18F-ML10 PET/CT demonstrates therapy-induced apoptosis in murine pancreatic adenocarcinoma

Objectives Autophagy inhibition in many solid tumors including pancreatic adenocarcinoma increases apoptotic cell death. We assessed chemotherapy-induced apoptosis with and without autophagy inhibition in mice bearing subcutaneous murine pancreatic tumors using the apoptotic PET imaging agent, 18F-labeled 2-(5-fluoro-pentyl)- 2-methyl-malonic acid (18F-ML-10), and compared results of treated mice with controls.

Methods Panc02 tumors cells were implanted in the right shoulder of 20 albino C57BL/6 mice. Tumor bearing mice were treated IP with gemcitabine (Gem; 40 mg/kg in 4 divided doses on days 0,4,6,9) alone (n=5), chloroquine (CQ; 50 mg/kg daily) alone (n=5), gemcitabine + chloroquine (Gem+CQ) (n=5), or no treatment at all (n=5). PET/CT imaging at 25 min post injection of 18F-ML-10 was performed at 0 h and at 5 and 10 d post-treatment on a Siemens Inveon PET/CT scanner. Evaluation of imaging characteristics was performed using Inveon Research Workplace (IRW) software. Tumor volume, mean standard uptake values (SUVmean), and maximum standard uptake values (SUVmax) were measured on PET/CT scans in all subjects. A measure of tracer uptake in tumor volume (ML-10 index) was calculated as the product of tumor volume and SUVmean in an effort to account for variable tumor sizes in each group. Following each imaging session, at least one subject from each group was euthanized and tumors were removed and frozen for histopathologic analysis, including TUNEL staining. Paired student’s t-test was used to evaluate pre and post therapeutic PET parameters for statistical significance and two-sample t-test was used to evaluate the difference between groups.

Results Ten tumor-bearing mice were imaged at all 3 time points (control=2, Gem=3, Gem+CQ=3, CQ=2). Greater than 2-fold increase in tumor volume was observed in non-treated controls. Mice treated with Gem alone and CQ alone also showed a greater than 2-fold increase; however, a similar tumor volume increase was not found following Gem+CQ therapy. All tumors showed 18F-ML-10 uptake (SUV: 1.7-12.0) and increase in ML-10 index from the baseline scan. Controls had a 2-fold increase in ML-10 index, attributed to an increase in size. In treated mice, the difference between pre and post therapeutic measures of tumor cell death by 18F-ML-10 PET/CT at post therapy day 5 was towards significance (ML-10 index: p=0.07) and became statistically significant by post therapy day10 (ML-10 index: p=0.03). In treated mice versus non-treated controls, the difference in % change in ML-10 index was toward significance at post therapy day 10 (p=0.07). There was a greater % change in ML-10 index in mice undergoing Gem or CQ therapy alone (4-fold increase) compared to non-treated controls. The greatest increase in ML-10 index was seen in the Gem+CQ group (~5-fold increase).

Conclusions Greater changes in ML-10 index in the treated groups compared with tumor controls suggest imaging evidence of therapy-induced cell death. Inhibition of further tumor growth and greatest % change in ML-10 index in the Gem+CQ group suggest enhanced apoptotic tumor cell killing with autophagy inhibition in addition to chemotherapy. These results show the potential of 18F-ML-10 PET/CT for early detection and monitoring of therapy-induced apoptosis and autophagy inhibition. Imaging results will be correlated with TUNEL staining of tumor specimens.