Non-Invasive Bioluminescence Imaging to Monitor the Immunological Control of a Plasmablastic Lymphoma-Like B Cell Neoplasia after Hematopoietic Cell Transplantation

To promote cancer research and to develop innovative therapies, refined pre-clinical mouse tumor models that mimic the actual disease in humans are of dire need. A number of neoplasms along the B cell lineage are commonly initiated by a translocation recombining c-myc with the immunoglobulin heavy-chain gene locus. The translocation is modeled in the C.129S1-Ighatm1(Myc)Janz/J mouse which has been previously engineered to express c-myc under the control of the endogenous IgH promoter. This transgenic mouse exhibits B cell hyperplasia and develops diverse B cell tumors. We have isolated tumor cells from the spleen of a C.129S1-Ighatm1(Myc)Janz/J mouse that spontaneously developed a plasmablastic lymphoma-like disease. These cells were cultured, transduced to express eGFP and firefly luciferase, and gave rise to a highly aggressive, transplantable B cell lymphoma cell line, termed IM380. This model bears several advantages over other models as it is genetically induced and mimics the translocation that is detectable in a number of human B cell lymphomas. The growth of the tumor cells, their dissemination, and response to treatment within immunocompetent hosts can be imaged non-invasively in vivo due to their expression of firefly luciferase. IM380 cells are radioresistant in vivo and mice with established tumors can be allogeneically transplanted to analyze graft-versus-tumor effects of transplanted T cells. Allogeneic hematopoietic stem cell transplantation of tumor-bearing mice results in prolonged survival. These traits make the IM380 model very valuable for the study of B cell lymphoma pathophysiology and for the development of innovative cancer therapies

Tumor Necrosis Factor Induces Tumor Promoting and Anti-Tumoral Effects on Pancreatic Cancer via TNFR1

Multiple activities are ascribed to the cytokine tumor necrosis factor (TNF) in health and disease. In particular, TNF was shown to affect carcinogenesis in multiple ways. This cytokine acts via the activation of two cell surface receptors, TNFR1, which is associated with inflammation, and TNFR2, which was shown to cause anti-inflammatory signaling. We assessed the effects of TNF and its two receptors on the progression of pancreatic cancer by in vivo bioluminescence imaging in a syngeneic orthotopic tumor mouse model with Panc02 cells. Mice deficient for TNFR1 were unable to spontaneously reject Panc02 tumors and furthermore displayed enhanced tumor progression. In contrast, a fraction of wild type (37.5%), TNF deficient (12.5%), and TNFR2 deficient mice (22.2%) were able to fully reject the tumor within two weeks. Pancreatic tumors in TNFR1 deficient mice displayed increased vascular density, enhanced infiltration of CD4+ T cells and CD4+ forkhead box P3 (FoxP3)+ regulatory T cells (Treg) but reduced numbers of CD8+ T cells. These alterations were further accompanied by transcriptional upregulation of IL4. Thus, TNF and TNFR1 are required in pancreatic ductal carcinoma to ensure optimal CD8+ T cell-mediated immunosurveillance and tumor rejection. Exogenous systemic administration of human TNF, however, which only interacts with murine TNFR1, accelerated tumor progression. This suggests that TNFR1 has basically the capability in the Panc02 model to trigger pro-and anti-tumoral effects but the spatiotemporal availability of TNF seems to determine finally the overall outcome

Generation of the malignantIgH-myc-driven plasmablastic lymphoma-like B cell line IM380.

<p><b>A</b>: Photomicrograph of the initial tumor in a five months old C.129S1-<i>Igha</i>^tm1(Myc)Janz/J mouse. The abdominal tumor mass shows starry sky-like areas indicative of widespread apoptosis and infiltrating macrophages. The left picture shows 100× and the right picture 400× magnification, H and E staining. <b>B</b>: Malignant splenocytes were cultured <i>in vitro</i> and gave rise to the IM380 cell line that was characterized for its expression of various B cell markers and activation-associated proteins by flow cytometry. (Representative results from at least two independent experiments). <b>C</b>: IM380 cells were treated <i>in vitro</i> with different chemotherapeutics for 48 h before being subjected to annexin V/propidium iodide staining. Upper panel: Exemplary flow cytometry data for etoposide treatment. Lower panel: The graph shows the sensitivity of the cells towards the different compounds, expressed as their respective IC₅₀-values. (Mean ± SEM; combined data from four independent experiments). <b>D</b>: Luciferase-transgenic IM380 tumor cells were co-cultured with activated T cells for 72 h. Tumor cell numbers were assessed by their <i>in vitro</i> bioluminescence (upper panel and graphic evaluation in lower panel). Co-cultures were set up in triplicates each and compared to the 1∶1 culture. Flow cytometric assessment of MHC expression on IM380 cells. (Representative results from two independent experiments).</p

Non-invasive assessment of <i>in vivo</i> tumor growth and dissemination.

<p><b>A</b>: 10⁵ luciferase-transgenic IM380 tumor cells were injected i.v. into the lateral tail vein into syngeneic BALB/c mice. Tumor growth was assessed by non-invasive <i>in vivo</i> BLI at the indicated time points. <b>B</b>: Representative BLI pictures of tumor-bearing mice. <b>C</b>: Tumor dissemination was determined by counting individual light-emitting tumor foci. <b>D</b>: Upper panel: Representative <i>ex vivo</i> BLI picture of a tumor bearing mouse (lu: lung, cLN: cervical lymph nodes, thy: thymus, hea: heart, ki: kidney, iLN: inguinal lymph nodes, li: liver, fe: femur, ti: tibia, sb: small bowel, lb: large bowel, mLN: mesenteric lymph nodes, st: stomach, cae: caecum, spl: spleen). Lower panel: Evaluation of tumor cell infiltration in individual organs. A–D: (Mean ± SEM; n = 5; shown is one representative experiment out of two). <b>E</b>: Representative eosin and hematoxylinstainings of organs from tumor bearing mice shown in 200× magnification.</p

Allogeneic hematopoietic stem cell transplantation of tumor-bearing mice results in prolonged survival.

<p>10⁵ luciferase-transgenic IM380 tumor cells were injected i.v. via the lateral tail vein into syngeneic BALB/c mice. Six days after tumor cell inoculation, mice were lethally irradiated with 8 Gy and transplanted with 5×10⁶ bone marrow cells and 0.5×10⁶ enriched splenic T cells from C57Bl/6 mice. <b>A and B</b>: Tumor growth was assessed by non-invasive <i>in vivo</i> BLI at the indicated time points (Mean ± SEM; n = 5; shown is one representative experiment out of two). <b>C</b>: Survival after allogeneic transplantation (n = 9–10; combined data from two independent experiments).</p

Tumor detection by <i>in vivo</i> BLI correlates to M315 IgA serum measurement by ELISA.

<p>(<b>A</b>) M315 serum levels of melphalan treated (n = 5) and vehicle control (n = 5) on day 14 of treatment and simultaneous BLI measurement of the same mice. The treatment schedule is depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052398#pone-0052398-g003" target="_blank">Figure 3A</a>. Measurement of idiotype specific M315 IgA significantly differed between the groups (two-tailed p value 0.0171) as it did with BLI (ventral+dorsal signal) (two-tailed p value 0.0221). (<b>B</b>) Scheme indicating time points for BLI measurement and serum collection for data presented in (C–G). (<b>C–G</b>) Idiotype specific M315 IgA serum levels of 5 untreated mice constantly increased over time correlating with progressing tumor burden as measured with BLI (ventral+dorsal signal). Of note, BLI measurements provided signals in early disease stages starting from day +9, whereas M315 IgA levels were not detectable at this time point. The left y-axis displays BLI signal intensity (black circles, solid lines); the right y-axis displays serum M315 IgA (red triangles, dashed lines).</p

Engraftment and growth dynamics of MOPC-315.BM luc⁺ myeloma cells in vivo.

<p>BALB/c wild type mice were injected with 1×10⁵ MOPC-315.BM luc⁺ cells via the tail vein. Tumor growth and spread was regularly monitored by BLI. (<b>A</b>) BLI images of one representative mouse at indicated time points after MM injection from ventral (top) and dorsal (bottom) view. Additional emerging tumor foci over time are marked with black or white arrows. (<b>B</b>) Number of skeletal spots per mouse on days +11 (n = 51), 19 (n = 56) and 33 (n = 25) and (<b>C</b>) percentage of mice presenting signals from liver and spleen. (<b>D</b>) Quantification of single tumor foci over time as marked in (A): 1 and 2 = BM compartment of femur/tibia, 3 = spleen, 4 = BM compartment of shoulder. (<b>E</b>) Absolute signal quantification by whole body BLI from ventral and dorsal views. (<b>F</b>) Representative osteolytic lesion in the neck of femur 42 days after MM injection. Corticalis is marked as c which is destroyed (arrow) by MOPC-315.BM luc⁺ cells marked with T. Original magnification 40X, scale bar is 200 µm. Insert: original magnification 200X, scale bar is 100 µm.</p

Flow cytometric measurement of surface receptors associated with BM homing and infiltration of myeloma cells.

<p>BALB/c wild type mice were injected with 1×10⁵ MOPC-315.BM luc⁺ cells via the tail vein. (<b>A</b>) 42 days after MM injection mice showed high BLI signals from hematopoietic compartments such as femur/tibia and spleen. Shown are two representative mice from ventral and dorsal view immediately before cells from BM and spleens were harvested for flow cytometry. (<b>B–D</b>) Besides BM and spleen derived MM cells, we also analysed MOPC-315.BM luc+ cells from culture. Dead cells were excluded by propidium iodide staining and MOPC cells identified as CD138⁺CD4⁺ double positive cells. (<b>B</b>) α4β1 integrin positive MOPC-315.BM luc⁺ cells were identified by flow cytometry as α4⁺ (CD49d⁺) and α4β7⁻. Representative FACS plots and the corresponding graph are shown, stating the frequency within CD138⁺CD4⁺ MOPC-315.BM luc⁺ cells expressing α4β1. For CXCR4 (<b>C</b>) and CD44 (<b>D</b>) representative histograms for each organ and cell line, including unstained fluorescence minus one (FMO) sample are displayed. Corresponding graphs state the fold difference in mean fluorescence intensity (MFI) related to the unstained FMO sample. BM and spleen: two independent experiments, n = 10, cells from cell culture: n = 4 for CXCR4 and CD44, n = 3 for α4β1. * indicates p<0.05 and ** indicates p<0.01 as determined by Kruskal-Wallis test with Dunn post test. (<b>E</b>) MOPC-315.BM luc⁺ cells were sorted for CXCR4^low and CXCR4^high expression. After 2 days in cell culture sorted cells regained the original CXCR4 expression level of the cell line. (<b>F</b>) 5×10⁵ sorted cells were i.v. injected into 4 female BALB/c mice each and BLI from ventral, lateral and dorsal was performed 10 days later. Sorted CXCR4^low as well as CXCR4^high CXCR4 cells readily homed to the BM compartment as well as to the spleen. (<b>G</b>) After BLI the mice were sacrificed, cells from left and right femur/tibia (separately) and the spleen extracted, and percentage as well as absolute numbers for CD138⁺CD4⁺ MM cells determined. From these values a ratio of spleen/BM was calculated to determine the homing capacity of the sorted populations. (<b>H</b>) Comparison of CXCR4 expression levels of sorted CXCR4^low and CXCR4^high cells immediately before injection and MM cells from BM and spleen after 10 days <i>in vivo</i> revealed a dynamic CXCR4 regulation.</p