Effect of the two peptides from the skin secretion of <i>Phrynomantis microps</i> applied to termite, <i>Macrotermes bellicosus</i>, soldiers and delaying the aggressive behaviour and stinging of <i>Paltothyreus tarsatus</i> ants.

<p>Maximum observation time was 20</p

Fractionation of the skin secretion from <i>Phrynomantis microps</i> by reversed-phase HPLC on a LiChroCART column, 125×4 mm (Merck, Darmstadt) which was eluted with a linear gradient from 0 to 60% acetonitrile in 0.1% trifluoroacetic acid (dotted line) over 60 min at a flow rate of 0.5 ml/min.

<p>Absorbance was monitored at 220(above). Below: Total ion chromatogram (TIC) of liquid chromatography-time-of-flight mass spectrometry (LC/TOF MS) analysis of the HPLC fraction containing two peptides: <i>m/z</i> 1029.506 and <i>m/z</i> 1143.548 (non-protonated). <i>De novo</i> sequencing of the peptides suggested the tentative sequences as indicated in the chromatograms. Inlet picture: adult <i>Phrynomantis microps</i> examined by <i>Paltothyreus tarsatus</i> workers.</p

Time from first ant, <i>Paltothyreus tarsatus</i>, contact with termites (left; inlet A) or mealworms (right), coated with the skin secretion of <i>Phrynomantis microps</i>, until stinging (inlet B).

<p>Control groups are termites or mealworms coated with water. Boxplots show the median and the interquartiles of time from first ant contact with a termite or mealworm until stinging. Coated insects were stung significantly later than control insects.</p

Histopathological analysis of organs from melphalan, vehicle control treated and untreated mice confirms <i>in vivo</i> BLI data.

<p>(<b>A</b>) Representative H&E stainings from organs harvested at day +33 after tumor inoculation from the different treatment groups. The hematopoietic compartments BM and spleen displayed clear infiltration of MOPC-315.BM luc⁺ cells, while MOPC-315.BM luc⁺ cells in lung and liver resided within the blood vessels as indicated by arrows. Scale bar is 50 µm for all shown sections (original magnification 200X/0.70 NA) and 20 µm for inserts (original magnification 400X/0.85 NA). (<b>B</b>) Determination of the percentage of MOPC-315.BM luc⁺ cells within BM and spleen and (<b>C</b>) number of cells per high power field (HPF) in lung and liver significantly correlates with the melphalan treatment and thereby confirms <i>in vivo</i> and <i>ex vivo</i> imaging data.</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

Detection of residing MOPC-315.BM luc⁺ cells and <i>in vivo</i> monitoring of reduced myeloma progression due to melphalan treatment.

<p>BALB/c wild type mice were injected with 1×10⁵ MOPC-315.BM luc⁺ cells via the tail vein. On day +19 after inoculation, tumors were established in all mice and readily detected by BLI. Then treatment was started ( = day 0 of treatment). Mice received 5 mg/kg melphalan (n = 9, two independent experiments) or mock treatment (vehicle control, n = 13, three independent experiments) intraperitoneally. One control group of MOPC-315.BM luc⁺ tumor bearing mice did not receive any treatment (untreated control, n = 14, three independent experiments). (<b>A</b>) Schematic study design, indicating treatment time points in respect to time after MM injection and end of experiment. Red arrows pinpoint melphalan treatment. (<b>B</b>) BLI images of two representative mice per group at selected time points in ventral (left) and dorsal (right) view. (<b>C</b>) Quantification of bioluminescence signal intensities over time from ventral or dorsal. Signals at day +19 were set as starting point and the following measurements were calculated as fold change of this initial signal intensity. Mice were treated at time points as indicated by arrows. Significant difference between melphalan treated mice vs vehicle control or vs untreated animals starting on day 10 of treatment for both, ventral (untreated vs melphalan p<0.0001, vehicle vs melphalan p = 0.0032) and dorsal (untreated vs melphalan p = 0.0006, vehicle vs melphalan p = 0.0024). (<b>D</b>) Quantification of skeletal tumor foci in untreated, vehicle control and melphalan treated mice on day 0 and 14 of drug treatment.</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

Loss of host TNFR1 perturbs the immunologic control of Panc02 tumors.

<p>Panc02-tumors were explanted one month after tumor cell inoculation, sectioned, and stained for different immune cells and blood vessels (B6.WT n = 4, B6.TNF KO n = 5, B6.TNFR1 KO n = 5, B6.TNFR2 KO n = 6, B6.TNFR1R2 KO n = 4).</p>*<p>p≤0.05, ** p≤0.01.</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