Development and Application of Reversible and Irreversible Covalent Probes for Human and Mouse Cathepsin-K Activity Detection, Revealing Nuclear Activity.

Cathepsin-K (CTSK) is an osteoclast-secreted cysteine protease that efficiently cleaves extracellular matrices and promotes bone homeostasis and remodeling, making it an excellent therapeutic target. Detection of CTSK activity in complex biological samples using tailored tools such as activity-based probes (ABPs) will aid tremendously in drug development. Here, potent and selective CTSK probes are designed and created, comparing irreversible and reversible covalent ABPs with improved recognition components and electrophiles. The newly developed CTSK ABPs precisely detect active CTSK in mouse and human cells and tissues, from diseased and healthy states such as inflamed tooth implants, osteoclasts, and lung samples, indicating changes in CTSK's activity in the pathological samples. These probes are used to study how acidic pH stimulates mature CTSK activation, specifically, its transition from pro-form to mature form. Furthermore, this study reveals for the first time, why intact cells and cell lysate exhibit diverse CTSK activity while having equal levels of mature CTSK enzyme. Interestingly, these tools enabled the discovery of active CTSK in human osteoclast nuclei and in the nucleoli. Altogether, these novel probes are excellent research tools and can be applied in vivo to examine CTSK activity and inhibition in diverse diseases without immunogenicity hazards.

Particle uptake in cancer cells can predict malignancy and drug resistance using machine learning.

Tumor heterogeneity is a primary factor that contributes to treatment failure. Predictive tools, capable of classifying cancer cells based on their functions, may substantially enhance therapy and extend patient life span. The connection between cell biomechanics and cancer cell functions is used here to classify cells through mechanical measurements, via particle uptake. Machine learning (ML) was used to classify cells based on single-cell patterns of uptake of particles with diverse sizes. Three pairs of human cancer cell subpopulations, varied in their level of drug resistance or malignancy, were studied. Cells were allowed to interact with fluorescently labeled polystyrene particles ranging in size from 0.04 to 3.36 µm and analyzed for their uptake patterns using flow cytometry. ML algorithms accurately classified cancer cell subtypes with accuracy rates exceeding 95%. The uptake data were especially advantageous for morphologically similar cell subpopulations. Moreover, the uptake data were found to serve as a form of "normalization" that could reduce variation in repeated experiments.

Outcomes of extracranial stereotactic body radiation therapy for induced oligometastatic non-small cell lung cancer on novel systemic therapy.

Background: Stereotactic body radiation therapy (SBRT) is often delivered in patients with oligometastatic disease (OMD). However, the specific subset of patients with polymetastatic non-small cell lung cancer (NSCLC) on novel systemic therapies who develop induced oligopersistant disease (OpersisD) or oligoprogressive disease (OprogD), as defined by the European Organisation for Research and Treatment of Cancer (EORTC) OMD classification, has not been well described. This study explores the outcomes of patients treated with this strategy. Methods: Patients with stage IV NSCLC being treated with osimertinib or immune checkpoint inhibitors (ICIs) who received extracranial SBRT for OpersisD or OprogD were identified in our retrospective analysis. Outcomes reported include progression-free survival (PFS), time to change of systemic treatment (TTCST), overall survival (OS), local control (LC) and treatment-related toxicity. Results: Forty-nine patients received SBRT for OpersisD (34.7%) or OprogD (65.3%) at a median of 5.8 and 15.3 months after start of systemic therapy, respectively. 55.1% received concurrent osimertinib and 44.9% received ICI. Seventy-seven extracranial lesions were treated with various fractionation schemas. At a median of 18.8 months follow-up from first SBRT, LC was achieved in 92.2% of total lesions treated (71). The 1-year OS was 91.7% for OpersisD and 83.3% for OprogD. OpersisD compared to OprogD had a longer median PFS (18.3 vs. 6.1 months) and longer median TTCST (23.6 vs. 13.5 months), median OS was not reached for either cohort. On multivariate analysis, patients treated with osimertinib had shorter PFS (HR: 2.20; 95% CI: 1.01-4.82; P=0.048) and shorter TTCST (HR: 2.83; 95% CI: 1.09-7.33; P=0.032). One patient (2%) experienced grade 3 pneumonitis after SBRT, and no grade 4-5 toxicities were reported with SBRT treatment. Conclusions: This study indicates that SBRT for OpersisD or OprogD in Stage IV NSCLC patients on osimertinib or ICIs is safe, very well tolerated, and may prolong the time before needing a shift in systemic therapy. Further prospective research is needed to validate and expand upon these findings.

The DNA methylome of human vascular endothelium and its use in liquid biopsies.

BACKGROUND: Vascular endothelial cells (VECs) are an essential component of each tissue, contribute to multiple pathologies, and are targeted by important drugs. Yet, there is a shortage of biomarkers to assess VEC turnover. METHODS: To develop DNA methylation-based liquid biopsies for VECs, we determined the methylome of VECs isolated from freshly dissociated human tissues. FINDINGS: A comparison with a human cell-type methylome atlas yielded thousands of loci that are uniquely unmethylated in VECs. These sites are typically gene enhancers, often residing adjacent to VEC-specific genes. We also identified hundreds of genomic loci that are differentially methylated in organotypic VECs, indicating that VECs feeding specific organs are distinct cell types with a stable epigenetic identity. We established universal and lung-specific VEC markers and evaluated their presence in circulating cell-free DNA (cfDNA). Nearly 2.5% of cfDNA in the plasma of healthy individuals originates from VECs. Sepsis, graft versus host disease, and cardiac catheterization are associated with elevated levels of VEC-derived cfDNA, indicative of vascular damage. Lung-specific VEC cfDNA is selectively elevated in patients with chronic obstructive pulmonary disease (COPD) or lung cancer, revealing tissue-specific vascular turnover. CONCLUSIONS: VEC cfDNA biomarkers inform vascular dynamics in health and disease, potentially contributing to early diagnosis and monitoring of pathologies, and assessment of drug activity. FUNDING: This work was supported by the Beutler Research Program, Helmsley Charitable Trust, JDRF, Grail and the DON Foundation (to Y.D.). Y.D holds the Walter & Greta Stiel Chair in heart studies. B.G., R.S., J.M., D.N., T.K., and Y.D. filed patents on cfDNA analysis.

The DNA damage response pathway regulates the expression of the immune checkpoint CD47.

CD47 is a cell surface ligand expressed on all nucleated cells. It is a unique immune checkpoint protein acting as "don't eat me" signal to prevent phagocytosis and is constitutively overexpressed in many tumors. However, the underlying mechanism(s) for CD47 overexpression is not clear. Here, we show that irradiation (IR) as well as various other genotoxic agents induce elevated expression of CD47. This upregulation correlates with the extent of residual double-strand breaks (DSBs) as determined by γH2AX staining. Interestingly, cells lacking mre-11, a component of the MRE11-RAD50-NBS1 (MRN) complex that plays a central role in DSB repair, or cells treated with the mre-11 inhibitor, mirin, fail to elevate the expression of CD47 upon DNA damage. On the other hand, both p53 and NF-κB pathways or cell-cycle arrest do not play a role in CD47 upregualtion upon DNA damage. We further show that CD47 expression is upregulated in livers harvested from mice treated with the DNA-damage inducing agent Diethylnitrosamine (DEN) and in cisplatin-treated mesothelioma tumors. Hence, our results indicate that CD47 is upregulated following DNA damage in a mre-11-dependent manner. Chronic DNA damage response in cancer cells might contribute to constitutive elevated expression of CD47 and promote immune evasion.

CDC25C Protein Expression Correlates with Tumor Differentiation and Clinical Outcomes in Lung Adenocarcinoma.

Given that, even after multimodal therapy, early-stage lung cancer (LC) often recurs, novel prognostic markers to help guide therapy are highly desired. The mRNA levels of cell division cycle 25C (CDC25C), a phosphatase that regulates G2/M cell cycle transition in malignant cells, correlate with poor clinical outcomes in lung adenocarcinoma (LUAD). However, whether CDC25C protein detected by immunohistochemistry can serve as a prognostic marker in LUAD is yet unknown. We stained an LC tissue array and a cohort of 61 LUAD tissue sections for CDC25C and searched for correlations between CDC25C staining score and the pathological characteristics of the tumors and the patients' clinical outcomes. Clinical data were retrieved from our prospectively maintained departmental database. We found that high expression of CDC25C was predominant among poorly differentiated LUAD (p < 0.001) and in LUAD > 1cm (p < 0.05). Further, high expression of CDC25C was associated with reduced disease-free survival (p = 0.03, median follow-up of 39 months) and with a trend for reduced overall survival (p = 0.08). Therefore, high expression of CDC25C protein in LUAD is associated with aggressive histological features and with poor outcomes. Larger studies are required to further validate CDC25C as a prognostic marker in LUAD.

A DNA methylation atlas of normal human cell types.

DNA methylation is a fundamental epigenetic mark that governs gene expression and chromatin organization, thus providing a window into cellular identity and developmental processes1. Current datasets typically include only a fraction of methylation sites and are often based either on cell lines that underwent massive changes in culture or on tissues containing unspecified mixtures of cells2-5. Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing, allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 205 healthy tissue samples. Replicates of the same cell type are more than 99.5% identical, demonstrating the robustness of cell identity programmes to environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny and identifies methylation patterns retained since embryonic development. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hypermethylated loci are rare and are enriched for CpG islands, Polycomb targets and CTCF binding sites, suggesting a new role in shaping cell-type-specific chromatin looping. The atlas provides an essential resource for study of gene regulation and disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies.

SARS-CoV-2 Omicron Induces Enhanced Mucosal Interferon Response Compared to other Variants of Concern, Associated with Restricted Replication in Human Lung Tissues.

SARS-CoV-2 Omicron variant has been characterized by decreased clinical severity, raising the question of whether early variant-specific interactions within the mucosal surfaces of the respiratory tract could mediate its attenuated pathogenicity. Here, we employed ex vivo infection of native human nasal and lung tissues to investigate the local-mucosal susceptibility and innate immune response to Omicron compared to Delta and earlier SARS-CoV-2 variants of concern (VOC). We show that the replication of Omicron in lung tissues is highly restricted compared to other VOC, whereas it remains relatively unchanged in nasal tissues. Mechanistically, Omicron induced a much stronger antiviral interferon response in infected tissues compared to Delta and earlier VOC-a difference, which was most striking in the lung tissues, where the innate immune response to all other SARS-CoV-2 VOC was blunted. Notably, blocking the innate immune signaling restored Omicron replication in the lung tissues. Our data provide new insights to the reduced lung involvement and clinical severity of Omicron.

Universal lung epithelium DNA methylation markers for detection of lung damage in liquid biopsies.

BACKGROUND: Circulating biomarkers for lung damage are lacking. Lung epithelium-specific DNA methylation patterns can potentially report the presence of lung-derived cell-free DNA (cfDNA) in blood, as an indication of lung cell death. METHODS: We sorted human lung alveolar and bronchial epithelial cells from surgical specimens, and obtained their methylomes using whole-genome bisulfite sequencing. We developed a PCR sequencing assay determining the methylation status of 17 loci with lung-specific methylation patterns, and used it to assess lung-derived cfDNA in the plasma of healthy volunteers and patients with lung disease. RESULTS: Loci that are uniquely unmethylated in alveolar or bronchial epithelial cells are enriched for enhancers controlling lung-specific genes. Methylation markers extracted from these methylomes revealed that normal lung cell turnover probably releases cfDNA into the air spaces, rather than to blood. People with advanced lung cancer show a massive elevation of lung cfDNA concentration in blood. Among individuals undergoing bronchoscopy, lung-derived cfDNA is observed in the plasma of those later diagnosed with lung cancer, and to a lesser extent in those diagnosed with other lung diseases. Lung cfDNA is also elevated in patients with acute exacerbation of COPD compared with patients with stable disease, and is associated with future exacerbation and mortality in these patients. CONCLUSIONS: Universal cfDNA methylation markers of normal lung epithelium allow for mutation-independent, sensitive and specific detection of lung-derived cfDNA, reporting on ongoing lung injury. Such markers can find broad utility in the study of normal and pathologic human lung dynamics.

Deep dive into the immune response against murine mesothelioma permits design of novel anti-mesothelioma therapeutics.

Given the need to improve the efficacy of standard-of-care immunotherapy (anti-CTLA-4 + anti-PD-1) in human malignant pleural mesothelioma (hMPM), we thoroughly characterized the immunobiology of the AB12 murine mesothelioma (MM) model, aiming to increase its accuracy in predicting the response of hMPM to immunotherapy and in designing novel anti-hMPM treatments. Specifically, we used immunologic, transcriptomic and survival analyses, to synchronize the MM tumor growth phases and immune evolution with the histo-molecular and immunological characteristics of hMPM while also determining the anti-MM efficacy of standard-of-care anti-hMPM immunotherapy as a benchmark that novel therapeutics should meet. We report that early-, intermediate- and advanced- AB12 tumors are characterized by a bell-shaped anti-tumor response that peaks in intermediate tumors and decays in advanced tumors. We further show that intermediate- and advanced- tumors match with immune active ("hot") and immune inactive ("cold") hMPM respectively, and that they respond to immunotherapy in a manner that corresponds well with its performance in real-life settings. Finally, we show that in advanced tumors, addition of cisplatin to anti CTLA-4 + anti PD-1 can extend mice survival and invigorate the decaying anti-tumor response. Therefore, we highlight this triple combination as a worthy candidate to improve clinical outcomes in hMPM.

Human Nasal and Lung Tissues Infected Ex Vivo with SARS-CoV-2 Provide Insights into Differential Tissue-Specific and Virus-Specific Innate Immune Responses in the Upper and Lower Respiratory Tract.

The nasal mucosa constitutes the primary entry site for respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While the imbalanced innate immune response of end-stage coronavirus disease 2019 (COVID-19) has been extensively studied, the earliest stages of SARS-CoV-2 infection at the mucosal entry site have remained unexplored. Here, we employed SARS-CoV-2 and influenza virus infection in native multi-cell-type human nasal turbinate and lung tissues ex vivo, coupled with genome-wide transcriptional analysis, to investigate viral susceptibility and early patterns of local mucosal innate immune response in the authentic milieu of the human respiratory tract. SARS-CoV-2 productively infected the nasal turbinate tissues, predominantly targeting respiratory epithelial cells, with a rapid increase in tissue-associated viral subgenomic mRNA and secretion of infectious viral progeny. Importantly, SARS-CoV-2 infection triggered robust antiviral and inflammatory innate immune responses in the nasal mucosa. The upregulation of interferon-stimulated genes, cytokines, and chemokines, related to interferon signaling and immune-cell activation pathways, was broader than that triggered by influenza virus infection. Conversely, lung tissues exhibited a restricted innate immune response to SARS-CoV-2, with a conspicuous lack of type I and III interferon upregulation, contrasting with their vigorous innate immune response to influenza virus. Our findings reveal differential tissue-specific innate immune responses in the upper and lower respiratory tracts that are specific to SARS-CoV-2. The studies shed light on the role of the nasal mucosa in active viral transmission and immune defense, implying a window of opportunity for early interventions, whereas the restricted innate immune response in early-SARS-CoV-2-infected lung tissues could underlie the unique uncontrolled late-phase lung damage of advanced COVID-19. IMPORTANCE In order to reduce the late-phase morbidity and mortality of COVID-19, there is a need to better understand and target the earliest stages of SARS-CoV-2 infection in the human respiratory tract. Here, we have studied the initial steps of SARS-CoV-2 infection and the consequent innate immune responses within the natural multicellular complexity of human nasal mucosal and lung tissues. Comparing the global innate response patterns of nasal and lung tissues infected in parallel with SARS-CoV-2 and influenza virus, we found distinct virus-host interactions in the upper and lower respiratory tract, which could determine the outcome and unique pathogenesis of SARS-CoV-2 infection. Studies in the nasal mucosal infection model can be employed to assess the impact of viral evolutionary changes and evaluate new therapeutic and preventive measures against SARS-CoV-2 and other human respiratory pathogens.

Outcomes of sublobar resection vs lobectomy for invasive clinical stage T1N0 non-small-cell lung cancer: A propensity-match analysis.

BACKGROUND: The role of sub lobar resection (SLR; either segmentectomy or wedge resection) vs lobectomy (LBCT) for invasive clinical stage T1N0 non-small-cell-lung-cancer (NSCLC) has not been fully established yet. AIM: We aimed to characterize the preoperative parameters leading to selecting SLR and compare the overall survival (OS) and disease-free survival (DFS) of these two surgical approaches. METHODS: Clinical data on 162 patients (LBCT-107; SLR-55) were prospectively entered in our departmental database. Preoperative parameters associated with the performance of SLR were identified using univariate and multivariate cox regression analysis. The Kaplan-Meier method was used to compute OS and DFS. Comparison between LBCT and SLR groups and 32 propensity-matched groups was performed using Log-rank test. RESULTS: Median follow-up time for the LBCT and SLR groups was 4.76 (Inter-quartile range [IQR] 2.96 to 8.23) and 3.38 (IQR 2.9 to 6.19) years respectively. OS and DFS rates were similar between the two groups in the entire cohort (OS-LBCT vs SLR P = .853, DSF-LBCT vs SLR P = .653) and after propensity matching (OS-LBCT vs SLR P = .563 DSF-LBCT vs SLR P = .632). Specifically, Two- and five-year OS rates for LBCT and SLR were 90.6.% vs 92.7%, 71.8% vs 75.9% respectively. Independent predictors of selecting for SLR included older age (P < .001), reduced FEV1% (P = .026), smaller tumor size (P = .025), smaller invasive component (P = .021) and higher American Society of Anesthesiology scores (P = .014). CONCLUSIONS: In 162 consecutive and 32 matched cases, SLR and lobar resection had similar overall and disease-free survival rates. SLR may be considered as a reasonable oncological procedure in carefully selected T1N0 NSCLC patients that present with multiple comorbidities and relatively small tumors.

11-year experience with Chest Wall resection and reconstruction for primary Chest Wall sarcomas.

BACKGROUND & OBJECTIVES: Primary chest wall sarcomas are rare and therapeutically challenging tumors. Herein we report the outcomes of a surgery-based multimodality therapy for these pathologies over an 11-year period. In addition, we present a case that illustrates the surgical challenges that extensive chest wall resection may pose. METHODS: Using the Society of Thoracic Surgeons general thoracic surgery database, we have prospectively collected data in our institute on all patients undergoing chest wall resection and reconstruction for primary chest wall sarcomas between June 2008-October 2019. RESULTS: We performed 28 surgical procedures on 25 patients aged 5 to 91 years (median age 33). Eleven tumors were bone- and cartilage-derived and 14 tumors originated from soft tissue elements. Seven patients (7/25, 28%) received neo-adjuvant therapy and 14 patients (14/25, 56%) received adjuvant therapy. The median number of ribs that were resected was 2.5 (range 0 to 6). In 18/28 (64%) of surgeries, additional skeletal or visceral organs were removed, including: diaphragm [1], scapula [2], sternum [2], lung [2], vertebra [1], clavicle [1] and colon [1]. Chest wall reconstruction was deemed necessary in 16/28 (57%) of cases, polytetrafluoroethylene (PTFE) Gore-Tex patches was used in 13/28 (46%) of cases and biological flaps where used in 4/28 (14%) of cases. R0, R1 and R2 resection margins were achieved in 19/28 (68%), 9/28 (32%) and 0/28 (0%) of cases, respectively. The median follow up time was 33 months (range 2 to 138). During the study period, disease recurred in 8/25 (32%) of patients. Of these, 3 were re-operated on and are free of disease. At date of last follow up, 5/25 (20%) of patients have died due to their disease and in contrast, 20/25 (80%) were alive with no evidence of disease. CONCLUSIONS: Surgery-based multimodality therapy is an effective treatment approach for primary chest wall sarcomas. Resection of additional skeletal or visceral organs and reconstruction with synthetic and/or biological flaps is often required in order to obtain R0 resection margins. Ultimately, long-term survival in this clinical scenario is an achievable goal.

Benefits of medical clowning in the treatment of young children with autism spectrum disorder.

We investigated the contribution of group therapy delivered by a medical clown to young children diagnosed with autism spectrum disorder (ASD). So far, scientific publications regarding medical clowning focus on general health advantages. The current study is the first controlled research examining the use of medical clowning in the therapy for children with ASD. Twenty-four children aged 2-6 years old with ASD enrolled in our special education intensive program were examined before and after group sessions with clown intervention (CI) and other intervention (OI). We tested stereotypic behaviors, verbal expression, play reciprocity, and social smiles. Data was collected during 12 weeks of intervention, and the trajectory of change was evaluated in addition to the pre-/post-intervention.Conclusion: improvement over time in all measures: Significant increase in word production, play reciprocity, and amount of social smiles during CI as compared with OI. We also found a reduction in frequency of stereotypic behaviors during and following CI as compared with before CI. These preliminary results indicate that medical clowning may be beneficial for young children with ASD, since it promotes communication and social reciprocity in a fun and lively interventional setting. What is Known: • Many therapies are used and proven as efficacious interventions for children with ASD. • So far, medical clowning was not tested as an intervention or therapy for ASD. What is New: • Medical clowning sessions with children with ASD elicited enhanced communication during the interventions as compared with other interventions. • Medical clowning sessions contributed to a decrease in frequency of stereotypic movements over time, in children with ASD.

CXCR4 Based Therapeutics for Non-Small Cell Lung Cancer (NSCLC).

Lung cancer is the second most common malignancy. Unfortunately, despite advances in multimodality therapeutics for the disease, the overall five-year survival rate among newly diagnosed lung cancer patients remains in the range region of 15%. In addition, although immune checkpoint inhibitors are increasingly being incorporated into lung cancer treatment protocols, the proportion of patients that respond to these agents remains low and the duration of response is often short. Therefore, novel methodologies to enhance the efficacy of immunotherapy in lung cancer are highly desirable. Chemokines are small chemotactic cytokines that interact with their 7 transmembrane G-protein⁻coupled receptors, to guide immune cell trafficking in the body under both physiologic and pathologic conditions. Tumor cells highjack a small repertoire of the chemokine/chemokine receptor system and utilize it in a manner that benefits local tumor growth and distant spread. The chemokine receptor, CXCR4 is expressed in over 30 types of malignant tumors and, through interaction with its ligand CXCL12, was shown exert pleotropic pro-tumorigenic effects. In this review, the pathologic roles that CXCL12/CXCR4 play in lung cancer propagation are presented. Furthermore, the challenges and potential benefits of incorporating drugs that target CXCL12/CXCR4 into immune-based lung cancer therapeutic protocols are discussed.

Matrix metalloproteinase 12 promotes tumor propagation in the lung.

OBJECTIVE: Past studies are inconsistent with regard to the role of matrix metalloproteinase 12 in lung tumorigenesis. This is due, in part, to differential tumorigenesis based on tumor-derived versus immune-derived matrix metalloproteinase 12 expression. Our study aims to thoroughly dissect the role of matrix metalloproteinase 12 in lung tumorigenesis. METHODS: We tested matrix metalloproteinase 12 expression and the association with prognosis using a tissue array and a published non-small cell lung cancer gene expression database. In addition, we characterized the contribution of matrix metalloproteinase 12 to tumor propagation in the lung using a series of in vitro and in vivo studies. RESULTS: Tumor cells of a diverse set of human lung cancers stained positive for matrix metalloproteinase 12, and high matrix metalloproteinase 12 mRNA levels in the tumor were associated with reduced survival. The lung microenvironment stimulated endogenous production of matrix metalloproteinase 12 in lung cancer cells (human 460 lung cancer cell line, Lewis lung carcinoma). In vitro, matrix metalloproteinase 12 knockout Lewis lung carcinoma and Lewis lung carcinoma cells had the same proliferation rate, but Lewis lung carcinoma showed increased invasiveness. In vivo, deficiency of matrix metalloproteinase 12 in Lewis lung carcinoma cells, but not in the host, reduced tumor growth and invasiveness. CONCLUSIONS: We suggest that tumor cell-derived matrix metalloproteinase 12 promotes tumor propagation in the lung and that in the context of pulmonary malignancies matrix metalloproteinase 12 should further be tested as a potential novel therapeutic target.

CXCR4 Promotes Neuroblastoma Growth and Therapeutic Resistance through miR-15a/16-1-Mediated ERK and BCL2/Cyclin D1 Pathways.

CXCR4 expression in neuroblastoma tumors correlates with disease severity. In this study, we describe mechanisms by which CXCR4 signaling controls neuroblastoma tumor growth and response to therapy. We found that overexpression of CXCR4 or stimulation with CXCL12 supports neuroblastoma tumorigenesis. Moreover, CXCR4 inhibition with the high-affinity CXCR4 antagonist BL-8040 prevented tumor growth and reduced survival of tumor cells. These effects were mediated by the upregulation of miR-15a/16-1, which resulted in downregulation of their target genes BCL-2 and cyclin D1, as well as inhibition of ERK. Overexpression of miR-15a/16-1 in cells increased cell death, whereas antagomirs to miR-15a/16-1 abolished the proapoptotic effects of BL-8040. CXCR4 overexpression also increased miR-15a/16-1, shifting their oncogenic dependency from the BCL-2 to the ERK signaling pathway. Overall, our results demonstrate the therapeutic potential of CXCR4 inhibition in neuroblastoma treatment and provide a rationale to test combination therapies employing CXCR4 and BCL-2 inhibitors to increase the efficacy of these agents.Significance: These results provide a mechanistic rationale for combination therapy of CXCR4 and BCL-2 inhibitors to treat a common and commonly aggressive pediatric cancer.Cancer Res; 78(6); 1471-83. ©2017 AACR.

New Concepts in the Treatment of Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is a highly aggressive and generally incurable cancer. Current anti-MPM chemotherapy-based treatments are only marginally effective, and long-term survival remains an unmet goal. Nonetheless, in selected cases, personalized surgery-based multimodality treatments (MMT) have been shown to significantly extend survival. The design of MMT and selection of patients are challenging, and optimal results require accurate presurgical diagnosis, staging, and risk stratification. Further, meticulous surgical techniques and advanced radiation protocols must be applied. We review key principles and evolving concepts in the care of MPM patients with a focus on the expanding role of MMT in MPM.

Future directions in esophageal cancer therapy.

Resection techniques for esophageal carcinoma continue to evolve, from endoscopic mucosal resection or endoscopic submucosal dissection for early stage disease to standard and robot-assisted minimally invasive esophagectomy as part of multimodal therapy for locally advanced disease. Though currently limited to assessing conduit perfusion and sentinel lymph nodes, embedded technology in the robotic surgical platform will likely play an expanded role during esophagectomy in the future. The use of targeted therapies, checkpoint inhibitors, engineered immune cell therapy, and cancer vaccines show promise in the treatment of systemic disease. Radiation therapy techniques are becoming increasingly sophisticated and they may play a more active role in stage IV disease in the future.