Molecular and functional characterization of reversible-sunitinib-tolerance state in human renal cell carcinoma.

Therapy failure with the tyrosine kinase inhibitor (TKI) sunitinib remains a great challenge in metastatic renal cell carcinoma (mRCC). Growing evidence indicates that the tumour subpopulation can enter a transient, non-mutagenic drug-tolerant state to endure the treatment underlying the minimal residual disease and tumour relapse. Drug tolerance to sunitinib remains largely unexplored in RCC. Here, we show that sunitinib-tolerant 786-O/S and Caki-2/S cells are induced by prolonged drug treatment showing reduced drug sensitivity, enhanced clonogenicity, and DNA synthesis. Sunitinib-tolerance developed via dynamic processes, including (i) engagement of c-MET and AXL pathways, (ii) alteration of stress-induced p38 kinase and pro-survival BCL-2 signalling, (iii) extensive actin remodelling, which was correlated with activation of focal adhesion proteins. Remarkably, the acute drug response in both sensitive and sunitinib-tolerant cell lines led to dramatic fine-tuning of the actin-cytoskeleton and boosted cellular migration and invasion, indicating that the drug-response might depend on cell state transition rather than pre-existing mutations. The drug-tolerant state was transiently acquired, as the cells resumed initial drug sensitivity after >10 passages under drug withdrawal, reinforcing the concept of dynamic regulation and phenotypic heterogeneity. Our study described molecular events contributing to the reversible switch into sunitinib-tolerance, providing possible novel therapeutic opportunities in RCC.

Gas6/TAM system as potential biomarker for multiple sclerosis prognosis.

Introduction: The protein growth arrest-specific 6 (Gas6) and its tyrosine kinase receptors Tyro-3, Axl, and Mer (TAM) are ubiquitous proteins involved in regulating inflammation and apoptotic body clearance. Multiple sclerosis (MS) is the most common inflammatory demyelinating disease of the central nervous system leading to progressive and irreversible disability if not diagnosed and treated promptly. Gas6 and TAM receptors have been associated with neuronal remyelination and stimulation of oligodendrocyte survival. However, few data are available regarding clinical correlation in MS patients. We aimed to evaluate soluble levels of these molecules in the cerebrospinal fluid (CSF) and serum at MS diagnosis and correlate them with short-term disease severity. Methods: In a prospective cohort study, we enrolled 64 patients with a diagnosis of clinical isolated syndrome (CIS), radiological isolated syndrome (RIS) and relapsing-remitting (RR) MS according to the McDonald 2017 Criteria. Before any treatment initiation, we sampled the serum and CSF, and collected clinical data: disease course, presence of gadolinium-enhancing lesions, and expanded disability status score (EDSS). At the last clinical follow-up, we assessed EDSS and calculated MS severity score (MSSS) and age-related MS severity (ARMSS). Gas6 and TAM receptors were determined using an ELISA kit (R&D Systems) and compared to neurofilament (NFLs) levels evaluated with SimplePlex™ fluorescence-based immunoassay. Results: At diagnosis, serum sAxl was higher in patients receiving none or low-efficacy disease-modifying treatments (DMTs) versus patients with high-efficacy DMTs (p = 0.04). Higher CSF Gas6 and serum sAXL were associated with an EDSS <3 at diagnosis (p = 0.04; p = 0.037). Serum Gas6 correlates to a lower MSSS (r2 = -0.32, p = 0.01). Serum and CSF NFLs were confirmed as disability biomarkers in our cohort according to EDSS (p = 0.005; p = 0.002) and MSSS (r2 = 0.27, p = 0.03; r2 = 0.39, p = 0.001). Results were corroborated using multivariate analysis. Conclusions: Our data suggest a protective role of Gas6 and its receptors in patients with MS and suitable severity disease biomarkers.

Polyfluoroalkyl Tag Decoration Enables Significantly Enhanced Tumor Penetration Ability of a PTK7 Targeting Aptamer.

Aptamers are widely used molecular recognition tools in targeted therapy, but their ability to effectively penetrate deep into solid tumors remains a significant challenge, leading to suboptimal treatment efficacy. Here, we developed a polyfluoroalkyl (PFA) decoration strategy to enhance aptamer recognition, cell internalization, and solid tumor penetration. Our results indicate that PFA with around 11 fluorine atoms significantly improves aptamer internalization both in vitro and in vivo settings. However, we also observed that the use of PFA tags containing 19 and 23 fluorine atoms on aptamers resulted in nonspecific cell anchoring in control cell lines, affecting the specificity of aptamers. Overall, we found that using a chemical modification strategy could enhance the deep tumor penetration ability of aptamers and validate their effectiveness in vivo. This approach has significant practical applications in targeted drug delivery for cancer treatment.

Building, Breaking, and Repairing Neuromuscular Synapses.

A coordinated and complex interplay of signals between motor neurons, skeletal muscle cells, and Schwann cells controls the formation and maintenance of neuromuscular synapses. Deficits in the signaling pathway for building synapses, caused by mutations in critical genes or autoantibodies against key proteins, are responsible for several neuromuscular diseases, which cause muscle weakness and fatigue. Here, we describe the role that four key genes, Agrin, Lrp4, MuSK, and Dok7, play in this signaling pathway, how an understanding of their mechanisms of action has led to an understanding of several neuromuscular diseases, and how this knowledge has contributed to emerging therapies for treating neuromuscular diseases.

FOXP4-mediated induction of PTK7 activates the Wnt/ß-catenin pathway and promotes ovarian cancer development.

Ovarian cancer (OV) poses a significant challenge in clinical settings due to its difficulty in early diagnosis and treatment resistance. FOXP4, belonging to the FOXP subfamily, plays a pivotal role in various biological processes including cancer, cell cycle regulation, and embryonic development. However, the specific role and importance of FOXP4 in OV have remained unclear. Our research showed that FOXP4 is highly expressed in OV tissues, with its elevated levels correlating with poor prognosis. We further explored FOXP4's function through RNA sequencing and functional analysis in FOXP4-deficient cells, revealing its critical role in activating the Wnt signaling pathway. This activation exacerbates the malignant phenotype in OV. Mechanistically, FOXP4 directly induces the expression of protein tyrosine kinase 7 (PTK7), a Wnt-binding receptor tyrosine pseudokinase, which causes abnormal activation of the Wnt signaling pathway. Disrupting the FOXP4-Wnt feedback loop by inactivating the Wnt signaling pathway or reducing FOXP4 expression resulted in the reduction of the malignant phenotype of OV cells, while restoring PTK7 expression reversed this effect. In conclusion, our findings underscore the significance of the FOXP4-induced Wnt pathway activation in OV, suggesting the therapeutic potential of targeting this pathway in OV treatment.

A retrospective multicenter study on clinical and serological parameters in patients with MuSK myasthenia gravis with and without general immunosuppression.

Introduction: Muscle-specific kinase (MuSK)- myasthenia gravis (MG) is caused by pathogenic autoantibodies against MuSK that correlate with disease severity and are predominantly of the IgG4 subclass. The first-line treatment for MuSK-MG is general immunosuppression with corticosteroids, but the effect of treatment on IgG4 and MuSK IgG4 levels has not been studied. Methods: We analyzed the clinical data and sera from 52 MuSK-MG patients (45 female, 7 male, median age 49 (range 17-79) years) from Italy, the Netherlands, Greece and Belgium, and 43 AChR-MG patients (22 female, 21 male, median age 63 (range 2-82) years) from Italy, receiving different types of immunosuppression, and sera from 46 age- and sex-matched non-disease controls (with no diagnosed diseases, 38 female, 8 male, median age 51.5 (range 20-68) years) from the Netherlands. We analyzed the disease severity (assessed by MGFA or QMG score), and measured concentrations of MuSK IgG4, MuSK IgG, total IgG4 and total IgG in the sera by ELISA, RIA and nephelometry. Results: We observed that MuSK-MG patients showed a robust clinical improvement and reduction of MuSK IgG after therapy, and that MuSK IgG4 concentrations, but not total IgG4 concentrations, correlated with clinical severity. MuSK IgG and MuSK IgG4 concentrations were reduced after immunosuppression in 4/5 individuals with before-after data, but data from non-linked patient samples showed no difference. Total serum IgG4 levels were within the normal range, with IgG4 levels above threshold (1.35g/L) in 1/52 MuSK-MG, 2/43 AChR-MG patients and 1/45 non-disease controls. MuSK-MG patients improved within the first four years after disease onset, but no further clinical improvement or reduction of MuSK IgG4 were observed four years later, and only 14/52 (26.92%) patients in total, of which 13 (93.3%) received general immunosuppression, reached clinical remission. Discussion: We conclude that MuSK-MG patients improve clinically with general immunosuppression but may require further treatment to reach remission. Longitudinal testing of individual patients may be clinically more useful than single measurements of MuSK IgG4. No significant differences in the serum IgG4 concentrations and IgG4/IgG ratio between AChR- and MuSK-MG patients were found during follow-up. Further studies with larger patient and control cohorts are necessary to validate the findings.

Phosphatidylserine and Tyro3-Axl-Mertk Receptor Tyrosine Kinase level detection in plasma and on plasma-derived extracellular vesicle surface in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a chronic lymphoproliferative disorder characterized by monoclonal B cell proliferation. Studies carried out in recent years suggest that extracellular vesicles (EVs) may be a potential biomarker in cancer. Tyro3-Axl-Mertk (TAM) Receptor Tyrosine Kinases (RTKs) and Phosphatidylserine (PS) have crucial roles in macrophage-mediated immune response under normal conditions. In the tumor microenvironment, these molecules contribute to immunosuppressive signals and prevent the formation of local and systemic antitumor immune responses. Based on this, we aimed to evaluate the amount of PS and TAM RTK in plasma and on the surface of EVs in CLL patients and healthy volunteers in this study. In this study, 25 CLL (11 F/14 M) patients in the Rai (O-I) stage, newly diagnosed or followed up without treatment, and 15 healthy volunteers (11 F/4 M) as a control group were included. For all samples, PS and TAM RTK levels were examined first in the plasma and then in the EVs obtained from the plasma. We detected a significant decrease in plasma PS, and TAM RTK levels in CLL patients compared to the control. Besides, we determined a significant increase in TAM RTK levels on the EV surface in CLL, except for PS. In conclusion, these receptor levels measured by ELISA in plasma may not be effective for the preliminary detection of CLL. However, especially TAM RTKs on the surface of EVs may be good biomarkers and potential targets for CLL therapies.

[Muscle-Specific Receptor Tyrosine Kinase Antibody Positive Myasthenia Gravis].

Reportedly, patients with muscle-specific kinase (MuSK) antibody-positive myasthenia gravis (MG) account for approximately 3.0% of all patients with MG in Japan. Compared with patients who have acetylcholine receptor antibody-positive MG, those with MuSK antibody-positive MG show young-onset disease with female predominance, a low rate of ocular involvement (5.9%), and greater severity of dysphagia. The aforementioned types of MG are indistinguishable based on clinical symptoms and electrophysiological tests, and measurement of MuSK antibodies is essential for diagnosis. Thymectomy and complement inhibitors are not indicated for treatment, and acetylcholinesterase inhibitors, steroids, immunosuppressants, plasma exchange, intravenous immunoglobulin therapy, and neonatal Fc receptor inhibitors are used.

Elucidating the Functional Mechanism of PTK7 in Cancer Development through Spatial Assembly Analysis Using Super Resolution Imaging.

Protein tyrosine kinase-7 (PTK7) has been reported as a vital participant in the Wnt signaling pathway, influencing tumorigenesis and metastasis. However, their specific roles in the mechanisms underlying cancer development and progression remain elusive. Here, using direct stochastic optical reconstruction microscopy (dSTORM) with aptamer-probe labeling, we first revealed that a weakening clustering distribution of PTK7 on the basal membranes happened as cellular migration increased during cancer progression. This correspondence was further supported by a diminished aggregated state of PTK7 caused by direct enhancement of cell migration. By comparing the alterations in PTK7 distribution with activation or inhibition of specific Wnt signaling pathway, we speculated that PTK7 could modulate cell migration by participating in the interplay between canonical Wnt (in MCF7 cells) and noncanonical Wnt signals (in MDA-MB-231 cells). Furthermore, we discovered that the spatial distribution morphology of PTK7 was also subject to the hydrolysis ability and activation state of the related hydrolase Matrix metallopeptidase14 (MMP14). This function-related specific assembly of PTK7 reveals a clear relationship between PTK7 and cancer. Meanwhile, potential molecular interactions predicted by the apparent assembly morphology can promote a deep understanding of the functional mechanism of PTK7 in cancer progress.

Molecular dissection of PI3Kß synergistic activation by receptor tyrosine kinases, GßGγ, and Rho-family GTPases.

Phosphoinositide 3-kinase (PI3K) beta (PI3Kß) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kß prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kß localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kß when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kß membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kß prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GßGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kß to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GßGγ or pY/Rac1(GTP), PI3Kß activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kß is synergistically activated by pY/GßGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.

Tobacco Smoke Condensate Induces Morphologic Changes in Human Papillomavirus-Positive Cervical Epithelial Cells Consistent with Epithelial to Mesenchymal Transition (EMT) with Activation of Receptor Tyrosine Kinases and Regulation of TGFB.

High-risk human papillomavirus (HR-HPV; HPV-16) and cigarette smoking are associated with cervical cancer (CC); however, the underlying mechanism(s) remain unclear. Additionally, the carcinogenic components of tobacco have been found in the cervical mucus of women smokers. Here, we determined the effects of cigarette smoke condensate (CSC; 3R4F) on human ectocervical cells (HPV-16 Ect/E6E7) exposed to CSC at various concentrations (10-6-100 µg/mL). We found CSC (10-3 or 10 µg/mL)-induced proliferation, enhanced migration, and histologic and electron microscopic changes consistent with EMT in ectocervical cells with a significant reduction in E-cadherin and an increase in the vimentin expression compared to controls at 72 h. There was increased phosphorylation of receptor tyrosine kinases (RTKs), including Eph receptors, FGFR, PDGFRA/B, and DDR2, with downstream Ras/MAPK/ERK1/2 activation and upregulation of common EMT-related genes, TGFB SNAI2, PDGFRB, and SMAD2. Our study demonstrated that CSC induces EMT in ectocervical cells with the upregulation of EMT-related genes, expression of protein biomarkers, and activation of RTKs that regulate TGFB expression, and other EMT-related genes. Understanding the molecular pathways and environmental factors that initiate EMT in ectocervical cells will help delineate molecular targets for intervention and define the role of EMT in the initiation and progression of cervical intraepithelial neoplasia and CC.

Single-cell SERS imaging of dual cell membrane receptors expression influenced by extracellular matrix stiffness.

Membrane receptors perform a diverse range of cellular functions, accounting for more than half of all drug targets. The mechanical microenvironment regulates cell behaviors and phenotype. However, conventional analysis methods of membrane receptors often ignore the effects of the extracellular matrix stiffness, failing to reveal the heterogeneity of cell membrane receptors expression. Herein, we developed an in-situ surface-enhanced Raman scattering (SERS) imaging method to visualize single-cell membrane receptors on substrates with different stiffness. Two SERS substrates, Au@4-mercaptobenzonitrile@Ag@Sgc8c and Au@4-pethynylaniline@Ag@SYL3c, were employed to specifically target protein tyrosine kinase-7 (PTK7) and epithelial cell adhesion molecule (EpCAM), respectively. The polyacrylamide (PA) gels with tunable stiffness (2.5-25 kPa) were constructed to mimic extracellular matrix. The simultaneous SERS imaging of dual membrane receptors on single cancer cells on substrates with different stiffness was achieved. Our findings reveal decreased expression of PTK7 and EpCAM on cells cultured on stiffer substrates and higher migration ability of the cells. The results elucidate the heterogeneity of membrane receptors expression of cells cultured on the substrates with different stiffness. This single-cell analysis method offers an in-situ platform for investigating the impacts of extracellular matrix stiffness on the expression of membrane receptors, providing insights into the role of cell membrane receptors in cancer metastasis.

Narazaciclib, a novel multi-kinase inhibitor with potent activity against CSF1R, FLT3 and CDK6, shows strong anti-AML activity in defined preclinical models.

CSF1R is a receptor tyrosine kinase responsible for the growth/survival/polarization of macrophages and overexpressed in some AML patients. We hypothesized that a novel multi-kinase inhibitor (TKi), narazaciclib (HX301/ON123300), with high potency against CSF1R (IC50 ~ 0.285 nM), would have anti-AML effects. We tested this by confirming HX301's high potency against CSF1R (IC50 ~ 0.285 nM), as well as other kinases, e.g. FLT3 (IC50 of ~ 19.77 nM) and CDK6 (0.53 nM). An in vitro proliferation assay showed that narazaciclib has a high growth inhibitory effect in cell cultures where CSF1R or mutant FLT3-ITD variants that may be proliferation drivers, including primary macrophages (IC50 of 72.5 nM) and a subset of AML lines (IC50 < 1.5 µM). In vivo pharmacology modeling of narazaciclib using five AML xenografts resulted in: inhibition of MV4-11 (FLT3-ITD) subcutaneous tumor growth and complete suppression of AM7577-PDX (FLT3-ITD/CSF1Rmed) systemic growth, likely due to the suppression of FLT3-ITD activity; complete suppression of AM8096-PDX (CSF1Rhi/wild-type FLT3) growth, likely due to the inhibition of CSF1R ("a putative driver"); and nonresponse of both AM5512-PDX and AM7407-PDX (wild-type FLT3/CSF1Rlo). Significant leukemia load reductions in bone marrow, where disease originated, were also achieved in both responders (AM7577/AM8096), implicating that HX301 might be a potentially more effective therapy than those only affecting peripheral leukemic cells. Altogether, narazaciclib can potentially be a candidate treatment for a subset of AML with CSF1Rhi and/or mutant FLT3-ITD variants, particularly second generation FLT3 inhibitor resistant variants.

Dabrafenib mitigates the neuroinflammation caused by ferroptosis in experimental autoimmune encephalomyelitis by up regulating Axl receptor.

Multiple sclerosis is an autoimmune disease that causes inflammatory damage to the central nervous system. At present, the pathogenesis of the disease is unknown. There is a lack of few effective therapy medications available. Therefore, it is necessary to further explore the pathogenesis of this illness and develop potential therapeutic drugs. Dabrafenib is potential therapeutic medicine for nervous system disease. In this study, we preliminarily studied the possible mechanism of dabrafenib in the treatment of multiple sclerosis from the perspective of ferroptosis. First, we observed that dabrafenib significantly improved symptoms of gait abnormalities, limb weakness or paralysis, and down-regulated levels of spinal cord inflammation in an experimental autoimmune encephalitis (EAE) model. Meanwhile, we also observed that dabrafenib could inhibit the proteins of ferroptosis in spinal cord tissue of EAE mice by Western blot. The results of immunohistochemical analysis showed that the effect of dabrafenib on ferroptosis mainly occurred in microglia. Second, dabrafenib was demonstrated to be able to inhibit the S phase of the cell cycle, reduce ROS levels, and reinstate mitochondrial activity in the LPS-induced BV2 inflammatory cell model. Futhermore, we found that dabrafenib inhibits P-JAK2 and P-STAT3 activation by acting Axl receptor, which in turn prevents neurogenic inflammation in microglia. The co-stimulated BV2 cell model with LPS and Erastin also verified these findings. Ultimately, the Axl knockout mice used to construct the EAE model allowed for the confirmation that dabrafenib prevented ferroptosis in microglia by up-regulating Axl receptor, which reduced the inflammatory demyelination associated with EAE. In summary, our research demonstrates the advantages of dabrafenib in multiple sclerosis treatment, which can prevent ferroptosis in microglia in multiple sclerosis through up-regulating Axl receptor, thus halting the progression of multiple sclerosis.

Serum GAS6, sAXL, IL-10, NO, and BCL-2 levels are decreased in patients with Behçet's disease.

PURPOSE: Behçet's disease (BD) is an autoimmune chronic systemic inflammatory disease characterized by a versatile clinical spectrum. Growth arrest specific protein 6 (GAS6)/soluble AXL (sAXL) signaling pathway draws attention in the resolution of inflammation, and its deficiency is associated with chronic inflammatory, autoimmune diseases, as well as clearance of apoptotic cells by phagocytes - efferocytosis. In this study, it was aimed to investigate whether GAS6/sAXL, interleukin (IL)-10, nitric oxide (NO), and BCL-2 levels were associated with inflammation and efferocytosis contributes to the pathogenesis of BD. METHODS: A total of 37 Behçet patients with ocular involvement and 30 healthy control subjects were included in this study. GAS6, sAXL, IL-10, NO, and BCL-2 levels were quantified using enzyme-linked immunosorbent assay (ELISA) method. RESULTS: Serum GAS6, sAXL, IL-10, NO, and BCL-2 levels were significantly lower in patients with BD compared to the controls (P < 0.005, P < 0.001, P < 0.001, P < 0.001, and P < 0.001, respectively). In correlation analysis, research parameters decreased in patients with BD was significantly correlated with each other: GAS6-IL-10 (r = 0.585, P < 0.001), GAS6-BCL-2 (r = 0.541, P < 0.001), sAXL-BCL-2 (r = 0.696, P < 0.001), IL-10-NO (r = 0.717, P < 0.001), IL-10-BCL-2 (r = 0.759, P < 0.001), and NO-BCL-2 (r = 0.541, P < 0.001). CONCLUSION: In conclusion, decreased serum BCL-2 level may be an indicator of increased apoptosis in these patients and decreased levels of GAS6/sAXL, IL-10, and NO may indicate insufficient clearance of apoptotic bodies released as a result of increased apoptosis in BD patients.

Comparative study of imaging and pathology of primary mucinous adenocarcinoma with different imaging manifestations.

BACKGROUND: Pulmonary mucinous adenocarcinoma is a special type of lung cancer. Its imaging manifestations are diverse, which brings challenges to clinical diagnosis. However, its formation mechanism is unclear. OBJECTIVE: The objective of this study is to analyse the relevant mechanisms of the formation of pulmonary mucinous adenocarcinoma by observing its different imaging and pathological manifestations. DATA AND METHODS: Retrospective analysis was conducted on imaging manifestations and pathological data of 103 patients with pulmonary mucinous adenocarcinoma confirmed intraoperatively or pathologically. RESULTS: Forty-three patients had pulmonary mucinous adenocarcinoma with a solitary nodule/mass, 41 patients with localized pneumonia and 19 patients with diffuse pneumonia. Their CT manifestations included 'falling snowflake sign', ground-glass opacity close to the heart, vacuous signs/honeycombing and withered tree branches. Under the microscope, all the three types of pulmonary mucinous adenocarcinoma had visibly formed mucus lakes but were made of tumour cells with totally different shapes, which included the goblet-like shape (tall column-like shape) and quasi-circular shape. Tall column-shaped tumour cells were negative or weakly positive for thyroid transcription factor-1 (TTF-1) and strongly positive for ALK mutation, whereas quasi-circular tumour cells were positive for TTF-1 and less positive for ALK mutation. CONCLUSION: The different imaging manifestations of mucinous adenocarcinoma are possibly due to the different amounts or viscosity of mucus produced, and the mechanisms of its formation may include (1) tumour cells in different shapes have different abilities to produce mucus; (2) tumours in different stages produce different amounts or viscosity of mucus; and (3) the TTF-1 and ALK genes affect the production of mucus.

Alectinib in Resected ALK-Positive Non-Small-Cell Lung Cancer.

BACKGROUND: Platinum-based chemotherapy is the recommended adjuvant treatment for patients with resectable, ALK-positive non-small-cell lung cancer (NSCLC). Data on the efficacy and safety of adjuvant alectinib as compared with chemotherapy in patients with resected ALK-positive NSCLC are lacking. METHODS: We conducted a global, phase 3, open-label, randomized trial in which patients with completely resected, ALK-positive NSCLC of stage IB (tumors ≥4 cm), II, or IIIA (as classified according to the seventh edition of the Cancer Staging Manual of the American Joint Committee on Cancer and Union for International Cancer Control) were randomly assigned in a 1:1 ratio to receive oral alectinib (600 mg twice daily) for 24 months or intravenous platinum-based chemotherapy in four 21-day cycles. The primary end point was disease-free survival, tested hierarchically among patients with stage II or IIIA disease and then in the intention-to-treat population. Other end points included central nervous system (CNS) disease-free survival, overall survival, and safety. RESULTS: In total, 257 patients were randomly assigned to receive alectinib (130 patients) or chemotherapy (127 patients). The percentage of patients alive and disease-free at 2 years was 93.8% in the alectinib group and 63.0% in the chemotherapy group among patients with stage II or IIIA disease (hazard ratio for disease recurrence or death, 0.24; 95% confidence interval [CI], 0.13 to 0.45; P<0.001) and 93.6% and 63.7%, respectively, in the intention-to-treat population (hazard ratio, 0.24; 95% CI, 0.13 to 0.43; P<0.001). Alectinib was associated with a clinically meaningful benefit with respect to CNS disease-free survival as compared with chemotherapy (hazard ratio for CNS disease recurrence or death, 0.22; 95% CI, 0.08 to 0.58). Data for overall survival were immature. No unexpected safety findings were observed. CONCLUSIONS: Among patients with resected ALK-positive NSCLC of stage IB, II, or IIIA, adjuvant alectinib significantly improved disease-free survival as compared with platinum-based chemotherapy. (Funded by F. Hoffmann-La Roche; ALINA ClinicalTrials.gov number, NCT03456076.).

LVING reveals the intracellular structure of cell growth.

The continuous balance of growth and degradation inside cells maintains homeostasis. Disturbance of this balance by internal or external factors cause state of disease, while effective disease treatments seek to restore this balance. Here, we present a method based on quantitative phase imaging (QPI) based measurements of cell mass and the velocity of mass transport to quantify the balance of growth and degradation within intracellular control volumes. The result, which we call Lagrangian velocimetry for intracellular net growth (LVING), provides high resolution maps of intracellular biomass production and degradation. We use LVING to quantify the growth in different regions of the cell during phases of the cell cycle. LVING can also be used to quantitatively compare the effect of range of chemotherapy drug doses on subcellular growth processes. Finally, we applied LVING to characterize the effect of autophagy on the growth machinery inside cells. Overall, LVING reveals both the structure and distribution of basal growth within cells, as well as the disruptions to this structure that occur during alterations in cell state.

Phylogenetic and transcriptomic characterization of insulin and growth factor receptor tyrosine kinases in crustaceans.

Receptor tyrosine kinases (RTKs) mediate the actions of growth factors in metazoans. In decapod crustaceans, RTKs are implicated in various physiological processes, such molting and growth, limb regeneration, reproduction and sexual differentiation, and innate immunity. RTKs are organized into two main types: insulin receptors (InsRs) and growth factor receptors, which include epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR). The identities of crustacean RTK genes are incomplete. A phylogenetic analysis of the CrusTome transcriptome database, which included all major crustacean taxa, showed that RTK sequences segregated into receptor clades representing InsR (72 sequences), EGFR (228 sequences), FGFR (129 sequences), and PDGFR/VEGFR (PVR; 235 sequences). These four receptor families were distinguished by the domain organization of the extracellular N-terminal region and motif sequences in the protein kinase catalytic domain in the C-terminus or the ligand-binding domain in the N-terminus. EGFR1 formed a single monophyletic group, while the other RTK sequences were divided into subclades, designated InsR1-3, FGFR1-3, and PVR1-2. In decapods, isoforms within the RTK subclades were common. InsRs were characterized by leucine-rich repeat, furin-like cysteine-rich, and fibronectin type 3 domains in the N-terminus. EGFRs had leucine-rich repeat, furin-like cysteine-rich, and growth factor IV domains. N-terminal regions of FGFR1 had one to three immunoglobulin-like domains, whereas FGFR2 had a cadherin tandem repeat domain. PVRs had between two and five immunoglobulin-like domains. A classification nomenclature of the four RTK classes, based on phylogenetic analysis and multiple sequence alignments, is proposed.