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Large cell carcinoma (LCC) is a rare and aggressive lung cancer subtype with poor prognosis and no targeted therapies. Tumor-associated fibroblasts (TAFs) derived from LCC tumors exhibit premature senescence, and coculture of pulmonary fibroblasts with LCC cell lines selectively induces fibroblast senescence, which in turn drives LCC cell growth and invasion. Here we identify MMP1 as overexpressed specifically in LCC cell lines, and we show that expression of MMP1 by LCC cells is necessary for induction of fibroblast senescence and consequent tumor promotion in both cell culture and mouse models. We also show that MMP1, in combination with TGF-ß1, is sufficient to induce fibroblast senescence and consequent LCC promotion. Furthermore, we implicate PAR-1 and oxidative stress in MMP1/TGF-ß1-induced TAF senescence. Our results establish an entirely new role for MMP1 in cancer, and support a novel therapeutic strategy in LCC based on targeting senescent TAFs.

Pulmonary grade-1 typical (TC) and grade-2 atypical (AC) carcinoids share molecular characteristics with grade-3 large-cell neuroendocrine carcinoma (LCNEC) despite the distinct clinical behaviors. Most carcinoids can be surgically resected, however, limited treatment options exist for metastatic disease, present in 10-23% of TC and 40-50% of AC. Comprehensive genomic studies could help identify better therapeutic opportunities, novel diagnostic markers, and provide insight on the mechanisms responsible for the increased aggressiveness of AC versus TC. Such studies are rare due to the limited availability of suitable material. We have established a multi-center collaboration that has given us access to a unique collection of samples. We have already characterized 40 TC and 60 LCNEC genomes/exomes, and 61 TC, 8 AC and 69 LCNEC trancriptomes (published data). In the present study, we have performed whole-exome and transcriptome sequencing on 20 AC patients. Methylation data from 850K Illumina arrays were also generated for these samples, and for a subset of 20 TC and 20 LCNEC previously mentioned. When comparing the mutational data on AC with that of TC and LCNEC, we have found that similar to TC, AC harbor recurrent alterations in chromatin remodeling genes (such as MEN1 and ARID1A). They also carry alterations in genes involved in other cancer-related pathways (based on STRING), such as cell motility and cell death explaining their more aggressive phenotype. Integrative clustering analysis (MOFA and iCLUSTER) based on expression and methylation data tends to classify carcinoids into four groups: groups 1 and 2 are mostly composed of females with TC, and differ by their age composition and smoking status (Fisher's exact test p=0.008 and 0.03, respectively). Groups 3 and 4 are mostly composed of males with AC (Fisher's exact test for tumor type p=8x10-5). When including the LCNEC data, the samples from group 3 cluster with LCNEC, suggesting that AC can display a variety of expression and methylation patterns that may be linked to aggressiveness. This result was supported by the better survival of groups 1 and 2 compared to groups 3 and 4 (log-rank p=0.02), for which survival was similar to that of patients with LCNEC. Here, we present for the first time: (i) a multi-omics study on AC; (ii) the methylome characterization of TC, AC, and LCNEC; and (iii) the results of a comparative analysis of TC, AC, and LCNEC based on their molecular characteristics. We have identified the genes and pathways that might explain the progression from low-grade TC to intermediate-grade AC. Our expression and methylation data also supports the existence of a “super-AC” group, which clusters with LCNEC. Finally, we have identified a panel of molecular alterations that may help pathologist distinguishing between these three entities. NL and NA contributed equally. LFC and MF jointly supervised this work. Citation Format: Noémie Leblay, Nicolas Alcala, David Hervás Marin, Tiffany M. Delhomme, Théo Giffon, Akram Ghantous, Amélie Chabrier, Cyrille Cuenin, Janine Altmueller, Geoffroy Durand, Catherine Voegele, Philippe Lorimier, Anne-Claire Toffart, Jules Derks, Odd Terje Brustugun, Joachim H. Clement, Joerg Saenger, John K. Field, Alex Soltermann, Gavin M. Wright, Luca Roz, Lucia Anna Muscarella, Paolo Graziano, Zdenko Herceg, Ernst-Jan Speel, Peter Nuernberg, James McKay, Nicolas Girard, Sylvie Lantuejoul, Juan Sandoval, Elisabeth Brambilla, Matthieu Foll, Lynnette Fernandez-Cuesta. Multi-omics comparative analyses of pulmonary typical carcinoids, atypical carcinoids, and large-cell neuroendocrine carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5358.

In lung cancer CD133+ cells have properties of cancer initiating cells (CICs) as stemness features, high tumorigenic potential and chemoresistance. Within the CD133+ CICs, we have recently identified a specific subset, defined as CD133+CXCR4+EpCAM-, endowed with high dissemination and metastatic potential (metastasis initiating cells, MICs). In normal tissues stem cells reside in specialized niches composed of both stromal cells and extracellular matrix proteins (ECM) while factors responsible for CICs maintenance and modulation are relatively unknown. Collagen, the most abundant protein of ECM, plays a pivotal role in mediating regulation of adhesion, survival and proliferation of tumor cells. The relevance of collagen glycosylation, a fundamental post-translational modification controlling several biological processes, remains however largely unexplored. To investigate the effects of interactions between tumor cells and differentially glycosylated ECM epitopes, we cultured primary (LT73) and established lung cancer cell lines (A549, H460) on type I collagen films neo-glycosylated with glucose, galactose or sialic acid residues. Our results showed a general increase of CICs subsets, evaluated by flow cytometry, in tumor cells cultured on glycosylated collagen compared to pristine collagen or tissue culture plates with a concomitant increased expression of stemness-related genes. Particularly, we observed that collagen functionalized with glucose had the highest efficiency in enriching for MICs (3-fold change). Analysis of proliferation and viability of tumor cells cultured on collagen films showed that glucose residues were particularly proficient in causing G1 phase growth arrest and cell death compared to pristine collagen resulting in an overall decrease in the bulk population and CICs enrichment. In PKH label-retention assays, glucose-glycosylated collagen also selected and increased the fraction of PHK-labeled quiescent tumor cells, enriched for MICs component, confirming the preferential selection/survival of MICs subset. Using LT73 cell line depleted for CD133+ cells, we then proved that CICs increase was also due to a de novo generation of CD133+ cells and in particular of CD133+CXCR4+EpCAM- metastatic subset through non-CICs to CICs conversion. The immunophenotypic increase of CD133+ cells was functionally validated in vivo by a limiting dilution tumorigenic assay that estimated a 4.6 higher frequency of CICs in LT73 cells cultured on glucose-glycosylated collagen compared to control group. Moreover tumors derived from cells exposed to glucose residues retained a higher contents of MICs associated with an increased dissemination potential compared to controls. Our results suggest that differential collagen glycosylation could play an essential role in the creation of a niche favorable for the generation and selection/survival of lung metastasis initiating cells

Purpose: We aimed to maximize the performance of detecting genetic alterations in lung cancer using high-throughput sequencing for patient-derived xenografts (PDXs). Experimental Design: We undertook an integrated RNA and whole-exome sequencing of 14 PDXs. We focused on the genetic and functional analysis of beta 2-microglobulin (B2M), a component of the HLA class-I complex. Results: We identified alterations in genes involved in various functions, such as B2M involved in immunosurveillance. We extended the mutational analysis of B2M to about 230 lung cancers. Five percent of the lung cancers carried somatic mutations, most of which impaired the correct formation of the HLA-I complex. We also report that genes such as CALR, PDIA3, and TAP1, which are involved in the maturation of the HLA-I complex, are altered in lung cancer. By gene expression microarrays, we observed that restitution of B2M in lung cancer cells upregulated targets of IFN alpha/IFN gamma. Furthermore, one third of the lung cancers lacked the HLA-I complex, which was associated with lower cytotoxic CD8(+) lymphocyte infiltration. The levels of B2M and HLA-I proteins correlated with those of PD-L1. Finally, a deficiency in HLA-I complex and CD8(+) infiltration tended to correlate with reduced survival of patients with lung cancer treated with anti-PD-1/anti-PD-L1. Conclusions: Here, we report recurrent inactivation of B2M in lung cancer. These observations, coupled with the mutations found at CALR, PDIA3, and TAP1, and the downregulation of the HLA-I complex, indicate that an abnormal immunosurveillance axis contributes to lung cancer development. Finally, our observations suggest that an impaired HLA-I complex affects the response to anti-PD-1/anti-PD-L1 therapies. (C) 2016 AACR.

The European Early Lung Cancer (EUELC) project aims to determine if specific genetic alterations occurring in lung carcinogenesis are detectable in the respiratory epithelium. In order to pursue this objective, nonsmall cell lung cancer (NSCLC) patients with a very high risk of developing progressive lung cancer were recruited from 12 centres in eight European countries: France, Germany, southern Ireland, Italy, the Netherlands, Poland, Spain and the UK. In addition, NSCLC patients were followed up every 6 months for 36 months. A European Bronchial Tissue Bank was set up at the University of Liverpool (Liverpool, UK) to optimise the use of biological specimens. The molecular–pathological investigations were subdivided into specific work packages that were delivered by EUELC Partners. The work packages encompassed mutational analysis, genetic instability, methylation profiling, expression profiling utilising immunohistochemistry and chip-based technologies, as well as in-depth analysis of FHIT and RARb genes, the telomerase catalytic subunit hTERT and genotyping of susceptibility genes in specific pathways. The EUELC project engendered a tremendous collaborative effort, and it enabled the EUELC Partners to establish protocols for assessing molecular biomarkers in early lung cancer with the view to using such biomarkers for early diagnosis and as intermediate end-points in future chemopreventive programmes.

The loss of genetic material on chromosome 10q is frequent in different tumors and particularly in malignant gliomas. We analyzed 90 of these tumors and found loss of heterozygosity (LOH) in >90% of the informative loci in glioblastoma multiforme (GBM). Initial studies restricted the common LOH region to 10q24-qter. Subsequently, the study of a pediatric GBM suggested D10S221 and D10S209, respectively, as centromeric and telomeric markers of a 4-cM LOH region. It is interesting to note that, in one subset of cells from this tumor, locus D10S209 seems involved in the allelic imbalance of a larger region, with D10S214 as telomeric marker. This 17-cM region contains the D10S587-D10S216 interval of common deletion recently defined on another set of gliomas.

According to World Health Organization guidelines, Atypical Carcinoids (ACs) are well-differentiated lung neuroendocrine tumors with 2-10 mitoses/2 mm2 and/or foci of necrosis (usually punctate). Besides morphological criteria no further tools in predicting AC clinical outcome are proposed. Aim of this work was to identify novel factors able to predict AC disease aggressiveness and progression. Three hundred-seventy lung carcinoids were collected and centrally reviewed by two expert pathologists. Morphology and immunohistochemical markers (Ki-67, TTF-1, CD44, OTP, SSTR2A, Ascl1, p53 and Rb1) were studied and correlated with disease free survival (DFS) and overall survival (OS). Fifty-eight of 370 tumors were defined as AC. Survival analysis showed that patients with Ascl1+ACs and those with OTP-ACs had a significantly worse DFS than patients with Ascl1-ACs and OTP+ACs, respectively. Combining Ascl1 and OTP expressions, groups were formed reflecting the aggressiveness of disease (p=0.0005). Ki-67 ≥10% patients had a significantly worse DFS than patients with Ki-67

The generation of 3D-bioprintable and biocompatible hydrogels based on elastin and hyaluronic acid is described. The procedure is based on a biocompatible click reaction between maleimide and thiol for the final crosslinking, employable with living cells due to fast kinetics and absence of side products. To this end, both α-elastin and hyaluronic acid were functionalized with linkers ending with maleimide groups, at controlled functionalization intensities, and crosslinked with a dithiol−PEG linker. Varying the equivalents of the three reagents, four different hydrogels were obtained and their biocompatibility, swelling capacity and printability were tested. Biological experiments were performed with human lung fibroblast, human bronchial epithelial and human endothelial cell lines cultured in the hydrogels. Fibroblasts and epithelial cells can survive and proliferate. Epithelial cells showed an increased expression of CD44 and integrin αvβ3. Gene expression analysis revealed up-regulation of metalloproteinases both in normal fibroblast and epithelial cells.

WHO classification of Thoracic Tumours defines lung carcinoid tumours (LCTs) as well-differentiated neuroendocrine neoplasms (NENs) classified in low grade typical (TC) and intermediate grade atypical carcinoids (AC). Limited data exist concerning protein expression and morphologic factors able to predict disease aggressiveness. Though Ki-67 has proved to be a powerful diagnostic and prognostic factor for Gastro-entero-pancreatic NENs, its role in lung NENs is still debated. A retrospective series of 370 LCT from two oncology centers was centrally reviewed. Morphology and immunohistochemical markers (Ki-67, TTF-1, CD44, OTP, SSTR-2A, Ascl1, and p53) were studied and correlated with Overall Survival (OS), Cancer-specific survival (CSS) and Disease-free survival (DFS). Carcinoid histology was confirmed in 355 patients: 297 (83.7%) TC and 58 (16.3%) AC. Ki-67 at 3% was the best value in predicting DFS. Ki-67 ≥ 3% tumours were significantly associated with AC histology, stage III-IV, smoking, vascular invasion, tumour spread through air spaces OTP negativity, and TTF-1, Ascl1 and p53 positivity. After adjustment for center and period of diagnosis, both Ki-67 (≥3 versus

In lung cancer, CD133+ cells represent the subset of cancer stem cells (CSC) able to sustain tumor growth and metastatic dissemination. CSC function is tightly regulated by specialized niches composed of both stromal cells and extracellular matrix (ECM) proteins, mainly represented by collagen. The relevance of collagen glycosylation, a fundamental post‐translational modification controlling several biological processes, in regulating tumor cell phenotype remains, however, largely unexplored. To investigate the bioactive effects of differential ECM glycosylation on lung cancer cells, we prepared collagen films functionalized with glucose (Glc‐collagen) and galactose (Gal‐collagen) exploiting a neoglycosylation approach based on a reductive amination of maltose and lactose with the amino residues of collagen lysines. We demonstrate that culturing of tumor cells on collagen determines a glycosylation‐dependent positive selection of CSC and triggers their expansion/generation. The functional relevance of CD133+ CSC increase was validated in vivo, proving an augmented tumorigenic and metastatic potential. High expression of integrin β1 in its active form is associated with an increased proficiency of tumor cells to sense signaling from glycosylated matrices (glyco‐collagen) and to acquire stemness features. Accordingly, inhibition of integrin β1 in tumor cells prevents CSC enrichment, suggesting that binding of integrin β1 to Glc‐collagen subtends CSC expansion/generation. We provide evidence suggesting that collagen glycosylation could play an essential role in modulating the creation of a niche favorable for the generation and selection/survival of lung CSC. Interfering with this crosstalk may represent an innovative therapeutic strategy for lung cancer treatment., Glycans are well known for their involvement in recognition phenomena between cells; however, the role of small glycan epitopes in cell–extracellular matrix (ECM) interactions needs to be further elucidated. Here we exploit bioactive ECM mimetics, functionalizing type I collagen films with different glycans, to investigate the relevance of specific ECM glycosignatures in tumor‐ECM interactions. We show that in vitro culturing of lung cancer cells on glycosylated collagen films results in differential modulation of cancer stem cells (CSC), associated in vivo with an enhanced tumor initiation ability and increased metastatic potential. Interactions of CSC with glycosylated collagen are regulated through the active form of integrin β1. Interfering with integrin β1 signaling results in abrogation of CSC enrichment induced by glycosylated collagen.

Platinum-based chemotherapy remains widely used in advanced non-small cell lung cancer (NSCLC) despite experimental evidence of its potential to induce long-term detrimental effects, including the promotion of pro-metastatic microenvironments. In this study, we investigated the interconnected pathways underlying the promotion of cisplatin-induced metastases. In tumor-free mice, cisplatin treatment resulted in an expansion in the bone marrow of CCR2+CXCR4+Ly6Chigh inflammatory monocytes (IMs) and an increase in lung levels of stromal SDF-1, the CXCR4 ligand. In experimental lung metastasis assays, cisplatin-induced IMs promoted the extravasation of tumor cells and the expansion of CD133+CXCR4+ metastasis-initiating cells (MICs). Peptide R, a novel CXCR4 inhibitor designed as an SDF-1 mimetic peptide, prevented cisplatin-induced IM expansion, the recruitment of IMs into the lungs, and the promotion of metastasis. At the primary tumor site, cisplatin treatment reduced tumor size while simultaneously inducing tumor release of SDF-1, MIC expansion, and recruitment of pro-invasive CXCR4+ macrophages. Co-recruitment of MICs and CCR2+CXCR4+ IMs to distant SDF-1-enriched sites also promoted spontaneous metastases that were prevented by CXCR4 blockade. In clinical specimens from NSCLC patients SDF-1 levels were found to be higher in platinum-treated samples and related to a worse clinical outcome. Our findings reveal that activation of the CXCR4/SDF-1 axis specifically mediates the pro-metastatic effects of cisplatin and suggest CXCR4 blockade as a possible novel combination strategy to control metastatic disease.

miRNAs play a central role in the complex signaling network of cancer cells with the tumor microenvironment. Little is known on the origin of circulating miRNAs and their relationship with the tumor microenvironment in lung cancer. Here, we focused on the cellular source and relative contribution of different cell types to circulating miRNAs composing our risk classifier of lung cancer using in vitro/in vivo models and clinical samples. A cell‐type specific expression pattern and topography of several miRNAs such as mir‐145 in fibroblasts, mir‐126 in endothelial cells, mir‐133a in skeletal muscle cells was observed in normal and lung cancer tissues. Granulocytes and platelets are the major contributors of miRNAs release in blood. miRNAs modulation observed in plasma of lung cancer subjects was consistent with de‐regulation of the same miRNAs observed during immunosuppressive conversion of immune cells. In particular, activated neutrophils showed a miRNA profile mirroring that observed in plasma of lung cancer subjects. Interestingly mir‐320a secreted by neutrophils of high‐risk heavy‐smokers promoted an M2‐like protumorigenic phenotype through downregulation of STAT4 when shuttled into macrophages. These findings suggest a multifactorial and nonepithelial cell‐autonomous origin of circulating miRNAs associated with risk of lung cancer and that circulating miRNAs may act in paracrine signaling with causative role in lung carcinogenesis and immunosuppression., What's new? microRNAs play a central role in the complex signaling network of cancer cells with the tumor microenvironment. However, little is known on the origin of circulating miRNAs and their mechanisms of action. This study found a multifactorial and non‐epithelial cell‐autonomous origin of circulating miRNAs associated with lung cancer risk. The findings also suggest a link between an immunosuppressive and pro‐tumorigenic microenvironment and modulation of circulating miRNAs associated with lung cancer risk. The authors propose a novel mechanism whereby miRNA released by neutrophils induce macrophage polarization to support lung cancer growth, highlighting the potential for reprogramming macrophages toward an anti‐tumor polarization.

PURPOSE: Lung cancer heterogeneity makes response to therapy extremely hard to predict. Patient-derived xenografts (PDXs) represent a reliable preclinical model that closely recapitulates the main characteristics of the primary tumor and could represent a useful asset to test new therapies. Here, using PET imaging, we verify how lung cancer PDXs reproduce the metabolic features of the corresponding primary tumors. METHODS: We performed longitudinal [18F]FDG-PET studies on nine different PDXs, obtained by implants of primary cancer fragments harvested from patients. Max [18F]FDG uptake values of the lesion for each group were calculated and compared to corresponding patient's uptake. RESULTS: Different PDXs showed variable tumor growth rate and [18F]FDG uptake confirming the preservation of individual characteristics. A good intra-group reproducibility of PET measurements was observed. Furthermore, the subgroup of PDXs originating from primary tumors with higher metabolic rate displayed a rank order of [18F]FDG uptake similar to that of patients' original SUV. CONCLUSION: PDXs reproduced the original glucose metabolism of primary lesions and represent therefore a promising preclinical model also for the early assessment of therapy efficacy.

Inactivation of the FHIT and TP53 genes is frequently observed in primary non-small cell lung cancers (NSCLC) and cell lines and may contribute to resistance to apoptotic stimuli elicited by various anti-tumor drugs. To evaluate a possible relationship between FHIT and TP53 status and response to platinum-analogue regimens, we retrospectively selected 55 NSCLC patients treated with carboplatin/gemcitabine. Pre-treatment formalin fixed biopsies were analyzed for FHIT and p53 protein expression by immunohistochemistry and representative micro dissected tissue for TP53 mutations by DG-DGGE/sequencing. The FHIT-negative immunophenotype (FHIT-, pathologic) was found in 33 patients (60%) and p53 over expression/mutation (p53+, pathologic) in 25 patients (45%). The FHIT-/p53+ combination was present in 12 patients (22%). Overall, there was partial response in 21 patients (38%), with subgroup response rates of 33% in FHIT+/p53-, 46% in FHIT+/p53+, 38% in FHIT-/p53- and 33% in FHIT-/p53+ patients. Median progression-free survival (PFS) was 9.6, 7.9, 6.8 and 5.9 months and median overall survival (OS) was 12.8, 11.9, 10.5 and 8.7 months in the four groups, respectively. The Group comparison showed significantly worse PFS (p=0.04) in FHIT-/p53+ than the other groups. There was no significant difference in OS between the groups. A trend (p=0.07) for shorter OS was found in FHIT- cases suggesting that NSCLC tumors carrying this feature are less responsive to treatment. This retrospective study indicates that FHIT-/p53+ status might be a biological variable influencing the efficacy of carboplatin/gemcitabine treatment in NSCLC.