Identifying the molecular mechanisms responsible for the resistance of gliomas to anticancer treatments is an issue of great therapeutic interest. Δ⁹-Tetrahydrocannabinol (THC), the major active ingredient of marijuana, and other cannabinoids inhibit tumor growth in animal models of cancer, including glioma, an effect that relies, at least in part, on the stimulation of autophagy-mediated apoptosis in tumor cells. Here, by analyzing the gene expression profile of a large series of human glioma cells with different sensitivity to cannabinoid action, we have identified a subset of genes specifically associated to THC resistance. One of these genes, namely that encoding the growth factor midkine (Mdk), is directly involved in the resistance of glioma cells to cannabinoid treatment. We also show that Mdk mediates its protective effect via the anaplastic lymphoma kinase (ALK) receptor and that Mdk signaling through ALK interferes with cannabinoid-induced autophagic cell death. Furthermore, in vivo Mdk silencing or ALK pharmacological inhibition sensitizes cannabinod-resistant tumors to THC antitumoral action. Altogether, our findings identify Mdk as a pivotal factor involved in the resistance of glioma cells to THC pro-autophagic and antitumoral action, and suggest that selective targeting of the Mdk/ALK axis could help to improve the efficacy of antitumoral therapies for gliomas.

Identification of the gene expression profile characteristic of THC-resistant glioma cells

To gain insight into the molecular features associated with the resistance of glioma cells to cannabinoid action, we initially characterized 10 human glioma cell lines on the basis of their different sensitivity to THC-induced cell death (Figure 1a). Thus, cell lines were classified as cannabinoid sensitive (IC₅₀ for THC lower than 2.5μM) and cannabinoid resistant (IC₅₀ for THC higher than 2.5μM). To note, we did not find any correlation between the sensitivity of these cell lines to THC and to other two chemotherapeutic agents (carmustine and temozolomide) frequently used in glioma treatment (Supplementary Figures 1A, B and C), which indicates that the resistance to THC action is determined by specific mechanisms distinct from those promoting resistance to other drugs. In addition, differences in the levels of CB₁ or CB₂ receptors of glioma cells did not correlate with a higher or lower sensitivity to THC-induced cell death (Figure 1b), and pharmacological blockade of CB₁ receptors with the selective antagonist SR141716 or CB₂ receptors with the selective antagonist SR144528 abrogated THC-induced death in both sensitive and resistant cell lines (Figure 1c and Supplementary Figure 1D). This supports that mechanisms other than changes in the expression or the functionality of CB receptors are responsible for the differences in THC sensitivity of glioma cells. Analysis of some of the genes most frequently altered in gliomas (p16, PTEN, p53 and EGFR) did not reveal association between the presence of mutations, amplifications or deletions on those genes and sensitivity to cannabinoid treatment (data not shown). Therefore, in order to identify the molecular factors that determine the different sensitivity of glioma cells to THC, we analyzed by DNA microarrays the gene expression profile of the 10 glioma cell lines. After selection of those genes whose expression was significantly changed (FDR<0.2) at least 2.5-fold in more than three sensitive or resistant cell lines with respect to their sensitive or resistant counterparts (Figure 2a, Supplementary Figure 2A and Supplementary Tables 1 and 2) and real-time quantitative PCR validation of the array data (Figure 2b, Supplementary Figure 2B and Supplementary Tables 3 and 5), we selected 20 genes as potential candidates to predict the resistance of glioma cells to cannabinoid action. Next, we analyzed the expression of these genes in primary cultures of glioma cells obtained from tumor samples of GBM patients (in which we confirmed that THC promoted cell death via stimulation of cannabinoid receptors; Supplementary Figure 3D). Data revealed that 8 of the 20 initially selected genes were differentially upregulated in the cultures that were more resistant to THC-induced cell death (Figures 2c and d, Supplementary Figure 3A and Supplementary Table 4). These observations support that this set of genes is strongly associated with a higher resistance of glioma cells to THC-induced death.

Figure 1

Classification of human glioma cell lines according to their sensitivity to THC-induced cell death. (a) Effect of THC on the viability (72h) of 10 human glioma cells lines (cells with IC₅₀<2.5μM (left panel) and cells …

Figure 2

Identification of the gene expression profile associated with resistance to THC-induced cell death. (a) Array data: selection of genes that exhibit different basal expression levels between THC-resistant and THC-sensitive human glioma cell lines. Data …

The growth factor Mdk confers resistance to THC

One of the genes whose upregulation was more strongly associated with the resistance of glioma cells to cannabinoid treatment was the growth factor Mdk; Mdk mRNA levels were 30- to 80-fold higher in the resistant than in the sensitive cell lines (Figures 2b and d and Supplementary Figures 4A and C). Mdk is a member of the pleiotrophin (PTN)/Mdk family and was originally described as a developmentally regulated, heparin-bound, neurotrophic factor.^16, 17 Mdk has been proposed to modulate the proliferation and migration of various types of cancer cells and is often overexpressed in malignant tumors.^18, 19 Of interest, analysis of samples from 201 glioma patients (including 115 GBMs) revealed that Mdk expression is associated to a lower overall survival of glioma, high-grade glioma and GBM patients (Figure 3a and Supplementary Figure 5). These analyses were validated with external data obtained from The Cancer Genome Atlas³ (Supplementary Figure 5E and Supplementary Table 6).

Figure 3

Increased Mdk expression confers human glioma cells resistance to THC. (a) Kaplan–Meier survival curves for 115 glioblastoma tumors from patients collected at the Virgen de la Salud Hospital (Toledo) and classified according to Mdk expression. …

We found that the levels of Mdk are increased in the supernatants of the cannabinoid-resistant glioma cell lines (Figure 3b). We therefore investigated whether this growth factor participates in the mechanism of resistance to cannabinoid antitumoral action of gliomas. In agreement with this possibility, Mdk silencing sensitized two cannabinoid-resistant cell lines (Figure 3c and Supplementary Figure 6A) as well as a primary culture of glioma cells (Figure 3d) to THC-induced cell death. Moreover, incubation with conditioned medium derived from the cannabinoid resistant cell line T98 (which was enriched in Mdk) (Figure 3e) or with exogenous Mdk (Figure 3f) rendered the cannabinoid-sensitive line U87 resistant to THC treatment, an effect that was abrogated by Mdk neutralization with an anti-Mdk antibody (Figure 3e and Supplementary Figure 6B). These observations strongly support that Mdk is a factor for resistance to THC-induced glioma cell death.

Of note, the cannabinoid-resistant pancreatic cancer cell line Panc1 exhibited much higher levels of Mdk than the cannabinoid-sensitive pancreatic cancer cell line MiaPaca2 (Supplementary Figures 7A and B). Furthermore, Mdk knockdown rendered Panc1 cells sensitive to THC, suggesting that a similar mechanism of resistance to cannabinoid-induced cell death also operates in pancreatic cancer cells (Supplementary Figure 7C).

Mdk promotes resistance to THC through stimulation of the ALK receptor

The mechanism by which Mdk mediates its different biological actions has not been completely clarified¹⁶ and may involve the stimulation of several receptors, including ALK, protein-tyrosine phosphatase, receptor-type, zeta-1 (RPTPZ), and low-density lipoprotein receptor-related protein 1 (LRP1).¹⁶ It has been proposed that the pro-survival and pro-angiogenic effect of Mdk on tumor cells relies on the stimulation of the ALK receptor.²⁰ ALK was expressed in all the cell lines and primary cultures of human glioma cells used in this study (Supplementary Figures 8A, B and C), and therefore, we asked whether Mdk binding to ALK has a role in the resistance of glioma cells to THC-induced cell death. In agreement with this idea: (i) ALK knockdown sensitized T98 and HG2 glioma cells to THC-induced cell death (Figure 4a and Supplementary Figure 9), whereas silencing of RPTPZ and LRP1 only modestly enhanced the response to THC treatment of T98 (Supplementary Figure 9C) and HG2 (Supplementary Figure 9D) cells, respectively; (ii) incubation with T98 cell-conditioned medium or addition of exogenous Mdk increased ALK phosphorylation of U87 cells (Supplementary Figures 10C and D); (iii) silencing of ALK – but not of RPTPZ or LRP1 – abrogated the resistance to THC-induced cell death exerted by the addition of exogenous Mdk to U87 cells (Figure 4b and Supplementary Figure 10A); (iv) knockdown of ALK abolished the protective action exerted by T98-conditioned medium on U87 cells (Supplementary Figure 10B); and (v) incubation with the ALK-selective inhibitor NVP-TAE-684 reduced ALK phosphorylation (Figure 4c) and enhanced THC action in T98 cells (Figure 4d). Altogether, these observations strongly support that Mdk promotes resistance to THC-induced cell death via ALK stimulation.

Figure 4

Mdk promotes resistance to THC-induced cell death via ALK. (a) Effect of THC on the viability of T98 (left panel) or HG2 (right panel) cells transfected with control (siC) or ALK-selective (siALK) siRNA (mean±S.D.; n=5 for T98 and HG2 cells; …

Mdk inhibits the activation of the cannabinoid-evoked autophagy-mediated cell death pathway

We have recently found that THC stimulates autophagy in glioma cells and that the activation of this cellular process is required for the pro-apoptotic and antitumoral action of cannabinoids.¹⁵ We therefore asked whether the mechanism by which Mdk promotes resistance to THC-induced cell death could be based on the modulation of the signaling route that regulates autophagy in response to cannabinoid treatment. In line with this hypothesis, knockdown of Mdk or ALK in T98 or HG2 cells enhanced THC-induced autophagy, as determined by the presence of cells with LC3 dots as well as by the accumulation of the lipidated form of LC3 (LC3-II; two hallmarks of autophagy;²¹ Figures 5a and b and Supplementary Figure 11A), as well as THC-induced apoptosis (Figure 5c and Supplementary Figure 11B). Likewise, pharmacological inhibition of ALK-enhanced THC-induced autophagy and apoptosis of T98 cells (Supplementary Figure 12). Moreover, incubation of U87 cells with T98 cell-conditioned medium or with exogenous Mdk decreased THC-induced autophagy (Figure 5d) and apoptosis (Figure 5e) in response to THC. These data strongly support that the Mdk/ALK axis modulates THC-induced autophagy.

Figure 5

Mdk promotes resistance to THC-induced autophagy and apoptosis. (a) Effect of THC (2.5μM, 24h) on LC3 immunostaining of siC, siMdk or siALK-transfected T98 cells. Values in the upper right corner at each photomicrograph correspond …

Induction of autophagy in many cellular settings relies on the inhibition of the mammalian mTORC1, which in turn regulates the activity of several autophagy proteins involved in the early triggering of this cellular process.²¹ Accordingly, THC stimulates an ER stress-related pathway that leads to the upregulation of the transcription factor p8 and its downstream target TRB3. This latter protein triggers the inhibition of the Akt/mTORC1 axis, thus leading to the stimulation of autophagy-mediated cell death.¹⁵ On the other hand, ALK stimulation has been shown to activate the phosphatidylinositol 3-kinase (PI3K)/Akt pathway.²⁰Therefore, we investigated whether the resistance to THC-induced autophagy exerted by Mdk relied on the modulation of p8/TRB3 expression and/or on the activation of the Akt/mTORC1 axis. In line with this idea, THC-triggered p8 and TRB3 induction in T98 and HG2 cells was strongly enhanced by Mdk or ALK knockdown (Figure 6a and Supplementary Figure 11C). Furthermore, a decrease in the phosphorylation of Akt and S6 (a well-established readout of mTORC1 activity) was observed upon THC treatment of Mdk- or ALK-silenced T98 and HG2 cells (Figure 6b and Supplementary Figure 11D). On the other hand, incubation of U87 cells with T98 cell-conditioned medium or with exogenous Mdk decreased THC-induced p8 and TRB3 upregulation (Figure 6c) and attenuated THC inhibition of Akt and S6 phosphorylation (Figure 6d). Taken together, these observations strongly support that Mdk (via ALK) modulates p8/TRB3 expression as well as the activity of the Akt/mTORC1 axis to prevent the stimulation of autophagy-mediated cell death by cannabinoids.

Figure 6

Mdk promotes resistance to THC-induced autophagy by preventing p8 and TRB3 upregulation and AKT/mTORC1 inhibition. (a) Effect of THC (2.5μM, 6h) on p8 and TRB3 mRNA levels (as determined by quantitative real-time PCR) of siC- …

In vivo silencing of Mdk sensitizes cannabinoid-resistant tumors to THC anticancer action

To analyze the in vivo relevance of our findings, we generated tumor xenografts by subcutaneous injection of T98 or U87 cells in nude mice. In agreement with our in vitro data, T98 cell-derived tumors exhibited higher levels of Mdk and were more resistant to THC treatment than the ones generated with U87 cells (Supplementary Figures 13A, B and C). To investigate whether Mdk is responsible for the resistance to THC antitumoral action we analyzed the effect of THC on the growth of T98 tumor xenografts in which Mdk expression had been knocked-down (Figure 7b and Supplementary Figure 13D). As shown in Figure 7a and in Supplementary Figure 13E, in vivo silencing of Mdk rendered established T98 cell-derived tumors sensitive to THC treatment, although it did not affect tumor growth by itself. Subsequent analysis of tumor samples revealed that Mdk silencing correlated with a decrease in ALK phosphorylation (Figure 7c). Moreover, THC treatment increased TRB3 expression (Figure 7d, upper panels), decreased S6 phosphorylation (Figure 7d, middle panels), enhanced autophagy (as determined by LC3 immunostaining (Figure 7d, lower panels)) and induced apoptosis (as determined by active caspase-3 immunostaining (Figure 7e)) only in those tumors in which Mdk expression had been knocked-down. Finally, we investigated the participation of ALK on the resistance of T98 tumors to THC antitumoral action. To this aim we analyzed the effect of the coadministration of THC and NVP-TAE-684 on the growth of T98 tumor xenografts. Similar to what we observed on Mdk silencing, administration of NVP-TAE-684 decreased ALK phosphorylation and sensitized established T98 cell-derived tumors to THC treatment, but did not affect tumor growth by itself (Figures 8a and b). Furthermore, THC treatment increased TRB3 expression (Figure 8c, upper panels), decreased S6 phosphorylation (Figure 8c, middle panels), enhanced autophagy (Figure 8c, lower panels) and induced apoptosis (Figure 8d) only in those tumors in which ALK had been inhibited. Altogether, these results strongly support that the Mdk/ALK axis promotes resistance to THC antitumoral action in vivo by modulating the activation of the autophagy mediated-cell death pathway.

Figure 7

In vivo silencing of Mdk sensitizes T98 cell-derived tumors to THC antitumoral action. (a) Effect of Mdk silencing and THC (15mg/kg per day) treatment on the growth of tumor xenografts derived from T98 cells (mean±S.D.;n=5 for each condition, …

Figure 8

Pharmacological inhibition of ALK sensitizes T98 cell-derived tumors to THC antitumoral action. (a) Effect of NPVTAE684 (TAE; 1 mg/kg per day) and and THC (15mg/kg per day) treatment on the growth of tumor xenografts derived from T98 cells (mean±S.D.; …

Reagents

THC was kindly provided by GW Pharmaceuticals (Salisbury, UK). The cannabinoid antagonists SR141716 (SR1) and SR144528 (SR2) were kindly donated by Sanofi-Aventis (Montpellier, France). Recombinant human Midkine was from Sigma (St. Louis, MO, USA). Double-stranded RNA duplexes corresponding to human Mdk, human ALK (smart-pool) and control siRNA were synthesized by Dharmacon (Lafayette, CO, USA). The RNA sequence targeting human Mdk is described in ref. 39 The ALK inhibitor NVP-TAE 684 was synthesized by Sergey A Lakatosh at the Gause Institute of New Antibiotics (Moscow, Russia). BCNU was purchased from Sigma and TMZ was kindly provided by Schering-Plough (Whitehouse Station, NJ, USA).

Cell culture and viability

The human glioma cell lines GOS3, U87 MG (U87), A172, SW1783, U118 MG (U118), U373 MG (U373), T98G (T98), SW1088, CCF-STTG1 (CCF) and LN405 were from ATCC (Middlesex, UK), DSMZ (Braunschweig, Germany) or were kindly provided by Dr. Javier Castresana (Universidad Pública de Navarra, Pamplona, Spain). Primary cultures of brain tumor cells were obtained from biopsies donated by the Tumor Bank Network coordinated by the Spanish Cancer Research Centre and from the Hospital Clínico Universitario (Madrid, Spain). Human pancreatic cancer cell lines MiaPaca2 and Panc1 were kindly provided by Dr. JL Iovanna (Marseille, France).

Human glioma or pancreatic cancer cell lines were cultured in DMEM containing 10% FBS. The preparation of adherent primary culture of brain tumor cells was as follows: tumor samples were homogenized, digested with type Ia collagenase (Sigma) for 90min at 37°C and incubated on ice for 10min. The supernatant was collected and, after centrifugation to discard the remaining death-floating cells, resuspended in DMEM containing 15% FBS. Finally, cells were seeded at a density of 400000 cells per cm² and kept in culture for 2 weeks in DMEM containing 15% FBS and 1% glutamine. Cells were transferred to a serum-free medium (human glioma cell lines), 0.5% FBS medium (primary cultures of glioma cells) or conditioned medium 18h before performing the different treatments. When indicated, cells were preincubated with exogenous Mdk for 2h or with NVP-TAE-684, SR1 or SR2 for 1h before treatment with THC. Cell viability was determined after 72h of THC treatment by using the MTT test (Promega, Madison, WI, USA) according to manufacturer’s instructions. THC stock solution was prepared in dimethylsulfoxide. Control incubations contained the same amount of dimethylsulfoxide and no significant effect was observed in any of the parameters determined throughout this study at the final concentration used (0.1–0.2%, v/v).

Analysis of gene expression by cDNA microarrays

Total RNA was isolated from human glioma cells lines with RNeasy Mini Kit (Qiagen, Hilden, Germany). Double-stranded cDNA was amplified with Superscript Choice System and T7-(deoxythymidine)24 oligo-primers (Life Technologies, Inc., Gaithersburg, MD, USA) and in vitro transcription was carried out with Megascript T7 (Ambion, Austin, TX, USA). Samples were labeled with Cy3-dUTP (Amersham, Uppsala, Sweden) and Universal Human Reference RNA (Stratagene, La Jolla, CA, USA) with Cy5-dUTP (Amersham) and hybridized on an advanced version of the Spanish National Cancer Research Centre (CNIO) Oncochip (http://bioinfo.cnio.es/data/oncochip) as described in the MIAME. Slides were scanned in a Scanarray 5000 XL scanner (GSI Lumonics Kanata, Ontario, Canada) and the data matrices were processed with GenePix 6.0 software (Axon, Instruments Inc., Union City, CA, USA).

Human Mdk ELISA bioassay

Soluble Mdk levels were determined by using the Human Midkine ELISA (Biovendor, Heidelberg, Germany).

Conditioned medium experiments

U87 or T98 cells were seeded in complete medium at a density of 30000 cells per cm² and, after 8h, transferred to a serum-free medium for 18h. Media were subsequently recovered, centrifuged and incubated alone or with an anti-Mdk antibody (20μg/ml; Ref: sc-20715, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or with an isotype antibody (rabbit IgG; 20μg/ml; Amersham Biosciences Corp., Piscataway, NJ, USA) for 1h at 37°C. To study the influence of the conditioned media (obtained as described above) on cell viability, U87 cells were seeded in complete medium at a density of 5000 cells per cm² and, after 8h, transferred to the corresponding conditioned medium 18h before performing the different treatments. Finally, cell viability was determined as described above.

Transfections

T98, LN405, HG2, or Panc1 (75% confluent) cells were transfected with control (siC), human Mdk (siMdk) or human ALK (siALK) siRNAs using the X-tremeGENE siRNA Transfection Reagent (Roche, Basel, Switzerland) according to manufacturer’s instructions. After 24h of transfection, cells were trypsinized and seeded in complete medium at a density of 5000 cells per cm². After 8h, cells were transferred to a serum-free (cell lines) or 0.5% FBS (primary culture) medium for 18h and the different treatments were performed. Transfection efficiency was monitored using a control fluorescent siRNA (Qiagen). The efficacy of transfection was higher than 80% in all the experiments.

Reverse transcription- and real-time quantitive-PCR analysis

RNA was isolated using the RNeasy Protect kit (Qiagen) including a DNase digestion step using the RNase-free DNase kit (Qiagen). cDNA was subsequently obtained using Transcriptor Reverse Transcriptase (Roche). PCR reactions were performed using the following parameters: 95°C for 5min, 94°C for 30s, 58°C for 30s, and 72°C for 1min followed by a final extension step of 72°C for 5min. The number of cycles (23–25 cycles for human Mdk and 22–25 cycles for GAPDH) was adjusted to allow detection in the linear range. Finally, PCR products were separated on 1.5% agarose gels. Real-time quantitive PCR assays were performed using the FastStart Master Mix with Rox (Roche) and probes were obtained from the Universal Probe Library Set (Roche). Amplifications were run in a 7900 HT-Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). Each value was adjusted using 18S RNA levels as reference. Primers sequences and probes used in this study can be found in Supplementary Table 8.

Tumor xenografts

Tumors were induced by subcutaneous injection in nude mice of 10 × 10⁶ U87 or T98 cells in PBS supplemented with 0.1% glucose. When tumors had reached an average size of 250mm³ (an average of 2 weeks for U87 tumors and 14 weeks for T98 tumors), animals were assigned randomly to various groups and injected peritumorally for 15 days with the corresponding treatment (THC (15mg/kg per day), NVP-TAE-684 (1mg/kg per day) or THC (15mg/kg per day) + NVP-TAE-684 (1mg/kg per day)) or vehicle in 100μl of PBS supplemented with 5mg/ml defatted and dialyzed BSA. Tumors were measured with external caliper, and volume was calculated as (4π/3) × (width/2)² × (length/2). At the end of the treatment, animals were killed, all tumors were excised and their weights were determined.

In vivo silencing

In vivo Mdk silencing was performed by using a 1:1 mixture of AteloGeneTM (Koken Co. Ltd., Tokyo, Japan) and 10μM HPLC-purified double-stranded siRNA (synthesized by Dharmacon), according to manufacturer’s instructions. Briefly, the mix of AteloGene and siRNA (1nmol of control or Mdk-selective siRNA per injection) was administered in each tumor at days 1 and 7 of vehicle or THC treatment. RNA sequence targeting human Mdk was 5′-GUUUGGAGCCGACUGCAAGdTdT-3′.³⁹

Western blot analysis

Western blot analysis was performed following standard methods. The antibodies used were: anti-phospho-S6 ribosomal protein Ser 235/236 and anti-total-Akt, anti-phospho-Akt Ser473 (1:1000; Cell Signaling Technology, Inc., Lake Placid, NY, USA), anti-α-tubulin (1:4000; Sigma), anti-LC3 (1:1000; polyclonal, MBL, Naka-Ku Nagoya, Japan, ref PM036), anti-CB₁ and anti-CB₂ (1:1000; Affinity Bioreagents, Golden, CO, USA); anti-Mdk (1:200; Santa Cruz Biotechnology Inc.), anti-total ALK (1:250; rabbit polyclonal ab75953; Abcam, Cambridge, UK) and anti-phospho ALK (Tyrosine 1604) (1:500; rabbit monoclonal (EP661-Y) ab51030, Abcam).

Imunofluorescence from cell cultures

Cell cultures grown on 12mm-coverslips were washed in PBS, fixed with 4% paraformaldehyde (20min at room temperature), permeabilized and blocked to avoid non-specific binding with 10% goat antiserum and 0.25% Triton X-100 in PBS for 45min, and subsequently incubated with the following primary antibodies (for 1h at room temperature): rabbit polyclonal anti-CB₁ antibody and anti-CB₂ antibody (1:500; Affinity Bioreagents), rabbit polyclonal anti-Mdk antibody (1:100; Santa Cruz Biotechnology), rabbit monoclonal anti-phospho ALK antibody (1/200; Abcam), rabbit polyclonal anti-cleaved caspase-3 Asp175 antibody (1:100; Cell Signaling Technology) and mouse monoclonal anti-LC3 antibody (1:100; Nanotools Antikörpertechnik GmbH & Co. Antikörpertechnik GmbH & Co., Teningen, Germany; clone 5F10). Incubation with appropriate Alexa-488 or Alexa-594-conjugated secondary antibodies (Invitrogen, Eugene, OR, USA) was performed in the dark at room temperature for 1h. Cell nuclei were stained with Hoechst 33342 (Invitrogen). Finally, coverslips were mounted in ProLong Gold antifade reagent (Invitrogen) and visualized in a Leica TCS SP2 confocal microscope (Leica, Wetzlar, Germany).

Immunofluorescence from tumor samples

Samples from tumors xenografts were dissected and frozen. Sections (8μm) were permeabilized and blocked to avoid nonspecific binding with 10% goat antiserum and 0.25% Triton X-100 in PBS for 45min, and subsequently incubated with rabbit polyclonal anti-Mdk antibody (1:100; Santa Cruz Biotechnology; 4°C, o/n), rabbit monoclonal anti-phospho ALK antibody (1:200; Abcam; 4°C, o/n), mouse monoclonal anti-LC3 antibody (1:100; Nanotools Antikörpertechnik GmbH & Co; 4°C, o/n), rabbit polyclonal anti-cleaved caspase-3 Asp175 (1:100; Cell Signaling Technology; 90min, room temperature), anti-TRB3 (1:800; Calbiochem, San Diego, CA, USA; 90min, room temperature) or anti-phospho S6 (1:200; Cell Signaling; 90min, room temperature) antibodies, washed, further incubated with the corresponding Alexa-488 or Alexa-594-conjugated secondary antibodies (Invitrogen; 90min, room temperature). Nuclei were stained with Hoechst 33342 (Invitrogen; 10min, room temperature) before montage with Mowiol (Merck, Darmstadt, Germany) was performed. Fluorescence images were acquired using an Axiovert 135 microscope (Carl Zeiss, Thornwood, NY, USA).

TUNEL

Tumor samples were fixed, blocked and permeabilized and TUNEL was performed as described.⁹

Tissue microarray immunohistochemistry

Tissue microarrays were constructed as described previously⁴⁰ using formalin-fixed, paraffin-embedded archival tissue blocks from five non-tumoral tissues (four normal brains and one tonsil) and 201 glioma samples (115 glioblastomas WHO grade IV, GBs; 14 anaplastic astrocytomas WHO grade III, AAs; 12 diffuse astrocytomas WHO grade II, DAs; 12 pilocytic astrocytomas WHO grade I, PAs; 4 anaplastic oligoastrocytomas WHO grade III, AOAs; 8 oligoastrocytomas WHO grade II, OAs; 19 anaplastic oligodendrogliomas WHO grade III, AOGs; and 17 oligodendrogliomas WHO grade II, OGs). All samples were reviewed by means of full tissue sections stained with hematoxylin and eosin to verify tumor viability, select the representative areas of each tumor and confirm the diagnosis according to WHO guidelines.²

For immunohistochemical staining, 4-μm-thick paraffin-embedded tissue sections were subsequently dewaxed, rehydrated and subjected to antigen retrieval using EnVision FLEX Target Retrieval solution at pH10 and heated at 95°C. The slides were cooled and treated with the EnVision FLEX Peroxidase-Blocking Reagent solution (Dako, Glostrup, Denmark) for 5min. Sections were then immunostained with Mdk mouse monoclonal antibody A-9 (1:50; Santa Cruz Biotechnology Inc.) using the Dako EnVision FLEX/HRP Technique (Dako), counterstained with hematoxylin and mounted. Mdk expression was considered positive in presence of cytoplasmic staining was observed.

Survival analyses

Survival curves were plotted according to the Kaplan–Meier method, and the log-rank test was used to establish the statistical significance of the differences between curves. Differences of overall survival based on MDK positive- or negative-protein expression were analyzed. Survival analysis of the MDK microarray expression TCGA (The Cancer Genome Atlas) data reported by Verhaak et al.⁴ was performed by categorizing patients into two groups according to the median MDK expression (high expression ≥0.12; low expression <0.12). The association between continuous MDK expression levels and overall survival was determined by a Cox regression univariate model. The threshold for statistical significance in all cases was taken to be P<0.05. Statistical analyses were carried out with SPSS v18.0 (SPSS, Chicago, IL, USA).

Statistics

Unless otherwise specified, results shown represent mean±S.D. Statistical analysis was performed by ANOVA with a post hoc analysis by the Student-Neuman–Keuls test.

This work was supported by grants from Spanish Ministry of Science and Innovation (MICINN) (PS09/01401; HF2005/0021, FR2009-0052 and IT2009-0053 to GV; SAF2006/00918 to MG), Santander-Complutense (PR34/07-15856 to GV), Comunidad de Madrid (S-SAL/0261/2006 to MG). ML was sequentially the recipient of a ‘Juan de la Cierva’ contract, a postdoctoral contract from Spanish Ministry of Education and Science (MEC) and a postdoctoral contract from Comunidad de Madrid. ST was recipient of a research formation contract from Comunidad de Madrid, MS was recipient of a fellowship from MEC and of a research formation contract from Comunidad de Madrid, AC was recipient of fellowships from Gobierno Vasco, FEBS and EMBO. SH-T has a technician contract from MICINN and Fondo Social Europeo. We thank Horacio Zimman and Carmen Moreno from Hospital Clínico San Carlos as well as Leyre Urigüen from Universidad del Pais Vasco for their kind collaboration in the processing and delivery of glioma samples; Miguel Ángel Piris, Raquel Villuendas, Paloma Cueva and Rosa Pérez for technical advice in the gene expression experiments and other members of our lab for their continuous support.

THC

Δ⁹-tetrahydrocannabinol

GBM

glioblastoma multiforme

ER

endoplasmic reticulum

TRB3

tribbles homolog 3

mTORC1

mammalian target of rapamycin complex 1m

ALK

anaplastic lymphoma kinase receptor

Mdk

midkine

PTN

pleiotrophin

PI3K

phosphatidylinositol 3-kinase

RPTP

protein-tyrosine phosphatase, receptor-type, zeta-1

LRP1

low-density lipoprotein receptor-related protein 1

Supplementary Information accompanies the paper on Cell Death and Differentiation website (http://www.nature.com/cdd)

Edited by P Mehlen

Supplementary Figures 1–13

Supplementary Table 1

Supplementary Table 2

Supplementary Table Legends 1 and 2

Supplementary Table 3

Supplementary Table 4

Supplementary Table 5

Supplementary Table 6

Supplementary Table 7

Supplementary Table 8

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