Resistance to anti-VEGF therapy mediated by autocrine IL … · 2016. 2. 26. · Author Manuscript...
Transcript of Resistance to anti-VEGF therapy mediated by autocrine IL … · 2016. 2. 26. · Author Manuscript...
Resistance to anti-VEGF therapy mediated by autocrine IL-6/STAT3 signaling and overcome by
IL-6 blockade
Alexandra Eichten*, Jia Su*, Alexander P. Adler*, Li Zhang*, Ella Ioffe, Asma A. Parveen,
George D. Yancopoulos, John Rudge, Israel Lowy, Hsin Chieh Lin, Douglas MacDonald,
Christopher Daly, Xunbao Duan, Gavin Thurston#
Regeneron Pharmaceuticals, 777 Old Saw Mill River Rd, Tarrytown, NY 10591
* authors contributed equally to this work
# corresponding author:
Gavin Thurston
Phone: 914-847-7575
Fax: 914-847-7544
Running title: IL-6/STAT3 signaling mediates anti-VEGF resistance
Disclosure of potential conflicts of interest: All authors are current or former employees and
shareholders of Regeneron Pharmaceuticals
Word count (excluding references, figure legends and figures): 5669
Number of figures: 7
Number of Tables: 0
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ABSTRACT
Anti-VEGF therapies benefit several cancer types, but drug resistance that limits therapeutic
response can emerge. We generated cell lines from anti-VEGF-resistant tumor xenografts to
investigate the mechanisms by which resistance develops. Of all tumor cells tested, only A431
(A431-V) epidermoid carcinoma cells developed partial resistance to the VEGF inhibitor
aflibercept. Compared to the parental tumors, A431-V tumors secreted greater amounts of IL-6
and exhibited higher levels of phospho-STAT3. Notably, combined blockade of IL-6 receptor
(IL-6R) and VEGF resulted in enhanced activity against A431-V tumors. Similarly, inhibition of
IL-6R enhanced the antitumor effects of aflibercept in DU145 prostate tumor cells that displays
high endogenous IL-6R activity. In addition, post-hoc stratification of data obtained from a
clinical trial investigating aflibercept efficacy in ovarian cancer showed poorer survival in
patients with high levels of circulating IL-6. These results suggest that the activation of the IL-
6/STAT3 pathway in tumor cells may provide a survival advantage during anti-VEGF treatment,
suggesting its utility as a source of response biomarkers and as a therapeutic target to heighten
efficacious results.
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INTRODUCTION
Vascular endothelial growth factor (VEGF) plays a key role in physiological and
pathological angiogenesis, including tumor angiogenesis. Blockade of the VEGF pathway is
effective at inhibiting angiogenesis in many tumors (1-3). Several VEGF pathway inhibitors,
including the monoclonal anti-VEGF antibody bevacizumab, the soluble receptor aflibercept
(VEGF Trap, known as ziv-aflibercept in the USA), and the monoclonal antibody to VEGF
receptor 2 (ramucirumab) delay tumor growth in preclinical tumor models (4-6) and extend
survival of cancer patients (7-11).
Despite showing broad activity in both preclinical and clinical settings, not all tumors
respond to anti-VEGF therapies, and those that do may eventually become resistant. Clinical and
preclinical studies suggest that resistance of tumors to anti-VEGF therapies can occur via several
mechanisms: (a) changes in the tumor microenvironment resulting in upregulation of various
pro-angiogenic factors, which lead to vessels that are less sensitive to VEGF blockade and/or (b)
changes in the characteristics of cancer cells, such as mutations and epigenetic changes, which
provide the cells with a survival advantage and/or increased invasive potential in the
environment of reduced tumor vessels (12-14). These resistance mechanisms cause insensitivity
of tumor vasculature to anti-VEGF treatments and/or decreased dependence of tumors on
angiogenesis. Combination treatments targeting VEGF and other angiogenic pathways such as
angiopoietin-2 (Ang2) or Delta-like 4 (Dll4) are being evaluated to increase the efficacy of
angiogenic blockade. However, the underlying mechanisms of tumor cells becoming less
dependent on angiogenesis in response to anti-VEGF therapies also need to be investigated.
To begin addressing this, we aimed to identify molecules involved in mediating anti-
VEGF resistance using xenograft tumor models with acquired resistance to VEGF blockade.
Aflibercept is a recombinant fusion protein that potently binds all isoforms of human and murine
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VEGF-A, VEGF-B and Placental Growth Factor (PlGF) (4). A431 human epidermoid cell tumor
xenografts are sensitive to aflibercept, but can acquire resistance when treated for longer time
periods (several weeks or more). We established a stable variant cell line (A431-V), which is
partially resistant to aflibercept and allowed the direct comparison of resistant tumors to their
sensitive parental A431 (A431-P) counterparts. Studies using these two cell lines revealed that
A431-V tumors have increased levels of pro-inflammatory mediators including IL-6, as well as
activation of the STAT3 signaling pathway. The activation of the IL-6/STAT3 pathway led us to
investigate the effects of an anti-IL-6R antibody (sarilumab), which blocks IL-6 receptor
signaling on human cells. Importantly, sarilumab does not bind or block murine IL-6R.
Treatment of A431-V tumor cells with sarilumab reduced the phosphorylation of STAT3, and
enhanced the anti-tumor activity of aflibercept against A431-V tumors in vivo. Similarly, IL-6R
inhibition enhanced the anti-tumor activity of aflibercept in another tumor model with high
endogenous IL-6R activity, namely Du145 prostate tumors. In addition, IL-6 overexpression
rendered sensitive A431 cells resistant to aflibercept. Finally, IL-6R blockade decreased the
number of A431 tumors that escaped long-term aflibercept treatment. To assess potential clinical
relevance, we determined whether circulating levels of IL-6 were associated with poorer
outcome in a Phase 2 clinical trial of ovarian cancer patients treated with aflibercept. A
correlation between high levels of IL-6 and poorer tumor response to anti-VEGF therapy was
observed. Taken together these data suggest that resistance to VEGF blockade can be mediated
at least in part by increased IL-6/STAT3 signaling in tumor cells, and that blockade of IL-6
signaling on tumor cells can overcome this resistance.
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MATERIALS AND METHODS
Tumor cells and reagents
Tumor cells including A431 human epidermoid cell skin carcinoma and Du145 human
prostate carcinoma were obtained from the American Type Culture Collection (ATCC) and
grown according to ATCC guidelines. All cell lines were authenticated between 2012 and 2015
using the STR Profile Testing by ATCC. A431 parental (P) and variant (V) cells were obtained
by in vivo passaging, as described in Figure 1.
Aflibercept (also known as VEGF Trap or ziv-aflibercept in the United States) is a
recombinant fusion protein that potently binds all isoforms of human and murine VEGF-A,
VEGF-B and Placental Growth Factor (PlGF). Sarilumab (REGN88) is a fully-human IgG1
monoclonal antibody that binds human IL-6R and prevents binding of IL-6. Cetuximab is a
chimeric mouse-human (30:70) IgG1 monoclonal antibody that competitively inhibits the
binding of epidermal growth factor (EGF) to its receptor EGFR.
Cell culture and in vitro cell growth
A431 and Du145 cells were cultured in 10% FBS DMEM and MEM, respectively.
Preparation of conditioned media: A431-P and A431-V cells were grown in serum-free media
for 16 hours followed by media collection. A431-P and A431-V cells grown on a 6-well plate at
a density of 1x106 cells/well or on 8-well chamber slides at a density of 30,000 cells/well were
serum starved for 16 hours followed by exposure to conditioned media for 1 hour. Cells were
either lysed to detect p-STAT3 status by Western blot or fixed for immunocytochemistry. Cell
lysates preparation for the RTK array: A431-V and A431-P cells were lysed in modified RIPA
buffer (1% NP-40, 0.1% deoxycholate, 50 mM Tris.HCl pH 7.4, 150 mM NaCl, EDTA 1mM,
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Na3VO4 1 mM, NaF 5 mM, glycerophosphate 5 mM and Roche complete protease inhibitor
cocktail tablets).
In vitro cell growth: 1x105 A431-P or A431-V cells were seeded and cell numbers were
assessed after a 3 day growth period. Doubling time (DT): DT (h) = (t - t0)log2/(logN - logN0),
where t and t0 are the times at which the cells were counted, and N and N0 are the cell numbers
at times t and t0, respectively.
RTK signaling antibody array
PathScan RTK Signaling array kit (Cell Signaling) was used following manufacturer’s
instructions.
Luminex analysis
A431-P and A431-V conditioned media analysis: Milliplex human cytokine/chemokine
42plex Kit (Millipore) following manufacturer’s instructions. Cytokines/chemokines assessed:
EGF, Eotaxin, FGF-2, Flt-3 ligand, fractalkine, G-CSF, GM-CSF, GRO, IFNα2, IFNγ, IL-1α,
IL-1β, IL-1ra, IL-2, sIL-2ra, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12 (p40), IL-12(p70),
IL-13, IL-15, IL-17, IP-10, MCP-1, MCP-3, MDC, MIP-1α, MIP-1β, PDGF-AA, PDGF-AB/BB,
RANTES, sCD40L, TGFα, TNFα, TNFβ, VEGFA. Samples were run using a Flexmap3D
instrument (Luminex), and data was analyzed with Masterplex software.
Phospho-STAT3 (Tyr705) levels in A431-P and A431-V cell lysates were assessed using
Bio-Plex phosphoprotein detection kit (Bio-Rad) following manufacturer’s instructions. The
“relative fluorescent units” readout was used to compare the phosphorylation levels.
Flow cytometry
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A431-P and –V cells were treated with enzyme free cell dissociation solution Hanks
based solution (Millipore, catalog # S-004-C), blocked in 2% FBS/PBS and human truStain FcX
(BioLegend, catalog # 422302), incubated with anti-human IL-6R-APC (catalog # 352805,
BioLegend) for 1 hour at RT and assessed by flow cytometry.
In vivo tumor studies
Tumor studies were performed in accordance with Regeneron’s Institutional Animal Care
and Use Committee guidelines. 1x106 A431-P or A431-V cells, or 5x106 Du145 cells were
implanted s.c. into CB.17 SCID mice (Taconic). Tumor volume was assessed with calipers using
the LxW2/2 formula. 100-200 mm3 tumor-bearing mice were randomized into groups and treated
s.c. with 25 mg/kg hFc (control protein), aflibercept (VEGF Trap), sarilumab, combination of
aflibercept and sarilumab (25 mg/kg each) or cetuximab 2x per week. Tumor growth curves:
average mean +/- standard deviation (SD).
Generation of IL-6 overexpressing A431 tumor cells
Lentiviral pLOC vector encoding human IL-6 and TurboGFP(nuc) reporter genes were
purchased (Thermo Scientific) and packaged per manufacturer's instructions. A431 carcinoma
cells were virally transduced with pLOC-IL-6 lentivirus particles isolated by two rounds of flow
cytometry cell sorting.
Immunohistochemistry and immunocytochemistry
Cryosections: OCT embedded tissue cut into 5 µm sections, air dried and fixed in acetone
(-20 °C) for 10 minutes, avidin-biotin blocking kit (Vector), blocked in 2.5 % normal goat serum
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/ 1 % BSA/PBS, Ki-67 Ab (BD; clone B56), biotinylated antibody (Vector), 3,3’-
diaminobenzidine (DAB, Sigma). Tissue sections were counterstained with methyl green.
In Situ Cell Death Detection Kit’ (Sigma) according to manufacturers’ recommendations
and apoptotic index was determined using HALO software (Indica Labs).
FFPE tissue sections: tumors were fixed in 10% neutral-buffered formalin, dehydrated
through graded ethanol and xylenes, embedded in paraffin, cut into 8 µm sections and
deparaffinized. p-STAT3 immunohistochemistry: antigen retrieval, block with 4% BSA,
incubation with p-STAT3 antibody (Cell Signaling, catalogue # 9145), HRP-conjugated
secondary antibody (Vector, catalogue # PI1000), 3,3’-diaminobenzidine (DAB, Sigma). Ki-67
immunohistochemistry: antigen retrieval, block with 0.1% Triton X-100, 5% goat serum, 2.5%
BSA in PBS, Ki-67 antibody (BD, catalogue # 556003), biotinylated antibody (Vector, catalogue
# BA-2001), ABC-ELITE (Vector; ABC VectaStain Elite), 3,3’-diaminobenzidine (DAB,
Sigma). Tissue sections were counterstained with hematoxylin.
Immunocytochemistry: cells were fixed in 4% PFA for 20 min, blocked for 45 min in
10% donkey serum / 0.3% Triton X-100, incubated with rabbit anti-human phopho-STAT3
(Y705) antibody (R&D, catalog # AF4607), anti-rabbit NL557-conjugated Ab (R&D, catalog #
NL004) and DAPI. Slides were mounted in ProLong Gold.
Immunoblotting analysis and ELISA assay
Tumor cells or tissues were harvested in lysis buffer (50mM Hepes pH 8.0, 10%
Glycerol, 1% Triton X-100, 150mM NaCl, 1mM EDTA, 1.5mM MgCl2, 100mM NaF, 10mM
NaP2O7, freshly supplemented with protease inhibitor tablet (Roche catalogue # 1836153001)
and 1mM Na3VO4). Tissues were homogenized on ice and tissue or cell lysates were run on a
SDS-PAGE gel and Western Blotted with the following antibodies: Cell Signaling: p-STAT3
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(catalogue # 9145), STAT3 (catalogue # 9139), p44/42 MAPK (ERK1/2) (catalogue # 4348),
Phospho-p44/42 MAPK (ERK1/2) (catalogue # 4370), tubulin (catalogue # 2125), Santa Cruz:
NGAL (catalogue # sc-50350), S100A7 (catalogue # sc52948), SAA (catalogue # sc-52214),
IL36G (catalogue # sc-80056) and β-actin (catalogue # sc-69879), fibronectin (BD Transduction,
catalogue # 610077) overnight at 4°C. HRP-conjugated anti-rabbit (Cell Signaling, catalogue #
7074), anti-mouse (Pierce, catalogue # 31437), and anti-rat (Santa Cruz, catalogue # sc-2006)
antibodies were applied for 1 hour at room temperature. Blots developed with ECL.
Densitometry analysis was performed using Carestream Software (Molecular Imaging).
IL-6 levels in conditioned medium of cultured cells, tumor lysates and patient plasma
were measured using Quantikine High Sensitivity IL-6 ELISA kit according to manufacturers’
instructions (R&D, catalogue # HS600B). VEGF levels of tumor lysates were measured using
mouse and human VEGF Quantikine ELISA Kit (R&D, catalogue # MMV00, DVE00)
according to manufacturers’ instructions.
Patient Samples
Collection and testing of plasma for exploratory biomarker analysis was specified in the
amended clinical trial protocol for ARD6122/AVE0005, a phase 2, multicenter, randomized,
double-blind, parallel-arm, two-stage study of aflibercept in patients with platinum-resistant and
topotecan- and/or liposomal doxorubicin-resistant advanced ovarian cancer. Informed consent
for sample testing was obtained following protocol approval by local Institutional Review
Boards.
Statistics
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Tumor sizes at the different time points from control and treatment groups were
compared using two-way ANOVA and Bonnferoni’s multiple comparison tests (Prism software
version 5).
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RESULTS
Generation and characterization of an aflibercept-resistant A431 tumor cell line
Human xenograft tumors grown in mice show different degrees of response to VEGF
blockade. In order to study resistance mechanisms, we aimed to derive tumor cell lines with
acquired resistance to aflibercept from aflibercept-sensitive tumors. For this aim, we selected
four aflibercept-sensitive tumor lines, namely A498, Colo205, 786-0 and A431, and treated
tumor-bearing mice with high dose aflibercept (25 mg/kg, twice weekly), for up to seven weeks.
For three of the four tumor lines, we observed complete growth stasis (Colo205, A498) or
gradual incremental tumor growth (786-0), but no outgrowth of resistant tumors based on our
criteria (Supplemental Figure S1). However, A431 human epidermoid cell carcinoma tumors
exposed to long-term high dose aflibercept showed complete and prolonged tumor growth
inhibition for approximately 6 weeks followed by conspicuous outgrowth of some of the tumors
(Figure 1B, Supplemental Figure S1 for individual tumor growth).
One of the outgrowing A431 tumors was harvested, and fragments of viable tumor tissue
were re-implanted subcutaneously into other SCID mice (the procedure is outlined in Figure 1A).
In some cases, re-implanted A431 tumors that were treated with aflibercept continued to grow.
This re-implantation and selection procedure was repeated once more, and we again observed
tumor growth in the presence of aflibercept. In parallel, we performed a similar re-implantation
procedure with A431 tumors treated with human control protein (Fc fragment only, Figure 1A).
After in vivo passaging and selection, the aflibercept and control treated tumors were harvested,
minced and transferred to tissue culture dishes for propagation in vitro. The cell lines derived
from control and aflibercept-treated tumors were called A431-P (parental) and A431-V (variant)
respectively (Figure 1A).
To confirm that the isolated A431-V cells retained resistance to aflibercept, tumors from
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A431-V and A431-P cell lines were studied for growth kinetics and response to aflibercept. We
observed that control treated A431-V tumors grew slightly faster than control treated A431-P
tumors (Figure 1C). In addition, A431-V tumors continued to grow during treatment with
aflibercept, albeit at a slower rate (Figure 1C), suggesting that A431-V tumors are inherently
resistant to VEGF blockade. These differences in tumor growth were mirrored when assessing
cell proliferation in tissue sections by detecting the proliferation marker Ki-67 (Figure 1D).
Control treated A431-V tumors had higher numbers of proliferating cells in the tumor center than
control treated A431-P tumors, consistent with the faster growth rate of A431-V tumors (Figure
1C and D). Aflibercept treatment dramatically reduced the number of proliferating cells in A431-
P tumors (Figure 1D), with changes being apparent as early as 24 hours after aflibercept
administration (Supplemental Figure S2A). In contrast, aflibercept treatment had a much smaller
effect on cell proliferation in A431-V tumors (Figure 1D). Similarly, TUNEL staining revealed
that aflibercept treatment increased apoptotic cells in A431-P tumors, but not in A431-V tumors
(Figure 1E).
Interestingly, the growth rates of A431-P and A431-V cells in vitro did not differ
significantly (Figure 2A). In addition, A431-V tumors had increased vessel density compared to
A431-P tumors, although vessel density after VEGF blockade was similar in A431-P and A431-
V tumors (Supplemental Figure S2B and S2C). A431-P and A431-V tumor lysates did not show
differences in murine (20.5 +/- 2 and 20.8 +/- 5 pg/mg, respectively) or human VEGF (13 +/- 1.9
and 12.1 +/- 4 ng/mg, respectively), suggesting that VEGF levels were not responsible for the
difference in vessel density.
We next wanted to determine whether the resistance of A431-V tumors was specific to
anti-VEGF agents, or whether it was a more generalized resistance to targeted therapy. A431
cells express high levels of EGFR and are normally sensitive to EGFR antibodies. Variants of
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A431 that are resistant to EGFR blockers have been previously described (15). EGFR activation
was not altered in A431-V cells, as no difference was observed in a phospho-protein array
(Figure 3A). To determine EGFR activity we assessed phospho-ERK1/2 levels by Western blot
and observed that P-ERK1/2 in A431-V was somewhat lower compared to A431-P (Figure 2B),
suggesting similar EGFR activity in both cell lines. To determine whether A431-V tumors,
which were selected for resistance to aflibercept, maintained sensitivity to EGFR blockade, mice
bearing A431 variant or parental tumors were treated with the EGFR antibody cetuximab. Both
A431-V and –P tumors were similarly responsive to cetuximab, whereas, as expected the variant
tumors were resistant to aflibercept (Figure 2C and D). Together, these findings indicate that the
aflibercept resistance of A431-V tumors is a specific and stable trait, which provides a model to
study the underlying molecular mechanisms of VEGF resistant phenotype.
Identification of pathways/factors mediating aflibercept resistance in A431-V tumors
To explore the mechanisms of aflibercept resistance in A431-V tumors, we first utilized a
phospho-protein array to compare the activities of 28 receptor tyrosine kinases and 11
downstream signaling molecules in A431-V and A431-P cell lines (Figure 3A). The most distinct
difference between the resistant and sensitive tumor cells was a marked increase in the
phosphorylation of STAT3 at Tyr705 (Figure 3A). The increase in phospho-STAT3 was
confirmed by Luminex as well as Western blot analysis (Figure 3B and C, respectively). STAT3
can be phosphorylated in response to various secreted cytokines and chemokines, including IL-6,
IL-10 and LIF (16). To determine whether the phosphorylation of STAT3 in A431-V cells is
mediated by secreted factors, we exposed A431-P cells to conditioned media (CM) from A431-V
cells. We observed that phospho-STAT3 was increased, and was localized in the nucleus, in
A431-P cells treated with CM from A431-V cells (Figure 3D (third lane) and E). In contrast,
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phospho-STAT3 in A431-V cells was notably reduced after incubation with A431-P CM (Figure
3D (fourth lane) and E), suggesting that secreted factor(s) mediate the phosphorylation of
STAT3 in A431-V cells.
To identify the factors secreted by A431-V cells that could mediate STAT3 activation,
we compared the relative expression levels of secreted proteins in A431-P and A431-V cells
using a quantitative proteomics technique called SILAC (stable isotope labeling with amino
acids in cell culture) (Supplemental Figure S3A and S3B). We observed increased expression of
80 proteins and decreased expression of 98 proteins in the secretome of A431-V cells compared
to A431-P cells (Supplemental Table S1). Since many secreted signaling proteins, such as
cytokines, are below the current detection limit of mass spectrometry, a multiplex Luminex assay
was performed in parallel to measure the concentrations of 42 cytokines in A431-P and A431-V
CM. A total of 13 cytokines could be detected (Supplemental Table S2), of which IL-8, CXCL1,
GM-CSF, IL-1α, and IL-6 were significantly increased in CM from A431-V cells compared to
that from A431-P cells (Figure 3F).
Of these cytokines, CXCL-1 and IL-6 are known to activate STAT3 in various cell types
(16-20). To assess the role of CXCL-1 in aflibercept resistance, we generated A431 cells
overexpressing CXCL-1 and assessed the response to aflibercept in vivo. CXCL-1
overexpression provided a slight growth advantage to A431 cells, but did not confer resistance to
aflibercept (Supplemental Figure S4A and S4B), suggesting that CXCL-1 is not a key player in
mediating aflibercept resistance in this model.
We next focused on IL-6 and Ingenuity Pathway Analysis (IPA) of the 186 dysregulated
proteins (SILAC plus Luminex) revealed the IL-6 network as one of the most activated networks
(in addition to IL-8 and IL-1α networks) in A431-V cells (Supplemental Figure S5A and S5B).
In addition, increased protein expression of serum amyloid A (SAA), S100 calcium-binding
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protein A7 (S100A7), fibronectin (FN1) and interleukin-36 (IL36G) was observed (Figure
3G), all of which have been reported to be downstream of the IL-6/STAT3 pathway (21-24).
Upregulation of SAA and NGAL mRNA was also observed in A431-P cells exposed to A431-V
CM (Supplemental Figure S3C). Based on these data, we hypothesized that secreted IL-6 might
be mediating phosphorylation of STAT3 in A431-V cells.
Blockade of IL-6 signaling by anti-IL-6R antibody partially attenuates STAT3 activity in
vitro and completely overcomes aflibercept resistance in vivo
To assess whether elevated STAT3 activation in A431-V cells was due to increased IL-6
signaling, we determined whether A431-V cells and tumors have higher IL-6 levels than their
A431-P counterparts. Indeed, IL-6 protein levels in both A431-V cell CM and A431-V tumor
lysates were more than 3-fold higher than those in A431-P counterparts (Figure 4A). In contrast
to IL-6, levels of IL-6R were similar on A431-P and -V tumor cells (Figure 4B). To determine if
increased phosphorylation of STAT3 in A431-V tumors was due to the increased IL-6, we used
an IL-6 receptor antibody (sarilumab) that blocks IL-6 binding to IL-6R (25). As shown in
Figure 4C, STAT3 phosphorylation in cultured A431-V cells was noticeably reduced by
blocking IL-6R with sarilumab, although not to quite the same level as seen in A431-P cells.
To test the role of elevated IL-6/STAT3 signaling in aflibercept resistance of A431-V
tumors, we treated tumor-bearing mice with sarilumab and/or aflibercept to assess the effects on
A431-V and A431-P tumor growth. Sarilumab binds and blocks human IL-6R but not murine IL-
6R, so its actions are specific to the A431 human tumor cells. Sarilumab had no effect on
parental A431-P tumor growth and did not enhance the effects of aflibercept, indicating that the
growth of A431-P tumors is not dependent on IL-6 signaling, irrespective of whether VEGF
activity is inhibited (Figure 4D, left). While single agent sarilumab had no effect on the growth
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of A431-V tumors, the combination of sarilumab plus aflibercept had a significantly greater
effect than aflibercept alone, completely inhibiting A431-V tumor growth (Figure 4D, right).
This finding indicates that IL-6 signaling limits the effectiveness of aflibercept in A431-V
tumors. The changes in growth kinetics of A431-V tumors upon aflibercept, sarilumab or
aflibercept plus sarilumab were reflected in differences in proliferation, as assessed by Ki-67
immunohistochemistry (Figure 4E). Consistent with a more prominent role for IL-6/STAT3
signaling in A431-V tumors, Western blot analysis revealed that A431-V tumors have elevated
levels of phospho-STAT3 compared to A431-P tumors, and that this STAT3 phosphorylation
can be blocked by sarilumab treatment (Figure 4F). These findings confirm that aflibercept
resistance in A431-V cells is attributable to the increased STAT3 phosphorylation and IL-6
signaling.
To extend our findings on the role of IL-6/STAT3 signaling in limiting aflibercept
efficacy, we tested the effects of sarilumab as a single agent and in combination with aflibercept
on the growth of Du145 prostate tumor xenografts. Cultured Du145 cells exhibit constitutive
STAT3 phosphorylation that can be inhibited by blocking IL-6R with sarilumab, indicating the
presence of an autocrine IL-6/STAT3 signaling pathway (Figure 5A) similar to that in A431-V
cells (Figure 4C). As shown in Figure 5B, sarilumab as a single agent caused a significant
growth delay of Du145 tumors, suggesting a role for autocrine IL-6 signaling in Du145 tumor
growth. Aflibercept as a single agent had a more dramatic effect than single agent sarilumab,
causing some tumor regression (Figure 5B). However, the combination of aflibercept plus
sarilumab caused a more rapid and pronounced tumor regression than aflibercept alone (Figure
5B), further supporting the possibility that IL-6 signaling limits the effectiveness of VEGF
blockade in some tumors. Consistent with active IL-6/STAT3 signaling in Du145 tumors,
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sarilumab decreased STAT3 phosphorylation as assessed by immunohistochemistry of tumor
sections (Figure 5C).
Overexpression of IL-6 promotes aflibercept resistance, whereas long-term blockade of
both IL-6 and VEGF decreases the emergence of aflibercept resistance
To determine whether high IL-6 expression is sufficient to induce resistance to
aflibercept, A431 cells were transduced with a lentiviral vector expressing human IL-6 or an
empty control vector. Ectopic IL-6 expression resulted in accelerated tumor growth compared to
control tumors (Supplemental Figure S6A). The high levels of ectopic IL-6 expression also
resulted in increased circulating IL-6 protein level (~100-200 ng/ml; data not shown), which was
associated with body weight loss and early morbidity of tumor-bearing mice (Supplemental
Figure S6B). Aflibercept treatment starting when tumors were ~100 mm3 had no effect on tumor
growth in IL-6 overexpressing A431 tumors, while being very effective in A431 empty vector
control tumors (Figure 6A and B). These data indicate that elevated IL-6 expression in tumor
cells is sufficient to induce resistance to aflibercept in A431 xenograft tumors.
To investigate if blockade of the IL-6 pathway could prevent the emergence of
aflibercept-resistant A431 tumors, we treated A431 tumor bearing mice with control protein,
aflibercept, sarilumab or a combination of aflibercept plus sarilumab when the tumors reached a
volume of ~100 mm3. Single agent sarilumab treatment did not delay A431 tumor growth,
whereas both aflibercept single agent and aflibercept plus sarilumab combination treatments
resulted in prolonged growth stasis (Figure 6C and D) for 6 weeks. As observed before, a subset
of A431 tumors (5 of 7 tumors; Figure 6D top panel, Supplemental Figure S1) began to grow
significantly after 6 weeks of continuous aflibercept treatment. In contrast, no tumors showed
steady growth during the latter phases of continuous treatment with aflibercept plus sarilumab (0
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of 7 tumors; Figure 6D lower panel). These results suggest that changes in the IL-6 pathway are
not only relevant after aflibercept resistance occurred, but also play a role in the development of
anti-VEGF resistance in A431 xenograft tumors.
IL-6 serum levels may be predictive for outcome in ovarian cancer patients treated with
aflibercept
Taken together, our preclinical results show that activation of the IL-6/STAT3 pathway
can provide evasive resistance to treatment with VEGF inhibitors such as aflibercept. To assess
whether this finding is clinically relevant, we examined the levels of IL-6 in ovarian cancer
patients treated with single agent aflibercept (26). Serum levels of IL-6 were measured in a group
of patients in an international, double blind, phase II study of advanced ovarian cancer, in which
two different doses of aflibercept were used as monotherapy. 96 patients were stratified into
those with high and low circulating IL-6 (cutoff 3.86 pg/ml). Although the study showed only a
modest overall response rate to aflibercept, we observed that patients with high IL-6 serum levels
(> median) show significantly poorer overall survival compared to those with low IL-6 levels (<
median) (Figure 7). These results suggest a correlation between high levels of IL-6 and poorer
tumor response to anti-VEGF therapy.
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DISCUSSION
To uncover mechanisms underlying resistance to anti-VEGF therapies, we used serial
passaging and selection in vivo to generate a variant A431 tumor cell line (A431-V) that is
partially resistant to aflibercept, and compared this line to the aflibercept-sensitive parental
counterpart A431-P. Our studies found that increased levels of pro-inflammatory mediators, such
as IL-6, were associated with STAT3 pathway activation in A431-V tumors, and can contribute
to aflibercept resistance. Blockade of the IL-6/STAT3 pathway with an anti-IL-6R antibody
(sarilumab), which is specific to human IL-6R, rendered A431-V tumors more sensitive to
aflibercept. Similarly, IL-6R inhibition enhanced the activity of aflibercept in another tumor
model with strong endogenous IL-6/STAT3 activity, namely Du145 prostate tumors. In addition,
IL-6 overexpression rendered A431 tumors resistant to aflibercept, suggesting that elevated IL-6
expression is sufficient to provide a survival advantage to tumor cells during aflibercept
treatment. In these experiments, activation of IL-6R/STAT3 signaling in cancer cells is mediated
by human IL-6 because mouse IL-6 does not activate the human IL-6R (27), and murine IL-6R is
not inhibited by sarilumab, a human specific IL-6R antibody. Limitations of working with
human tumor cells in immunocompromised mice include the lack of species cross-activity of
some factors such as IL-6, thus potentially limiting tumor–stromal interactions that could induce
STAT3 signaling in other tumor compartments (43). Nevertheless, based on these findings we
conclude that in A431-V tumors, aflibercept resistance is mediated to a large extent by increased
IL-6/STAT3 signaling in tumor cells, and that blockade of IL-6R can overcome resistance to
aflibercept.
Various clinical studies have correlated high pretreatment serum IL-6 level with poor
outcome in patients with different types of cancer including head and neck squamous cell
carcinoma (28), stage II and III gastric carcinoma (29), prostate cancer (30), metastatic renal cell
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carcinoma (31), metastatic breast cancer (32) and ovarian cancer (33). Elevated levels of IL-6 in
cancer patients could be due to increased expression in normal immune cells as a consequence of
chronic (systemic) inflammation, and/or from stromal or cancer cells within tumors. The
resulting activation of the STAT3 transcription factor in cancer cells is essential for IL-6-
dependent oncogenic activities such as promoting cancer cell proliferation and survival (34).
STAT3 activation has been shown to support cancer cell survival in the conditions of growth
factor deprivation (35) and to promote chemoresistance in cancer cells exposed to hypoxia (36),
suggesting that STAT3 may be a potentially important cancer cell survival factor in tumors
treated with anti-angiogenic therapies. Our findings that patients with high IL-6 serum levels
showed poorer survival than those with low IL-6 levels in a phase II study of advanced ovarian
cancer where aflibercept was used as monotherapy (26), suggests that our preclinical findings
could have clinical significance and warrant further investigation.
Resistance to anti-VEGF therapies can occur via several mechanisms: (a) upregulation of
various pro-angiogenic factors resulting in vessels that are less sensitive to VEGF blockade
and/or (b) changes in the characteristics of cancer cells, which provide the cells with a survival
advantage and/or increased invasive potential in the environment of reduced tumor vessels (12-
14). Our approach to induce variants of A431 tumor cells that are resistant to aflibercept allowed
direct comparisons between parental and variant tumors. For example, screening of phospho-
protein arrays found that phospho-STAT3 was the major difference between parental and variant
A431 tumor cells. We also used unbiased SILAC screens to test for overall differences in the
secreted protein profile (secretome) of parental and variant cells. Detailed expression analysis
revealed upregulation of various potentially proangiogenic factors, including IL-8 and CXCL1 in
A431-V tumors, which could contribute to the increased vessel density. A key role for CXCL1 in
mediating aflibercept resistance was ruled out in our model system. Given the STAT3 activation
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21
we observed in A431-V tumors, we decided to focus on IL-6, rather than IL-8 in subsequent
studies. A large body of literature suggests that IL-6 activates STAT3 (37,38) and, in addition,
our studies implicate a key role for the IL-6/STAT3 pathway in resistance to anti-VEGF
therapies.
Notably, our studies on the role of IL-6 in resistance to anti-VEGF therapies focus on
autocrine IL-6/STAT3 signaling in the cancer cells. Autocrine IL-6 signaling has been reported
in various cancer types, including multiple myeloma (39), prostate cancer (40), lung cancer (41)
and breast cancer (42). In our studies on A431-V and Du145 xenograft tumors, activation of IL-
6R/STAT3 signaling in cancer cells is solely mediated by IL-6 derived from the human cancer
cells, but not the murine stromal cells, because mouse IL-6 does not activate the human IL-6R
(27). Although human IL-6 activates mouse IL-6R on stromal cells, this interaction is not
inhibited by sarilumab, which is a human specific IL-6R antibody. Therefore, the effects of
sarilumab reported here are due to the interruption of autocrine IL-6/STAT3 signaling in cancer
cells. Although other studies have reported that increased activation of tumor or stromal Stat3
can lead to enhanced STAT3 signaling in tumor endothelial cells (43), such a mechanism is
apparently not at play in the A431-V model. Instead, our data highlight a mechanism of
resistance to anti-VEGF therapy in which the cancer cell characteristics change and provide a
survival advantage in the environment of reduced tumor vessels.
Although IL-6 has previously been implicated as a potential biomarker for targeted anti-
VEGF therapy, the results have been inconsistent. When treated with bevacizumab plus a
chemotherapeutic regimen, a better outcome was reported for metastatic colorectal cancer
(mCRC) patients with a signature that included lower than average baseline IL-6 serum levels
(44), whereas in another study of mCRC a worse outcome was associated with a signature that
included lower than average IL-6 serum levels (45). High baseline IL-6 levels were also reported
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to predict shorter progression-free survival (PFS) and overall survival (OS) in patients with
advanced hepatocellular carcinomas (46). In line with these later studies, we observed in an
ovarian cancer trial that aflibercept-treated patients with high IL-6 had poorer survival than
patients with low IL-6 serum levels. However, caution should be taken when using serum IL-6
levels as a biomarker, since our studies suggest that IL-6R signaling on tumor cells is the
relevant feature that confers resistance to anti-VEGF therapy.
IL-6 could be a potential biomarker for anti-VEGF therapies, but our data now suggest
that IL-6 can also mediate anti-VEGF resistance and thus might be a valid therapeutic target
when combined with anti-VEGF therapies in cancer patients. Although the data presented in this
manuscript do not provide details on how IL-6/STAT3 activation in the tumor cells can confer
resistance to aflibercept treatment, we hypothesize that a double hit of targeting the vasculature,
thus diminishing oxygen and nutrient supply for the tumor cells, and blocking IL-6, thereby
shutting down the IL-6/STAT3 regulated survival and proliferation pathway in the tumor cells
and limiting IL-6 mediated inflammation in the tumor microenvironment, can overcome anti-
VEGF resistance in tumors with measurable IL-6/STAT3 signaling. Further studies and clinical
trials are necessary to validate or refute the role of IL-6 in anti-VEGF resistance in different
types of cancer.
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23
ACKNOWLEDGEMENT
The authors would like to thank Bo Luan, Thomas Nevins, Benjamin Strober, Bradley Hagan
and Jennifer Espert for technical assistance, Cristina Abrahams for critical comments and
suggestions, and Alshad Lalani and Bo Gao for assistance with processing clinical samples and
data analysis.
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24
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FIGURE LEGENDS
Figure 1: Generation and characterization of aflibercept-resistant A431 variants
(A) Schematic of A431 parental and variant tumor cell line generation
(B) SCID mice bearing A431 tumors (50-100 mm3) were treated with aflibercept or human
Fc control protein for up to 7 weeks.
(C) Growth kinetics and response to aflibercept treatment of tumors grown from in vivo
passaged A431-V and A431-P cell lines. The average tumor volume ± SD is plotted over
the course of treatment, **** P < 0.0001 two-way ANOVA, Bonferroni post-hoc test.
(D) Representative images of cell proliferation assessed by Ki-67 immunohistochemistry in
A431-P and A431-V tumors treated with human Fc control protein or aflibercept for 14
days. Scale bar = 50 µm.
(E) Representative images of cell apoptosis assessed by TUNEL staining in A431-P and
A431-V tumors treated with human Fc control protein or aflibercept for 24h. Scale bar =
10µm. Quantitative analysis of apoptotic index based on TUNEL-positive (green) and –
negative nuclei in A431-P and A431-V tumor tissue. *** P < 0.001 one-way ANOVA
with Bonferroni’s post-hoc test.
Figure 2: Additional characterization of aflibercept-resistant A431 variants
(A) In vitro growth kinetics of A431-P and A431-V cell lines as assessed by population
doubling time (28.6 h for A431-P and 27.9 h for A431-V). Triplicates of each cell line
were analyzed.
(B) Total levels as well as phosphorylation levels ERK1/2 were assessed in A431-P and
A431-V cells by Western blot.
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(C) Tumor growth kinetics of A431-P and A431-V tumors (100-150mm3) treated with human
Fc control protein, aflibercept or cetuximab for 14 days. The average tumor volume ± SD
is plotted over the course of treatment.
(D) Tumor growth changes of A431-P and A431-V tumors treated with control protein,
aflibercept or cetuximab from start of treatment. ** P < 0.01, **** P < 0.0001 one-way
ANOVA, Bonferroni post-hoc test.
Figure 3. Constitutive STAT3 activation in A431-V cells
(A) Phospho-RTK signaling antibody array was used to examine phosphorylation status of 28
receptor tyrosine kinases and 11 downstream signaling molecules in A431-P and A431-V
cell lysates. Each RTK or signaling node is spotted in duplicate.
(B) Tyr705 phosphorylation level of STAT3 in A431-P and A431-V cells assessed by
Luminex.
(C) Tyr705 phosphorylation level of STAT3 in A431-P and A431-V cells assessed by
Western blot.
(D) A431-P and A431-V cells were exposed to conditioned media from A431-P and A431-V
cells as indicated by the red arrows and cell lysates were analyzed for phospho-STAT3
detection by Western blot.
(E) A431-P and A431-V cells were exposed to conditioned media from A431-P and A431-V
cells as indicated and analyzed for phospho-STAT3 by IHC. Scale bar = 10µm
(F) Top five cytokines with higher concentrations in A431-V conditioned media compared to
A431-P conditioned media, as measured by Luminex.
(G) Increased expression of the IL-6/STAT3-dependent protein SAA, S100A7, fibronectin
and IL36G in A431-V cell conditioned media was confirmed by Western blot.
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30
Figure 4. Effect of blocking cancer cell IL-6 signaling pathway with sarilumab on the
response of A431-P and A431-V tumors to aflibercept treatment
(A) Concentration of human IL-6 was measured in conditioned medium from cultured A431-
P or A431-V cells (left panel) and lysates from A431-P or A431-V tumor xenografts
(right panel) by ELISA. ** P < 0.01, unpaired t test.
(B) IL-6R was detected on A431-P and A431-V cells by flow cytometry.
(C) A431-P and A431-V cells were treated with human Fc control or sarilumab in vitro and
cell lysates were used for Western blot analysis detecting STAT3 and phospho-STAT3.
Densitometry analysis was performed to calculate the ratio of phospho-STAT3 to total
STAT3 (p/t STAT3).
(D) A431-P or A431-V tumor-bearing mice were treated with human Fc, sarilumab,
aflibercept or the combination of sarilumab plus aflibercept for 14 days. The average
tumor volume ± SD is plotted over the course of treatment, * P < 0.05, ** P < 0.01, ****
P < 0.0001, two-way ANOVA with Bonferroni post-hoc test.
(E) Representative images of cell proliferation assessed by Ki-67 immunohistochemistry in
A431-V tumors treated with control Fc, sarilumab, aflibercept or the combination of
sarilumab plus aflibercept for 14 days. Scale bar = 10 µm; S - Stroma, T - Tumor, N -
Necrosis.
(F) A431-P or A431-V tumor-bearing mice were treated with a single dose of human Fc or
sarilumab and tumor lysates were prepared 18 hours after treatment. Phospho-STAT3 (p-
STAT3) and total STAT3 (t-STAT3) levels were determined by Western blot analysis.
Densitometry analysis was performed to calculate the ratio of phospho-STAT3 to total
STAT3 (p/t STAT3). *P<0.05, one-way ANOVA with Bonferroni post-hoc test.
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31
Figure 5. Blocking the cancer cell IL-6 signaling pathway with sarilumab affects the
response of Du145 tumors to aflibercept treatment
(A) Du145 prostate cancer cells were cultured in complete medium and treated with 10 ug/ml
of human Fc control protein or 10 µg/ml of sarilumab for 18 hours (lanes 1 - 4), and
followed by a treatment of 10 ng/ml of hIL6 for 30 minutes (lanes 3 and 4). Cell lysates
were harvested after treatment and western analysis was performed to assess the levels of
phospho-STAT3 and total-STAT3.
(B) Du145 tumor-bearing mice were treated with human Fc control protein, sarilumab,
aflibercept or the combination of sarilumab plus aflibercept. The average tumor volume ±
SD is plotted over the course of treatment. * P < 0.05, ** P < 0.01, **** P < 0.0001 vs
hFc treated group, two-way ANOVA with Bonferroni post-hoc test.
(C) Representative images of phospho-STAT3 immunohistochemistry on FFPE Du145
tumors treated with human Fc or sarilumab. Scale bar = 50 µm.
Figure 6. Overexpression of IL-6 or long-term blockade of the IL-6 pathway affects the
response of A431 tumor xenografts to aflibercept treatment
(A) A431 tumor cells were engineered to overexpress IL-6 and tumor growth kinetics and
aflibercept response was compared to A431 empty vector tumors for 7 days. The average
tumor volume ± SD is plotted over the course of treatment.
(B) Tumor growth changes of IL-6 or empty vector expressing tumors in response to human
Fc control protein or aflibercept from start of treatment. **** P < 0.0001 one-way
ANOVA, Bonferroni post-hoc test.
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32
(C) SCID mice bearing A431 tumors (100-150mm3) were treated with human Fc control
protein, sarilumab, aflibercept or a combination of aflibercept + sarilumab for up to 7
weeks.
(D) Individual tumor growth kinetics for the aflibercept and aflibercept + sarilumab treated
groups. The tumors that exhibit a ‘late escape’, defined as a tumor showing 40% or more
increase in tumor volume between day 42 and day 49, are indicated by a red star next to
the tumor growth curve. One combo treated tumor, as indicated by a black # symbol,
showed intermittent growth throughout the experiment, but did not show escape as
defined above. One combo treated tumor grew at the end of the experiment, but only
showed an increase in tumor volume of 33% between day 42 and 49 – indicated by a blue
star next to the tumor growth curve
Figure 7. Aflibercept treatment is more effective in increasing overall survival in ovarian
cancer patients with low IL-6 levels
In an international, double blind, phase II study of advanced ovarian cancer, aflibercept was used
as a monotherapy. Patients received either a 2 mg/kg or 4 mg/kg of aflibercept every two weeks
and were monitored for overall response. The study showed a modest response rate of 5% in
both arms, and the ovarian indication has not been further vigorously pursued, in part due to this
data. When the data was stratified based on low (< median) and high (> median) IL-6 serum
levels, it became apparent that patients with high IL-6 levels show a worse response compared to
those with low IL-6 levels.
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0 20 40 600
200
400
600
800
Days after treatment start
Tu
mo
r Vo
lum
e (m
m3 )
Days post treatment start
Tum
or
Volu
me (
mm
3)
0 5 10 150
500
1000
1500 A431 P - ctrl
A431 P - aflibercept
A431 V - ctrl
A431 V - aflibercept****
****
****
A431 V afl vs V ctrl
A431 P afl vs P ctrl
A431 V ctrl vs P ctrl
ctrl
aflibercept
A
B C
E D
ctrl aflibercept
A431-P
A
431-V
Long-term aflibercept treatment of A431
tumor-bearing mice
Response to aflibercept treatment of in vivo
passaged A431 tumors
Cell proliferation in vivo – Ki-67 staining
Generation of aflibercept-resistant A431 cells re-implant (1x)
A431 variant
(A431-V)
A431
cells
. . . . . .
. . . . . .
A431 parental
(A431-P)
implant
50-100 mm3
A431 tumor
bearing mice
Tumor growth
stasis for 6-7
weeks, then
regrowth
Continuous
tumor growth
re-
implant
re-
implant
14 day
treatment
14 day
treatment
100-150 mm3
tumor-bearing mice
Cell line
isolation
Cell line
isolation
100-150 mm3
tumor-bearing mice
Figure 1: Generation and characterization of aflibercept-resistant A431 variants
Cell apoptosis in vivo – TUNEL staining
ctrl aflibercept
A431-P
A
431-V
A43
1-P h
Fc
A43
1-P a
flib
A43
1-V h
Fc
A43
1-V a
flib
0
5
10
15
20
% T
UN
EL
-po
sitiv
e n
uc
lei ***
NS
A43
1-P h
Fc
A43
1-P a
flib
A43
1-V h
Fc
A43
1-V a
flib
0
500
1000
1500
To
tal n
uc
lei
an
aly
ze
d
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0 5 10 150
200
400
600
800
1000
1200
Tu
mo
r vo
lum
e (m
m3
)
Days post treatment start
A431 P - ctrl
A431 P - aflib
A431 P - cetux
A431 V - ctrl
A431 V - aflib
A431 V - cetux
A
C D
A431
P -
ctrl
A431
P - af
lib
A431
P -
cetu
x
A431
V -
ctrl
A431
V - af
lib
A431
V -
cetu
x 0
200
400
600
800
1000
1200
Tu
mo
r vo
lum
e (m
m3
) A431 P - ctrl
A431 P - aflib
A431 P - cetux
A431 V - ctrl
A431 V - aflib
A431 V - cetux
**** **** ****
**
**, **** vs A431 V - ctrl
**** vs A431 P - ctrl
Cell growth in vitro
Tumor growth after cetuximab or
aflibercept
Final tumor size after cetuximab or
aflibercept
0
1
2
3
4
5
6
7
A431-P A431-V
Ce
lls p
er
we
ll (1
05)
Day 0
Day 3
Figure 2: Additional characterization of aflibercept-resistant A431 variants
B ERK1/2 phosphorylation
ERK1/2
P-ERK1/2
A43
1-P
A43
1-V
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A B
D
p-STAT3 (Tyr705)
A431-P A431-V0
100
200
300
400
500
MF
I
P-Stat3
Stat3
A431-P A431-V
F G
P-Stat3
Stat3
A431-P A431-V
A431-P A431-V
β-actin
SAA
A431-P A431-V
S100A7
FN1
IL36G
Figure 3. Constitutive STAT3 activation in A431-V cells
C Phospho-RTK antibody array STAT3 phosphorylation in
cells - Luminex STAT3 phosphorylation in
cells – Western blot
STAT3 phosphorylation in cells upon
addition of conditioned media – Western blot
Cytokines increased in A431-V cells IL-6/STAT3 dependent protein expression
Stat3
(Tyr705)
A431-P
A431-V
Positive
control
EGFR
(pan-Tyr)
E STAT3 phosphorylation in cells upon
addition of conditioned media - IHC
A431-P cells A431-V cells
A431-P conditioned media
A431-V conditioned media
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p/t
STAT3
1.0 1.3 3.8 5.2 1.0 1.0 2.5 2.6
p-
STAT3
β-actin
hFc
Sarilumab
t-STAT3
A431-V A431-P A431-P A431-V
+ + + +
+ + + +
p-STAT3
β-actin
t-STAT3
A431-P (Fc)
A431-P (sarilumab)
A431-V (Fc)
A431-V (sarilumab)
A431-P(Fc)
A431-P(sarilumab)
A431-V(Fc)
A431-V(sarilumab)
0.0
0.5
1.0
1.5
2.0
2.5*
p-STAT3/t-STAT3(Densitomery)
C
D
E F
A
Effect of sarilumab +/- aflibercept on
proliferation (Ki-67 staining) in A431-V tumors
IL-6 expression - ELISA Effect of sarilumab on cell STAT3
phosphorylation
Effect of sarilumab +/- aflibercept on tumor growth
Effect of sarilumab on tumor STAT3 phosphorylation
Fc
aflibercept
sarilumab
Combo
T
S
S S
S
T
T T
N
Figure 4. Effect of blocking cancer cell IL-6 signaling pathway with sarilumab on the response of A431-
P and A431-V tumors to aflibercept treatment
A431-P - unstained
A431-V - unstained
A431-P - anti-IL6R-APC
A431-V – anti-IL6R-APC
B IL-6R expression in
A431-P and –V cells
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A
Fc Sarilumab
B
C
Figure 5. Blocking the cancer cell IL-6 signaling pathway with sarilumab affects the response of
Du145 tumors to aflibercept treatment
STAT3 phosphorylation in Du145 cells
Fc sarilumab Fc
+ IL6
sarilumab
+ IL6
p-STAT3
STAT3
Phospho-STAT3 staining of Du145 tumors
Effect of sarilumab on Du145 tumor growth
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Figure 6. Overexpression of IL-6 or long-term blockade of the IL-6 pathway affects the response of
A431 tumor xenografts to aflibercept treatment
A
A43
1 IL
-6 c
trl
A43
1 IL
-6 a
fl
A43
1 em
pty v
ecto
r ct
rl
A43
1 em
pty v
ecto
r af
l-100
0
100
200
300
400
Tu
mo
r g
row
th (m
m3)
****ns
Tumor growth response of control and IL-6 overexpressing tumors to aflibercept
B
Days post treatment start
Tu
mo
r V
olu
me
(m
m3)
0 2 4 6 80
100
200
300
400
500
A431 IL-6 ctrl
A431 IL-6 aflA431 empty vector ctrl
A431 empty vector afl
Final tumor size of IL-6 overexpression after aflibercept
0 20 40 600
200
400
600
800
1000
Days post treatment start
Tu
mo
r V
olu
me
(m
m3)
A431 Fc (25 mg/kg)
A431 sarilumab (25 mg/kg)
A431 aflibercept (25 mg/kg)
A431 combo (25 mg/kg/25 mg/kg)
C Long-term treatment in A431 tumor-
bearing mice
0 20 40 600
100
200
300
400
500
Days post treatment start
Tu
mo
r V
olu
me (
mm
3)
0 20 40 600
100
200
300
400
500
Days post treatment start
Tu
mo
r V
olu
me
(m
m3)
D Individual tumor growth curves
aflibercept
combo
*
* *
*
* 5/7 tumors escaped
0/7 tumors escaped
*
#
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Figure 7. Aflibercept treatment is more effective in increasing overall survival in
ovarian cancer patients with low IL-6 levels O
vera
ll su
rviv
ing
frac
tio
n
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Published OnlineFirst February 26, 2016.Cancer Res Alexandra Eichten, Jia Su, Alexander Adler, et al. IL-6/STAT3 signaling and overcome by IL-6 blockadeResistance to anti-VEGF therapy mediated by autocrine
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