In Vitro Validation of the Therapeutic Potential of Dendrimer-Based Nanoformulations against Tumor Stem Cells.

Tumor cells with stem cell properties are considered to play major roles in promoting the development and malignant behavior of aggressive cancers. Therapeutic strategies that efficiently eradicate such tumor stem cells are of highest clinical need. Herein, we performed the validation of the polycationic phosphorus dendrimer-based approach for small interfering RNAs delivery in in vitro stem-like cells as models. As a therapeutic target, we chose Lyn, a member of the Src family kinases as an example of a prominent enzyme class widely discussed as a potent anti-cancer intervention point. Our selection is guided by our discovery that Lyn mRNA expression level in glioma, a class of brain tumors, possesses significant negative clinical predictive value, promoting its potential as a therapeutic target for future molecular-targeted treatments. We then showed that anti-Lyn siRNA, delivered into Lyn-expressing glioma cell model reduces the cell viability, a fact that was not observed in a cell model that lacks Lyn-expression. Furthermore, we have found that the dendrimer itself influences various parameters of the cells such as the expression of surface markers PD-L1, TIM-3 and CD47, targets for immune recognition and other biological processes suggested to be regulating glioblastoma cell invasion. Our findings prove the potential of dendrimer-based platforms for therapeutic applications, which might help to eradicate the population of cancer cells with augmented chemotherapy resistance. Moreover, the results further promote our functional stem cell technology as suitable component in early stage drug development.

Delayed development of chronic lymphocytic leukemia in the absence of macrophage migration inhibitory factor.

UNLABELLED: Survival of chronic lymphocytic leukemia (CLL) cells depends on stimuli provided by a suitable microenvironment. The factors and mechanisms providing this growth support for CLL cells are not fully understood. We found that plasma levels of macrophage migration inhibitory factor (MIF), a proinflammatory and immunoregulatory chemokine, were elevated in CLL patients. Therefore, we characterized the functional role of MIF in a CLL mouse model. For this purpose, we crossed Eµ-TCL1 mice with MIF knockout (MIF-/-) mice. The resulting TCL1+/wtMIF/ mice showed a delayed onset of leukemia, reduced splenomegaly and hepatomegaly, and a longer survival than TCL1+/wtMIFwt/wt controls. Immunohistochemical examination of the lymphoid organs showed that the numbers of macrophages were significantly reduced in the spleen and bone marrow of TCL1+/wtMIF/ mice compared with TCL1+/wtMIFwt/wt controls. Mechanistic studies in vitro revealed that the absence of MIF rendered CLL cells more susceptible to apoptosis. Accordingly, incubation with an anti-MIF antibody reduced the survival of CLL cells on a macrophage feeder layer. In addition, the migratory activity of TCL1+/wtMIF/ macrophages was decreased compared with TCL1+/wtMIFwt/wt macrophages. Taken together, our results provide evidence that MIF supports the development of CLL by enhancing the interaction of CLL cells with macrophages. KEY POINTS: Targeted deletion of the gene for macrophage migration inhibitory factor (MIF) delays development of chronic lymphocytic leukemia and prolongs survival in mice. MIF recruits leukemia-associated macrophages to spleen or liver.

Impact of leukemia-associated macrophages on the progression and therapy response of chronic lymphocytic leukemia.

The treatment landscape of chronic lymphocytic leukemia (CLL) has advanced remarkably over the past decade. The advent and approval of the BTK inhibitor ibrutinib and BCL-2 inhibitor venetoclax, as well as monoclonal anti-CD20 antibodies rituximab and obinutuzumab, have resulted in deep remissions and substantially improved survival outcomes for patients. However, CLL remains a complex disease with many patients still experiencing relapse and unsatisfactory treatment responses. CLL cells are highly dependent on their pro-leukemic tumor microenvironment (TME), which comprises different cellular and soluble factors. A large body of evidence suggests that CLL-associated macrophages shaped by leukemic cells play a pivotal role in maintaining CLL cell survival. In this review, we summarize the pro-survival interactions between CLL cells and macrophages, as well as the impact of the current first-line treatment agents, including ibrutinib, venetoclax, and CD20 antibodies on leukemia-associated macrophages.

Role of the tumor microenvironment in CLL pathogenesis.

Chronic lymphocytic leukemia (CLL) cells extensively interact with and depend on their surrounding tumor microenvironment (TME). The TME encompasses a heterogeneous array of cell types, soluble signals, and extracellular vesicles, which contribute significantly to CLL pathogenesis. CLL cells and the TME cooperatively generate a chronic inflammatory milieu, which reciprocally reprograms the TME and activates a signaling network within CLL cells, promoting their survival and proliferation. Additionally, the inflammatory milieu exerts chemotactic effects, attracting CLL cells and other immune cells to the lymphoid tissues. The intricate CLL-TME interactions also facilitate immune evasion and compromise leukemic cell surveillance. We also review recent advances that have shed light on additional aspects that are substantially influenced by the CLL-TME interplay.

Headache and neurological deficits with cerebrospinal fluid lymphocytosis (HaNDL) syndrome with reversible cytotoxic lesion of the corpus callosum (CLOCC) on MRI of the brain.

Headache and neurological deficits with cerebrospinal fluid (CSF) lymphocytosis (HaNDL) is a rare condition characterised by recurrent episodes of headache and transient neurological deficits. This case report presents a young patient initially diagnosed with hemiplegic migraine, having a normal brain CT, with focal cerebral perfusion mismatch not restricted to a single vascular territory on CT angiography. Brain MRI revealed a cytotoxic lesion of the splenium in the corpus callosum (CLOCC), a feature also reported in migraine. However, recurrent headaches with neurological deficits prompted further investigations with CSF analysis and brain MRI, confirming HaNDL and demonstrating reversibility of CLOCC. Recognising HaNDL as a differential diagnosis is essential in patients with recurrent headaches with focal neurological deficits, given the differences in therapeutic approach. The relationship between migraine and HaNDL is not fully understood, but they may share a pathophysiological link. Awareness of this is crucial for accurate diagnosis.

Carriers for hydrophobic drug molecules: lipid-coated hollow mesoporous silica particles, and the influence of shape and size on encapsulation efficiency.

Hydrophobic drugs, while designed to interact with specific receptors or enzymes located in lipid-rich cell membranes, often face challenges of limited bioavailability and insufficient circulation time due to their insolubility in aqueous environments. One plausible pathway to increase their blood circulation time is to load these drugs into biocompatible and hydrophilic carriers to enhance their uptake. In this study, mesoporous silica (mSiO2) nanocarriers of various morphologies (including cubes, capsules, and spheres) were synthesized. These nanocarriers were then surface-functionalized with alkyl chain hydrocarbons, specifically octadecyl-trimethoxysilane, (OCH3)3Si(CH2)17CH3, to render them hydrophobic. The resulting nanocarriers (((OCH3)3Si(CH2)17CH3)@mSiO2) showed up to 80% uptake for hydrophobic drugs. However, a significant drawback was observed as most of the drugs were prone to uncontrollable release within 6 h. This challenge of premature drug release was successfully mitigated by effectively sealing the drug-loaded nanocarriers with a pH-sensitive lipid overlayer. The lipid-coated nanocarriers prolonged drug containment and sustained release up to 72 h, compared to 6 h for uncoated nanocarriers, thereby facilitating longer blood circulation times. Moreover, the shape and size of nanocarriers were found to influence both drug entrapment capacity and release behavior with cubic forms exhibiting superior loading capacity due to higher surface area and porosity. Additionally, it was observed that the molecular weight and chemical structure of the drug molecules played a crucial role in determining their uptake and release profiles. Furthermore, the influence of different morphologies of nanocarriers on cell uptake and cytotoxicity in immune cells was elucidated. These findings underscore the importance of nanocarrier morphology and drug properties to enhance loading capacities and controlled release profiles, for designing drug delivery systems tailored for hydrophobic drugs.

Eph receptor B4 is a regulator of estrogen receptor alpha in breast cancer cells.

BACKGROUND: Estrogen receptor alpha (ER-α) plays an important role in breast cancer initiation and progression and represents a major target in cancer therapy. The expression and activity of ER-α is regulated by multiple mechanisms at the transcriptional and post-translational level. Interaction of tyrosine kinase receptor-activated signaling pathways with ER-α function has been reported. We previously performed a kinome-wide small interfering RNA high-throughput screen to identify novel protein kinases involved in the regulation of ER-α transcriptional activity in human breast cancer cells. Our screening analysis identified the Eph receptor tyrosine kinases (Eph) as potential positive regulators of ER-α. RESULTS: In this study, we demonstrate Eph receptor B4 (EphB4), a member of Eph kinase family, a positive regulator of ER-α in human breast cancer cell lines (MCF-7, T-47D and BT-474). Down-regulation of EphB4 by RNA interference technology impairs estrogen-dependent ER-α transcriptional activity in breast cancer cells. Decreased activity of ER-α after EphB4 knockdown is the consequence of diminished ER-α messenger RNA and protein expression. Furthermore, phosphorylation of Akt, a downstream mediator of EphB4, is reduced following EphB4 silencing. CONCLUSIONS: Our data suggests EphB4 as an upstream regulator of ER-α in human breast cancer cells by modulating ER-α transcription. The results also suggest Akt as a relevant downstream signaling molecule in this novel EphB4-ER-α pathway.

Non-viral TRAC-knocked-in CD19KICAR-T and gp350KICAR-T cells tested against Burkitt lymphomas with type 1 or 2 EBV infection: In vivo cellular dynamics and potency.

Introduction: The ubiquitous Epstein-Barr virus (EBV) is an oncogenic herpes virus associated with several human malignancies. EBV is an immune-evasive pathogen that promotes CD8+ T cell exhaustion and dysregulates CD4+ T cell functions. Burkitt lymphoma (BL) is frequently associated with EBV infections. Since BL relapses after conventional therapies are difficult to treat, we evaluated prospective off-the-shelf edited CAR-T cell therapies targeting CD19 or the EBV gp350 cell surface antigen. Methods: We used CRISPR/Cas9 gene editing methods to knock in (KI) the CD19CAR.CD28z or gp350CAR.CD28z into the T cell receptor (TCR) alpha chain (TRAC) locus. Results: Applying upscaled methods with the ExPERT ATx® MaxCyte system, KI efficacy was ~20% of the total ~2 × 108 TCR-knocked-out (KO) generated cells. KOTCRKICAR-T cells were co-cultured in vitro with the gp350+CD19+ BL cell lines Daudi (infected with type 1 EBV) or with Jiyoye (harboring a lytic type 2 EBV). Both types of CAR-T cells showed cytotoxic effects against the BL lines in vitro. CD8+ KICAR-T cells showed higher persistency than CD4+ KICAR-T cells after in vitro co-culture with BL and upregulation of the activation/exhaustion markers PD-1, LAG-3, and TIM-3. Two preclinical in vivo xenograft models were set up with Nod.Rag.Gamma mice injected intravenously (i.v.) with 2 × 105 Daudi/fLuc-GFP or with Jiyoye/fLuc-GFP cells. Compared with the non-treated controls, mice challenged with BL and treated with CD19KICAR-T cells showed delayed lymphoma dissemination with lower EBV DNA load. Notably, for the Jiyoye/fLuc-GFP model, almost exclusively CD4+ CD19KICAR-T cells were detectable at the endpoint analyses in the bone marrow, with increased frequencies of regulatory T cells (Tregs) and TIM-3+CD4+ T cells. Administration of gp350KICAR-T cells to mice after Jiyoye/GFP-fLuc challenge did not inhibit BL growth in vivo but reduced the EBV DNA load in the bone marrow and promoted gp350 antigen escape. CD8+PD-1+LAG-3+ gp350KICAR-T cells were predominant in the bone marrow. Discussion: The two types of KOTCRKICAR-T cells showed different therapeutic effects and in vivo dynamics. These findings reflect the complexities of the immune escape mechanisms of EBV, which may interfere with the CAR-T cell property and potency and should be taken into account for future clinical translation.

LYN kinase programs stromal fibroblasts to facilitate leukemic survival via regulation of c-JUN and THBS1.

Microenvironmental bystander cells are essential for the progression of chronic lymphocytic leukemia (CLL). We have discovered previously that LYN kinase promotes the formation of a microenvironmental niche for CLL. Here we provide mechanistic evidence that LYN regulates the polarization of stromal fibroblasts to support leukemic progression. LYN is overexpressed in fibroblasts of lymph nodes of CLL patients. LYN-deficient stromal cells reduce CLL growth in vivo. LYN-deficient fibroblasts show markedly reduced leukemia feeding capacity in vitro. Multi-omics profiling reveals that LYN regulates the polarization of fibroblasts towards an inflammatory cancer-associated phenotype through modulation of cytokine secretion and extracellular matrix composition. Mechanistically, LYN deletion reduces inflammatory signaling including reduction of c-JUN expression, which in turn augments the expression of Thrombospondin-1, which binds to CD47 thereby impairing CLL viability. Together, our findings suggest that LYN is essential for rewiring fibroblasts towards a leukemia-supportive phenotype.

Distinct Genetically Determined Origins of Myd88/BCL2-Driven Aggressive Lymphoma Rationalize Targeted Therapeutic Intervention Strategies.

Genomic profiling revealed the identity of at least 5 subtypes of diffuse large B-cell lymphoma (DLBCL), including the MCD/C5 cluster characterized by aberrations in MYD88, BCL2, PRDM1, and/or SPIB. We generated mouse models harboring B cell-specific Prdm1 or Spib aberrations on the background of oncogenic Myd88 and Bcl2 lesions. We deployed whole-exome sequencing, transcriptome, flow-cytometry, and mass cytometry analyses to demonstrate that Prdm1- or Spib-altered lymphomas display molecular features consistent with prememory B cells and light-zone B cells, whereas lymphomas lacking these alterations were enriched for late light-zone and plasmablast-associated gene sets. Consistent with the phenotypic evidence for increased B cell receptor signaling activity in Prdm1-altered lymphomas, we demonstrate that combined BTK/BCL2 inhibition displays therapeutic activity in mice and in five of six relapsed/refractory DLBCL patients. Moreover, Prdm1-altered lymphomas were immunogenic upon transplantation into immuno-competent hosts, displayed an actionable PD-L1 surface expression, and were sensitive to antimurine-CD19-CAR-T cell therapy, in vivo. SIGNIFICANCE: Relapsed/refractory DLBCL remains a major medical challenge, and most of these patients succumb to their disease. Here, we generated mouse models, faithfully recapitulating the biology of MYD88-driven human DLBCL. These models revealed robust preclinical activity of combined BTK/BCL2 inhibition. We confirmed activity of this regimen in pretreated non-GCB-DLBCL patients. See related commentary by Leveille et al., p. 8. This article is highlighted in the In This Issue feature, p. 1.

The scaffold protein NEDD9 is necessary for leukemia-cell migration and disease progression in a mouse model of chronic lymphocytic leukemia.

The scaffold protein NEDD9 is frequently upregulated and hyperphosphorylated in cancers, and is associated with poor clinical outcome. NEDD9 promotes B-cell adhesion, migration and chemotaxis, pivotal processes for malignant development. We show that global or B-cell-specific deletion of Nedd9 in chronic lymphocytic leukemia (CLL) mouse models delayed CLL development, markedly reduced disease burden and resulted in significant survival benefit. NEDD9 was required for efficient CLL cell homing, chemotaxis, migration and adhesion. In CLL patients, peripheral NEDD9 expression was associated with adhesion and migration signatures as well as leukocyte count. Additionally, CLL lymph nodes frequently expressed high NEDD9 levels, with a subset of patients showing NEDD9 expression enriched in the CLL proliferation centers. Blocking activity of prominent NEDD9 effectors, including AURKA and HDAC6, effectively reduced CLL cell migration and chemotaxis. Collectively, our study provides evidence for a functional role of NEDD9 in CLL pathogenesis that involves intrinsic defects in adhesion, migration and homing.

Spleen tyrosine kinase mediates innate and adaptive immune crosstalk in SARS-CoV-2 mRNA vaccination.

Durable cell-mediated immune responses require efficient innate immune signaling and the release of pro-inflammatory cytokines. How precisely mRNA vaccines trigger innate immune cells for shaping antigen specific adaptive immunity remains unknown. Here, we show that SARS-CoV-2 mRNA vaccination primes human monocyte-derived macrophages for activation of the NLRP3 inflammasome. Spike protein exposed macrophages undergo NLRP3-driven pyroptotic cell death and subsequently secrete mature interleukin-1ß. These effects depend on activation of spleen tyrosine kinase (SYK) coupled to C-type lectin receptors. Using autologous cocultures, we show that SYK and NLRP3 orchestrate macrophage-driven activation of effector memory T cells. Furthermore, vaccination-induced macrophage priming can be enhanced with repetitive antigen exposure providing a rationale for prime-boost concepts to augment innate immune signaling in SARS-CoV-2 vaccination. Collectively, these findings identify SYK as a regulatory node capable of differentiating between primed and unprimed macrophages, which modulate spike protein-specific T cell responses.

Stromal cells support the survival of human primary chronic lymphocytic leukemia (CLL) cells through Lyn-driven extracellular vesicles.

Introduction: In chronic lymphocytic leukemia (CLL), the tumor cells receive survival support from stromal cells through direct cell contact, soluble factors and extracellular vesicles (EVs). The protein tyrosine kinase Lyn is aberrantly expressed in the malignant and stromal cells in CLL tissue. We studied the role of Lyn in the EV-based communication and tumor support. Methods: We compared the Lyn-dependent EV release, uptake and functionality using Lyn-proficient (wild-type) and -deficient stromal cells and primary CLL cells. Results: Lyn-proficient cells caused a significantly higher EV release and EV uptake as compared to Lyn-deficient cells and also conferred stronger support of primary CLL cells. Proteomic comparison of the EVs from Lyn-proficient and -deficient stromal cells revealed 70 significantly differentially expressed proteins. Gene ontology studies categorized many of which to organization of the extracellular matrix, such as collagen, fibronectin, fibrillin, Lysyl oxidase like 2, integrins and endosialin (CD248). In terms of function, a knockdown of CD248 in Lyn+ HS-5 cells resulted in a diminished B-CLL cell feeding capacity compared to wildtype or scrambled control cells. CD248 is a marker of certain tumors and cancer-associated fibroblast (CAF) and crosslinks fibronectin and collagen in a membrane-associated context. Conclusion: Our data provide preclinical evidence that the tyrosine kinase Lyn crucially influences the EV-based communication between stromal and primary B-CLL cells by raising EV release and altering the concentration of functional molecules of the extracellular matrix.

An international survey of different transcranial magnetic stimulation (TMS) protocols for patients with obsessive-compulsive disorder (OCD).

This study aims to evaluate current preferences and trends in the delivery of Transcranial Magnetic Stimulation (TMS) for Obsessive-Compulsive Disorder (OCD). A 10-item online questionnaire was developed and conducted online between April to June 2020, surveying providers of TMS for patients with OCD internationally. A total of 27 valid responses were analysed from 10 countries. The most common target for TMS was the supplementary motor area and stimulation was commonly given bilaterally, but techniques differed between centres. Exposure tasks were not commonly used during TMS. The study calls for more research clarifying the best mode of TMS delivery for OCD.

CD30-Positive Extracellular Vesicles Enable the Targeting of CD30-Negative DLBCL Cells by the CD30 Antibody-Drug Conjugate Brentuximab Vedotin.

CD30, a member of the TNF receptor superfamily, is selectively expressed on a subset of activated lymphocytes and on malignant cells of certain lymphomas, such as classical Hodgkin Lymphoma (cHL), where it activates critical bystander cells in the tumor microenvironment. Therefore, it is not surprising that the CD30 antibody-drug conjugate Brentuximab Vedotin (BV) represents a powerful, FDA-approved treatment option for CD30+ hematological malignancies. However, BV also exerts a strong anti-cancer efficacy in many cases of diffuse large B cell lymphoma (DLBCL) with poor CD30 expression, even when lacking detectable CD30+ tumor cells. The mechanism remains enigmatic. Because CD30 is released on extracellular vesicles (EVs) from both, malignant and activated lymphocytes, we studied whether EV-associated CD30 might end up in CD30- tumor cells to provide binding sites for BV. Notably, CD30+ EVs bind to various DLBCL cell lines as well as to the FITC-labeled variant of the antibody-drug conjugate BV, thus potentially conferring the BV binding also to CD30- cells. Confocal microscopy and imaging cytometry studies revealed that BV binding and uptake depend on CD30+ EVs. Since BV is only toxic toward CD30- DLBCL cells when CD30+ EVs support its uptake, we conclude that EVs not only communicate within the tumor microenvironment but also influence cancer treatment. Ultimately, the CD30-based BV not only targets CD30+ tumor cell but also CD30- DLBCL cells in the presence of CD30+ EVs. Our study thus provides a feasible explanation for the clinical impact of BV in CD30- DLBCL and warrants confirming studies in animal models.

Constitutive activation of Lyn kinase enhances BCR responsiveness, but not the development of CLL in Eµ-TCL1 mice.

The treatment of chronic lymphocytic leukemia (CLL) has been improved dramatically by inhibitors targeting B-cell receptor (BCR)-associated kinases. The tyrosine kinase Lyn is a key modulator of BCR signaling and shows increased expression and activity in CLL. To evaluate the functional relevance of Lyn for CLL, we generated a conditional knockin mouse model harboring a gain-of-function mutation of the Lyn gene (LynY508F), which was specifically expressed in the B-cell lineage (Lynup-B). Kinase activity profiling revealed an enhanced responsiveness to BCR stimulation in Lynup-B B cells. When crossing Lynup-B mice with Eµ-TCL1 mice (TCL1tg/wt), a transgenic mouse model for CLL, the resulting TCL1tg/wt Lynup-B mice showed no significant change of hepatomegaly, splenomegaly, bone marrow infiltration, or overall survival when compared with TCL1tg/wt mice. Our data also suggested that TCL1 expression has partially masked the effect of the Lynup-B mutation, because the BCR response was only slightly increased in TCL1tg/wt Lynup-B compared with TCL1tg/wt. In contrast, TCL1tg/wt Lynup-B were protected at various degrees against spontaneous apoptosis in vitro and upon treatment with kinase inhibitors targeting the BCR. Collectively, and consistent with our previous data in a Lyn-deficient CLL model, these data lend further suggest that an increased activation of Lyn kinase in B cells does not appear to be a major driver of leukemia progression and the level of increased BCR responsiveness induced by Lynup-B is insufficient to induce clear changes to CLL pathogenesis in vivo.

Meta-Analysis Reveals Significant Sex Differences in Chronic Lymphocytic Leukemia Progression in the Eµ-TCL1 Transgenic Mouse Model.

The Eµ-TCL1 transgenic mouse model represents the most widely and extensively used animal model for chronic lymphocytic leukemia (CLL). In this report, we performed a meta-analysis of leukemia progression in over 300 individual Eµ-TCL1 transgenic mice and discovered a significantly accelerated disease progression in females compared to males. This difference is also reflected in an aggressive CLL mouse model with additional deletion of Tp53 besides the TCL1 transgene. Moreover, after serial adoptive transplantation of murine CLL cells, female recipients also succumbed to CLL earlier than male recipients. This sex-related disparity in the murine models is markedly contradictory to the human CLL condition. Thus, due to our observation we urge both careful consideration in the experimental design and accurate description of the Eµ-TCL1 transgenic cohorts in future studies.

CD74 is dispensable for development of chronic lymphocytic leukemia in Eµ-TCL1 transgenic mice.

CD74 is a surface protein expressed on immune cells, which acts as receptor for the chemokine macrophage migration inhibitory factor (MIF). Signaling via the MIF/CD74-axis has been reported to be important for the pathogenesis of chronic lymphocytic leukemia (CLL). We wanted to clarify the role of CD74 in MIF-induced signaling/leukemic development. In Eµ-TCL1 transgenic mice, occurrence of the leukemic phenotype was associated with increased surface CD74 expression. Eµ-TCL1+/+Cd74-/- mice showed similar kinetics and clinical features of CLL development as Eµ-TCL1+/+ mice. MIF stimulation of leukemic splenocytes led to AKT activation in a CD74-dependent manner. AKT activation was reduced in Cd74-deficient splenocytes in the presence of the oncogenic TCL1-transgene. Tumor cell apoptosis/proliferation were unaffected in Eµ-TCL1+/+Cd74-/- mice. Our data suggest that the need for active CD74 signaling is overcome in the leukemic context of TCL1-driven CLL, and that CD74 may have a dispensable role for CLL pathogenesis in this model.

New roles for B cell receptor associated kinases: when the B cell is not the target.

Targeting of B cell receptor associated kinases (BAKs), such as Bruton's tyrosine kinase (BTK) or phosphoinositol-3-kinase (PI3K) delta, by specific inhibitors has revolutionized the therapy of B lymphoid malignancies. BAKs are critical signaling transducers of BCR signaling and seem relevant in B cell lymphoma pathogenesis. The functional relevance of BTK for lymphoid malignancies is strongly supported by the observation that resistance to therapy in CLL patients treated with BTK inhibitors such as ibrutinib is often associated with mutations in genes coding for BTK or Phospholipase-C gamma (PLCÉ£). In some contrast, next generation sequencing data show that BAKs are mutated at very low frequency in treatment-naïve B cell lymphomas. Therefore, it remains debatable whether BAKs are essential drivers for lymphoma development. In addition, results obtained by targeted deletion of BAKs such as Lyn and Btk in murine CLL models suggest that BAKs may be essential to shape the dialogue between malignant B cells and the tumor microenvironment (TME). Since BAKs are expressed in multiple cell types, BAK inhibitors may disrupt the lymphoma supportive microenvironment. This concept also explains the typical response to BAK inhibitor treatment, characterized by a long-lasting increase of peripheral blood lymphoid cells, due to a redistribution from the lymphoid homing compartments. In addition, BAK inhibitors have shown some efficacy in solid tumors, probably through mediator cells in the TME. This review summarizes and validates the evidence for BAK inhibitors being part of a class of agents that modulate the (hematopoietic) microenvironment of cancers.

Extracellular vesicle measurements with nanoparticle tracking analysis - An accuracy and repeatability comparison between NanoSight NS300 and ZetaView.

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 (% DTEM range: 79.5-134.3) compared to ZetaView (% DTEM 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.