Evaluating the mechanism underlying antitumor effect of interleukin 27 on B cells of chronic lymphocytic leukemia patients.

Chronic lymphocyte leukemia (CLL) is a B-cell malignancy resisted to apoptosis. Recently, some studies indicated that cytokines such as interleukin 27 (IL-27) can reduce B-cell proliferation. The aim of this study is to evaluate the mechanism underlying the proapoptotic effect of IL-27 on B cells of patients with CLL in comparison with B cells of normal subjects. The effect of IL-27 on the antitumor activity of natural killer (NK) and T cells was also evaluated. Peripheral blood mononuclear cells (PBMCs) were isolated from 35 patients with CLL and 15 normal subjects. B cells and PBMCs were cocultured with IL-27 and B cells apoptosis to evaluate proliferation. Both messenger RNA and protein expression of IL-27 and IL-27 receptor were determined using flow cytometry and real-time polymerase chain reaction analysis. To evaluate the apoptotic effect of IL-27 on B cells of patients with CLL, Annexin V-FITC and 7-AAD (BioLegend) fluorescent dyes were used. In addition, the IL-27 effect on activation of T cell and NK cell was determined by determining CD96 molecule expression. IL-27 and IL-27 receptor expression in patients with CLL was significantly lower than that of normal subjects (p < .05). IL-27 enhanced apoptosis of B cells in patients with CLL (p < .05) but this effect was not significantly observed in B cells of normal subjects (p > .05). Consequently, IL-27 reduced the proliferation of B cells and enhanced NK cell activity (p < .05). IL-27, through inducing apoptosis, can exert an inhibitory effect on cancer B cells of CLL patients with minimal effect on normal B cells.

IL-25 Impact on Malignant B Cells Survival and T Cells Activation in Chronic Lymphocytic Leukemia.

T cell dysregulation and shift to T helper 2 responses, boosting tumor microenvironment support, contributes to the survival of leukemic B cells in Chronic Lymphocytic Leukemia. Interleukin (IL)-25 is involved in the initiation of T helper 2 cell responses. Signal transduction of IL-25 begins with the heterodimer receptor (IL-17RA/IL-17RB). The presence of IL-25 in the tumor microenvironment may affect the supportive effects of T cells in the surrounding tumor cell environment. The purpose of this study was to evaluate the role of IL-25 in the biology of CLL. IL-17RB expression in CD3+ and CD19+ cells was assessed in isolated peripheral blood mononuclear cells (PBMCs) of nine CLL patients and nine healthy subjects by real-time polymerase chain reaction and flow cytometry. B cells were positively enriched from PBMCs using magnetic-activated cell sorting (MACS). PBMCs and purified leukemic B cells were cultured with recombinant human IL-25 (20ng/ml) for 72 hours, then the viability and apoptosis of cultured cells were measured by MTT assay and AnnexinV/7AAD. Furthermore, the levels of CD69 expression on T lymphocytes and IL-17RB in T and B cells were determined by flow cytometry. The basal level of IL-17RB expression in CLL patients was significantly higher than that in control individuals. In addition, the percentage of IL-17RB+/CD3+, IL-17RB+/CD19+ cells and CD69+/CD3+ cells increased after 72 hours of culture with IL-25 in CLL patients compared to healthy subjects. IL-25 also reduces the apoptosis rate of tumor cells. We found that IL-25 could stimulate T cells in CLL patients and lower B cell death. This suggests that IL-25 might have a role in enhancing the survival of tumor cell by expressing receptors for inflammation, such as IL-17RB, and might be involved in the development of CLL.

A Small Molecule Targeting the Intracellular Tyrosine Kinase Domain of ROR1 (KAN0441571C) Induced Significant Apoptosis of Non-Small Cell Lung Cancer (NSCLC) Cells.

The ROR1 receptor tyrosine kinase is expressed in embryonic tissues but is absent in normal adult tissues. ROR1 is of importance in oncogenesis and is overexpressed in several cancers, such as NSCLC. In this study, we evaluated ROR1 expression in NSCLC patients (N = 287) and the cytotoxic effects of a small molecule ROR1 inhibitor (KAN0441571C) in NSCLC cell lines. ROR1 expression in tumor cells was more frequent in non-squamous (87%) than in squamous (57%) carcinomas patients, while 21% of neuroendocrine tumors expressed ROR1 (p = 0.0001). A significantly higher proportion of p53 negative patients in the ROR1+ group than in the p53 positive non-squamous NSCLC patients (p = 0.03) was noted. KAN0441571C dephosphorylated ROR1 and induced apoptosis (Annexin V/PI) in a time- and dose-dependent manner in five ROR1+ NSCLC cell lines and was superior compared to erlotinib (EGFR inhibitor). Apoptosis was confirmed by the downregulation of MCL-1 and BCL-2, as well as PARP and caspase 3 cleavage. The non-canonical Wnt pathway was involved. The combination of KAN0441571C and erlotinib showed a synergistic apoptotic effect. KAN0441571C also inhibited proliferative (cell cycle analyses, colony formation assay) and migratory (scratch wound healing assay) functions. Targeting NSCLC cells by a combination of ROR1 and EGFR inhibitors may represent a novel promising approach for the treatment of NSCLC patients.

Anti-tumor effect of berberine on chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) is a blood malignancy that is characterized by remarkable expression of CD69 and Ki67 in CLL cells. Elevated levels of Cleaved-Poly (ADP-ribose) polymerase-1 (PARP1) and microRNA-155 (MiR-155) are related to poor prognosis of disease. Berberine as a natural isoquinoline alkaloid, has shown an anti-tumor potential in tumor cells. The objective of present study was to explore some aspects of molecular mechanisms of berberine effect in CLL cells. To analyze the expression of CD69 and Ki67 using flow cytometry, 16 peripheral blood samples and seven bone marrow aspirates were collected from CLL patients. Isolated peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) were treated with 25 µM of berberine for 24 h. The level of miR-155 expression was subsequently evaluated by real-time PCR. Furthermore, western blot was used for assessment of cleaved PARP1. Our results demonstrated a significant reduction in CD69 and Ki67 expression on CD19+ cells when the cells were treated by berberine. Interestingly, the expression level of miR-155 was reduced after berberine treatment in compare to the control group. Furthermore, western blotting revealed an increased level of cleaved PARP1 in dose-dependently manner in CLL cells. The results confirmed the anti-tumor impact of berberine on CLL cells through reducing CD69, Ki67, and miR-155 expression and increasing cleaved PARP1 may be considered as an option for future clinical studies.

Platelet-Derived PDGFB Promotes Recruitment of Cancer-Associated Fibroblasts, Deposition of Extracellular Matrix and Tgfß Signaling in the Tumor Microenvironment.

Platelets constitute a major reservoir of platelet-derived growth factor B (PDGFB) and are continuously activated in the tumor microenvironment, exposing tumors to the plethora of growth factors contained in platelet granules. To address the specific role of platelet-derived PDGFB in the tumor microenvironment, we have created a mouse model with conditional knockout of PDGFB in platelets (pl-PDGFB KO). Lack of PDGFB in platelets resulted in 10-fold lower PDGFB concentration in the tumor microenvironment, fewer cancer-associated fibroblasts and reduced deposition of the extracellular matrix (ECM) molecules fibronectin and collagen I in the orthotopic RIP1-Tag2 model for pancreatic neuroendocrine cancer. Myosin light chain phosphorylation, promoting cell contraction and, consequently, the mechano-induced release of active transforming growth factor (TGF) ß from extracellular compartments, was reduced in tumors from pl-PDGFB KO mice. In agreement, TGFß signaling, measured as phosphorylated Smad2, was significantly hampered in tumors from mice lacking PDGFB in their platelets, providing a plausible explanation for the reduced deposition of extracellular matrix. These findings indicate a major contribution of platelet-derived PDGFB to a malignant transformation of the tumor microenvironment and address for the first time the role of PDGFB released specifically from platelets in the remodeling of the ECM in tumors.

Surface protein profiling of prostate-derived extracellular vesicles by mass spectrometry and proximity assays.

Extracellular vesicles (EVs) are mediators of intercellular communication and a promising class of biomarkers. Surface proteins of EVs play decisive roles in establishing a connection with recipient cells, and they are putative targets for diagnostic assays. Analysis of the surface proteins can thus both illuminate the biological functions of EVs and help identify potential biomarkers. We developed a strategy combining high-resolution mass spectrometry (HRMS) and  proximity ligation assays (PLA) to first identify and then validate surface proteins discovered on EVs. We applied our workflow to investigate surface proteins of small EVs found in seminal fluid (SF-sEV). We identified 1,014 surface proteins and verified the presence of a subset of these on the surface of SF-sEVs. Our work demonstrates a general strategy for deep analysis of EVs' surface proteins across patients and pathological conditions, proceeding from unbiased screening by HRMS to ultra-sensitive targeted analyses via PLA.

Extracellular Vesicle Capture by AnTibody of CHoice and Enzymatic Release (EV-CATCHER): A customizable purification assay designed for small-RNA biomarker identification and evaluation of circulating small-EVs.

Circulating nucleic acids, encapsulated within small extracellular vesicles (EVs), provide a remote cellular snapshot of biomarkers derived from diseased tissues, however selective isolation is critical. Current laboratory-based purification techniques rely on the physical properties of small-EVs rather than their inherited cellular fingerprints. We established a highly-selective purification assay, termed EV-CATCHER, initially designed for high-throughput analysis of low-abundance small-RNA cargos by next-generation sequencing. We demonstrated its selectivity by specifically isolating and sequencing small-RNAs from mouse small-EVs spiked into human plasma. Western blotting, nanoparticle tracking, and transmission electron microscopy were used to validate and quantify the capture and release of intact small-EVs. As proof-of-principle for sensitive detection of circulating miRNAs, we compared small-RNA sequencing data from a subset of small-EVs serum-purified with EV-CATCHER to data from whole serum, using samples from a small cohort of recently hospitalized Covid-19 patients. We identified and validated, only in small-EVs, hsa-miR-146a and hsa-miR-126-3p to be significantly downregulated with disease severity. Separately, using convalescent sera from recovered Covid-19 patients with high anti-spike IgG titers, we confirmed the neutralizing properties, against SARS-CoV-2 in vitro, of a subset of small-EVs serum-purified by EV-CATCHER, as initially observed with ultracentrifuged small-EVs. Altogether our data highlight the sensitivity and versatility of EV-CATCHER.