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BCL2+IL6+ plasma cell tumors
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Supplemental Methods
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Mice
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All studies were performed on the BALB/c (C) background and approved under IACUC Protocol
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0006A56361. BCL2+IL6+ congenics harbor the EμSV-Bcl-2-22 1 and H2-Ld-IL6 2 transgenes. CD45.1+
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congenics carry the SJL-derived allotype of CD45, CD45.1, instead of the C allotype, CD45.2.
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Adoptive B-cell transfer
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Splenic B cells were obtained from 5-8 weeks old BCL2+IL6+ mice using the MACS B220 Mouse B-cell
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kit (Miltenyi, Cambridge, MA). Two days later, 2 million of these cells were transferred to normal C host
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mice, using retroocular intravenous (IV) injection. Prior to the adoptive transfer, B cells were
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maintained in vitro in RPMI1640-based cell culture medium supplemented with 10% inactivated fetal
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calf serum, 1% penicillin/ streptomycin, 200 M L-glutamine and 50 M 2-mercaptoethanol. B cells
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were stimulated in culture (37 °C; 5% CO2) with 10 g/ml LPS and 12.5 ng/ml mIL-4. Two hours prior
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to cell transfer, 2-4 months old hosts were conditioned with either 450 cGy whole-body irradiation
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(sublethal dose not requiring HSC rescue) or 1,100 cGy whole-body irradiation (lethal dose requiring
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HSC rescue). The latter was accomplished by transferring 3 x 106 normal bone marrow cells (obtained
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from inbred C mice) together with the BCL2+IL6+ B cells.
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DNA constructs and viral stock
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All PCR products were cloned into pCRII-TOPO (Invitrogen) and confirmed by DNA sequencing. To
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generate a retrovirus that co-expressed tGFP (turbo green fluorescence protein) and firefly luciferase
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(fLuc), we cloned the respective cDNA genes into MSCV (murine stem cell virus). Using an intervening
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picornoviral 2A segment to accommodate both genes, we thus produced MSCV-tGFP-2A-fLUC. All
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DNA preparations were purified using Zymo Research DNA plasmid purification kits (Irvine, CA, USA).
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Sequences and maps are available upon request. Preparation of the retrovirus was as follows: 293T
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cells (Clontech Laboratories, Mountain View, CA, USA) were co-transfected with 15 μg retroviral vector
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DNA and 15 μg Psi2 (2) packaging vector DNA, using the CaCl2 method. Medium was changed 4-6
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hrs post transfection. Supernatant was harvested 48 hrs after transfection. Supernatant was filtered
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using PVDF with a pore size of 0.45 μm. Virus was freshly prepared for each use.
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Viral gene transduction
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For retroviral reporter gene transduction, a spinfection protocol was used. Briefly, B cells were plated in
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6-well cell culture dishes (3.5 × 106 cells per well) that contained one-third viral supernatant and two-
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BCL2+IL6+ plasma cell tumors
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thirds cell culture medium. The latter was supplemented with 7.5 g/ml polybrene. Plates were then
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subjected to centrifugation (500 x g, 2 hrs, 30 C) followed by transfer of plates to ambient temperature
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(30 min). Cells (still in viral supernatant) were then pelleted (300 x g, 10 min), re-suspended at 1 x 106
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cells/ml in cell culture medium, and incubated overnight (37 °C; 5% CO2). The next morning, cells were
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harvested and transferred to host mice as described above. Transduction efficiency, based on flow
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cytometric evaluation of fluorescent reporter protein expression, was ≥90%.
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Diagnosis and histopathology of plasma cell neoplasms (PCNs)
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Incipient tumors were detected by monitoring mice for health status parameters, including occurrence
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of hind limb weakness or paralysis. The diagnosis of PCN was established at necropsies of tumor-
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bearing mice and confirmed histologically using criteria described in the Bethesda classification of
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mouse hematopoietic tumors 3. Four- icrometer sections of paraffin-embedded tissues were stained
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with hematoxylin and eosin (H&E) and evaluated by a board-certified hematopathologist (Carol
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Holman). For detection and enumeration of osteoclasts in situ, the activity of tartrate-resistant acid
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phosphatase (TRAP) was used as biomarker. Briefly, bone sections were fixed with formaldehyde
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followed by decalcification in 10% EDTA for 6 days at 4 °C. The decalcified bones were rinsed with
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PBS three times (3 x 5 min), postfixed in cold 70% ethanol, and embedded in paraffin. Tissue sections
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were deparaffinized and stained for TRAP activity, using an acid phosphatase leukocyte kit from Sigma
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according to the manufacture’s suggestions. Specimens were counter-stained with eosin and mounted
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for imaging and histomorphometry.
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Flow-cytometric analysis of PCNs
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Antibodies to B220-PE-Cy7 (6B2), CD45.1-FITC (A20) and CD45.2-PE (104) were purchased from
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eBioscience (San Diego, CA). Antibody to CD138-APC (281-2) was purchased from BD Biosciences
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(San Jose, CA). To obtain single cell suspensions of lymphocytes, lymphoid tissues were harvested
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and minced between frosted glass slides. ACK lysis (Lonza, Radnor, PA) was used to remove red
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blood cells. For flow analysis, one million cells were washed and re-suspended in staining buffer that
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consisted of balanced salt solution, 5% bovine calf serum and 0.1% sodium azide. Non-specific
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binding of antibody was blocked using 10 μl rat serum (Jackson Immunoresearch, West Grove, PA)
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and 10 μg 2.4G2 (BioXCell, West Lebanon, NH). Cells were labeled on wet ice in the dark. Samples
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were run on a FACSCANTO II (Becton Dickinson, San Jose, CA) and data were analyzed using FlowJo
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(Tree Star, Ashland, OR).
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Detection and isotyping of serum paraproteins and measurement of serum cytokines
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Whole blood was collected from mice at necropsy, using heart puncture. Blood was transferred to
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EDTA-coated Microtainer tubes (Becton Dickinson) and spun for 5 min at 14,000 RPM to obtain serum.
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After centrifugation, serum was removed and frozen until the time of analysis. Serum protein
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elecrophoresis was used to detect paraproteins (M-spikes). Serum proteins were fractionated on
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Hydragel Protein(e) K20 gels using a Sebia elecrophoresis chamber (90 V constant; 40 min migration
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time; 12±3 mA). Paraproteins were isotyped using the Mouse Immunoglobulin Isotyping ELISA from
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BD Pharmingen according to the manufacturer’s recommendations. Modifications included dilution of
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serum samples and HRP-labeled antibodies in blocking buffer containing 0.05% Tween, and using
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HRP-labeled antibodies at 1:200. ELISA 96-well microplates were read at 450 nm using the Multiskan
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Spectrum from Thermo Scientific. The Bio-Plex 200 system (BioRad, Hercules, CA) was used to
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determine serum cytokine and chemokine levels, according to the manufacturer's instructions. Plates
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were washed on a Bio-Plex Pro II device and analyzed on a Bio-Plex 200 reader.
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In vivo bioluminescence imaging (BLI) analyses of tumor growth rates and tumor dissemination
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patterns
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Tumor formation was monitored using serial BLI on the IVIS100 system. Mice were anesthetized by
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isoflurane inhalation maintained at 2.5% through a nose cone, and images were obtained in dorsal and
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ventral presentations 5 min and 10 min after D-luciferin injection, respectively. Subsequently, a
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rectangular region of interest was placed around the dorsal and ventral images for each mouse and
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total photon flux (photons per second) was quantified using Living Image software v2.50 (Caliper Life
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Sciences). Dorsal and ventral values were summed and compared to previous values to estimate the
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whole-body tumor growth rate. Animals were observed twice weekly for adverse events associated
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with tumor growth (e.g., >15% body weight loss, immobility, loss of grooming, paralysis), at which time
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mice were euthanized.
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Integrated FDG-PET and CT scanning and image analysis
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We exactly followed the protocol described elsewhere 4. Briefly, for PET/CT scanning, mice were
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anesthetized, administered 18F-FDG (8.65±2.7 MBq) via the lateral tail vein and placed prone in a
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heated (36°C) multimodality chamber (M2M Imaging, Cleveland, OH) in the PET scanner’s gantry.
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PET list mode data were acquired for 15 min, using an Inveon small-animal PET/CT/SPECT imaging
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system (Preclinical Solutions, Siemens Healthcare Molecular Imaging, Knoxville, TN). In the same
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workflow, a CT image was acquired for attenuation correction purposes. Images were reconstructed
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using a 3D OP-MAP algorithm. Images were analyzed using PMOD v3.2 software (PMOD
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Technologies, Zurich, Switzerland).
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Supplemental Figure Legends
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Supplemental Figure 1: Bioluminescence imaging (BLI) of plasma cell neoplasms (PCNs)
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genetically tagged with firefly luciferase (fLuc).
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(A) BLI images of 2 tumor-bearing host mice (#19 and 20) in dorsal (left) and ventral (right) position
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evaluated on days 96 (top) and 129 (bottom) following reconstitution with luciferase-transduced (Luc+)
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B cells. Two additional tumor-bearing hosts (#8 and 12) reconstituted with donor-type B cells not
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transduced with (Luc-) were included as control. The increased photon flux scales next to the heat
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maps at the bottom reflect the incrased tumor burden at the later time point.
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(B) Second set of host mice bearing either Luc+ tumors (#13, 17, 45 and 47) or a Luc- tumor (#14).
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Images were taken on days 112 (top) and 202 (bottom) after B-cell reconstitution.
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(C) Third set of host mice bearing either Luc+ tumors (#30 and 49) or Luc- tumors (#48). Images were
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taken in a relatively short interval, on days 132 (top) and 144 (bottom) after B-cell reconstitution. The
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reason for the lack of signal in case of mouse 30 remained unclear.
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Supplemental Figure 2: Fast-onset tumors do not produce paraprotein.
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(A) Presented is a serum protein electropherogram that contains samples from 6 different tumor-
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bearing mice (lanes 2-7). M-spikes (paraproteins) are not detected. Included as a control is a serum
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sample from a host mouse carrying a late-onset tumor (lane 1). The massive M-spike in that case is
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indicated by red arrowhead pointing right.
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(B) H&E-stained tissue section (100x) of a representative tumor included in lanes 2-7 of panel A. The
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tumor was classified as diffuse large B-cell lymphoma (DLBCL) based on the following
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histopathological criteria: large lymphoid tumor cells with nuclear size equal to or exceeding normal
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macrophage nuclei and/or twice the size of a normal lymphocyte; diffuse growth pattern effacing normal
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tissue architecture; and abundance of immunoblast- and centroblast-like cells. Note that hind limb
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paralysis also occurred in some DLBCL-carrying mice, indicating that this diagnostic feature is not
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specific for PCN in our adoptive-transfer mouse model of myeloma.
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Supplemental Figure 3: Coexistent PCN and DLBCL in the bone marrow of the same mouse.
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Shown is a H&E-stained bone marrow section (original magnification 100x) in which the approximate
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borderline between the two tumors is indicated by a wavy yellow vertical line that bisects the image.
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Blowups of the areas marked by yellow rectangles reveal distinct cytological features: immunoblast-like
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cells in case of DLBCL (left) and atypical plasma cells with eccentric nuclei and large amounts of
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cytoplasm in case of PCN (right).
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Supplemental Figure 4: Isotyping of paraproteins using ELISA. Serum M-spikes in 6 different host
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mice carrying adoptive-transfer BCL2+IL6+ tumors were detected using protein electrophoresis (left; M-
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spikes are indicated by red arrowheads) and then isotyped using ELISA (right). For the latter, serum
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samples were logarithmically diluted, from 10-4 to 10-8, and probed with antibodies to immunoglobulin
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heavy-chains and light-chains indicated vertically to the left of the 3 photographic images of 96-well
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microplates. The 2 rightmost columns in these plates were used for positive and negative controls (Co)
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supplied by the manufacturer. M-spikes in sera that contained single paraproteins were readily
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isotyped: 2b in case of 1395, 1397 and 1403, and2a in case of 1162. Mouse 1161 harbored a
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major and a minor M-spike, which were isotyped as  and3. Because of its unusual electrophoretic
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mobility near the -globulin fraction and our past experience with paraprotein typing in mice, it is likely
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that the major M-spike indicates the IgM+ tumor. However, this has not been demonstrated. Because
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usage of light-chains is unusual in mice, it is also possible that the IgM+ and IgG3+ tumors that
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coexisted in mouse 1161 are clonally related, by virtue of isotype switching. But this has not yet been
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shown. Mouse 1270 also harbored two M-spikes: one was isotyped as 2b (likely indicating the larger
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spike) but it remained unclear whether the second spike was 1+ or +.
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Supplemental Figure 5: Myeloma-like kidney disease. The kidneys in the host mice bearing
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adoptive-transfer BCL2+IL6+ transgenic tumors exhibit morphologic changes consistent with myeloma
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cast nephropathy. A representative example is shown. Numerous voluminous laminated eosinophilic
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tubular casts are seen (top panel). The tubular cells appear flattened and show varying degrees of
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necrosis and denudation of tubular basement membranes. There are significant acute (center panel,
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left) and chronic (center panel, right) interstitial inflammatory cell infiltrates. The latter include
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occasional multinucleated giant cells (bottom panel, right, and inset below) and focal collections of
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neoplastic plasma cells (bottom panel, left, and inset below).
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Supplemental Figure 6: Massive hypergammaglobulinemia and emerging M-spikes in double-
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transgenic BCL2+IL6+ mice ~3-5 months of age. Serum protein electropherograms demonstrating
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striking polyclonal elevations of immunoglobulins (hypergammaglobulinemia), emerging M-spikes in 2
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cases (indicated by arrowheads pointing left in the top and bottom panels) and a pronounced M-spike
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(monoclonal Ig a.k.a. paraprotein, indicated by arrowhead pointing up in the center panel) in one case
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in a cohort of untreated BCL2+IL6+ mice (n = 15) ranging from ~3 months to ~5 months in age. A
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serum sample from a 4-month-old inbred C mouse (not transgenic, not treated) was used as control.
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The albumin and globulin fractions of serum proteins are labeled.
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Supplemental Figure 7: Flow-cytometric evaluation of a PCN that arose spontaneously in a
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double-transgenic BCL2+IL6+ mouse ~5 months of age. Cells were gated on forward and side
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scatter (not shown) and evaluated for expression of the B cell marker, B220, and the plasma cell (PC)
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marker, CD138. B cells (B220+CD138+) were distinguished from PCs (B220+CD138+). Plasmablasts
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(B220+CD138+) were not detected. Note the high frequency of PCs in the bone marrow (49%) relative
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to the spleen (6.8%). However, in this case, another secondary (extramedullary) lymphoid tissue, the
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perithymic lymph node, was as heavily infiltrated with neoplastic PCs as the bone marrow.
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Supplemental Figure 8: Elevated serum levels of cyto- and chemokines. Presented are results of
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Bio-Plex analyses of 3 groups of PCN-bearing BALB/c mice: (1) BCL2+IL6+ B-cell reconstituted mice
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harboring myeloma-like tumors, as described in the main text (n = 6; center columns); (2) Myc+IL6+ B-
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cell reconstituted mice containing peritoneal plasmacytomas (n = 6; left columns) that developed in
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pristane-induced inflammatory granulomas, as described in a previous Letter 6 and (3) double-
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transgenic BCL2+IL6+ mice harboring spontaneously arising tumors without adoptive B cell transfer or
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any other manipulation (n = 7; right columns). Mean values and standard errors of the mean are
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plotted. Statistical significance was determined with the help of the unpaired t test (in case of equal
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variance) or the Mann-Whitney test (different variance). All 5 cytokines included in the figure have
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been previously implicated in myeloma by other investigators. Thus, interleukin (IL)-5 and IL-13 are
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TH2 cytokines that may promote myeloma cells in the bone marrow niche 7. Elevated IL-17, produced
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by pro-inflammatory TH17 cells, promotes myeloma cell growth via a complex mechanism that includes
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inhibition of immune functions 8. Monocyte chemotactic protein (MCP)-1 and tumor necrosis factor
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alpha (TNF) play important roles in myeloma bone disease 9. See Supplemental Tables 1 and 2 for
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additional cyto- and chemokines. More work is warranted before it can be decided which cyto- and
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chemokines, if any, are important for the development of myeloma-like tumors in the BCL2+IL6+
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adoptive transfer model of human myeloma.
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Supplemental Figure 9: General bone loss in lumbar vertebrae of host mice harboring BCL2+IL6+
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driven PCNs. CT images depicting bone thickness in false color are shown. The strong bone (white
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to yellow) in the two normal mice (used as controls; left) compared to the weak bone (blue-to-red) in the
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two tumor-bearing mice (right) can be readily appreciated upon visual inspection of images. Statistical
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comparison of quantitative data (not shown) demonstrated a significant bone loss in diseased mice (p <
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0.01).
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Supplemental References
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Strasser, A., Harris, A.W. & Cory, S. E mu-bcl-2 transgene facilitates spontaneous
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Duncan, K. et al. (18)F-FDG-PET/CT imaging in an IL-6- and MYC-driven mouse model of
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human multiple myeloma affords objective evaluation of plasma cell tumor progression and
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Tompkins et al. – Supplemental Information
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