Citations

AbstractBackgroundThe development of phenotypic biomarkers to aid the selection of treatment for patients with castrate‐resistant prostate cancer (CRPC) is an important priority. Plasma exosomes have excellent potential as real‐time biomarkers to characterize the tumor because they are easily accessible in the blood and contain DNA, RNA, and protein from the parent cell. This study aims to investigate the characteristics of putative prostate‐specific plasma extracellular vesicle (EV) markers and their relationship with clinical outcomes.Methods and PatientsWe investigated plasma EVs in a total of 89 patients with prostate cancer (PCa) at different stages of disease progression. EVs were isolated using both precipitation and ultracentrifugation methods; physical characterization was performed using dynamic light scattering, acetylcholinesterase (AChE) activity, and velocity gradients. An immunocapture method was developed for the evaluation of prostate‐specific membrane antigen (PSMA)‐positive exosomes. Exosomal messenger RNA (mRNA) was quantified using droplet digital polymerase chain reaction for the expression of KLK3 and androgen receptor splice variant 7 (AR‐V7) genes, which code prostate‐specific antigen (PSA) and AR‐V7, respectively. Serum sex steroids were measured using liquid chromatography‐tandem mass spectroscopy.ResultsIsolated exosomes from patients with CRPC had a smaller hydrodynamic size than those isolated from localized patients with PCa, while AChE activity showed no difference. Moreover, no differences were observed after initiation of androgen deprivation therapy in serial patient samples. Velocity gradients identified that PSMA‐positive exosomes occupied a specific fraction of isolated EVs. A total of 35 patients with CRPC had mRNA analyzed from isolated plasma exosomes. Detectable exosomal KLK3 corresponded with higher concomitant serum PSA measurements, as expected (mean, 112.6 vs 26.61 ng/mL; P = .065). Furthermore, detectable levels of AR‐V7 mRNA were associated with a shorter time to progression (median, 16.0 vs 28.0 months; P = .0499). Furthermore, detectable exosomal AR‐V7 was significantly associated with testosterone levels below the lower limit of quantification (<0.1 nM).ConclusionsOur results suggest that exosomal AR‐V7 is correlated with lower sex steroid levels in CRPC patients with a poorer prognosis. PSMA immunocapture does not appear sufficient to isolate PCa‐specific exosomes.

Abstract Over 30% of patients with renal cell carcinoma (RCC) present with metastases, with median survival of 2 years. Kidney injury molecule 1 (KIM-1) is a cell-surface glycoprotein expressed by &gt;85% of RCC tumours and may serve as an early biomarker for RCC detection. Here we sought to determine if tumour KIM-1 plays a role in RCC cell extravasation and metastasis to lungs. Methods: We overexpressed KIM-1 in Renca cells (murine RCC) using stable transfection of an expression plasmid, or silenced endogenous KIM-1 in human 769-P and 786-O RCC cells using shRNA. We studied extravasation using the chorioallantoic membrane (CAM) model. We injected ~1x105 Renca or 769-P cells into blood vessels of chicken embryos (n=6), and measured percentage of cells that exited the capillary bed into surrounding spaces after 24 hours. To study lung metastasis, we injected ~5x105 Renca or 786-O cells intravenously through the tail vein of syngeneic BALB/c (n=10/group) or immune deficient Rag1-/- [BALB/c] (n=5/group) mice, and manually counted metastatic nodules in excised lungs after 17 days. Finally, we analyzed the publicly available Cancer Genome Atlas Illumina RNA-Seq dataset to determine if KIM-1 mRNA expression predicts overall survival in patients with clear cell RCC. Results: In CAM experiments, extravasation efficiency was significantly decreased in KIM-1pos 769-P cells compared to KIM-1neg 769-P cells (30.08% vs. 47.94%; p=0.0004), and in KIM-1pos Renca cells compared to KIM-1neg Renca cells (47.78% vs 60.73%; p=0.042). BALB/c mice injected with Kim-1pos Renca cells or Kim-1pos 786-O cells had significantly fewer lung metastases compared to mice injected with Kim-1neg Renca cells or Kim-1neg 786-O cells, respectively (257 vs 429, p=0.02; 131 vs 339 p=0.001). The inhibitory effect of KIM-1 expression in Renca and 786-O cells on numbers of lung metastases was similar in Rag1-/- mice, suggesting that the observed effects were not dependent on adaptive immunity. In the TCGA database, patients with the lowest vs. highest tertile of KIM-1 RCC expression has significantly higher mortality (unadjusted HR 1.82, 95% CI 1.23-2.70; p=0.0034). Conclusion: KIM-1 is a negative regulator of cell extravasation and inhibits the development of lung metastases in mice, and KIM-1 expression in human RCC may predict better survival. Deciphering the underlying mechanisms may lead to novel therapies. Citation Format: Jasper C. Lee, Marie A. Sarabusky, Audrey Champagne, Fabrice Lucien, Lakshman Gunaratnam. Kidney injury molecule-1 regulates metastasis in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4604.

Circulating extracellular vesicles (EVs) are a novel and emerging biomarker for nonalcoholic steatohepatitis (NASH). It has been demonstrated that total circulating EVs and hepatocyte‐derived EVs are elevated in male mice with diet‐induced NASH. How hepatocyte‐derived EVs change over time and other cellular sources of EVs in NASH have not been determined. Our objective was to define the quantitative evolution of hepatocyte‐derived, macrophage‐derived, neutrophil‐derived, and platelet‐derived EVs in male and female mice with dietary NASH. Fluorescently labeled antibodies and a nanoscale flow cytometer were used to detect plasma levels of EVs. Asialoglycoprotein receptor 1 (ASGR1) and cytochrome P450 family 2 subfamily E member 1 (CYP2E1) are markers of hepatocyte‐derived EVs; galectin 3 is a marker of macrophage‐derived EVs; common epitope on lymphocyte antigen 6 complex, locus G/C1 (Ly‐6G and Ly‐6C) is a marker of neutrophil‐derived EVs; and clusters of differentiation 61 (CD61) is a marker of platelet‐derived EVs. Nonalcoholic fatty liver disease activity score (NAS) was calculated using hematoxylin and eosin‐stained liver sections, and magnetic resonance imaging (MRI) was used for measurement of the fat fraction and elastography. Hepatocyte‐derived EVs increased in both male and female mice at 12 and 10 weeks of feeding, respectively, and remained elevated at 24 weeks in both male and female mice and at 48 weeks in male mice and 36 weeks in female mice. Macrophage‐ and neutrophil‐derived EVs were significantly elevated at 24 weeks of dietary feeding concomitant with the histologic presence of inflammatory foci in the liver. In fat‐, fructose‐, and cholesterol‐ (FFC) fed male mice, platelet‐derived EVs were elevated at 12, 24, and 48 weeks, whereas in female mice, platelet derived EVs were significantly elevated at 24 weeks. Hepatocyte‐, macrophage‐ and neutrophil‐derived EVs correlated well with the histologic NAS. Conclusion: Circulating cell‐type‐specific EVs may be a novel biomarker for NASH diagnosis and longitudinal follow up.

Background: Unprecedented clinical outcomes were reported after CD19 chimeric antigen receptor T-cell (CART19) therapy. However, the complete response rate in chronic lymphocytic leukemia (CLL) is much lower, at approximately 20%–30%. Several immune defects have been identified in CLL that result from the complex interaction between CLL cells and the microenvironment. This leukemic microenvironment is rich with extracellular vesicles (EVs) secreted by B-CLL cells. Here, we aimed to investigate the role of EVs play in the diminished CART response seen in some CLL patients. Methods: EVs were isolated from peripheral blood of 16 patients with untreated CLL at different Rai stages and risk profile by FISH. Cytometry was used to determine size, number of particles per μl, and CD19 expression. To investigate the impact of EVs on CAR T-cell functions, CART19 cells were stimulated with either CLL B cells or the CD19-positive cell line JEKO and different effector functions were analyzed. Results: Two patterns of EVs in CLL patients were identified; a single versus 2 distinct EV populations, characterized by size (small [EVssmall] and large [EVslarge], Fig 1A). In 25% of patients, EVs were CD19 positive (EVCD19+). CD19 positivity was detected only in patients with the EVslarge (Fig 1B). The EVs concentration, CD19 expression (EVsCD19+ vs EVsCD19-), or the size (EVssmall vs EVslarge) did not correlate with disease stage (early vs advanced Rai stage) or risk profile of CLL (low vs high risk). To investigate our hypothesis that EVs could modulate CART19 function, CART19 cell effector functions were examined in the presence of EVsCD19+, EVsCD19-, EVssmall, or EVslarge. EVs alone were insufficient to stimulate CART19 cells. However when CART19 cells were stimulated with the CD19-positive cell line JEKO, their effector functions were reduced only in the presence of EVsCD19+ but not EVsCD19-. This included a significant reduction in CART-specific killing (Fig 1C) and a reduction in cytokine production. The impairment of CART cell functions was independent of the size of EVs, ie, there was no impairment of CART functions with large or small size EVCD19- in co-culture. Conclusion: We identify CD19-positive large size EVs from patients with CLL and demonstrate that these EVs play a role in the leukemic microenvironment by reducing CAR T-cell activity. Studies are ongoing to define the mechanism(s).

Abstract The concept of vesicles or cell debris released by cancer cells to promote metastasis is not new, but the mechanisms used to currently ascribe their impact in metastasis are of intense debate. A significant increase in reports describing the role of cancer-derived EVs in cancer metastasis has been followed by a growing amount of uncertainty behind these claims. This review will delve into the role of EVs in promoting cancer metastasis by relying on a balanced perspective that looks at challenges faced previously by extracellular vesicle biologists, current technical limitations in the field, and overlooked physiologic mechanisms that may play a confounding role. This review will also discuss how certain experimental approaches are misleading which ultimately lead to overly optimistic mechanisms that have minimally contributed to the pathophysiology of metastasis.

Background Platelet microparticles (PMPs) and their abundance in the blood are a prognostic biomarker in thrombotic disorders and cancer. Nanoscale flow cytometry (nFC) is ideal for high-throughput analysis of PMPs but these clinical assays have not been developed previously. Objective This article demonstrates that nFC is a suitable technology to enumerate PMPs present in plasma samples in a clinical setting. Materials and Methods nFC was performed using the Apogee A50-Micro instrument. Instrument settings and acquisition parameters were developed with the use of fluorescent beads and plasma samples. Sample preparation and handling was also optimized. Results nFC allows for linear detection of particles between approximately 200 and 1,000 nm based on calibration beads and was dependent on dilution factor and flow rate. Linearity in event analysis as samples became more diluted was lost when events approximately 100 nm were gated while linearity was maintained despite dilution of sample in events larger than 200 nm in diameter. Higher flow rates lead to an under-estimation of events analysed per microlitre of analyte and this was more pronounced when plasma samples were not diluted more than 1/20×. Conclusion nFC offers multi-parametric analysis of PMPs when optimal calibration of acquisition and sample processing settings is performed. Analysis of plasmas from metastatic prostate cancer patients and leukaemia patients revealed that PMP levels were larger than 100 nm and were equally abundant in patients that responded to or failed androgen deprivation therapy or between patients representing different stages of leukaemia.

BackgroundThe ability to isolate extracellular vesicles (EVs) such as exosomes or microparticles is an important method that is currently not standardized. While commercially available kits offer purification of EVs from biofluids, such purified EV samples will also contain non‐EV entities such as soluble protein and nucleic acids that could confound subsequent experimentation. Ideally, only EVs would be isolated and no soluble protein would be present in the final EV preparation.MethodsWe compared commercially available EV isolation kits with immunoaffinity purification techniques and evaluated our final EV preparations using atomic force microscopy (AFM) and nanoscale flow cytometry (NFC). AFM is the only modality capable of detecting distinguishing soluble protein from EVs which is important for downstream proteomics approaches. NFC is the only technique capable of quantitating the proportion of target EVs to non‐target EVs in the final EV preparation.ResultsTo determine enrichment of prostate derived EVs relative to non‐target MPs, anti‐PSMA (Prostate Specific Membrane Antigen) antibodies were used in NFC. Antibody‐based immunoaffinity purification generated the highest quality of prostate derived EV preparations due to the lack of protein and RNA present in the samples. All kits produced poor purity EV preparations that failed to deplete the sample of plasma protein.ConclusionsWhile attractive due to their ease of use, EV purification kits do not provide substantial improvements in isolation of EVs from biofluids such as plasma. Immunoaffinity approaches are more efficient and economical and will also eliminate a significant portion of plasma proteins which is necessary for downstream approaches.