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

Loss of host TNFR1 affects the expression of immunosuppressive genes and IL-4.

<p>Panc02-tumors were explanted one month after tumor cell inoculation and total RNA was isolated from the tumor tissue. RNA was reverse transcribed and amplified by qRT-PCR. Data is presented as relative expression within tumors derived from B6.TNFR1 KO mice compared to tumors derived from wild type (WT) mice (B6.WT n = 3, B6.TNFR1 KO n = 4). * p≤0.05.</p

Loss of host TNFR1 perturbs the immunologic control of pancreatic ductal carcinoma.

<p>Panc02-tumors were explanted one month after tumor cell inoculation, consecutive histological sections were stained for indicated immune cell populations and blood vessels (CD31). Pancreatic tumors resulted in an influx of immune cell populations of the innate and adaptive immune system that were not observed in healthy pancreatic tissue under steady-state conditions. Of note, deficiency of TNFR1 resulted in a reduced cytotoxic CD8⁺ T cell infiltration but increased T_reg cell infiltration. Exemplary photomicrographs are shown. Scale bar indicates 100 µm.</p

Panc02 cells show little <i>in vitro</i> capabilities for metastasis.

<p>Panc02 cells were treated with 1.67: Adhesion to different extracellular matrix proteins (n = 4). B: Flow cytometric determination of the expression of proteins involved in adhesion and migration (n = 3). C: Invasive capabilities of Panc02 cells. Left panel: <i>In vitro</i> invasion of the basement membrane (n = 3). Right panel: Gelatin zymography of tumor cell samples. Infarcted mouse heart lysate was used as a positive control (n = 4).</p

Primer sequences used for qRT-PCR.

<p>Primer sequences used for qRT-PCR.</p

Exogenous TNF treatment perturbs the immunologic control of Panc02 tumors.

<p>Panc02-tumors from untreated and human TNF treated mice were explanted 23 days after tumor cell inoculation, sectioned, and stained for different immune cells and blood vessels (untreated n = 7, TNF n = 9). * p≤0.05.</p

Exogenous TNF treatment increases orthotopic Panc02 tumor growth.

<p>10⁴ tumor cells were injected into the spleen of albino B6.WT mice. The mice were either left untreated or were treated every other day with 5 µg of recombinant human TNF. Tumor growth was determined by <i>in vivo</i> BLI. A: Tumor growth displayed as total radiance (untreated n = 11, TNF n = 10). B: <i>Ex vivo</i> imaging 23 days after tumor cell inoculation. Internal organs were imaged for the presence of tumor cells. Pancreatic tumor size is displayed as total radiance (untreated n = 11, TNF n = 10). ** p≤0.01. Combined data from two independent experiments.</p

Loss of host TNFR1 abrogates spontaneous rejection of orthotopic Panc02 tumors.

<p>Murine pancreatic ductal adenocarcinoma (Panc02) cells were transduced to stably express eGFP and firefly luciferase and 10⁴ tumor cells were injected orthotopically into albino C57Bl/6 mice. Tumor growth in wild type mice (B6.WT) and mice that were deficient for TNF or its receptors was determined by <i>in vivo</i> BLI. A: Tumor growth displayed as total radiance (B6.WT n = 8, B6.TNF KO n = 8, B6.TNFR1 KO n = 6, B6.TNFR2 KO n = 9, B6.TNFR1R2 KO n = 7). B: Exemplary pictures of the imaging time course of a mouse that spontaneously rejected the tumor (left) and a mouse that could not control tumor progression (right). C: <i>Ex vivo</i> imaging one month after tumor cell inoculation. Internal organs were imaged for the presence of tumor cells. Exemplary pictures of a mouse that spontaneously rejected the tumor (I), a mouse with low tumor burden (II), and a mouse with high tumor burden (III). D: Pancreatic tumor size one month after Panc02 inoculation is displayed as total radiance (B6.WT n = 8, B6.TNF KO n = 8, B6.TNFR1 KO n = 6, B6.TNFR2 KO n = 9, B6.TNFR1R2 KO n = 5). * p≤0.05, ** p≤0.01. Combined data from four independent experiments.</p