Search

Summary of metastatic assay on sorted CD133/CXCR4 cells in bone-bearing mice.

Supplementary Methods, References, Figure Legends from Microenvironment-Modulated Metastatic CD133+/CXCR4+/EpCAM− Lung Cancer–Initiating Cells Sustain Tumor Dissemination and Correlate with Poor Prognosis

Figure S1. Integrated strategies to detect disseminated tumor cells in murine lung. Figure S2. Effect of cisplatin and CTCE-9908 combination therapy on lung tumor growth and dissemination. Figure S3. Time course analysis of metastatic CICs during lung colonization. Figure S4. Functional studies of lung-DTCs from PDX models. Figure S5. EMT induction provides tumor cells with migratory and stemness features.

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

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

The 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.

// Patrizia Gasparini 1, * , Michela Casanova 2, * , Raffaella Villa 3 , Paola Collini 4 , Rita Alaggio 5 , Angelica Zin 6 , Paolo Bonvini 6 , Cristina R Antonescu 8 , Renata Boldrini 9 , Roberto Caserini 1 , Massimo Moro 1 , Giovanni Centonze 1 , Cristina Meazza 2 , Maura Massimino 2 , Luca Bergamaschi 2 , Roberto Luksch 2 , Stefano Chiaravalli 2 , Gianni Bisogno 10 , Nadia Zaffaroni 7 , MariaGrazia Daidone 3 , Gabriella Sozzi 1, # , Andrea Ferrari 2, # 1 Tumor Genomics Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 2 Department of Pediatric Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 3 Biomarkers Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 4 Soft Tissue and Bone Pathology, Histopathology and Pediatric Pathology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 5 Department of Pathology, University Hospital of Padova, Padova, Italy 6 Institute of Pediatric Research Citta della Speranza, Padova, Italy 7 Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 8 Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA 9 Department of Pathology, Bambino Gesu Children Hospital-Research Institute, Rome, Italy 10 Hematology Oncology Division, Department of Mother and Child’s Health, University Hospital of Padova, Padova, Italy * These authors contributed equally to this work # Co-last authors Correspondence to: Gabriella Sozzi, email: gabriella.sozzi@istitutotumori.mi.it Keywords: anaplastic lymphoma kinase, rhabdomyosarcoma, EML4-ALK, chromosomal rearrangement, metastasis Received: February 11, 2016 Accepted: June 12, 2016 Published: July 01, 2016 ABSTRACT Rhabdomyosarcoma (RMS) is the most frequent soft tissue tumor in childhood and arises from immature mesenchymal cells committed to skeletal muscle differentiation. Anaplastic Lymphoma Kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in several cancers. Moreover, ALK full-length receptor protein has been observed in RMS, although its clinical and functional significance is yet controversial. The role of ALK and its clinical relevance were investigated in a selected cohort of 74 FFPE pediatric RMS and a panel of RMS cell lines, evaluating its gene and protein status, utilizing Fluorescent In Situ Hybridization (FISH), immunohistochemistry (IHC) and Western blot approaches. Moreover, to get insight into its possible therapeutic relevance, effects of ALK silencing on cell proliferation, invasion and apoptosis were studied in RMS cells. ALK IHC positivity was significantly correlated with gene copy number gain, the alveolar subtype, PAX3/7-FOXO1 rearrangements, the presence of metastasis at diagnosis and a worse overall outcome. Furthermore, EML4-ALK fusion gene associated with higher protein expression was identified in an embryonal RMS. ALK silencing in RH30 ALK positive cells strongly inhibited invasion capability. Overall, our data suggest a potential role of ALK in pediatric RMS.

Introduction Genetic alterations suitable for targeted therapy are poorly known issues in pulmonary sarcomatoid carcinoma (PSC), an uncommon and life-threatening family of non–small cell lung cancers. Methods Ninety-eight PSCs were assessed for MNNG HOS Transforming gene ( MET ) and anaplastic lymphoma receptor tyrosine kinase gene ( ALK ) status by fluorescence in situ hybridization (FISH) and for relevant protein expression by immunohistochemical analysis, also taking advantage of phosphorylated (p-) antibodies. Moreover, levels of ALK and MET mRNA were also determined by real-time polymerase chain reaction and Western blot analysis for downstream activation pathways involving p-MET, p–protein kinase B, p–mitogen-activated protein kinase, p-SRC proto-oncogene tyrosine-protein kinase, and p–focal adhesion kinase (p-FAK). Results MET amplification was detected by FISH in 25 of 98 PSCs (25.6%) and ALK amplification (but not the relevant rearrangement) was found in 16 of 98 (16.3%), with all ALK -amplified tumors also showing MET amplification ( p MET amplification without any ALK gene alteration. ALK protein expression was always lacking, whereas MET and p-MET were confined to the relevant amplified tumors only. Increased levels of ALK and MET mRNA were detectable in tumors with no direct relationship between mRNA content, protein expression, or alterations detected by FISH. Western blot assays showed complete activation of downstream signal pathways up to p-SRC proto-oncogene tyrosine-protein kinase, and p–focal adhesion kinase recruitment in MET and ALK– coamplified tumors only, whereas isolated MET amplification, MET and ALK borderline amplification (5% – 10% of tumor cells with ≥15 copies of the relevant gene), or negative tumors showing eusomy or chromosome polysomy were confined to p–mitogen-activated protein kinase, p–protein kinase B, and/or p-MET activation. Multivariate survival analysis pushed a higher percentage of MET altered cells or a higher value of MET copy gain per cell to marginally emerge for overall survival ( p = 0.140) and disease-free survival ( p = 0.060), respectively. Conclusions ALK and MET seemed to act as synergistic, nonrandom coactivators of downstream signal when coamplified in a subset of patients with PSC, thus likely suggesting a combined mechanism of oncogene addiction. These alterations could be a suitable target for therapy based on specific inhibitors.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

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.

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

Cancer initiating cells (CICs) have been shown to be responsible for primary tumor formation, drug resistance and metastatic dissemination. We previously demonstrated that the subset of CD133+ lung CICs co-expressing the chemokine receptor CXCR4, is resistant to chemotherapy and endowed with high metastasis initiating ability using in vivo experimental metastasis assay. To investigate the role of the subset of CD133+CXCR4+ cells in the in vivo spontaneous tumor dissemination process, we analyzed lungs of mice bearing subcutaneous implants of lung cancer patients derived xenografts (PDXs). Using FACS and Real Time PCR analysis a small fraction of lung disseminated tumor cells (DTCs: 0.001% to 0.01%) was detected in 9/11 different PDX models, likely remaining at single cells state as suggested by the lack of histologically apparent metastasis. In particular, compared to parental subcutaneous tumors, lung DTCs were enriched for CD133+CXCR4+ CICs not expressing the epithelial antigen EpCAM. Modifying an innovative cancer cells culture method that allows the recovery of highly purified and viable tumor cells (Kondo J. et al. 2011, PNAS), we also generated murine lung tissue spheroids that were 100 fold enriched in DTCs. Real Time PCR analysis of lung spheroids consistently revealed an up-regulation of stemness and EMT-associated genes compared to the parental tumor, in accordance with the enrichment for disseminating CD133+CXCR4+EpCAM- CICs subset observed by FACS. Subcutaneous injection in SCID mice of lung spheroids containing approximately 200 DTCs, generated tumors resembling the original PDX, but also showing an increased fraction of CD133+ stem like cells and particularly of the CD133+CXCR4+EpCAM- subset. Moreover, cells recovered from DTCs-originated tumors were able to grow in vitro as spheres in anchorage independent condition, up-regulated EMT-genes and showed an enhanced tumorigenic potential and increased ability to disseminate to murine lung compared with original PDX in in vivo serial transplantation assay, indicating that the fraction of CD133+CXCR4+EpCAM- CICs enriched in DTCs was able to efficiently originate tumors with distinctive traits. We also found that the fraction of CD133+ cells and particularly the CD133+CXCR4+EpCAM- subset were increased in patient's metastatic lymph nodes compared to primary tumors, confirming in a clinical setting the role of CD133+CXCR4+EpCAM- cells as metastasis initiating cells. Our data functionally proves that lung DTCs contain a specific subset of tumor initiating cells that is able to generate tumors showing distinct properties and enrichment in the mesenchymal-like CICs compartment. The relevance of this finding is confirmed in clinical setting, identifying a new possible target for novel therapies aimed at interfering with metastatic dissemination, which represents the main reason for lung cancer poor outcome.. Citation Format: Giulia Bertolini, Massimo Moro, Monica Tortoreto, Roberto Caserini, Ugo Pastorino, Luca Roz, Gabriella Sozzi. Disseminated cells from primary lung cancers contain a distinct cancer initiating subpopulation with mesenchymal-like features. [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 3898. doi:10.1158/1538-7445.AM2014-3898

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

Metformin is an oral antidiabetic drug of the biguanide class. Several epidemiological and case-control studies reported that diabetics using metformin may have lower cancer risk in comparison to those using other antidiabetic medications. Recent studies in experimental models suggest that another biguanide, phenformin, has higher anticancer activity compared with metformin. The aim of this study was to evaluate the effects of metformin and phenformin in Non-Small Cell Lung Cancer (NSCLC) models. In particular, the effects of these compounds in LKB1 or LKB1/KRAS mutated compared to wild type models have been investigated. NSCLC cell lines (A549, H460, H1299, LT73) have been treated with either metformin or phenformin to evaluate in vitro dose/response depending on the different mutational status of KRAS and LKB1 genes. The effects of the two biguanide have been investigated also in vivo models, exploiting the panel of Patient Derived Xenografts (PDXs) directly generated from NSCLC patients in our Institute. In vitro experiments indicated that cell lines with already impaired metabolism (LKB1 and/or KRAS mutated) were more sensitive to phenformin treatment. Interestingly, treatments of these cell lines with r phenformin resulted in a 50% decrease in the amount of CD133+ Tumor Initiating Cells (TIC). Moreover, high doses of phenformin (LD75) did not result in the increase of TIC previously observed after Cisplatin treatment. In vivo, early treatments (Tumor Volume = 50-100mm3) of an LKB1 mutated PDX model resulted in a significant reduction of tumor volume (300 ± 50 mm3 in phenformin treated vs 750 ± 100 mm3 in vehicle treated tumors) already after three weeks of treatment (100mg/Kg/day). Importantly, no regrowth was observed in responding tumors even after the end of treatments. No effects were observed in LKB1 wild type PDX treated with phenformin. Taken together, these preliminary data indicate a selective action of phenformin on LKB1 mutated lung adenocarcinoma models in vitro and in vivo. Interestingly, this compound seems to be more active in TIC than normal chemotherapeutic drugs (i.e. cisplatin). Effects of biguanide on TIC could be the cause of the lack of tumor regrowth after treatment, thus making these compounds potentially useful in counteracting disease recurrence and/or metastasis formation. Further investigations are needed to elucidate the different effects of metformin and phenformin in double mutated (LKB1 and KRAS) PDXs models. Citation Format: Massimo Moro, Luca Roz, Roberto Caserini, Ugo Pastorino, Gabriella Sozzi. Effects of phenformin in LKB1/KRAS mutated non-small cell lung cancer patient derived xenograft. [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 1216. doi:10.1158/1538-7445.AM2014-1216

Aim: CD133 positive cells have been identified as tumor initiating cells in non-small cell lung cancer (NSCLC) and have been shown to be resistant to conventional cytotoxic chemotherapy in experimental studies, suggesting a possible role in chemoresistance and disease relapse. However their prognostic relevance in the context of clinical treatment of NSCLC has not been fully investigated. Methods: We analyzed tumor samples from 60 consecutive NSCLC patients with stage IIIA-B undergoing neo-adjuvant chemotherapy with platinum-based regimens followed by radically surgery. Tumor specimen collection included pre-treatment mediastinal lymph-node biopsies (n = 23) and post-treatment surgical specimens (25 from lymph-nodes and 52 from primary tumors). CD133 status was determined by immunohistochemistry and correlations with clinico-pathological variables were evaluated using univariable and multivariable Cox models. Results: CD133 positive cells (mean 5% of total tumor cells, range 1-40%) were detected in 35% of pre-treatment samples and after chemotherapy in 38% and 32% of resected primary tumors and metastatic lymph-nodes, respectively. When both lymph nodes and primary tumor were available (24 patients) no correlation was found between CD133 status, possibly indicating different dynamics in primary tumor maintenance and dissemination. CD133 positive cells were detected more frequently in tumors with best response to chemotherapy (by ≥30% size reduction: 14/28, 50% vs. 6/24, 25%) suggesting enrichment of chemoresistant cells after treatment. CD133 positivity after induction chemotherapy was correlated with higher risk of local and distant recurrence (73% vs 53%), especially in patients with pre-treatment CD133+ lymph nodes (87% vs. 53%). Finally, positivity for CD133 after treatment seems to be correlated with worse overall survival (HR 2.42, 95% CI: 0.93-6.32, p = 0.184). Conclusions: Investigation of tumors after chemotherapy provides indirect evidence of chemoresistance of CD133+ cells. CD133 positive cells are correlated with worse prognosis in neo-adjuvant treatment of locally advanced NSCLC and could provide clinical relevant information for personalized therapies. Disclosure: All authors have declared no conflicts of interest.

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