Hybrid epithelial-mesenchymal status of lung cancer dictates metastatic success through differential interaction with NK cells.

BACKGROUND: Epithelial to mesenchymal transition (EMT) endows cancer cells with pro-metastatic properties, which appear most effective when cells enter an intermediate hybrid (H) state, characterized by integrated mesenchymal (M) and epithelial (E) traits. The reasons for this advantage are poorly known and, especially, it is totally unexplored whether the interplay between H-cells and NK cells could have a role. Here we characterize the pro-metastatic mechanics of non-small cell lung cancer (NSCLC) H-cells and their subset of cancer-initiating cells (CICs), dissecting crucial interactions with NK cells. METHODS: Human lung cancer cell lines and sublines representative of E, M, or H states, assessed by proteomics, were analyzed in vivo for their tumor-forming and disseminating capabilities. Interactions with NK cells were investigated in vitro using migration assays, cytotoxic degranulation assays, and evaluation of CD133+ CICs modulation after coculture, and validated in vivo through NK cell neutralization assays. Correlation between EMT status, NK cell infiltration, and survival data, was evaluated in a cohort of surgically resected NSCLC cases (n=79). RESULTS: We demonstrated that H-cells, have limited dissemination capability but show the highest potential to initiate metastases in vivo. This property was related to their ability to escape NK cell surveillance. Mechanistically, H-cells expressed low levels of NK-attracting chemokines (CXCL1 and CXCL8), generating poorly infiltrated metastases. Accordingly, proteomics and GO enrichment analysis of E, H, M cell lines showed that the related secretory processes could change during EMT.Furthermore, H-CICs uniquely expressed high levels of the inhibitory ligand B7-H3, which protected H-CIC from NK cell-mediated clearance. In vivo neutralization assays confirmed that, indeed, the pro-metastatic properties of H-cells are poorly controlled by NK cells.Finally, the analysis of patients revealed that detection of hybrid phenotypes associated with low NK infiltration in NSCLC clinical specimens could identify a subset of patients with poor prognosis. CONCLUSIONS: Our study demonstrates that H-cells play a central role in the metastatic spread in NSCLC. Such pro-metastatic advantage of H-cells is supported by their altered interaction with NK cells and by the critical role of B7-H3 in preserving their H-CIC component, indicating B7-H3 as a potential target in combined NK-based therapies.

Soluble galectin-3 as a microenvironment-relevant immunoregulator with prognostic and predictive value in lung adenocarcinoma.

Despite the success of therapies in lung cancer, more studies of new biomarkers for patient selection are urgently needed. The present study aims to analyze the role of galectin-3 (GAL-3) in the lung tumor microenvironment (TME) using tumorspheres as a model and explore its potential role as a predictive and prognostic biomarker in non-small cell lung cancer patients. For in vitro studies, lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC) primary cultures from early-stage patients and commercial cell lines were cultured, using tumorsphere-forming assays and adherent conditions for the control counterparts. We analyzed the pattern of secretion and expression of GAL-3 using reverse transcription-quantitative real-time PCR (RTqPCR), immunoblot, immunofluorescence, flow cytometry, and immunoassay analysis. Our results using three-dimensional (3D) models of lung tumor cells revealed that soluble GAL-3 (sGAL-3) is highly expressed and secreted. To more accurately mimic the TME, a co-culture of tumorspheres and fibroblasts was used, revealing that GAL-3 could be important as an immunomodulatory molecule expressed and secreted in the TME, modulating immunosuppression through regulatory T cells (TREGS ). In the translational phase, we confirmed that patients with high expression levels of GAL-3 had more TREGS , which suggests that tumors may be recruiting this population through GAL-3. Next, we evaluated levels of sGAL-3 before surgery in LUAD and LUSC patients, hypothesizing that sGAL-3 could be used as an independent prognostic biomarker for overall survival and relapse-free survival in early-stage LUAD patients. Additionally, levels of sGAL-3 at pretreatment and first response assessment from plasma to predict clinical outcomes in advanced LUAD and LUSC patients treated with first-line pembrolizumab were evaluated, further supporting that sGAL-3 has a high efficiency in predicting durable clinical response to pembrolizumab with an area under curve of 0.801 (P = 0.011). Moreover, high levels might predict decreased progression-free survival and OS to anti-PD-1 therapy, with sGAL-3 being a prognosis-independent biomarker for advanced LUAD.

Leveraging Tissue-Specific Enhancer-Target Gene Regulatory Networks Identifies Enhancer Somatic Mutations That Functionally Impact Lung Cancer.

Enhancers are noncoding regulatory DNA regions that modulate the transcription of target genes, often over large distances along with the genomic sequence. Enhancer alterations have been associated with various pathological conditions, including cancer. However, the identification and characterization of somatic mutations in noncoding regulatory regions with a functional effect on tumorigenesis and prognosis remain a major challenge. Here, we present a strategy for detecting and characterizing enhancer mutations in a genome-wide analysis of patient cohorts, across three lung cancer subtypes. Lung tissue-specific enhancers were defined by integrating experimental data and public epigenomic profiles, and the genome-wide enhancer-target gene regulatory network of lung cells was constructed by integrating chromatin three-dimensional architecture data. Lung cancers possessed a similar mutation burden at tissue-specific enhancers and exons but with differences in their mutation signatures. Functionally relevant alterations were prioritized on the basis of the pathway-level integration of the effect of a mutation and the frequency of mutations on individual enhancers. The genes enriched for mutated enhancers converged on the regulation of key biological processes and pathways relevant to tumor biology. Recurrent mutations in individual enhancers also affected the expression of target genes, with potential relevance for patient prognosis. Together, these findings show that noncoding regulatory mutations have a potential relevance for cancer pathogenesis and can be exploited for patient classification. SIGNIFICANCE: Mapping enhancer-target gene regulatory interactions and analyzing enhancer mutations at the level of their target genes and pathways reveal convergence of recurrent enhancer mutations on biological processes involved in tumorigenesis and prognosis.

Extracellular vesicles from subjects with COPD modulate cancer initiating cells phenotype through HIF-1α shuttling.

Chronic obstructive pulmonary disease (COPD) is a risk factor for lung cancer development. COPD induces activation of hypoxia-induced signaling, causing remodeling of surrounding microenvironmental cells also modulating the release and cargo of their extracellular vesicles (EVs). We aimed to evaluate the potential role of circulating EVs from COPD subjects in lung cancer onset. Plasma-EVs were isolated by ultracentrifugation from heavy smoker volunteers with (COPD-EVs) or without (heavy smoker-EVs, HS-EV) COPD and characterized following MISEV guidelines. Immortalized human bronchial epithelial cells (CDK4, hTERT-HBEC3-KT), genetically modified with different oncogenic alterations commonly found in lung cancer (sh-p53, KRASV12), were used to test plasma-EVs pro-tumorigenic activity in vitro. COPD-EVs mainly derived from immune and endothelial cells. COPD-EVs selectively increased the subset of CD133+CXCR4+ metastasis initiating cells (MICs) in HBEC-sh-p53-KRASV12high cells and stimulated 3D growth, migration/invasion, and acquisition of mesenchymal traits. These effects were not observed in HBEC cells bearing single oncogenic mutation (sh-p53 or KRASV12). Mechanistically, hypoxia-inducible factor 1-alpha (HIF-1α) transferred from COPD-EVs triggers CXCR4 pathway activation that in turn mediates MICs expansion and acquisition of pro-tumorigenic effects. Indeed, HIF-1α inhibition or CXCR4 silencing prevented the acquisition of malignant traits induced by COPD-EVs alone. Hypoxia recapitulates the effects observed with COPD-EVs in HBEC-sh-p53-KRASV12high cells. Notably, higher levels of HIF-1α were observed in EVs from COPD subjects who subsequently developed cancer compared to those who remained cancer-free. Our findings support a role of COPD-EVs to promote the expansion of MICs in premalignant epithelial cells through HIF-1α-CXCR4 axis activation thereby potentially sustaining lung cancer progression.

The metastatic niche formation: focus on extracellular vesicle-mediated dialogue between lung cancer cells and the microenvironment.

Lung cancer is the deadliest cancer in the world, with the majority of patients presenting with advanced or metastatic disease at first diagnosis. The lungs are also one of the most common sites of metastasis from lung cancer and other tumors. Understanding the mechanisms that regulate metastasis formation from primary lung cancer and in the lungs is therefore fundamental unmet clinical need. One of the first steps during the establishment of lung cancer metastases includes the formation of the pre-metastatic niche (PMN) at distant organs, which may occur even during the early phases of cancer development. The PMN is established through intricate cross-talk between primary tumor-secreted factors and stromal components at distant sites. Mechanisms controlling primary tumor escape and seeding of distant organs rely on specific properties of tumor cells but are also tightly regulated by interactions with stromal cells at the metastatic niche that finally dictate the success of metastasis establishment. Here, we summarize the mechanisms underlying pre-metastatic niche formation starting from how lung primary tumor cells modulate distant sites through the release of several factors, focusing on Extracellular Vesicles (EVs). In this context, we highlight the role of lung cancer-derived EVs in the modulation of tumor immune escape. Then, we illustrate the complexity of Circulating Tumor Cells (CTCs) that represent the seeds of metastasis and how interactions with stromal and immune cells can help their metastatic dissemination. Finally, we evaluate the contribution of EVs in dictating metastasis development at the PMN through stimulation of proliferation and control of disseminated tumor cell dormancy. Overall, we present an overview of different steps in the lung cancer metastatic cascade, focusing on the EV-mediated interactions between tumor cells and stromal/immune cells.

Ascl1 and OTP tumour expressions are associated with disease-free survival in lung atypical carcinoids.

According to World Health Organization guidelines, atypical carcinoids (ACs) are well-differentiated lung neuroendocrine tumours with 2-10 mitoses/2 mm2 and/or foci of necrosis (usually punctate). Besides morphological criteria, no further tools in predicting AC clinical outcomes are proposed. The aim of this work was to identify novel factors able to predict AC disease aggressiveness and progression. METHODS AND RESULTS: 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 tumours 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 <10%. At multivariable analysis, Ascl1 (present versus absent, hazard ratio [HR] = 3.42, 95% confidence interval [CI] 1.35-8.65, P = 0.009) and OTP (present versus absent, HR = 0.26, 95% CI 0.10-0.68, P = 0.006) were independently associated with DFS. The prognosis of patients with Ki-67 ≥10% tended to be worse compared to that with Ki-67 <10%. On the contrary, OTP (present versus absent, HR = 0.28, 95% CI 0.09-0.89, P = 0.03), tumour stage (III-IV versus I-II, HR = 4.25, 95% CI 1.42-12.73, P = 0.01) and increasing age (10-year increase, HR = 1.67, 95% CI 1.04-2.68, P = 0.03) were independently associated with OS. CONCLUSION: This retrospective analysis of lung ACs showed that Ascl1 and OTP could be the main prognostic drivers of postoperative recurrence.

Lung carcinoid tumours: histology and Ki-67, the eternal rivalry.

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 <3) and histology (AC versus TC) alone significantly added prognostic information to OS and CSS multivariable model with age, stage and OTP; addition of both variables did not provide further prognostic information. Conversely, an improved significance of the DFS prediction model at multivariate analysis was seen by adding Ki-67 (≥3 versus <3, P adj = 0.01) to TC and AC histological distinction, age, lymph node involvement, residual tumour and OTP. Ki-67 ≥ 3% plays a potentially pivotal role in LCT prognosis, irrespective of histological grade.

Eating away T cell responses in lung cancer.

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.

Single-Cell Phenotypic and Molecular Characterization of Circulating Tumor Cells Isolated from Cryopreserved Peripheral Blood Mononuclear Cells of Patients with Lung Cancer and Sarcoma.

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.

Circulating extracellular vesicles from individuals at high-risk of lung cancer induce pro-tumorigenic conversion of stromal cells through transfer of miR-126 and miR-320.

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.

A novel CXCR4 antagonist counteracts paradoxical generation of cisplatin-induced pro-metastatic niches in lung cancer.

Platinum-based chemotherapy remains widely used in advanced non-small cell lung cancer (NSCLC) despite experimental evidence of its potential to induce long-term detrimental effects, including the promotion of pro-metastatic microenvironments. In this study, we investigated the interconnected pathways underlying the promotion of cisplatin-induced metastases. In tumor-free mice, cisplatin treatment resulted in an expansion in the bone marrow of CCR2+CXCR4+Ly6Chigh inflammatory monocytes (IMs) and an increase in lung levels of stromal SDF-1, the CXCR4 ligand. In experimental lung metastasis assays, cisplatin-induced IMs promoted the extravasation of tumor cells and the expansion of CD133+CXCR4+ metastasis-initiating cells (MICs). Peptide R, a novel CXCR4 inhibitor designed as an SDF-1 mimetic peptide, prevented cisplatin-induced IM expansion, the recruitment of IMs into the lungs, and the promotion of metastasis. At the primary tumor site, cisplatin treatment reduced tumor size while simultaneously inducing tumor release of SDF-1, MIC expansion, and recruitment of pro-invasive CXCR4+ macrophages. Co-recruitment of MICs and CCR2+CXCR4+ IMs to distant SDF-1-enriched sites also promoted spontaneous metastases that were prevented by CXCR4 blockade. In clinical specimens from NSCLC patients SDF-1 levels were found to be higher in platinum-treated samples and related to a worse clinical outcome. Our findings reveal that activation of the CXCR4/SDF-1 axis specifically mediates the pro-metastatic effects of cisplatin and suggest CXCR4 blockade as a possible novel combination strategy to control metastatic disease.

MMP1 drives tumor progression in large cell carcinoma of the lung through fibroblast senescence.

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-ß1, is sufficient to induce fibroblast senescence and consequent LCC promotion. Furthermore, we implicate PAR-1 and oxidative stress in MMP1/TGF-ß1-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.

Differential glycosylation of collagen modulates lung cancer stem cell subsets through ß1 integrin-mediated interactions.

In lung cancer, CD133+ cells represent the subset of cancer stem cells (CSC) able to sustain tumor growth and metastatic dissemination. CSC function is tightly regulated by specialized niches composed of both stromal cells and extracellular matrix (ECM) proteins, mainly represented by collagen. The relevance of collagen glycosylation, a fundamental post-translational modification controlling several biological processes, in regulating tumor cell phenotype remains, however, largely unexplored. To investigate the bioactive effects of differential ECM glycosylation on lung cancer cells, we prepared collagen films functionalized with glucose (Glc-collagen) and galactose (Gal-collagen) exploiting a neoglycosylation approach based on a reductive amination of maltose and lactose with the amino residues of collagen lysines. We demonstrate that culturing of tumor cells on collagen determines a glycosylation-dependent positive selection of CSC and triggers their expansion/generation. The functional relevance of CD133+ CSC increase was validated in vivo, proving an augmented tumorigenic and metastatic potential. High expression of integrin ß1 in its active form is associated with an increased proficiency of tumor cells to sense signaling from glycosylated matrices (glyco-collagen) and to acquire stemness features. Accordingly, inhibition of integrin ß1 in tumor cells prevents CSC enrichment, suggesting that binding of integrin ß1 to Glc-collagen subtends CSC expansion/generation. We provide evidence suggesting that collagen glycosylation could play an essential role in modulating the creation of a niche favorable for the generation and selection/survival of lung CSC. Interfering with this crosstalk may represent an innovative therapeutic strategy for lung cancer treatment.

CXCR4 Inhibition Counteracts Immunosuppressive Properties of Metastatic NSCLC Stem Cells.

Cancer stem cells (CSCs) are functionally defined as the cell subset with greater potential to initiate and propagate tumors. Within the heterogeneous population of lung CSCs, we previously identified highly disseminating CD133+CXCR4+ cells able to initiate distant metastasis (metastasis initiating cells-MICs) and to resist conventional chemotherapy. The establishment of an immunosuppressive microenvironment by tumor cells is crucial to sustain and foster metastasis formation, and CSCs deeply interfere with immune responses against tumors. How lung MICs can elude and educate immune cells surveillance to efficiently complete the metastasis cascade is, however, currently unknown. We show here in primary tumors from non-small cell lung cancer (NSCLC) patients that MICs express higher levels of immunoregulatory molecules compared to tumor bulk, namely PD-L1 and CD73, an ectoenzyme that catalyzes the production of immunosuppressive adenosine, suggesting an enhanced ability of MICs to escape immune responses. To investigate in vitro the immunosuppressive ability of MICs, we derived lung spheroids from cultures of adherent lung cancer cell lines, showing enrichment in CD133+CXCR4+MICs, and increased expression of CD73 and CD38, an enzyme that also concurs in adenosine production. MICs-enriched spheroids release high levels of adenosine and express the immunosuppressive cytokine IL-10, undetectable in an adherent cell counterpart. To prevent dissemination of MICs, we tested peptide R, a novel CXCR4 inhibitor that effectively controls in vitro lung tumor cell migration/invasion. Notably, we observed a decreased expression of CD73, CD38, and IL-10 following CXCR4 inhibition. We also functionally proved that conditioned medium from MICs-enriched spheroids compared to adherent cells has an enhanced ability to suppress CD8+ T cell activity, increase Treg population, and induce the polarization of tumor-associated macrophages (TAMs), which participate in suppression of T cells. Treatment of spheroids with anti-CXCR4 rescued T cell cytotoxic activity and prevented TAM polarization, likely by causing the decrease of adenosine and IL-10 production. Overall, we provide evidence that the subset of lung MICs shows high potential to escape immune control and that inhibition of CXCR4 can impair both MICs dissemination and their immunosuppressive activity, therefore potentially providing a novel therapeutic target in combination therapies to improve efficacy of NSCLC treatment.

Beyond Traditional Morphological Characterization of Lung Neuroendocrine Neoplasms: In Silico Study of Next-Generation Sequencing Mutations Analysis across the Four World Health Organization Defined Groups.

Lung neuroendocrine neoplasms (LNENs) represent a rare and heterogeneous population of lung tumors. LNENs incidence rate has increased dramatically over the past 30 years. The current World Health Organization LNENs classification (WHO 2015), distinguished four LNENs prognostic categories, according to their morphology, necrosis amount and mitotic count: typical carcinoid (TC), atypical-carcinoid (AC), large cell neuroendocrine carcinoma (LCNEC) and small cell lung cancer (SCLC). At present, due to their rarity and biological heterogeneity there is still no consensus on the best therapeutic approach. Next-generation-sequencing analysis showed that WHO 2015 LNENs classes, could be characterized also by specific molecular alterations: frequently mutated genes involving chromatin remodeling and generally characterized by low mutational burden (MB) are frequently detected in both TC and AC; otherwise, TP53 and RB1 tumor suppressor genes alterations and high MB are usually detected in LCNEC and SCLC. We provide an overview concerning gene mutations in each WHO 2015 LNENs class in order to report the current LNENs mutational status as potential tool to better understand their clinical outcome and to drive medical treatment.

Paradoxical effects of chemotherapy on tumor relapse and metastasis promotion.

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.

Circulating mir-320a promotes immunosuppressive macrophages M2 phenotype associated with lung cancer risk.

miRNAs play a central role in the complex signaling network of cancer cells with the tumor microenvironment. Little is known on the origin of circulating miRNAs and their relationship with the tumor microenvironment in lung cancer. Here, we focused on the cellular source and relative contribution of different cell types to circulating miRNAs composing our risk classifier of lung cancer using in vitro/in vivo models and clinical samples. A cell-type specific expression pattern and topography of several miRNAs such as mir-145 in fibroblasts, mir-126 in endothelial cells, mir-133a in skeletal muscle cells was observed in normal and lung cancer tissues. Granulocytes and platelets are the major contributors of miRNAs release in blood. miRNAs modulation observed in plasma of lung cancer subjects was consistent with de-regulation of the same miRNAs observed during immunosuppressive conversion of immune cells. In particular, activated neutrophils showed a miRNA profile mirroring that observed in plasma of lung cancer subjects. Interestingly mir-320a secreted by neutrophils of high-risk heavy-smokers promoted an M2-like protumorigenic phenotype through downregulation of STAT4 when shuttled into macrophages. These findings suggest a multifactorial and nonepithelial cell-autonomous origin of circulating miRNAs associated with risk of lung cancer and that circulating miRNAs may act in paracrine signaling with causative role in lung carcinogenesis and immunosuppression.

Diagnostic role of circulating extracellular matrix-related proteins in non-small cell lung cancer.

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

MiR-16 regulates the pro-tumorigenic potential of lung fibroblasts through the inhibition of HGF production in an FGFR-1- and MEK1-dependent manner.

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

Author Correction: Establishment of patient derived xenografts as functional testing of lung cancer aggressiveness.

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.