysis is shown at bottom right. (d) Photomicrograph of a PCT found in an enlarged CLN of ∼4-month-old C.IL6Myc mouse (hematoxylin and eosin (H&E); original magnification × 63). (e) Detection of paraproteins in sera of PCT-bearing C.IL6Myc mice (lanes 2–9). Lane 1 contains a sample from a normal control C mouse. Paraproteins (M-components, M-spikes) are indicated by red arrowheads pointing right, for example, IgG1/κ in lane 5 and IgA/κ in lane 7. Of 10 M-spikes isotyped by enzyme-linked immunosorbent assay (ELISA), 5 were found to be IgG1, 3 were IgG2b, 1 was IgG3 and 1 was IgA. The κ light chains were used in all cases. (f) Heatmap of differentially expressed genes in IL6Myc-dependent PCT (n=11) compared with normal B cells. Up- and down-regulated genes are indicated by red and blue, respectively. All tumor samples were obtained from enlarged MLNs. B cells were isolated from the spleens of 5-month-old C mice using B220 magnetic columns (Milteny, Auburn, CA, USA). The B220+ splenocytes were either left untreated (n=3) or activated by treatment with lipopolysaccharide (LPS) and IL-4 (n=3). Illumina microarray data were processed using GenomeStudio v1.9.0 (Illumina). Raw expression values were normalized using the quantile method and assessed for genes with differential expression based on analysis of variance (ANOVA). Probeset values were normalized to a mean of zero (that is, mean centered) and a s.d. of 1. The false discovery rate (FDR) was then applied to adjust P-values for multiple test correction. Differentially expressed genes were selected with thresholds based on FDR-adjusted P-value <0.05 and fold change >1.5 or <−1.5. (g) Shown to the left is a PCA (principal component analysis) plot of global gene expression of the tumor and control samples included in the heatmap presented above: A, quiescent B cells; B, proliferating B cells; C, PCT. Shown to the right is a Venn diagram that depicts the number of gene probes found to be significantly variable by ANOVA in pairwise comparisons using a cut-off threshold of FDR 0.05 and a fold expression change that is either >1.5 or <−1.5. The 831 gene probes intersecting all four comparison groups are included in the heatmap in (a). (h) Diagram showing expression levels of six genes found to be up- or down-regulated in PCT (n=11) compared with normal B cells (n=3+3). Mean gene expression levels (columns) and s.d. of the mean (vertical lines) are plotted. All differences between PCT and either unstimulated or stimulated B cells were significant (P<0.05) using Student’s t-test. (i) Infiltration of the bone marrow with malignant PCs. The left top panel depicts flow data on the presence of malignant, high forward scatter-gated CD138+B220- PCs (10.8%) in the femur of a C.IL6Myc mouse harboring a mid-stage PCT. B220 and CD138 were detected using phycoerythrin- and allophycocyanin-conjugated antibodies 6B2 (eBioscience, San Diego, CA, USA) and 281–2 (BD Biosciences, San Jose, CA, USA), respectively. Shown in the right top and bottom panels is a tissue section of a skull base bone of another mouse bearing a late-stage PCT. The normal marrow has been replaced entirely with malignant PCs (H&E; original magnification × 2 and × 63, respectively).
