TrkA and Glioma Invasion
We first examined the expression of the CTGF binding receptor, TrkA, in mouse xenografts generated by intracranial injection with human glioma–derived 0827 TIC/TSCs. Immunohistochemistry of xenograft sections revealed considerable TrkA expression at the tumor margin () and in the infiltrative cells extending from the tumor mass () compared with the noninvasive bulk tumor (), as shown by immunofluorescence staining. An independent human glioma–derived TIC/TSC (0206 TIC/TSCs) demonstrated the same pattern of TrkA staining with greater TrkA expression at the invasive front of intracranial xenograft tumor compared with cells in the bulk region of the tumor (Supplementary Figure 1
, A, available online). By contrast, xenograft tumors derived from injection with the established glioma cell lines U87MG () and U251MG () showed no invasion and no TrkA staining. These data indicate that TrkA expression is relegated largely to glioma cells and TIC/TSCs at the infiltrating front of the xenograft tumors in vivo. Furthermore, immunohistochemical localization of CTGF in sections of xenograft tumors established by intracranial injection of 0827 TIC/TSCs that stably express red fluorescent protein (RFP) suggests that gliomas derived from injection of 0827-RFP TIC/TSCs secrete CTGF (Supplementary Figure 1, B
, available online): Measurement of the fluorescence intensity of CTGF within the tumor and the surrounding parenchyma is consistent with an increasing gradient of CTGF from the center of the tumor out to the infiltrative front (Supplementary Figure 1, C
, available online).
Figure 1 Tyrosine kinase receptor type A (TrkA) expression in sections of glioma-derived tumor-initiating or tumor stem cell (TIC/TSC) intracranial xenograft tumors. Sections of xenograft tumors derived from intracranial injection of neonatal SCID mice with glioma-derived (more ...)
Identification of a Complex Between CTGF, ITGB1, and TrkA
ITGB1 can partner with different membrane protein receptors, such as platelet-derived growth factor receptor and epidermal growth factor receptor, to form a trimolecular complex between ITGB1, the growth factor receptor, and the ligand to the receptor (26
). To test the hypothesis that ITGB1 and TrkA form a receptor complex for CTGF binding, we first examined whether we could obtain evidence for an interaction between endogenous ITGB1 and TrkA in TIC/TSCs via coimmunoprecipitation. Lysates prepared from 0206 TIC/TSCs and 0827 TIC/TSCs were subjected to an immunoprecipitation assay. ITGB1 and TrkA coimmunoprecipitated from lysates of both TIC/TSCs, regardless of whether an antibody to TrkA or ITGB1 was used for immunoprecipitation (). To provide further support for an in vivo interaction between ITGB1 and TrkA, we performed immunohistochemistry on sections of 0827 TIC/TSC–derived intracranial xenografts using an Alexa 488 green fluorescent dye–labeled antibody to ITGB1 and an Alexa 594 red fluorescent dye–labeled antibody to TrkA and looked for areas of antibody colocalization. ITGB1 and TrkA colocalized in infiltrating TIC/TSCs in vivo ().
Figure 2 In vitro and in vivo evidence for a TrkA–ITGB1–CTGF complex. Lysates of glioma TIC/TSCs (0206 and 0827) were subjected to immunoprecipitation with a control IgG or anti-TrkA antibody (A) or anti-ITGB1 antibody (B). The immunoprecipitates (more ...)
To further verify the in vivo interaction of ITGB1 with TrkA, xenograft tumors derived from intracranial injection of 0827 TIC/TSCs were serially sectioned and labeled in the same manner as the colocalization studies and used for fluorescence resonance energy transfer experiments. Fluorescence resonance energy transfer analysis can be used to detect a protein–protein interaction based on the transfer of energy (a photon) of a fluorescent dye conjugated to one protein (the donor protein) to the conjugated fluorescent dye of the other protein (the acceptor protein). When two proteins are in close proximity (ie, when the proteins interact with each other), light excites the donor protein and triggers the energy transfer to the acceptor protein. The energy transfer will allow the acceptor to emit light and produce an image that can be visualized with a confocal microscope, indicating the transfer of fluorescence resonance energy. However, if the two proteins are not close enough for energy transfer (ie, if they do not physically interact) no image is produced. As shown in , fluorescence resonance energy transfer was achieved in the labeled xenograft sections, suggesting a physical interaction between ITGB1 and TrkA. Results of the coimmunoprecipitation experiments, the colocalization studies, and the fluorescence resonance energy transfer analysis indicate that ITGB1 and TrkA interact in TIC/TSCs in gliomas in vivo.
We next conducted a competitive binding assay to examine whether CTGF binding to TrkA could compete with TrkA binding to NGF or NT-3, two known ligands for TrkA (27
). Rat pheochromocytoma PC12 cells that overexpress TrkA were incubated with 125
I-labeled NGF, then washed with increasing concentrations of unlabeled NGF, NT-3, or CTGF, and the 125
I-labeled NGF that remained bound to cells was quantified using a gamma counter. The amount of 125
I-labeled NGF that remained bound to cells decreased when cells were washed with increasing concentrations of unlabeled NGF and NT-3 but not when they were washed with increasing concentrations of unlabeled CTGF (). These data suggest that CTGF may bind to TrkA at a site different from that of NGF and NT-3. However, we cannot rule out the possibility that CTGF binds to TrkA with very low affinity.
Mechanism of CTGF Signaling
To identify downstream effectors of CTGF signaling in TIC/TSCs, we first performed coimmunoprecipitation experiments, which demonstrated that CTGF binds to both TrkA and to ITGB1 (Supplementary Figure 2, A
, available online). Furthermore, CTGF binding to TrkA induces its activation through TrkA phosphorylation (Supplementary Figure 2, A
To further elucidate the necessary components for CTGF activation of the TrkA receptor via phosphorylation, we selectively knocked down expression of the components of the putative receptor complex by stably transfecting 0827 TIC/TSCs with shRNAs targeting either TrkA or ITGB1 and examined TrkA phosphorylation in cells cultured in the presence or absence of purified recombinant CTGF (200 ng/mL). CTGF-mediated TrkA phosphorylation was reduced by knockdown of either TrkA or ITGB1 (). These data demonstrate that CTGF induces TrkA phosphorylation in 0827 TIC/TSCs that express TrkA and ITGB1.
Effect of TrkA Activation on NF-κB Activity
Given that NF-κB has been previously implicated in tumor cell migration and metastasis (29
), and TrkA activation can lead to NF-κB activation (30
), we examined the effect of TrkA activation on NF-κB activity in TIC/TSCs. The 0827 TIC/TSCs were transiently transfected with an NF-κB luciferase reporter plasmid and then incubated in the absence or presence of increasing concentrations of recombinant purified CTGF. NF-κB transcriptional activation was then assessed by measuring the relative luciferase activity (compared with a minimal luciferase promoter plasmid that served as a baseline for luciferase activity).
Increasing concentrations of CTGF resulted in increasing NF-κB luciferase reporter activity in 0827 TIC/TSCs (mean relative luciferase activity [arbitrary units], untreated vs CTGF200 ng/mL
: 0.42 vs 2.35, difference = 1.93, 95% CI = 1.53 to 2.33, P
< .001; untreated vs CTGF400 ng/mL
: 0.42 vs 4.5, difference = 4.08, 95% CI = 3.82 to 4.34, P
< .001; untreated vs CTGF600 ng/mL
: 0.42 vs 8.3, difference = 7.8, 95% CI = 5.3 to 10.5, P
< .001). To determine if TrkA was required for CTGF-mediated NF-κB transcriptional activity, 0827 TIC/TSCs were transiently transfected with an NF-κB luciferase reporter plasmid and then treated with increasing concentrations of GW441756, a TrkA inhibitor that blocks TrkA phosphorylation and subsequent activation, in the presence of a constant concentration of purified recombinant CTGF (200 ng/mL). The cells were then assayed for NF-κB transcriptional activation by measuring relative luciferase activity. Treatment of cells with the TrkA inhibitor reversed NF-κB transcriptional activity (mean relative luciferase activity, GW400 nM
vs CTGF 200 ng/mL
: 2.6 vs 2.35, difference = 0.25, 95% CI = −0.25 to 0.75, P
= .12; GW800 nM
vs CTGF 200 ng/mL
: 2 vs 2.35, difference = 0.35, 95% CI = −0.17 to 0.87, P
= .5; GW1100 nM
vs CTGF 200 ng/mL
: 1.2 vs 2.35, difference = 1.15, 95% CI = 0.67 to 1.63, P
= .19). Similar results were seen in 0206 TIC/TSCs (relative luciferase activity, untreated vs CTGF200 ng/mL
: 0.53 vs 1.87, difference = 1.34, 95% CI = 0.69 to 2, P
< .001). These data suggest that TrkA signals through NF-κB. Therefore, NF-κB might be a potential downstream activator of CTGF-mediated tumor cell infiltration. Importantly, addition of up to 250 ng/mL of CTGF to 0827 TIC/TSCs or 0206 TIC/TSCs did not result in any cytotoxic effects or a noticeable increase in cell proliferation as determined by cell viability assays using Alamar blue (Supplementary Figure 2, D
, available online).
Identification of Effector Molecules Downstream of NF-κB
The observation that TrkA activation is associated with a trimolecular complex containing ITGB1, TrkA, and the CTGF ligand with subsequent NF-κB activation led us to explore possible effector molecules downstream of NF-κB. Given the role of the ZEB-1 transcription factor in tumor cell migration and its plausible relationship with NF-κB (31
), we investigated whether NF-κB signaled through ZEB-1.
To determine if CTGF-mediated NF-κB activation induced the transcription factor ZEB-1, 0827 TIC/TSCs were transiently transfected with a ZEB-1 promoter luciferase reporter or a control minimal promoter reporter plasmid and then incubated with recombinant purified CTGF. ZEB-1 transcriptional activation was then determined by measuring the difference in luciferase activity between the ZEB-1 promoter and the control promoter reporter. Similarly, the luciferase activity of recombinant CTGF–treated 0827 TIC/TSCs cotransfected with the ZEB-1 luciferase reporter plasmid, and an NF-κB expression plasmid was compared with the luciferase activity of recombinant CTGF–treated 0827 TIC/TSCs cotransfected with the ZEB-1 luciferase reporter plasmid and an IκB (an inhibitor of NF-κB) expression plasmid. Overexpression of NF-κB p65 or exposure of 0827 TIC/TSCs to purified recombinant CTGF resulted in induction of the ZEB-1 luciferase reporter (mean relative luciferase activity, untreated vs CTGF200 ng/mL: 1.0 vs 5.00, difference = 4, 95% CI = 3.4 to 4.6, P < .001; untreated vs NF-κB overexpression: 1.0 vs 4.6, difference = 3.6, 95% CI = 3.4 to 3.8, P < .001) (). By contrast, CTGF treatment of 0827 TIC/TSCs transiently transfected with a plasmid expressing the NF-κB inhibitor IκB resulted in a decrease in ZEB-1 promoter activation compared with TIC/TSCs treated with CTGF or transfected with NF-κB p65 expression plasmid (mean relative luciferase activity, CTGF200 ng/mL vs CTGF/IκB: 5.00 vs 2.8, difference = 2.2, 95% CI = 1.69 to 2.71, P = .11; NF-κB overexpression vs CTGF/IκB: 4.6 vs 2.8, difference = 1.8, 95% CI = 1.71 to 1.89, P = .035).
Figure 3 Identification of effector molecules downstream of nuclear factor kappa B (NF-κB). A) ZEB-1 luciferase reporter assay. The 0827 tumor-initiating or tumor stem cells (TIC/TSCs) were transfected with a ZEB-1 luciferase reporter alone or in combination (more ...)
We used two approaches to examine whether NF-κB could bind directly to the ZEB-1 promoter. We first performed an oligonucleotide precipitation assay (19
) in which nuclear lysates made from 0827 TIC/TSCs (untreated or treated with 200 ng/mL purified recombinant CTGF) were incubated with biotinylated double-stranded oligonucleotides containing a putative NF-κB binding sequence located within the ZEB-1 promoter region (, top).
Immunoblot analysis of DNA–protein complexes precipitated with streptavidin–agarose beads revealed the p65 subunit of NF-κB bound to the oligonucleotides corresponding to the ZEB-1 promoter to a much greater extent when the assay was conducted with lysates made from CTGF-treated 0827 TIC/TSCs compared with lysates made from untreated 0827 TIC/TSCs, or when a NF-κB mutant double-stranded oligonucleotide control sequence was used in the precipitation assay (, bottom).
We next used an antibody to NF-κB in a ChIP assay to examine the direct binding of NF-κB to the ZEB-1 promoter in 0827 TIC/TSCs that were treated with CTGF. CTGF-treated 0827 TIC/TSCs displayed an increase in NF-κB binding at the ZEB-1 promoter region compared with untreated 0827 TIC/TSCs as shown by ChIP RT-PCR (fold increase in NF-κB binding to the ZEB-1 promoter region: Untreated + NF-κB antibody vs CTGF200 ng/mL + NF-κB antibody: 0.375 vs 2.45, difference = 2.075, 95% CI = 2.04 to 2.11, P < .001) (, left panel). Taken together, these data demonstrate that exposure of TIC/TSCs to CTGF results in increased transcriptional activation of ZEB-1 and the direct binding of NF-κB to the ZEB-1 promoter.
CTGF-Dependent NF-κB Activation and E-cadherin Expression
The development of invasive tumors is often associated with the decreased expression of E-cadherin, which plays an important role in epithelial cell adhesion and cell mobility. Interactions among integrins, the extracellular matrix, and cell surface receptors are also involved in regulating cell-to-cell contacts and cell mobility (32
It has been reported (31
) that NF-κB can affect E-cadherin expression, possibly via the transcriptional repressor ZEB-1. Given that ZEB-1 has been shown to bind to E-box regions of E-cadherin (E-box regions are found within promoter regions and can either increase or decrease transcriptional activity depending on which specific transcription factor binds to the E-box region), we surmised that CTGF binding to a TrkA–ITGB1 complex would induce NF-κB activation and subsequent ZEB-1 activation, ultimately leading to decreased expression of E-cadherin (33
). We used a ChIP assay with an antibody to ZEB-1 to examine whether CTGF signaling could result in ZEB-1 binding to the E-box region of the E-cadherin promoter in glioma 0827 TIC/TSCs. CTGF-treated 0827 TIC/TSCs displayed increased binding of ZEB-1 to the E-box region of the E-cadherin promoter compared with untreated 0827 TIC/TSCs as shown by ChIP RT-PCR (fold increase in ZEB-1 binding to the E-cadherin promoter region: Untreated + ZEB-1 antibody vs CTGF200 ng/mL
+ ZEB-1 antibody: 1.5 vs 6.4, difference = 4.9, 95% CI = 4.8 to 5.0, P < .001) (, right panel).
To provide further evidence that CTGF mediates decreased expression of E-cadherin through NF-κB and ZEB-1, we examined E-cadherin promoter activity in 0827 TIC/TSCs that were transfected with an E-cadherin luciferase reporter plasmid alone or in combination with an NF-κB or ZEB-1 expression plasmid. Cells that overexpressed either NF-κB or ZEB-1 had decreased E-cadherin luciferase activity compared with cells that overexpressed neither protein (). Conversely, transfection of 0206 TIC/TSCs with the E-cadherin luciferase reporter plasmid and either of two shRNAs targeting ZEB-1 resulted in transcriptional activation of the E-cadherin promoter (Supplementary Figure 3
, available online). Together, these data indicate that exposure to CTGF leads to decreased expression of E-cadherin through a signaling pathway involving the activation of TrkA, which leads to activation of NFkB and subsequent ZEB-1-mediated transcriptional repression of E-cadherin.
Effect of CTGF on E-cadherin Expression in Glioma TIC/TSCs
We examined the effects of CTGF on E-cadherin expression in TIC/TSCs obtained from three adult glioblastoma patients because these cells more closely mimic the infiltrative nature of primary human glioblastomas compared with established glioma cell lines (5
). Exposure of the three TIC/TSC lines to purified recombinant CTGF resulted in a considerable decrease in the number of E-cadherin–positive cells as demonstrated by immunofluorescence staining with an antibody to E-cadherin (, left) and quantitative fluorescence-activated cell sorting analysis (, right). Similarly, CTGF exposure resulted in a considerable decrease in E-cadherin luciferase reporter activity in 0827 TIC/TSCs and 0206 TIC/TSCs (Supplementary Figure 4, A
, available online). RT-PCR further confirmed decreased expression of E-cadherin mRNA in 0827 TIC/TSCs treated with CTGF (Supplementary Figure 4, B
, available online). By contrast, CTGF exposure of 0827 TIC/TSCs and 0206 TIC/TSCs that were transfected with an shRNA-targeting TrkA resulted in increased E-cadherin luciferase activity compared with cells transfected with a control shRNA (Supplementary Figure 4, C
, available online).
Figure 4 Effect of connective tissue growth factor (CTGF) on E-cadherin expression in tumor-initiating or tumor stem cells (TIC/TSCs). A) Indirect immunofluorescence detection (left) and Fluorescence-activated cell sorting analysis (right) of E-cadherin expression (more ...)
In addition, 0827 TIC/TSCs treated with CTGF and either a TrkA inhibitor (GW441756) or an ITGB1 inhibitor (MAB225Z) showed no CTGF-mediated inhibition of E-cadherin protein expression compared with cells treated with CTGF alone (, right; Supplementary Figure 4, D
, available online), further confirming the involvement of a ITGB1–TrkA complex in E-cadherin regulation. It is of interest to note that exposure of 0206 TIC/TSCs to pharmacological concentrations (ie, 10 ng/mL) of either NGF or NT-3 did not affect E-cadherin protein expression as detected by immunofluorescence staining, indicating the specificity of the CTGF–TrkA complex for decreasing expression of E-cadherin ().
Effect of the CTGF–ITGB1–TrkA Complex on Tumor Cell Migration and Invasion
Although the effects of CTGF have been previously studied in a number of established cancer cell lines, to our knowledge, the effects on patient-derived primary cancer stem cells have yet to be explored. Thus, we evaluated the contribution of each member of the CTGF–ITGB1–TrkA complex to glioma TIC/TSC invasion by using an automated in vitro real-time cell migration and invasion assay. The 0206 TIC/TSCs and 0827 TIC/TSCs stably expressing shRNA-TrkA, shRNA-ITGB1, or shRNA-CTGF were seeded in the upper chamber of a 16-chamber microtiter plate containing purified recombinant full-length CTGF or a truncated CTGF182–250 (200 ng/mL) in NBE. The upper chamber was coated with laminin to form a barrier for invasion of TIC/TSCs into the lower chamber. The lower chamber contained NBE with no growth factors (serum free). The 16-chamber microtiter plates were placed into a RT-CIM apparatus for measurement of cell invasion and migration. Treatment of 0827 TIC/TSCs and 0206 TIC/TSCs with purified recombinant full-length CTGF or CTGF182–250 (a TrkA-binding truncated form of CTGF) induced a statistically significant increase in the amount of tumor cell migration compared with untreated cells (mean electrical impedance [arbitrary units], 0827 TIC/TSCs, untreated vs full-length CTGF: 3.88 vs 10.32, difference = 6.44, 95% CI = 6.2 to 6.7, P < .001; 0206 TIC/TSCs, untreated vs full-length CTGF: 1.38 vs 4.1, difference = 2.72, 95% CI = 2.5 to 3, P < .001; 0206 TIC/TSCs, untreated vs CTGF182–250: 1.38 vs 6.26, difference = 4.88, 95% CI = 4.6 to 5.1, P < .001).
By contrast, shRNA-mediated knockdown of endogenously produced CTGF decreased cell migration and invasiveness of TIC/TSCs compared with untransfected (wild-type) cells, suggesting a potential paracrine and/or autocrine mechanism of CTGF-mediated TIC/TSC invasion and migration (mean electrical impedance [arbitrary units], 0827 TIC/TSCs, WT vs shCTGF: 5.3 vs 1.2, difference = 4.1, 95% CI = 3.9 to 4.3, P < .001; 0206 TIC/TSCs, WT vs shCTGF: 1.4 vs 0.38, difference = 1.02, 95% CI = 1.01 to 1.03, P < .001).
To examine the importance of TrkA and ITGB1 in transducing the CTGF promigratory signal, we assayed migration of TIC/TSCs transfected with shRNAs that target TrkA or ITGB1 or a scrambled-sequence control shRNA (Scr). shRNA-mediated knockdown of either TrkA or ITGB1 resulted in a decrease in CTGF-induced cell migration compared with CTGF-induced Scr control (mean electrical impedance [arbitrary units], 0827 TIC/TSCs, WT vs Scr: 4.3 vs 7.7, difference = 3.4, 95% CI = 3.1 to 3.6, P < .001; 0827 TIC/TSCs, Scr vs shTrkA59: 7.7 vs 4.6, difference = 3.1, 95% CI = 2.97 to 3.23, P < .001; 0206 TIC/TSCs, WT vs Scr: 0.88 vs 10.6, difference = 9.72, 95% CI = 9.71 to 9.73, P < .001; 0206 TIC/TSCs, Scr vs shTrkA59: 10.6 vs 0.82, difference = 9.78, 95% CI = 9.77 to 9.79, P < .001; 0827 TIC/TSCs, WT vs shITGB1: 5.0 vs 1.1, difference = 3.9, 95% CI = 3.8 to 4.0, P < .001; 0206 TIC/TSCs, WT vs shITGB1: 1.5 vs 0.78, difference = 0.72, 95% CI = 0.71 to 0.73, P < .001).
Finally, to examine the effect of a different type of knockdown of E-cadherin expression—siRNA-mediated gene silencing—on TIC/TSC invasion and migration in vitro, we assayed migration of TIC/TSCs that were transfected with four siRNAs targeting E-cadherin (designated No. 8, 9, 10, and 11) or a nontargeting siRNA as a negative control. siRNA-mediated knockdown of E-cadherin expression increased TIC/TSC invasion and migration in vitro compared with nontargeting siRNA negative control–transfected TIC/TSCs (mean electrical impedance [arbitrary units], 0827 TIC/TSCs, control vs E-cadherin siRNA No. 8: 0.6 vs 3.8, difference = 3.2, 95% CI = 3.1 to 3.3, P < .001; 0827 TIC/TSCs, control vs E-cadherin siRNA No. 9: 0.6 vs 12, difference = 11.4, 95% CI = 11.39 to 11.4, P < .001; 0827 TIC/TSCs, control vs E-cadherin siRNA No. 10: 0.6 vs 7.6, difference = 7, 95% CI = 6.8 to 7.2, P < .001; 0827 TIC/TSCs, control vs E-cadherin siRNA No. 11: 0.6 vs 8.6, difference = 8, 95% CI = 7.5 to 8.5, P < .001; 0206 TIC/TSCs, control vs E-cadherin siRNA No. 8: 1.0 vs 7.0, difference = 6.0, 95% CI = 5.9 to 6.1, P < .001; 0206 TIC/TSCs, control vs E-cadherin siRNA No. 9: 1.0 vs 5.1, difference = 4.1, 95% CI = 3.5 to 4.7, P < .001; 0206 TIC/TSCs, control vs E-cadherin siRNA No. 10: 1.0 vs 6.7, difference = 5.7, 95% CI = 5.69 to 5.71, P < .001; 0206 TIC/TSCs, control vs E-cadherin siRNA No. 11: 1.0 vs 6.4, difference = 5.4, 95% CI = 5.1 to 5.6, P < .001). This finding supports our hypothesis that the promigratory effect of CTGF is transduced downstream through its repression of E-cadherin.
Effect of TrkA on Glioma TIC/TSC Infiltration In Vivo
To directly evaluate the importance of TrkA expression for TIC/TSC invasion in vivo, we created 0827 TIC/TSCs that stably express one of two shRNAs targeting TrkA (sh58 or sh59) or a control scrambled-sequence shRNA. Stable expression of sh58 and sh59 resulted in considerable knockdown of TrkA protein expression by immunoblot analysis compared with uninfected 0827 TIC/TSCs or 0827 TIC/TSCs expressing a scrambled-sequence shRNA (more so for sh59 than for sh58; ). However, shRNA-mediated knockdown of TrkA did not affect the proliferation or clonogenicity of the TIC/TSCs in vitro (data not shown).
Figure 5 Effect of tyrosine kinase receptor type A. TrkA expression on invasiveness of glioma tumor–initiating or tumor stem cell (TIC/TSC)–derived xenografts. The 0827 TIC/TSCs were stably transfected with one of two short hairpin RNA (shRNA) (more ...)
To examine the effect of shRNA-mediated knockdown of TrkA on TIC/TSC infiltration in vivo, we studied intracranial xenograft tumors generated by injection of mice with patient-derived glioblastoma 0827 TIC/TSCs stably expressing scrambled sequence shRNA, sh58, sh59, or wild-type cells. The tumors were sectioned and stained with hematoxylin and eosin for examination of tumor cell infiltration. We observed extensive tumor cell infiltration of the cerebral cortex in mice injected with wild-type 0827 TIC/TSCs or scrambled sequence shRNA-expressing 0827 TIC/TSCs (, upper left and right panels, respectively). By contrast, mice injected with sh58-expressing 0827 TIC/TSCs had considerably less tumor cell invasion in vivo, and those injected with sh59-expressing 0827 TIC/TSCs, which showed the greatest TrkA knockdown by immunoblot analysis, had no tumor cell invasion into normal cortex (, lower left and right panels, respectively). These data demonstrate the requirement for TrkA expression in glioma invasion in this model system.
Characterization of Reactive Astrocytes in an Orthotopic Glioma Model
A profound glial reaction is often seen within and around the invading front of malignant gliomas in situ. The glial reaction is characterized by the presence of reactive astrocytes that are likely to be responding to an endogenous brain injury signal and/or to the release of cytokine(s) by glioma cells and associated inflammatory cells. Given that injured brain can release CTGF (16
), and given the intimate proximity of reactive astrocytes to the invading glioma front, we first examined whether reactive astrocytes provide a promigratory stimulus to glioma cells through the release of CTGF. Intracranial xenograft tumors generated by injection of mice with patient-derived glioblastoma 0206 TIC/TSCs were serially sectioned and stained for GFAP, a marker of reactive astrocytes. We observed large numbers of GFAP-positive reactive host astrocytes surrounding the growing tumor mass, as is typically seen in human gliomas in situ (, left panel). We used a mouse-specific probe to detect mouse CTGF mRNA by in situ hybridization on serial mouse brain sections containing the intracranial human glioma xenograft. There were very high levels of expression of mouse-specific CTGF mRNA in reactive astrocytes adjacent to the infiltrating tumor front (, middle panel).
Figure 6 Characterization of reactive astrocytes in orthotopic gliomas derived from intracranial injection of mice with 0206 tumor-initiating or tumor stem cells (TIC/TSCs). Xenograft tumors derived from intracranial injection of SCID mice with 0206 TIC/TSCs were (more ...)
To confirm that reactive astrocytes not only produce CTGF mRNA but also secrete biologically active CTGF protein, we cultured human reactive astrocytes in vitro, collected the culture medium, and used a CTGF antibody to immunodeplete CTGF from this rACM. First, we demonstrated by immunoblot analysis of control IgG–treated rACM (lane 1) that CTGF was present in rACM. This was confirmed by the selective depletion of CTGF in the CTGF antibody–treated rACM (lane 2). Finally, total rACM, with no antibody treatment, demonstrated that CTGF is secreted by rACM (lane 3, ). Next, to verify the biological activity of the immunoblot-detected CTGF, we examined the effect of rACM collected from cultures of human reactive astrocytes transfected with CTGF shRNA and from mock-transfected human reactive astrocytes on the in vitro migratory and invasive activity of 0206 TIC/TSCs. The 0206 TIC/TSCs exposed to rACM from mock-transfected reactive astrocytes had more migrating cells compared with 0206 TIC/TSCs exposed to rACM from CTGF shRNA–infected reactive astrocytes (mean electrical impedance: 0206 TIC/TSC, mock-transfected vs CTGF shRNA–tranfected: 9.75 vs 1.25, difference = 8.5, 95% CI = 7.7 to 9.3, P < .001) (). Together, these results demonstrate that reactive astrocytes secrete biologically active CTGF, which potentiates the proinfiltrative phenotype of the glioma cells via a novel host-tumor signaling pathway ().