-1 -0.5 0 0.5 1 Tumor A1 Tumor A2 Tumor A3 Tumor B1 Tumor B2 Tumor B3 Tumor C1 Tumor C2 Tumor C3 Tumor A1 Tumor A2 Tumor A3 Tumor B1 Tumor B2 Tumor B3 Tumor C1 Tumor C2 Tumor C3 Tumor A1 Tumor A2 Tumor A3 Tumor B1 Tumor B2 Tumor B3 Tumor B4 Tumor C1 Tumor C2 Tumor C3 Tumor A1 Tumor A2 Tumor A3 Tumor A4 Tumor B1 Tumor B2 Tumor B3 Tumor C1 Tumor C2 Tumor C3 Tumor D1 Tumor D2 Tumor D3 Tumor E1 Tumor E2 Tumor E3 Tumor F1 Tumor F2 Tumor F3 Tumor B1 Tumor B2 Tumor B3 Tumor B4 Tumor D1 Tumor D2 Tumor D3 Tumor E1 Tumor E2 Tumor E3 Tumor A1 Tumor A2 Tumor A3 Tumor B1 Tumor B2 Tumor B3 Tumor C1 Tumor C2 Tumor C3 Tumor E1 Tumor E2 Tumor E3 SNU5 + pazopanib SNU5 + vehicle MKN45 + Pazopanib Tivantinib MKN45 + pazopanib MKN45 + Tivantinib MKN45 + vehicle Vimentin:E-Cadherin (Log) Quantitative immunofluorescence assessment of MET and epithelial-to-mesenchymal transition (EMT) biomarker modulation by antiangiogenic inhibitors in xenograft tumor tissues Tony Navas 1 , Scott M. Lawrence 1 , Donna Butcher 2 , Lindsay M. Dutko 2 , Melinda G. Hollingshead 3 , Robert J. Kinders 1 , Ralph E. Parchment 1 , Donald P. Bottaro 4 , W. Marston Linehan 4 , Joseph E. Tomaszewski 5 , Apurva K. Srivastava 1 , and James H. Doroshow 5 1 Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702; 2 Pathology/Histotechnology Laboratory, Laboratory Animal Sciences Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702; 3 Biological Testing Branch, Developmental Therapeutics Program, National Cancer Institute, Frederick MD 21702; 4 Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda MD 20810; 5 Division of Cancer Treatment and Diagnosis and Center for Cancer Research, National Cancer Institute, Bethesda MD 20810 MET IFA • We have developed a specific and sensitive multiplex pY1235-MET and total c-Met IFA on FFPE tissues. • We have demonstrated assay “fit for purpose” application of the multiplex MET IFA using SNU5 xenograft tissues treated with crizotinib as well as with pazopanib, tivantinib, or the drug combination. • We have shown by IFA that tivantinib had no activity on its own in inhibiting pY1235-MET in SNU5 xenografts, that pazopanib induced pY1235-MET compared to vehicle; and that the drug combination inhibited pY1235-MET compared to pazopanib. • We have used Definiens analysis to enumerate % pY1235-MET/total Met levels in tissue sections and have shown good concordance with ELISA data taken from correlated tumor samples from the same sets of experiments. EMT IFA • EMT IFA analysis of gastric tumors showed that vehicle-treated MKN45 has a relatively epithelial phenotype, while vehicle-treated SNU5 showed a mesenchymal phenotype. • Pazopanib-treated MKN45 (4/6 tumors) appears to show varying levels of epithelial-to-mesenchymal transition compared to vehicle. • Pazopanib/tivantinib combination–treated groups (3/3 evaluable tumors) showed a generally more epithelial phenotype, while 2/5 samples had no tumor content by H&E. 1. Sennino and Mcdonald (Oct 2012) Controlling escape from angioganesis inhibitors. Nature Reviews Cancer 12, 699-709 2. Srivastava et. al. (ASCO 2011) Development and validation of biomarker assays to assess pharmacodynamic modulation of MET 3. Srivastava et al. (AACR 2012) Preclinical assessment of MET modulation by a VEGFR inhibitor/MET inhibitor combination that shows additive antitumor efficacy 4. Srivastava et al. (2014) Development and Validation of MET Assays to Assess Pharmacodynamic Response in Tumor Tissues (submitted) Introduction Abstract # 1049 Summary and Conclusions Results GAPDH Methods Reagents. Mouse monoclonal antibody against total MET (clone Met4) was from Van Andel Research Institute Technologies, Inc. Two rabbit monoclonal antibodies specific to pY1235-MET (clone 23111; with undetectable reactivity to pY1234-MET) and pY1356-MET were developed at Epitomics Inc., by using peptide antigens corresponding to the MET sequence surrounding these amino acids . E-Cadherin-AF488 mouse monoclonal antibody (clone 32) was from BD Biosciences, and Vimentin-AF647 (clone V9) was from Santa Cruz Biotechnology. Animal models and drug administration. Athymic nude mice (NCr; Animal Production Program, FNLCR) were implanted with the human cancer cell lines A549 (lung carcinoma), MDA-MB-231 (breast carcinoma), HT-29 (colon carcinoma), GTL-16 (gastric carcinoma; MET amplified), MKN 45 (gastric carcinoma; MET amplified), and SNU5 (gastric carcinoma; MET amplified) by subcutaneous injection. The NCI Animal Production Program, FNLCR, is accredited by Association for Assessment and Accreditation of Laboratory Animal Care International and follows the USPHS Policy for the Care and Use of Laboratory Animals. All the studies were conducted according to an approved animal care and use committee protocol. MET inhibitors. PF 02341066 (NSC D749769-Y, crizotinib), tivantinib (NSC 758242), and VEGFR inhibitor pazopanib (NSC 737754) were synthesized at the Developmental Therapeutics Program (DTP), NCI. PF02341066 and pazopanib were administered by oral gavage in a saline vehicle, and PHA 665752 by intraperitoneal (IP) injections in a vehicle composed of 10% DMSO in saline. Tivantinib was administered orally in a PEG 400:20% vitamin E TPGS solution (60:40) vehicle. Xenograft biopsy and tumor quarter collection. Whole xenograft tumors were collected on the same schedule as tumor biopsies by standard dissection methods. Specimens were cut into two to four equal pieces with fine-point scissors and placed into Sarstedt tubes that were pre-cooled in liquid nitrogen and was immediately flash frozen in an o-ring-sealed, conical bottom screw-capped 1.5-mL Sarstedt cryovial by touching the biopsy to the inside of the tube within one minute of excision. Tubes were pre-cooled on liquid nitrogen. MET immunofluorescence analysis. HT29, GTL16, and A549 cancer cell lines (FNLCR Repository) were treated with DMSO (0.1% w/v), 100 nM crizotinib, or 100 nM sorafenib for 24 h at 37°C. Frozen tissues or cells were fixed in 10% neutral buffered formalin for 24 h (Sigma Aldrich), and then embedded in paraffin. Paraffin-embedded tissue was cut into 5 μm sections and placed on slides, and staining was performed in a Bond- max Autostainer (Leica Microsystems). Antigen retrieval was performed using Bond Epitope Retrieval Solution 2 (Leica) at 100°C for 10 min. For antigen detection, 10 g/mL rabbit monoclonal anti-pY1235–MET (clone 23111) was used as the primary antibody, followed by 10 μg/mL goat anti- rabbit AF488 (Life Technologies) as the secondary antibody. Multiplex immunofluorescence was performed on formalin-fixed paraffin-embedded tumor tissues; 10 g/mL rabbit monoclonal anti-pY1235–MET (clone 23111) and 5 g/mL mouse monoclonal anti-intact MET (clone MET4, Van Andel Research Institute) were used as primary antibodies, followed by 10 μg/mL goat anti-rabbit AF488 and 10 μg/mL anti-mouse AF660 (Life Technologies) secondary antibodies. Image acquisition and analysis were performed on a wide-field fluorescent and confocal microscope (Nikon 90i Andor Camera, NIS Elements Software). This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. This research was supported [in part] by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. Frederick National Laboratory for Cancer Research is accredited by AAALAC International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the "Guide for Care and Use of Laboratory Animals" (National Research Council; 1996; National Academy Press; Washington, D.C.). Demonstration of pY1235-MET antibody specificity by Western Analysis Anti-pY1235-MET (clone 23111) 145 kD, MET ~145 kD 145 kD, MET 145 kD, MET 170 kD, Pro-MET 145 kD, MET 0 5 25 crizotinib (nm) 23111 + pY1235 peptide 23111 + pY1234 peptide 23111 + rRON 23111 Figure 2. The rabbit monoclonal 23111 was highly specific for pY1235-MET, with undetectable cross-reactivity to pY1234-MET and to cytoplasmic RON. Specificity of pY1235-MET antibody clone 23111 demonstrated by Western blot staining of lysates from GTL-16 cells treated with increasing amounts of the MET inhibitor crizotinib (top panel). Similar blots pre-incubated with pY1235-MET peptide (2 nd panel), pY1234-MET peptide (3 rd panel), and recombinant cytoplasmic RON (4 th panel). The bottom panel shows total intact MET staining with C-terminal antibody; similar total intact MET staining was observed with all blots (Ref 4). Demonstration of pY1235-MET IFA staining specificity using specific MET and non-specific TK inhibitors on FFPE tissues by confocal microscopy: GTL16 HT29 A549 pY1235-MET GTL1 6 HT29 A549 Total MET DMS O 100 nM crizotinib (PF02341066) 100 nM sorafenib Figure 3. Specificity of the pY1235-MET monoclonal antibody is demonstrated in formalin-fixed, paraffin-embedded GTL-16 (MET-amplified), A549, and HT-29 (paracrine or autocrine) cancer cells treated in vitro with 100 nM crizotinib (specific MET-kinase inhibitor) or 100 nM sorafenib (nonspecific TK inhibitor) for 4 h. Total MET staining of the same tissue sections using Met4 Mab shows differences in expression levels among the various cell lines, but no changes in expression were observed with drug treatment. Staining was performed in a Bond-max Autostainer (Leica Microsystems), and slides were imaged on a confocal microscope (Nikon 90i Andor Camera, NIS Elements Software). HGF specifically induces pY1235-MET IFA staining, which was completely inhibited by crizotinib in MET-expressing cell lines in vitro A549 + HGF + 100 nM crizotinib A549 + HGF 15 min A549 10 2ndary only pY1235-MET (ug/ml) 2.5 1.0 HT29 HT29 + HGF 24h HT29 + HGF + 100 nM crizotinib DAPI pY1235-MET Merge Figure 4. Immunofluorescence staining of pY1235-MET on A549 and HT29 cell lines was specifically induced by HGF stimulation but completely blocked by treatment with crizotinib in vitro. A549 or HT29 cells were grown on chamber slides to 80% confluency before being serum starved for 24 h. Cells were incubated with or without 100 nM crizotinib for 4 h prior to stimulation with 20 ng/ml HGF for 15 min (A549) or for 24 h (HT29) in serum-free media. Cells were fixed in 10% NBF for 15 min, permeabelized in 0.3% Triton X-100, and stained with increasing concentrations of pY1235-MET antibody (A549), followed by 10 ug/ml anti-rabbit AF546 secondary. Slides were imaged with a Nikon 90i Andor Camera, and analyzed using NIS Elements Software. Multiplex IFA staining and Definiens quantitation analysis of pY1235-MET and total MET on FFPE tissues GTL16 HT29 MDA-MB-231 SNU1 pY1235- MET AF488 Total MET AF660 Merge GTL16 HT29 SNU1 Marker Masks Nuclear Masks pY1235-MET (Green) total Met (Red) Marker Masks DAPI (Blue) Nuclear Mask and Segmentation MDA-MB-231 0 200 400 600 800 1000 1200 GTL16 HT29 MDA-MB-231 SNU1 Marker Area (um 2 )/ 100 cells pY1235-MET total MET 0 10 20 30 40 50 60 70 GTL16 HT29 MDA-MB-231 SNU1 % pY1235-MET/total MET (Marker Area / Cell) % pY1235-MET/total Met Figure 5. Quantitative assessment of pY1235‐MET was determined in xenograft tissue models that demonstrate very high IFA expression of pY1235‐MET and total MET via gene amplification (GTL‐16), lower expression in HGF-induced paracrine or autocrine cell lines (A549 and HT29), and no detectable staining for pY1235‐MET in negative controls (SNU‐1 and MDA‐MB‐231). pY1235-MET and total MET IFA staining of FFPE renal cell carcinoma tissue Merge pY1235-MET DAPI total MET 40x Renal Patient (20x) H1112R c-Met germline mutation Figure 6. MET multiplex immunofluorescence staining of excised formalin-fixed, paraffin- embedded HPRCC tumor tissue from a patient harboring an H1112R c-Met germline mutation (left panel). Merged and individual fluorescent images are shown on the right, with total MET (pink; primarily membrane and cytoplasmic), pY1235-MET (green; primarily nuclear), and DAPI (blue; nuclear). Magnification, 40x. Inhibition of VEGF signaling by pazopanib reduces tumor growth in some preclinical models. However, angiogenic inhibition is often accompanied by enhanced hypoxic conditions in tumors that lead to increased c-Met expression and activity as well as to an epithelial-to-mesenchymal transition (EMT)-like phenotype, which leads ultimately to tumor invasion and metastasis (Fig.1A, Ref 1). Co-inhibition of c-Met can block these effects, providing a potential mechanism to overcome increased invasion in the face of antiangiogenic therapy. We have previously reported the development of ELISA assays for intact MET, pY1235-MET, and pY1356-MET in tumor lysates (Fig. 1B) (Ref. 2) and measured drug-related biomarker changes induced by the anti-VEGF and anti- MET combination therapy in gastric xenograft models (Ref 3). Here, we report the development of a multiplex quantitative immunofluorescence assay (qIFA) for simultaneous detection of pY1235-MET and total MET in formalin-fixed paraffin-embedded (FFPE) tissues to determine whether pY1235-MET or total MET is induced by anti-VEGF inhibitors due to increased EMT transition and whether this effect could be reversed by the drug combination with a MET inhibitor, tivantinib (ARQ197) in FFPE tissue samples. Preclinical study designs of MET inhibitor and VEGFR/MET inhibitor combination studies A. SNU5 xenografts treated with crizotinib B. SNU5 and MKN45 xenografts treated with pazopanib, tivantinib (ARQ197) or the drug combination C. PD sample collection diagram Doses 4 hr Post-dose 4 Biopsy collection Day4 Predose 1………….4 Group # Mice Drug Dose Schedule Route 1 8 water 0.1 ml/10 grams BW QDx4 PO 2 8 crizotinib 25 mg/kg QDx4 PO 3 8 crizotinib 12.5 mg/kg QDx4 PO 4 8 untreated tumors untreated ELISA IFA Tissue microarray multiplex staining of pY1235-MET and total MET on FFPE tissues from SNU5 xenografts treated with crizotinib in vivo • pY1235-MET (Green) • total MET (Pink) • DAPI (Blue) Definiens quantitation analysis of pY1235-MET and total MET on FFPE SNU5 xenograft tissues Vehicle (Water) 12.5 mg/kg crizotinib 25 mg/kg crizotinib 17 18 19 20 21 22 23 24 25 26 27 28 pY1235-MET (Green) total Met (Red) Marker Masks ***P<0.0001 Regression analysis showing concordance between % pY1235-MET/total MET IFA and ELISA data for SNU5 xenograft tissues treated with crizotinib in vivo MET multiplex IFA vs ELISA analysis of %pY1235-MET/total MET in FFPE tissues from SNU5 xenografts treated with VEGF inhibitor (pazopanib), MET inhibitor (tivantinib; ARQ197) or the drug combination ELISA IFA Definiens analysis of EMT IFA multiplex-stained SNU5 and MKN45 FFPE tissues treated with VEGF inhibitor (pazopanib), MET inhibitor (tivantinib) or the drug combination in vivo EMT multiplex IFA staining and Definiens analysis of control FFPE xenograft tissues b-Catenin- AF546 Vimentin AF647 E-Cadherin- AF488 DAPI Direct-Conjugate Mab Source/Vendor Mab Clone Direct- Conjugate Total mg Total Vol ml Direct Conjugate Mab Conc. (mg/ml) Moles Alexa-Fluor per mole protein Catalog # Lot # E-Cadherin-AF488 BD Bioscience 36 Mouse Mab 0.2 mg 4.0 ml 0.05 mg/ml 4.0 560061 39472 Betacatenin-AF546 Epitomics Custom Conjugation E247 Rabbit Mab 3.69 mg 3.0 ml 1.23 mg/ml 7.0 C47215 96780A Lot #2 Vimentin-AF647 Santa Cruz Biotechnology V9 Mouse Mab 5.0 mg 5 ml 1 mg/ml 3.0 sc-6260 AF647 KO813 EMT Multiplex IFA Assay Panel Figure 8. Tissue microarray cores derived from SNU5 xenografts treated with vehicle (water), 12.5 mg/kg crizotinib, or 25 mg/kg crizotinib (QDx4, PO) 4 h after the last dose. Three TMA cores per tumor were collected from 4 different animals per treatment group. Tumors were stained with pY1235-MET (AF488) and total MET (AF647), and scanned by Aperio. Figure 7. Preclinical study designs of (A) MET inhibitor (crizotinib) or (B) VEGFR (pazopanib)/MET inhibitor (tivantinib) single or combination studies in SNU5 and MKN45 gastric xenograft models for MET and EMT IFA assay “fit for purpose” biomarker studies. (C) PD sample collection diagram of tumor samples from preclinical studies for MET and EMT IFA and ELISA assays. Figure 11. Scatter graphs (left) showing a comparison of % pY1235-MET/total MET IFA and ELISA values from 5-6 tumor quadrants from each treatment group of SNU5 xenografts treated with vehicle, pazopanib, tivantinib or the drug combination (QD or BID) based on the preclinical study plan shown in Figure 7B. Each point in the IFA graph represents the mean % pY1235- MET/total MET of 12 regions of interest (ROIs). Each ROI , containing 2–3,000 non-necrotic tumor cells, was randomly selected per tumor sample. Regression analysis (above) shows correlation between IFA values expressed as the ratio of marker area/# of nuclei vs. ELISA values expressed as pM/ug total protein, from each of the treatment groups Vehicle (Water) 25 mg/kg crizotinib 17-1 17-2 17-3 18-1 18-2 18-3 19-1 19-2 19-3 20-1 20-2 20-3 12.5 mg/kg crizotinib 25-1 25-2 25-3 26-1 26-2 26-3 27-1 27-2 27-3 28-1 28-2 28-3 21-1 21-2 21-3 26-1 26-2 26-3 27-1 27-2 27-3 28-1 28-2 28-3 0 500 1000 1500 2000 2500 3000 3500 MB231 SNU5 SNU1 GTL16 HT29 Relative Marker Area per ROI per 1000 cells E-Cadherin Vimentin -4 -2 0 2 4 MB231 SNU5 SNU1 GTL16 HT29 Log10 V:E Ratio Mesenchymal (Red) = Log (V:E) >0 Epithelial (Green) = Log (V:E) < 0 Vimentin and E-Cadherin marker area per ROI of 1000 tumor cells or nuclei (LLQ < or 1 = 1 ) Vimentin E-Cadherin MDA-MB-231 SNU5 SNU1 GTL16 HT29 MDA-MB-231 SNU5 SNU1 GTL16 HT29 B. A. Epithelial Mesenchymal Figure 13. EMT multiplex IFA staining and Definiens analysis of FFPE xenograft control tissues. (A) E-Cadherin (green) and Vimentin (red) IFA levels expressed as mean marker area per 1,000 cells for 3 ROIs examined per tissue. (B) Ratio of Vimentin and E-Cadherin (log scale). Mesenchymal phenotype (red) is shown as Log (V:E) > 0 and Epithelial phenotype (green) is expressed as Log (V:E) < 0. Left set of images shows RGB image files collected by Aperio, while Definiens-derived marker masked images of E-Cadherin (green) and Vimentin (red) are shown on the right. Figure 14. EMT multiplex IFA staining and Definiens analysis of FFPE tissues from SNU5 and MKN45 xenografts treated with either vehicle, pazopanib, or the combination with tivantinib (QDx8) based on the preclinical study plan shown in Fig 7B. Representative images from different ROIs taken from each tumor in various treatment groups are shown in (A), while the Vimentin:E-Cadherin ratios (log scale) for 3 representative tumor ROIs per tumor selected from each treatment group are shown in (B). Based on the V:E log ratio (range from only -1 to 1 is shown ), vehicle-treated MKN45 shows a relatively epithelial phenotype, while vehicle-treated SNU5 shows a mesenchymal phenotype. These tissue controls were used to set the threshold for each biomarker channel that was applied to all tissue ROIs in each treatment group. ROIs for analysis were selected based on the H&E staining of non-necrotic tumor tissue and human mitochondrial- positive staining in adjacent tumor tissue sections. A total of 4/6 tumors in the pazopanib-treated MKN45 group appear to show varying levels of epithelial-to- mesenchymal transition compared to vehicle or the combination group. Pazopanib/tivantinib combination–treated groups (3/3 evaluable tumors) showed a generally epithelial phenotype, while 2/5 samples had no tumor content by H&E. SNU5 + pazopanib MKN45 + pazopanib SNU5 + vehicle MKN45 + vehicle MKN45 + pazopanib tivantinib QDx8 A1 A2 A3 A1 A2 A3 C1 C2 C3 A1 A2 A3 A1 A2 A3 D1 E1 F1 E2 D2 A1 C1 B1 C1 A3 References Clone # AF276 Figure 1B IFA vs. ELISA % pY1234-35 MET/total MET ELISA (Ratio of pM / ug protein) Left A Tumor Quadrant % Phos/Total MET IFA Ratio of Marker Area / # Nuclei Right H Tumor Quadrant IFA vs. ELISA % pY1235-MET/Total MET IFA Ratio of Marker Area / # Nuclei Right H Tumor Quadrant % pY1235-MET/total MET ELISA (Ratio of pM/ug protein) Left A Tumor Quadrant Figure 9. Definiens quantitation analysis showing marker masks for pY1235-MET (green) and total MET (red) (left panel) in representative tissue microarray cores derived from SNU5 xenografts treated with vehicle or crizotinib. Scatter graphs (right panel) show pY1235-MET (green) and total-MET (red) IFA levels expressed as mean marker area/# of nuclei for 3 representative cores per tumor, with 4 tumors examined per treatment group. Figure 10. Scatter graphs show a comparison between IFA vs. ELISA values for % pY1235-MET/total MET measured from different tumor quadrants derived from SNU5 xenografts treated with vehicle or crizotinib. Regression analysis shows correlation between the IFA values expressed as the ratio of marker area/# of nuclei vs. ELISA values expressed as pM/ug total protein. Figure 12. EMT multiplex IFA assay developed using directly conjugated monoclonal antibodies to E-Cadherin, b-catenin, and Vimentin. Nature Reviews Cancer 12, 699-709 (October 2012) Figure 1A Epithelial Mesenchymal A. B.