In vitro and in vivo investigation of matrix metalloproteinase expression in metastatic tumor models

Abstract 

Introduction

Overexpression of matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, has been correlated with poor prognosis in several cancer types including lung, colon and breast. Noninvasive detection of MMP expression might allow physicians to better determine when more aggressive cancer therapy is appropriate. The peptide CTT (CTTHWGFTLC) was identified as a selective inhibitor of MMP-2/9 that inhibits the growth of MDA-MB-435 human breast cancer xenografts.

Methods

CTT was conjugated with the bifunctional chelator DOTA (1,4,7,10-tetraazacyclotetradecane-N,N′,N″,N‴-tetraacetic acid) for radiolabeling with ⁶⁴Cu (t_1/2=12.7 h, 17.4% β⁺, 39% β⁻), a radionuclide suitable for positron emission tomography (PET). In vitro affinity was determined in a fluorogenic substrate assay. Tumor gelatinase targeting was evaluated in both biodistribution and microPET imaging studies.

Results

Cu(II)-DOTA-CTT inhibited hMMP-2 (EC₅₀=8.7 μM) and mMMP-9 (EC₅₀=18.2 μM) with similar affinity to CTT (hMMP-2 EC₅₀=13.2 μM; mMMP-9 EC₅₀=11.0 μM). In biodistribution and microPET imaging studies, ⁶⁴Cu-DOTA-CTT was taken up by MMP-2/9-positive B16F10 murine melanoma tumors. Subsequently, imaging studies using ⁶⁴Cu-DOTA-CTT were performed on MDA-MB-435 tumor-bearing mice. With zymography, tumor MMP-2/9 expression in this model was shown to be inconsistent, resulting in microPET detection of the MDA-MB-435 tumor in only 1 of 24 imaged mice. Following limited imaging success, ⁶⁴Cu-DOTA-CTT was shown to have poor in vivo stability.

Conclusions

Despite some evidence for selective uptake of ⁶⁴Cu-DOTA-CTT by gelatinase-expressing tumors, the low affinity for MMP-2 and MMP-9 and in vivo instability make this an inadequate radioligand for in vivo tumor evaluation.

Abbreviations: % ID, percent injected dose, ACN, acetonitrile, APMA, 4-aminophenylmercuric acetate, CTT, CTTHWGFTLC, DIEA, diisopropyl ethylamine, DMF, dimethyl formamide, DOTA, 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid, CB-TE2A, 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane, EC₅₀, 50% effective concentration, ECM, extracellular matrix, ES-MS, electrospray mass spectrometry, Fmoc, fluorenylmethyl, HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro phosphate, HOBt, N-hydroxybenzotriazole, HPLC, high-performance liquid chromatography, HWGF, His-Trp-Gly-Phe, IC₅₀, 50% inhibitory concentration, mAb, monoclonal antibody, MeOH, methanol, MMP, matrix metalloproteinase, OSEM, ordered subsets expectation maximization, PET, positron emission tomography, ROI, region of interest, SDS, sodium dodecyl sulfate, SPECT, single photon emission tomography, STT, STTGHFWTLS, SUV, standard uptake value, TETA, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid, TFA, trifluoroacetic acid, TLC, thin layer chromatography

Keywords: PET imaging, Cancer, MMP