Supplemental Data. Integrin Alpha 6 Regulates Glioblastoma Stem Cells. Supplemental Data Inventory Supplemental Data

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1 Cell Stem Cell, Volume 6 Supplemental Data Integrin Alpha 6 Regulates Glioblastoma Stem Cells Justin D. Lathia, Joseph Gallagher, John M. Heddleston, Jialiang Wang, Christine E. Eyler, Jennifer MacSwords, Qiulian Wu, Amit Vasanji, Roger E. McLendon, Anita B. Hjelmeland, and Jeremy N. Rich Supplemental Data Inventory Supplemental Data Supplemental Experimental Procedures Supplemental Figure These data relate to Figure which shows integrin alpha 6 expression in GBM surgical specimens. The distance of an integrin alpha 6 positive cell was calculated relative to a blood vessel in comparison to all cells within a GBM surgical biopsy specimen (Supplementary Fig. A). Also included is double staining with integrin alpha 6 and nestin in GBM surgical biopsy specimens (Supplementary Fig. B,C) as well as integrin alpha 6 and CD33 staining in parental GBM specimens 3359 and 369 (Supplementary Fig. D, E) of which xenografts were established and used for this study. Supplementary Table and Supplementary Figure These data relate to Figure which shows integrin alpha 6 expression in GBM xenografts and the potential overlap of expression with CD33. Flow cytometry data for expression of CD33 and integrin alpha 6 in xenografted GBM cells is shown as a table (Supplementary Table ) and as a histogram (Supplementary Fig. ). Supplementary Figure 3 These data relate to Figures 3 and 4 which demonstrate integrin alpha 6 coexpression with putative GSC markers and appropriate co-receptor and ligand in tumorsphere cultures GSCs. Supplementary Figure 3 expands on these observations and demonstrates that integrin alpha 6 expression correlates with cells in active m-phase as marked by phospho histone H3 and does not overlap with the neuronal marker, Map, or dying cells as assessed by the TUNEL assay in the core of the tumorspheres. Supplementary Figure 4 These data relate to Figure 5 which demonstrates that integrin alpha 6 alone or in combination with CD33 can be used to enrich for cells with GSC properties (proliferation, tumorsphere formation. Supplementary Figure 4 shows the ability for integrin alpha 6 to enrich for cells which possess GSC properties (proliferation, tumorsphere formation) in a tumor with little to no CD33 expression. Supplementary Figure 5 and Supplementary Table These data relate to Figure 5 which shows integrin alpha 6 positive cells have potential to form tumorspheres and generate tumors in vivo at a greater rate than integrin alpha 6 negative cells. The ability for an integrin alpha 6 positive GBM cell to undergo multilineage differentiation is shown in Supplementary Figure 5. Supplementary Figure 6 and Supplementary Table 3 - These data relate to Figure 7 which shows a loss of GSC properties when integrin alpha 6 is targeted with an delivered lentivirus. Supplementary Figure 6 expands on the finding shown in Figure 7 and demonstrates various aspects related to the integrin alpha 6 knockdown. In Supplementary Figure 6A, the efficiency of knockdown of integrin alpha 6 is demonstrated in three separate GSC populations using flow cytometry. Various aspects of cell behavior are demonstrated to change with integrin alpha 6 knockdown in GSCs: tumorsphere formation decrease (Supplementary Fig. 6B, C), an increase in G and decrease in the S phase of the cell cycle (Supplementary Fig. 6D, E), an increase in cell death as assessed by a Caspase3/7 assay (Supplementary Fig. 6F, G). A similar phenotype is seen when integrin alpha 6 is knocked down in GSC derived from integrin alpha 6+/CD33- cells: decrease in tumorsphere formation (Supplementary Fig. 6H), decrease in proliferation as assessed by an ATP assay (Supplementary Fig. 6I), an increase in G and decrease in the S phase of the cell cycle (Supplementary Fig. 6J), an increase in cell death as assessed by a Caspase3/7 assay (Supplementary Fig. 6K)

2 or a DNA fragmentation assay (Supplementary Fig. 6L). Finally, integrin alpha 6 expression is detected in tumors forming from integrin alpha 6 knockdown cells (Supplementary Fig. 6M). Supplementary Table 3 displays the in vivo study results when integrin alpha 6 is knocked down in GSCs and then transplanted intracranially into mice. Supplementary Figure 7 and Supplementary Table 4 - These data relate Figure 8 which show that an integrin alpha 6 blocking antibody can be used to induce a loss of GSC properties. Supplementary Figure 7 expands on this finding and demonstrates that integrin alpha 6 expression inversely correlates with patient survival using a patient expression database (REMBRANDT, Supplementary Fig. 7). In addition, Supplementary Table 4 displays the in vivo study results when GSCs are treated with the integrin alpha 6 blocking antibody and then transplanted intracranially into mice.

3 Supplemental Experimental Procedures Isolation of Glioblastoma Stem Cells All tumor specimens were derived from primary glioblastoma surgical biopsy specimens obtained from patients undergoing resection for newly diagnosed or recurrent GBM in accordance with protocols approved by the Duke University Medical Center or Cleveland Clinic Foundation Institutional Review Boards. Written consent to utilize excess tissue for research was obtained from each patient, and de-identified tissues were used for all studies. Tumors were dissociated to single cells by a papain dissociation kit (Worthington Biochemical) as per manufacturer s protocol and cultured in vitro prior to use using previously reported culturing methods (Lee et al., 6a; Li et al., 9). Cells under passage were used to all analysis and the majority of cells used were immediately after dissociation. PCR analyses Total RNA was extracted from CD33-positive and CD33-negative cell fractions using the RNeasy kit (Qiagen), and reverse transcribed into cdna by iscript cdna synthesis kit (BioRad). Reverse transcription PCR was done using cdna transcribed from RNA as described above. Following the manufacturer s cdna amplification protocol, a 55 C annealing temperature was used. PCR products were verified by melting curves. As a control, no cdna was added to the reaction to ensure accuracy. The PCR products were separated on a.5% agarose gel and imaged using ethidium bromide and a transilluminator. Immunofluorescence For immunostaining analysis at the single cell level, CD33-positive and CD33- negative fractions were separated by magnetic beads and plated on tissue culture plastic chamber slides (Nunc) for 8 hours to allow sufficient attachment. Cells were then fixed with 4% PFA for 5 minutes at room temperature, washed 3 times with PBS, and then blocked with a PBS based solution containing % normal goat serum (Sigma) and.% triton x- (Sigma). Cells were incubated overnight with the appropriate antibody (:5): rat monoclonal anti-integrin α6 (MAB378, Millipore), rabbit polyclonal anti-cd33 (ab9898, abcam or CD33/, Miltenyi), or rabbit polyclonal Olig (kind gift from Drs. John Alberta and Charles Stiles, Harvard University). Cells were washed 3 times with PBS and incubated with the appropriate secondary antibody (:5): goat anti-rat Alexa 488 IgG or goat anti-rabbit Alexa 568 IgG (Invitrogen). Nuclei were counterstained with hoescht 3334 (: dilution from a 5mg/ml stock solution, Invitrogen). For immunostaining analysis on tumorspheres sections, tumorspheres were fixed as described above and cryoprotected with 3% sucrose prior to embedding in OCT (Sakura). Frozen sections of microns were cut using a Leica (CM9) cryostat. Immunostaining, as described above, was done by blocking sections and incubating with the appropriate primary antibody (:): antiintegrin α6, mouse monoclonal anti-integrin β (MAB959, Millipore), mouse monoclonal anti-map (Sigma), anti-cd33, anti-olig, rabbit polyclonal anti-phospho histone H3 (6-57, Millipore), rabbit polyclonal anti-nestin (ab5968, abcam), or rabbit polyclonal anti-pan laminin (L-9393, Sigma). Sections were washed 3 times with PBS

4 and incubated with the appropriate secondary antibody (:5): goat anti-rat Alexa 488 IgG, goat anti-rabbit Alexa 568 IgG (Invitrogen), or goat anti-mouse Alexa 568 IgGa (Invitrogen). Nuclei were counterstained with Hoechst 3334 (: of a 5mg/ml stock solution, Invitrogen). The TUNEL assay was done per manufacturer s protocol (7-4, Millipore). For immunostaining analysis on human GBM surgical biopsy specimens, micron sections frozen sections were obtained from the Duke University Brain Tumor Center Tissue Bank. Sections were fixed for with 4% PFA for minutes at room temperature and processed as described in the section above. Differentiation studies was conducted by allowing whole tumorspheres to attach to tissue culture plastic chamber slides (Nunc) and cultured for a week in media void of EGF and bfgf or in DMEM containing % fetal calf serum (Sigma). After cells had attached, spread out, and undergone distinct morphological changes, they were fixed in % PFA, washed 3 times in PBS and processed as described above with the following antibodies: mouse monoclonal anti-map (:4, M994, Sigma), mouse monoclonal anti-β3 tubulin (:, T576, Sigma), mouse monoclonal anti-gfap (:4, 6565, BD), mouse monoclonal anti-o4 (:4, MAB345, Millipore), and mouse monoclonal anti-cnpase (:4, C59, Sigma). Appropriate goat secondary antibodies were used as described above. Nuclei were counterstained with Hoescht 3334 (: dilution from a 5mg/ml stock solution, Invitrogen). All imaging done using a Leica SP-5 confocal microscope as described previously (Wang et al., 8b) and images were processed and assembled in photoshop (Adobe). For integrin α6 perivascular assessment, confocal microscopy images comprised of integrin α6 positive cells (nuclei counterstained with Hoechst 3334) and blood vessels (labeled with an antibody against CD3) were imported into Image-Pro Plus (v6., Media Cybernetics, Silver Spring, MD). Proximity analysis of cell nuclei to blood vessels for each image was performed in an automated fashion (batch mode) using customized visual basic Image-Pro Plus macros. Briefly, cell nuclei and Alexa 568 blood vessels channels were extracted from each image. The cell nuclei channel was flattened to remove any uneven illumination followed by a spectral filter to equalize brightness of pixels within each nucleus. The nuclei were then segmented using intensity and morphological thresholds and touching nuclei were split using a watershed filter. Subsequently, the user was prompted to draw a region of interest (ROI) around each vessel present within a given image. Each ROI was then converted to single pixel width perimeter (Sobel filter) forming a point boundary for each vessel. Finally a minimum distance was calculated between the centroid coordinate of each nucleus to the set of perimeter points around each vessel, exporting each cell distance to Excel. For a visual representation of distance profiles, the calculated distances were mapped to a LUT with nuclei centroids pseudo-colored to represent their respective distance to the nearest vessel boundary. Growth curve and tumorsphere formation assays All FACS sorted cell fractions of interest were plated at a density of cells/well in a 96-well plate in at least triplicate for growth curve analysis. Cell number was measured for every other day and normalized to x -7 M of ATP using the CellTiter-Glo assay kit (Promega). For tumorsphere formation assessment, 4- or 96-well plates were seeded at a density of or cells per condition. Reported numbers represent a minimum of 8 wells per condition. Ten days after plating, tumorspheres containing more than cells

5 were scored. For studies with the integrin α6 blocking antibody (GoH3, MAB378, Millipore), doses of either μg/ml or μg/ml were used and compared to a rat isotype control antibody (μg/ml Invitrogen). Lentiviral construct production All lentiviral constructs used were manufactured as previously reported (Li et al., 9, Wang et al., 8). In short, using lipofectamine (invitrogen), 93FT cells were co-transfected with packaging vectors pspax and pci-vsvg (Addgene) and lentiviral vectors directing expression of specific to integrin α6 (TRCN57774 ( ) and TRCN57775 ( )) or a nontargeting control (SHC) (Sigma) to produce virus. Media on the 93FT cells was changed 8 hours after transfection and two days later, viral supernatants were collected, filtered, and used or frozen at -8 C. Caspase 3/7 assay For caspase 3/7 assays, CD33-positive or integrin α6-positive/cd33-negative cells were infected for days, selected for days and sorted using flow cytometry based on negative PI incorporation. Sorted fractions were plated in triplicate, allowed to recover overnight, and measured three separate times after the caspase 3/7 substrate (Promega) was added. Relative luciferase was calculated based on cell numbers from three separate wells normalized to x -7 M of ATP calculated using the CellTiter-Glo assays as described above. Cell cycle analysis Following integrin α6 knockdown, 5, cells from each treatment group were filtered through a 3μm filter before fixation in 3 ml of 7% Ethanol at 4 degrees overnight. After fixation, the cells were pelleted and resuspended in.5ml of PBS. In order to remove any traces of free RNA that could bind with propidium iodide and interfere with data interpretation, each sample was incubated with 5 μl of RNAse A ( μg/ml) for 3 minutes at 37 C. The RNAse was then inactivated by incubation for 5 minutes at 4 C. Following addition of μl of PI, cells were analyzed by flow cytometry using a FACScan for DNA content. Detection of apoptosis by DNA fragmentation Apoptosis was assessed in the GSCs infected with lentiviral knock down constructs after days of puromycin selection (total of 4 days post infection) by the DNA fragmentation assay. The assay was performed as described in (Benhar et al., 8) with slight modifications. In brief, the cells were pelleted from suspension culture and washed with PBS. The pellet was then lysed using ml of lysis buffer ( mm Tris, mm EDTA,.5% Triton X-, ph 8.), and the solution was centrifuged for min at, g. The protein content was measured using the BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL) and was used to standardize the number of cells processed for each condition. All volumes were adjusted to 5 µl and treated with µg RNAse A (Invitrogen) for hours at 37 C. Following this, each sample was treated with µg Proteinase K (Roche) for hour at 37 C. DNA was precipitated from the samples by adding volume of isopropanol and incubating at - C for hours. The

6 DNA was pelleted by centrifuging at, g for min. After removal of the isopropanol the pellets were redissovled in 5 µl buffer TE. A 3 µl aliquot of the solution was separated by electrophoresis on a.% agarose gel stained with ethidium bromide. A staurosporine positive control (Sigma, loaded at a : dilution) was used and for size comparisons, kb and bp ladders were used (Invitrogen). In vivo tumor initiation analysis In vivo tumor initiation analysis was done as previously described (Bao et al., 6b, Wang et al., 8). For integrin α6 high vs low studies, cells (,, 5, or cells) were transplanted after hours of recovery post-facs. For integrin α6 studies, cells were used 4 days after selection and cells (5 or ) were transplanted into the brains of BALB/c nu/nu mice. For integrin α6 blocking antibody studies, cells were incubated with integrin a6 blocking antibody ( μg/ml) or isotype control antibody for five days prior to transplantation ( cells) into the brains of BALB/c nu/nu mice. Brains of euthanized mice were collected, fixed in 4% PFA, paraffin embedded, sectioned, and subjected to hematoxylin and eosin staining. Supplemental References Bao, S., Wu, Q., Sathornsumetee, S., Hao, Y., Li, Z., Hjelmeland, A. B., Shi, Q., McLendon, R. E., Bigner, D. D., and Rich, J. N. (6b). Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor. Cancer Res 66, Benhar, M., Forrester, M. T., Hess, D. T., and Stamler, J. S. (8). Regulated protein denitrosylation by cytosolic and mitochondrial thioredoxins. Science 3, Lee, J., Kotliarova, S., Kotliarov, Y., Li, A., Su, Q., Donin, N. M., Pastorino, S., Purow, B. W., Christopher, N., Zhang, W., et al. (6a). Tumor stem cells derived from glioblastomas cultured in bfgf and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer cell 9, Li, Z., Bao, S., Wu, Q., Wang, H., Eyler, C., Sathornsumetee, S., Shi, Q., Cao, Y., Lathia, J., McLendon, R. E., et al. (9). Hypoxia-inducible factors regulate tumorigenic capacity of glioma stem cells. Cancer cell 5, Wang, J., Wang, H., Li, Z., Wu, Q., Lathia, J. D., McLendon, R. E., Hjelmeland, A. B., and Rich, J. N. (8b). c-myc is required for maintenance of glioma cancer stem cells. PloS one 3, e3769.

7 A Distance (μm) Integrin a6+ cell Analyzed cell 5 6% % 68% % 5 77% 3% 5 8% 5% p <. HP 444 HP 456 GBM Surgical Biopsy B C B C Nestin Hoechst HP 3359 HP 369 GBM Surgical Biopsy D E D E CD33 Hoechst Figure S, related to Figure Integrin α6 + cells are preferentially located in the perivascular compartment in GBM biopsy specimens and co-express nestin

8 Summary of immunofluorescence analysis of GBM surgical biopsies (A) double stained with int α6 (green) and CD3 (red) calculated based on 6 specimens (n=8 intα6 positive cells and 75 total cells,, p <. with ANOVA comparison between populations). Example analyzed micrograph (A, right) shows several intα6 positive cells (white arrows) located adjacent to a blood vessel and an int α6 positive cell (pink arrow) not adjacent to the blood vessel. Immunostaining analysis of GBM surgical biopsies (HP 444, HP 456) shows that co-expression of int α6 (green) and nestin (red) is maintained in the perivascular compartment while nestin expression appears to be nonperivascular cells (B, C). Blood vessels marked with an, regions of interest marked with a white arrow, and enlarged regions of interest marked with yellow arrow and shown in B -C. Immunostaining analysis of GBM surgical biopsies (HP 3359 and HP 369) passaged as xenografts and used for analysis shows that co-expression of intα6 (green) and CD33 (red) is maintained in the perivascular compartment (D, E). Blood vessels marked with an, regions of interest marked with a white arrow, and enlarged regions of interest marked with yellow arrow and shown in D -E. All nuclei counterstained with Hoechst in blue. Scale bar represents 5 μm.

9 % of integrin α6 expression CD33+ GSC Enriched CD33 - GSC Depleted Figure S, related to Figure Integrin α6 co-segregates with CD33 in GBM cells The majority of CD33 + cells (red) are int α6 + as compared to CD33 - cells (black) displayed as a bar graph calculated based on flow cytometry analysis (data contained in Supplementary Table, n=7, +/- S.E.M.,, p <.).

10 T3359 T369 T43 A B C Hoechst ph3 D ph3 E ph3 F Map Map Map G TUNEL T43 Figure S3, related to Figures 3 and 4 Integrin α6 co-segregates proliferating cells in the periphery of tumorspheres but not in the center which contains differentiated and dead cells

11 Immunostaining analysis of cryosections from tumorspheres generated from GSC enriched populations (T3359, T369, T43) show two distinct regions of expression, the peripheral region and the inner region. Photomicrographs show int α6 (green) is coexpressed with the m-phase marker phospho histone H3 (ph3, red, A, B, C) in the peripheral region (yellow arrow) but not in the inner region (white arrow). The center of the tumorspheres which contains differentiated cells as assessed by a neuronal marker (Map, positive center cell marked with blue arrow) expression (red, C, D, E) is int α6 low. Furthermore, the center of tumorsphere contains dead cells as assessed by TUNEL staining (G, white box indicates enlarged area in right panels). All nuclei counterstained with Hoechst in blue. Scale bar represents 5 μm.

12 A CD33 - APC Antibody control D3MG.%.6% 39.% B Antibody control 3 α6 low α6 medium 3 α6 high ATP x ^-7M C Integrin α6- FITC 4 3 ### high medium low D3MG ### Day ### D Tumorspheres per well 4 3 D3MG low medium high # Figure S4, related to Figure 5 Selection of cells based on integrin α6 expression levels in the absence of CD33 enriches for a population which display GSC properties Flow cytometry plots (A) show int α6 expression levels in the xenograft D3MG, which has a low level on CD33 expression. GSC enrichment was based on the adjusted intα6 expression alone as shown on this histogram (B), region contains low levels of intα6 (α6 low, black), region represents the lower 5% of int α6 expression level (α6 medium, red/white), and region 3 represents the upper 5% of int α6 expression level (int α6 high, red). (C) Cell enrichment based on intα6 expression level correlates a greater cell proliferation profile as assessed using the cell titer assay., p<. with ANOVA comparison to int α6 low cells at the same timepoint; ###, p<. with ANOVA comparison of int α6 high to int α6 medium cells at the same timepoint. (D) Cell enrichment based on int a6 expression level correlates with a greater ability to form tumorspheres, p<.5 with ANOVA comparison to int α6 low expressing cells; #, p<.5 with ANOVA comparison to int α6 medium cells. Error bars represent ± SEM.

13 GFAP O4 Map β3 tubulin CNPase Figure S5, related to Figure 5 Integrin α6 + cells are multipotent and capable of giving rise to all three major central nervous system lineages Immunostaining analysis of int α6 + cells derived from xenografted GBMs (T43, T4 data not shown) demonstrate that int α6 high cells have the capacity to give rise to all three central nervous system lineages (green): neurons (indicated by Map and β3 tubulin), astrocytes (indicated by GFAP), and oligodendrocytes (indicated by O4 and CNPase). All nuclei counterstained with Hoechst in blue. Scale bar represents μm.

14 A T3359 T4 T43 8% % 8% 83% % 3% 85% 3% 8% B F Tumorspheres per well Relative Caspase 3/7 Activity Control 3 NT T3359 T NT C Tumorspheres per well G Relative Caspase 3/7 Activity T4 T4597 D 6 4 Integrin α 6 - FITC T43 ### NT Control Control NT H Tumorspheres per well Percentage ## G phase S phase Cell cycle stage E Percentage T4597 Non-targeting Int α 6 Int α 6 T43, α6+/cd33-.5 NT ATP x ^-7 M I ### 4 3 ## G phase S phase Cell cycle stage T43, α6+/cd33- Non-targeting Int α 6 Int α Days J Percentage T4597, α6+/cd33- ### 4 3 ## G phase S phase Cell cycle stage Non-targeting Int α 6 Int α 6 Relative Caspase 3/7 Activity K T43, α6+/cd33- NT L T43, α6+/cd M Un NT sh sh Ctrl M M Integrin α6 Figure S6, related to Figure 7 Integrin α6 knockdown can be achieved using lentiviral constructs and compromises GSC properties in CD33 + and integrin α6 + /CD33 - GSCs was knocked down in GSCs using two separate lentiviral delivered constructs ( targeting exon 4 and targeting exon ). Flow cytometry analysis (A, xenografted tumor specimens T3359, T4, T43) show int α6

15 expression levels (blue) were reduced in targeting constructs but not in a control nontargeting construct. Percentage of int α6 + cells was calculated relative to an antibody control (red). was knocked down in GSCs (T3359, 4, T43, T4597) using two separate lentiviral delivered constructs (black, grey). Self-renewal was impaired in T3359 (B) and T4 (C) xenografted cells targeted with the lentivial constructs (, ) directed against intα6 in comparison to non-targeting control (NT ) as assessed by tumorsphere formation assays., p<. with ANOVA comparison to non-targeting control. In addition, cell cycle changes (increase in G and decrease in S-phases) were detected when T43 (D) and T4597 (E) xenografted cells were targeted with the lentivial constructs directed against intα6 in comparison to a non-targeting control as assessed by flow cytometry cell cycle based analysis., p<. with ANOVA comparison to non-targeting control, ###, p <. and ## p <. with ANOVA comparison to. Cell death was also increased when T43 (F) and T4597 (G) xenografted cells were targeted with the lentivial construct () directed against int α6 in comparison to a nontargeting control (NT ) as assessed by Caspase 3/7 activity., p<.. Intα6 was knocked down in intα6 +/CD33 - GSCs (T43, T4597) using two separate lentiviral delivered constructs (black, grey). Self-renewal was impaired in T43 (H) xenografted cells targeted with the lentivial constructs (, ) directed against intα6 in comparison to non-targeting control (NT ) as assessed by tumorsphere formation assays., p<. with ANOVA comparison to nontargeting control. Knockdown of int α6 using two separate lentiviral constructs results in a decreased cell proliferation profile as assessed by the cell titer assay in T43 (I) xenograft tumor cells., p<. with ANOVA comparison to nontargeting at the same timepoint. In addition, cell cycle changes (increase in G and decrease in S-phases) was detected when T597(J) xenografted cells were targeted with the lentivial constructs directed against int α6 in comparison to a nontargeting control as assessed by flow cytometry cell cycle based analysis., p<. with ANOVA comparison to non-targeting control, ###, p <. and ## p <. with ANOVA comparison to. Cell death was also increased when T43 (K) xenografted cells were targeted with the lentivial constructs (, ) directed against intα6 in comparison to a non-targeting control (NT ) as assessed by flow cytometry cell cycle based analysis., p<.5 with ANOVA comparison to non-targeting control. Induction of cell death upon intα6 targeting was also confirmed by a DNA fragmentation assay (L) on T43 xenograft tumor cells (M=kb ladder plus from Invitrogen, Un = uninfected control, NT = non-targeting control, sh=, sh=, Ctrl = staurosporine control loaded at : dilution, M=bp ladder from Invitrogen). Immunostaining analysis (M) for intα6 shows positive cells (yellow arrow indicated inset) in a tumor generated from in vivo intracranial transplantation of 5 CD33 + GSC (T3359) infected with int α6 targeting construct ( ). Scale bar represents 5 μm. Error bars represent ± SEM.

16 All Glioma Up-Reg. >= 3.X Down-Reg. >= 3.X Intermediate Figure S7, related to Figure 8 Elevated integrin a6 expression in GBMs correlates with poor patient prognosis Kaplan-Meier survival plot for glioblastoma patients with differential tumor int α6 expression calculated by NCI REpository for Molecular BRAin Neoplasia DaTa (REMBRANDT) bioinformatics database ( The log-rank P-value for significance of difference of survival between intα6 upregulated group and int α6 intermediate group was.7. The log-rank P-value for significance of difference of survival between int α6 up-regulated group and all other groups was.9 ( p <.5). Numbers of samples in group were 7 (int α6 Up- Regulated), (int α6 Down-Regulated), and 84 (int α6 Intermediate).

17 CD Total - Integrin α Total Tumor ID T T T T T CCF CCF Table S, related to Figure Summary of integrin α6 and CD33 flow cytometry analysis Summary of flow cytometry analysis based on int α6 and CD33 expression on GBM xenografts (T369, T3359, T3, T7, T66) and primary surgical GBM biopsies (CCF58, CCF585). Percentage positive was calculated relative to relevant antibody controls.

18 Integrin α6 low Integrin α6 high T8-387 Integrin α6 Sorted Cells Cell number Incidence Median Survival /3 N.A. 5 /4 N.A. 5/5 6 /5 N.A. 5 5/5 5/5 4, ## 35 Integrin α6 low Integrin α6 high CCF 966 Integrin α6 Sorted Cells Cell number Incidence Median Survival /4 N.A. /5 N.A. 3/ /5 56 4/5 34 4/4 4.5 Table S, related to Figure 6 Summary of in vivo intracranial transplantation of integrin α6 high and low cells Summary of incidence and median survival of intracranial transplantation of limiting dilutions of int α6 high or low cells from tumor specimens T8-387 and CCF966. p <.67, p <.5 and p <. with log-rank analysis of survival curves for same cell concentration and ## p <. with log-rank analysis of survival curves of T intα6 high vs int α6 low cells). N.A. represents median survival not achieved.

19 Non-Targeting Control Integrin α6 Integrin α6 T3359 CD33+ Lentivirus Infected Cells Cell number Incidence Median Survival 4/ /5 34 4/ /5 48 4/ /5 6 Table S3, related to Figure 7 Summary of in vivo intracranial transplantation of GSCs infected with integrin α6 targeting or control non-targeting constructs Summary of incidence and median survival of intracranial transplantation of 5 or T3359 CD33 + infected with int α6 targeting constructs of a control nontargeting construct. p <.5 and p <. with log-rank analysis of survival curves for same cell concentration as compared to non-targeting control.

20 Isotype control antibody α6 integrin blocking antibody T4597 α6+/cd33- Antibody Treated Cells Cell number Incidence Median Survival 5/5 8 5/5 46 Isotype control antibody α6 integrin blocking antibody T43 α6+/cd33- Antibody Treated Cells Cell number Incidence Median Survival 5/5 36 5/5 45 Table S4, related to Figure 8 Summary of in vivo intracranial transplantation of GSCs treated with an integrin α6 blocking antibody Summary of incidence and median survival of intracranial transplantation of T4597 of T43 int α6 + /CD33 - cells treated with an int α6 blocking antibody or isotype control antibody. p <.5, p <. with log-rank analysis of survival curves for same cell concentration. N.A. represents median survival not achieved.