resistance to anti-vegf therapy mediated by autocrine il-6...

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

[email protected]

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

on June 8, 2019. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 26, 2016; DOI: 10.1158/0008-5472.CAN-15-1443

http://cancerres.aacrjournals.org/

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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




15

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




16

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,




17

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




18

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




20

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




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




22

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.




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.




24

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28

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.




29

(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.




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.




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.




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.




0 20 40 600

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Generation of aflibercept-resistant A431 cells re-implant (1x)

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Figure 1: Generation and characterization of aflibercept-resistant A431 variants

Cell apoptosis in vivo – TUNEL staining

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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




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




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




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




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


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


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


Tu

mo

r V

olu

me (

mm

3)

0 20 40 600

100

200

300

400

500


Tu

mo

r V

olu

me

(m

m3)

D Individual tumor growth curves

aflibercept

combo

*

* *

*

* 5/7 tumors escaped

0/7 tumors escaped

*

#




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




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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