In Vivo c-Met Pathway
Inhibition Depletes Human
Glioma Xenografts of
Prakash Rath*,3, Bachchu Lal*,3, Olutobi Ajala*,
Yunqing Li*, Shuli Xia*, Jin Kim†and John Laterra*
*Department of Neurology, The Hugo W. Moser Research
Institute at Kennedy Krieger Inc and The Johns Hopkins
University School of Medicine, Baltimore, MD;†Galaxy
Biotech, LLC, Mountain View, CA
Solid malignancies contain sphere-forming stem-like cells that are particularly efficient in propagating tumors. Iden-
tifying agents that target these cells will advance the development of more effective therapies. Recent converging
evidence shows that c-Met expression marks tumor-initiating stem-like cells and that c-Met signaling drives human
glioblastoma multiforme (GBM) cell stemness in vitro. However, the degree to which tumor-propagating stem-like
cells depend on c-Met signaling in histologically complex cancers remains unknown. We examined the effects of
in vivo c-Met pathway inhibitor therapy on tumor-propagating stem-like cells in human GBM xenografts. Animals
bearing pre-established tumor xenografts expressing activated c-Met were treated with either neutralizing anti–
hepatocyte growth factor (HGF) monoclonal antibody L2G7 or with the c-Met kinase inhibitor PF2341066 (Crizotinib).
c-Met pathway inhibition inhibited tumor growth, depleted tumors of sphere-forming cells, and inhibited tumor
expression of stem cell markers CD133, Sox2, Nanog, and Musashi. Withdrawing c-Met pathway inhibitor therapy
resulted in a substantial rebound in stem cell marker expression concurrent with tumor recurrence. Cells derived from
xenografts treated with anti-HGF in vivo were depleted of tumor-propagating potential as determined by in vivo serial
dilution tumor-propagating assay. Furthermore, daughter xenografts that did form were 12-fold smaller than controls.
These findings show that stem-like tumor-initiating cells are dynamically regulated by c-Met signaling in vivo and that
c-Met pathway inhibitors can deplete tumors of their tumor-propagating stem-like cells.
Translational Oncology (2013) 6, 104–111
Glioblastoma multiforme (GBM) is a nearly universally fatal brain
tumor with an associated median survival of approximately 14 months
despite aggressive surgical resection, radiation therapy, and chemo-
therapy. GBM is highly heterogeneous at the histopathologic, cellular,
and molecular levels and its cell subpopulations display varying sensi-
tivities to cytotoxic agents and emerging therapies designed to target
specific oncogenic pathways. Advances over the past decade have found
that GBM contains subpopulations of multipotent stem-like cells
characterized by their capacity to grow as nonadherent spheres in
defined serum-free medium, differentiate along multiple neural cell
lineages, and efficiently propagate tumor xenografts that recapitulate
the invasive and histopathologic features of clinical GBM . The
tumor-propagating capacity of these stem-like cells along with their
relative resistance to DNA-damaging agents predicts that therapies
directed against stem-like neoplastic cells will delay tumor relapse and
prolong patient survival [2,3]. Multiple autocrine and paracrine signal-
ing pathways andmicroenvironmentalcueshavebeenfoundtosupport
tumor stem-like cell self-renewal and regulate their transition to more
differentiated progenitors [4–6].
Address all correspondence to: JohnLaterra,MD,PhD,The Kennedy KriegerInstitute,
707 N. Broadway, Baltimore, MD 21205. E-mail: firstname.lastname@example.org
1This research was funded by the National Institutes of Health through grants
NS43987 (J.L.), CA129192 (J.L.), and NS073611 (J.L.), by the Maryland Stem Cell
Research Fund Fellowship (P.R.), and by the James S. McDonnell Foundation (J.L.).
B.L., J.L., and J.K. have financial interests related to the anti-HGF mAb L2G7 used in
2This article refers to supplementary material, which is designated by Table W1 and is
available online at www.transonc.com.
3These authors contributed equally.
Received 16 January 2013; Revised 16 January 2013; Accepted 17 January 2013
Copyright © 2013 Neoplasia Press, Inc. All rights reserved 1944-7124/13/$25.00
Volume 6 Number 2April 2013pp. 104–111
The c-Met receptor tyrosine kinase (RTK) and its ligand hepato-
cyte growth factor (HGF) are strongly implicated in the malignant
progression of many solid neoplasms and expression levels correlate
with poor prognosis in multiple malignancies including GBM [7–9].
We and others have reported that inhibitors of c-Met pathway acti-
vation inhibit the growth of c-Met+tumor xenografts by inducing
apoptosis and inhibiting tumor cell proliferation and angiogenesis
[7,10–12]. Evidence has recently emerged from multiple laboratories
showing that c-Met is a marker for stem-like tumor-initiating cell
subsets and that c-Met signaling induces stemness in human GBM
invitro [13–15].These findings suggestthat c-Met signaling enhances
tumor malignancy by preventing differentiation or inducing de novo
formation of dynamically regulated stem-like cells through reprogram-
ming mechanisms. However, the degree to which tumor-propagating
stem-like cells depend on c-Met signaling in histologically complex
cancersremains unknown and ithas yet to bedeterminediftherapeutic
in vivo c-Met pathway inhibition can target neoplastic stem-like cell
subsets. Determining the effects of c-Met pathway inhibition on cancer
stem-like cells in vivo will aid the clinical translation of c-Met inhibitors
and the development of treatment strategies designed to target cancer
This present study examines the effects of in vivo therapy with two
mechanistically distinct c-Met pathway inhibitors currently in clini-
cal development, neutralizing anti-HGF monoclonal antibody (mAb)
L2G7 and the small molecule c-Met kinase inhibitor PF2341066
(Crizotinib), on the stem-like cell phenotype in GBM xenografts.
We demonstrate that glioma xenograft growth inhibition in response
to c-Met pathway inhibition in vivo is accompanied by reductions in
the tumor-propagating stem-like phenotype based on molecular
marker expression, neurosphere-forming capacity, and the capacity
of primary xenograft-derived cells to propagate aggressive intracranial
tumors. Our results show for the first time that c-Met pathway inhib-
itor therapy can deplete tumors of their stem-like tumor-initiating
Materials and Methods
U87 cells were originally obtained from American Type Culture
Collection (Manassas, VA) and cultured in Dulbecco’s modified
eagle’s medium supplemented with 10% FBS (Gemini Bio-Products,
Sacramento, CA), nonessential amino acids and penicillin and strep-
tomycin (Quality Biological Inc, Gaithersburg, MD). The human
GBM xenograft lines, Mayo 39 and Mayo 59, were originally obtained
from the Mayo Clinic (Rochester, MN) . Primary human brain
neural stem cells were isolated from discarded human abortuses as pre-
viously described and kindly provided by Dr Alfredo Quinones (Johns
Hopkins School of Medicine) . All cells were grown at 37°C in a
humidified incubator with 5% CO2.
subcutaneous tumor xenograft tissue was minced, enzymatically dis-
sociated by trypsinization, and seeded at 4 × 105cells per 10-cm dish
factor (EGF) and fibroblast growth factor (FGF) (neurosphere medium)
as described [15,18]. Free-floating tumor spheres were passaged by
mechanical dissociation through a 1- to 200-μl pipette tip, and passages
5 to 8 were used for experimentation. Neurosphere cells derived from
U87 xenografts are referred to as U87-NS cells.
For HGF stimulation studies in vitro, neurospheres were disso-
ciated to single-cell suspension, plated at 4 × 105cells per 10-cm
dish, and cultured in EGF/FGF-free medium overnight before the
addition of HGF (50 ng/ml final concentration, kindly provided by
Genentech, Inc, San Francisco, CA) for 30 minutes. For determining
effects of c-Met kinase inhibition in vitro, dissociated cells were plated
in GBM stem cell medium and PF02341066 (kindly provided by
Pfizer, Inc, New York, NY) or buffer only was added daily (150 or
300 nM final concentration) for 7 days before embedding cells in
1% agarose. Neurosphere numbers and diameters were quantified
by computer-assisted analyses as previously described .
(MAPK) (rabbit, 1:1000), phospho-AKT (rabbit, 1:1000), total AKT
Cell Signaling Technology (Danvers, MA); CD133 (rabbit, 1:400),
Nestin (mouse, 1:1000) and neurofilament medium (NF, rabbit, 1:1000)
were from Abcam (Cambridge, MA); Musashi (rabbit, 1:1000),
βIII-tubulin (mouse, 1:1000), O1 (mouse, 1:500), and glial fibrillary
acidic protein (GFAP) (rabbit, 1:250) were from Millipore (Billerica,
MA); total Met (rabbit, 1:500) and phospho-Met Tyr1349 (rabbit,
1:200) were from Santa Cruz Biotechnology (Santa Cruz, CA); Nanog
(rabbit, 1:500) was from Kamiya Biomedical Company (Seattle, WA);
actin (mouse, 1:15,000; rabbit, 1:25,000) was from Sigma-Aldrich
(Franklin Lakes, NJ).
Phospho–mitogen-activated protein kinase
1:15,000) and IRDye 680CW (goat anti-rabbit, 1:20,000) were
from LI-COR Biosciences (Lincoln, NE); AF 488 (goat anti-rabbit,
1:800) and AF 488 (goat anti-mouse, 1:400) were from Invitrogen
(Carlsbad, CA); and Cy3 (goat anti-mouse, 1:800) was from Jackson
ImmunoResearch Laboratories (West Grove, PA).
IR dyes IRDye 800CW (goat anti-mouse,
Subcutaneous and Intracranial Xenografts
All animal experiments were performed according to Johns Hopkins
University Animal Care and Use Committee–approved protocols.
Subcutaneous U87 glioma xenografts were generated in 6- to 8-week-old
nude mice as previously described . Volumes of subcutaneous xeno-
grafts were estimated by measuring the length and width with calipers
and calculated with the equation: Volume = (length × width2)/2
. Intracranial tumor volumesweredetermined bymeasuring tumor
cross-sectional areas in hematoxylin and eosin (H&E)–stained brain
sections by computer-assisted image analysis and then applying the
Anti-HGF mAb (L2G7) or isotype control antibody (5G8) were
kindly provided by Galaxy Biotech (Sunnyvale, CA) and adminis-
tered by intraperitoneal injection (5 or 10 mg/kg) every other day
for a total of four injections. Met kinase inhibitor PF2341066 (Pfizer
Inc) or solute as control (DMSO) was resuspended in phosphate-
buffered saline (PBS) and administered by oral gavage (40 mg/kg)
everyday for 7 continuous days.
For the serial dilution tumor-propagating experiments (see Fig-
ure 4A), animals bearing U87 subcutaneous xenografts were treated
Translational Oncology Vol. 6, No. 2, 2013In Vivo c-Met Inhibition Depletes GBM Stem CellsRath et al.
with anti-HGF L2G7 or isotype control 5G8 mAb as described above.
Xenografts were removed, minced, and trypsinized to dissociate cells
as previously described [20,21]. Cells were then passed through 40-μm
mesh filters, washed with PBS, labeled with trypan blue and immedi-
PA) before intracranial injections. Cells were implanted intracranially
using 3 × 102to 1 × 104viable cells per animal as indicated. Animals
were sacrificed for histopathologic analyses when the corresponding
5G8-treated control groups developed neurological signs indicative of
near terminal tumor growth.
Histology, Immunohistochemistry, and Immunofluorescence
Brains were placed in 4% paraformaldehyde for 72-hour fixation,
cryoprotected in 30% sucrose, sectioned on a cryostat, and stained
with H&E as previously described . Tumor volumes were quan-
tified by computer-assisted image analysis as previously described
[21,22]. For immunofluorescence, dissociated neurosphere cells were
plated and stained according to a protocol outlined in the Human
Neural Stem Cell Characterization Kit (Millipore). Undifferentiated
neurosphere cells were embedded and sectioned on a cryostat at 5 μm
as previously described . Sections were counterstained with
Vectashield (Vector Laboratories, Burlingame, CA) mounting medium
containing the nuclear counterstain 4′,6-diamidino-2-phenylindole
(DAPI). Nonimmune IgG was used as primary IgG in negative controls.
Semiquantitative immunoblot analysis was performed according to
standard procedures using the specific antibodies shown above, as
described previously . Briefly, total protein was extracted from
glioma xenografts or cells using RIPA lysis buffer containing protease
Tissue protein extracts were also sonicated. Proteins were quantified
using Coomassie Protein Assay (Thermo Scientific, Waltham, MA)
according to the manufacturer’s protocol. Sodium dodecyl sulfate–
polyacrylamide gel electrophoresis and transfer to nitrocellulose mem-
branes were performed electrophoretically and membranes were
blocked with Odyssey LI-COR Blocking Buffer (LI-COR Biosciences)
before antibody incubations. In selected circumstances, membranes
were stripped and reprobed with anti–β-actin antibodies as a loading
control. Proteins were detected and quantified using the Odyssey Infra-
red Imager (LI-COR Biosciences).
Quantitative Reverse Transcription–Polymerase
Total RNA was extracted from tissues and cells with QIAzol
reagent and RNeasy Mini Kit according to the manufacturer’s protocol
(Qiagen, Hilden, Germany). Reverse transcription was performed
using MuLV Reverse Transcriptase and Oligo (dT) primers (Applied
Biosystems, Grand Island, NY), and quantitative real-time polymerase
Sequence Detection System and iQ5 System (Bio-Rad). Samples were
to 18S RNA and generated in the Bio-Rad iQ5 software. The following
primers were used: Nestin: forward, 5-aagacttccctcagctttcag-3′, reverse,
5-agcaaagatccaagacgcc-3′; βIII-tubulin: forward, 5-tttggacatctcttcaggcc-3′,
reverse, 5-tttcacactccttccgcac-3′; 18S: forward, 5-acaggattgacagattga-
tagctc-3′, reverse, 5-caaatcgctccaccaactaagaa-3′.
Forced Differentiation and Neurosphere Assays
U87 cells grown as neurospheres (U87-NS) were cultured in
serum-free neurobasal medium containing EGF/FGF (neurosphere
medium) as described above. Forced differentiation was performed
according to previously published methods [15,24,25]. Briefly,
U87-NS cells were dissociated to single-cell suspensions and cultured
on Matrigel in neurosphere medium lacking FGF for 2 days and then
grown in medium containing 1% FBS without EGF/FGF for 5 days.
Forced differentiated cells (referred to as U87-FD) were then pro-
cessed for immunofluorescence as described above.
To quantify neurosphere formation by cells derived from subcuta-
neous tumor xenografts, primary xenograft-derived neurospheres
were dissociated and plated at 4 × 105cells per 10-cm dish and al-
lowed to grow in neurosphere medium for 1 week before embedding
in 1% agarose for quantification. Neurosphere numbers were
counted by computer-assisted image analysis. For HGF stimulation
studies in vitro, U87-NS cells were dissociated to single-cell suspen-
sion, plated at 4 × 105cells per 10-cm dish, and cultured in neuro-
sphere medium lacking EGF/FGF overnight before the addition of
purified recombinant HGF (50 ng/ml, kindly provided by Genentech,
Inc) for 30 minutes. For c-Met inhibition conditions, after cell plating,
PF02341066 was added daily (150 or 300 nM final concentration) for
7 days before 1% agarose embedding and analysis. Neurospheres
greater than 100 μm were quantified and compared to control cells
treated with DMSO only.
Systemic Anti-HGF Therapy Depletes HGF-Dependent
GBM Xenografts of Stem-Like Cells
We established that U87 xenografts contain sphere-forming multi-
potent stem-like cells as an initial step toward asking if c-Met activity
modulates stem-like GBM cell phenotypes in vivo. Single-cell suspen-
sions obtained from U87 xenografts (established by serial subcutaneous
passage) and immediately cultured in neurosphere medium readily
formed large spheres (referred to as U87-NS cells) demonstrating the
U87-NS). These U87-NS cells maintained in neurosphere medium
self-renewed under sphere-forming culture conditions for >40 passages
and expressed the stem cell molecular markers GFAP, Nestin, Musashi,
and Sox2 (Figure 1B). U87-NS cells adopted an adherent growth pat-
tern and expressed differentiation molecular markers βIII-tubulin, O1,
and intermediate neurofilament, consistent with multilineage neural
differentiation potential, in response to forced differentiation induced
by growth factor withdrawal and serum (Figure 1B).
We evaluated levels of stem cell marker mRNA and protein in
U87, U87-NS, and U87-NS cells subjected to forced differentiation
in multipotent human neural stem cells (hNSCs), was expressed mini-
mally in U87 cells maintained under standard adherent culture condi-
tions, was upregulated in U87-NS cells, and decreased in the U87-FD
βIII-tubulin, which is low in hNSCs, was also low in U87 cells and
U87-NS cells but upregulated in U87-FD cells (Figure 1C). We exam-
ined the expression levels of various other stem cell and reprogramming
U87-FD cells (Figure 1, D–F). The capacity of the xenograft-derived
In Vivo c-Met Inhibition Depletes GBM Stem CellsRath et al. Translational Oncology Vol. 6, No. 2, 2013
U87 cells to 1) self-renew as spheres in neural stem cell medium, 2)
differentiate along multiple neural lineages, and 3) upregulate multi-
ple markers and transcription factors associated with neural stem cells
and GBM stem cells provides rigorous in vitro evidence for their stem-
U87-NS cells were found to have a functioning c-Met signaling
cells with the c-Met kinase inhibitor PF2341066 (150–300 nM)
inhibited their capacity to form spheres consistent with a role for c-Met
as previously shown by us in other human GBM stem cell isolates
(Figure 1H) . Thus, U87 xenografts contain a subset of stem-like
cells with a functional endogenously c-Met signaling cascade.
To test the hypothesis that therapeutic stem-like cell subpopula-
tions within GBM are sensitive to in vivo c-Met pathway inhibition,
we evaluated the effects of treating pre-established U87 xenografts
with the neutralizing anti-HGF mAb L2G7 [5 mg/kg, every other
Figure 1. U87 xenografts contain multipotent neoplastic stem-like cells. (A) Xenograft-derived U87 cells form neurospheres (U87-NS,
neurospheres) in serum-free stem neurosphere medium containing EGF and FGF. (B) Undifferentiated U87-NS cells express neural pro-
genitor and stem cell markers GFAP (green), Nestin (red), Musashi (green), and Sox2 (green) (left panel). U87-NS cells subjected to
conditions of forced differentiation (U87-FD) extend processes and exhibit morphologies consistent with neuronal lineage differentiation
and express mature cell markers GFAP (green), βIII-tubulin (red), O1 (red), and neurofilament medium (NF) (green) (right panel). Nuclear
counterstain DAPI is shown in blue. (C) Nestin and βIII-tubulin expression normalized to 18S RNA in hNSCs, U87, U87-NS, and U87-FD cells
as determined by quantitative reverse transcription–polymerase chain reaction. (D–F) Immunoblot analysis of U87, U87-NS, and U87-FD
*P < .05, **P < .001, as compared with controls.
Translational Oncology Vol. 6, No. 2, 2013In Vivo c-Met Inhibition Depletes GBM Stem CellsRath et al.
day, intraperitoneally (i.p.)] or with isotype mAb 5G8 as control under
conditions previously shown to induce tumor regression . L2G7
treatment resulted in a robust antitumor response (P < .0001;
Figure 2A). Stopping L2G7 treatment in a subset of mice (referred
to as L2G7 withdrawal, N = 6) resulted in tumor regrowth at a rate
comparable to untreated controls (Figure 2A). The effects of L2G7
treatment on biologic and molecular markers of the stem-like cell
phenotype were evaluated in tissues and cells obtained from post-
implantation day (PID) 14 L2G7- and 5G8-treated xenografts and
PID 28 xenografts 14 days after L2G7 withdrawal. L2G7 treatment
inhibited tumor c-Met phosphorylation by ∼69% (P < .001), concur-
rent with tumor growth arrest, as determined by immunoblot analysis
of tumor protein (Figure 2B). Xenografts treated with L2G7 up to
the time of sacrifice were depleted of sphere-forming stem-like cells
(P < .001; Figure 2C). Expression of the stem cell markers CD133,
Musashi, Sox2, and Nanog was statistically significantly lower in
L2G7-treated xenografts (Figure 2, D–G). These results show that
tumor stem-like cells are among the cell subpopulations targeted by
c-Met pathway inhibition. The disproportionate loss of neurosphere-
forming stem-like cells and the downregulated expression of stem cell
markers and transcription factors in response to anti-HGF therapy are
consistent with preferential targeting of stem-like cell subsets. With-
drawing L2G7 therapy resulted in a rebound in c-Met phosphorylation
(Figure 2B), tumor sphere-forming capacity (Figure 2C), and tumor
expression of molecular stem cell markers to magnitudes at least as high
asthosefound in5G8-treatedcontrols (Figure 2,D–G).The transcrip-
tion factors Sox2 and Nanog rebounded to levels approximately twice
as high as those found in control tumors (P < .001 and P < .05, re-
spectively; Figure 2, F and G). These results show that the neoplastic
stem-like phenotype is dynamically regulated by the c-Met pathway
activity in vivo.
Systemic c-Met Kinase Inhibitor Therapy Depletes
HGF-Independent Human GBM Xenograft
Lines of Stem-Like Cells
Glioma cells derived from the HGF−/c-Met+Mayo 39 and Mayo
59 GBM xenograft lines express readily detectable levels of activated
phospho–c-Met in the absence of HGF expression (Table W1). We
used the small molecule c-Met kinase inhibitor PF2341066 to exam-
ine the effects of in vivo c-Met pathway inhibition on the stem-like
phenotype in these HGF-independent glioma models. Subcutaneous
xenografts were allowed to grow for approximately 23 days after
which animals received PF2341066 (40 mg/kg daily by gavage) or
carrier only as control for 7 days. PF2341066 statistically significantly
inhibited the growth of both Mayo 59 (P < .01) and Mayo 39 (P <
.05) xenografts (Figure 3A) concurrent with statistically significant
inhibition of tumor c-Met activation by 50% to 80% as determined
by immunoblot analysis of total tumor protein (Figure 3B). Similar to
that observed in cells obtained from anti–HGF-treated U87 xeno-
grafts, systemic treatment with PF2341066 depleted xenografts of
their stem-like sphere-forming cells (Figure 3, C and D). We exam-
ined the effects of systemic PF2341066 treatment on xenograft expres-
sion of stem cell–associated markers. Immunoblot analyses showed
decreased expression of Nanog and Musashi in Mayo 59 xenografts
and decreased expression of CD133 and Sox2 in Mayo 39 xenografts
(Figure 3, E and F). These results show that in vivo c-Met inhibition
Figure 2. Systemic anti-HGF therapy inhibits tumor growth and modulates the stem-like phenotype in U87 xenografts. (A) Growth of
subcutaneous tumor xenografts established from U87 cells and treated i.p. from PID 7 to 13 with control 5G8 mAb or with anti-HGF L2G7.
L2G7 withdrawal depicts tumors treated with L2G7 for PID 7 to 13 followed by no therapy for PID 14 to 27 (N = 6). (B) Immunoblot
analysis of tumor phospho–(tyr-1349)– and total Met levels in PID 14 U87 xenografts treated with 5G8 mAb or with L2G7 from PID 7
to 13 or in xenografts isolated on PID 27, 14 days after withdrawing L2G7 treatment (N = 4). (C) Neurosphere formation by cells derived
from subcutaneous xenografts treated with 5G8 and L2G7 for PID 7 to 13 and following L2G7 withdrawal (N = 6). (D–G) Immunoblot
analysis of tumor xenograft protein for CD133, Musashi, Sox2, and Nanog expression following L2G7 treatment and L2G7 withdrawal (N = 4).
*P < .05, **P < .001, ***P < .0001 compared with control (5G8 treatment).
In Vivo c-Met Inhibition Depletes GBM Stem CellsRath et al. Translational Oncology Vol. 6, No. 2, 2013
reduces tumor cell stemness in HGF-independent xenografts, similar
to results in the HGF-dependent U87 xenograft model.
In Vivo c-Met Pathway Inhibition Depletes Glioma
Xenografts of Tumor-Propagating Cells
Neurosphere-forming capacity and stem cell marker expression
have been found to correlate with tumor-propagating potential in
multiple solid tumor cell types including glioma. Therefore, our results
presented above predict that c-Met pathway inhibition depletes tumor
xenografts of tumor-propagating cells, one of the most clinically rele-
vant neoplastic stem cell phenotypes. We asked if inhibiting c-Met sig-
naling in vivo alters the capacity of tumor-derived cells to propagate
orthotopic glioma xenografts (see Figure 4A for experimental schema).
Pre-established subcutaneous U87 xenografts were treated with either
anti-HGF L2G7 or control mAb 5G8 (5 mg/kg, i.p., every other day)
for 1 week, the time at which c-Met was found to be inhibited by
∼69% by anti-HGF (see Figure 2B). Tumors were dissected and
single-cell suspensions of serially diluted tumor-derived cells (1 × 104,
3 × 103, or 3 × 102viable cells per animal) were injected to the brain.
This serial dilution tumor-propagatingassay indirectlyevaluates relative
numbers of stem-like tumor-propagating cells within heterogeneous
cell populations based on the efficiency of daughter xenograft forma-
tion. Animals in each group were sacrificed when the respective
5G8-treated control animals developed behavioral signs of impending
death from tumor burden (i.e., PID day 19 for 1 × 104cells/animal,
PID 19 for 3 × 103cells/animal, and PID 34 for 3 × 102cells/animal).
Histopathologic examination of H&E-stained brain sections revealed a
reduction in tumor-propagating capacity by cells derived from anti–
HGF-treated tumors (Figure 4B). In addition, the sizes of daughter
xenografts that formed from anti–HGF-treated tumors were 12-fold
smaller than controls (Figure 4C). Control animals consistently devel-
inhibition depletes tumor xenografts of their most aggressive tumor-
propagating stem-like cells.
Therapies that target neoplastic stem-like cells offer promising new
approaches for combating solid malignancies. Identifying and taking full
advantage of stem cell–based cancer therapy requires a deeper under-
standing of the role for neoplastic stem-like cells in specific cancers and
the molecular pathways driving the formation and tumor-propagating
potential of neoplastic stem-like cells. We and others recently found
Figure 3. Systemic c-Met kinase inhibitor therapy inhibits tumor growth and depletes xenografts of stem-like cells. (A) Effects of
PF2341066 (PF) on growth of Mayo 59 and Mayo 39 subcutaneous xenografts (N = 5). (B) Immunoblot analysis of phospho–(tyr-1349)–
PF-treated Mayo 59 and Mayo 39 xenografts. (D) Quantitation of neurosphere formation by cells derived from control and PF-treated xeno-
grafts (N = 4). (E) Immunoblot analysis of Nanog and Musashi expression levels in control and PF-treated Mayo 59 xenografts (N = 4).
(F) Immunoblot analysis of CD133 and Sox2 expression in control and PF-treated Mayo 39 xenografts (N = 4). *P < .05, **P < .001.
Translational Oncology Vol. 6, No. 2, 2013 In Vivo c-Met Inhibition Depletes GBM Stem CellsRath et al.
that c-Met correlates with the stem-like phenotype in human glio-
blastoma cells and that c-Met signaling induces GBM cell stemness
in in vitro model systems [13–15]. To determine if these previous find-
ings have therapeutic implications, we asked if clinically translatable
c-Met pathway inhibitors administered in vivo to glioblastoma-bearing
animals target the pool of stem-like tumor-propagating cells. We
investigated this question in three human glioblastoma xenograft
models representing ligand-dependent and ligand-independent mecha-
nisms of c-Met activation using two functionally distinct c-Met pathway
inhibitors currently in clinical development. While the effects of c-Met
pathway inhibitors are not limited to the tumor stem-like cell sub-
cells as evidenced by reductions in multiple molecular and biologic
markers associated with cancer cell stemness and tumor-propagating
potential. Numerous aspects of our experimental design and results
make it unlikely that nonspecific antitumor effects (e.g., general cell
“morbidity”) explain the observed effects of c-Met pathway inhibi-
tion on tumor cell stemness. 1) We were very careful to input equal
numbers of viable cells for all functional endpoints including in vitro
neurosphere-forming capacity and in vivo tumor-propagating capacity.
2) Stem cell protein markers were quantified using methods rigorously
normalized to housekeeping proteins and the results independently
supported reduced stemness observed using biologic endpoints depen-
dent on cell viability. These normalized biochemical endpoints would
not be affected by nonspecific differences in the general state of the cell.
3) We observed that tumors regrow rapidly after withdrawing c-Met
pathway inhibition, a behavior much more consistent with the reacti-
vation of oncogenic RTK signaling and inconsistent with a general
moribund state of the residual viable tumor cells. 4) c-Met inhibition
also depleted the Mayo xenograft models of their stem-like cells even
though there was less robust tumor growth inhibition in these models.
We and others have previously reported that neutralizing anti-
HGF mAbs and c-Met kinase inhibitors can substantially inhibit
the growth of glioblastoma xenografts derived from tumor cell lines
consistent with the well-documented oncogenic effects of HGF/c-Met
present the novel findings that human glioblastoma xenograft lines,
which express high levels of activated c-Met and more accurately rep-
licate the histopathologic features of GBM, are also sensitive to c-Met
inhibition. The different degrees of tumor growth inhibition achieved
by anti-HGF mAb in U87 xenografts and PF2341066 in the Mayo
xenograft lines are not likely to be due simply to different magnitudes
of c-Met inhibition since we found that both agents similarly reduced
tumor levels of phospho-Met in the current experiments. There are
multiple potential reasons why the HGF-negative xenograft lines were
foundto beless sensitive than U87xenografts toc-Metpathwayinhibi-
tion. Xie et al. recently found that ligand-dependent c-Met activation
predicts greater sensitivity to c-Met inhibitors than ligand-independent
mechanisms of c-Met activation known to result from, for example,
c-met gene amplification and RTK cross talk . In addition, Mayo
39 and Mayo 59 cells express the constitutively activated EGF receptor
(EGFR) deletion mutant EGFRvIII that supplies an alternate parallel
is possible that simultaneously inhibiting EGFR and c-Met will be par-
ticularly affective against these xenograft lines as previously observed in
U87 xenografts engineered to express EGFRvIII [21,28]. Experiments
designed to answer this hypothesis are currently underway.
Our finding that c-Met pathway inhibitor therapy depletes three
distinct xenograft models of multiple biologic and molecular markers
of GBM cell stemness is consistent with a role for c-Met signaling in
the generation and/or maintenance of GBM stem-like cells within
Figure 4. c-Met pathway inhibition depletes glioma xenografts of tumor-propagating cells. (A) Schematic of experimental design for
testing the effect of anti-HGF (L2G7) therapy on the tumor-propagating capacity of U87 xenograft–derived cells. Animals bearing sub-
cutaneous U87 xenografts were treated with 5G8 or L2G7 (N = 5). Tumors were recovered and tumor-derived cells were serially diluted
and implanted intracranially using either 3 × 102(N = 8), 3 × 103(N = 5), and 1 × 104(N = 5) viable cells/animal as shown. Animals
were sacrificed and brains were assessed for daughter xenograft formation by analysis of H&E-stained histologic section. (B) The table
shows the percentage of animals with detectable intracranial tumor xenografts. (C) Volumes of detectable tumors were quantified by
computer-based image analysis as described in Materials and Methods section. *P < .05, **P < .001, ***P < .0001 compared with
control (5G8 treatment).
In Vivo c-Met Inhibition Depletes GBM Stem Cells Rath et al. Translational Oncology Vol. 6, No. 2, 2013
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Translational Oncology Vol. 6, No. 2, 2013In Vivo c-Met Inhibition Depletes GBM Stem CellsRath et al.
Table W1. HGF Concentration in the Cells.
Cell LineHGF (pg/μg)
U87wt, Mayo 39, and Mayo 59 cells were grown under serum starvation conditions for 24 hours
and then cells were washed with cold PBS three times. Proteins were extracted using RIPA buffer
containing protease and phosphate inhibitors (Calbiochem) at 4°C and proteins were quantified
using Coomassie Protein Assay (Pierce Thermo Scientific, Rockford, IL). HGF was quantified by using
ELISA Kit (R&D Systems, Minneapolis, MN).