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. Author manuscript; available in PMC: 2016 May 1.
Published in final edited form as: J Nucl Med. 2015 Apr 3;56(5):758–763. doi: 10.2967/jnumed.115.154690

PET of c-Met in cancer with 64Cu-labeled Hepatocyte Growth Factor

Haiming Luo 1, Hao Hong 1, Michael R Slater 2, Stephen A Graves 3, Sixiang Shi 4, Yunan Yang 1, Robert J Nickles 4, Frank Fan 2, Weibo Cai 1,3,4,5,*
PMCID: PMC4417426  NIHMSID: NIHMS678338  PMID: 25840981

Abstract

The hepatocyte growth factor (HGF) and its receptor, c-Met, are actively involved in tumor progression/metastasis and associated closely with poor prognostic outcome of cancer patients. Thus developing positron emission tomography (PET) agents for assessing c-Met expression would be extremely useful for diagnosis of cancer and subsequent monitoring of responses to c-Met-targeted therapies. Here we report the characterization of recombinant human hepatocyte growth factor (rh-HGF) as a PET tracer for detection of c-Met expression in vivo.

Methods

rh-HGF was expressed in human embryonic kidney (HEK) 293 cells and purified by nickel-nitrilogriacetic acid (Ni-NTA) affinity chromatography. The concentrated rh-HGF was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and labeled with 64Cu. c-Met binding evaluation by flow cytometry was performed in both U87MG and MDA-MB-231 cell lines, which have high and low level of c-Met, respectively. PET imaging and biodistribution studies were performed in nude mice bearing U87MG and MDA-MB-231 xenografted tumors.

Results

The rh-HGF expression yield was 150–200 µg protein per 5 × 106 cells after 48 h transfection with purity of 85% ~ 90%. Flow cytometry examination confirmed strong and specific binding capacity of rh-HGF to c-Met. After labeled with 64Cu, PET imaging revealed specific and prominent uptake of 64Cu-NOTA-rh-HGF in c-Met positive U87MG tumors (6.7 ± 1.8 %ID/g at 9 h post-injection) and significantly lower uptake in c-Met negative MDA-MB-231 tumors (1.8 ± 0.6 %ID/g at 9 h post-injection). The fact that sonicated-denatured rh-HGF (termed as dnrh-HGF) had significantly lower uptake in U87MG tumors, along with histology analysis, confirmed the c-Met specificity of 64Cu-NOTA-rh-HGF.

Conclusion

The study provided the initial evidence to confirm that 64Cu-NOTA-rh-HGF is applicable for visualizing c-Met expression in vivo, which may also find potential applications in treatment monitoring of c-Met-targeted cancer therapy.

Keywords: c-Met, hepatocyte growth factor (HGF), Positron emission tomography (PET), 64Cu, cancer

INTRODUCTION

c-Met is a tyrosine kinase receptor for hepatocyte growth factor (HGF), overexpressed or aberrantly activated in a variety of cancers, including renal, ovarian, lung, colorectal, breast, gastric, cervical, stomach, pancreatic, esophageal, and melanoma (1, 2). The overexpression of c-Met from gene amplification or mutation in cancer results in aberrant activation of the c-Met axis, leading to proliferation, apoptosis inhibition, angiogenesis and invasion of cancer cells (3). c-Met overexpression is also associated with poor clinical prognosis, elevated metastasis incurrence, and increased drug resistance of cancer (4, 5). Together, these characteristics suggest that c-Met is an active participant in cancer initiation and progression, thus the assessment of c-Met expression in real time could potentially aid in the diagnosis and monitoring of responses to relevant therapies.

Numerous studies also suggest that c-Met is an attractive drug target in cancer therapy because blocking of c-Met can usually result in the inhibition of tumor growth and metastasis (2, 6). Multiple clinical trials have adopted tyrosine kinase inhibitors and monoclonal antibodies (mAbs) against c-Met pathways as cancer therapeutic agents (7–9). Diagnostic methods that can help to identify the suitable patient population for c-Met targeted therapy are fundamental to produce improved clinic outcome. The current patient selection standard is usually based on immunohistochemistry or fluorescent in situ hybridization (FISH). Despite the fact that these methods can provide quantitative information about c-Met abundance, they are not applicable in two scenarios: reflecting c-Met expression fluctuation over time, and dealing with c-Met heterogeneity in different tumor sites. Positron emission tomography (PET) imaging can overcome these limitations due to its high sensitivity to detect molecular events in a real time manner. Several radiolabeled antibodies against c-Met were already used for in vivo tumor targeting. One candidate named PRS-110, an anticalin with monovalent specificity for c-Met, was labeled with 89Zr (t1/2 = 78.4 h) for imaging U87MG glioblastoma. Moderate tumor accumulation (4 percentage injected dose per gram [%ID/g] for U87MG) was obtained in this study (10). Other choices such as DN30 and onartuzumab were also labeled with 89Zr for imaging c-Met expression in vivo with good tumor uptake (11, 12). However, the prolonged circulation time and slow clearance can lead to low tumor-to-background ratio and limit their clinical application as diagnostic agents.

HGF, the only known ligand for c-Met, is usually secreted as an inactive polypeptide and cleaved by serine proteases into the bioactive format composed of a 69-kDa alpha chain and 34-kDa beta-chain, linked by a disulfide bond (9). There have been several earlier attempts to express biologically active recombinant human HGF (rh-HGF) in both Escherichia coli and insect cells (13, 14). Although biological activity of rh-HGF produced from E.coli has been reported to be equivalent to its native form (15), the absence of the disulfide bond formation and lack of molecular glycosylation can potentially compromise its applicability in vivo. Furthermore, production of rh-HGF in E.coli inclusion bodies involves a refolding process (15), which is time-consuming and usually causes low protein production yield. Due to the similar reasons, insect cells are not ideal hosts for expressing human glycoproteins due to different glycosylation levels (16). To attain rh-HGF with an equivalent structure to their native form, expression in mammalian cells including COS-1, rat hepatocytes and Chinese hamster ovary (CHO) cells is preferred (17–19).

In this study, we use mammalian HEK293 cells to express 10His tagged rh-HGF. After attaining rh-HGF with high purity, we used PET imaging to investigate the in vivo distribution pattern and c-Met targeting efficacy of 64Cu-labeled rh-HGF (named 64Cu-NOTA-rh-HGF). Two human cancer cell lines were selected, i.e. U87MG glioblastoma with high c-Met expression and MDA-MB-231 breast cancer cells with low c-Met expression. Region-of-interest (ROI) analysis of PET images was also carried out for uptake quantification of 64Cu-NOTA-rh-HGF in major tissues/organs. Histology evaluation was also provided to confirm the uptake of 64Cu-NOTA-rh-HGF in tumors is relevant to c-Met expression.

MATERIALS AND METHODS

Plasmids and Cell lines

The plasmid pCMV-hHGF-10his (Sino Biological Inc., Beijing, China) was used in the cloning and expression procedure. Large-scale plasmid DNA was extracted with an EZNA Plasmid Mini Kit II (Omega, Goraville, GA). The E.coli DH5α (Invitrogen, Carlsbad, CA) was used as a host for cloning of pCMV-hHGF-10his.

U87MG human glioblastoma and MDA-MB-231 human breast cancer lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured according to the supplier’s instructions. All animal studies were conducted under a protocol approved by the University of Wisconsin Institutional Animal Care and Use Committee. U87MG and MDA-MB-231 tumor models were prepared using a similar method as previously described (20).

Expression and purification of rh-HGF

HEK293 cells (5 × 106) were seeded onto 150 cm2 cell culture flasks and transfection was carried out with 46.9 µg of pCMV-hHGF-10his DNA using the FuGENE HD (Promega, Madison, WI) when cell density reached 70% according to the manufacturer’s protocol. After 2 days, the cells were collected via Cellstripper (Corning, Manassas, VA) and lysed by 1 × mammalian lysis buffer (Promega, Madison, WI). Repeated “freezing and thawing” steps were carried out in the cell suspension for releasing the recombinant proteins.

The rh-HGF encoded by pCMV carries 10 histidine residues at its C-terminus. The Ni-NTA was balanced by binding buffer before the supernatant of cell lysis containing rh-HGF was added to the column, and the proteins associated with the resin were eluted by increasing concentrations of imidazole in the buffer. The details of purification procedures were performed using a method previously described (21). The purity of rh-HGF was evaluated with 8% SDS-PAGE gel under non-reducing conditions stained with Coomassie brilliant blue R-250.

rh-HGF conjugation and Radiolabeling

NOTA-rh-HGF was prepared using a similar method as previously described (20). Briefly, NOTA conjugation was carried out at pH 9.0 with the reaction ratio of p-SCN-Bn-NOTA:rh-HGF being 10:1at room temperature for 1 h. PD-10 columns were used to purify NOTA-rh-HGF with PBS as the mobile phase. Similar reaction conditions were adopted for the conjugation of rh-HGF with NHS-Fluorescein (Thermo Scientific, Pierce, USA). 64Cu labeling and purification followed the routine protocol described by our group previously (20). 64CuCl2 (37 MBq) was diluted in 300 µL of 0.1M sodium acetate buffer (pH 6.5) and added to 20 µg of NOTA-rh-HGF.

Flow cytometry

The biological activity of purified rh-HGF was evaluated by flow cytometry. Briefly, U87MG and MDA-MB-231 cells were incubated with 20 µg/mL rh-HGF for 1 h. Subsequently, the cells was incubated with mouse anti-human HGF antibody (Thermo Scientific, Hudson, NH) for 4 h and FITC-labeled goat anti-mouse secondary antibody for 1 h. To evaluate the c-Met expression levels, U87MG and MDA-MB-231 cells was fixed by cold 4% paraformaldehyde before the two-step incubation with rat anti-human c-Met antibody (eBioscience, San Diego, CA) at 4 °C for overnight and Cy3 conjugated donkey anti-rat secondary antibody for 1 h.

The c-Met binding specificity of rh-HGF to U87MG and MDA-MB-231 cells was further confirmed by incubation of cells with 50 nM FITC-rh-HGF or FITC-NOTA-rh-HGF for 30 min. Sonicated-denatured FITC-dnrh-HGF was used as a control. All cells were washed and analyzed with a FACSCalibur 4-color analysis cytometer (Becton-Dickinson) with FlowJo analysis software (Three Star, Inc.)

PET imaging and Biodistribution studies

PET scans, image reconstruction, and ROI analysis of each PET scan were performed using an Inveon microPET/microCT rodent model scanner (Siemens Medical Solutions USA, Inc.) as described previously (20). Each tumor-bearing mouse was intravenously injected with 5–10 MBq of 64Cu-NOTA-rh-HGF and 5- to 10-min static PET scans were performed at various time points post-injection (p.i.). The tracer uptake was calculated as percentage injected dose per gram of tissue (%ID/g) (mean ± SD; ≥3 mice per group).

Denature studies were carried out to evaluate c-Met specificity of 64Cu-NOTA-rh-HGF in vivo, where a group of 3 mice were each injected with 5–10 MBq of sonicated-denatured tracer, 64Cu-NOTA-dnrh-HGF. Biodistribution studies were performed after the last PET scans at 24 h p.i. to validate the PET data. The tumors, liver and muscle were also frozen and cryosectioned for histologic analysis. Quantitative data were expressed as mean ± SD. Means were compared using the Student t test. P values of less than 0.05 were considered statistically significant.

Histology

After blocking with 10% donkey serum, frozen tissue slices of 5 µm thickness were incubated with rh-HGF (20 µg/mL) for 1 h at 4 °C. After washing with PBS, the slices were incubated with mouse anti-human HGF MAb for 4 h, followed by FITC-labeled goat anti-mouse IgG for 1 h. The tissue slices were also stained with rat anti-human c-Met antibody and Cy3-labeled donkey anti-rat IgG. All images were acquired with a Nikon Ti Eclipse confocal microscope (Nikon, Tokyo, Japan).

RESULTS

Expression and purification of rh-HGF

To attain high level of rh-HGF, the human cytomegalovirus (CMV) promoter was chosen due to its potent expression efficiency for downstream gene. As shown in Figure 1A, under CMV driven constitutive promoter, human HGF gene can be expressed in mammalian cells as a tagged protein with a C-terminal 10His tag, which can be used for further product purification. Transient transfections were performed using a mammalian cell line HEK293 using the procedures described above. After 2 days of incubation with Fugene HD and recombinant plasmid, protein production yield was initially tested by SDS-PAGE. After the confirmation of high-yield protein production in the cells, large-scale purification was carried out. After cell lysis, Ni-NTA resin was used to capture the 10His tagged rh-HGF. The schematic workflow for rh-HGF protein expression and purification is illustrated in Figure 1B. There was one clear band at 90 kDa on SDS-PAGE under non-reducing conditions, which represented the bioactive rh-HGF, and its purity was 85% ~ 90%. Two weak bands at the molecular weight of 60 and 34 kDa were also shown on SDS-PAGE, corresponding to α chain and β chain of HGF respectively (Figure 1C).

Figure 1.

Figure 1

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Expression and purification of rh-HGF. (A) Schematic representation of the pCMV-human HGF construct. The functional elements of the expression vector are the following. pCMV: Human cytomegalovirus immediate early I promoter/enhancer; T7 primer: T7 promoter priming site; Kozak: a Kozak translation-initiation sequence and an initiation codon (ATG) for proper initiation of translation; human HGF-10his tag: human HGF protein fused to 10his tag; (B) Schematic representation of steps involved in the expression of secreted proteins from stable mammalian cell lines; (C) SDS-PAGE (8%) analysis of purified rh-HGF transiently expressed in HEK293 cells. Molecular weight markers are shown on the left.

Characterization of rh-HGF

FITC conjugated anti-human HGF antibody was used to visualize rh-HGF binding to U87MG and MDA-MB-231 cells. The vast majority of U87MG cells showed high levels of cell surface-bound rh-HGF based on enhanced cellular fluorescence (Figure 2A). To confirm the cellular expression level of c-Met, Cy3 labeled anti-c-Met antibody was used in U87MG and MDA-MB-231 cells. The flow cytometry results showed that c-Met expression level of U87MG was significantly higher than that of MDA-MB-231 (Figure 2B). Together, these results indicated that purified rh-HGF protein had high binding affinity to cellular c-Met. The binding level of rh-HGF to U87MG and MDA-MB-231 cells was in accordance with their c-Met expression levels.

Figure 2.

Figure 2

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Evaluation of c-Met binding capability of rh-HGF by flow cytometry. (A) Assessment of rh-HGF binding to c-Met positive U87MG and c-Met negative MDA-MB-231 cells. rh-HGF was incubated with the cells first, followed by mouse anti-human HGF antibody and FITC-conjugated goat anti-mouse IgG for flow cytometry analysis; (B) Assessment of c-Met expression level in U87MG and MDA-MB-231 cells by flow cytometry with rat anti-human c-Met antibody and Cy3-labeled donkey anti-rat IgG; (C) Flow cytometry analysis in U87MG and MDA-MB-231 cells confirmed the specificity of rh-HGF and NOTA-rh-HGF for c-Met. dnrh-HGF does not bind to c-Met.

To further confirm cellular binding specificity of rh-HGF, c-Met positive U87MG cells were incubated with 50 nM of FITC conjugated rh-HGF, NOTA-rh-HGF, or sonicated-denatured dnrh-HGF. Figure 2C showed that there were no observable differences in cellular uptake between FITC-rh-HGF and FITC-NOTA-rh-HGF, while the uptake of FITC-rh-HGF was significantly higher than that of FITC-dnrh-HGF. Combined with the fact that uptake of FITC-rh-HGF was extremely low in MDA-MB-231, these results confirmed that NOTA conjugation did not compromise the binding affinity or specificity of rh-HGF for c-Met.

PET and Biodistribution Studies

Time points of 0.5, 3, 9, 15 and 24 h p.i. were chosen for serial PET scans after intravenous injection of 64Cu-NOTA-rh-HGF into tumor-bearing mice. The coronal slices that contained the tumors are shown in Figure 3 and the quantitative data obtained from region-of-interest analysis of PET results are shown in Figure 4. In the U87MG tumor, which expresses high level of c-Met, 64Cu-NOTA-rh-HGF had a rapid accumulation and tumor uptake was clearly visible at 0.5 h p.i., peaked at 9 h p.i., and remained stable over time (5.5 ± 2.0, 6.7 ± 1.5, 6.8 ± 1.8, 5.9 ± 1.4 and 5.4 ± 0.9 %ID/g at 0.5, 3, 9, 15 and 24 h p.i. respectively; n = 4; Figure 4A). The liver uptake (36.0 ± 0.3, 32.7 ± 4.5, 26.0 ± 3.0, 24.5 ± 4.3 and 21.3 ± 2.7 %ID/g at 0.5, 3, 9, 15 and 24 h p.i., respectively) and kidney radioactivity (30.5 ± 1.0, 26.6 ± 1.3, 19.6 ± 1.0, 14.3 ± 1.1 and 10.0 ± 2.2 %ID/g at 0.5, 3, 9, 15 and 24 h p.i., respectively; Figure 4A) was similar to those in c-Met negative MDA-MB-231 tumor models. The uptake in most other organs was at a very low level. As a comparison, uptake of 64Cu-NOTA-rh-HGF in MDA-MB-231 tumor was low (1.5 ± 2.0, 1.6 ± 0.5, 1.8 ± 0.6, 1.7 ± 0.6 and 1.6 ± 0.6 %ID/g at 0.5, 3, 9, 15 and 24 h after injection, respectively; n = 3; Figure 4B). These values were significantly lower than those of U87MG tumors at all-time points (P < 0.05), indicating that c-Met targeting is the primary factor for the prominent uptake of 64Cu-NOTA-rh-HGF in the U87MG tumor.

Figure 3.

Figure 3

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Small animal PET imaging of c-Met expression in U87MG and MDA-MB-231 tumor-bearing mice. Serial coronal PET images at 0.5, 3, 9, 15 and 24 h post-injection of 64Cu-NOTA-rh-HGF or 64Cu-NOTA-dnrh-HGF are shown and tumors are indicated by arrows.

Figure 4.

Figure 4

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Quantitative analysis of the PET data. (A) Time-activity curves of the U87MG tumor, blood, liver, kidney and muscle upon intravenous injection of 64Cu-NOTA-rh-HGF; (B) Time-activity curves of the MDA-MB-231 tumor, blood, liver, kidney and muscle upon intravenous injection of 64Cu-NOTA-rh-HGF; (C) Time-activity curves of the U87MG tumor, blood, liver, kidney and muscle upon intravenous injection of 64Cu-NOTA-dnrh-HGF; (D) Comparison of tumor uptake in all three groups. n ≥ 3.

64Cu-NOTA-dnrh-HGF (which was sonicated denatured hence does not bind to c-Met) had significantly lower U87MG tumor uptake than 64Cu-NOTA-rh-HGF, with 1.0 ± 0.2, 1.2 ± 0.3, 1.4 ± 0.5, 1.6 ± 0.6 and 1.6 ± 0.3 %ID/g at 0.5, 3, 9, 15 and 24 h p.i. respectively (n = 3; P < 0.05 at all time points; Figure 4C, D). Liver uptake (34.9 ± 1.6, 30.3 ± 1.8, 25.3 ± 0.5, 22.2 ± 0.7 and 19.9 ± 1.2 %ID/g at 0.5, 3, 9, 15 and 24 h p.i. respectively; n = 3; Figure 4C) and kidney uptake (30.7 ± 0.4, 25.5 ± 1.4, 19.6 ± 1.0, 16.3 ± 1.0 and 13.7 ± 1.0 %ID/g at 0.5, 3, 9, 15 and 24 h p.i. respectively; n = 3; Figure 4C) of 64Cu-NOTA-dnrh-HGF were comparable to those of mice injected with 64Cu-NOTA-rh-HGF, since both liver and kidneys are the expected clearance organs for a tracer of this size. Overall, tracer uptake in all major organs was similar between 64Cu-NOTA-rh-HGF and 64Cu-NOTA-dnrh-HGF except the U87MG tumor (significantly higher in the former), further confirming the c-Met specificity of the tracer.

After the terminal PET scans at 24 h p.i., all mice were euthanized for biodistribution studies to validate quantitative tracer uptake values based on PET imaging data. The U87MG tumor uptake of 64Cu-NOTA-rh-HGF was 5.9 ± 1.3 %ID/g at 24 h p.i., significantly higher than 64Cu-NOTA-dnrh-HGF in U87MG tumor (1.4 ± 0.4 %ID/g) and 64Cu-NOTA-rh-HGF in MDA-MB-231 tumor (0.7 ± 0.3 %ID/g) (Figure 5). As a result, U87MG tumor uptake of 64Cu-NOTA-rh-HGF was the highest and provided excellent tumor contrast with 13.5 ± 6.1 (n = 4) tumor-to-muscle ratios at 24 h p.i. Overall, quantification data obtained from biodistribution studies was consistent with PET scans, indicating that ROI analysis of non-invasive PET scans truly reflected tracer distribution in vivo, as well as c-Met specificity of 64Cu-NOTA-rh-HGF.

Figure 5.

Figure 5

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Biodistribution of 64Cu-NOTA-rh-HGF in the U87MG and MDA-MB-231 tumors, as well as 64Cu-dnrh-HGF in the U87MG tumors at 24 h post-injection. n ≥ 3.

Histology

Based on fluorescence staining of c-Met, c-Met expression was prominent on the U87MG tumor cells but absent on the MDA-MB-231 cells and rh-HGF can target c-Met with high specificity (Figure 6). c-Met staining of mouse liver and muscle gave a low signal, indicating that these tissues do not express high level of c-Met. Thus, uptake of 64Cu-NOTA-rh-HGF in the liver was mostly due to hepatic clearance of the tracer rather than c-Met binding. The fluorescence from FITC-rh-HGF provided excellent overlay with c-Met in the U87MG tumors but not in MDA-MB-231 tumors.

Figure 6.

Figure 6

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Immunofluorescence rh-HGF/c-Met/DAPI triple staining of U87MG and MDA-MB-231 tumors, liver and muscle tissue sections. Tissues were incubated with rh-HGF first, and mouse anti-human HGF antibody and FITC-conjugated goat anti-mouse IgG were used for rh-HGF staining (red). Rat anti-human c-Met antibody and Cy3-labeled donkey anti-rat IgG were used for c-Met staining (green). DAPI staining was used to reveal the location of cell nuclei. Scale bar: 20 µm.

DISCUSSION

The HGF/c-Met signaling pathway has emerged as a promising therapeutic target for inhibiting tumor growth (22). Activation or upregulation of c-Met is a negative prognostic indicator for various carcinomas, multiple myeloma, or glioma (9). Several strategies to inhibit c-Met activation are currently under clinical evaluation, such as the use of tyrosine kinase inhibitors, monoclonal antibodies (against the receptor or ligand), and compounds against c-Met/HGF (8, 23). To obtain more accurate clinic outcome, suitable biomarkers for non-invasive monitoring and predicting response to those therapies are needed. 18F-labeled FDG and FLT for PET imaging (24, 25) and gadolinium labeled anti-c-Met antibody for magnetic resonance imaging (26) have been investigated. However, as the only known ligand of c-Met, HGF has not been used for PET imaging to quantify c-Met abundance. Since HGF is a multifunctional growth factor involved in stimulation of hepatocyte proliferation, inhibition of apoptosis, and invasive growth of tumor cells (27), rh-HGF has been used in a clinical trial for treatment of fulminant hepatic failure (FHF) or late-onset hepatic failure (LOHF) (28). Developing a HGF-based PET tracer for imaging of c-Met expression in cancer patients may significantly facilitate patient stratification in clinical trials, as well as provide surveillance for the efficacy of c-Met-targeted therapies.

To avoid proteolytic digestion and complexity in downstream processes including disulfide bonds and unique glycosylation patterns, the production of HGF requires eukaryotic or even mammalian expression systems to achieve high yields of functional active protein (29). A simple but robust protocol was established in this study using pCMV standard vector with 10His tag and transient expression in HEK293 cells to produce rh-HGF with high purity and quantity. After purification, 150–200 µg of rh-HGF per 150 cm2 flask could be obtained after a 48 h transfection. SDS-PAGE gel analysis showed a major constituent with a molecular weight of 90 kDa, standing for purified rh-HGF. Two weak bands about 60 and 30-kDa were also seen on SDS-PAGE, which was consist with the reported result of rh-HGF in CHO expression system since there is an equilibrium between rh-HGF and its α/β chain (19). 10His Tag was used to simplify the production and characterization of rh-HGF, which did not affect the function of rh-HGF, confirmed by potent and specific binding capacity of rh-HGF to c-Met based on flow cytometry (Figure 2). The 10His tag can also be a potential binding site for 99mTc (30), which can be used for future SPECT imaging.

To the best of our knowledge, imaging of c-Met with rh-HGF has not been reported to date. In this study, we have successfully developed and characterized 64Cu-NOTA-rh-HGF for PET imaging of tumor c-Met expression in vivo. c-Met specificity of 64Cu-NOTA-rh-HGF was demonstrated by a number of control experiments in vitro, in vivo, and ex vivo, which makes it a promising PET tracer with broad potential applications in clinical diagnosis and treatment monitoring of cancer. With a molecular weight of ~90 kDa, rh-HGF is significantly smaller than mAbs (typically 150 kDa). Therefore, 64Cu-NOTA-rh-HGF displayed a faster circulation clearance than radiolabeled intact mAbs. When compared with 89Zr-labeled onartuzumab, a c-Met-targeting humanized 1-armed mAb (which has a similar molecular weight of ~99 kDa) which had a tumor/muscle ratio of 7.0 in the U87MG tumor at 120 h p.i. (12), 64Cu-NOTA-rh-HGF uptake in the U87MG tumor was much faster with significantly higher tumor/muscle ratio (13.5 ± 6.1 at 9 h p.i.; n = 4).

HGF contains two c-Met binding sites including a high affinity constitutively active site on the α chain and a low affinity site on the β chain (31). While α chain of HGF activates c-Met and induces its dimerization, β chain residues bind c-Met when the receptor binding site is located on the α chain and subsequently induce phosphorylation and downstream signaling (31). Our future work will focus on the design and purification of α chain of rh-HGF with other labeling strategies, such as the use of shorter-lived PET isotopes with higher positron ranching ratio (61Cu, 44Sc, etc), and testing the resulting PET tracers in different tumor models. We believe that non-invasive PET imaging with radiolabeled rh-HGF, upon future clinical translation, could support the selection of patients for c-Met targeting drugs and identify the responding/non-responding patients for such therapeutics.

CONCLUSION

Herein we report the purification of rh-HGF, the characterization and in vitro/in vivo investigation of 64Cu-NOTA-rh-HGF for PET imaging of c-Met expression in tumor models. Fast, prominent and c-Met-specific uptake of 64Cu-NOTA-rh-HGF in the U87MG tumors was observed, which was further validated by various in vivo and ex vivo experiments. Upon further optimization and development, rh-HGF based PET tracers may be translated into the clinic for cancer diagnosis/prognosis based on c-Met targeting.

ACKNOWLEDGEMENTS

This work is supported, in part, by the University of Wisconsin - Madison, the National Institutes of Health (NIBIB/NCI 1R01CA169365, P30CA014520, and T32CA009206), the Department of Defense (W81XWH-11-1-0644 and W81XWH-11-1-0648), and the American Cancer Society (125246-RSG-13-099-01-CCE).

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