Extracellular Vesicles Reflect the Efficacy of Wheatgrass Juice Supplement in Colon Cancer Patients During Adjuvant Chemotherapy

Abstract

Introduction: Colorectal cancer (CC) is the third most common type of cancer, accounting for 10% of all cancer cases. Adjuvant chemotherapy is recommended in stages II-III CC. Wheatgrass juice (WGJ) from wheat seeds has high nutritional values, may induce synergistic benefits to chemotherapy and may attenuate chemotherapy-related side effects. Extracellular vesicles (EVs) are subcellular membrane blebs. EVs include exosomes (generated in the endosome, in size <150 nm) and microvesicles (shed from the plasma cell membrane) provide information on their parental cells and play a role in intercellular communication. We aimed to elucidate the effects of chemotherapy administration with supportive treatment of WGJ on CC patients' EVs characteristics.

Methods: EVs were isolated from the blood samples of 15 healthy controls (HCs) and 50 CC patients post-surgery, treated by chemotherapy, with or without additional daily WGJ. Blood samples were taken before, during, and at the end of chemotherapy. EVs were characterized by size, concentration, membrane antigens and cytokine content using nanoparticle-tracking analysis, western blot, flow cytometry, and protein array methods.

Results: EVs were found to be similar by size and concentration with reduced levels of exosome markers (CD81) on samples at the end of combined treatment (chemotherapy and WGJ). Higher levels of endothelial EVs, which may indicate impairment of the vascular endothelial cells during treatment, were found in CC patients treated by chemotherapy only compared to those with chemotherapy and daily WGJ. Also, EVs thrombogenicity was lower in patients added WGJ compared to patients who had only chemotherapy (levels of tissue factor p = 0.029 and endothelial protein C receptor p = 0.005). Following treatments, levels of vascular endothelial growth factor receptors (VEGFR-1) and the majority of growth-factors/pro-inflammatory cytokines were higher in EVs of patients treated by chemotherapy only than in EVs obtained from patients with the combined treatment.

Conclusion: Daily consumption of WGJ during chemotherapy may reduce vascular damage and chemotherapy-related thrombogenicity, growth factors and cytokines, as reflected by the characteristics of patient's EVs.

Keywords: adjuvant chemotherapy; colon cancer; cytokines; extracellular vesicles; thrombogenicity; wheatgrass juice.

FIGURE 1

Study design flow chart. Patients diagnosed with colon cancer at stages II–III toward adjuvant chemotherapy following curative surgery. Patients who were interested in participating were admitted to the WGJ (CC-W) group. Blood samples were collected from CC-W and from non-WGJ patients, following consent. Blood samples were obtained at three time points: before first chemotherapy (time point I – BT), after 3 months of chemotherapy (time point 2), and at the end of chemotherapy administration (time point 3). One blood sample was collected from each healthy control (HC). PPP EVs were analyzed.

FIGURE 2

EVs size distribution, concentration and exosome markers. EVs were obtained from the study groups: healthy controls (HC) and colon cancer patients during adjuvant chemotherapy only CC-C or adjuvant chemotherapy with WGJ (CC-W). Samples obtained before treatment (BT, time point 1), during treatment (time point 2) and at the end of treatment (time point 3). EVs were isolated by a series of centrifugations Nanoparticle-tracking analysis-determined EV size distribution (A) and concentration (B). N = 5 for each study group. WB analysis with antibodies against exosome markers CD63 and CD81. Representative gel with molecular weight marker (MWM) and representative sample of each study cohort (C) graph summaries the bends area divided with protein concentration of each sample that were loaded to the gel. N = 3 (D).

FIGURE 3

EVs tumor cell markers. Antigen levels of tumorigenic markers were measured on EVs obtained from healthy controls and on EVs obtained from patients (CC-C and CC-W) before chemotherapy (BT, time point 1), during treatment (time point 2), and at the end of chemotherapy treatment (time point 3). The percentage of labeled EVs was calculated from the total number of EVs using FACS analysis. The graph presents significant changes (>50% increase or >30% decrease) in patient EVs levels BT compared to HC and the change during treatment and at the end of chemotherapy treatment compared to the levels BT of EVSA33 antigen (A); EVs CD66 (B).

FIGURE 4

EVs endothelial cell markers. Antigen levels of endothelial markers were measured on EVs obtained from healthy controls and on EVs obtained from patients (CC-C and CC-W) before chemotherapy (BT, time point 1), during treatment (time point 2), and at the end of chemotherapy treatment (time point 3). The percentage of labeled EVs was calculated from the total number of EVs using FACS analysis. The graph presents significant changes (>50% increase or >30% decrease) in patient EVs levels BT compared to HC and the change during treatment and at the end of chemotherapy treatment compared to the levels BT of EVs CD31 + /CD41 (A); EVs CD144 (B).

FIGURE 5

EVs thrombogenicity. Antigen levels of coagulation markers were measured on EVs obtained from healthy controls and from patients before chemotherapy (BT, time point 1), during treatment (time point 2), and at the end of chemotherapy treatment (time point 3), using specific fluorescent antibodies. The percentage of labeled EVs was calculated from the total number of EVs using FACS analysis. The graph presents significant changes (>50% increase or >30% decrease) in patient EVs levels BT compared to HC and the change during treatment and at the end of chemotherapy treatment compared to the levels BT related to% TF labeled EVs (A); % TFPI labeled EVs (B); TF/TFPI EVs ratio (C); % EPCR labeled EVs (D); % Annexin V (E); % EVs procoagulant activity was measured by factor X chromogenic assay (F).

FIGURE 6

EVs expression of VEGF-receptors (Flt-1, KDR) and cytokine contents. Levels of growth factors receptors were measured on EVs obtained from healthy controls and on EVs obtained from patients at three time points, using specific fluorescent antibodies; The graph presents significant changes (>50% increase or >30% decrease) in patient EVs levels BT compared to HC and the change during treatment and at the end of chemotherapy treatment compared to the levels BT related to A. VEGFR1 (Flt-1) (A), VEGFR2 (KDR) (B). EV proteins extract, obtained from a pool of five specimens within each patient subgroup, were validated by Human Angiogenesis Protein Antibody Array. The graph presented the change in specific EVs cytokine and growth factors content during treatment compared the EVs content before treatment. Growth factors (C) and inflammatory cytokine (D).