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 carboplatingemcitabine 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.

Vitamin D ameliorates asthma‐induced lung injury by regulating HIF‐1α/Notch1 signaling during autophagy

“…Studies have confirmed that several genes are involved and regulate the pathogenesis of asthma, including some autophagy‐related genes, such as autophagy‐related 5 (ATG5) and sequestosome 1 (SQSTM1; Kasembeli et al., 2018 ). In the present study, immunofluorescence and immunohistochemistry results revealed that autophagy marker proteins LC 3B (Abdel Karim et al., 2019 ), Beclin1 (Chen et al., 2020 ), and P62 (Li et al., 2015 ) were unaltered in lung tissues of RSV‐infected mice; however, expression levels of LC 3B and Beclin1, both at the gene and protein levels, were significantly increased following Vit D treatment. The P62 gene and protein levels were significantly decreased, suggesting that the mechanism of Vit D in treating RSV‐induced asthma may involve enhanced autophagy in lung tissue.…”

Impaired Autophagy in Krabbe Disease: The Role of BCL2 and Beclin-1 Phosphorylation

“…LC3B-II appeared to not increase further after 4 h of culture in EBSS with CQ in comparison to 2 h of culture in EBSS with CQ and in KD fibroblasts, particularly, it seemed to decrease. This effect could be possibly explained by hypothesizing that LC3B-II could also be partially lost through other routes such as the exosome release related to autophagy induction, as previously demonstrated [ 26 , 27 ]. However, given these problematics arose considering only LC3B-II formation and to better clarify LC3B-II behaviour, we calculated the ratio between normalized LC3B-II content in cells treated in EBSS with CQ and LC3B-II content in cells in EBSS without CQ, in order to monitor the autophagic flux.…”

Chloroquine and Chemotherapeutic Compounds in Experimental Cancer Treatment

“…), a combination of CQ or HCQ with CPT increased progressive-free disease and overall survival (OS), although some side effects were reported [96]. Importantly, in the exosomes obtained from the blood plasma of patients who received such treatment, both LC3-B isoforms were detected at advanced time points of the second and third cycles [97].…”