Platelet activation and reactivity in a large cohort of patients with Gaucher disease

Patients with Gaucher disease (GD) are at increased risk of bleeding and have varying degrees of thrombocytopenia, making the analysis of platelet function difficult. This study aimed to provide a clinically relevant quantitative assessment of platelet function and determine its relationship with bleeding and GD-related data. Methods: Unstimulated and stimulated platelet function was measured by whole blood flow cytometry of platelet surface activated αIIbβ3 integrin (detected with monoclonal antibody PAC1), P-selectin (CD62P), and lysosomal-associated membrane protein (LAMP3/CD63) in 149 GD patients. Results: GD patients had a higher level of unstimulated CD63 expression than healthy subjects, which was mildly correlated with glucosylsphingosine (lyso-Gb1) levels (r 0.17, p-value 0.042). Splenectomized GD patients had a higher level of unstimulated αIIbβ3 integrin and P-selectin expression. Reduced platelet reactivity (-2 SD of reference range) was found in 79 (53%, 95% CI 44%-61%) patients, of whom 10 (6.7%, 95% CI 3.3%-12%) had more severe platelet dysfunction. In a multivariate model, only lyso-Gb1 levels were associated with the more severe platelet dysfunction. Fifty-four (49%) of 128 adult patients who completed the bleeding tendency questionnaire reported positive bleeding history. In a multivariate logistic model, older age (OR (95% CI), 1.05 (1.01-1.1)) and low P-selectin reactivity (OR (95% CI), 2.03 (1.25-3.35)) were associated with more than one bleeding manifestation. Conclusion: Flow cytometry enables the study of platelet function in thrombocytopenic GD patients. A platelet degranulation defect, but not αIIbβ3 integrin activation defect, is associated with clinical bleeding. In vivo increased CD63 expression may be related to GD-related inflammation.

CD63 and Dna Mismatch Repair Protein Expression in Prostate Cancer

Protein expression levels in immunohistochemistry and molecular biomarkers have been reported for their ability to predict recurrence, progression, development of metastases, and patient survival. The molecular features in low- and high-grade prostate cancer can differ and influence treatment decision and prognosis. The objective of the current study was to compare the expression of exosomal biomarkers CD63 and mismatch repair proteins (MSH2, MSH6, MLH1, and PMS2) by immunohistochemistry (IHC) in tissue of patients with prostate cancer and benign hyperplasia. Altogether, 62 patients with prostate acinar adenocarcinoma and 20 patients with prostate benign hyperplasia were enrolled in this retrospective study. CD63, MSH2, MSH6, MLH1, and PMS2 expression was analysed by immunohistochemistry. The obtained results showed that CD63 expression was significantly higher in patients with Grade III–V prostate cancer compared to Grade I–II, respectively; 2.23 (1–3) vs 0.92 (0–2) score, p = 0.001. In addition, a significant positive correlation between CD63 expression and grade groups was revealed (Rho = +0.54; p < 0.0001). Furthermore, progression-free survival was significantly higher in patients with low CD63 expression, compared to high CD63 expression (p = 0.0007). MMR expression was absent in 14 patients (four patients with Grade I–II cancer and 10 patients with Grade III–cancer). MMR was present in all cases of benign prostate hyperplasia (mild to moderate staining). The conclusion was that high grade prostate cancer (Grade groups III–V) was characterised by increased CD63 expression, which correlated with progression-free survival.

Circulating exosomes from patients with Graves’ disease induce an inflammatory immune response

Exosomes are extracellular vesicles that can participate in autoimmune diseases. The purpose of this study was to explore whether circulating exosomes are involved in Graves’ disease (GD) pathogenesis. In this study, serum exosomes were extracted from 26 healthy controls (HC-EXO), 26 GD patients (GD-EXO), and 7 Graves’ ophthalmopathy patients (GO-EXO). For each group, the total protein content was detected, and thyrotropin receptor (TSHR), insulin-like growth factor 1 receptor (IGF-1R), HSP60, and CD63 expression were analyzed by Western blotting (WB). Healthy volunteer-derived peripheral blood mononuclear cells (PBMCs) and HC-EXO or GD-EXO were cocultured for 24 h, and immunofluorescence was used to observe the locations of the exosomes and Toll-like receptor (TLR) 2/3. CD11c+TLR2+ and CD11c+TLR3+ cell percentages were determined by flow cytometry. Myeloid differentiation factor 88 (MyD88), Toll/IL-1 receptor domain-containing adaptor inducing interferon-β (TRIF) and p-P65 expression were analyzed by WB. IL-6 and IL-1β supernatant levels were detected using enzyme-linked immunosorbent assay (ELISA). The results showed that the total protein concentration was similar among GD-EXO, GO-EXO and HC-EXO. IGF-1R and HSP60 expression was significantly higher in GD-EXO and GO-EXO than in HC-EXO. After coculturing PBMCs with GD-EXO or HC-EXO for 24 h, GD-EXO could bind to TLR2/3. GD-EXO significantly increased CD11c+TLR2+ and CD11c+TLR3+ cell percentages; MyD88, TRIF, and p-P65 protein expression; and IL-6 and IL-1β levels. In conclusion, we first demonstrated that GD-EXO and GO-EXO highly expressed IGF-1R and HSP60. GD-EXO may induce an inflammatory response through the TLR/NF-κB signaling pathway and be involved in the pathogenesis of GD.

Exosomal marker CD63 expression pattern using immunohistochemistry (IHC) in patients with rectal adenocarcinoma in comparison with left-sided colon cancer.

e16096 Background: Adenocarcinomas arising from the distal one third of the transverse colon, splenic flexure, descending colon, sigmoid colon and rectum are often grouped together due to their hindgut embryologic origin and referred to as left-sided colorectal cancer (CRC). Rectal cancer represents a subset of CRC that has distinct differences in anatomical location, clinical behavior, prognosis and molecular background. In patients with left sided colon cancer (LSCC), the expression of exosomal marker CD63 was reported to be higher in the adjacent normal mucosa (ANM) compared to the tumor (224 vs 154, p = 0.0001). Here, we explored the pattern of CD63 expression using immunohistochemistry in patients with rectal cancer in comparison with patients with LSCC. Methods: Between 2015 and 2018, 53 patients underwent rectal cancer biopsy/resection and had available tissues for CD63 IHC staining. Two pathologists independently scored CD63 expression in the tumor and ANM. Staining intensity was graded from 1-3. Staining percentage was estimated in 10% increments. Mean quick-score (Q-score) was calculated (intensity x percentage). Paired t test was used for statistical analysis. Results: Median age was 60 (range 34-80). Females represented 26%. Caucasians and African Americans represented 74% and 26%, respectively. In patients with rectal cancer, the mean CD63 expression was higher in ANM compared to their expression in the tumor (147 vs 113, p = 0.0012). Compared to patients with LSCC (N = 30), the mean CD63 expression in patients with rectal cancer was lower in the ANM (224 vs 147, p < 0.0001) and in the tumor (154 vs 113, p = 0.01). Conclusions: In our cohort of patients with rectal cancer, exosomal marker CD63 expression was lower in tumor compared to ANM. This observation was similar to our previously reported findings in patients with LSCC. Compared to patients with LSCC, patients with rectal cancer had lower expression of CD63 in the tumor and ANM. To our knowledge, this is the first study to explore exosomal marker CD63 expression using IHC in patients with rectal cancer.

Inhibition of a v b 3 integrin impairs adhesion and uptake of tumor-derived small extracellular vesicles

Background: Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from cells and mediate cell-cell communication. Integrin adhesion receptors are enriched in small EVs (SEVs) and SEV-carried integrins have been shown to promote cancer cell migration and to mediate organ-specific metastasis; however, how integrins mediate these effects is not entirely clear and could represent a combination of EV binding to extracellular matrix and cells.Methods: To probe integrin role in EVs binding and uptake, we employed a disintegrin inhibitor (DisBa-01) of integrin binding with specificity for avb3 integrin. EVs were purified from MDA-MB-231 cells conditioned media by serial centrifugation method. Isolated EVs were characterized by different techniques and further employed in adhesion, uptake and co-culture experiments.Results: We find that SEVs secreted from MDA-MB-231 breast cancer cells carry avb3 integrin and bind directly to fibronectin-coated plates, which is inhibited by DisBa-01. SEV coating on tissue culture plates also induces adhesion of MDA-MB-231 cells, which is inhibited by DisBa-01 treatment. Analysis of EV uptake and interchange between cells reveals that the amount of CD63-positive EVs delivered from malignant MDA-MB-231 breast cells to non-malignant MCF10A breast epithelial cells is reduced by DisBa-01 treatment. Inhibition of avb3 integrin decreases CD63 expression in cancer cells suggesting an effect on SEV content.Conclusion: In summary, our findings demonstrate for the first time a key role of avb3 integrin in cell-cell communication through SEVs.

Exosomal markers (CD63 and CD9) expression and their prognostic significance using immunohistochemistry in right-sided and left-sided colon cancer.

182 Background: Exosomes play pivotal roles in cancer progression, metastasis and chemoresistance. CD63 and CD9 are widely accepted exosomal markers. Their pattern of expression and prognostic significance in patients with RSCC and LSCC is unknown. This study explored CD63 and CD9 expression and prognostic significance in patients with RSCC and LSCC using immunohistochemistry (IHC). Methods: Between 2015 and 2018, 63 patients underwent surgical resection of colon cancer for whom we had available tissues for CD63 and CD9 IHC staining. Two pathologists independently scored the CD63 and CD9 expression in the tumor and adjacent normal mucosa (ANM). Staining intensity was graded 1-3 and staining percentage was estimated in 10% increments. Mean Quick-score (Q-score) (intensity X percentage of staining) was calculated. Results: RSCC and LSCC represented 52% and 48% of the patients respectively. The ANM and Tumor CD63 Q-scores were 225 vs 191 (p = 0.009) in RSCC and 224 vs 154 (p = 0.0001) in LSCC, respectively. The ANM and Tumor CD9 Q-scores were 134 vs 152 (p = 0.142) in RSCC and 135 vs 154 (p = 0.137) in LSCC, respectively. In patients with RSCC and LSCC, the mean Tumor CD63 Q-score was 191 vs 154 (p = 0.024), while the mean ANM CD63 Q-score was 225 vs 224 (p = 0.920). The mean Tumor CD9 Q-score was 152 and 154 (p = 0.883), and the mean ANM CD9 Q-score was 134 vs 135 (p = 0.926). In our cohort, there was no difference in progression free survival (PFS) between patients with RSCC and LSCC (p = 0.2349). In all patients, there was no difference in PFS in patients with CD63 expression < 100 and ≥100 (p = 0.8284). Among patients with RSCC, there was a significantly lower PFS in patients with CD63 expression < 100 vs. ≥100 (p = 0.0259). However, among patients with LSCC, there was no difference in PFS in patients with CD63 expression < 100 vs. ≥100 (p = 0.3494). Conclusions: To our knowledge, this is the first study to show a difference in exosomal marker (CD63) expression pattern and its prognostic significance in patients with RSCC and LSCC. There was a significant positive correlation between progression free survival in patients with RSCC and higher exosomal expression.