Semapimod removes microglia-induced radioprotection on GL261 <i>in vitro</i>.

<p>GL261 cells were cultured in the presence or absence of microglia and in the presence or absence of 200 nM of semapimod, followed by a colony formation assay. (A) Colony formation assay of GL261 after treatment with microglia and semapimod. (B) Determination of survival of GL261 after treatment with 3 Gy irradiation, microglia and semapimod. Data shown represent the average +/− SEM of 3 independent experiments. *: p<0.05 student's 2 tailed t-test.</p

Semapimod does not affect microglia-stimulated growth of glioblastoma cells <i>in vitro</i>.

<p>GL261 cells were cultured in the presence or absence of microglia at the indicated ratio's, in the presence and absence of 200 nM semapimod. Cell growth over a period of 3 days was determined using the SRB method. Data shown represent the average +/− SEM of 3 independent experiments. *: p<0.05, **: p<0.01 student's 2 tailed t-test.</p

Semapimod inhibits glioblastoma cell invasion <i>in vivo</i>.

<p>GL261 cells were orthotopically implanted into C57Bl/6 mice. Starting 7 days after cell inoculation, the mice were treated intracranially for 1 week with semapimod, delivered via an osmotic pump. (A) Micrographs of tumor sections illustrating inhibition of GL261 cell invasion by semapimod. GL261 cells were visualized using Ki67 staining. The tumor borders are outlined. Scale bar represents 200 µm. (B) Quantification of the number of invaded GL261 cells normalized to the length of the tumor border (expressed in mm). Data shown represent the average +/− SEM of 5 different tumors. **: p<0.01 student's 2 tailed t-test. (C) Micrographs illustrating infiltration of microglia into GL261 tumors. Activated microglia were visualized using Iba1 staining. (D) Quantification of Iba1⁺ microglia infiltrated into the tumor. Scale bar represents 100 µm. Data shown represent the average +/− SEM of 5 different tumors.</p

Semapimod inhibits tumor growth <i>in vivo</i>.

<p>GL261 cells were implanted and mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095885#pone-0095885-g004" target="_blank">Fig. 4</a>. (A) Quantification of tumor volume. Data shown represent the average +/− SEM of 5 different tumors. (B) Quantification of tumor cell density. Data shown represent the average +/− SEM of 5 different tumors. (C) Quantification of total tumor cell number. Data shown represent the average +/− SEM of 5 different tumors. *: p<0.05 student's 2 tailed t-test.</p

Semapimod increases apoptosis induced by radiation <i>in vivo</i>.

<p>GL261 tumors were generated in mice and treatments were carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095885#pone-0095885-g006" target="_blank">Fig. 6</a>, except that the total radiation was 8 Gy. The mice were euthanized the day after the last radiation treatment. Frozen brain sections were analyzed for apoptosis using TUNEL staining as described in Materials and Methods. (A) Micrographs of tumor sections illustrating TUNEL staining. (B) Quantification of TUNEL staining. The percentage of apoptotic cells was calculated using 20–60 micrographs per tumor section. Data shown represent the average +/− SEM of 3–4 different tumors. *: p<0.05 student's 2 tailed t-test.</p

Semapimod inhibits microglia-stimulated glioblastoma invasion <i>in vitro</i>.

<p>(A) Semapimod inhibits microglia-stimulated glioblastoma cell invasion. GL261 cells were embedded in basement membrane extract (BME) with or without microglia in the presence of the indicated concentrations of semapimod, layered in the transwell and incubated for 48 h. The total number of invading GL261 cells was determined and normalized to that of GL261 cells in monoculture. Data shown represent the average +/− SEM of 3 independent experiments, performed in duplicate. (B) Semapimod does not affect serum-stimulated glioblastoma cell invasion. GL261 cells embedded in BME in the presence of the indicated concentration of semapimod, were layered in the transwell and incubated for 48 h. Serum (10% FBS) was added to the bottom well. The total number of invading GL261 cells was determined and normalized to that of GL261 cells in the absence of drug. Data shown represent the average +/− SEM of 3 independent experiments. (C) Semapimod inhibits glioblastoma cell-stimulated microglia invasion. Glioblastoma cells were plated in the bottom well and microglia were embedded in BME in the presence or absence of 200 nM semapimod, layered in the transwell and incubated for 48 h. The total number of invading microglia cells was determined. Data shown represent the average +/− SEM of 3 independent experiments. *: p<0.05, **: p<0.01 student's 2 tailed t-test.</p

Semapimod increases survival of glioblastoma-bearing mice in conjunction with ionizing radiation.

<p>C57Bl/6 mice were injected orthotopically with GL261 cells. Starting 7 days after cell inoculation, the mice were treated intracranially for 2 weeks with semapimod, delivered via an osmotic pump. Starting on day 8, animals were given 2 Gy whole brain irradiation every other day over a period of 10 days (10 Gy total). *: p<0.05. Chi square test, with 3 degrees of freedom.</p