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Supplementary Figure 1, 2,3 4,5

Supplementary Table 1A, B, C, D and E

Supplementary Table S3

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.

This file contains 28 Supplementary Figures dealing with: frequency of T cell subsets in tumor and non neoplastic lung tissue (S1), activation and naive to memory profile of T cells at tumor site (S2), functional competence of T cells at tumor site (S3), frequency of PD-1+ T cells in tumor according to clinical stage and patients' survival according to PD-1+ T cell frequency in tumor (S4), Ki-67 vs eomes profile of PD-1+ T cells (S5), expression of Eomes in distinct T cell subsets at tumor site (S6), PD-1 modulation and IFN-g production in PD-1(lo) and PD-1(Hi) T cell subsets (S7), functional competence of T cell subsets according to IRs profile (S8), immunohistochemistry for CD8+FOXP3+ T cells at tumor site (S9-S12), immune profile by immunohistochemistry of lesions containing high frequency of CD8+FOXP3+ T cells (S13-S23), frequency of CD8+FOXP3+ cells at tumor site according to clinical parameters (S24), relationship of CD8+FOXP3+ cells with clinical stage and patients' survival (S25), maturation profile of CD8+FOXP3+ cells (S26), IRs expression and competence for differentiation of CD8+FOXP3+ cells (S27), co-expression of activation/proliferation markers in different T cell subsets at tumor site (S28)

This file contains two Supplementary Tables. Table S1 lists antibodies used in flow cytometry experiments and Table S2 shows demographic and clinical parameters of the patients.

Purpose: We aimed to maximize the performance of detecting genetic alterations in lung cancer using high-throughput sequencing for patient-derived xenografts (PDXs).Experimental Design: We undertook an integrated RNA and whole-exome sequencing of 14 PDXs. We focused on the genetic and functional analysis of β2-microglobulin (B2M), a component of the HLA class-I complex.Results: We identified alterations in genes involved in various functions, such as B2M involved in immunosurveillance. We extended the mutational analysis of B2M to about 230 lung cancers. Five percent of the lung cancers carried somatic mutations, most of which impaired the correct formation of the HLA-I complex. We also report that genes such as CALR, PDIA3, and TAP1, which are involved in the maturation of the HLA-I complex, are altered in lung cancer. By gene expression microarrays, we observed that restitution of B2M in lung cancer cells upregulated targets of IFNα/IFNγ. Furthermore, one third of the lung cancers lacked the HLA-I complex, which was associated with lower cytotoxic CD8+ lymphocyte infiltration. The levels of B2M and HLA-I proteins correlated with those of PD-L1. Finally, a deficiency in HLA-I complex and CD8+ infiltration tended to correlate with reduced survival of patients with lung cancer treated with anti-PD-1/anti-PD-L1.Conclusions: Here, we report recurrent inactivation of B2M in lung cancer. These observations, coupled with the mutations found at CALR, PDIA3, and TAP1, and the downregulation of the HLA-I complex, indicate that an abnormal immunosurveillance axis contributes to lung cancer development. Finally, our observations suggest that an impaired HLA-I complex affects the response to anti-PD-1/anti-PD-L1 therapies. Clin Cancer Res; 23(12); 3203–13. ©2016 AACR.

Contains the legends for all the supplementary figures and legends

Supplementary methods and materials

Supplementary Figure 6. Immunohistochemistry studies for KAT6B and acetylated K23-H3 staining according to KAT6B deletion status

Supplementary Figure 8. Effect of KAT6B shRNA-mediated depletion in ATM expression and H2AX phosphorylation

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

Supplementary Figure 4. Effect of KAT6B shRNA-mediated depletion in the growth rate according to the XTT assay

Supplementary Figure 1. KAT6B copy number and DNA methylation status in SCLC cell lines

Supplementary Figure 7. Effect of twenty-eight histone deacetylase inhibitors and two classical drugs in SCLC chemotherapy, such as cisplatin and etoposide, in the IC50 values of KAT6B shRNA-transfected vs scramble shRNA-transfected NCI-N417 cells.

Supplementary Table 1. List of antibodies and primer sets used in the study.

Supplementary Table 3. The twenty-eight HDAC inhibitors used in the study and their specificity

Supplementary Figure Legends

Supplementary Figure 3. Expression of KAT6B isoforms in SCLC cell lines.

Supplementary Table 2. Genes that significantly change expression upon KAT6B depletion. Normalized intensities from the array in each sample and log Fold Changes from the analysis are depicted.

Figure S1. PARD3-inactivation in the H157 lung cancer cell line. Figure S2. Ratio charts of the multiplex ligation-dependent probe amplification (MLPA) depicting a large intragenic deletion (from exon 4 to 23) in one of the LSCCs but not a normal DNA. Figure S3. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) to determine the presence of promoter hypermethylation of PARD3. Figure S4. Relative abundance of the PARD3 transcriptsFigure S5. Western blot showing ectopic and transient expression of the indicated PAR3 proteins in the H157 cell line, immunoblotted with HA or PAR3 antibodies. Figure S6. Immunofluorescence with the anti-PAR3 antibody in the indicated T98G-derived cells, after (Dox+) induction of PAR3 expression with doxycycline (1 ng/µl, 24h). Scale bar, 50 µm. Figure S7. Western blot depicts the endogenous PAR3 protein in the indicated lung cancer cells and the ectopic expression of PAR3 in the H157tr-pD41_R74del cells (dox, 1 ng/µl; 24 h). Figure S8. Immunostaining of PAR3 in lung primary tumors and head and neck squamous cell carcinomas (HNSCCs).

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.

Correct apicobasal polarization and intercellular adhesions are essential for the appropriate development of normal epithelia. Here, we investigated the contribution of the cell polarity regulator PARD3 to the development of lung squamous cell carcinomas (LSCC). Tumor-specific PARD3 alterations were found in 8% of LSCCs examined, placing PARD3 among the most common tumor suppressor genes in this malignancy. Most PAR3-mutant proteins exhibited a relative reduction in the ability to mediate formation of tight junctions and actin-based protrusions, bind atypical protein kinase C, activate RAC1, and activate STAT3 at cell confluence. Thus, PARD3 alterations prevented the formation of contacts between neighboring cells and the subsequent downstream signaling. Notably, reconstituting PAR3 activity in vivo reduced tumor-invasive and metastatic properties. Our findings define PARD3 as a recurrently inactivated cell polarity regulator in LSCC that affects tumor aggressiveness and metastasis. Cancer Res; 75(7); 1287–97. ©2015 AACR.

Supplementary Materials and Methods

Table S1. List of lung cancer cell lines and histopathological type used to determine the status of the PARD3. Table S2. List of primers used for cloning, PCR and Sanger sequencing of PARD3. Table S3. List of single nucleotide polymorphisms (SNPs) and variants of unknown biological significance identified in lung cancer cell lines and lung primary tumors tested. Table S4. Clinical and pathological parameters of the NSCLC patients and tumors included in the PARD3 gene alterations screening. Table S5. List of genes selected from those genes that fulfilled the criteria: i) adjusted p

Supplementary Figure 2. Copy number and expression levels of six KATs in SCLC cell lines

According to World Health Organization guidelines, Atypical Carcinoids (ACs) are well-differentiated lung neuroendocrine tumors with 2-10 mitoses/2 mm2 and/or foci of necrosis (usually punctate). Besides morphological criteria no further tools in predicting AC clinical outcome are proposed. Aim of this work was to identify novel factors able to predict AC disease aggressiveness and progression. Three hundred-seventy lung carcinoids were collected and centrally reviewed by two expert pathologists. Morphology and immunohistochemical markers (Ki-67, TTF-1, CD44, OTP, SSTR2A, Ascl1, p53 and Rb1) were studied and correlated with disease free survival (DFS) and overall survival (OS). Fifty-eight of 370 tumors were defined as AC. Survival analysis showed that patients with Ascl1+ACs and those with OTP-ACs had a significantly worse DFS than patients with Ascl1-ACs and OTP+ACs, respectively. Combining Ascl1 and OTP expressions, groups were formed reflecting the aggressiveness of disease (p=0.0005). Ki-67 ≥10% patients had a significantly worse DFS than patients with Ki-67

The generation of 3D-bioprintable and biocompatible hydrogels based on elastin and hyaluronic acid is described. The procedure is based on a biocompatible click reaction between maleimide and thiol for the final crosslinking, employable with living cells due to fast kinetics and absence of side products. To this end, both α-elastin and hyaluronic acid were functionalized with linkers ending with maleimide groups, at controlled functionalization intensities, and crosslinked with a dithiol−PEG linker. Varying the equivalents of the three reagents, four different hydrogels were obtained and their biocompatibility, swelling capacity and printability were tested. Biological experiments were performed with human lung fibroblast, human bronchial epithelial and human endothelial cell lines cultured in the hydrogels. Fibroblasts and epithelial cells can survive and proliferate. Epithelial cells showed an increased expression of CD44 and integrin αvβ3. Gene expression analysis revealed up-regulation of metalloproteinases both in normal fibroblast and epithelial cells.

Despite evidence for clinical benefit in patients suffering from lung cancer following treatment with immune checkpoint inhibitors (ICI), it is still uncertain how to predict which patients are likely to experience a significant response. In their work, Valencia et al. (2022. J. Exp. Med.https://doi.org/10.1084/jem.20220726) identify the DSTYK kinase as a cancer cell–intrinsic modulator of response to immunotherapy. Through regulation of the mTOR pathway and stimulation of protective autophagy, DSTYK blunts CD8+ T cell–mediated killing of cancer cells. Accordingly, lung cancers with increased expression of DSTYK are less responsive to ICI treatment. These observations could be useful in the clinic towards the development of predictive biomarkers and novel therapeutic strategies.

In developed countries, colorectal cancer is the third leading cause of mortality. Despite significant advances in therapeutic approaches, metastases remain the primary concern in the treatment of colorectal cancer. Ion channels have an important role in the development of aggressive behavior, but the majority of them are involved in regular physiological activities. Chloride Intracellular Channel 1 (CLIC1) is a promising contender among the new possible biomarkers. CLIC1 is expressed as a cytosolic monomer by all cell types during physiological homeostasis. The main characteristic of this protein is that it can exist in both cytosolic and transmembrane forms (tmCLIC1), with the latter being involved in a variety of clinical conditions, including cancer. CLIC1 mRNA overexpression in colorectal cancer cells has been linked to a poor prognosis. The primary goal of this study is to understand the role of tmCLIC1 in the carcinogenesis, invasion, and migratory potential of colorectal cancer. We initially investigated the level of CLIC1 protein expression and the activity of its transmembrane form as an ion channel in numerous colorectal cancer cells at various stages of development. Our results show that tmCLIC1 protein expression and activity are related to cell aggressiveness, and that its role in cellular proliferation, migration, and invasion is critical. The role of tmCLIC1 activity in colorectal cancer was confirmed also in in vivo models. Experiments in zebrafish and mouse models have shown that silencing CLIC1 impairs primary tumor development and significantly reduces the generation of distal metastases. Furthermore, when the CLIC1 protein is missing, many distinct metastatic genes are downregulated, particularly those associated to oxidative stress, typical of cancer cells. As a matter of fact, CLIC1's peculiar activity and its interaction with glutathione make this protein critical for the redox equilibrium of colorectal cancer cells. Overall, the findings could be considered as a beginning point for elucidating a mechanism of action that could constitute a new milestone in anticancer research. Citation Format: Francesca Cianci, Ivan Verduci, Carlotta Tacconi, Gaetano Cannavale, Matteo Ranucci, Giulia Bertolini, Paolo Malatesta, Federica Barbieri, Alessandro Fantin, Luca Roz, Tullio Florio, Michele Mazzanti. Oxidative stress promotes colorectal cancer aggressiveness through transmembrane chloride intracellular channel 1 (tmCLIC1) antioxidant activity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4788.

Introduction: Cancer associated fibroblasts (CAF) are increasingly recognized as central players in the regulation of the tumor immune microenvironment. In lung cancer however little is known about specific fibroblast subpopulations or mechanisms subtending their immunoregulatory potential. We previously identified the nuclear transporter XPO1 as a master controller of lung fibroblast protumorigenic potential. Here we evaluated the potential of fibroblasts to modulate interactions between immune and cancer cells. Material and Methods: Cultures of primary lung fibroblasts isolated from tissues resected during lung cancer surgery were characterized by multiparametric flow-cytometry and used for experiments with lung cancer cells and monocytes. Co-cultures, conditioned media and decellularized extracellular matrices deposited by fibroblasts (dECM) were employed to investigate different modes of interactions. Modulation of stemness and EMT properties were evaluated by flow cytometry and Real-Time PCR. Co-injection of dECM and lung cancer cell lines were performed in immunocompromised mice and heterotypic tumors were evaluated for their growth, local microenvironments and disseminating potential. Results and Discussions: Using fibroblast cultures form both cancer and normal lung tissue we detected protumorigenic potential also in cultures form normal lungs, in line with presence of classical activation markers (alpha-SMA, FAP). Bioactivity of fibroblasts include the ability to induce EMT and modulate stemness potential (increase of CD133+ cancer stem cells and CD133+/CXCR4+ metastasis initiating cells). Interestingly dECM displayed similar potential compared to co-cultures. Exosomes isolated from both CAF and normal fibroblasts also showed potential to induce conversion of normal monocytes into myeloid derived suppressor cells. In co-injection experiments, dECM from pro-tumorigenic fibroblasts regulated the local immune microenvironment of ensuing tumors inducing generation of a stroma with high content of COL1A and COL6A3, reducing the infiltration from NK cells and increasing neutrophil content, thereby favoring dissemination of cancer cells to distant organs. Pharmacological inhibition of fibroblast activation through compounds targeting XPO1 activity was able to reduce these effects and counteract fibroblast protumorigenic action. Conclusion: Fibroblasts play a regulatory role in tumor stroma and dissection of their activities could pave the way for rationale development of treatments to reverse immunosuppressive microenvironments. Citation Format: Giulia Bertolini, Elvira Pantano, Giuliana Pollaci, Federica Facchinetti, Ugo Pastorino, Gabriella Sozzi, Luca Roz. Fibroblasts play an immunoregulatory role in lung cancer through exosome secretion and by controlling extracellular matrix composition. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5844.

Background The isolation of circulating tumor cells (CTCs) requires rapid processing of the collected blood due to their inherent fragility. The ability to recover CTCs from peripheral blood mononuclear cells (PBMCs) preserved from cancer patients could allow for retrospective analyses or multicenter CTC studies. Methods We compared the efficacy of CTC recovery and characterization using cryopreserved PMBCs vs fresh whole blood from patients with non-small cell lung cancer (NSCLC; n = 8) and sarcoma (n = 6). Two epithelial cellular adhesion molecule (EpCAM)-independent strategies for CTC enrichment, based on Parsortix® technology or immunomagnetic depletion of blood cells (AutoMACS®) were tested, followed by DEPArray™ single-cell isolation. Phenotype and genotype, assessed by copy number alterations analysis, were evaluated at a single-cell level. Detection of target mutations in CTC-enriched samples from frozen NSCLC PBMCs was also evaluated by digital PCR (dPCR). Results The use of cryopreserved PBMCs from cancer patients allowed for the retrospective enumeration of CTCs and their molecular characterization, using both EpCAM-independent strategies that performed equally in capturing CTC. Cells isolated from frozen PBMCs were representative of whole blood-derived CTCs in terms of number, phenotype, and copy number aberration profile/target mutations. Long-term storage (≥3 years) did not affect the efficacy of CTC recovery. Detection of target mutations was also feasible by dPCR in CTC-enriched samples derived from stored PBMCs. Conclusions Isolating CTCs from longitudinally collected PBMCs using an unbiased selection strategy can offer a wider range of retrospective genomic/phenotypic analyses to guide patients’ personalized therapy, paving the way for sample sharing in multicenter studies.

WHO classification of Thoracic Tumours defines lung carcinoid tumours (LCTs) as well-differentiated neuroendocrine neoplasms (NENs) classified in low grade typical (TC) and intermediate grade atypical carcinoids (AC). Limited data exist concerning protein expression and morphologic factors able to predict disease aggressiveness. Though Ki-67 has proved to be a powerful diagnostic and prognostic factor for Gastro-entero-pancreatic NENs, its role in lung NENs is still debated. A retrospective series of 370 LCT from two oncology centers was centrally reviewed. Morphology and immunohistochemical markers (Ki-67, TTF-1, CD44, OTP, SSTR-2A, Ascl1, and p53) were studied and correlated with Overall Survival (OS), Cancer-specific survival (CSS) and Disease-free survival (DFS). Carcinoid histology was confirmed in 355 patients: 297 (83.7%) TC and 58 (16.3%) AC. Ki-67 at 3% was the best value in predicting DFS. Ki-67 ≥ 3% tumours were significantly associated with AC histology, stage III-IV, smoking, vascular invasion, tumour spread through air spaces OTP negativity, and TTF-1, Ascl1 and p53 positivity. After adjustment for center and period of diagnosis, both Ki-67 (≥3 versus

Several lines of compelling pre-clinical evidence identify chemotherapy as a potentially double-edged sword: therapeutic efficacy on the primary tumor may in fact be counterbalanced by the induction of tumor/host reactive responses supportive for survival and dissemination of cancer cell subpopulations. This paradoxical effect of chemotherapy can affect different districts such as the primary tumor, the circulation and distant organs by simultaneously shaping properties and composition of tumor and stromal cells. At the primary tumor site, chemotherapy has been reported to promote selection of chemoresistant and disseminating tumor cells endowed with properties of cancer stem cells (CSCs) through activation of autocrine and paracrine self-renewing/survival pathways promoted jointly by therapy-selected tumor and stromal cells. Resistant CSCs represent seeds for tumor relapse and increased infiltration by immune cells, together with enhanced vascular permeability induced by chemotherapy, facilitates tumor cells intravasation, the first step of the metastatic cascade. As a consequence of primary tumor/metastasis re-shaping induced by chemotherapy, circulating tumor cells (CTCs) detected during therapy can display a shift towards a more mesenchymal and stem-like phenotype, conductive to increased ability to survive in the circulation and seed distant organs. At the metastatic site, host responses to therapy activate inflammatory pathways that ultimately facilitate tumor cells extravasation and metastatic colonization. Finally, cooperation of immune cells and endothelial cells at perivascular niches favors the extravasation of tumor cells endowed with high potential for metastasis initiation and protects them from chemotherapy. This review highlights the paradoxical pro-metastatic effects of chemotherapy linking reactive responses to treatment to tumor relapse and metastasis formation through primary tumor remodeling and generation of a favorable pro-metastatic niche.

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

Large cell carcinoma (LCC) is a rare and aggressive lung cancer subtype with poor prognosis and no targeted therapies. Tumor-associated fibroblasts (TAFs) derived from LCC tumors exhibit premature senescence, and coculture of pulmonary fibroblasts with LCC cell lines selectively induces fibroblast senescence, which in turn drives LCC cell growth and invasion. Here we identify MMP1 as overexpressed specifically in LCC cell lines, and we show that expression of MMP1 by LCC cells is necessary for induction of fibroblast senescence and consequent tumor promotion in both cell culture and mouse models. We also show that MMP1, in combination with TGF-β1, is sufficient to induce fibroblast senescence and consequent LCC promotion. Furthermore, we implicate PAR-1 and oxidative stress in MMP1/TGF-β1-induced TAF senescence. Our results establish an entirely new role for MMP1 in cancer, and support a novel therapeutic strategy in LCC based on targeting senescent TAFs., This work was further supported by grants from the Agencia Estatal de Investigaci´on (AEI/FEDER) (PI13/02368, SAF2016-79527-R and PID2019-110944RB-I00 (AEI/ 10.13039/501100011033) to JA, PI16/ 00890 to NR), Fundaci´o Privada Cellex (to JA), National Institutes of Health (grant R01 GM132100 to ER), Generalitat de Catalunya (AGAUR SGR661 and CERCA Programme to JA), Junta Provincial de Barcelona de l’Associaci´o Espanyola Contra el C`ancer (AECC-B16-917 to JA), Italian Ministry of Health (RF-2016-02362946 to LR), Italian Association for Cancer Research (AIRC-IG21431 to LR), Sociedad Espa˜nola de Neumología y Cirugía Tor´acica – SEPAR (SEPAR 437 to NR), COST Action (PROTEOSTASIS BM1307), and by fellowships from Ciˆencia sem Fronteiras CNPq (to RI), and Universitat de Barcelona/beca APIF (to PD).

Introduction: Epithelial to mesenchymal transition (EMT) is essential for cancer dissemination but its role in overt metastasis establishment remains an open issue. Multiple EMT states and great phenotypic plasticity have also been described, indicating the need for in-depth characterization of biological properties associated with different EMT phenotypes. The so-called hybrid phenotype, characterized by presence of both mesenchymal and epithelial markers, might in fact identify cancer cells with enhanced metastatic propensity. Materials and Methods: To assess the metastatic potential of different EMT phenotypes, we exploited two lung cancer cell lines with different properties (A549/epithelial and LT73/hybrid) and generated their respective sublines through four sequential rounds of in vitro migration assay (named M4). TGFβ treatment was also used to induce EMT. With a previously validated score based on the ratio between expression of epithelial (CDH1) or mesenchymal genes (SNAI2), cells were classified as: epithelial (A549)>hybrid (A549-M4, LT73)>mesenchymal (LT73-M4, A549-TGFβ). The disseminating/metastatic properties of the different lines were tested in vivo by subcutaneous and tail-vein injections in SCID mice. Interaction with NK cells (NKs), known to specifically target EMT cancer cells, was also assessed. Results and Discussion: Compared to parental cells, M4 cells showed up-regulation of EMT-associated genes, expansion of the subset of CD133+ lung cancer stem cells (CSC) and displayed in vivo higher ability to disseminate from primary tumors to murine lungs (2-fold change, p=0.004). However, hybrid cells (LT73 and A549-M4) showed an increased ability to generate overt lung metastases in experimental metastasis assays compared to either epithelial (A549) or mesenchymal cells (LT73-M4 and A549-TGFβ). The higher metastatic ability of hybrid cells was correlated with CSCs expansion and reduced NKs infiltration to the metastatic lungs. Expression of IL-8, a cytokine involved in NKs recruitment, was decreased in hybrid cells and they were less prone to chemoattract NKs in vitro compared to both epithelial and mesenchymal cells. Finally, in vivo prevention of NKs recruitment to the lungs using a neutralizing antibody, massively increased metastasis formation and expansion of CSCs in mice injected i.v. with mesenchymal A549-TGFβ (p=0.0006), but had limited effects in groups injected with both epithelial A549 and hybrid A549-M4. Conclusions: Our data indicate that the hybrid phenotype correlates with the highest metastatic potential related to efficient cancer cell dissemination, increased induction of stemness features and ability to avoid NKs recruitment. Conversely, fully mesenchymal cells show limited metastatic potential, possibly due the control exerted by NKs on their CSC subsets. Citation Format: Giulia Bertolini, Monica Parodi, Massimo Milione, Fabio Murianni, Giovanni Centonze, Giulia Taiè, Gabriella Sozzi, Massimo Vitale, Luca Roz. A hybrid epithelial-mesenchymal phenotype identifies lung cancer cells with high metastatic potential due to increased stemness properties and escape from NK immunosurveillance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 962.