The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods to characterize single EVs. Nanoparticle Tracking Analysis (NTA) is commonly used to determine EV concentration and diameter. As the EV field is lacking methods to easily confirm and validate NTA data, questioning the reliability of measurements remains highly important. In this regard, a comparison addressing measurement quality between different NTA devices such as Malvern’s NanoSight NS300 or Particle Metrix’ ZetaView has not yet been conducted. To evaluate the accuracy and repeatability of size and concentration determinations of both devices, we employed comparative methods including transmission electron microscopy (TEM) and single particle interferometric reflectance imaging sensing (SP-IRIS) by ExoView. Multiple test measurements with nanospheres, liposomes and ultracentrifuged EVs from human serum and cell culture supernatant were performed. Additionally, serial dilutions and freeze-thaw cycle-dependent EV decrease were measured to determine the robustness of each system. Strikingly, NanoSight NS300 exhibited a 2.0–2.1-fold overestimation of polystyrene and silica nanosphere concentration. By measuring serial dilutions of EV samples, we demonstrated higher accuracy in concentration determination by ZetaView (% BIAS range: 2.7–8.5) in comparison with NanoSight NS300 (% BIAS range: 32.9–36.8). The concentration measurements by ZetaView were also more precise (% CV range: 0.0–4.7) than measurements by NanoSight NS300 (% CV range: 5.4–10.7). On the contrary, quantitative TEM imaging indicated more accurate EV sizing by NanoSight NS300 (% D TEM range: 79.5–134.3) compared to ZetaView (% D TEM range: 111.8–205.7), while being equally repeatable (NanoSight NS300% CV range: 0.8–6.7; ZetaView: 1.4–7.8). However, both devices failed to report a peak EV diameter below 60 nm compared to TEM and SP-IRIS. Taken together, NTA devices differ strongly in their hardware and software affecting measuring results. ZetaView provided a more accurate and repeatable depiction of EV concentration, whereas NanoSight NS300 supplied size measurements of higher resolution.
Despite the undeniable vast improvement in CLL treatment strategies, resistance to novel compounds such as ibrutinib and venetoclax already emerged and posed a challenge in many aggressive cases. The fundamental role of the homing process in CLL progression and presumably relapse prompted us to analyze the impact of a crucial regulator of chemokine response, migration and lymphocyte homing - namely NEDD9 - on CLL pathogenesis. The scaffold protein NEDD9 is frequently upregulated and hyperphosphorylated in different cancer entities, with its deregulation being associated with poor clinical outcome and therapy resistance. In B cells, activation of integrin- and the B cell receptor signaling pathways leads to hyperphosphorylation of NEDD9, predominantly by Src family kinases, promoting cell adhesion, migration and chemotaxis. To elucidate the functional relevance of NEDD9 in CLL pathogenesis in vivo,Eµ-TCL1 transgenic mice were crossbred with Nedd9 deficient mice. CLL burden was monitored in the peripheral blood of Nedd9-proficient (TCL1tg/wt Nedd9wt/wt)versus Nedd9-deficient (TCL1tg/wt Nedd9-/-) mice every two months over a year, revealing a significantly lower proportion of CLL per total B cells (CLL/B cells) in the peripheral blood in TCL1tg/wt Nedd9-/- mice at four and six months of age. CLL onset was clearly delayed in TCL1tg/wt Nedd9-/- mice in comparison to TCL1tg/wt Nedd9wt/wt mice. The infiltration of CLL cells into the spleen and bone marrow was significantly reduced in TCL1tg/wt Nedd9-/- mice at three and ten months, accompanied by significantly longer overall survival of the TCL1tg/wt Nedd9-/- group. Particularly, this eminent role of Nedd9 in CLL pathogenesis could be largely attributed to Nedd9 expression in B cells. Using a conditional Nedd9 knockout mouse exclusively in B cells (TCL1tg/wt CD19Cretg/wt Nedd9flfl (TCN)), we observed a highly similar phenotype of TCN mice to the TCL1tg/wt Nedd9-/- mice, including significantly delayed CLL onset, lower proportion of CLL per total B cells (CLL/B cells) in the peripheral blood, and reduced hepatosplenomegaly in TCN mice compared to the control TCL1tg/wt CD19Crewt/wt Nedd9flfl (TN) mice. In summary, our mouse data suggest that Nedd9 deficiency significantly delayed CLL onset and progression, particularly in the early stages of CLL. Moreover, Nedd9 deficiency significantly decreased the accumulation of CLL cells both in typical leukemic homing organs such as spleen and bone marrow as well as in the peripheral blood in two independent mouse models and significantly prolonged survival of the TCL1tg/wt Nedd9-/- mice. The strongly reduced capacity of Nedd9-deficient CLL cells to migrate and home to the lymphoid niche prompted us to investigate the underlying mechanistic signaling pathway upon Nedd9 loss. For this purpose, we examined surface expression levels of prominent cell trafficking mediators on Nedd9-proficient and -deficient CLL cells, and found a consistently reduced level of CXCR4 on the surface of TCL1tg/wt Nedd9-/-cells. In the transwell assay, CLL cells isolated from TCL1tg/wt Nedd9-/- mice showed a dramatic reduction in migration towards CXCL12 compared to their wild type counter parts. In line with the results of the murine CLL cells, NEDD9-depleted MEC1 cells (shNedd9) also showed decreased CXCR4 levels and dramatic reduction in migration towards CXCL12 compared to the control cell line (shNT). Collectively, we provide the first direct evidence that genetic targeting of Nedd9 in vivo impairs CLL cell adhesion, migration and chemotaxis, resulting in decreased CLL cell infiltration into secondary lymphoid organs and the bone marrow. These observations could serve as basis for the development of new treatment strategies, targeting a scaffold protein to impair the homing process of CLL cells, a prerequisite for their survival and expansion within the microenvironment of protective niches. Disclosures No relevant conflicts of interest to declare.
Growth of chronic lymphocytic leukemia cells strongly depends on a nurturing microenvironmental niche that is specifically primed by diverse, bi-directional interactions to promote leukemic homing, proliferation and progression. The Src-family kinase Lyn was previously identified by our group as a key factor for the formation of this pro-leukemic niche and for the expansion of CLL cells, using the Eµ-TCL1 mouse model. In order to attribute the pro-leukemic function of Lyn to a specific cell type, chimeric mice with lineage-specific defects of Lyn within hematopoietic or non-hematopoietic compartments were generated by irradiating BL6J-mice lethally and restoring their hematopoietic system with Lyn-WT or Lyn-KO stem cells. Consecutively mice were xenotransplanted with TCL1+-malignant cells. Lyn deficiency within the non-hematopoietic compartment decelerated leukemic expansion to a higher degree than did Lyn deficiency within the hematopoietic compartment. Completely Lyn deficient mice showed a more prominent retardation of leukemic expansion compared with both lineage specific Lyn deficient mouse strains, suggesting an additive effect of the two distinct compartments for leukemic expansion. In focusing on the non-hematopoietic fibroblastic bystander cells, primary human CLL cells were cocultured in vitro with Lyn-deficient mouse embryonic fibroblasts as well as Lyn-KO human HS5 cells, generated via the CRISPR-Cas9 system, and leukemic cell survival was assessed over time. All Lyn-deficient fibroblasts showed a significantly reduced feeding capacity for CLL cells compared to WT stroma, indicating the functional relevance of Lyn in leukemia-associated fibroblasts. Subsequently, transcriptomic, proteomic and phosphoproteomic alterations related to Lyn-KO in HS5 cells were comprehensively analyzed, revealing a surprisingly extensive change in gene and protein expression pattern that appeared to be regulated mainly at the transcriptional level. The differentially expressed genes were remarkably often extracellular matrix (ECM)-, cytoskeleton- or cytokine-associated. GO-term enrichment analysis additionally suggested a correlation with ECM processes. Therefore, we hypothesized that Lyn-deficiency might induce transcriptional changes of the cancer-associated fibroblast (CAF)-like phenotype, thus leading to a reduction in leukemic feeding capacity. The diminished expression of several CAF-makers congruent with this reduced activation status was validated in Lyn-KO fibroblasts, as well as the transcriptionally regulated differential expression of chosen target genes. Amongst those, the deubiquitinating enzyme UCHL1 was most abundantly reduced in Lyn-KO HS5 cells, showing an almost complete loss of mRNA and protein expression. Application of a specific UCHL1-inhibitor -in a dose without toxic effects on CLL cells - to CLL-stroma coculture resulted in a significantly hampered feeder effect and reduced CLL cell survival, implying a functional relevance of microenvironmental UCHL1 for stromal support in our system. Additionally, stroma cell death induced by higher drug concentrations in WT cells was completely prohibited in Lyn-KO stroma, illustrating the importance of Lyn for regulating UCHL1 expression and function. In summary, we propose that the Lyn kinase contributes to the formation of a supportive microenvironment via the transcriptional reprogramming of stroma fibroblasts into a "CAF-like" phenotype, which echances viability of CLL cells. In addition, UCHL1 might be a potentially druggable mediator of this activation process. Disclosures Hallek: Roche, Gilead Sciences, Inc., Mundipharma, Janssen, Celgene, Pharmacyclics, AbbVie: Honoraria, Research Funding, Speakers Bureau.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.