Noninvasive assessment and therapeutic monitoring of drug-resistant colorectal cancer by MR molecular imaging of extradomain-B fibronectin.

Antineoplastic resistance represents a multifaceted challenge for cancer therapy and diagnostics. Extensive molecular heterogeneity, even within neoplasms of the same type, can elicit distinct outcomes of administering therapeutic pressures, frequently leading to the development of drug-resistant populations. Improved success of oncotherapies merits the exploration of precise molecular imaging technologies that can detect not only anatomical but also molecular changes in tumors and their microenvironment, early on in the treatment regimen. To this end, we developed magnetic resonance molecular imaging (MRMI) strategies to target the extracellular matrix oncoprotein, extradomain-B fibronectin (EDB-FN), for non-invasive assessment and therapeutic monitoring of drug-resistant colorectal cancer (CRC). Methods: Two drug-resistant CRC lines generated from parent DLD-1 and RKO cells by long-term treatment with 5'-FU and 5'-FU plus CB-839 respectively, were characterized for functional and gene expression changes using 3D culture, transwell invasion, qRT-PCR, and western blot assays. Contrast-enhanced MRMI of EDB-FN was performed in athymic nu/nu mice bearing subcutaneous tumor xenografts with 40 µmol/kg dose of macrocyclic ZD2-targeted contrast agent MT218 [ZD2-N3-Gd (HP-DO3A)] on a 3T MRS 3000 scanner. Immunohistochemistry was conducted on patient specimens and xenografts using anti-EDB-FN antibody G4. Results: Analyses of TCGA and GTEx databases revealed poor prognosis of colon cancer patients with higher levels of EDB-FN. Similarly, immunohistochemical staining of patient specimens showed increased EDB-FN expression in primary colon adenocarcinoma and hepatic metastases, but none in normal adjacent tissues. Drug-resistant DLD1-DR and RKO-DR cells were also found to demonstrate enhanced invasive potential and significantly elevated EDB-FN expression over their parent counterparts. MRMI of EDB-FN with 40 µmol/kg dose of MT218 (60% lower than the clinical dose) resulted in robust signal enhancement in the drug-resistant CRC xenografts with 84-120% increase in their contrast-to-noise ratios (CNRs) over the non-resistant counterparts. The feasibility of non-invasive therapeutic monitoring using MRMI of EDB-FN was also evaluated in drug-resistant DLD1-DR tumors treated with a pan-AKT inhibitor MK2206-HCl. The treated drug-resistant tumors failed to respond to therapy, which was accurately detected by MRMI with MT218, demonstrating higher signal enhancement and increased CNRs in the 4-week follow-up scans over the pre-treatment scans. Conclusions: EDB-FN is a promising molecular marker for assessing drug resistance. MRMI of EDB-FN with MT218 at a significantly reduced dose can facilitate effective non-invasive assessment and treatment response monitoring of drug-resistant CRC, highlighting its translational potential for active surveillance and management of CRC and other malignancies.

Preclinical Assessment of the Effectiveness of Magnetic Resonance Molecular Imaging of Extradomain-B Fibronectin for Detection and Characterization of Oral Cancer.

PURPOSE: Oral squamous cell carcinoma (OSCC) has not seen a substantial improvement in patient survival despite therapeutic advances, making accurate detection and characterization of the disease a clinical priority. Here, we aim to demonstrate the effectiveness of magnetic resonance imaging (MRI) with the targeted MRI contrast agent MT218 specific to extradomain-B fibronectin (EDB-FN) in the tumor microenvironment for detection and characterization of aggressive OSCC tumors. PROCEDURES: EDB-FN expression was evaluated in human normal tongue and OSCC specimens with immunohistochemistry. Invasiveness of human CAL27, HSC3, and SCC4 OSCC cells was analyzed with spheroid formation and transwell assays. EDB-FN expression in the cells was analyzed with semiquantitative real-time PCR, western blotting, and a peptide binding study with confocal microscopy. Contrast-enhanced MRI with MT218 was performed on subcutaneous OSCC mouse models at a dose of 0.04 mmol/kg, using gadoteridol (0.1 mmol/kg) as a control. RESULTS: Strong EDB-FN expression was observed in human untreated primary and metastatic OSCC, reduced expression in treated OSCC, and little expression in normal tongue tissue. SCC4 and HSC3 cell lines demonstrated high invasive potential with high and moderate-EDB-FN expression, respectively, while CAL27 showed little invasive potential and low-EDB-FN expression. In T1-weighted MRI, MT218 produced differential contrast enhancement in the subcutaneous tumor models in correlation with EDB-FN expression in the cancer cells. Enhancement in the high-EDB-FN tumors was greater with MT218 at 0.04 mmol/kg than gadoteridol at 0.1 mmol/kg. CONCLUSIONS: The results suggest EDB-FN has strong potential as an imageable biomarker for aggressive OSCC. MRMI results demonstrate the effectiveness of MT218 and the potential for differential diagnostic imaging of oral cancer for improving the management of the disease.