Emil Lou

Tunneling Nanotubes Provide a Unique Conduit for Intercellular Transfer of Cellular Contents in Human Malignant Pleural Mesothelioma

Tunneling nanotubes are long, non-adherent F-actin-based cytoplasmic extensions which connect proximal or distant cells and facilitate intercellular transfer. The identification of nanotubes has been limited to cell lines, and their role in cancer remains unclear. We detected tunneling nanotubes in mesothelioma cell lines and primary human mesothelioma cells. Using a low serum, hyperglycemic, acidic growth medium, we stimulated nanotube formation and bidirectional transfer of vesicles, proteins, and mitochondria between cells. Notably, nanotubes developed between malignant cells or between normal mesothelial cells, but not between malignant and normal cells. Immunofluorescent staining revealed their actin-based assembly and structure. Metformin and an mTor inhibitor, Everolimus, effectively suppressed nanotube formation. Confocal microscopy with 3-dimensional reconstructions of sectioned surgical specimens demonstrated for the first time the presence of nanotubes in human mesothelioma and lung adenocarcinoma tumor specimens. We provide the first evidence of tunneling nanotubes in human primary tumors and cancer cells and propose that these structures play an important role in cancer cell pathogenesis and invasion.

Estimating Survival in Patients With Lung Cancer and Brain Metastases: An Update of the Graded Prognostic Assessment for Lung Cancer Using Molecular Markers (Lung-molGPA).

Importance Lung cancer is the leading cause of cancer-related mortality in the United States and worldwide. As systemic therapies improve, patients with lung cancer live longer and thus are at increased risk for brain metastases. Understanding how prognosis varies across this heterogeneous patient population is essential to individualize care and design future clinical trials. Objective To update the current Diagnosis-Specific Graded Prognostic Assessment (DS-GPA) for patients with non–small-cell lung cancer (NSCLC) and brain metastases. The DS-GPA is based on data from patients diagnosed between 1985 and 2005, and we set out to update it by incorporating more recently reported gene and molecular alteration data for patients with NSCLC and brain metastases. This new index is called the Lung-molGPA. Design, Setting, and Participants This is a multi-institutional retrospective database analysis of 2186 patients diagnosed between 2006 and 2014 with NSCLC and newly diagnosed brain metastases. The multivariable analyses took place between December 2015 and May 2016, and all prognostic factors were weighted for significance by hazard ratios. Significant factors were included in the updated Lung-molGPA prognostic index. Main Outcomes and Measures The main outcome was survival. Multiple Cox regression was used to select and weight prognostic factors in proportion to their hazard ratios. Log rank tests were used to compare adjacent classes and to compare overall survival for adenocarcinoma vs nonadenocarcinoma groups. Results The original DS-GPA was based on 4 factors found in 1833 patients with NSCLC and brain metastases diagnosed between 1985 and 2005: patient age, Karnofsky Performance Status, extracranial metastases, and number of brain metastases. The patients studied for the creation of the DS-GPA had a median survival of 7 months from the time of initial treatment of brain metastases. To design the updated Lung-molGPA, we analyzed data from 2186 patients from 2006 through 2014 with NSCLC and newly diagnosed brain metastases (1521 adenocarcinoma and 665 nonadenocarcinoma). Significant prognostic factors included the original 4 factors used in the DS-GPA index plus 2 new factors: EGFR and ALK alterations in patients with adenocarcinoma (mutation status was not routinely tested for nonadenocarcinoma). The overall median survival for the cohort in the present study was 12 months, and those with NSCLC-adenocarcinoma and Lung-molGPA scores of 3.5 to 4.0 had a median survival of nearly 4 years. Conclusions and Relevance In recent years, patient survival and physicians’ ability to predict survival in NSCLC with brain metastases has improved significantly. The updated Lung-molGPA incorporating gene alteration data into the DS-GPA is a user-friendly tool that may facilitate clinical decision making and appropriate stratification of future clinical trials.

Analysis of genetic variants in never-smokers with lung cancer facilitated by an Internet-based blood collection protocol: a preliminary report.

Purpose: Germ line polymorphisms may confer susceptibility to lung cancer in never smokers, but studies in the United States have been limited by the low number of cases seen at single institutions. We hypothesized that we could use the Internet to bolster the accrual of appropriate patients. Experimental Design: We established an Internet-based protocol to collect blood and information from patients throughout the United States. To illustrate the power of this approach, we used these samples, plus additional cases and age-matched controls from the Memorial Sloan-Kettering Cancer Center (New York, NY) and the Aichi Cancer Center (Nagoya, Japan), to analyze germ line DNA for genetic variants reportedly associated with lung cancer susceptibility. The genotypes for the polymorphisms rs763317 (intron 1) and T790M (exon 20) in the EGFR gene were determined by direct sequencing, and CHRNA3 nicotinic acetylcholine receptor single nucleotide polymorphisms (rs8034191 and rs1051730) were genotyped as part of a pilot genome-wide association study. Results: We successfully analyzed germ line DNA from 369 cases, including 45 obtained via the Internet, and 342 controls. A germ line EGFR T790M variant was identified in 2 of the 369 cases (0.54%; 95% confidence interval, 0.21-1.29%), and in none of the 292 controls (P = 0.21). No difference was observed in EGFR rs763317 frequency between cases and controls. Similarly, neither CHRNA3 rs8034191 nor rs1051730 were associated with lung cancer risk. Conclusions: The Internet provides a way to recruit patients throughout the country for minimal risk studies. This approach could be used to facilitate studies of germ line polymorphisms in specific groups of patients with cancer. Clin Cancer Res; 16(2); 755–63

Bevacizumab continuation beyond initial bevacizumab progression among recurrent glioblastoma patients

Background:Bevacizumab improves outcome for most recurrent glioblastoma patients, but the duration of benefit is limited and survival after initial bevacizumab progression is poor. We evaluated bevacizumab continuation beyond initial progression among recurrent glioblastoma patients as it is a common, yet unsupported practice in some countries.Methods:We analysed outcome among all patients (n=99) who received subsequent therapy after progression on one of five consecutive, single-arm, phase II clinical trials evaluating bevacizumab regimens for recurrent glioblastoma. Of note, the five trials contained similar eligibility, treatment and assessment criteria, and achieved comparable outcome.Results:The median overall survival (OS) and OS at 6 months for patients who continued bevacizumab therapy (n=55) were 5.9 months (95% confidence interval (CI): 4.4, 7.6) and 49.2% (95% CI: 35.2, 61.8), compared with 4.0 months (95% CI: 2.1, 5.4) and 29.5% (95% CI: 17.0, 43.2) for patients treated with a non-bevacizumab regimen (n=44; P=0.014). Bevacizumab continuation was an independent predictor of improved OS (hazard ratio=0.64; P=0.04).Conclusion:The results of our retrospective pooled analysis suggest that bevacizumab continuation beyond initial progression modestly improves survival compared with available non-bevacizumab therapy for recurrent glioblastoma patients require evaluation in an appropriately randomised, prospective trial.

Tunneling Nanotubes: A new paradigm for studying intercellular communication and therapeutics in cancer.

Tunneling nanotubes are actin-based cytoplasmic extensions that function as intercellular channels in a wide variety of cell types.There is a renewed and keen interest in the examination of modes of intercellular communication in cells of all types, especially in the field of cancer biology. Tunneling nanotubes –which in the literature have also been referred to as “membrane nanotubes,” “’intercellular’ or ‘epithelial’ bridges,” or “cytoplasmic extensions” – are under active investigation for their role in facilitating direct intercellular communication. These structures have not, until recently, been scrutinized as a unique and previously unrecognized form of direct cell-to-cell transmission of cellular cargo in the context of human cancer. Our recent study of tunneling nanotubes in human malignant pleural mesothelioma and lung adenocarcinomas demonstrated efficient transfer of cellular contents, including proteins, Golgi vesicles, and mitochondria, between cells derived from several well-established cancer cell lines. Further, we provided effective demonstration that such nanotubes can form between primary malignant cells from human patients. For the first time, we also demonstrated the in vivo relevance of these structures in humans, having effectively imaged nanotubes in intact solid tumors from patients. Here we provide further analysis and discussion on our findings, and offer a prospective ‘road map’ for studying tunneling nanotubes in the context of human cancer. We hope that further understanding of the mechanisms, methods of transfer, and particularly the role of nanotubes in tumor-stromal cross-talk will lead to identification of new selective targets for cancer therapeutics.

Tumor exosomes induce tunneling nanotubes in lipid raft-enriched regions of human mesothelioma cells.

Tunneling nanotubes (TnTs) are long, non-adherent, actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study, we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation. Mesothelioma cells co-cultured with exogenous mesothelioma-derived exosomes formed more TnTs than cells cultured without exosomes within 24-48 h; and this effect was most prominent in media conditions (low-serum, hyperglycemic medium) that support TnT formation (1.3-1.9-fold difference). Fluorescence and electron microscopy confirmed the purity of isolated exosomes and revealed that they localized predominantly at the base of and within TnTs, in addition to the extracellular environment. Time-lapse microscopic imaging demonstrated uptake of tumor exosomes by TnTs, which facilitated intercellular transfer of these exosomes between connected cells. Mesothelioma cells connected via TnTs were also significantly enriched for lipid rafts at nearly a 2-fold higher number compared with cells not connected by TnTs. Our findings provide supportive evidence of exosomes as potential chemotactic stimuli for TnT formation, and also lipid raft formation as a potential biomarker for TnT-forming cells.

Intercellular communication in malignant pleural mesothelioma: properties of tunneling nanotubes.

Malignant pleural mesothelioma is a particularly aggressive and locally invasive malignancy with a poor prognosis despite advances in understanding of cancer cell biology and development of new therapies. At the cellular level, cultured mesothelioma cells present a mesenchymal appearance and a strong capacity for local cellular invasion. One important but underexplored area of mesothelioma cell biology is intercellular communication. Our group has previously characterized in multiple histological subtypes of mesothelioma a unique cellular protrusion known as tunneling nanotubes (TnTs). TnTs are long, actin filament-based, narrow cytoplasmic extensions that are non-adherent when cultured in vitro and are capable of shuttling cellular cargo between connected cells. Our prior work confirmed the presence of nanotube structures in tumors resected from patients with human mesothelioma. In our current study, we quantified the number of TnTs/cell among various mesothelioma subtypes and normal mesothelial cells using confocal microscopic techniques. We also examined changes in TnT length over time in comparison to cell proliferation. We further examined potential approaches to the in vivo study of TnTs in animal models of cancer. We have developed novel approaches to study TnTs in aggressive solid tumor malignancies and define fundamental characteristics of TnTs in malignant mesothelioma. There is mounting evidence that TnTs play an important role in intercellular communication in mesothelioma and thus merit further investigation of their role in vivo.

Phase II trial of upfront bevacizumab and temozolomide for unresectable or multifocal glioblastoma

Patients with unresectable glioblastomas have a poor prognosis, with median survival of 6–10 months. We conducted a phase II trial of upfront 5‐day temozolomide (TMZ) and bevacizumab (BV) in patients with newly diagnosed unresectable or multifocal glioblastoma. Patients received up to four cycles of TMZ at 200 mg/m2 on days 1–5, and BV at 10 mg/kg on days 1 and 15 of a 28‐day cycle. Brain magnetic resonance imaging (MRI) was performed monthly. Therapy was continued as long as there was no tumor progression, grade 4 nonhematologic toxicity, or recurrent grade 4 hematologic toxicity after dose reduction. The primary end point was best tumor response as measured on MRI. Forty‐one patients were accrued over 12 months; 39 had a full set of MRI scans available for evaluation. Assessment for best radiographic responses was as follows: partial responses in 24.4%, stable disease in 68.3%, and progressive disease in 2.4%. Treatment‐related toxicities included seven grade 4 toxicities and one grade 5 toxicity (myocardial infarction). From this study, it was concluded that an upfront regimen of TMZ and BV for unresectable glioblastoma was well tolerated and provided a significant level of disease stabilization. Therapeutic toxicities were consistent with those seen in the adjuvant setting using these agents. The upfront approach to treatment of glioblastoma in the unresectable population warrants further investigation in randomized controlled phase III trials.

Tunneling nanotube formation is stimulated by hypoxia in ovarian cancer cells

In this study, we demonstrated that hypoxic conditions stimulated an increase in tunneling nanotube (TNT) formation in chemoresistant ovarian cancer cells (SKOV3, C200). We found that suppressing the mTOR pathway using either everolimus or metformin led to suppression of TNT formation in vitro, verifying TNTs as a potential target for cancer-directed therapy. Additionally, TNT formation was detected in co-cultures including between platinum-resistant SKOV3 cells, between SKOV3 cells and platinum-chemosensitive A2780 cells, and between SKOV3 cells cultured with benign ovarian epithelial (IOSE) cells; these findings indicate that TNTs are novel conduits for malignant cell interactions and tumor cell interactions with other cells in the microenvironment. When chemoresistant C200 and parent chemosensitive A2780 cells were co-cultured, chemoresistant cells displayed a higher likelihood of TNT formation to each other than to chemosensitive malignant or benign epithelial cells. Hypoxia-induced TNT formation represents a potential mechanism for intercellular communication in ovarian cancer and other forms of invasive refractory cancers.

Vaccinia virus GLV-1h153 in combination with 131I shows increased efficiency in treating triple-negative breast cancer

We investigated the therapeutic efficacy of a replication‐competent oncolytic vaccinia virus, GLV‐1h153, carrying human sodium iodide symporter (hNIS), in combination with radioiodine in an orthotopic triple‐negative breast cancer (TNBC) murine model. In vitro viral infection was confirmed by immunoblotting and radioiodine uptake assays. Orthotopic xenografts (MDA‐MB‐231 cells) received intratumoral injection of GLV‐1h153 or PBS. One week after viral injection, xenografts were randomized into 4 treatment groups: GLV‐1h153 alone, GLV‐1h153 and 131I (~5 mCi), 131I alone, or PBS, and followed for tumor growth. Kruskal‐Wallis and Wilcoxon tests were performed for statistical analysis. Radiouptake assay showed a 178‐fold increase of radioiodine uptake in hNIS‐expressing infected cells compared with PBS control. Systemic 131I‐iodide in combination with GLV‐1h153 resulted in a 6‐fold increase in tumor regression (24 compared to 146 mm3 for the virus‐only treatment group; P<0.05; d 40). We demonstrated that a novel vaccinia virus, GLV‐1h153, expresses hNIS, increases the expression of the symporter in TNBC cells, and serves both as a gene marker for noninvasive imaging of virus and as a vehicle for targeted radionuclide therapy with 131I.—Gholami, S., Chen, C‐H., Lou, E., Belin, L. J., Fujisawa, S., Longo, V. A. Chen, N. G., Gönen, M., Zanzonico, P. B., Szalay, A. A., Fong, Y. Vaccinia virus GLV‐1h153 in combination with 131I shows increased efficiency in treating triple‐negative breast cancer. FASEB J. 28, 676–682 (2014). www.fasebj.org