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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.

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.

BACKGROUND: Interactions between cancer cells and the surrounding microenvironment are crucial determinants of cancer progression. During this process, bi-directional communication among tumor cells and cancer associated fibroblasts (CAF) regulate extracellular matrix (ECM) deposition and remodeling. As a result of this dynamic process, soluble ECM proteins can be released into the bloodstream and may represent novel circulating biomarkers useful for cancer diagnosis. The aim of the present study was to measure the levels of three circulating ECM related proteins (COL11A1, COL10A1 and SPARC) in plasma samples of lung cancer patients and in healthy heavy-smokers controls and test whether such measurements have diagnostic or prognostic value. METHODS: Gene expression profiling of lung fibroblasts isolated from paired normal and cancer tissue of NSCLC patients was performed by gene expression microarrays. The prioritization of the candidates for the study of circulating proteins in plasma was based on the most differentially expressed genes in cancer associated fibroblasts. Soluble ECM proteins were assessed by western blot in the conditioned medium of lung fibroblasts and by ELISA assays in plasma samples. RESULTS: Plasma samples from lung cancer patients and healthy heavy-smokers controls were tested for levels of COL11A1 and COL10A1 (n = 57 each) and SPARC (n = 90 each). Higher plasma levels of COL10A1 were detected in patients (p ≤ 0.001), a difference that was driven specifically by females (p < 0.001). No difference in COL11A1 levels between patients and controls was found. SPARC levels were also higher in plasma patients than controls (p < 0.001) with good performance in discriminating the two groups (AUC = 0.744). No significant association was observed between plasma proteins levels and clinicopathological features or survival. CONCLUSION: Soluble factors related to proficient tumor-stroma cross-talk are detectable in plasma of primary lung cancer patients and may represent a valuable complementary diagnostic tool to discriminate lung cancer patients from healthy heavy-smokers individuals as shown for the SPARC protein.

Background Fibroblasts are crucial mediators of tumor-stroma cross-talk through synthesis and remodeling of the extracellular matrix and production of multiple soluble factors. Nonetheless, little is still known about specific determinants of fibroblast pro-tumorigenic activity in lung cancer. Here, we aimed at understanding the role of miRNAs, which are often altered in stromal cells, in reprogramming fibroblasts towards a tumor-supporting phenotype. Methods We employed a co-culture-based high-throughput screening to identify specific miRNAs modulating the pro-tumorigenic potential of lung fibroblasts. Multiplex assays and ELISA were instrumental to study the effect of miRNAs on the secretome of both primary and immortalized lung fibroblasts from lung cancer patients and to evaluate plasmatic levels of HGF in heavy smokers. Direct mRNA targeting by miRNAs was investigated through dual-luciferase reporter assay and western blot. Finally, the pro-tumorigenic activity of fibroblasts and their conditioned media was tested by employing in vitro migration experiments and mouse xenografts. Results We identified miR-16 as a master regulator of fibroblast secretome and showed that its upregulation reduces HGF secretion by fibroblasts, impairing their capacity to promote cancer cell migration. This effect is due to a pleiotropic activity of miR-16 which prevents HGF expression through direct inhibition of FGFR-1 signaling and targeting of HGF mRNA. Mechanistically, miR-16 targets FGFR-1 downstream mediator MEK1, thus reducing ERK1/2 activation. Consistently, chemical or genetic inhibition of FGFR-1 mimics miR-16 activity and prevents HGF production. Of note, we report that primary fibroblast cell lines derived from lungs of heavy smokers express reduced miR-16 levels compared to those from lungs not exposed to smoke and that HGF concentration in heavy smokers’ plasma correlates with levels of tobacco exposure. Finally, in vivo experiments confirmed that restoration of miR-16 expression in fibroblasts reduced their ability to promote tumor growth and that HGF plays a central role in the pro-tumorigenic activity of fibroblasts. Conclusions Overall, these results uncover a central role for miR-16 in regulating HGF production by lung fibroblasts, thus affecting their pro-tumorigenic potential. Correlation between smoking exposure and miR-16 levels could provide novel clues regarding the formation of a tumor-proficient milieu during the early phases of lung cancer development. Electronic supplementary material The online version of this article (10.1186/s13045-018-0594-4) contains supplementary material, which is available to authorized users.

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.

Background Extensive clinical experience has demonstrated the potential usefulness of autologous fat tissue (AFT) graft in tissue reconstruction, repair or regeneration. In the present study, we evaluated the feasibility and safety of AFT in the repair of surgically injured lung surface. Methods Eighty consecutive procedures of pulmonary metastasectomy by laser precision resection, were performed in 66 patients between March 2010 and December 2012. In the first 20 procedures, AFT graft was applied on the wounded pulmonary surface without closure of parenchymal surface. The following 40 procedures were carried on without AFT (20 leaving the resection margins open and 20 closing the resection margins with a running suture). In the remaining 20 procedures, AFT was applied and the resection margins closed. The efficacy of this technique was evaluated by comparing the AFT group with the non-AFT group, with respect to prolonged alveolar air leakage (PAAL), time to drain removal, length of hospital stay, and patient survival at four years. Results The occurrence of PAAL was lower in the AFT group as compared to non-AFT group (17.5% versus 42.5%, p = 0.027), and median time to drain removal shorter (4 versus 6 days respectively, p = 0.016). Overall 4-year survival was 70% for AFT group, and 59% for non-AFT group (p = 0.34). Conclusions This prospective cohort observational study demonstrated the feasibility and safety of AFT pulmonary grafting after laser metastasectomy. AFT graft improved pulmonary healing, by reducing the incidence and severity of PAAL. Moreover, there was no evidence of tumor promotion in the metastatic setting, with a similar overall survival at 4 years.

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.

Clinical efficacy of PD-1/PD-L1 targeting relies upon the reactivation of tumor-specific but functionally impaired PD-1+ T cells present before therapy. Thus, analyzing early-stage primary tumors may reveal the presence of T cells that are not yet functionally impaired. In this study, we report that activated (HLA-DR+) T cells with an effector memory (TEM) profile are enriched in such lesions. Tumor-infiltrating lymphocytes coexpressed PD-1 with the inhibitory receptors TIM-3, CTLA-4, LAG-3, and TIGIT, but also displayed a recently activated, nonexhausted phenotype. We also identified a subset of CD8+PD-1+FOXP3+ T lymphocytes at the earliest phase of functional differentiation after priming, termed “early effector cells” (EEC), which also exhibited an activated nonexhausted phenotype, but was less differentiated and associated with coexpression of multiple inhibitory receptors. In response to autologous tumor, EECs upregulated CD107a, produced IL2 and IFNγ, and were competent for differentiation. The identification of EECs marked by inhibitory receptor expression at tumor sites will enable investigations of early stages of adaptive antitumor immunity, as well as support the rationale for administering immunotherapy in early-stage non–small cell lung cancer. Cancer Res; 77(4); 851–61. ©2016 AACR.

Bi-directional interactions between cancer cells and the surrounding microenvironment actively contribute to cancer development and progression. Cancer-associated fibroblasts (CAFs) are one of the most prevalent cellular components in the tumor microenvironment and their metabolic reprogramming towards an activated phenotype reminiscent of fibroblasts found at sites of wound healing is involved in the acquisition of pro-tumorigenic properties. Specific determinants of tumor-promoting function of fibroblasts in lung cancer are however not well characterized. Here we used microarray gene expression profiling of fibroblasts isolated from lung cancer patients and functional in vitro and in vivo assays to identify enablers of fibroblasts pro-tumorigenic activity. Gene expression profiles of CAFs and normal fibroblasts (n=60) identified increased levels of the nuclear transporter Exportin 1 (XPO1/CRM1) in patient-derived fibroblasts endowed with an in vivo pro-tumorigenic phenotype in co-injection experiments with lung cancer cells. Elevated levels of XPO1, which controls cellular localization of numerous proteins including oncosuppressors, have been observed in cancer cells but the role of XPO1-related signaling in stromal cells has not been investigated. Pharmacological inhibition of XPO1 in fibroblasts resulted in anti-proliferative effects with a G2/M cell cycle arrest linked to p53 accumulation, increased CDKN1a transcription, induction of senescence (2-4 fold increase of β-galactosidase positive cells) and accumulation of γ-H2AX foci. Interestingly these effects were persistent only in activated fibroblasts compared to control fibroblasts suggesting a central role for XPO1 in mediating and sustaining permanent reprogramming of pro-tumorigenic fibroblasts. Additionally XPO1 inhibition was associated with cellular relocalization of p62/SQSTM1, a master regulator of energy metabolism in tumor stroma. Low levels of nuclear p62 were observed in activated fibroblasts and could be restored to normal levels through XPO1-activity inhibition. p62 nuclear accumulation inhibited DNA-damage repair and reduced extracellular acidification, indicating potential modification of metabolic properties of activated fibroblasts. Consistently, in heterotypic co-cultures experiments, we found that 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. Overall these evidences suggest that XPO1 is a potent enabler of the tumor promoting functions of fibroblasts and that XPO1-driven metabolic reprogramming in stromal cells could be targeted to affect cancer progression. Citation Format: Antonina Bruccoleri, Francesca Andriani, Federica Facchinetti, Ugo Pastorino, Gabriella Sozzi, Luca Roz. Exportin-1 enables pro-tumorigenic metabolic reprogramming of lung fibroblasts through relocalization of p53 and p62/SQSTM1 [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 5090.

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.

Loss of FHIT expression and p53 mutations are critical events in the early stages of lung carcinogenesis. The restoration of Fhit function in FHIT-negative cancer cells has been reported to cause tumour suppression by inhibition of cell proliferation and/or activation of apoptotic pathways. However, the studies designed to elucidate the biological role of Fhit and its potential interaction with p53 have produced conflicting results. We investigated here the effects of the simultaneous restoration of FHIT and p53 in Calu-1 cells by using a hormone-inducible gene expression system. We demonstrate that the restoration of FHIT expression reinforces the anti-proliferative effect associated with the simultaneous replacement of p53. Indeed, a more pronounced inhibition of cell proliferation associated with an earlier and higher induction of p21(waf1) mRNA and protein expression was observed in Fhit/p53-expressing cells compared with cells expressing p53 alone. This effect was not due to Fhit-mediated up-regulation of p53 expression; in fact p53 protein was expressed at the same level in both FHIT-positive and FHIT-negative cell clones. Consistent with this result, Fhit did not affect the expression of MDM2, a protein known to interact directly with p53 and target p53 for proteolytic degradation, thus down-regulating its activity.

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.

FHIT is a tumor suppressor gene that is frequently inactivated in human cancer. Although the Fhit protein is known to hydrolyze diadenosine triphosphate (Ap3A), this hydrolase activity is not required for Fhit-mediated oncosuppression. Indeed, the molecular mechanisms and the regulatory elements of Fhit oncosuppression are largely unknown. Here, we review physiological and pathological aspects of Fhit in the context of the ApnA family of signaling molecules, as well as the involvement of Fhit in apoptosis and the cell cycle in cancer models. We also discuss recent findings of novel Fhit interactions that may lead to new hypotheses about biochemical mechanisms underlying the oncosuppressor activity of this gene. J. Cell. Physiol. 208: 274–281, 2006. © 2006 Wiley-Liss, Inc.

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.

Abnormalities in the expression of the tumour suppressor fragile histidine triad (FHIT) gene have been reported in a variety of human tumours, including lung cancer and restoration of its expression in cancer cell lines resulted in the inhibition of proliferation and apoptosis induction. Most of the studies that have assigned a proapoptotic role to the FHIT gene were performed in adenoviral-FHIT-transduced cancer cells expressing high levels of the Fhit protein. The present work was the first study designed to investigate the effects of FHIT gene replacement in a human FHIT-negative non-small-cell lung cancer (NSCLC) cell line (Calu-1) by using a hormone-inducible expression system that allows tight modulation of the transgene expression. Through this approach, we demonstrated that a prolonged induction was required to accumulate the Fhit protein at levels adequate to promote a significant decrease of cell proliferation. Analysis of cell-cycle phase distribution showed an accumulation of cells in the G0/G1 phase and a concomitant decrease in the S phase. Moreover, an upregulation of p21waf1 transcript was found, which could account for the alteration of the cycling properties of the cells. The growth-inhibitory effects observed were not associated with apoptosis appearance, and although in these conditions the Fhit protein content was higher than in normal bronchial human epithelial cells (NHBE), it was still significantly lower than the level capable of inducing apoptosis in Calu-1 cells after adenoviral-mediated FHIT gene transfer. These results indicate that the tumour suppressor properties of Fhit are strictly related to its expression level and show that the Fhit protein has a dose-dependent antiproliferative effect on the Fhit-negative Calu-1 lung cancer cell line.

Recent studies have demonstrated the possibility to detect genetic changes in plasma DNA of cancer patients. The goal of this study was to validate a panel of molecular markers for lung cancer detection in plasma DNA. Three markers, p53, FHIT and microsatellite alterations at loci on chromosome 3, were used to detect mutations in tumor and plasma DNA of 64 stage I-III non small cell lung cancer patients. p53 mutations were studied by direct sequencing of exons 5 through 8 in tumor DNA and by plaque hybridization assay and sequencing in plasma DNA. Allelic losses were evaluated by fluorescent PCR in tumor and plasma DNA. p53 genomic mutations were detected in 26 (40.6%) of 64 tumor DNA samples and the identical mutation was identified in plasma of 19 (73.1%) of them. Microsatellite alterations at FHIT and 3p loci were observed in 40 (62.5%) tumors and in 23 (35.9%) plasma samples. Of the 40 patients showing microsatellite alterations in tumors, 19 (47.5%) displayed the same change in plasma DNA. At least 1 of the 3 genetic markers (p53, FHIT and 3p) was altered in plasma of 51.6% of all patients and 60.7% of stage I patients. Moreover, genetic markers in plasma identified 29 of 45 (64.4%) of all stages and 15 of 22 (68.2%) of stage I patients whose tumors had an alteration. These results provide the proof of principle that plasma DNA alterations are tumor-specific in most cases and support blood testing as a noninvasive strategy for early detection.

We have shown that adenovirus-mediated manipulation of apoptotic genes such as bax could be a therapeutic option for prostate cancer. Unfortunately, the response of experimental prostate tumors to a single therapeutic gene of the apoptotic pathway is short-lived, and most of these tumors relapse after a short period of time. In this investigation we present data generated with adenovirus AvARR(2)PB-Bad, in which the apoptotic gene bad was placed under the control of the dihydrotestosterone (DHT)-inducible third-generation probasin-derived promoter ARR(2)PB. This therapeutic virus was given alone or in combination with other therapeutic viruses to a variety of in vitro and in vivo experimental models of prostate cancer. On infection with AvARR(2)PB-Bad, DHT-induced Bad overexpression occurred specifically in androgen receptor-positive (AR(+)) cells of prostatic derivation. The apoptotic effect of AvARR(2)PB-Bad (group 1) was compared with that of AvARR(2)PB-Bax (which overexpresses the apoptotic protein Bax) (group 2), with that of the combination AvARR(2)PB-Bad plus AvARR(2)PB-Bax (group 3), and with that of the control virus AvARR(2)PB-CAT (group 4) in the cell line LNCaP. In addition to identifying the modality of apoptosis induction by overexpressed Bad, the results suggested that group 3 contained more apoptotic cells than any other group. In additional studies, AR(+) androgen-dependent LNCaP cells or AR(+) and androgen-independent C4-2 cells were injected subcutaneously into nude mice. Four groups of six LNCaP or C4-2 tumors were treated with the same combinations of viruses discussed above for groups 1, 2, 3, and 4. Treatment resulted in decreased tumor size in groups 1, 2, and 3 compared with group 4. There was a better response in group 3 compared with group 2, and in group 2 compared with group 1. A better response in group 3 was confirmed during a 8-week follow-up period, in which no treatment was administered. Two LNCaP and C4-2 tumors of group 3 disappeared at the end of treatment and did not recur after an 8-week follow-up period. The data suggest that polygene therapy with apoptotic molecules is more effective in experimental models of androgen-dependent or -independent prostate cancer than monogene therapy.

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.

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. Citation Format: Giulia Bertolini, Cecilia Gardelli, Francesca Andriani, Massimo Moro, Laura Russo, Laura Cipolla, Gabriella Sozzi, Luca Roz. Differential glycosylation of extracellular matrix specifically modulates lung cancer initiating cells subsets. [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 3351.

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.

Surgical removal is the mainstay for early lung cancer treatment and persistent air leaks represent one of the most common clinical complications after lung surgery. Adipose tissue transplantation has been proposed as a new strategy for regenerative therapy after breast cancer surgery; however its efficacy and safety of lung tissue healing after lung resections are unknown. The purpose of this study was to test the biological activity of adipose tissue to facilitate lung tissue healing and evaluate its effect on cancer cells growth, thus providing insight for a possible clinical application. Different in vitro cellular models were used to prove the potential biologic effect of autologous fat tissue (AFT) in repairing injured lung tissue, and in vivo xenograft models were used to evaluate tumor promoting potential of AFT on putative residual cancer cells. Treatment of both embryonic (WI-38) and adult lung fibroblasts and of normal bronchial epithelial cells (HBEC-KT) with AFT samples, harvested from subcutaneous tissue layer of 20 patients undergoing pulmonary metastasectomy, improved wound healing and cell proliferation indicating a trophic effect on both mesenchymal and epithelial cell types. Conversely AFT-conditioned medium was unable to stimulate in vitro proliferation of a lung adenocarcinoma reporter cellular system (A549). Moreover, co-injection of AFT and A549 cells in nude mice did not promote engraftment and progression of A549 cells. These preclinical findings provide preliminary evidence on the potential efficacy of AFT to accelerate lung tissue repair without undesired tumor promoting effects on putative residual cancer cells. J. Cell. Physiol. 228: 1166–1173, 2013. © 2012 Wiley Periodicals, Inc.

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.

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

The expression of the tumour suppressor protein fragile histidine triad (Fhit) is often impaired in many human cancers and its restoration in Fhit-negative cancer cell lines suppresses tumorigenicity and induces apoptosis. Although the proapoptotic function of Fhit is well documented, little is known about its precise mechanism of action and further studies are needed in order to elucidate the putative therapeutic properties of this protein. To this end, we have engineered the lung cancer cell line NCI-H460 in order to express different molecules involved in the control of apoptotic pathways. Infection of these cells with an adenoviral vector transducing the Fhit gene (Ad-Fhit) revealed that complete protection from apoptosis was conferred by the inhibitor of caspases Cytokine response modifier A (CrmA) and by a dominant-negative form of the adapter protein Fas-associated death domain (FADD) and partial protection by a dominant-negative form of caspase-8, while cells over expressing mitochondrial mediators of the apoptotic response such as Bcl-2 or Bcl-x(L) that are resistant to treatment with cisplatin, remained highly susceptible to cell death triggered by Fhit gene transfer. In line to what was observed in H460 cells, Ad-Fhit efficacy was not affected by Bcl-2 overexpression also in two other lung cancer cell lines (A549 and Calu-1). Analysis of cytochrome c release also confirmed that in Bcl-2- or Bcl-x(L)-expressing cells apoptosis could be detected by terminal deoxynucleotidyl-transferase mediated dUTP nick-end labelling (TUNEL) assay before any evidence of mitochondrial membrane perturbation. In conclusion, our analysis indicates that the Fhit protein exerts its oncosuppressor activity through induction of an apoptotic mechanism that seems to be FADD dependent, caspase-8 mediated and independent from mitochondrial amplification.

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

Epithelial cancers could be described as abnormal organs composed of cells with different phenotype and functional properties. It has been suggested that only a small fraction of cells defined as “cancer stem cells” (CSCs) is capable of initiating and maintaining a new tumor and that local microenvironment could be involved in modulation of stemness properties and metastatic colonization by induction of the epithelial-mesenchymal transition (EMT). To investigate how signals from stromal fibroblasts can influence stemness properties we used a primary lung adenocarcinoma cell line (LT73) containing a subpopulation of CD133+ CSCs (0.1%) and its derivative devoid of CD133+ cells (LT73CD133neg) which remains stable during short-term culturing. Medium conditioned by primary fibroblasts cell lines (CM) resulted in increase of CD133 positive cells, more pronounced in LT73CD133neg, indicating positive regulation of the stem cell pool and de novo generation of CSCs. Accordingly, expression of stemness related genes OCT4, NANOG and GA6 was increased (fold change 1.5-3). In vivo experiments also confirmed that tumors generated by injection of CM-treated cells had greater size compared to controls and maintained increased stemness features. Moreover stimulation with CM, associated to generation of CD133+ cells, was also often accompanied by gene expression changes consistent to EMT activation. To verify the importance of fibroblasts in providing the right cues also for successful colonization of the lungs, LT73 cells were injected in the tail vein of SCID mice. After 2 months LT73 cells co-injected with fibroblasts were present in the lungs in larger numbers (4 fold increase) compared to control mice indicating a possible role for fibroblasts in the preparation of the pre-metastatic niche Finally to establish whether the EMT process was required for acquisition of a stem like phenotype in presence of microenvironmental stimuli, we overexpressed in LT73 wt cell line miR200c which has been shown to prevent TGFbeta-induced EMT. Preliminary experiments showed that, although miR200c was able to partially prevent the EMT process by contrasting downregulation of the epithelial marker CDH1, its overexpression was insufficient to abrogate upregulation of the mesenchymal markers SNAI2, VIM, FN1 and de novo generation of stem-like cells (CD133+) after TGFbeta treatment. This indicates that even partial activation of the EMT process could be sufficient to modulate stemness features. Together these data demonstrate that stimuli from fibroblasts could de novo generate CD133+ cells modulating stem cells phenotype probably through EMT process and that the proficient cross-talk between fibroblasts and cancer stem cells could also be relevant for metastatic colonization. Citation Format: Francesca Andriani, Tiziana Caputo, Giulia Bertolini, Federica Facchinetti, Erika Baldoli, Massimo Moro, Roberto Caserini, Gabriella Sozzi, Luca Roz. Microenvironment stimuli elicited by fibroblasts contribute to epithelial mesenchymal transition and acquisition of stemness phenotype in 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 4867. doi:10.1158/1538-7445.AM2014-4867

Background: Adenovirus-mediated overexpression of the apoptosis-inducing protein Bax can induce apoptosis in prostate cancer cell lines. Constitutive overexpression of Bax could result in unwanted apoptosis in every site of accidental Bax accumulation in vivo. Therefore, we developed an adenoviral construct (Av-ARR 2 PB-Bax) in which the probasin promoter, modified to contain two androgen response elements, drives Bax expression. This promoter would be expected to limit expression of Bax to cells expressing the androgen receptor. Methods: A variety of androgen receptor (AR)-positive and -negative cell lines of prostatic or nonprostatic origin were infected with Av-ARR 2 PB-Bax or a control virus, Av-ARR 2 PB-CAT, in which the same promoter drives expression of the chloramphenicol acetyl transferase-reporter gene. Bax expression and apoptosis in vitro were assessed by western blot analysis. Tumor size and apoptosis in vivo were assessed after four weekly injections of Av-ARR 2 PB-Bax or Av-ARR 2 PB-CAT into subcutaneous LNCaP xenografts growing in uncastrated male mice. All statistical tests were two-sided. Results: Bax was overexpressed in an androgen-dependent way in AR-positive cell lines of prostatic origin but not in AR-positive cells of nonprostatic origin or in AR-negative cell lines of either prostatic or nonprostatic origin. The androgen dihydrotestosterone activated apoptosis in LNCaP cells infected with Av-ARR 2 PB-Bax but not in those infected with Av-ARR 2 PB-CAT. Av-ARR 2 PB-Bax-injected LNCaP xenograft tumors decreased in tumor size from 34.1 mm 3 (95% confidence interval [CI] = 25.1 mm 3 to 43.1 mm 3 ) to 24.6 mm 3 (95% CI = -2.5 mm 3 to 51.7 mm 3 ), but the difference was not statistically significant (P = .5). Tumors injected with Av-ARR 2 PB-CAT increased in size, from 28.9 mm 3 (95% CI = 12.7 mm 3 to 45.1 mm 3 ) to 206 mm 3 (95% CI = 122 mm 3 to 290 mm 3 ) (P = .002) and contained statistically significant more apoptotic cells (23.3% [95% CI = 21.1% to 25.6%] versus 9.5% [95% CI = 8.0% to 11.1]) (P

We previously reported the identification of the diagnostic and prognostic miRNA signatures in plasma samples of lung cancer patients detected by low dose computed tomography (LDCT) screening. Interestingly we also found miRNA profiles able to predict lung cancer development and its aggressiveness up to 2 years before lung cancer detection by LDCT. Circulating miRNAs associated with risk of aggressive disease onset showed to be concordantly regulated in corresponding normal lung tissues, rather than in tumor tissue. Recently, several papers suggest that miRNAs released by the microenvironment can also have a role in targeting tumor cells promoting their growth and invasiveness, and may act as hormones by functionally activating Toll-like receptors. In an effort to examine the components of the lung that are most involved in the generation of plasma miRNA signatures we looked at the expression of our 24 miRNAs in several cell components including fibroblasts, hematopoietic, endothelial, normal and tumor epithelial cells and in their respective culture medium. Our preliminary results indicated that every single miRNA is released differently and independently from the level of expression in respective cells across cell types and that tumor cells are less involved in the release of miRNAs. We also observed that several miRNAs are expressed and released from non-epithelial components only, whereas other miRNAs are expressed in all cellular components but released from specific cell types only. Among the 24 miRNA composing the signatures, we focused on two miRNAs (mir-486-5p, mir-660) that are strongly down-modulated in plasma samples of patients with an aggressive form of lung cancer. To evaluate if they have also a functional role in lung tumorigenesis, their activity was assessed in lung cancer (A549) and normal human bronchial epithelial (HBEC-KT1) cellular models using miRNA mimics or inhibitors. A new method for in vitro functional studies was settled-up. A549 and HBEC cells were infected with a lentivirus carrying the gene encoding Green Fluorescent Protein (GFP) (A549GFP and HBECGFP) and their biological features were tested using a fluorescence microplate reader. By using this methodology, it was possible to assess the biological endpoints by measuring the emission of fluorescent signal without interrupting cellular growth or migration. Our experiments using mir-486-5p and mir-660 mimics on A549GFP show an impairment of migratory and invasive capacity of these cells. Moreover, inhibition of miRNAs in HBECGFP resulted in an increased number of migrated and invaded cells compared to control. To evaluate the biological effect of these two miRNAs, in an in vivo model we injected A549 transiently transfected with mir-486 and mir-660 subcutaneously in both flanks of 4 to 6 weeks old female nude mice. Xenograft growth was monitored weekly by caliper measurement and data were shown as tumour growth rate or as Kaplan-Meyer curve (time required to reach tumor volume of 250 mm3 as endpoint). We demonstrated that miRNAs transiently over-expressed in A549 cells subcutaneously implanted in mice significantly reduce tumor growth and increase mice survival. Our preliminary findings on the origin of miRNAs support the conclusion that plasma miRNAs are heterogeneous and contributed by different cellular components, and not specifically by tumor cells, a result in keeping with the predicting value of the miRNA test up to two years before clinical detection of lung cancer. Moreover, functional studies on mir-486-5p and mir-660, strongly down-modulated in plasma of patients, suggested that may act as tumor suppressor miRNAs. Further analyses are in progress to identify the specific pathways in which they are involved. Nonetheless these preliminary results set the basis for a possible therapeutic use of miRNAs in lung cancer. Citation Format: Orazio Fortunato, Mattia Boeri, Carla Verri, Massimo Moro, Francesca Andriani, Luca Roz, Ugo Pastorino, Gabriella Sozzi. Origin and functional role of plasma circulating miRNAs. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A19.

There is increasing evidence of the central contribution of tumor microenvironment to cancer development and progression. The elucidation of the role of different cellular components to this mechanism might therefore provide novel insights on the transformation process and potentially indicate targets for innovative therapeutic intervention. In particular, it has been shown that fibroblasts isolated from invasive breast lesions can contribute to tumor growth through the CXCR4/SDF1 pathway and that human prostatic carcinoma-associated fibroblasts (CAFs) can drive the progression of initiated non-tumorigenic epithelial cells. Recently a gene-expression signature derived from lung CAFs was shown to have prognostic value in non-small cell lung cancer (NSCLC) patients. To further investigate the contribution of fibroblasts to lung cancer we established primary cultures of CAFs and matched normal fibroblasts (NFs) from resected NSCLC and performed co-injection experiments in nude mice with A549 lung cancer cells. Co-injection of fibroblasts generally resulted in tumors of bigger size and/or increased take rate when cells were injected at low doses. Time-course experiments revealed presence of human cells in the stromal component of xenografts up to one month after injection, indicating possible involvement also in the progression phase. To evaluate the contribution of fibroblasts to extracellular matrix composition of tumors we therefore analyzed by real-time PCR with human specific primers the expression levels of 14 extracellular matrix (ECM) related genes in 63 tumors generated by co-injection of lung cancer cells and fibroblasts (CAFs or NFs). Compared to tumors generated by injection of A549 cells alone, heterotypic tumors displayed strongly increased levels of COL6A3 and MMP2 (p≤0.03), slightly increased levels of SPARC and reduced CTSL and CTSC (p≤0.01) indicating influence of fibroblasts on ECM composition. Tumors generated by co-injections were also more likely to metastasize to the lungs as determined by flow cytometry and real-time PCR (31% vs. 17%; OR=2.56 95% CI 0.6-12.7). Moreover culturing of primary lung cancer cells with CAF conditioned medium (CM) also resulted in similar transcriptional regulations of ECM-related genes and increased levels of MMP2 were detected in tumors derived from injection of CM treated cells. Taken together these data demonstrate that cross-talk between fibroblasts and cancer cells can dictate ECM composition and regulate dissemination of lung cancer and suggest that identification of factors responsible for this cross-talk could be instrumental in devising novel therapeutic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1483. doi:1538-7445.AM2012-1483

Fibroblasts isolated from cancer specimens have been shown to possess pro-tumorigenic properties, often associated with traits reminiscent of myofibroblasts found at sites of wound healing, but little is known about the mediators of the interaction between fibroblasts and cancer cells especially in lung cancer. To evaluate the contribution of fibroblasts to lung cancer development we established 60 fibroblast cultures from cancerous and normal lung tissue from primary lung cancer patients, including 18 pairs of normal (NF) and cancer-associated fibroblasts (CAF) from the same patient. The mesenchymal nature of the cultures was confirmed by expression of surface markers CD90, CD166, CD105 and CD73 and negativity for epithelial and hematopoietic markers (ESA and CD45). Further immunophenotyping with markers specific for fibroblasts subpopulations (FSP, vimentin, alpha-SMA, FAP) identified frequent expression of activation markers, with up to 50% of alpha-SMA+ cells. Interestingly activation properties were identified also in cultures derived from normal lung tissue. Co-mingling of fibroblasts (NF or CAF) with A549 lung cancer cells generally resulted in increased tumor size after injection in nude mice and 2/4 of the CAF cultures tested also increased tumor take when cancer cells were injected at low doses. Human cells were identified by HLA staining in the stroma of tumors grown after co-injection confirming the contribution of the mixed fibroblasts to tumor development. Furthermore medium conditioned by fibroblasts promoted growth of lung cancer cells or immortalized bronchial epithelial cells. All the cultures tested displayed a normal karyotype suggesting that the functional properties of fibroblasts are likely to be the result of epigenetic changes.These results indicate that lung fibroblasts might play a central role in the growth and progression of lung neoplasia. Consistently with their activated phenotype also cultures from normal lung tissue of heavy smokers displayed pro-tumorigenic activity, indicating that the microenvironment of lungs heavily exposed to damage from carcinogenic substances might already be predisposed to foster the growth of transformed epithelial cells. The full dissection of this complex picture might reveal mediators of the cross-talk between stroma and cancer cells and provide novel therapeutic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 530. doi:10.1158/1538-7445.AM2011-530