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Background Extracellular vesicles (EVs) containing specific subsets of functional biomolecules are released by all cell types and analysis of circulating EVs can provide diagnostic and prognostic information. To date, little is known regarding the role of EVs both as biomarkers and potential key players in human lung cancer. Methods Plasma EVs were isolated from 40 cancer-free heavy-smokers classified according to a validated 24-microRNA signature classifier (MSC) at high (MSCpos-EVs) or low (MSCneg-EVs) risk to develop lung cancer. EVs origin and functional properties were investigated using in vitro 3D cultures and in vivo models. The prognostic value of miRNAs inside EVs was assessed in training and in validation cohorts of 54 and 48 lung cancer patients, respectively. Results Different membrane composition, biological cargo and pro-tumorigenic activity were observed in MSCpos vs MSCneg-EVs. Mechanistically, in vitro and in vivo results showed that miR-126 and miR-320 from MSCpos-EVs increased pro-angiogenic phenotype of endothelial cells and M2 polarization of macrophage, respectively. MSCpos-EVs prompted 3D proliferation of non-tumorigenic epithelial cells through c-Myc transfer. Moreover, hypoxia was shown to stimulate the secretion of EVs containing c-Myc from fibroblasts, miR-126-EVs from endothelial cells and miR-320-EVs from granulocytes. Lung cancer patients with higher levels of mir-320 into EVs displayed a significantly shorter overall survival in training [HR2.96] and validation sets [HR2.68]. Conclusion Overall our data provide a new perspective on the pro-tumorigenic role of circulating EVs in high risk smokers and highlight the significance of miR-320-EVs as a new prognostic biomarker in lung cancer patients.

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-β1, is sufficient to induce fibroblast senescence and consequent LCC promotion. Furthermore, we implicate PAR-1 and oxidative stress in MMP1/TGF-β1-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., This work was further supported by grants from the Agencia Estatal de Investigaci´on (AEI/FEDER) (PI13/02368, SAF2016-79527-R and PID2019-110944RB-I00 (AEI/ 10.13039/501100011033) to JA, PI16/ 00890 to NR), Fundaci´o Privada Cellex (to JA), National Institutes of Health (grant R01 GM132100 to ER), Generalitat de Catalunya (AGAUR SGR661 and CERCA Programme to JA), Junta Provincial de Barcelona de l’Associaci´o Espanyola Contra el C`ancer (AECC-B16-917 to JA), Italian Ministry of Health (RF-2016-02362946 to LR), Italian Association for Cancer Research (AIRC-IG21431 to LR), Sociedad Espa˜nola de Neumología y Cirugía Tor´acica – SEPAR (SEPAR 437 to NR), COST Action (PROTEOSTASIS BM1307), and by fellowships from Ciˆencia sem Fronteiras CNPq (to RI), and Universitat de Barcelona/beca APIF (to PD).

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.

Simple Summary Lung neuroendocrine neoplasms (LNENs) classes, as proposed by the World Health Organization 2015, do not provide properly prognostic and therapeutic indications. In fact, high-throughput molecular analysis, based on next-generation sequencing, identified novel molecular subgroups, associated with different genomic signatures, that could pave the way for alternative therapeutic approaches. The present review, coupled with in silico molecular analysis, could show the current genomic alterations state in actual LNENS groups. Interestingly our manuscript suggests that the molecular novelties could improve the LNENs therapeutics efficacy. In more detail, we reported the differences of gene alterations and mutational rate between LNENS, confirming the central pathogenetic role given by a different mutational rate in chromatin remodeling genes and tumor suppressors TP53-RB1. In conclusion, our results underlined that a further molecular layer is needed to improve the efficacy of LNENs medical treatment. Abstract Lung neuroendocrine neoplasms (LNENs) represent a rare and heterogeneous population of lung tumors. LNENs incidence rate has increased dramatically over the past 30 years. The current World Health Organization LNENs classification (WHO 2015), distinguished four LNENs prognostic categories, according to their morphology, necrosis amount and mitotic count: typical carcinoid (TC), atypical-carcinoid (AC), large cell neuroendocrine carcinoma (LCNEC) and small cell lung cancer (SCLC). At present, due to their rarity and biological heterogeneity there is still no consensus on the best therapeutic approach. Next-generation-sequencing analysis showed that WHO 2015 LNENs classes, could be characterized also by specific molecular alterations: frequently mutated genes involving chromatin remodeling and generally characterized by low mutational burden (MB) are frequently detected in both TC and AC; otherwise, TP53 and RB1 tumor suppressor genes alterations and high MB are usually detected in LCNEC and SCLC. We provide an overview concerning gene mutations in each WHO 2015 LNENs class in order to report the current LNENs mutational status as potential tool to better understand their clinical outcome and to drive medical treatment.

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.

Extracellular matrix (ECM) plays a crucial role in the regulation of tumor growth and progression. In the lungs, fibroblasts are involved in ECM homeostasis and are therefore crucial in lung tissue repair and remodeling. These activities are exploited by cancer cells during tumor growth by reprogramming fibroblast towards a tumor supportive phenotype including regulation of ECM-related proteins. We previously identified the nuclear transported XPO1 as a master regulator of fibroblast metabolic reprogramming. How cues from ECM can regulate the fate of cells disseminating from primary tumors is however poorly understood. To investigate the relevance of ECM remodeling during tumor development, primary lung fibroblasts were treated with TGF-β to mimic signals in the tumor microenvironment and Real-Time PCR was then performed to evaluate expression levels of ECM-related genes. Co-cultures and experiments with fibroblast conditioned medium or fibroblast-derived decellularized extracellular matrix (dECM) were performed with lung cancer cells and modulation of stemness phenotype evaluated by increase of CD133+ cancer stem cells. dECM from patient-derived fibroblast cultures was used in subcutaneous co-injections experiments with lung cancer cell lines in immunocompromised mice and dissemination to the lungs evaluated by flow-cytometry. Treatment with TGF-β induced over-expression of ECM-related genes (including COL1A and SPARC) which could be prevented by pharmacological inhibition of XPO1. Primary tumor growth was increased by dECM obtained from cultures of cancer associated fibroblasts. Interestingly analysis of tumors obtained by co-injection with different ECMs showed acquisition of specific phenotypes at late time points including relative content of CD133+ cancer stem cells and regulation of ECM-related genes in both cancer cells and stromal cells. Primary tumor microenvironments with high content of COL1A and COL6A3 were conductive to dissemination to distant sites which was associated by a local reduction of NK cells and an increase in macrophages. In vitro XPO1 inhibition prevented the ability of activated fibroblasts to stimulate migration of lung cancer cell lines and to increase the frequency of CD133+ cancer stem cells thereby reducing their pro-tumorigenic activity in vivo. Taken together these results reveal the activity of lung fibroblasts in dictating the metastatic phenotype by an ECM-regulated niche at the primary tumor site and suggest potential therapeutic value of pharmacological interventions blocking tumor-stroma interactions. Citation Format: Giuliana Pollaci, Francesca Giovinazzo, Ornella Rondinone, Antonina Bruccoleri, Federica Facchinetti, Monica Tortoreto, Ugo Pastorino, Giulia Bertolini, Gabriella Sozzi, Luca Roz. Cues from extracellular matrix produced by fibroblasts prime lung cancer cells for distant dissemination [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5098.

Background: Platinum-based treatment of locally advanced non-small cell lung cancer (NSCLC) provides some clinical benefit in controlling local disease but is ineffective on metastatic dissemination. Rising evidence suggests that chemotherapy can promote an inflammatory reaction supportive of metastasis growth.We previously demonstrated that cisplatin treatment of mouse xenografts enriches for the chemoresistant fraction of CD133+CXCR4+ lung cancer metastasis initiating cells (MICs), increasing distant metastasis development. We hypothesize here that the SDF-1/CXCR4 axis, implied in MICs migration and in stromal cells trafficking, could play a critical role in cisplatin-induced pro-metastatic effects. Results: To study the effects of cisplatin in promoting a pre-metastatic niche, tumor-free naïve SCID mice were treated with cisplatin plus/minus peptide R, a novel inhibitor of CXCR4. Cisplatin resulted in a rapid expansion of CCR2+CXCR4+Ly6Chigh inflammatory monocytes (IM) in the bone marrow, concomitantly with their recruitment to murine lungs guided by enhanced endothelial release of SFD-1. Tail-vein injection of H460 human lung cancer cells 72h after cisplatin administration resulted in augmented number of lung metastases enriched in CD133+CXCR4+ MICs. We proposed that the abundance of CXCR4+IM together with increased endothelial permeability caused by cisplatin favors tumor cells extravasations and the expansion of MICs through SDF-1/CXCR4 axis activation. Combination treatment with a CXCR4 inhibitor prevented IM recruitment, MICs expansion and consequently abolished metastasis overgrowth induced by cisplatin. Combination treatment of H460 subcutaneous xenografts in a regimen mimicking clinical setting revealed that cisplatin also caused tumor release of SDF-1 able to trigger local expansion of MICs subset and to recruit CXCR4+tumor associated macrophages involved in tumor cells intravasation. Stromal SDF-1 induced by cisplatin at distant site also co-recruited MICs and CCR2+CXCR4+IM, promoting spontaneous metastasis formation that can be counteracted by CXCR4 blockade. We confirmed in clinical setting that platinum-based neoadjuvant treatment of NSCLC patients significantly increases tumor SDF-1 expression. Moreover, an higher expression of tumor SDF-1 after cisplatin neo-adjuvant treatment was associated with patients' shorter DFS (p=0,0056; Hazards Ratio= 3,1) and poor OS (p=0,029; Hazards Ratio= 3,46). Conclusions: Our data reveal a paradoxical pro-metastatic effect of cisplatin that fosters MIC-IM recruitment and cross-talk via SDF-1/CXCR4 axis activation. A new combination strategy based on CXCR4 inhibition may disrupt these interactions, providing more effective results for lung cancer treatment Citation Format: Giulia Bertolini, Valeria Cancila, Orazio Fortunato, Giuseppe Lo Russo, Monica Tortoreto, Giovanni Centonze, Massimo Milione, Claudio Tripodo, Stefania Scala, Gabriella Sozzi, Luca Roz. Cisplatin-induced activation of SDF-1/CXCR4 axis sustains lung cancer metastasis by promoting co-recruitment of metastasis initiating cells and inflammatory monocytes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1106.

Lung cancer represents the leading cause of cancer-related deaths worldwide due to its high incidence and the lack of effective therapies. Current pharmacological strategies for the treatment of advanced stage disease are in fact largely ineffective mostly due to the emergence of drug resistance. The cancer stem cell (CSC) hypothesis suggests that treatment failure and tumor relapse may be explained by the existence of a subset of self-renewing cancer cells endowed with tumor-initiating potential which are able to escape conventional and targeted therapies and to regenerate tumors.

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.

Pulmonary large-cell neuroendocrine carcinomas (LCNECs) have similarities with other lung cancers, but their precise relationship has remained unclear. Here we perform a comprehensive genomic (n = 60) and transcriptomic (n = 69) analysis of 75 LCNECs and identify two molecular subgroups: “type I LCNECs” with bi-allelic TP53 and STK11/KEAP1 alterations (37%), and “type II LCNECs” enriched for bi-allelic inactivation of TP53 and RB1 (42%). Despite sharing genomic alterations with adenocarcinomas and squamous cell carcinomas, no transcriptional relationship was found; instead LCNECs form distinct transcriptional subgroups with closest similarity to SCLC. While type I LCNECs and SCLCs exhibit a neuroendocrine profile with ASCL1high/DLL3high/NOTCHlow, type II LCNECs bear TP53 and RB1 alterations and differ from most SCLC tumors with reduced neuroendocrine markers, a pattern of ASCL1low/DLL3low/NOTCHhigh, and an upregulation of immune-related pathways. In conclusion, LCNECs comprise two molecularly defined subgroups, and distinguishing them from SCLC may allow stratified targeted treatment of high-grade neuroendocrine lung tumors., The molecular nature of large-cell neuroendocrine lung carcinomas (LCNEC) has remained unclear. Here, the authors show LCNECs represent a distinct transcriptional subgroup among lung cancers and comprise two molecular subgroups, type I (TP53 and STK11/KEAP1 alterations) and type II (TP53 and RB1 inactivation).

Cancer cells within a tumor are functionally heterogeneous and specific subpopulations, defined as cancer initiating cells (CICs), are endowed with higher tumor forming potential. The CIC state, however, is not hierarchically stable and conversion of non‐CICs to CICs under microenvironment signals might represent a determinant of tumor aggressiveness. How plasticity is regulated at the cellular level is however poorly understood. To identify determinants of plasticity in lung cancer we exposed eight different cell lines to TGFβ1 to induce EMT and stimulate modulation of CD133+ CICs. We show that response to TGFβ1 treatment is heterogeneous with some cells readily switching to stem cell state (1.5–2 fold CICs increase) and others being unresponsive to stimulation. This response is unrelated to original CICs content or extent of EMT engagement but is tightly dependent on balance between epithelial and mesenchymal features as measured by the ratio of expression of CDH1 (E‐cadherin) to SNAI2. Epigenetic modulation of this balance can restore sensitivity of unresponsive models to microenvironmental stimuli, including those elicited by cancer‐associated fibroblasts both in vitro and in vivo. In particular, tumors with increased prevalence of cells with features of partial EMT (hybrid epithelial/mesenchymal phenotype) are endowed with the highest plasticity and specific patterns of expression of SNAI2 and CDH1 markers identify a subset of tumors with worse prognosis. In conclusion, here we describe a connection between a hybrid epithelial/mesenchymal phenotype and conversion to stem‐cell state in response to external stimuli. These findings have implications for current endeavors to identify tumors with increased plasticity., Highlights Signals from the microenvironment are involved in modulation of cancer initiating cells (CICs) in lung cancer.Balance between epithelial/mesenchymal features is a crucial determinant of proclivity to stemness phenotype acquisition.Epigenetic modification of epithelial/mesenchymal balance can regulate response to microenvironmental stimuli.A specific pattern of expression of E‐cadherin and SNAI2 is associated with worst prognosis in NSCLC.

Metastasis is the main reason for lung cancer–related mortality, but little is known about specific determinants of successful dissemination from primary tumors and metastasis initiation. Here, we show that CD133+/CXCR4+ cancer-initiating cells (CIC) directly isolated from patient-derived xenografts (PDX) of non–small cell lung cancer are endowed with superior ability to seed and initiate metastasis at distant organs. We additionally report that CXCR4 inhibition successfully prevents the increase of cisplatin-resistant CD133+/CXCR4+ cells in residual tumors and their metastatization. Immunophenotypic analysis of lung tumor cells intravenously injected or spontaneously disseminated to murine lungs demonstrated the survival advantage and increased colonization ability of a specific subset of CD133+/CXCR4+ with reduced expression of epithelial cell adhesion molecule (EpCAM−), which also shows the greatest in vitro invasive potential. We next prove that recovered disseminated cells from lungs of PDX-bearing mice enriched for CD133+/CXCR4+/EpCAM− CICs are highly tumorigenic and metastatic. Importantly, microenvironment stimuli eliciting epithelial-to-mesenchymal transition, including signals from cancer-associated fibroblasts, are able to increase the dissemination potential of lung cancer cells through the generation of the CD133+/CXCR4+/EpCAM− subset. These findings also have correlates in patient samples where disseminating CICs are enriched in metastatic lymph nodes (20-fold, P = 0.006) and their detection in primary tumors is correlated with poor clinical outcome (disease-free survival: P = 0.03; overall survival: P = 0.05). Overall, these results highlight the importance of specific cellular subsets in the metastatic process, the need for in-depth characterization of disseminating tumor cells, and the potential of therapeutic strategies targeting both primary tumor and tumor–microenvironment interactions. Cancer Res; 75(17); 3636–49. ©2015 AACR.

The existence of specific cellular subpopulations within primary tumors with increased tumorigenic potential and chemotherapy resistance (tumor-initiating cells, TICs) holds great therapeutic implications. Resistant cells can remain quiescent for long periods and be responsible for local relapses and metastasis. We and others have previously described in non–small-cell lung cancer the presence of cisplatin-resistant CD133+ cells with tumor-initiating potential and co-expression of CXCR4 as possible indicator of TICs with disseminating potential. In this study, we report, by in vitro cell fate tracing systems, heterogeneity within the TIC compartment with a highly quiescent pool and a slowly dividing subpopulation, both containing CD133+ cells but respectively enriched for CD133+/CXCR4− and CD133+/CXCR4+ cells. Pretreatment with differentiating agent all-trans retinoic acid counteracts cisplatin resistance specifically of the slowly dividing compartment indicating effect on CD133+/CXCR4+ cells. The same effects are appreciable also in vivo in patient-derived xenografts, where several cycles of all-trans retinoic acid and cisplatin treatment are able to stably reduce this fraction of TICs and tumor dissemination. Thus, partially affecting the heterogeneous TICs compartment, differentiating therapy has promising effects in counteracting cisplatin resistance of CD133+ cells, reducing both local tumor growth and dissemination. In addition, our approach discloses a further level of complexity of chemotherapy-resistant CD133+ TICs, revealing phenotypical and functional heterogeneity of the cancer stem cell compartment in lung cancer.

Development of molecular markers that help to identify high-risk individuals or diagnose indeterminate pulmonary nodules could have a major impact on lung cancer clinical management. In this study, we evaluated the diagnostic potential of a newly-developed ELISA that specifically detects complement C4d. We measured this marker in five independent cohorts of plasma and bronchoalveolar lavage samples from lung cancer patients and controls. In case-control studies, the area under the ROC curve for the diagnosis of lung cancer was 0.82 (95%CI = 0.72-0.92) in plasma samples, and 0.80 (95%CI = 0.69 to 0.90) in bronchoalveolar lavage fluids. In a set of plasma samples from the MILD CT-screening trial, the assay was unable to discriminate between asymptomatic high-risk individuals with or without early stage lung cancer. On the contrary, in two independent cohorts of individuals with indeterminate pulmonary nodules, plasma samples from patients with lung cancer nodules presented higher levels of C4d than those from patients with benign nodules. Using a target population of patients with 8 to 30 mm nodules, the test identified likely benign lung nodules with 84% negative predictive value and 54% positive predictive value, at 89% specificity and 44% sensitivity. In conclusion, the specific determination of C4d may serve as an adjunct to current clinical practice in the diagnosis of indeterminate pulmonary nodules.

Purpose Recent screening trial results indicate that low-dose computed tomography (LDCT) reduces lung cancer mortality in high-risk patients. However, high false-positive rates, costs, and potential harms highlight the need for complementary biomarkers. The diagnostic performance of a noninvasive plasma microRNA signature classifier (MSC) was retrospectively evaluated in samples prospectively collected from smokers within the randomized Multicenter Italian Lung Detection (MILD) trial. Patients and Methods Plasma samples from 939 participants, including 69 patients with lung cancer and 870 disease-free individuals (n = 652, LDCT arm; n = 287, observation arm) were analyzed by using a quantitative reverse transcriptase polymerase chain reaction–based assay for MSC. Diagnostic performance of MSC was evaluated in a blinded validation study that used prespecified risk groups. Results The diagnostic performance of MSC for lung cancer detection was 87% for sensitivity and 81% for specificity across both arms, and 88% and 80%, respectively, in the LDCT arm. For all patients, MSC had a negative predictive value of 99% and 99.86% for detection and death as a result of disease, respectively. LDCT had sensitivity of 79% and specificity of 81% with a false-positive rate of 19.4%. Diagnostic performance of MSC was confirmed by time dependency analysis. Combination of both MSC and LDCT resulted in a five-fold reduction of LDCT false-positive rate to 3.7%. MSC risk groups were significantly associated with survival (χ12 = 49.53; P < .001). Conclusion This large validation study indicates that MSC has predictive, diagnostic, and prognostic value and could reduce the false-positive rate of LDCT, thus improving the efficacy of lung cancer screening.

The contribution of stromal cells to tumor progression is widely established while less is known about stromal changes that may influence the early phases of tumorigenesis such as incipient growth of (pre)neoplastic lesions or seeding and awakening of metastatic cells. We have previously characterized the pro-tumorigenic properties of lung fibroblasts identifying increased activity of the nuclear transporter Exportin-1 (XPO1) as a key driver in modulating metabolic properties of fibroblasts towards a tumor promoting phenotype through subcellular re-localization of p53 and p62/SQSTM1. Consistently, XPO1 inhibition prevented the ability of activated fibroblasts to stimulate migration of lung cancer cells and to increase the frequency of CD133+ cancer stem cells, thereby also reducing their in vivo pro-tumorigenic activity. To test the potential of therapeutic strategies based on modification of pro-tumorigenic microenvironments we exploited a mouse model of lung fibrosis based on single intranasal administration of an adenoviral vector expressing activated TGFβ. Fibrosis was efficiently induced in both nude and SCID mice and was associated with the creation of a pro-tumorigenic milieu as tested by tail vein injection of microenvironment-responsive lung cancer cell line LT73 one month after fibrosis induction. Mice treated with AdTGFβ showed increased number of tumor cells and/or CD133+ lung cancer stem cells two months after cancer cell seeding (p Overall these findings suggest potential therapeutic efficacy of XPO1 inhibition to revert the pro-tumorigenic activity of fibrotic lung microenvironments. Citation Format: Antonina Bruccoleri, Ornella Rondinone, Francesca Andriani, Federica Facchinetti, Monica Tortoreto, Gabriella Sozzi, Giulia Bertolini, Luca Roz. Fondazione IRCCS Ist. Inhibition of Exportin-1 function reverses the pro-tumorigenic potential of lung fibrotic microenvironments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1912.

The efficacy of computed tomography (CT) screening for early lung cancer detection in heavy smokers is currently being tested by a number of randomized trials. Critical issues remain the frequency of unnecessary treatments and impact on mortality, indicating the need for biomarkers of aggressive disease. We explored microRNA (miRNA) expression profiles of lung tumors, normal lung tissues and plasma samples from cases with variable prognosis identified in a completed spiral-CT screening trial with extensive follow-up. miRNA expression patterns significantly distinguished: ( i ) tumors from normal lung tissues, ( ii ) tumor histology and growth rate, ( iii ) clinical outcome, and ( iv ) year of lung cancer CT detection. Interestingly, miRNA profiles in normal lung tissues also displayed remarkable associations with clinical features, suggesting the influence of a permissive microenvironment for tumor development. miRNA expression analyses in plasma samples collected 1–2 y before the onset of disease, at the time of CT detection and in disease-free smokers enrolled in the screening trial, resulted in the generation of miRNA signatures with strong predictive, diagnostic, and prognostic potential (area under the ROC curve ≥ 0.85). These signatures were validated in an independent cohort from a second randomized spiral-CT trial. These results indicate a role for miRNAs in lung tissues and plasma as molecular predictors of lung cancer development and aggressiveness and have theoretical and clinical implication for lung cancer management.

INTRODUCTION miRNAs play a role in the complex network of signaling between cancer cells and tumor microenvironment. We previously reported the identification of diagnostic miRNA signatures (MSC) based on 24-miRNAs in plasma samples of lung cancer patients detected by low dose computed tomography (LDCT) screening. MATERIAL and METHODS To evaluate the potential origin of the miRNAs of the diagnostic signature, we analyzed their expression by real-time or digital PCR in both cells and conditioned medium (CM) from different cell types of the lung microenvironment as well as in plasma samples of heavy smokers and patients. Lung tissues and cell-blocks were analyzed by miRNAs in situ hybridization. Modulation of miRNAs after in vitro treatments, known to induce changes associated with cancer progression, in different cell types was assessed and correlated to changes observed in circulating miRNAs signatures. RESULTS and DISCUSSION The analysis of 100 pre and post surgery plasma samples from 31 patients suggested a tumor-related origin of those miRNAs which significantly declined to basal levels after curative tumor resection. Other miRNAs (miR-126, miR-92a, miR-320, miR-28-3p, miR-486, miR-451, miR-16) remained deregulated afer surgery likely indicating a “host-related” origin and the persistence of a risk profile. Specific expression of mir-145 in fibroblasts, mir-126 in endothelial cells, mir-133 in skeletal muscle cells, mir-451 and 142-3p in hematopoietic cells was observed with a good degree of correlation between cellular and secreted miRNAs levels in each cell types (Pearson correlation range: 0.59-0.81). In vitro experiments comparing activated vs. resting neutrophils, showed that 17 out of the 24 miRNAs were concordantly modulated as in lung cancer patients' plasma (Pearson r=0.60, p CONCLUSION These findings suggest that circulating miRNAs are non cell-autonomous, may act in paracrine signalling and have a causative role in lung carcinogenesis and immunesuppression. Citation Format: Orazio Fortunato, Cristina Borzi, Massimo Milione, Giovanni Centonze, Davide Conte, Mattia Boeri, Massimo Moro, Federica Facchinetti, Luca Roz, Veronica Huber, Chiara Camisaschi, Chiara Castelli, Licia Rivoltini, Valeria Cancila, Claudio Tripodo, Ugo Pastorino, Gabriella Sozzi. Circulating mir-320 promotes immunosuppressive macrophages M2 phenotype associated with lung cancer progression [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 4981.

The identification of lung tumor-initiating cells and associated markers may be useful for optimization of therapeutic approaches and for predictive and prognostic information in lung cancer patients. CD133, a surface glycoprotein linked to organ-specific stem cells, was described as a marker of cancer-initiating cells in different tumor types. Here, we report that a CD133 + , epithelial-specific antigen-positive (CD133 + ESA + ) population is increased in primary nonsmall cell lung cancer (NSCLC) compared with normal lung tissue and has higher tumorigenic potential in SCID mice and expression of genes involved in stemness, adhesion, motility, and drug efflux than the CD133 − counterpart. Cisplatin treatment of lung cancer cells in vitro resulted in enrichment of CD133 + fraction both after acute cytotoxic exposure and in cells with stable cisplatin-resistant phenotype. Subpopulations of CD133 + ABCG2 + and CD133 + CXCR4 + cells were spared by in vivo cisplatin treatment of lung tumor xenografts established from primary tumors. A tendency toward shorter progression-free survival was observed in CD133 + NSCLC patients treated with platinum-containing regimens. Our results indicate that chemoresistant populations with highly tumorigenic and stem-like features are present in lung tumors. The molecular features of these cells may provide the rationale for more specific therapeutic targeting and the definition of predictive factors in clinical management of this lethal disease.

Cancer is a complex disease, driven by the accumulation of several somatic aberrations but fostered by a two-way interaction between tumour cells and the surrounding microenvironment. Cancer associated fibroblasts (CAFs) represent one of the major players in tumour-stroma crosstalk. Recent in vitro and in vivo studies, often conducted by employing high throughput approaches, have started unravelling the key pathways involved in their functional effects. This review focus on open challenges in the study of CAF properties and function, highlighting at the same time the existence of common mechanisms as well as peculiarities in different cancer types (breast, prostate and lung cancer). Although still limited by current experimental models, which are unable to deal with the full level of complexity of the tumour microenvironment, a better understanding of these mechanisms may enable the identification of new biomarkers and therapeutic targets, to improve current strategies for cancer diagnosis and treatment.

To evaluate the relevance of fragile histidine triad (FHIT) status in relation to drug treatment, we analyzed the sensitivity of the Fhit-negative non-small cell lung cancer (NSCLC) cell line NCI-H460 to different drugs, after treatment with an adenoviral vector expressing the FHITtransgene. Expression of Fhit resulted in reduced sensitivity to etoposide, doxorubicin, and topotecan. This feature was associated with Fhit-induced downregulation of DNA topoisomerases I and II. In contrast, expression of Fhit did not modulate sensitivity to Taxol, but produced a slight increase in sensitivity to cisplatin, as shown by colony-forming assays. Analysis of apoptosis revealed that, after cisplatin exposure, the number of apoptotic cells was two-fold higher in Fhitexpressing H460 cells. Moreover, it appeared that wildtype p53 was required for sensitization to cisplatin because the effect was marginal in A549 and Calu-1 cells, where the p53 pathway is altered and simultaneous restoration of p53 and Fhit in Calu-1 cells increased cisplatin sensitivity. Fhit could also partially restore sensitivity to cisplatin in Bcl-2- and BCl-xL-overexpressing H460 cells that are normally resistant to this drug. Our results support the possible relevance of FHIT in cisplatin-based chemotherapy as well as in the reversal of drug resistance in NSCLC.

We evaluated the aberrant promoter methylation profile of a panel of 3 genes in DNA from tumor and sputum samples, in view of a complementary approach to spiral computed tomography (CT) for early diagnosis of lung cancer. The aberrant promoter methylation of RARbeta2, p16(INK4A) and RASSF1A genes was evaluated by methylation-specific PCR in tumor samples of 29 CT-detected lung cancer patients, of which 18 had tumor-sputum pairs available for the analysis, and in the sputum samples from 112 cancer-free heavy smokers enrolled in a spiral CT trial. In tumor samples from 29 spiral CT-detected patients, promoter hypermethylation was identified in 19/29 (65.5%) cases for RARbeta2, 12/29 (41.4%) for p16(INK4A) and 15/29 (51.7%) for RASSF1A. Twenty-three of twenty-nine (79.3%) samples of the tumors exhibited methylation in at least 1 gene. In the sputum samples of 18 patients, methylation was detected in 8/18 (44.4%) for RARbeta2 and 1/18 (5%) for both RASSF1A and p16(INK4A). At least 1 gene was methylated in 9/18 (50%) sputum samples. Promoter hypermethylation in sputum from 112 cancer-free smokers was observed in 58/112 (51.7%) for RARbeta2 and 20/112 (17.8%) for p16, whereas methylation of the RASSF1A gene was found in only 1/112 (0.9%) sputum sample. Our study indicates that a high frequency of hypermethylation for RARbeta2, p16(INK4A) and RASSF1A promoters is present in spiral CT-detected tumors, whereas promoter hypermethylation of this panel of genes in uninduced sputum has a limited diagnostic value in early lung cancer detection.

Chromosomal deletions are often observed in lung cancers suggesting that inactivation of tumor suppressor genes plays an important role in the development of this neoplasm. The region around chromosome 8p22 is a frequent and early target of these deletions and has therefore been investigated for the presence of candidate genes. The FEZ1/LZTS1 gene, located at 8p22, is inactivated in many cancers with 8p deletions, including prostate, esophageal, gastric, bladder, and breast cancer and the Fez1 protein has been shown to suppress growth of cancer cells and to regulate mitosis. To elucidate the role of FEZ1 in lung cancer, we have analyzed its expression by immunohistochemistry in 103 primary lung cancer specimens including 98 non–small cell lung cancers (57 adenocarcinomas, 32 squamous cell carcinomas, 7 large cell carcinomas, and 2 others) and five small cell carcinomas. Absence of Fez1 protein expression was observed in 27 cases (26%) and additional 43 cases (42%) showed strong reduction in immunoreactivity. There was a positive association between loss of FEZ1 expression and tumor grading (P = 0.0345) and a tendency toward a reduction in the mortality rate in subjects with strong FEZ1 expression. Overall, these data indicate an important role for FEZ1 in lung cancer and suggest the possibility that it may serve as a novel prognostic indicator.

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We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Deltaex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.

Deletions on chromosome arm 8p, as defined by allelic imbalance, are a frequent event in many different types of malignant tumors, including those of the head and neck. These regions are thought to harbor tumor suppressor genes. In order to define a high-density deletion map of this chromosomal arm in oral and oropharyngeal squamous cell carcinomas, we have tested for allelic imbalance in 35 such tumors with 22 short tandem-repeat polymorphisms. Overall, 21 (60%) of the 35 tumors showed allelic imbalance at one or more loci on chromosome arm 8p. Interstitial deletions defined three discrete areas of deletion: at 8p23, 8p22, and 8p12-p21. Tumors of TNM stages II-IV showed a significantly higher frequency of allelic imbalance on 8p than did TNM stage I tumors. Our data suggest that there are least three tumor suppressor loci on chromosome arm 8p that may be implicated in oral carcinogenesis. Furthermore, inactivation of such genes may be associated with high-grade tumors.

Analysis of molecular markers in biological fluids has been proposed as a tool for early detection and monitoring of cancer. Circulating plasma DNA concentrations have been found to be higher in cancer patients than in cancer-free control subjects, but little is known about the effect of specimen storage on plasma DNA concentrations. Here we investigated the impact of long-term storage of both plasma samples and purified plasma DNA on the reproducibility of plasma DNA quantification as determined using real-time polymerase chain reaction analysis. The analysis was performed on samples from a subset of 34 lung cancer patients and 28 matched control subjects selected from 200 subjects in our previously published case-control study and from 117 cancer-free smokers enrolled in a lung cancer screening program. Two samples of plasma and isolated DNA were assessed for each patient, with a median of 41 months between the first and second assessments for participants in the case-control study and 9 months for participants in the screening study. DNA levels declined substantially between the two assessments at an average rate of approximately 30% per year. These data provide valuable information for the rational planning of retrospective studies of banked series of biological samples, particularly if collected over a long period of time, as can occur in large clinical trials.

Purpose Non–small-cell lung cancer (NSCLC) is a tumor in which only small improvements in clinical outcome have been achieved. The issue is critical for stage I patients for whom there are no available biomarkers that indicate which high-risk patients should receive adjuvant chemotherapy. We aimed to find DNA methylation markers that could be helpful in this regard. Patients and Methods A DNA methylation microarray that analyzes 450,000 CpG sites was used to study tumoral DNA obtained from 444 patients with NSCLC that included 237 stage I tumors. The prognostic DNA methylation markers were validated by a single-methylation pyrosequencing assay in an independent cohort of 143 patients with stage I NSCLC. Results Unsupervised clustering of the 10,000 most variable DNA methylation sites in the discovery cohort identified patients with high-risk stage I NSCLC who had shorter relapse-free survival (RFS; hazard ratio [HR], 2.35; 95% CI, 1.29 to 4.28; P = .004). The study in the validation cohort of the significant methylated sites from the discovery cohort found that hypermethylation of five genes was significantly associated with shorter RFS in stage I NSCLC: HIST1H4F, PCDHGB6, NPBWR1, ALX1, and HOXA9. A signature based on the number of hypermethylated events distinguished patients with high- and low-risk stage I NSCLC (HR, 3.24; 95% CI, 1.61 to 6.54; P = .001). Conclusion The DNA methylation signature of NSCLC affects the outcome of stage I patients, and it can be practically determined by user-friendly polymerase chain reaction assays. The analysis of the best DNA methylation biomarkers improved prognostic accuracy beyond standard staging.

Permanent cell cycle arrest through senescence has been previously regarded as a protective mechanism against tumor progression. In contrast, there is evidence that senescence in tumor-associated fibroblasts (TAFs) enhances tumor growth. Senescence has been reported in tumor associated fibroblasts (TAFs) from a growing list of selected cancer types. However, the presence of senescent TAFs in lung cancer remains undefined. To address this gap of knowledge, we examined common markers of senescence in primary TAFs from the 3 major non-small cell lung cancer (NSCLC) subtypes: adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC). Given the difficulties in gathering LCC-TAFs owing to the lower prevalence of LCC compared to the other subtypes, primary fibroblasts from 2 independent cell collections were used. We found an enrichment of the myofibroblast-like phenotype in TAFs regardless their histologic subtype, yet senescence was observed in LCC-TAFs only. Likewise, co-culturing normal lung fibroblasts with LCC (but not ADC or SCC) cancer cells was sufficient to induce senescence, and this induction was prevented in the presence of an antioxidant, indicating that it is mediated through oxidative stress. Of note, senescent fibroblasts provided growth and invasive advantages to LCC cells in culture and in vivo beyond those provided by control (non-senescent) fibroblasts. These results expand recent evidence that challenges the common assumption that lung TAFs are a heterogeneous myofibroblast-like cell population regardless their histologic subtype. Of note, because LCC often distinguishes itself in the clinic by its aggressive nature, our findings support that senescent or senescent-like TAFs may contribute to the selective aggressive behavior of LCC tumors. Citation Format: Roberto Lugo, Marta Gabasa, Francesca Andriani, Marta Puig, Federica Facchinetti, Josep Ramírez, Abel Gómez-Caro, Ugo Pastorino, Pere Gascón, Albert Davalos, Noemí Reguart, Luca Roz, Jordi Alcaraz. Cancer cell-stromal cell crosstalk drives fibroblast senescence and tumor progression in large cell carcinoma of the lung in culture and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4103.

Interactions between cancer cells and the surrounding microenvironment are crucial determinants of cancer progression. Fibroblasts isolated from carcinomas (cancer associated fibroblasts, CAF) have been shown to fuel the neoplastic process. While normal fibroblasts from different organs generally present very specific transcriptional programs, CAF from different tumors display a similar phenotype reminiscent of myofibroblasts found at sites of wound healing, indicating that common transcriptional programs could be induced by cancer cells on the surrounding microenvironment. To investigate the existence of common mechanisms in fibroblast activation across different cancer types, gene expression profiles of tumor stroma or cancer associated fibroblasts (CAF) derived from breast, prostate and lung cancer were previously evaluated performing an inter-pathology bio-informatics analysis. For each comparison, genes were ranked and subjected to Gene Set Enrichment Analysis to identify Canonical Pathways significantly enriched. Most of commonly enriched gene sets were related to extracellular matrix (ECM) structure and remodeling, cytoskeletal organization and cell-cell adhesion. We specifically evaluated a previously identified ECM-related signature (ECM3) defining a breast cancer subtype likely to progress. We found that ECM3 signature was significantly enriched (p-value < 0.05) in cancer associated fibroblasts compared to the normal counterpart across different pathologies supporting the implication of ECM-related genes in fibroblasts with activated status. Furthermore increased expression of some of the most ECM3 genes (COL10A1, COMP and COL11A1) was detected in fibroblasts co-cultured with breast cancer cells, both in cellular extracts and in the conditioned medium (CM), suggesting their potential importance as circulating markers for early detection and risk assessment. To explore the significance of ECM-related molecules also in lung, we finally selected some of enriched ECM3-related molecules in lung fibroblasts (SPARC, COL11A1, MMP11, FN1, COMP, COL10A1) and we evaluated their presence in the conditioned medium. The analysis showed that the ECM molecules could be detected in medium conditioned by lung fibroblasts and generally enriched in CAF_CM compared to NF_CM (SPARC p = 0.004, COMP p = 0.003, MMP11 p = 0.012). Interestingly, analysis of de-cellularized extracellular matrix showed that Fibronectin 1 (FN1) was preferentially deposited by CAFs compared to NFs (p = 0.003) denoting a possible role of this molecule in the modulation of extracellular matrix during tumor development and/or progression. These findings support the hypothesis that identification of common factors responsible for proficient tumor-stroma cross-talk could be instrumental in novel strategies for risk assessment in different diseases. Citation Format: Francesca Andriani, Marta Giussani, Federica Facchinetti, Maurizio Callari, Elda Tagliabue, Ugo Pastorino, Luca Roz, Gabriella Sozzi, Gabriella Sozzi. Cancer-associated fibroblasts transcriptional pattern reveals common changes in extracellular matrix-related genes across different cancer types. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5085.

Introduction FHIT and p53 are the two most commonly altered tumor suppressor genes in lung cancer, and their molecular status regulates sensitivity to anticancer drugs. Although their functions are independent, there is evidence that their pathways might be interconnected, but little is known at the molecular level. Methods Microarray profiling of FHIT-transduced lung cancer cells and modulation of FHIT levels by RNA interference in human bronchial cells were used to generate a signature of FHIT-regulated transcripts. Expression of these genes was evaluated by real-time polymerase chain reaction in 55 primary lung cancer samples characterized for FHIT and p53 expression by immunehistochemistry. Results A signature of FHIT-transcripts, particularly enriched in genes involved in cell cycle control, was identified. This signature showed overlap with p53-regulated genes, indicating possible crosstalk between these proteins. Consistently, transcriptional deregulation after FHIT modulation was higher in p53-negative cells. In primary lung cancers, inactivation of either gene was detected in 48 of 55 cases (87%) and both genes in 23 of 55 (42%) cases, confirming the central role of these pathways. Primary tumors with inactivation of both FHIT and p53 displayed the strongest deregulation of growth-related pathways with high levels of expression of CCNB1, BUB1, CDC6, TOP2A, MCM6, and CENPF. Conclusions FHIT and p53 seem to rely on common mediators, and inactivation of both genes results in prominent deregulation of growth-related pathways in lung cancer cell lines and primary tumors. This reveals crosstalk between these proteins and suggests a possible distinctive phenotype for tumors with inactivation of both genes.

Current chemotherapy regimens have unsatisfactory results in most advanced solid tumors. It is therefore imperative to devise novel therapeutic strategies and to optimize selection of patients, identifying early those who could benefit from available treatments. Mouse models are the most valuable tool for preclinical evaluation of novel therapeutic strategies in cancer and, among them, patient-derived xenografts models (PDX) have made a recent comeback in popularity. These models, obtained by direct implants of tissue fragments in immunocompromised mice, have great potential in drug development studies because they faithfully reproduce the patient’s original tumor for both immunohistochemical markers and genetic alterations as well as in terms of response to common therapeutics They also maintain the original tumor heterogeneity, allowing studies of specific cellular subpopulations, including their modulation after drug treatment. Moreover PDXs maintain at least some aspects of the human microenvironment for weeks with the complete substitution with murine stroma occurring only after 2-3 passages in mouse and represent therefore a promising model for studies of tumor-microenvironment interaction. This review summarizes our present knowledge on mouse preclinical cancer models, with a particular attention on patient-derived xenografts of non small cell lung cancer and their relevance for preclinical and biological studies.

Rationale: Fragile histidine triad (FHIT) is a tumor suppressor gene involved in the pathogenesis of lung cancer. Objectives: The purpose of this study was to investigate the different molecular alterations leading to the inactivation of FHIT gene function and to validate their use as biomarkers of risk for progression of the disease in patients belonging to the multicentric European study for the Early detection of Lung Cancer (EUELC) who were resected for early-stage lung tumors. Methods: FHIT immunostaining was performed on 305 tumor samples. Themethylation status of FHIT promoterwas assessed by nested methylation-specific polymerase chain reaction (MSP-PCR) in 232 tumor and 225 normal lung samples ofwhich a subset of 187 patients had available normal/tumorDNA pairs. Loss of heterozygosity (LOH) at the FHIT locus was analyzed in 202 informative cases by D3S1300 and D3S1234 microsatellite markers. Measurements and Main Results: Lost or reduced FHIT expression was found in 36.7 and 75.7% of the tumor samples, respectively. Methylation of the FHIT promoter was found in 36.7%of tumor and 32.7% of normal lung samples, whereas LOH was detected in 61.9% of the tumors. A strong association with complete loss of FHIT expression was presentwhenmethylation and LOHwere analyzed together (P5 0.0064). Loss of FHIT protein expression was significantly more frequent in squamous cell carcinoma histotype (P , 0.0001) and in smokers (P5 0.008). FHIT methylation in normal lung was associated with an increased risk of progressive disease (OR, 2.27; P 5 0.0415). Conclusions:Our results indicate thatdifferentmolecularmechanisms interplay to inactivate FHIT expression and support the proposition that FHIT methylation in normal lung tissue could represent a prognostic marker for progressive disease.

Free circulating plasma DNA has emerged as a potential biomarker for early lung cancer detection. In a previous case-control study we have shown that high levels of plasma DNA are a strong risk factor for lung cancer.To assess the diagnostic performance and prognostic value of plasma DNA levels in a cohort of 1,035 heavy smokers monitored by annual spiral computed tomography (CT) for 5 years.Plasma DNA levels were determined through real-time quantitative PCR at baseline and at time of lung cancer diagnosis. Screening performance of the assay was calculated through the area under the receiver-operating characteristic curve (AUC-ROC). Kaplan-Meier analyses were computed for association with prognosis.Median baseline concentration of plasma DNA was not different in individuals who developed CT-detected lung cancers in the 5-year period (n = 38) versus cancer-free control subjects (AUC-ROC, 0.496; P = 0.9330), and only slightly higher at the time of cancer diagnosis (AUC-ROC, 0.607; P = 0.0369). At surgery, plasma DNA was higher in tumors detected at baseline (AUC-ROC, 0.80; P0.0001) and in Stage II to IV tumors detected during the first 2 years of screening (AUC-ROC, 0.87; P0.0001). A longitudinal study of plasma DNA levels showed increased values approaching to lung cancer diagnosis (P = 0.0010). Higher plasma DNA was significantly associated with poorer 5-year survival (P = 0.0066).Baseline assessment of plasma DNA level does not improve the accuracy of lung cancer screening by spiral CT in heavy smokers. Higher levels of plasma DNA at surgery might represent a risk factor for aggressive disease.

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.

Epithelial cancers consist in heterogeneous populations of cells characterized by different phenotypic and biological features. Neoplastic progression is driven by a subpopulation of cells within the tumor bulk defined as “cancer initiating cells”(CICs) with stemness features. Modulation of cancer initiating properties is a consequence of genetic and epigenetic changes in cancer cells combined with their interaction with the surrounding microenvironment. To investigate how microenvironmental signals could influence the stemness properties and how these signals are integrated in tumors with different phenotypic features, eight non-small cell lung cancer cell lines were exposed to external stimuli such as TGFβ. The frequency of CD133, previously identified as a marker of stemness in lung cancer, was evaluated by flow cytometry and the expression of epithelial and mesenchymal genes was analyzed by Real-Time PCR and immunofluorescence (IF) or immunohystochemistry (IHC). TGFβ treatment resulted in heterogeneous increased of CD133+ cells and the extent of response was strictly dependent on the ratio between epithelial and mesenchymal features, estimated by the expression of E-cadherin and SNAI2 genes. Only cells that reached a critical threshold in the balance between epithelial and mesenchymal traits were able to generate CICs, acquiring increased in vivo tumorigenic properties in response to external stimuli. Consistently, in cell lines that reached the critical threshold by modulating the epithelial-mesenchymal balance through over expression of miR-200c or demethylation of E-cadherin promoter, the ability to acquire stemness phenotype under microenvironmental stimuli was restored. Single-cell analysis showed the presence of a sub-population of cells expressing both epithelial and mesenchymal markers that could be responsible of the phenotypic plasticity within different cell lines. Moreover, the evaluation of 60 primary lung tumors by IHC discovered a specific pattern of E-cadherin a SNAI2 that identify patients with worst prognosis. Together these findings provided new insight to understand how dynamic modulation of epithelial and mesenchymal properties determines the plasticity of lung cancer cells through the generation of cancer initiating cells in response to microenviromental stimuli. Citation Format: Francesca Andriani, Giulia Bertolini, Federica Facchinetti, Erika Baldoli, Massimo Moro, Patrizia Casalini, Roberto Caserini, Ugo Pastorino, Gabriella Sozzi, Luca Roz. Conversion to stem cell state in response to microenvironmental cues is regulated by balance between epithelial and mesenchymal features in lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1395. doi:10.1158/1538-7445.AM2015-1395

Purpose: Analysis of circulating DNA in plasma can provide a useful marker for earlier lung cancer detection. This study was designed to assess the sensitivity and specificity of a quantitative molecular assay of circulating DNA to identify patients with lung cancer and monitor their disease. Materials and Methods: The amount of plasma DNA was determined through the use of real-time quantitative polymerase chain reaction (PCR) amplification of the human telomerase reverse transcriptase gene (hTERT) in 100 non–small-cell lung cancer patients and 100 age-, sex-, and smoking-matched controls. Screening performance of the assay was calculated through the receiver operating characteristic (ROC) curve. Odds ratios were calculated using conditional logistic regression analysis. Results: Median concentration of circulating plasma DNA in patients was almost eight times the value detected incontrols (24.3 v 3.1 ng/mL). The area under the ROC curve was 0.94 (95% CI, 0.907 to 0.973). Plasma DNA was a strong risk factor for lung cancer; concentrations in the upper tertile were associated with an 85-fold higher risk than were those in the lowest tertile. Conclusion: This study shows that higher levels of free circulating DNA can be detected in patients with lung cancer compared with disease-free heavy smokers by a PCR assay, and suggests a new, noninvasive approach for early detection of lung cancer. Levels of plasma DNA could also identify higher-risk individuals for lung cancer screening and chemoprevention trials.

Loss of expression of the Fhit protein is often associated with the development of many human epithelial cancers, including lung and cervical carcinomas. Restoration of Fhit expression in cell lines derived from these tumors has however yielded conflicting results, prompting the need for careful evaluation of the oncosuppressive potential of FHIT . In the present study, we have investigated the effect of Fhit reintroduction in seven lung cancer and three cervical cancer cell lines. To achieve efficient gene transfer and high levels of transgene expression, we have used an adenoviral vector to transduce the FHIT gene. The induction of apoptosis was evaluated by using the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay and propidium iodide staining. Activation of caspases was detected by using Western blot analysis, and tumorigenic potential of transduced cells in the nude mouse was also assessed. Restoration of Fhit expression induced apoptosis in all Fhit-negative cell lines, with Calu-1, H460, and A549 being the most susceptible among the lung cancer cell lines and SiHa cells among cervical carcinomas. Activation of caspase-8 was always associated with Fhit-mediated apoptosis, and in vivo tumorigenicity was either abolished by FHIT gene transfer (in H460 and SK-Mes cells) or strongly suppressed (in A549 and SiHa cells). Our data demonstrate oncosuppressive properties and strong proapoptotic activity of the Fhit protein in lung and cervical cancer cell lines and strengthens the hypothesis of its possible use as a therapeutic tool.

Lung cancer represents the leading cause of cancer-related death in developed countries. Despite the advances in diagnostic and therapeutic techniques, the 5-year survival rate remains low. The research for novel therapies directed to biological targets has modified the therapeutic approach, but the frequent engagement of resistance mechanisms and the substantial costs, limit the ability to reduce lung cancer mortality. MicroRNAs (miRNAs) are small noncoding RNAs with known regulatory functions in cancer initiation and progression. In this study we found that mir-660 expression is downregulated in lung tumors compared with adjacent normal tissues and in plasma samples of lung cancer patients with poor prognosis, suggesting a potential functional role of this miRNA in lung tumorigenesis. Transient and stable overexpression of mir-660 using miRNA mimics reduced migration, invasion, and proliferation properties and increased apoptosis in p53 wild-type lung cancer cells (NCI-H460, LT73, and A549). Furthermore, stable overexpression using lentiviral vectors in NCI-H460 and A549 cells inhibited tumor xenograft growth in immunodeficient mice (95 and 50% reduction compared with control, respectively), whereas the effects of mir-660 overexpression were absent in H1299, a lung cancer cell line lacking p53 locus, both in in vitro and in vivo assays. We identified and validated mouse double minute 2 (MDM2) gene, a key regulator of the expression and function of p53, as a new direct target of mir-660. In addition, mir-660 expression reduced both mRNA and protein expression of MDM2 in all cell lines and stabilized p53 protein levels resulting in an upregulation of p21WAF1/CIP1 in p53 wild-type cells. Our finding supports that mir-660 acts as a tumor suppressor miRNA and we suggest the replacement of mir-660 as a new therapeutic approach for p53 wild-type lung cancer treatment.

The tumor microenvironment is increasingly recognized as a contributor to cancer development and progression since several stromal components, including fibroblasts, interact with cancer cells regulating their behavior and ultimately affecting tumor phenotype. Using primary cultures of human fibroblasts isolated from resected lung cancers we previously demonstrated that co-injection with cancer cells results in increased tumorigenicity and altered expression of ECM related genes in heterotypic tumors. To identify factors involved in the cross-talk between fibroblasts and lung cancer cells, microarray gene expression profiling was performed on 60 fibroblast lines from cancerous and normal lung tissue from primary lung cancer patients, including 25 pairs of normal (NF) and cancer-associated fibroblasts (CAF) from the same patients. Lines were also characterized for presence of the activation marker alpha-SMA and for in vivo pro-tumorigenic activity by co-injection with A549 adenocarcinoma cells. Class comparison analyses identified: (i) Factors highly expressed in cancer-associated fibroblasts (CAF) compared to normal fibroblasts (NF): RHOB, SNAI2, EDIL3, LRRN3, CHN1, CTSC, NOPE and DOCK10; (ii) Genes highly expressed in activated fibroblasts: POSTN, TNFAIP6, CLEC2B and (iii) Genes associated with an in vivo pro-tumorigenic phenotype: CNTN3, ELOVL6, MET, XPO1. To explore the potential prognostic value of these microenvironment related markers, we selected antibodies against 20 proteins differentially expressed in lung fibroblasts and evaluated their performance in immunohistochemistry on FFPE sections of 74 cases of NSCLC. Selection criteria to prioritize candidates for IHC were based on results from molecular analyses (both microarray hybridization and Real-Time PCR validation) and involvement in biological pathways identified as potentially relevant by Gene Set Enrichment Analysis (GSEA). The frequency of positive cells was evaluated on: cancer cells, cancer-associated fibroblasts (CAF) and fibroblasts in normal tissue adjacent (AF) or distant (NF) from the surgical margin. This in-depth analysis revealed heterogeneous expression of different proteins with factors predominantly expressed by cancer cells (LRRN3) and others by the stromal component (CLEC2B). Among factors expressed by fibroblasts, positivity for NOPE, MET, CTSC and CLEC2B was detected in the majority of cases (60-70%) and they were generally expressed at higher frequency by CAF compared to NF. In univariate Cox models, SNAI2 expression by CAF was shown to correlate with worst overall survival (p=0.0288) and we further identified association of CNTN3 expression by CAF with shorter disease free survival (p=0.0125).These findings strengthen the hypothesis that identification of factors responsible for proficient tumor-stroma cross-talk could be instrumental in innovative strategies for risk assessment. Citation Format: Francesca Andriani, Giorgia Leone, Elena Landoni, Federica Facchinetti, Tiziana Caputo, Erika Baldoli, Rosalba Miceli, Luigi Mariani, Ugo Pastorino, Giuseppe Pelosi, Gabriella Sozzi, Luca Roz. SNAI2 expression by cancer-associated fibroblasts is a negative prognostic factor in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2852. doi:10.1158/1538-7445.AM2014-2852

Recent screening trials indicate that low-dose computed tomography (LDCT) reduces lung-cancer mortality in high risk subjects. However, high false positive rates, costs and potential harms highlight the need for complementary biomarkers. MicroRNAs (miRNAs) are tissue and disease specific molecules, actively released by cells in the circulatory system, which are associated with protein complexes and/or packaged in exosomes and microvesicles. Circulating miRNAs are rather stable and easily detectable in body fluids suggesting the possibility of using miRNAs as a new promising class of biomarkers. Sensitivity, specificity, positive and negative predictive values (PPV and NPV, respectively) of a non-invasive plasma miRNA signature classifier (MSC) to identify lung cancer occurrence and mortality was retrospectively evaluated in samples prospectively collected from smokers within the randomized Multicentre Italian Lung Detection (MILD) trial. Plasma samples from 939 subjects including 69 lung cancer patients and 870 disease-free individuals were analyzed using a Real-Time quantitative PCR (qRT-PCR) based assay for MSC. Combined sensitivity and specificity of plasma MSC and LDCT were assessed in a blinded validation study. MSC correctly identified patients with lung cancer at 87% sensitivity, 81% specificity, and 99% NPV, and 18 of 19 lung cancer deaths at 95% sensitivity 81% specificity, and 99.86% NPV. MSC identified 43 of 49 (88%) LDCT-detected cancers, 8 of 9 (89%) interval cancers in the LDCT arm, and 9 of 11 (82%) cancers in the observational arm. Diagnostic performance of MSC was confirmed by time dependency analysis. MSC fell to negative after lung cancer resection in 16 of 22 evaluable subjects. In lung cancer patients, there was a trend in mortality across levels of MSC (p=0.0336) and survival rate significantly differed according to MSC in all subjects (X21=49.53, p This large validation study highlights the clinical diagnostic and prognostic value of plasma MSC for lung cancer screening in high-risk populations. The combined use of LDCT and MSC could reduce the false positive rate of LDCT improving the efficacy of lung cancer screening. Citation Format: Mattia Boeri, Carla Verri, Gabriella Sozzi, Luca Roz, Davide Conte, Paola Suatoni, Alfonso Marchiano, Carlo La Vecchia, Marta Rossi, Francesca Bravi, Eva Negri, Nicola Sverzellati, Ugo Pastorino. Clinical utility of a plasma microRNA biomarker within lung cancer screening. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 871. doi:10.1158/1538-7445.AM2014-871