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.

Detection of (pre)cancerous colorectal lesions in Lynch syndrome patients by microsatellite instability liquid biopsy.

Lynch syndrome (LS) is an inherited condition characterized by an increased risk of developing cancer, in particular colorectal cancer (CRC). Microsatellite instability (MSI) is the main feature of (pre)cancerous lesions occurring in LS patients. Close endoscopic surveillance is the only option available to reduce CRC morbidity and mortality. However, it may fail to intercept interval cancers and patients' compliance to such an invasive procedure may decrease over the years. The development of a minimally invasive test able to detect (pre)cancerous colorectal lesions, could thus help tailor surveillance programs in LS patients. Taking advantage of an endoscopic surveillance program, we retrospectively assessed the instability of five microsatellites (BAT26, BAT25, NR24, NR21, and Mono27) in liquid biopsies collected at baseline and possibly at two further endoscopic rounds. For this purpose, we tested a new multiplex drop-off digital polymerase chain reaction (dPCR) assay, reaching mutant allele frequencies (MAFs) as low as 0.01%. Overall, 78 plasma samples at the three time-points from 18 patients with baseline (pre)cancerous lesions and 18 controls were available for molecular analysis. At baseline, the MAFs of BAT26, BAT25 and NR24 were significantly higher in samples of patients with lesions but did not differ with respect to the grade of dysplasia or any other clinico-pathological characteristics. When all markers were combined to determine MSI in blood, this test was able to discriminate lesion-bearing patients with an AUC of 0.80 (95%CI: 0.66; 0.94).

Caloric restriction and metformin selectively improved LKB1-mutated NSCLC tumor response to chemo- and chemo-immunotherapy.

BACKGROUND: About 10% of NSCLCs are mutated in KRAS and impaired in STK11/LKB1, a genetic background associated with poor prognosis, caused by an increase in metastatic burden and resistance to standard therapy. LKB1 is a protein involved in a number of biological processes and is particularly important for its role in the regulation of cell metabolism. LKB1 alterations lead to protein loss that causes mitochondria and metabolic dysfunction that makes cells unable to respond to metabolic stress. Different studies have shown how it is possible to interfere with cancer metabolism using metformin and caloric restriction (CR) and both modify the tumor microenvironment (TME), stimulating the switch from "cold" to "hot". Given the poor therapeutic response of KRASmut/LKB1mut patients, and the role of LKB1 in cell metabolism, we examined whether the addition of metformin and CR enhanced the response to chemo or chemo-immunotherapy in LKB1 impaired tumors. METHODS: Mouse cell lines were derived from lung nodules of transgenic mice carrying KRASG12D with either functional LKB1 (KRASG12D/LKB1wt) or mutated LKB1 (KRASG12D/LKB1mut). Once stabilized in vitro, these cell lines were inoculated subcutaneously and intramuscularly into immunocompetent mice. Additionally, a patient-derived xenograft (PDX) model was established by directly implanting tumor fragments from patient into immunocompromised mice. The mice bearing these tumor models were subjected to treatment with chemotherapy or chemo-immunotherapy, both as standalone regimens and in combination with metformin and CR. RESULTS: Our preclinical results indicate that in NSCLC KRASmut/LKB1mut tumors, metformin and CR do enhance the response to chemo and chemo-immunotherapy, inducing a metabolic stress condition that these tumors are not able to overcome. Analysis of immune infiltrating cells did not bring to light any strong correlation between the TME immune-modulation and the tumor response to metformin and CR. CONCLUSION: Our in vitro and in vivo preliminary studies confirm our hypothesis that the addition of metformin and CR is able to improve the antitumor activity of chemo and chemoimmunotherapy in LKB1 impaired tumors, exploiting their inability to overcome metabolic stress.

Long-term outcomes of lung cancer screening in males and females.

BACKGROUND: This study explored female and male overall mortality and lung cancer (LC) survival in two LC screening (LCS) populations, focusing on the predictive value of coronary artery calcification (CAC) at baseline low-dose computed tomography (LDCT). METHODS: This retrospective study analysed data of 6495 heavy smokers enrolled in the MILD and BioMILD LCS trials between 2005 and 2016. The primary objective of the study was to assess sex differences in all-cause mortality and LC survival. CAC scores were automatically calculated on LDCT images by a validated artificial intelligence (AI) software. Sex differences in 12-year cause-specific mortality rates were stratified by age, pack-years and CAC score. RESULTS: The study included 2368 females and 4127 males. The 12-year all-cause mortality rates were 4.1 % in females and 7.7 % in males (p < 0.0001), and median CAC score was 8.7 vs. 41 respectively (p < 0.0001). All-cause mortality increased with rising CAC scores (log-rank test, p < 0.0001) for both sexes. Although LC incidence was not different between the two sexes, females had lower rates of 12-year LC mortality (1.0 % vs. 1.9 %, p = 0.0052), and better LC survival from diagnosis (72.3 % vs. 51.7 %; p = 0.0005), with a similar proportion of stage I (58.1 % vs. 51.2 %, p = 0.2782). CONCLUSIONS: Our findings demonstrate that female LCS participants had lower rates of all-cause mortality at 12 years and better LC survival than their male counterparts, with similar LC incidence rates and stage at diagnosis. The lower CAC burden observed in women at all ages might contribute to explain their lower rates of all-cause mortality and better LC survival.

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 new world of RNA diagnostics and therapeutics.

The 5th Workshop IRE on Translational Oncology was held in Rome (Italy) on 27-28 March at the IRCCS Regina Elena National Cancer Institute. This meeting entitled "The New World of RNA diagnostics and therapeutics" highlightes the significant progress in the RNA field made over the last years. Research moved from pure discovery towards the development of diagnostic biomarkers or RNA-base targeted therapies seeking validation in several clinical trials. Non-coding RNAs in particular have been the focus of this workshop due to their unique properties that make them attractive tools for the diagnosis and therapy of cancer.This report collected the presentations of many scientists from different institutions that discussed recent oncology research providing an excellent overview and representative examples for each possible application of RNA as biomarker, for therapy or to increase the number of patients that can benefit from precision oncology treatment.In particular, the meeting specifically emphasized two key features of RNA applications: RNA diagnostic (Blandino, Palcau, Sestito, Díaz Méndez, Cappelletto, Pulito, Monteonofrio, Calin, Sozzi, Cheong) and RNA therapeutics (Dinami, Marcia, Anastasiadou, Ryan, Fattore, Regazzo, Loria, Aharonov).

Metabolic classification of non-small cell lung cancer patient-derived xenografts by a digital pathology approach: A pilot study.

Introduction: Genetically characterized patient-derived tumor xenografts (PDX) are a valuable resource to understand the biological complexity of cancer and to investigate new therapeutic approaches. Previous studies, however, lack information about metabolic features of PDXs, which may limit testing of metabolism targeting drugs. Methods: In this pilot study, we investigated by immunohistochemistry (IHC) expression of five essential metabolism-associated markers in a set of lung adenocarcinoma PDX samples previously established and characterized. We exploited digital pathology to quantify expression of the markers and correlated results with tumor cell proliferation, angiogenesis and time of PDX growth in mice. Results: Our results indicate that the majority of the analyzed PDX models rely on oxidative phosphorylation (OXPHOS) metabolism, either alone or in combination with glucose metabolism. Double IHC enabled us to describe spatial expression of the glycolysis-associated monocarboxylate transporter 4 (MCT4) marker and the OXPHOS-associated glutaminase (GLS) marker. GLS expression was associated with cell proliferation and with expression of liver-kinase B1 (LKB1), a tumor suppressor involved in the regulation of multiple metabolic pathways. Acetyl CoA carboxylase (ACC) was associated with the kinetics of PDX growth. Conclusion: Albeit limited by the small number of samples and markers analyzed, metabolic classification of existing collections of PDX by this mini panel will be useful to inform pre-clinical testing of metabolism-targeting drugs.

Establishment of 6 pediatric rhabdomyosarcoma patient's derived xenograft models closely recapitulating patients' tumor characteristics.

INTRODUCTION: The prognosis for patients with metastatic and recurrent pediatric rhabdomyosarcoma (RMS) remains poor. The availability of preclinical models is essential to identify promising treatments We established a series of pediatric RMS patient derived xenografts (PDXs), all faithfully mirroring primary tumor characteristics and representing a unique tool for clarifying the biological processes underlying RMS progression and relapse. METHODS: Fresh tumor samples from 12 RMS patients were implanted subcutaneously in both flanks of immunocompromised mice. PDXs were considered as grafted after accomplishing three passages in mice. Characterization of tumor tissues and models was performed by comparing both morphology and immunoistochemical and fluorescence in situ hybridization (FISH) characteristics. RESULTS: Six PDXs were established, with a successful take rate of 50%. All models closely mirrored parental tumor characteristics. An increased grafting rate for tumors derived from patients with worse outcome (p = 0.006) was detected. For 50% PDXs grafting occurred when the corresponding patient was still alive. CONCLUSION: Our findings increase the number of available RMS PDX models and strengthen the role of PDXs as useful preclinical tools for patients with unmet medical needs and to develop personalized therapies.

The Educational Program of Macrophages toward a Hyperprogressive Disease-Related Phenotype Is Orchestrated by Tumor-Derived Extracellular Vesicles.

Hyperprogressive disease (HPD), an aggressive acceleration of tumor growth, was observed in a group of cancer patients treated with anti-PD1/PDL1 antibodies. The presence of a peculiar macrophage subset in the tumor microenvironment is reported to be a sort of "immunological prerequisite" for HPD development. These macrophages possess a unique phenotype that it is not clear how they acquire. We hypothesized that certain malignant cells may promote the induction of an "HPD-related" phenotype in macrophages. Bone-marrow-derived macrophages were exposed to the conditioned medium of five non-small cell lung cancer cell lines. Macrophage phenotype was analyzed by microarray gene expression profile and real-time PCR. We found that human NSCLC cell lines, reported as undergoing HPD-like tumor growth in immunodeficient mice, polarized macrophages towards a peculiar pro-inflammatory phenotype sharing both M1 and M2 features. Lipid-based factors contained in cancer cell-conditioned medium induced the over-expression of several pro-inflammatory cytokines and the activation of innate immune receptor signaling pathways. We also determined that tumor-derived Extracellular Vesicles represent the main components involved in the observed macrophage re-education program. The present study might represent the starting point for the future development of diagnostic tools to identify potential hyperprogressors.

MiR-223 Exclusively Impairs In Vitro Tumor Growth through IGF1R Modulation in Rhabdomyosarcoma of Adolescents and Young Adults.

Adolescents and young adults (AYA) with rhabdomyosarcoma (RMS) form a subgroup of patients whose optimal clinical management and best possible access to care remain a challenge and whose survival rates lag behind that of children diagnosed with histologically similar tumors. A better understanding of tumor biology that differentiates children (PEDS-) from AYA-RMS could provide critical information and drive new initiatives to improve their final outcome. We investigated the functional role of miRNAs implicated in AYA-RMS development, as they have the potential to lead to discovery of new targets pathways for a more tailored treatment in these age groups of young RMS patients. MiR-223 and miR-486 were observed de-regulated in nine RMS tissues compared to their normal counterparts, yet only miR-223 replacement impaired proliferation and aggressiveness of AYA-RMS cell lines, while inducing apoptosis and determining cell cycle arrest. Interestingly, IGF1R resulted in the direct target of miR-223 in AYA-RMS cells, as demonstrated by IGF1R silencing. Our results highlight an exclusive functional role of miR-223 in AYA-RMS development and aggressiveness.

Circulating CD81-expressing extracellular vesicles as biomarkers of response for immune-checkpoint inhibitors in advanced NSCLC.

PD-L1 in tumor cells is the only used biomarker for anti PD1/PD-L1 immune-checkpoints inhibitors (ICI) in Non Small Cell Lung Cancer (NSCLC) patients. However, this parameter is inaccurate to predict response, especially in patients with low tumor PD-L1. Here, we evaluated circulating EVs as possible biomarkers for ICI in advanced NSCLC patients with low tumoral PD-L1. EVs were isolated from plasma of 64 PD-L1 low, ICI-treated NSCLC patients, classified either as responders (R; complete or partial response by RECIST 1.1) or non-responders (NR). EVs were characterized following MISEV guidelines and by flow cytometry. T cells from healthy donors were triggered in vitro using patients' EVs. Unsupervised statistical approach was applied to correlate EVs' and patients' features to clinical response. R-EVs showed higher levels of tetraspanins (CD9, CD81, CD63) than NR-EVs, significantly associated to better overall response rate (ORR). In multivariable analysis CD81-EVs correlated with ORR. Unsupervised analysis revealed a cluster of variables on EVs, including tetraspanins, significantly associated with ORR and improved survival. R-EVs expressed more costimulatory molecules than NR-EVs although both increased T cell proliferation and partially, activation. Tetraspanins levels on EVs could represent promising biomarkers for ICI response in NSCLC.

Development of a Molecular Blood-Based Immune Signature Classifier as Biomarker for Risks Assessment in Lung Cancer Screening.

BACKGROUND: Low-dose CT (LDCT) screening trials have shown that lung cancer early detection saves lives. However, a better stratification of the screening population is still needed. In this respect, we generated and prospectively validated a plasma miRNA signature classifier (MSC) able to categorize screening participants according to lung cancer risk. Here, we aimed to deeply characterize the peripheral immune profile and develop a diagnostic immune signature classifier to further implement blood testing in lung cancer screening. METHODS: Peripheral blood mononuclear cell (PBMC) samples collected from 20 patients with LDCT-detected lung cancer and 20 matched cancer-free screening volunteers were analyzed by flow cytometry using multiplex panels characterizing both lymphoid and myeloid immune subsets. Data were validated in PBMC from 40 patients with lung cancer and 40 matched controls and in a lung cancer specificity set including 27 subjects with suspicious lung nodules. A qPCR-based gene expression signature was generated resembling selected immune subsets. RESULTS: Monocytic myeloid-derived suppressor cell (MDSC), polymorphonuclear MDSC, intermediate monocytes and CD8+PD-1+ T cells distinguished patients with lung cancer from controls with AUCs values of 0.94/0.72/0.88 in the training, validation, and lung cancer specificity set, respectively. AUCs raised up to 1.00/0.84/0.92 in subgroup analysis considering only MSC-negative subjects. A 14-immune genes expression signature distinguished patients from controls with AUC values of 0.76 in the validation set and 0.83 in MSC-negative subjects. CONCLUSIONS: An immune-based classifier can enhance the accuracy of blood testing, thus supporting the contribution of systemic immunity to lung carcinogenesis. IMPACT: Implementing LDCT screening trials with minimally invasive blood tests could help reduce unnecessary procedures and optimize cost-effectiveness.

Increased serum levels of KiSS1-derived peptides in non-small cell lung cancer patient liquid biopsies and biological relevance.

Background: The secreted products of the metastasis suppressor gene KiSS1 may represent useful biomarkers in non-small cell lung cancer (NSCLC) but their levels in patients have remained poorly investigated. We previously found that forced expression of KiSS1 decreased the invasive capability of NSCLC drug-resistant cells and a pro-apoptotic role for KiSS1 has been proposed in head and neck cancer. Thus, we designed a translational investigation including a pilot study to analyze KiSS1 levels in liquid biopsies, and in vitro experiments to explore the biological relevance of KiSS1 modulation. Methods: KiSS1-derived peptide levels in liquid biopsies from 60 NSCLC patients were assayed by ELISA. Preclinical experiments were carried out using quantitative real time polymerase chain reaction (qRT-PCR), ELISA, annexin V-binding and caspase activation assays. Results: We compared KiSS1 release in 3 different matrices (serum, plasma and urine) and the highest levels were detectable in serum (range, 0-4.5 ng/mL). We observed increased levels of seric KiSS1 in NSCLC patients as compared to healthy donors. KiSS1 serum concentrations, after surgical procedure and/or adjuvant therapy. We observed differences among disease stages in urine samples. In preclinical models, KiSS1 mRNA levels were increased by short term exposure to azacytidine, enhanced KiSS1 release was induced by the combination of azacytidine and cisplatin and KiSS1-derived peptides enhanced cisplatin-induced apoptosis. KiSS1 increase was observed upon exposure neurons-enriched cultures to tumor cell conditioned medium. Conclusions: Our results showing a peculiar modulation of KiSS1 levels in liquid biopsies of NSCLC patients and a regulation of cisplatin-induced apoptosis by KiSS1-derived peptides support an involvement of KiSS1 in cell response to treatment and highlight its promising features as a potential biomarker in NSCLC.

A Cross-Comparison of High-Throughput Platforms for Circulating MicroRNA Quantification, Agreement in Risk Classification, and Biomarker Discovery in Non-Small Cell Lung Cancer.

Background: Circulating microRNAs (ct-miRs) are promising cancer biomarkers. This study focuses on platform comparison to assess performance variability, agreement in the assignment of a miR signature classifier (MSC), and concordance for the identification of cancer-associated miRs in plasma samples from non-small cell lung cancer (NSCLC) patients. Methods: A plasma cohort of 10 NSCLC patients and 10 healthy donors matched for clinical features and MSC risk level was profiled for miR expression using two sequencing-based and three quantitative reverse transcription PCR (qPCR)-based platforms. Intra- and inter-platform variations were examined by correlation and concordance analysis. The MSC risk levels were compared with those estimated using a reference method. Differentially expressed ct-miRs were identified among NSCLC patients and donors, and the diagnostic value of those dysregulated in patients was assessed by receiver operating characteristic curve analysis. The downregulation of miR-150-5p was verified by qPCR. The Cancer Genome Atlas (TCGA) lung carcinoma dataset was used for validation at the tissue level. Results: The intra-platform reproducibility was consistent, whereas the highest values of inter-platform correlations were among qPCR-based platforms. MSC classification concordance was >80% for four platforms. The dysregulation and discriminatory power of miR-150-5p and miR-210-3p were documented. Both were significantly dysregulated also on TCGA tissue-originated profiles from lung cell carcinoma in comparison with normal samples. Conclusion: Overall, our studies provide a large performance analysis between five different platforms for miR quantification, indicate the solidity of MSC classifier, and identify two noninvasive biomarkers for NSCLC.

Cytisine Therapy Improved Smoking Cessation in the Randomized Screening and Multiple Intervention on Lung Epidemics Lung Cancer Screening Trial.

INTRODUCTION: Cytisine, a partial agonist-binding nicotine acetylcholine receptor, is a promising cessation intervention. We conducted a single-center, randomized, controlled trial (RCT) in Italy to assess the efficacy and tolerability of cytisine as a smoking cessation therapy among lung cancer screening participants. METHODS: From July 2019 to March 2020, the Screening and Multiple Intervention on Lung Epidemics RCT enrolled 869 current heavy tobacco users in a low-dose computed tomography screening program, with a randomized comparison of pharmacologic intervention with cytisine plus counseling (N = 470) versus counseling alone (N = 399). The primary outcome was continuous smoking abstinence at 12 months, biochemically verified through carbon monoxide measurement. RESULTS: At the 12-month follow-up, the quit rate was 32.1% (151 participants) in the intervention arm and 7.3% (29 participants) in the control arm. The adjusted OR of continuous abstinence was 7.2 (95% confidence interval: 4.6-11.2). Self-reported adverse events occurred more frequently in the intervention arm (399 events among 196 participants) than in the control arm (230 events among 133 participants, p < 0.01). The most common adverse events were gastrointestinal symptoms, comprising abdominal swelling, gastritis, and constipation. CONCLUSIONS: The efficacy and safety observed in the Screening and Multiple Intervention on Lung Epidemics RCT indicate that cytisine, a very low-cost medication, is a useful treatment option for smoking cessation and a feasible strategy to improve low-dose computed tomography screening outcomes with a potential benefit for all-cause mortality.

MiR-486-5p Targets CD133+ Lung Cancer Stem Cells through the p85/AKT Pathway.

Despite improvements in therapies and screening strategies, lung cancer prognosis still remains dismal, especially for metastatic tumors. Cancer stem cells (CSCs) are endowed with properties such as chemoresistance, dissemination, and stem-like features, that make them one of the main causes of the poor survival rate of lung cancer patients. MicroRNAs (miRNAs), small molecules regulating gene expression, have a role in lung cancer development and progression. In particular, miR-486-5p is an onco-suppressor miRNA found to be down-modulated in the tumor tissue of lung cancer patients. In this study, we investigate the role of this miRNA in CD133+ lung CSCs and evaluate the therapeutic efficacy of coated cationic lipid-nanoparticles entrapping the miR-486-5p miRNA mimic (CCL-486) using lung cancer patient-derived xenograft (PDX) models. In vitro, miR-486-5p overexpression impaired the PI3K/Akt pathway and decreased lung cancer cell viability. Moreover, miR-486-5p overexpression induced apoptosis also in CD133+ CSCs, thus affecting the in vivo tumor-initiating properties of these cells. Finally, we demonstrated that in vivo CCL-486 treatment decreased CD133+ percentage and inhibited tumor growth in PDX models. In conclusion, we provided insights on the efficacy of a novel miRNA-based compound to hit CD133+ lung CSCs, setting the basis for new combined 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.

Machine Learning Using Real-World and Translational Data to Improve Treatment Selection for NSCLC Patients Treated with Immunotherapy.

(1) Background: In advanced non-small cell lung cancer (aNSCLC), programmed death ligand 1 (PD-L1) remains the only biomarker for candidate patients to immunotherapy (IO). This study aimed at using artificial intelligence (AI) and machine learning (ML) tools to improve response and efficacy predictions in aNSCLC patients treated with IO. (2) Methods: Real world data and the blood microRNA signature classifier (MSC) were used. Patients were divided into responders (R) and non-responders (NR) to determine if the overall survival of the patients was likely to be shorter or longer than 24 months from baseline IO. (3) Results: One-hundred sixty-four out of 200 patients (i.e., only those ones with PD-L1 data available) were considered in the model, 73 (44.5%) were R and 91 (55.5%) NR. Overall, the best model was the linear regression (RL) and included 5 features. The model predicting R/NR of patients achieved accuracy ACC = 0.756, F1 score F1 = 0.722, and area under the ROC curve AUC = 0.82. LR was also the best-performing model in predicting patients with long survival (24 months OS), achieving ACC = 0.839, F1 = 0.908, and AUC = 0.87. (4) Conclusions: The results suggest that the integration of multifactorial data provided by ML techniques is a useful tool to select NSCLC patients as candidates for IO.