Transient treatment with ROCK inhibitors is sufficient to promote GSC-like cell expansion.

<p>The cells were treated with no inhibitor, with continuous exposure to the ROCK inhibitor (45 μM Y-27632 or 10 μM fasudil), or with transient exposure to the ROCK inhibitor (45 μM Y-27632 or 10 μM fasudil). (A) Representative micrographs of each experimental group on Day 3 (Scale bars = 100 μm). (B) The sphere diameter and number of spheres were analyzed for all experimental groups on Day 3 (mean ± SE; <i>n</i> = 100; * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001). The number of spheres per field of view in each experimental group were also quantified from the micrographs (mean ± SE; <i>n</i> = 20; * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001).</p

ROCK inhibitors enhance GSC-like stemness.

<p>(A) The clonogenicity of the cells was quantified using limiting dilution assay (steeper slope and lower value in x-intercept indicates increased clonogenic potential and stemness). The cells treated with 45 μM Y-27632 or 10 μM fasudil required fewer cells to form spheres indicating increased number of GSC-like cell than control. (B) The glioblastoma cells were grown as tumorspheres in two concentrations of Y-27632 (0 and 45μM) or two concentrations of fasudil (0 and 10 μM) for three days. Using flow cytometry, the percentage of the total population expressing the GSC marker SOX2 was quantified.</p

Knockdown of ROCK2 shows similar behavior to Y-27632 and Fasudil.

<p>U87-MG cells were transfected with ROCK2 siRNA and grown as tumorspheres for 3 days. The cells’ ability to form spheres was analyzed, and qRT-PCR was performed to confirm the success of the transfection. A) Representative micrographs of each experimental group on Day 1 (Scale bar = 100 μm). B) The sphere diameter (mean ± SE; <i>n</i> = 100; * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001) and number of spheres per field of view were analyzed for all experimental groups on Day 1 (mean ± SE; <i>n</i> = 20; * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001). C) qRT-PCR was performed on the transfected cells to confirm their gene expression levels of <i>ROCK2</i>, <i>CASP3</i>, and <i>CASP7</i>. Expression is reported as percentage of that of negative control (mean ± SE; <i>n</i> = 3; * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001).</p

ROCK inhibitors enhance GBM tumorsphere formation.

<p>The glioblastoma cells were grown as tumorspheres in two concentrations of Y-27632 (0 and 45 μM) or two concentrations of fasudil (0 and 10μM) for 6 days. (A) Sample micrographs of each experimental group on Day 6 (Scale bars = 100 μm). (B) The sphere diameter and number of spheres were analyzed for all experimental groups on Day 3 (mean ± SE; <i>n</i> = 100). It was found that the sphere diameter stayed relatively consistent between the experimental groups. The number of spheres per field of view in each experimental group were also quantified from the micrographs (mean ± SE; <i>n</i> = 20; * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001).</p

ROCK inhibitors are not toxic to GBM tumorspheres at low concentrations.

<p>The toxicities of Y-27632 and fasudil were measured using a water-soluble tetrazolium assay (WST-8 Cell Counting Kit 8). U87-MG, JX12, and SMC448 cells were exposed to varying concentrations of Y-27632 or fasudil for 48 hours. Cell viability was measured relative to 0 μM control (<i>n</i> = 10).</p

ROCK inhibitors protect GBM tumorspheres from apoptosis.

<p>Flow cytometry was used to quantify the late-stage apoptotic cells (Annexin V⁺/PI⁺) immediately after trituration. The cells that were treated with 45μM Y-27632 or 10 μM fasudil had decreased number of late-stage apoptotic cells in U87-MG, JX12, and SMC448 cell lines, indicating that the ROCK inhibitors Y-27632 and fasudil inhibited apoptosis in glioblastoma cells.</p

Flow cytometric evaluation of the γδ T cell receptor (TCR) repertoire in control vs. early and late stage tumor-bearing mice (distribution on left panels, x ± SD, right panels, * = p<0.05).

<p>There were no significant changes in the Vγ1, Vγ4, or Vγ7 subsets from controls either post-tumor injection day 11±1 or at end stage. Additionally, there were no changes in the proportions of Vδ1, Vδ4, and Vδ6.3 at day 10. A significant decrease of the %Vδ6.3 population at end stage was offset by a parallel increase in %Vδ4 cells. TCR subsets Vγ3 and Vδ3 were also measured, however, relative proportions were negligible <i>(data not shown)</i>.</p

Cytotoxicity and growth inhibition assessment of activated γδ T cells against GL261 tumors.

<p><i>(a)</i> Activated γδ T cells were incubated with GL261 cells either in suspension (closed circles) or attached to plastic (closed squares) show robust <i>in vitro</i> cytotoxicity against the non-adherent GL261 cells in a 4h standard assay (x ± SD, * = p<0.05). The insert is a high-power confocal micrograph of adherent GL261 cells cytoplasm stained with wheat germ agglutinin (green) showing microfilopodia spreading from the attached cells as well as tumor from an athymic nude mouse was harvested 10 days following GL261 cells, chopped and disaggregated in media, fixed/post-fixed in in glutaraldehyde and osmium tetroxide, dehydrated in ethanol, and embedded in epoxy resin. Filopodia (clear arrows) and microspikes (filled arrows) are seen on the cell surface in these transmission electron micrographs from tumor sections. <i>(b)</i> Injection of 5 x 10⁵ GL261 cells followed 15m later by 1.5 x 10⁶ activated γδ T cells (dashed line) did not improve median survival when compared to saline-injected mice (solid line). <i>(c)</i> Evidence of local tumor inhibition was noted in histologic examination. Representative histologic specimens show a much smaller tumor at the injection site (arrow) in the γδ T cells injected mouse (B) than from a control mouse (A) at 25 days.</p

Circulating γδ T cell enumeration and function in tumor-bearing mice.

<p><i>(a)</i> The circulating T cell count (distribution on left panels, x ± SD, right panels, * = p<0.05) was increased in tumor-bearing mice at 10 days post-GL261 injection independent of the total T cell count. Terminal γδ T cell counts in tumor-bearing mice also fell significantly lower than controls and day 10 glioma-bearing mice. <i>(b)</i> Approximately 12% of γδ T cells constitutively produced IFN-γ with no additional increase following PMA/Ionomycin stimulation. <i>(c)</i> Annexin V expression was upregulated on γδ T cells from glioma-bearing mice, also independently from the total T cell population indicating simultaneous γδ T cell proliferation and apoptosis likely due to activation-induced cell death (AICD). <i>(d)</i> The γδ T cell count and Annexin V expression were measured in unmanipulated (e.g. no intracranial injection) mice and 10 days following IC injection of the methylcellulose vehicle in which GL261 cells were suspended to determine if IC injection-related injury produced the same increase in γδ T cell count and Annexin V expression as tumor injection. There was no difference in either parameter between sham-injected mice and mice that received intracranial methylcellulose vehicle alone.</p