Research ArticleCancer

The mitotic kinesin KIF11 is a driver of invasion, proliferation, and self-renewal in glioblastoma

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Science Translational Medicine  09 Sep 2015:
Vol. 7, Issue 304, pp. 304ra143
DOI: 10.1126/scitranslmed.aac6762
  • Fig. 1. Expression of mitotic kinesins, including KIF11, is up-regulated in GBM.

    (A) Differential expression analysis by RNA-seq from radiographically localized biopsies of the NE and CE regions of human GBM surgical specimens. (B) Correlation of cell type–specific marker genes with KIF11 expression. (C) Quantitative PCR was performed for the listed mitotic regulators on RNA isolated from TICs and matched non-TICs from three patient-derived xenografts.

  • Fig. 2. KIF11 is increased in TICs because of attenuated protein turnover.

    (A) Whole-cell lysates from matched TICs and non-TICs from three patient-derived xenografts were probed for KIF11, OLIG2, and GFAP. (B) TICs and non-TICs from xenograft specimen 08-387 were synchronized at G1/S using a double-thymidine block. After release, whole-cell lysates were made every 2 hours over a 24-hour time course. Asynchronous (A) lysates were also harvested. Resulting lysates were probed for KIF11. (C) TICs and non-TICs from xenograft specimen 3691 were synchronized at M phase using a nocodazole block. Lysates were made 4 hours after release at late M and early G1, and APC/CCdh1 activity was evaluated using exogenous HA-securin as a substrate. Samples for immunoblot analysis were taken every 30 min over a 90-min time course and probed for HA. For all immunoblots, β-actin served as a loading control. The molecular weight (MW) of resulting bands is given in kilodaltons. (D) Quantification of band intensity, normalized to actin.

  • Fig. 3. KIF11 inhibition targets viability and self-renewal.

    (A) Matched TICs and non-TICs from patient-derived xenografts (n = 5, with three technical replicates per specimen) were exposed to increasing concentrations of ispinesib (0 to 32 nM) for 72 hours, followed by analysis of cell viability using an ATP-based assay. Shown are means ± SD; P = 0.0085, two-tailed t test. (B) Matched TICs and non-TICs from three patient-derived xenografts were evaluated for the percentage of sub-G1 (apoptotic) cells over a 4-day time course after a single exposure to vehicle [dimethyl sulfoxide (DMSO)] or 3 nM ispinesib at day 0. (C) Matched TICs and non-TICs from two patient-derived xenografts were monitored for cell viability with an ATP-based assay over a 5-day time course after a single exposure to vehicle (DMSO) or ispinesib. Shown are means ± SD; n = 3 biological replicates (with three technical replicates per biological replicate); P < 0.01 or P < 0.0001, one-way analysis of variance (ANOVA) with a Bonferroni posttest. (D) TICs were isolated from xenografts 3565, 3691, and 08-387 (three biological replicates for each xenograft) and plated from 50 down to 1 cell per well. Wells were scored 10 days later for the presence of a tumorsphere. Representative results from the third xenograft harvested for each specimen are shown.

  • Fig. 4. KIF11 inhibition blocks glioma cell motility and brain invasion.

    (A) Schematic of Transwell assay. TICs from patient-derived xenograft 08-387 were enriched within interphase using a thymidine arrest and release paradigm. Interphase-enriched cells were plated on a Matrigel-coated Transwell (125,000 per well) and given 8 hours to migrate in the presence of increasing concentrations of ispinesib (0 to 200 nM) before fixation and staining of the nuclei with DAPI. (B) Resulting membranes were scored for the movement of nuclei through the Transwell membrane. Shown are means ± SD; n = 3 biological replicates (with three technical replicates per biological replicate). (C) The Transwell assay was run as above with vehicle (DMSO) or 200 nM ispinesib, with data represented as the number of migrated cells per field. Shown are means ± SD; n = 3 biological replicates (with three technical replicates per biological replicate); P = 0.0009, unpaired t test. (D) Schematic of slice culture assay. A PDGF–IRES (internal ribosomal entry site)–GFP retrovirus was used to generate tumors in a rat glioma model. Resulting tumor-bearing brains were isolated to generate slice cultures. GFP-positive tumor cells were monitored by time-lapse video microscopy over a 10-hour time course. (E) Wind rose plots of tumor cell dispersion (55 cells per group). Individual cell tracks from time-lapse microscopy were plotted to a common origin to generate wind rose plots for displaying the dispersion of tumor cells in the presence of DMSO vehicle (left, black) or 200 nM ispinesib (right, red). (F) MSD versus time for vehicle (black) and 200 nM ispinesib (red) conditions. MSD was calculated for 55 cells each from control and ispinesib conditions for each time interval over 9.5 hours of observation. Black and red curves depict MSD (±1 SD) versus time, and superimposed white curves depict fitting of the data to a persistent random walk model using a nonlinear least-squares regression.

  • Fig. 5. KIF11 plays a role in cellular process formation.

    (A) Nascent cellular process formation was monitored for 6 hours by time-lapse microscopy in the presence of vehicle (DMSO) or ispinesib (200 nM) in 08-387 TICs plated on Geltrex and enriched in interphase. Representative images of the two treatment groups are shown at the 6-hour time point. Cell bodies are masked in red, and cell processes are marked in yellow. Scale bars, 10 μm. (B) Process length was measured over the time course. Shown are means ± SD; n > 500 cells per condition from three biological replicates; P < 0.0001, two-way ANOVA with a Bonferroni posttest. (C) Representative images from a modified scratch wound assay used to drive formation of a leading cellular process in 08-387 TICs enriched in interphase. Just before wound formation, medium was changed to that containing vehicle (DMSO) or ispinesib (200 nM). Six hours later, cells were fixed and processed for immunofluorescence to β-tubulin. (D) Anti-tubulin fluorescence signal from confocal images of the primary cell layer adjacent to the wound was quantified for both treatment groups and presented as signal over area. Horizontal bar represents the mean and error bars represent SD; n > 40 cells per condition; P < 0.0001, unpaired t test.

  • Fig. 6. KIF11 inhibition targets TICs in vivo and compromises tumor initiation.

    (A) Mice were injected in the flank with 100,000 3691 GBM cells. When tumors reached about 0.12 cm3, mice were randomized into one of two treatment groups: vehicle only (DMSO; n = 5) or ispinesib (10 mg/kg; n = 5), with daily administration over a 7-day time course. Tumor volume was measured daily. Shown are means ± SD; P < 0.01, P < 0.001, or P < 0.0001, one-way ANOVA with a Bonferroni posttest. (B) Tumors were removed at day 7 from both groups to calculate the final weight. Shown are means ± SD. (C) Tumors isolated on day 7 were processed for immunofluorescence with the stem cell marker SOX2 (green), and nuclei were counterstained with DAPI (blue). Scale bars, 20 μm. (D) TICs from patient-derived xenograft 3691 were pretreated in vitro for 18 hours with vehicle or 3 nM ispinesib. Viable cells (2500 or 25,000) were intracranially (IC) injected (DMSO, n = 10; ispinesib, n = 7). Mice were monitored for signs indicative of brain tumor development, at which time they were sacrificed. Three mice from each group were harvested at the time of first indication of neurological signs among the groups, which occurred in the vehicle group. Representative H&E images are shown. White arrowheads indicate tumor. P = 0.012 for the 2500 cohort and P = 0.020 for the 25,000 cohort (log-rank test). (E) Mice were injected in the flank with 100,000 3691 GBM cells. When tumors reached 0.2 to 0.6 cm3, mice were randomized into one of two treatment groups: vehicle only (DMSO; n = 5) or ispinesib (10 mg/kg; n = 5), with daily administration over a 3-day time course. Tumors were harvested and viable TICs were isolated. Viable cells (1000 or 10,000) were intracranially injected into mice, which were then monitored for signs indicative of brain tumor development, at which time they were sacrificed. P = 0.0019 for the 1000 cell cohort and P = 0.021 for the 10,000 cell cohort (log-rank test).

  • Fig. 7. KIF11 informs patient prognosis, and targeting improves survival in a preclinical model.

    (A) A mouse bearing an orthotopic tumor was injected with a single dose of ispinesib (10 mg/kg). The tumor was isolated 5 hours later and processed for immunofluorescence to β-tubulin with nuclei counterstained with DAPI. Arrowheads indicate cells with monoastral spindles. a′ and a″ represent enlarged regions of interest. Scale bars, 10 μm. (B) TICs (10,000) from patient-derived xenograft 3691 modified to express luciferase were intracranially implanted into mice. Seven days later, when positive luminescence signal indicated tumor burden, mice were randomized into one of two treatment groups: vehicle only (DMSO; n = 10) or ispinesib (10 mg/kg; n = 10), administered every 4 days for six doses. Arrows indicate day of vehicle or drug administration. Mice were monitored for signs indicative of brain tumor development, at which time they were sacrificed to generate a Kaplan-Meier survival curve. P < 0.001, log-rank test. (C) KIF11 protein expression was scored on a tissue microarray containing normal brain control (n = 9) and patient specimens from WHO grade II (n = 25), grade III (n = 22), and grade IV (n = 47) gliomas. P = 0.0001, one-way ANOVA with post hoc Tukey’s test. (D) Kaplan-Meier curves of patients with grade III or grade IV gliomas stratified by low or high KIF11 expression (anti-KIF11 score). P = 0.047, log-rank test. (E) Representative H&E and immunohistochemistry (IHC) for KIF11 in normal brain and a GBM specimen. Scale bars, 20 μm. (F) Representative signal for KIF11 within the mitotic spindle of GBM cells. Scale bars, 10 μm.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/304/304ra143/DC1

    Materials and Methods

    Fig. S1. iPAGE gene ontology analysis of the Spearman correlation for KIF11 in the NE and CE regions.

    Fig. S2. Subtype analysis for patient-derived xenograft specimens.

    Fig. S3. Synchronization of TICs and non-TICs.

    Fig. S4. Stem cell frequency after KIF11 inhibition.

    Fig. S5. Representative cell synchronization histograms.

    Fig. S6. Weights of mice during the orthotopic preclinical study.

    Table S1. Patient data correlating with the Kaplan-Meier survival curves.

    Movie S1. Representative movie from an untreated organotypic slice culture from a rodent glioma model.

    Movie S2. Representative movie from an ispinesib-treated organotypic slice culture from a rodent glioma model.

    References (5153)

  • Supplementary Material for:

    The mitotic kinesin KIF11 is a driver of invasion, proliferation, and self-renewal in glioblastoma

    Monica Venere, Craig Horbinski, James F. Crish, Xun Jin, Amit Vasanji, Jennifer Major, Amy C. Burrows, Cathleen Chang, John Prokop, Quilian Wu, Peter A. Sims, Peter Canoll, Matthew K. Summers, Steven S. Rosenfeld,* Jeremy N. Rich*

    *Corresponding author. E-mail: rosenfs{at}ccf.org (S.S.R.); richj{at}ccf.org (J.N.R.)

    Published 9 September 2015, Sci. Transl. Med. 7, 304ra143 (2015)
    DOI: 10.1126/scitranslmed.aac6762

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. iPAGE gene ontology analysis of the Spearman correlation for KIF11 in the NE and CE regions.
    • Fig. S2. Subtype analysis for patient-derived xenograft specimens.
    • Fig. S3. Synchronization of TICs and non-TICs.
    • Fig. S4. Stem cell frequency after KIF11 inhibition.
    • Fig. S5. Representative cell synchronization histograms.
    • Fig. S6. Weights of mice during the orthotopic preclinical study.
    • Table S1. Patient data correlating with the Kaplan-Meier survival curves.
    • Legends for movies S1 and S2
    • References (5153)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Representative movie from an untreated organotypic slice culture from a rodent glioma model.
    • Movie S2 (.mov format). Representative movie from an ispinesib-treated organotypic slice culture from a rodent glioma model.

    [Download Movie S1]

    [Download Movie S2]

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