p75^NTR Proteolytic Processing Occurs in Patient Specimens Endogenously Expressing p75^NTR

In a previous study, our laboratory identified p75^NTR as a potent mediator of invasion in human glioma using a novel invasive glioma mouse model generated by serial in vivo selection [15]. In that study, we found that p75^NTR was expressed in 22% mid-grade astrocytomas (two of nine) and 85% of glioblastoma multiforme (GBM) specimens (17 of 20), and that the p75^NTR-positive glioma cells in the patient tumor cell population were more migratory than the p75^NTR-negative glioma cells. Here, we investigate the mechanism underlying this p75^NTR-induced invasion. In neurons, p75^NTR is a substrate for sequential α- and γ-secretase–mediated intramembrane proteolysis generating 24 kDa CTF and 19 kDa ICD fragments, respectively, and the generation of these fragments are required for some of its biological functions [26,28,30–34]. We therefore sought to determine whether intramembrane proteolysis of p75^NTR occurred in malignant glioma patient specimens. To do this, we assessed whether the generation of the 24-kDa CTF and the 19-kDa ICD occurred in a panel of surgically resected human glioma specimens and normal human brain. Tumor and normal tissue taken at the time of surgery were immediately snap frozen in liquid nitrogen and stored at −80 °C. Frozen tumor tissue was digested in lysis buffer and analyzed by western blots using a p75^NTR cytoplasmic-specific antibody that not only detected the full-length p75^NTR protein, but also detected p75^NTR-positive fragments migrating at 24 and 19 kDa, respectively, in the p75^NTR-positive specimens (eight of nine GBMs and two of five Grade III glioma) (Figure 1A). Hence, p75^NTR processing occurs in human glioma tumors, and this suggested the possibility that p75^NTR processing is required for glioma invasion.

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Figure 1. p75^NTR Is Proteolytically Processed in Patient Specimens and Invasive Glioma Cells

(A) Western blot analysis detected p75^NTR-positive fragments migrating at 24 and 19 kDa in patient specimens expressing p75^NTR (eight of nine GBMs, two of five Grade III glioma) but was undetectable in tissue from normal human brain. U87 cells transfected with p75^NTR and grown in the presence of the proteasome inhibitor epoxomicin (1 μM) were used as a positive control (C). Western blots probed for beta-actin were used as a loading control. Molecular weight markers are indicated on the left.

(B) The highly invasive glioma cell lines U87R and U251R isolated by serial in vivo selection were treated with the proteasome inhibitor epoxomicin (Epo, 1 μM) and/or the γ-secretase inhibitor, Compound X (CompX, 2 μM) for 4 h. Western blots for p75^NTR were probed with an antibody specific to the cytoplasmic domain of p75^NTR which detects full-length (75 kDa), CTF (indicated by the less than symbol [<]; 25 kDa), and ICD (indicated by the asterisk [*]; 19 kDa) peptides. The ICD is derived from the cleavage of the CTF, which is shown to visibly accumulate in the presence of a γ-secretase inhibitor.

(C) p75^NTR proteolytic processing is a global event in human glioma cells. pcDNA3.1 encoding human p75^NTR or the empty pcDNA3.1 vector were stably transfected into U87, U251, U118, and U343. Cells expressing p75^NTR (U87p75, U251p75, U118p75, and U343p75) were treated as described above. Western blot analysis showed that all glioma cell lines tested cleaved the full-length p75^NTR to generate first the 24-kDa CTF and then the 19-kDa ICD.

https://doi.org/10.1371/journal.pbio.0060289.g001

p75^NTR Is Proteolytically Processed in Invasive Glioma Cells

To address the possible role(s) of p75^NTR proteolytic processing in glioma cells, we assessed whether the appearance of the p75^NTR-positive fragments at 24 and 19 kDa was the result of proteolytic processing of the full-length p75^NTR in invading glioma cells. We have previously established the highly invasive glioma cell lines U87R and U251R for which p75^NTR accounts for their invasive behavior [15]. The U87R and U251R invasive glioma cells were grown in the absence or presence of the proteasome inhibitor epoxomicin, a compound used to inhibit rapid degradation of proteins often associated with RIP-mediated proteins [26,35]. Western blot analysis showed that in addition to the 75-kDa full-length p75^NTR protein, 24-kDa and 19-kDa fragments (Figure 1B, lane 1 and 5) were present and stabilized in the presence of 1 μM epoxomicin (Figure 1B, lane 2 and 6). These results are in agreement with the model that the full-length p75^NTR protein is cleaved, releasing the ECD, CTF, and intracellular fragments. Next, we verified that the appearance of the 24-kDa and 19-kDa fragments was the result of sequential cleavage of p75^NTR by an α-secretase and then a γ-secretase. First, we determined whether treatment of p75^NTR glioma cells (U87p75^NTR) using the TNF-α protease inhibitor (TAPI)-2, known to inhibit metalloproteases and ADAMs such as tumor necrosis factor-α converting enzyme (TACE) [36–38] and previously shown to inhibit the proteolytic processing of p75^NTR in neurons [26,32,33,39], could inhibit p75^NTR processing in glioma cells. TAPI-2 inhibited the proteolytic processing of p75^NTR as indicated by the lack of CTF and ICD, and abrogated p75^NTR-mediated invasion (Figure S1). Since TAPI-2 has broad specificity, and glioblastomas are known to produce high levels of many proteases, including members of the MMP, ADAM, and ADAMTS families, leaving the exact identity of the α-secretase unclear, we focused our efforts on the second cleavage event. To determine whether the generation of the 19-kDa fragment was the result of cleavage of p75^NTR by a γ-secretase, U87R and U251R cells were treated with 2 μM Compound X (Calbiochem), a specific inhibitor of γ-secretase, for 4 h in the absence or presence of epoxomicin. Western blot analysis of p75^NTR revealed that in the presence of the γ-secretase inhibitor, an accumulation of the 24-kDa fragment occurred without subsequent cleavage to the 19-kDa ICD, consistent with the release of the ICD of p75^NTR by γ-secretase (Figure 1B, lanes 3, 4, 7, and 8). The role of processing of p75^NTR was not limited to a single glioma cell line and was a general mechanism observed in glioma cells established from genetically distinct individuals (U87p75, U251p75, U343p75, and U118p75).We found that in all p75^NTR-positive glioma cell lines, full-length p75^NTR was cleaved to generate two fragments of 19 and 24 kDa: ICD and CTF, respectively (Figure 1C). These results demonstrate that regulated intramembrane proteolysis of p75^NTR is a global event occurring in highly invasive p75^NTR-positive human glioma cells.

γ-Secretase Inhibition Significantly Abrogated p75^NTR-Induced Glioma Migration and Invasion In Vitro

In neurons, ectodomain shedding of p75^NTR by α-secretase and then γ-secretase cleavage to generate an ICD fragment can result in the activation of downstream events [26–28,30–34]. To test whether the processing of p75^NTR resulting in the release of the ICD fragment has a functional role in glioma invasion, we analyzed in vitro migration and invasion of U87R, U251R, U87p75^NTR, and U251p75^NTR glioma cell lines using circular monolayer migration assays (Figure 2A and 2B) and 3D-collagen invasion assays (Figure 2C and 2D) in the absence and presence of the γ-secretase inhibitor, Compound X. p75^NTR-mediated glioma migration and invasion were significantly inhibited in the presence of Compound X. In contrast, when the proteasome inhibitor epoxomicin was used to stabilize p75^NTR-ICD, a significant increase in migration and invasion was seen (unpublished data), consistent with increased invasion observed when a cDNA construct mimicking the ICD fragment was ectopically expressed in U87 glioma cells (Figure 3Aand 3B). To determine whether γ-secretase inhibition was confined to glioma invasion or had effects on other biological processes, we assessed the effect of γ-secretase inhibition on survival and proliferation of p75^NTR-positive glioma cells. No significant change was observed on either survival or proliferation in vitro (Figure S2).

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Figure 2. γ-Secretase Inhibitor Significantly Inhibited p75^NTR-Induced Migration and Invasion in Glioma Cells

(A and B) Monolayer circular migration assays of U87R and U251R cells, which endogenously express p75^NTR, and U87p75 and U251p75 cells, which ectopically express p75^NTR, were performed. Briefly, cells were seeded in monolayer wells through a cell sedimentation manifold. p75^NTR-negative U87T, U87pcDNA, U251T, and U251pcDNA cells were used for comparison. Once the sedimentation manifolds were removed, cells were given complete medium containing 2 μM Compound X (CompX). Digital images of the cells were taken before migration at 0 h and then again at 72 h. Best-fit circles were drawn around the area covered by the cells at the 0 h and 72 h time points, and the actual cell area was determined using Axiovision 4.2 imaging software. Quantitative migration scores were calculated as the increase in the area covered by the cells beyond the initial area of the cells. γ-Secretase inhibitor Compound X significantly inhibited p75^NTR-induced migration. Values shown are the mean ± s.e.m. from three independent experiments; triple asterisks (∗∗∗) indicate p < 0.001 as compared to control (one-way analysis of variance [ANOVA] with the Neuman-Keuls post-test).

(C and D) 3D-collagen invasion assays of U87R, U87p75, U251R, and U251p75 cells were performed by mixing cells with 3D-collagen matrix (collagen, fibronectin, and laminin) and then seeding them into 8.0-μm pore size transwell chambers in the presence or absence of 2 μM Compound X for 6 h. Cells were fixed and stained, and invasive cells were counted. U87T, U87pcDNA, U251T, and U251pcDNA cells were used for comparison. Inhibition of γ-secretase by Compound X significantly inhibited p75^NTR-induced glioma invasion. Values shown are the mean ± s.e.m. from three independent experiments; triple asterisks (***) indicate p < 0.001 as compared to control (one-way ANOVA with the Neuman-Keuls post-test).

https://doi.org/10.1371/journal.pbio.0060289.g002

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Figure 3. Expression and Proteolytic Processing of p75^NTR Chimeric Alleles

(A) p75^NTR ICD is sufficient to induce invasion of U87 glioma cells in vitro. 3D-collagen invasion assays of U87p75ICD cells were performed by mixing cells with 3D-collagen matrix (collagen, fibronectin, and laminin) and then seeding them into 8.0-μm pore size transwell chamber for 6 h. Cells were fixed and stained, and invasive cells were counted. U87pcDNA and U87p75 cells were used for comparison. Values shown are the mean ± s.e.m. from three independent experiments; triple asterisks (***) indicate p < 0.001 as compared to U87pcDNA (one-way ANOVA with the Neuman-Keuls post-test).

(B) Western blot analysis detected a p75^NTR-positive fragment migrating at 19 KDa in U87 cells expressing the p75-ICD construct. U87p75^NTR glioma cells treated with the proteasome inhibitor epoxomicin (Epo, 2 μM) were used as a positive control (U87-p75+Epo; the less than symbol [<] indicates CTF; the asterisk [*] indicates ICD).

(C) Schematic diagram of the p75^NTR constructs. Chimeric proteins were created by replacing either the transmembrane (TM) or the extracellular stalk (S) domain of p75^NTR with equivalent domains from the Fas receptor. The proteins were designated p75FasTM (FasTM) and p75FasS (FasS), respectively. Other p75^NTR constructs were generated, including the neurotrophin-binding mutant in which the ligand-binding site was mutated by inserting four amino acids after amino acid residue 130, designated p75CRD130 (CRD130), and the p75^NTR intracellular domain construct consisting of amino acids 238 to 399 (p75-ICD).

(D) Western blot analysis using a polyclonal p75^NTR antibody to the intracellular domain of p75^NTR confirmed the proper expression and processing of all p75^NTR constructs (p75FL, p75FasTM, p75FasS, or p75CRD130) in both U87 (left panel) and U251 (right panel) human glioma cell lines (the less than symbol [<] indicates CTF; the asterisk [*] indicates ICD). To examine comparable levels of chimeric proteins, lysates were adjusted accordingly.

https://doi.org/10.1371/journal.pbio.0060289.g003

Biochemical Characterization of U87 and U251 Glioma Cells Expressing p75^NTR Wild-Type or Cleavage-Resistant Chimeric p75^NTR Alleles

It is well known that γ-secretase has many substrates [40,41]. To directly test the role of p75^NTR processing in glioma invasion, we constructed cleavage-resistant chimeric proteins of p75^NTR by replacing either the transmembrane (p75FasTM) or the extracellular stalk domain of p75^NTR (p75FasS) with equivalent domains from the Fas receptor [39] (Figure 3C). Both p75^NTR and Fas receptors are members of the TNF receptor superfamily, and although they each contain similar domains, Fas, unlike p75^NTR, does not undergo RIP. Since ectopic expression of p75^NTR in the human glioma cells lines U87 and U251 was sufficient to mediate glioma invasion [15], these cell lines were used as a model system to assess the p75^NTR chimeric mutants. U87 and U251 were therefore stably transfected with the cleavage-resistant p75^NTR constructs (p75FasS and p75FasTM). To ensure proper function of all p75^NTR protein constructs, we assessed their location, topography, and ability to bind neurotrophin in both U87 and U251 glioma cell lines. Receptor orientation and localization at the plasma membrane was confirmed by flow cytometric analysis using a monoclonal antibody specific to the ECD domain of p75^NTR (Figure S3A). As expected, all p75^NTR constructs were expressed at the plasma membrane with the correct topography. Next, we assessed whether the chimeric constructs could still bind neurotrophin. Previously, we demonstrated that in the absence of p75^NTR, glioma cells secrete high levels of brain-derived neurotrophic factor (BDNF) protein into the culture medium in vitro. When these same cells express p75^NTR, the majority of the BDNF is found to be cell associated, presumably bound to p75^NTR [15]. To confirm that the p75^NTR cleavage-resistant chimeric forms retained the ability to bind neurotrophin, ELISA assays were performed to detect BDNF expression in the conditioned medium and total cell lysates of U87 and U251 cells expressing p75FL, p75FasTM, p75FasS, and p75CRD130 (Figure S3B). p75CRD130 is a neurotrophin-binding mutant created by inserting four amino acids after amino acid residue 130 [15,42–47]. Expression of the chimeric p75^NTR proteins (p75FasTM and p75FasS), just like the p75 wild type, resulted in a shift in BDNF localization from the conditioned medium to the cell lysate. This was in contrast to the cells expressing the neurotrophin-binding mutant (p75CRD130) or the empty vector (pcDNA) where the bulk of BDNF was detected in the culture medium. These data demonstrate that p75 ^NTR cleavage-resistant chimeric constructs p75FasTM and p75FasS retained their ability to bind neurotrophin (Figure S3C). Once we confirmed the correct expression and binding of the various p75^NTR constructs, western blots using a p75^NTR cytoplasmic domain-specific antibody were performed to evaluate proteolytic processing of the various p75^NTR receptors (Figure 3D). In cells expressing p75FasS, only the full-length protein was detected, consistent with inhibition of the α-secretase cleavage, whereas the full-length 75 kDa and the 24-kDa fragment were detected in cells expressing the p75FasTM construct corresponding to the ectodomain shedding of p75^NTR by α-secretase but with inhibition of the γ-secretase cleavage. Moreover, in the presence of epoxomicin, no additional p75^NTR fragments were observed (Figure S4). These results demonstrate the cleavage-resistant chimeric p75^NTR alleles were expressed with correct biochemical characteristics in U87 and U251 glioma cells. In addition, and consistent with the hypothesis that proteolytic processing of p75^NTR is required for glioma invasion, only the full-length 75 kDa band was detected in lysates from U87 cells expressing p75CRD130, a p75^NTR construct that was unable to induce glioma invasion [15].

Cleavage-Resistant Chimeric Forms of p75^NTR Do Not Induce Glioma Migration and Invasion In Vitro or In Vivo

Since we have shown that neurotrophin binding is required for p75^NTR-mediated glioma invasion [15], and the neurotrophin-binding mutant p75CRD130 does not undergo RIP, it would appear that RIP of p75^NTR is required for glioma invasion. To determine whether this is in fact true, U87 and U251 cells expressing the p75^NTR cleavage-resistant constructs were assessed for their invasive ability using 3D-collegen invasion assays. We found that expression of cleavage-resistant forms of p75^NTR (p75FasS, p75FasTM, and p75CRD130), which prevented receptor proteolysis, blocked p75^NTR-mediated glioma invasion (Figure 4A and 4B), providing evidence to support a role for γ-secretase–dependent release of p75^NTR ICD in mediating glioma invasion.

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Figure 4. Cleavage-Resistant Chimeric p75^NTR Proteins Do Not Induce Migration and Invasion In Vitro or In Vivo

(A and B) The invasive ability of U87 (A) and U251 (B) stably transfected with p75^NTR or the p75^NTR cleavage-resistant chimeric constructs (p75FasS and p75FasTM) were assessed using 3D-invasion assays. Expression of p75^NTR significantly increased invasion in the genetically distinct glioma cell lines U87 and U251, whereas neither p75FasS nor p75FasTM stimulated glioma invasion as compared to the p75^NTR-negative control cells (pcDNA). Values shown are the mean ± s.e.m. from three independent experiments; (the less than symbol [<] indicates CTF; the asterisk [*] indicates ICD; triple asterisks [∗∗∗] indicate p < 0.001 vs. p75^NTR wild type, one-way ANOVA with Neuman-Keuls post-test).

(C) U87 cells stably expressing p75^NTR or the cleavage-resistant p75FasS and p75FasTM were implanted into the brains of SCID mice and allowed to grow for 28 d. The mice were sacrificed, and frozen brain sections were stained with antibodies against human nuclei (top row, brown) and human p75^NTR (bottom row, brown). Sections were counterstained with toluidine blue (blue). Scale bars in (C) represent 100 μm. Implantation of U87 glioma cells stably transfected with the empty pcDNA vector or p75^NTR cleavage-resistant chimeras (p75FasTM and p75FasS), led to the formation of well-circumscribed tumors. In marked contrast, animals implanted with U87 glioma cells ectopically expressing p75^NTR (U87p75) developed tumors with highly infiltrative edges. Similar results were seen in three independent experiments with three animals in each group.

(D) p75^NTR cleavage-resistant chimeras do not generate ICD in vivo. Brain cryosections from SCID mice implanted with U87 glioma cells expressing p75^NTR, p75FasTM, or p75FasS were lysed in 2× loading buffer. Proteins were resolved on 10% SDS-PAGE gels and probed with a p75 cytoplasmic-specific antibody. U87R, U251R, and U87p75, which express high levels of p75^NTR and exhibit increased invasive activity, process full-length p75^NTR to generate both CTF and ICD in vivo. In contrast, and consistent with the in vitro data, the p75^NTR ICD peptide was not detected in tumors from animals expressing p75FasS or p75FasTM. Cell lysates from U87 cells expressing p75^NTR and grown in the presence of epoxomicin (p75^NTR+Epo) or expressing pcDNA (pcDNA) were used as positive and negative controls, respectively.

https://doi.org/10.1371/journal.pbio.0060289.g004

To determine whether p75^NTR processing was required for glioma invasion in vivo, U87 glioma cell lines ectopically expressing p75FasS and p75FasTM were implanted into the brains of immunocompromised (SCID) mice. U87 glioma cells expressing full-length p75^NTR (U87p75) or control vector (U87pcDNA) were used for comparison. Twenty-eight days after implantation, the mice were sacrificed, and frozen brain sections were stained with antibodies against human nuclei, to visualize all glioma cells (Figure 4C, upper panel) or with anti-human p75^NTR (Figure 4C, bottom panel). Implantation of U87 glioma cells stably transfected with the control pcDNA vector led to the formation of well-circumscribed tumors, while U87 glioma cells ectopically expressing p75^NTR formed tumors with highly infiltrative edges. In sharp contrast to the p75^NTR-expressing U87 tumors, tumors expressing either p75FasTM or p75FasS formed well-circumscribed tumors similar to the p75^NTR-negative tumors (U87pcDNA). Comparable results were seen in three independent experiments. In conjunction with the in vitro data, these data suggest that proteolytic processing of p75^NTR is required for glioma invasion in vivo (Figure 4C).

It is well known that the microenvironment of tumors can change the biochemical characterization and function of cells. We have demonstrated in vitro that glioma cells expressing p75^NTR undergo proteolytic processing to generate first the 24-kDa CTF and then the 19-kDa ICD. To provide evidence that RIP of p75^NTR occurs in vivo, 7–9-μm cryosections from mice implanted for 3–4 wk with in vivo–selected U87R and U251R, or ectopically expressing p75^NTR, p75FasS, p75FasTM, and pcDNA, were assessed for p75^NTR processing by western blot. The 24- and 19-kDa fragments were found in the highly invasive glioma cells, U87R, U251R, and U87p75^NTR. In contrast, neither the 24-kDa nor the 19-kDa fragment was seen in cells expressing p75FasS, and as expected, only the 24-kDa fragment was detected in cells expressing the p75FasTM, consistent with their in vitro characterization (Figure 4D). These data further support a role for RIP of p75^NTR in glioma invasion.

p75^NTR Proteolytic Processing Occurs in BTICs from Patient Specimens That Endogenously Express p75^NTR

Our data demonstrated that 85% of GBM specimens (17/20) express p75^NTR, that the p75^NTR-positive glioma cells in the original patient tumor cell population were more migratory [15], and that 24- and 19-kDa p75^NTR-positive fragments are present in p75^NTR-positive primary Grade III and GBM patient specimens (Figure 1). We therefore wanted to determine whether the appearance of these fragments in the malignant glioma patient specimens was the result of intramembrane proteolysis of p75^NTR. To do this, we established primary cultures from human glioma patient tumors. The recent discovery that human stem cell-like tumor cells, termed BTICs, retain characteristics that closely recapitulate the original patient tumor [3–6,48,49] prompted us to establish the primary patient tumor cultures under neural stem cell–promoting conditions. BTICs share characteristics with neural stem cells (NSCs) such as continuous self-renewal, extensive brain parenchymal migration and infiltration, and potential for full or partial differentiation, properties not found in established glioma cell lines [50,51]. Operative samples of human GBM were obtained at the time of surgery, and brain tumor sphere cultures were established in NS-A basal medium plus epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) (EF medium). Immunocytochemical analysis of BTICs established from five glioma patients expressed the early neural cell progenitor proteins Nestin, Musashi, hSox2, and CD133 (J. J. P. Kelly, S. Weiss, P. A. Forsyth, and D. L. Senger; unpublished data). In addition, four out of five glioma tumor progenitor cells in vitro expressed high levels of p75^NTR as detected by immunocytochemistry (Figure 5A) and western blot (Figure 5B). To determine whether p75^NTR expressed on the BTICs undergoes RIP, BTICs were grown in the absence and presence of γ-secretase and/or 2 μM epoxomicin. Similar to the glioma cell lines, full-length p75^NTR, CTF, and ICD were detected, and the 19-kDa ICD fragment was dependent on γ-secretase cleavage (Figure 5B). We next determined whether the BTICs retained their expression of p75^NTR in vivo: BTIC cells were implanted into the brains of SCID mice and allowed to establish for 4–8 wk. Animals were sacrificed, and frozen sections were stained with either an anti-human nuclear antibody (to identify the human BTICs) or anti-p75^NTR (Figure 5C). All tumors established from BTICs cells showed highly infiltrative tumors, and consistent with the in vitro data, four out of five tumors showed high expression of p75^NTR in vivo.

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Figure 5. Regulated Intramembrane Proteolysis Occurs in Brain Tumor-Derived Stem-Like Cells (BTICs) from Glioma Patient Specimens

(A and B) Primary cultures from human glioma patient tumor grown under brain tumor stem cell-promoting conditions (BTIC) express high levels of p75^NTR (red) in vitro as detected by immunocytochemistry (A) and western blot (B). U87 cells stably expressing pcDNA and p75^NTR were used as negative and positive controls, respectively. Cultures were counterstained with DAPI (blue) to visualize the cell nucleus. (B) p75^NTR expressed on the BTICs undergoes RIP. BTICs were grown in the absence and presence of 2 μM γ-secretase and/or 1 μM epoxomicin. Western blot analysis detected full-length p75^NTR, CTF, and ICD with the accumulation of the 24-kDa CTF in the presence of the γ-secretase inhibitor (CompX).

(C) Brain tumor-initiating cells (BTIC) express p75^NTR in vivo. BTIC cells were implanted into the brains of SCID mice and allowed to establish for 4–8 wk. Animals were sacrificed, and frozen sections were stained with anti-p75^NTR (brown). Sections were counterstained with toluidine blue (blue). Tumors established from BTICs express high levels of p75^NTR and form highly infiltrative tumors. M1 and M2 are individual mice.

https://doi.org/10.1371/journal.pbio.0060289.g005

γ-Secretase Inhibitor Significantly Abrogated p75^NTR-Induced Migration and Invasion In Vivo

p75^NTR as a substrate for γ-secretase [26,33,39] adds to a growing list of proteins shown to be substrates for γ-secretase cleavage, including APP [52–54], Notch [55,56], and Notch ligands Delta1 and Jagged2 [57], ErbB4 [58], CD44 [59,60], and E-cadherin [61,62]. Our in vitro data and the recent application of γ-secretase inhibitors (egs.LY-450139 and LY-411575) in advanced clinical trials for Alzheimer disease [63–65] prompted us to investigate the use of γ-secretase inhibitors to treat highly invasive gliomas. Using an intracranial glioma model, we assessed the therapeutic potential of γ-secretase inhibitors. Parallel experiments were performed using the genetically distinct U87p75^NTR and U251p75^NTR glioma cell lines and the p75^NTR-positive BTICs established from a patient GBM. Cells were implanted intracerebrally into SCID mice; and 3 d after implantation for U87p75^NTR and U251p75^NTR or 5 d after for BTICs, mice were administered subcutaneously (s.c.) either 10 mg/kg γ-secretase inhibitor or vehicle control (corn oil) once/day for 2–3 wk (three to five mice/group). Tumors were allowed to grow for a total of 4–6 wk, at which time, all animals were sacrificed, their brains removed, frozen in O.C.T. compound, and sectioned. Immunohistochemical staining of the frozen brain sections for anti-human nuclei (unpublished data) or p75^NTR (Figure 6A–6C) showed that animals implanted with U87p75^NTR-, U251p75^NTR-, or p75^NTR-positive BTICs formed tumors with highly infiltrative edges (Figure 6A–6C; upper panels). In sharp contrast, animals implanted with U87p75^NTR-, U251p75^NTR-, or p75^NTR-positive BTICs and given the γ-secretase inhibitor DAPT developed localized tumors with highly demarcated edges (Figure 6A–6C; lower panels). These results strongly suggested γ-secretase inhibition that results in blocking the generation of the p75^NTR ICD substantially inhibited the invasive ability of glioma cells in vivo. To establish whether the effects of the γ-secretase inhibition were confined to glioma invasion or had consequences on other biological functions, we assessed the effect of γ-secretase inhibition on glioma cell proliferation in vivo and found no significant change (Figure S5). The γ-secretase inhibitor had negliable effects on the self-renewal capability of the BTIC line used in Figure 5 (unpublished data).

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Figure 6. γ-Secretase Inhibitor Significantly Blocked p75^NTR Induced Glioma Migration and Invasion In Vivo

(A–C) u87p75^NTR (A), U251p75^NTR (B), or p75^NTR-positive BTICs established from a patient GBM specimen (C) were implanted intracerebrally into SCID mice (m1–m3 and m7–m9 represent individual mice). Three days (U87p75^NTR and U251p75^NTR) or 5 d (BTIC) later, mice were administered s.c. 10 mg/kg γ-secretase inhibitor DAPT (γ-SI) or vehicle (corn oil) alone, once/day for 2–3 wk (three to five mice/group). The mice were sacrificed, and frozen brain sections were stained with an antibody against human p75^NTR (brown). Sections were counterstained with toluidine blue to visualize the cell nucleus (blue). Control animals given vehicle alone (corn oil; middle row) developed tumors with highly infiltrative edges (top panels). In marked contrast, animals that received daily injections of the γ-secretase inhibitor DAPT (bottom panels) developed localized tumors with demarcated edges. Similar results were seen in two independent experiments with three to five animals in each group.

(D) γ-Secretase inhibitor blocks p75^NTR processing in vivo. Brain cryosections from three individual SCID mice implanted with u87p75^NTR-, U251p75^NTR-, or p75^NTR-positive BTICs and administered s.c. 10 mg/kg γ-secretase inhibitor DAPT or vehicle (corn oil) alone were lysed in 2× loading buffer. Proteins were resolved on 10% SDS-PAGE gels and probed with a p75 cytoplasmic-specific antibody. In animals given vehicle alone, western blot analysis detected p75^NTR-positive fragments migrating at 75, 24, and 19 kDa, whereas in cryosections from animals given the γ-secretase inhibitor DAPT, only the full-length p75^NTR and 25-kDa CTF were detected. U87 cells expressing p75^NTR and grown in the presence of epoxomicin (control; C) were used as a positive control. (The less than symbol [<] indicates CTF; the asterisk [*] indicates ICD.)

(E) γ-Secretase inhibitor increases survival of animals bearing U87p75^NTR xenografts. Kaplan-Meier survival curves of SCID mice harboring U87p75^NTR intracranial tumors given s.c. injections of 10 mg/kg γ-secretase inhibitor DAPT or vehicle (corn oil) alone, once/day beginning on day 3. Animals given the γ-secretase inhibitor (GSI) survived significantly longer than control animals (p < 0.0001).

https://doi.org/10.1371/journal.pbio.0060289.g006

To confirm that administration of DAPT inhibited p75^NTR ICD generation in vivo, 7–9 μm cryosections were lysed as described previously, and western blots for p75^NTR were performed (Figure 6D). These experiments show the presence of full-length p75^NTR, CTF, and ICD in control animals that received s.c. administration of vehicle (corn oil) alone. In contrast, western blots of cryosections from animals that received daily injections of the γ-secretase inhibitor DAPT detected only the full-length and CTF fragments of p75^NTR. Subcutaneous administration of γ-secretase inhibitor DAPT inhibited the generation of the p75ICD and resulted in a visible accumulation of the CTF. In addition and most importantly, animals bearing U87p75^NTR orthotopic xenographs and given daily s.c. injections of the γ-secretase inhibitor DAPT survived significantly longer (p < 0.0001) than control animals (Figure 6E), further highlighting the potential use of γ-secretase inhibitors in the clinical treatment of malignant glioma.

Cell culture.

The human glioma cell lines U87, U118, and U343 were obtained from the American Type Culture Collection. The human glioma cell line U251N was a kind gift from V. W. Yong (University of Calgary, Calgary, Alberta, Canada). All cells were maintained in complete media (Dulbecco's modified eagle's medium [DMEM] F12 supplemented with 10% heat-inactivated fetal bovine serum [FBS], 0.1 mM nonessential amino acids, 2 mM l-glutamine, and 1 mM sodium pyruvate [Gibco BRL, http://www.invitrogen.com]) at 37 °C in a humidified 5% CO₂ incubator. Cells were passaged by harvesting with trypsin (Gibco BRL) at 80%–90% confluence. Stable transfectants of U87, U251, U118, and U343 cells were maintained in identical media with the exception of the addition of 400 μg/ml of G418 (Invitrogen, http://www.invitrogen.com).

Construction of p75^NTR cleavage-resistant plasmids.

The human p75^NTR expression vector was constructed as described previously [98]. The expression plasmids containing the p75^NTR mutants were constructed by subcloning of PCR fragments containing the desired p75^NTR sequences. Chimeric proteins were created by replacing either the transmembrane (p75FasTM) or the extracellular stalk domain of p75^NTR (p75FasS) with equivalent domains from the Fas receptor [Figure 3C] as described by Domeniconi et al. 2005 [33]. The neurotrophin-binding mutant that was mutated by a four-amino acid (ARRA) insertion after amino acid residue130 was termed p75CRD130 [15,42,43,47]. The p75^NTR intracellular domain construct was created using amino acids 236–399 of the wild-type receptor plus a methionine at the amino terminus (p75-ICD). The original p75^NTR templates were from B. Hempstead (p75WT; Weill Medical College of Cornell University, New York, New York) and M. Chao (pT3/T7-p75; New York University School of Medicine, New York, New York). All constructs were inserted into pcDNA 3.1 expression vectors (Invitrogen). The sequences of all the mutant expression plasmids were confirmed prior to stable transfection.

Transfection of glioma cell lines.

Transfection of glioma cell lines was performed as described previously [15]. Briefly, cells to be transfected were seeded at 2 × 10⁵ cells/well in six-well plates, and incubated at 37 °C overnight. Vector DNA was introduced to the cells using FuGENE 6 transfection reagent (Roche Diagnostic, http://www.roche.com) according to the manufacturer's instructions. The following day, the medium was changed to fresh complete medium containing the antibiotic G418 (concentration determined by toxicity curve for each cell line) to select for those cells that had taken up the vector. The cells were then grown under antibiotic selection until the cells were at confluence. For U87p75^NTR, U251p75^NTR, U118p75^NTR, U343p75^NTR, U87p75FasTM, U87p75FasS, U87p75CRD130, U251p75FasTM, and U251p75FasS transfection, transfected cells were identified by flow cytometry and western blot.

Western blotting.

The desired cells were washed in ice-cold PBS and lysed by gentle rocking in lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10 mM NaF, 0.02% NaN₃, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet P-40, 1% Triton X-100, 1 mM EDTA, 60 mM β-octyl glucoside, 25 μg/ml aprotinin, 10 μg/ml leupeptin, 3 mM sodium orthovanadate, 1 mM PMSF) at 4 °C. Cellular debris was removed by centrifugation, and protein quantification was performed using the bicinchoninic acid (BCA) assay (Pierce Biotechnology, http://www.piercenet.com). Proteins were resolved on 12% SDS-PAGE gels, and western blots were performed using the following primary antibodies: rabbit polyclonal anti-human p75^NTR intracellular domain (Promega, http://www.promega.com), mouse monoclonal anti-human p75^NTR ECD (Upstate Biotechnology), mouse monoclonal anti-β-tubulin (Sigma-Aldrich, http://www.sigmaaldrich.com), or mouse monoclonal anti-β-actin (Cell Signaling Technology, http://www.cellsignal.com). The appropriate HRP-conjugated secondary antibody (Pierce Biotechnology) was used, and blots were visualized using enhanced chemiluminescence (Amersham Biosciences).

Flow cytometric analysis of p75^NTR.

A total of 1 × 10⁶ cells stably transfected with p75^NTR wild type or p75^NTR cleavage-resistant constructs p75FasTM and p75FasS were collected using Puck's EDTA at 37 °C and then washed in PBS containing 1 mM EDTA (PBS/EDTA). Cells were exposed to the monoclonal anti-p75^NTR, clone ME20.4 (which recognizes the extracellular domain; Upstate Biotechnology, http://www.upstate.com), diluted 1:250 in PBS/EDTA for 30 min on ice. Cells transfected with pcDNA vector alone were used as negative controls. After washing with PBS/EDTA, cells were incubated with Alexa-488 conjugated goat anti-mouse IgG (Invitrogen/Molecular Probes, http://www.probes.invitrogen.com) diluted 1:500 in PBS/EDTA for 30 min on ice. Cells were then washed with PBS/EDTA, resuspended in PBS/EDTA, and analyzed using a FACScan flow cytometer (Becton, Dickinson and Company, http://www.bdbiosciences.com).

ELISA.

U87 and U251 glioma cells stably transfected with p75^NTR wild type or the cleavage-resistant constructs were allowed to condition culture medium for 5 d. The conditioned medium was then collected, centrifuged, and filtered through a 0.2-μm syringe filter (VWR International, http://www.vwr.com). The remaining cells were washed with ice-cold PBS, and total cellular lysates were extracted as described for western blot. Protein quantification was performed using the BCA assay (Pierce Biotechnology), and BDNF, nerve growth factor (NGF), or neurotrophic factor 3 (NT-3) ELISA (R&D Systems, http://www.rndsystems.com) was performed as per the company protocol. Briefly, MaxiSorp ELISA plates (Nalge Nunc International, http://www.nalgenunc.com.com) were coated with monoclonal anti-human BDNF, NGF, or NT-3 (R&D Systems), nonspecific binding was blocked, and then the standards of serial dilutions of recombinant human BDNF, NGF, or NT-3 (Sigma-Aldrich) and equal volumes of conditioned medium or equal quantities of lysate were added. Bound antigen was detected using the corresponding biotinylated antibody, streptavidin HRP, and a tetramethylbenzidine substrate (R&D Systems). Absorbance was measured at 450 nm.

In vitro p75^NTR proteolysis.

For in vitro p75^NTR cleavage assessment, the desired cells were treated for 4 h at 37 °C and 5% CO₂ with the proteasome inhibitor epoxomicin (1 μM) (Calbiochem, http://www.emdbiosciences.com) and/or γ-secretase inhibitor Compound X (2 μM) (Calbiochem). DMSO was used as the vehicle control. Cells were then washed one time with cold PBS on ice, lysed in lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10 mM NaF, 0.02% NaN₃, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet P-40, 1% Triton X-100, 1 mM EDTA, 60 mM β-octyl glucoside, 25 μg/ml aprotinin, 10 μg/ml leupeptin, 3 mM sodium orthovanadate, 1 mM PMSF) at 4 °C with protease inhibitors, and centrifuged for 5 min at 14,000×g; supernatants were quantified by BCA assay (Pierce Biotechnology) for use in SDS-PAGE. The CTF (24 kDa) and ICD (19 kDa) fragments were detected using an antibody specific to the p75^NTR ICD (Promega).

Circular Monolayer Migration Assay.

Migration assays were performed using a microliter-scale radial monolayer migration assay as described previously [15]. Briefly, ten-well Teflon-masked microscope slides were coated with 20 μg/ml laminin, followed by the addition of 50 μl of medium to each well. Sedimentation manifold (Creative Scientific Methods, http://www.creative-sci.com) was placed over the laminin-coated slide. Cells were seeded through the central lumen of the cell sedimentation cylinder at 2,000 cells/well (five wells per cell type/condition) to establish a circular 1-mm diameter confluent monolayer. Slides were placed on ice for 60 min and then incubate at 37 °C for approximately 6 h. Attachment of the cells was confirmed prior to removing the sedimentation manifold. Once the sedimentation manifolds were removed, cells were given complete medium containing the γ-secretase inhibitor (Compound X, 2 μM). A digital image of the cells was taken (before migration = 0 h) using a Zeiss Axiovert 200M inverted fluorescent microscope (Carl Zeiss, http://www.zeiss.com). The cells were then incubated in a humidified chamber at 37 °C and 5% CO₂, and a second digital image was taken 48 h later. Best-fit circles were drawn around the area covered by the cells at the 0 h and 48 h time points, and the actual cell area was determined using Axiovision 4.2 imaging software (Carl Zeiss). Quantitative migration scores were calculated as the increase in the area covered by the cells beyond the initial area of the cells.

3D-invasion assay.

To test the invasive ability of the p75^NTR cleavage-resistant constructs, actively growing glioma cells U87 and U251 stably transfected with p75^NTR and p75^NTR cleavage-resistant constructs were suspended in a collagen gel solution and plated in transwell chambers with 8.0-μm pore size polycarbonate membrane (Costar, http://www.costar.com). The collagen gel was prepared by mixing collagen solution (Chemicon International 3D Collagen cell culture system, Cat# ECM675, http://www.chemicon.com) with 5× DMEM F12 medium on ice. Neutralization solution (40:1) and extracellular matrix (ECM) proteins were added at a concentration of 10 μg/ml (laminin, fibronectin, chondroitin sulfate proteoglycan, Chemicon). A total of 1 × 10⁵ cells were suspended in 350 μl of collagen gel solution, and 70 μl of the collagen/cell mixture was pipetted into the Transwell chambers (five chambers for each cell line). Chambers were immediately transferred to a 37 °C incubator for 60 min to allow the matrix to polymerize. Once polymerized, 100 μl of serum-free DMEM was added to the upper chamber and 1.0 ml of 10% FBS complete medium with the γ-secretase inhibitor Compound X (2 μM) was added to the lower chamber. Transwell chambers were kept at 37 °C for 6 h, at which time the chamber was washed with PBS, fixed with acid-alcohol for 15 min at room temperature, and then stained with hematoxylin. Any cells remaining in the top chamber were removed, and membranes were mounted on glass slides. Four different fields were counted for each membrane.

Animals.

Six- to 8-wk-old female SCID mice were purchased from Charles River Laboratories (http://www.criver.com). The animals were housed in groups of three to five and maintained on a 12-h light/dark schedule with a temperature of 22 °C ± 1 °C and a relative humidity of 50% ± 5%. Food and water were available ad libitum. All procedures were reviewed and approved by the University of Calgary Animal Care Committee.

In vivo studies using an intracranial glioma model.

Actively growing glioma cells stably transfected with p75^NTR and p75^NTR cleavage resistant constructs were harvested by trypsinization, washed, and resuspended in sterile PBS(137 mM NaCl, 8.1 mM Na₂HPO₄, 2.68 mM KCl, and 1.47 mM KH₂PO₄ [pH 7.5]). These cells were implanted intracerebrally into the right putamen of SCID mice (1 × 10⁵ cells/mouse) at a depth of 3 mm through a scalp incision and a 0.5-mm burr hole made 1.5–2 mm right of the midline and 0.5–1 mm posterior to the coronal suture. All mice were anaesthetized by intraperitoneal administration of ketamine (85 mg/kg) plus xylazine (15 mg/kg) (MTC Pharmaceuticals). The stereotactic injection used a 5-μl syringe (Hamilton Co., www.hamiltoncompany.com) with a 30-g needle mounted on a Kopf stereotactic apparatus (Kopf Instruments). After withdrawal of the needle, the incision was sutured. Animals were sacrificed at specific time points (generally weekly, from 2–6 wk postinjection) or when they lost 20% of their body weight or had difficulty ambulating, feeding, or grooming. For some experiments, BrdU was given by intraperitoneal injection 24 h prior to sacrifice. Following sacrifice, the brains were removed, frozen in Tissue-Tek O.C.T. compound (Electron Microscopy Sciences, http://www.emsdiasum.com), and cryo-sectioned into 7–9-μm sections for examination by immunohistochemistry and in vivo p75^NTR proteolysis assessment.

Immunohistochemistry.

Frozen sections were air-dried at room temperature, fixed with cold acetone, and then rinsed with PBS. Endogenous peroxidases in the sections were inactivated with 0.075% H₂O₂/methanol, and nonspecific binding was blocked with 10% normal goat serum in PBS. The sections were incubated with rabbit polyclonal anti-human p75^NTR ICD antibody (Promega) or mouse monoclonal anti-human nuclei (Chemicon) in blocking buffer overnight at 4 °C. Following washing with PBS, the appropriate biotinylated secondary antibody (Vector Laboratories, http://www.vectorlabs.com) was applied. Avidin-biotin peroxidase complexes were then formed using the VECTASTAIN Elite ABC kit (Vector Laboratories) and detected by addition of SIGMAFAST DAB (3,3′-diaminobenzidine tetrahydrochloride) (Sigma-Aldrich), which was converted to a brown reaction product by the peroxidase. Toluidine blue (for frozen sections) was used as a nuclear counterstain. Sections were then dehydrated in an ethanol/xylene series and mounted with Entellan (Electron Microscopy Sciences).

In vivo p75^NTR proteolysis.

For detection of p75^NTR proteolytic processing in vivo, the desired cells were implanted intracerebrally into SCID mice as described previously. Mice were sacrificed 3–4 wk later. Following sacrifice, the brains were removed, frozen in Tissue-Tek O.C.T. compound (Electron Microscopy Sciences), cryosectioned into 7–9-μm sections, and alternating sections were stained with toluidine blue. Based on the size of tumor, cryosections were lysed in 2× loading buffer (0.1 M Tris-HCl [pH 6.8], 4% SDS, 20% glycerol, 10% β-mercaptoethanol, 0.02% bromphenol blue). Proteins were resolved on 12% SDS-PAGE gels, and western blots were performed using an anti-p75^NTR cytoplasmic specific antibody (Promega).

Detection of p75^NTR proteolysis in patient specimens.

Tumor and normal tissues were obtained from the Canadian Brain Tumor Tissue Bank in London, Ontario, and the Brain Tumor Tissue Bank at the Clark Smith Brain Tumor Center within the Southern Alberta Cancer Research Institute. Briefly, tissue was taken during surgery while patients were under a general anesthetic, and was placed immediately in liquid nitrogen and stored at −80 °C. An institutional ethics board approved the collection and use of all of the surgical tissue used, and all of the patients gave signed informed consent. Frozen sections of patient tumor tissue were lysed in lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10 mM NaF, 0.02% NaN₃, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet P-40, 1% Triton X-100, 1 mM EDTA, 60 mM β-octyl glucoside, 25 μg/ml aprotinin, 10 μg/ml leupeptin, 3 mM sodium orthovanadate, 1 mM PMSF) with protease inhibitors on ice using a homogenizer (Life Technologies). Lysates were centrifuged for 5 min at 14,000×g to remove debris, and supernatants were quantified by BCA assay (Pierce Biotechnology). In this study, based on our previous immunohistochemistry results of p75^NTR expression in GBM patient specimens, western blot analysis was performed on nine GBMs and five mid-grade glioma samples using an antibody specific for the intracellular domain of p75^NTR.

Primary culture of brain tumor-initiating cells (BTICs).

Tumor and normal tissues were obtained from the Tumor Tissue Bank in Foothills Hospital, Calgary, Alberta. Operative samples of human gliomas were obtained during brain tumor surgery and transported to the laboratory in serum-free DMEM-F12. Primary cultures of brain tumor-initiating cells (BTICs) were established. Briefly, necrotic and connective tissue and any blood clots were removed using forceps, and the remaining tissue was washed in PBS and cut into pieces of approximately 1 mm³. The tissue was then incubated for 5–10 min at 37 °C in an enzyme cocktail of trypsin (0.25%) and DNase I (10 μg/ml) in PBS. The digested tissue was strained through a 100-μm mesh and washed with PBS. Following lysis of the red blood cells, the remaining cells were washed with PBS and strained through a 40-μm mesh. After spinning at 1,000 rpm for 5 min, cells were resuspended in stem cell medium (M medium) or stem cell medium plus EGF and bFGF (EF medium). M medium is NeuroCult NS-A basal medium (human) 450 ml plus NeuroCult NS-A Proliferation Supplements (human) 50 ml (StemCell Technologies). EF media is M media plus human recombinant EGF (20 ng/ml; Sigma) and bFGF (20 ng/ml; Chemicon).

Immunocytochemistry of BTICs.

Eight-well LAB-TEK chamber slides (Nalgel Nunc, http://www.nuncbrand.com) were coated with poly-l-ornithine (Sigma) and incubated at 37 °C for 1 h. The desired BTICs were plated into chambers with stem cell culture medium to equilibrate overnight at 37 °C, 5% CO₂. Chambers were then rinsed with PBS, fixed in 3.7% paraformaldehyde diluted in PBS for 20 min, and rinsed twice with PBS. Anti-p75^NTR cytoplasmic specific antibody (1:3,000, Promega) and antibodies to the progenitor markers: Nestin (1:1,000), hSOX2 (1:5,000), and mushashi (1:200) (R&D System) were diluted in 0.3% Triton X-100/PBS/10% goat serum, and 200 μl of these solutions were added to each chamber, incubated overnight at 4 °C. Following washing with PBS, the appropriate Cy-3– or FITC-conjugated secondary antibodies (1:2,000) (Cedarlane, http://www.cedarlanelabs.com) were applied and incubated for 30 min in the dark at room temperature. The chambers were removed, and slides were mounted with DAPI counterstained mounting medium (Vector Laboratories) and imaged with an Olympus IX70 Delta Vision RT microscope and the SoftWoRx software package.

Detection of p75^NTR proteolysis in BTICs.

Based on the immunocytochemistry staining results, BTICs were collected, dissociated by polished glass pipette, aliquoted into six-well plates, and treated with the proteasome inhibitor epoxomicin (1 μM) (Calbiochem) and/or the γ-secretase inhibitor Compound X (2 μM) (Calbiochem), or DMSO vehicle alone, for 4 h at 37 °C and 5% CO₂. Cells were then washed twice with cold PBS on ice, lysed in lysis buffer with protease inhibitors, and quantified by BCA assay. Western blots for p75^NTR using a p75^NTR cytoplasmic-specific antibody were performed.

In vivo γ-secretase studies using intracranial glioma models.

Actively growing glioma cell line U87p75 and BTICs were implanted intracerebrally into SCID mice as described previously. Three days later for the U87p75 cell line, and 5 d later for BTICs, mice were administered s.c. vehicle (corn oil) or 10 mg/kg γ-secretase in corn oil once/day for 2 wk (U87p75^NTR) or for 3 wk (BTIC). At 3 wk for animals bearing U87p75^NTR tumors or 4 wk for animals bearing BTIC tumors, animals were sacrificed, the brains were removed, frozen in Tissue-Tek O.C.T. compound, and cryosectioned into 7–9-μm sections. The cryosections were used for tumor immunohistochemistry staining and for assessment of in vivo p75^NTR cleavage. For survival studies (eight animals per group), U87p75^NTR glioma cells were implanted intracerebrally into SCID mice. Animals were given daily s.c. injections of 10 mg/kg γ-secretase inhibitor DAPT or vehicle (corn oil) alone, once/day beginning on day 3. Animals were followed until sacrifice was required.

Statistical analysis.

Statistical analysis of data was performed using GraphPad Prism software (GraphPad Software). Survival curves were generated using the Kaplan-Meier method. The log-rank test was used to compare the distributions of survival times. A p-value of less than 0.05 was considered statistically significant.