Noninvasive Evaluation of Multidrug Resistance via Imaging of ABCG2/BCRP Multidrug Transporter in Lung Cancer Xenograft Models

Abstract

Chemotherapy is an important method for the treatment of lung cancer, but multidrug resistance (MDR) greatly reduces the efficacy. The superfamily of ATP-binding cassette (ABC) transport proteins is related to MDR. As a subfamily of ABC proteins, ABCG2/BCRP (breast cancer resistance protein, BCRP) is considered a major player in the development of cancer MDR. For the stratification of chemotherapeutic choices, we constructed Cy5.5- or 89Zr-labeled ABCG2-targeted monoclonal antibody (mAb) ABCG2-PKU1 for noninvasive evaluation of ABCG2 expression in lung cancer xenograft models. ABCG2 expression was screened in H460/MX (mitoxantrone resistant), H460, and H1299 human lung cancer cell lines using Western blotting. ELISA, flow cytometry, and cell immunofluorescent staining were used to evaluate the binding ability of ABCG2-PKU1 to ABCG2 antigen. Lung cancer murine xenograft models were built for in vivo experiments. ABCG2-PKU1 was labeled with Cy5.5 (Cy5.5-ABCG2) for fluorescent imaging and radiolabeled with 89Zr (89Zr-DFO-ABCG2) for immunoPET imaging following the conjugation with p-SCN-deferoxamine (DFO). In vivo imaging was performed in lung cancer models at 2, 24, 48, 72, 96, 120, 144, and 168 h postinjection. Ex vivo biodistribution was conducted after the terminal time point of imaging. Finally, tissue immunohistochemical staining was used to evaluate the tumor expression of ABCG2. Western blotting showed that the H460/MX cells had a high ABCG2 expression level whereas H460 and H1299 had moderate and low levels. ELISA, flow cytometry, and cell immunofluorescent staining results validated the good binding affinity between ABCG2-PKU1 and ABCG2. The H460/MX and H460 cells were used to build positive lung cancer models, and H1299 cells were used to build negative models. The fluorescent imaging showed that the tumor average radiant efficiency of Cy5.5-ABCG2 reached the maximum at 72 and 120 h in H460/MX and H460 respectively (n = 3, P < 0.01). The tumor uptake of Cy5.5-ABCG2 in H1299 (n = 3) was significantly lower than H460/MX and H460 (P < 0.01). ImmunoPET imaging showed that the tumor uptake of 89Zr-DFO-ABCG2 in H460/MX was significantly higher than H460, with a maximum of 4.15 ± 0.41 %ID/g and 2.81 ± 0.24 %ID/g at 168 and 144 h, respectively (n = 5, P < 0.01). The H1299 tumors showed significantly lower uptake than H460/MX and H460 (n = 5, P < 0.01). The radioactive uptake of 89Zr-DFO-ABCG2 among three groups in the heart, liver, and kidney gradually decreased over time. Ex vivo biodistribution verified the differential tumor uptake among the three groups (P < 0.01). Immunohistochemical staining revealed that the H460/MX tumor had the highest expression of ABCG2, whereas H460 and H1299 had the moderate and lowest expression, respectively. Therefore, in this study, fluorescent and immunoPET imaging of lung cancer MDR models using Cy5.5-ABCG2 and 89Zr-DFO-ABCG2 noninvasively evaluated the differential expression of ABCG2, which are expected to be used for the diagnosis and the selection for clinical treatment opti...

Theranostic application of 64Cu/177Lu-labeled anti-Trop2 monoclonal antibody in pancreatic cancer tumor models

“…The tumor volume of high- 177 Lu-IMB1636 groups reached the maximum at 4 days p.i. and then gradually decreased with time, while the tumor volume of the other three control groups gradually increased, which may be related to the large molecular weight (~ 150 kDa) and the long blood circulation cycle of intact antibody [11]. The decay half-life of 177 Lu is 6.7 d, which is enough for the tumor to produce su cient RIT effect.…”

“…Proteins were collected, electrophoresed and transferred to the nitrocellulose (NC) membrane following a standard protocol [11].After being blocked with 5% skimmed milk dissolved in TBST buffer, the NC membrane was incubated with rabbit anti-human monoclonal Trop2 (Abcam) and rabbit anti-human βactin (ZSGB-BIO) primary antibodies (1:1000) overnight at 4 °C. Then the NC membrane was incubated with goat anti-rabbit IgG H&L (HRP, ZSGB-BIO) secondary antibodies (1:1500) for 40 min at room temperature.…”

Progress in the studies on the molecular mechanisms associated with multidrug resistance in cancers

“…It is quite easy to achieve anti-tumor reagents such as inhibitors, however, as to how to improve the properties of these reagents as drugs, their biological availability, half-life, biocompatibility, and so on, are highly concerned as common issues that might make them more efficient and less harmful to the patients during clinical applications. Till now, hundreds of studies surrounding clinical application of the anti-tumor drugs for curation of MDR cancers, or those focusing on the anti-tumor drug systems with high potential for clinical translation have been published 1 , 5 , 11 , 188 , 189 , 190 , 191 , 192 , 193 , 194 , 195 , 196 , 197 .…”