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.

Summary statistics on primary NSCLC population.

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

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Despite many years of research efforts, lung cancer still remains the leading cause of cancer deaths worldwide. Objective of this study was to set up a platform of non-small cell lung cancer patient derived xenografts (PDXs) faithfully representing primary tumour characteristics and offering a unique tool for studying effectiveness of therapies at a preclinical level. We established 38 PDXs with a successful take rate of 39.2%. All models closely mirrored parental tumour characteristics although a selective pressure for solid patterns, vimentin expression and EMT was observed in several models. An increased grafting rate for tumours derived from patients with worse outcome (p = 0.006), higher stage (p = 0.038) and higher CD133+/CXCR4+/EpCAM− stem cell content (p = 0.019) was observed whereas a trend towards an association with SUVmax higher than 8 (p = 0.084) was detected. Kaplan Meier analyses showed a significantly worse (p = 0.0008) overall survival at 5 years in patients with grafted vs not grafted PDXs also after adjusting for tumour stage. Moreover, for 63.2% models, grafting was reached before clinical recurrence occurred. Our findings strengthen the relevance of PDXs as useful preclinical models closely reflecting parental patients tumours and highlight PDXs establishment as a functional testing of lung cancer aggressiveness and personalized therapies.

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, pmitogen-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 < 0.0001). Nine PSCs, however, showed 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. (C) 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Hypothesis. Gene4c altera4ons suitable for targeted therapy are poorly known issues in pulmonary sarcomatoid carcinoma (PSC), an uncommon and life-threatening family of nonsmall cell lung cancer. Methods. Ninety-eight PSC were assessed for MET and ALK status by fluorescence in situ hybridiza4on (FISH) and relevant protein expression by immunohistochemistry (IHC) also taking advantage of phosphorylated (p-) an4bodies. Moreover, ALK and MET mRNA levels by real-4me PCR and western blot analysis for downstream ac4va4on pathways involving p-MET, p-AKT, p-MAPK, p-SRC and p-FAK were also determined. Results. MET amplifica4on upon FISH was found in 25/98 (25.6%) and ALK amplifica4on (but not the relevant rearrangement) in 16/98 (16.3%) PSC, with all ALK-amplified tumors also showing MET amplifica4on (p<0.0001). Nine PSC, however, showed MET amplifica4on without any ALK gene altera4on. ALK protein expression was always lacking, while MET and p-MET were confined to the relevant amplified tumors only. Increased ALK and MET mRNA levels were detectable in tumors, with no direct rela4onship between mRNA content, protein expression or FISH altera4ons. Western blot assays showed complete ac4va4on of downstream signal pathways up to p-SRC and p-FAK recruitment in MET&ALK co-amplified tumors only, whereas isolated MET amplifica4on, MET&ALK borderline amplifica4on (5-10% tumor cells with ≥15 copies of the relevant gene) or nega4ve tumors showing eusomy or chromosome polysomy were confined to p-MAPK, p-AKT and/or p-MET ac4va4on. Mul4variate survival analysis pushed a higher percentage of MET altered cells or a higher value of MET copy gain per cell to marginally emerge for OS (p=0.140) and DFS (p=0.060), respec4vely. Conclusions. ALK and MET seemed to act as synergic, non-random co-ac4vators of downstream signal when co-amplified in a subset of PSC pa4ents, thus likely sugges4ng a 4 Pelosi et al, MET and ALK altera4ons in pulmonary sarcomatoid carcinoma, JTO-D-15-01313-R1 combined mechanism of oncogene addic4on. These altera4ons could be a suitable target for therapy upon specific inhibitors.

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.

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.

Abstract 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.

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

ABSTRACT 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.

Abstract 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

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.

in primary breast tumours correlates with improved disease-free survival and overall survival in a large cohort of patients.Conclusion: Activation of the BMP4 signalling pathway is a potential therapeutic approach for patients with advanced breast cancer.