Exosomes as a Surrogate Marker for Autophagy in Peripheral Blood, Correlative Data from Phase I Study of Chloroquine in Combination with Carboplatin/Gemcitabine in Advanced Solid Tumors

Abstract

Background: Autophagy is a catabolic process, utilized constitutionally by body cells to recycle nutrients and to remove unwanted/damaged intracellular constituents. It is enhanced during periods of stress, such as starvation and hypoxia, aiding in cell survival and it is linked to major cellular processes, such as apoptosis and antigen expression. The process has been extensively studied in vitro models or tumor tissue samples with rare application on human subjects.

Methods: Plasma samples from 24 advanced solid tumor patients were collected at different time points before and after chemotherapy. Their exosomes were isolate and blotted for microtubule-associated protein-1 light chain-3 (LC-3B) protein as a marker for autophagy. All the subjects received a standard chemotherapy regimen of carboplatin- gemcitabine with chloroquine (CQ)/ hydroxychloroquine (HCQ) in chronic doses throughout their treatment period as an autophagy modulator. CQ/HCQ was given in 50 mg increments as guided by their tolerability to treatment.

Results: A total of 267 plasma samples were obtained for the 24 patients and processed. Each sample corresponds to a single time point. The first group included 6 patients, all received 50 mg of CQ with chemotherapy. LC-3B I was detected in their isolated exosomes, while LC3-BII was not detected in their samples. The second cohort of patients included 3 subjects who re-ceived 100mg of HCQ. They demonstrated both LC3-BI and II on day 15 after chemotherapy in one patient, and on third cycle after 24 hours in the second patient. The third cohort included 3 subjects who received 150 mg of HCQ. All cases demonstrated LC3-BI and II on first cycle of treatment after less than 24 hours. The last cohort included 8 subjects, who received a fixed dose of 100 mg of HCQ with treatment. In this cohort, we were able to detect both LC3-B isoforms on advanced time points of second and third cycles.

Conclusion: Detection of autophagy protein LC3-B in exosomes serves as a dynamic method to monitor autophagy. It can be utilized to study the effects of anti-neoplastic agents on autophagy and mechanisms of drug resistance, however, to standardize our results a larger specimen of patients should be included.

Keywords: Autophagy; Biomarker; Western blotting; cancer; exosomes.

Figure 1

Showing the Results of LC-3B Blotting. 1.1a exosomes extracted from the cell culture media of HEK293 cells, 1.1b U251 cells, 1.2a cycle 1 of patient who received CQ 150 mg starting 1 week before chemotherapy and after collection of day -7 sample collection, and 1.2b exosomes collected from U251 cell culture media and U251 cells, and exosomes from cell culture media of HEK293 cells and HEK293 cells

Figure 2

showing Results of LC-3B I and II Blotting in Exosomes Extracted from the Cell Culture Media of MiaPaCa 2 Cell-Line

Figure 3

Showing Results of LC-3B Blotting of Exosomes Extracted from the Cell Culture Media of U251 Cells. Gem treatment resulted in marked reduction in the intensity of the bands, while reducing the dose of CQ resulted in thinner bands while the intensity of the bands were the same

Figure 4

Showing LC-3B Blotting of Exosomes Extracted from the Plasma of a Patient who Received 50 mg of CQ with Treatment. The faint bands post-treatment can be observed only in the first cycle. The second, third and fourth cycles showed LC-3B II bands with increased intensity

Figure 5

Showing LC-3B Blotting of Exosomes Extracted from the Plasma of a Patient who Received 100 mg of HCQ. The patient showed no expression of LC-3B II at baseline. LC-3b II protein was expressed on day 15 of the third cycle

Figure 6

Showing LC-3B Blotting of Exosomes Extracted from the Plasma of a Patient who Received 150 mg of HCQ. LC-3B II band can be seen at 24h time point. This the earliest time point post-treatment that LC-3B II was detected

Figure 7

Showing the Annexin V Assay for the MiaPaCa 2 Cells Treated with Gem and CQ at Different Concentrations. Compared to controls and Gem treatment, CQ resulted increase in apoptotic and pre-apoptotic cell percentage. Increased CQ concentration resulted in marked increase in necrosis