Mouse weights by treatment regimen.

<p>Average mouse weights with standard deviations are reported according to treatment regimen, where weights were normalized to day zero of treatment (100%): <b>A</b>) mouse weights on saline, continuous-treatment (green line); <b>B</b>) mouse weights on 2mg/kg YM155, continuous-treatment (purple line); and <b>C</b>) mouse weights on 4mg/kg YM155, continuous-treatment (orange line). Mouse weights were adjusted to remove the weight of tumors prior to normalization. Weights from mice with significant liver metastases were not included as metastatic-tumor weights could not be determined during the course of treatment. </p

Various chemotherapeutics combined with YM155 induce MCC cell death in an additive manner, <i>in</i><i>vitro</i>.

<p>CellTiter-GLO assays were performed using multiple MCC cell lines as well as the control primary human fibroblast, BJ. Corresponding dose-response curves are shown for the following chemotherapeutic agents and drug combinations: <b>A</b>) YM155; <b>B</b>) Bortezomib; <b>C</b>) Bortezomib + 3nM YM155; <b>D</b>) Docetaxel; <b>E</b>) Docetaxel + 3nM YM155; <b>F</b>) Etoposide; <b>G</b>) Etoposide + 3nM YM155 <b>H</b>) Topotecan; and <b>I</b>) Topotecan + 3nM YM155. </p

Time-to-Palpability.

<p>The length of time lapsed after initial cell line injection to detection of palpable tumors (~2mm x 2mm) is indicated for each of the four MCC cell lines tested (MKL-1, WaGa, MKL-2, and MS-1). </p

Immunohistochemistry of MCV-LT in a MKL-1 xenograft primary tumor and a liver metastasis.

<p>Shown are paired hemotoxylin & eosin (H&E) stained slides and adjacent sections stained with CM2B4, the MCV-LT antibody (LT-IHC), in mice with MKL-1 xenografts: <b>A</b>) MKL-1 xenograft primary tumor, H&E; <b>B</b>) MKL-1 xenograft primary tumor, LT-IHC; <b>C</b>) MKL-1 xenograft liver metastasis, H&E; and <b>D</b>) MKL-1 xenograft liver metastasis, LT-IHC. MKL-1 cells contains nuclear staining of LT, consistent with an intact nuclear localization signal (NLS). Original magnification = 200X; insets = 600X. </p

MCC mouse xenograft treatment groups and experimental outline.

<p><b>A</b>) NSG mice were subcutaneously injected in the right flank with 2x10⁷ MCV-positive, MCC cells (MKL-1, MS-1, WaGa, or MKL-2). <b>B</b>) NSG mice were monitored for palpable tumors (~2mm x 2mm) to determine start of treatment. <b>C</b>) Mice with palpable tumors were randomly assigned to either saline treatment, YM155 treatment for 3-weeks at 2mg/kg, YM155 continuous treatment at 2mg/kg, or YM155 continuous treatment at 4mg/kg. Each week of treatment consisted of a single intraperitoneal injection per day for 5 days, followed by 2 days of rest. </p

Kaplan-Meier curves of multiple MCC mouse xenograft models on different treatments.

<p><b>A</b>) Estimated survival means and 95% confidence intervals are reported along compressed survival summaries per cell line and treatment arm, where open circles correspond survival of individual mice. <b>B</b>) Mice with MKL-1 xenografts exhibit significantly prolonged survival (****P < 0.0001) on any of the three YM155 treatment groups (3-weeks at 2mg/kg = red; continuous treatment at 2mg/kg = purple; continuous treatment at 4mg/kg = orange) relative to saline treatment (green). Increasing the duration of YM155 treatment from 3-weeks to continuous treatment at the 2mg/kg dose significantly prolongs survival (****P < 0.0001). Increasing the dose of YM155 from 2mg/kg to 4mg/kg on continuous treatment significantly prolongs survival (****P < 0.0001). <b>C</b>) Mice with MS-1 xenografts do not exhibit prolonged survival with YM155 continuous treatment (either at 2mg/kg or 4mg/kg) relative to saline treatment (NS = not significant). One mouse on saline treatment spontaneously regressed for over 5-weeks and was euthanized early (as indicated by <b>x</b>). <b>D</b>) Mice with WaGa xenografts exhibit significantly prolonged survival (**P = 0.0034) with continuous YM155 treatment at 4mg/kg relative to saline treatment. <b>E</b>) Mice with MKL-2 xenografts exhibit significantly prolonged survival (****P < 0.0001) with continuous YM155 treatment at 4mg/kg relative to saline treatment. Two mice did not reach the final 20mm tumor dimension by day 105 and were euthanized early (as indicated by <b>##</b>). </p

TLR7 and TLR9 antagonist blocks transient IFN-α production by pDC in lymph nodes without suppressing recruitment.

<p>(A) Percent of pDC in lymph nodes (left) and percent of pDC labeling with antibody to BrdU (right) in untreated (n = 5) and DV056-treated (n = 4) macaques. Horizontal lines represent means. **<i>P</i><.01. (B) Immunofluorescence of lymph node sections taken from naive and SIV-infected macaques with and without DV056 treatment at days 14 and 56 post infection stained with antibody to IFN-α or TNF-α (red) and co-stained with Hoescht dye (blue) to identify nuclei. Insets in naïve tissue sections represent immunofluorescence after labeling with isotype control antibody. Insets in SIV-infected sections show higher magnification images to highlight details of individual cells. Original magnification 400×. (C) Frequency of IFN-α and TNF-α producing cells in paracortex and parafollicular cortex from naive macaques (n = 2) and macaques at day 14 and 56 post infection with (n = 4 for IFN-α, n = 3 for TNF-α) and without (n = 5 for IFN-α, n = 3 for TNF-α) DV056 treatment. **<i>P</i><.01. (D) Immunofluorescence of lymph nodes from untreated and DV056-treated macaques at day 14 post infection stained with antibody against CD123, CD1a or CD163 (green) and IFN-α or TNF-α (red) and co-stained with Hoescht dye (blue). Arrowheads indicate cells co-labeling with cytokine- and cell-specific markers. Original magnification 400×.</p

mDC gain the capacity to secrete IFN-α upon virus stimulation following SIV infection.

<p>(A) Dot plots showing intracellular IFN-α expression in blood mDC from untreated and DV056-treated macaques at day 0 and day 14 post infection after iSIV stimulation. Data for all animals are shown, along with a representative dot plot of cells exposed to control microvesicles. Flow cytometric gating to define mDC was done as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003530#ppat-1003530-g001" target="_blank">Figure 1</a>. (B) Percent of blood mDC producing IFN-α after iSIV stimulation at the indicated times after infection in untreated and DV056-treated macaques. *<i>P</i><.05, **<i>P</i><.01.</p

TLR7 and TLR9 blockade does not impact virus load or pDC kinetics.

<p>(A) SIV RNA copies/ml of plasma for untreated (n = 5) and DV056-treated (n = 4) macaques over time as determined by quantitative RT-PCR. Shown are virus loads for each animal in untreated and DV056-treated groups as well as geometric means for each group. (B) Absolute counts of pDC in blood for untreated and DV056-treated macaques over time. Shown is the percent change in cell counts relative to preinfection levels for each animal in untreated and DV056-treated groups as well as means for each group. (C) Percent of CCR7+ pDC in blood over time. Shown is the percent pDC expressing CCR7 before and after SIV infection for each animal in untreated and DV056-treated groups as well as the means for each group.</p

In vivo administration of TLR7 and TLR9 antagonist to rhesus macaques blocks pDC responses to virus stimulation.

<p>DV056 was administered by weekly subcutaneous injection at 2 mg/kg to SIV-naive rhesus macaques, and PBMC and lymph node cell suspensions collected 3 days after administration were stimulated with iSIV and influenza virus to assess TLR7 and TLR9 blockade. (A) pDC production of IFN-α and TNF-α in PBMC and lymph node cell suspensions from untreated and DV056-treated macaques. Flow cytometric gating was done as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003530#ppat-1003530-g001" target="_blank">Figure 1</a>. (B) The percent of pDC in PBMC (left) and lymph node cell suspensions (right) from DV056-treated macaques (n = 4) compared to untreated macaques (n = 12 for PBMC, n = 8 for lymph node) producing IFN-α and TNF-α in response to iSIV and influenza virus stimulation. Each symbol represents an individual animal and horizontal lines represent means. **<i>P</i><.01.</p