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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 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.

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.

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 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

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