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Supplementary Tables S1-S5. Table S1: Antiproliferative effects of PI3K inhibitors on SKMES-1 and PIK3CA amplified/mutated clones Tables S2-S4: densitometric analysis of Western blotting of Figure 1D Table S5: Antiproliferative effects of PI3K inhibitors on H596 and HCC2450 cells

Supplementary Figures S1-S5. Figure S1: p-AKT levels in PIK3CA transfected clones Figure S2: Induction of cell death after treatment with PI3K inhibitors Figure S3: Time course evaluation of PI3K/AKT/mTOR pathway activity after exposure to PI3K inhibitors Figure S4: Evaluation of cell migration after treatment with PI3K inhibitors Figure S5: CD133 expression in tumor spheres

Purpose: The success of immune checkpoint inhibitors strengthens the notion that tumor growth and regression are immune regulated. To determine whether distinct tissue immune microenvironments differentially affect clinical outcome in non–small cell lung cancer (NSCLC), an extended analysis of PD-L1 and tumor-infiltrating lymphocytes (TIL) was performed.Experimental Design: Samples from resected adenocarcinoma (ADC 42), squamous cell carcinoma (SCC 58), and 26 advanced diseases (13 ADC and 13 SCC) treated with nivolumab were analyzed. PD-L1 expression and the incidence of CD3, CD8, CD4, PD-1, CD57, FOXP3, CD25, and Granzyme B TILs were immunohistochemically assessed.Results: PD-L1 levels inversely correlated with N involvement, although they did not show a statistically significant prognostic value in resected patients. The incidence and phenotype of TILs differed in SCC versus ADC, in which EGFR and KRAS mutations conditioned a different frequency and tissue localization of lymphocytes. NSCLC resected patients with high CD8pos lymphocytes lacking PD-1 inhibitory receptor had a longer overall survival (OS: HR = 2.268; 95% CI, 1.056–4.871, P = 0.03). PD-1-to-CD8 ratio resulted in a prognostic factor both on univariate (HR = 1.952; 95% CI, 1.34–3.12, P = 0.001) and multivariate (HR = 1.943; 95% CI, 1.38–2.86, P = 0.009) analysis. Moreover, low PD-1 incidence among CD8pos cells was a distinctive feature of nivolumab-treated patients, showing clinical benefit with a prolonged progression-free survival (PFS: HR = 4.51; 95% CI, 1.45–13.94, P = 0.004).Conclusions: In the presence of intrinsic variability in PD-L1 expression, the reservoir of PD-1–negative effector T lymphocytes provides an immune-privileged microenvironment with a positive impact on survival of patients with resected disease and response to immunotherapy in advanced NSCLC. Clin Cancer Res; 24(2); 407–19. ©2017 AACR.

Clinicopathological correlations. Figure S1: PD-L1 Expression and Clinicopathologic Parameters; Figure S2: PD-L1 Expression and TILs; Figure S3: TILs and Clinicopathological Parameters; Figure S4: NSCLC Immune Microenvironments Based on PD-L1 and TILs.

Table S1. Antibodies for Immunohistochemistry; Table S2. Magnitude of Sampling; Table S3. Magnitude of Sampling

Background: In situ cardiac tissue engineering aims to heal the infarcted myocardium by guiding tissue regeneration within the patient body. A key step in this approach is the design of a bioactive scaffold, able to stimulate tissue repair at the site of damage. In the development of bioactive scaffolds, molecular imprinting nanotechnology has been recently proposed as a new functionalization strategy. Objectives: In this work, Molecularly Imprinted Particles (MIP) with recognition properties towards the stromal-derived factor-1 (SDF-1) were synthesized, characterized and used for the functionalization of a biomimetic scaffold. MIP are expected to favor the enrichment of the SDF-1 bioactive molecule within the scaffold, thereby promoting myocardial regeneration. Methods: MIP were obtained by precipitation polymerization, using the SDF-1 molecule as a template. Alginate/gelatin/elastin sponges were fabricated by freeze-drying and functionalized by MIP deposition. Morphological, physicochemical and functional analyses were performed both on MIP and on MIP-modified scaffolds. A preliminary biological in vitro investigation was also carried out using rat cardiac progenitor cells (rCPCs). Results: Imprinted nanoparticles with an average diameter between 0.6 and 0.9 µm were obtained. Infrared analysis of MIP confirmed the expected chemical structure. Recognition and selectivity tests showed that MIP were able to selectively recognize and rebind the template, even after their deposition on the scaffold. In vitro biological tests showed that cell adhesion to the scaffold was promoted by MIP functionalization. Conclusion: Results obtained in the present study suggest that biomimetic alginate/gelatin/elastin sponges, functionalized by MIP with recognition properties towards SDF-1, could be successfully used for tissue engineering approaches to repair the infarcted heart.

The mechanisms underlying the success of propranolol in the treatment of infantile hemangioma (IH) remain elusive and do not fully explain the rapid regression of hemangiomatous lesions following drug administration. As autophagy is critically implicated in vascular homeostasis, we determined whether β-blockers trigger the autophagic flux on infantile hemangioma-derived endothelial cells (Hem-ECs) in vitro.Fresh tissue specimens, surgically removed for therapeutic purpose to seven children affected by proliferative IH, were subjected to enzymatic digestion. Cells were sorted with anti-human CD31 immunolabeled magnetic microbeads. Following phenotypic characterization, expanded Hem-ECs, at P2 to P6, were exposed to different concentrations (50 μM to 150 μM) of propranolol, atenolol or metoprolol alone and in combination with the autophagy inhibitor Bafilomycin A1. Rapamycin, a potent inducer of autophagy, was also used as control. Autophagy was assessed by Lysotracker Red staining, western blot analysis of LC3BII/LC3BI and p62, and morphologically by transmission electron microscopy.Hem-ECs treated with either propranolol, atenolol or metoprolol displayed positive LysoTracker Red staining. Increased LC3BII/LC3BI ratio, as well as p62 modulation, were documented in β-blockers treated Hem-ECs. Abundant autophagic vacuoles and multilamellar bodies characterized the cytoplasmic ultrastructural features of autophagy in cultured Hem-ECs exposed in vitro to β-blocking agents. Importantly, similar biochemical and morphologic evidence of autophagy were observed following rapamycin while Bafilomycin A1 significantly prevented the autophagic flux promoted by β-blockers in Hem-ECs.Our data suggest that autophagy may be ascribed among the mechanisms of action of β-blockers suggesting new mechanistic insights on the potential therapeutic application of this class of drugs in pathologic conditions involving uncontrolled angiogenesis.

Qualitative and quantitative CT imaging features might intercept the multifaceted tumor immune microenvironment (TIME), providing a non-invasive approach to design new prognostic models in NSCLC patients.Our study population consisted of 100 surgically resected NSCLC patients among which 31 served as a validation cohort for quantitative image analysis. TIME was classified according to PD-L1 expression and the magnitude of Tumor Infiltrating Lymphocytes (TILs) and further defined as hot or cold by the tissue analysis of effector (CD8-to-CD3Specific CT-SFs (texture [TXT], effect [EFC] and margins [MRG]) strongly correlated to PD-L1 and TILs status and showed significant impact on survival outcome (TXT, HR:3.39, 95 % CI 1.12-10-27, P 0.05; EFC, HR:0.41, 95 % CI 0.18-0.93, P 0.05; MRG, HR:1.93, 95 % CI 0.88-4.25, P = 0.09). Seven CT derived radiomic features were able to sharply discriminate cases with hot (inflamed) vs cold (desert) TIME, which also exhibited opposite OS (long vs short, HR:0.09, 95 % CI 0.04-0.23, P 0.001) and DFS (long vs short, HR:0.31, 95 % CI 0.16-0.58, P 0.001). Moreover, we identified 6 prognostic radiomic features among which ClusterProminence displayed the highest statistical significance (HR:0.13, 95 % CI 0.06-0.31, P 0.001). These findings were independently validated in an additional cohort of NSCLC (HR:0.11, 95 % CI 0.03-0.40, P = 0.001). Finally, in our training cohort we developed a multiparametric prognostic model, interlacing TIME and clinico-pathological characteristics with CT-SFs (ROC curve AUC:0.83, 95 % CI 0.71-0.92, P 0.001) or CT-RFs (AUC: 0.91, 95 % CI 0.83-0.99, P 0.001), which appeared to outperform pTNM staging (AUC: 0.66, 95 % CI 0.51-0.80, P 0.05) in the risk assessment of NSCLC.Higher order CT extracted features associated with specific TIME profiles may reveal a radio-immune signature with prognostic impact on resected NSCLC.

Introduction: ALK tyrosine kinase inhibitors (TKIs) are the standard treatment for advanced ALK-positive NSCLC. Nevertheless, drug resistance inevitably occurs. Here, we report a case of a patient with metastatic ALK-positive lung adenocarcinoma with an impressive resistance to sequential treatment with ALK TKIs mediated by YES1 and MYC amplification in a contest of epithelial-to-mesenchymal transition and high progressive chromosomal instability. Methods: The patient received, after chemotherapy and 7 months of crizotinib, brigatinib and lorlatinib with no clinical benefit to both treatments. A study of resistance mechanisms was performed with whole exome sequencing on different biological samples; primary cell lines were established from pleural effusion after lorlatinib progression. Results: At whole exome sequencing analysis, YES1 and MYC amplifications were observed both in the pericardial biopsy and the pleural effusion samples collected at brigatinib and lorlatinib progression, respectively. Increasing chromosomal instability from diagnostic biopsy to pleural effusion was also observed. The addition of dasatinib to brigatinib or lorlatinib restored the sensitivity in primary cell lines; data were confirmed also in H3122_ALK-positive model overexpressing both YES1 and MYC. Conclusions: In conclusion, YES1 and MYC amplifications are candidates to justify a rapid acquired resistance to crizotinib entailing primary brigatinib and lorlatinib resistance. In this context, a combination strategy of ALK TKI with dasatinib could be effective to overcome a rapid resistance.

Background The third generation Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor (TKI) osimertinib has been initially approved for T790M positive Non-Small Cell Lung Cancer (NSCLC) and more recently for first-line treatment of EGFR-mutant T790M negative NSCLC patients. Similarly to previous generation TKIs, despite the high response rate, disease progression eventually occurs and current clinical research is focused on novel strategies to delay the emergence of osimertinib resistance. In this study we investigated the combination of osimertinib with pemetrexed or cisplatin in EGFR-mutated NSCLC cell lines and xenografts. Methods Tumor growth was evaluated in a PC9T790M xenograft model and tissue composition was morphometrically determined. PC9, PC9T790M and HCC827 cell lines were employed to test the efficacy of osimertinib and chemotherapy combination in vitro. Cell viability and cell death were evaluated by MTT assay and fluorescence microscopy. Protein expression and gene status were analysed by Western blotting, fluorescence in situ hybridization analysis, next-generation sequencing and digital droplet PCR. Results In xenograft models, osimertinib significantly inhibited tumor growth, however, as expected, in 50% of mice drug-resistance developed. A combination of osimertinib with pemetrexed or cisplatin prevented or at least delayed the onset of resistance. Interestingly, such combinations increased the fraction of fibrotic tissue and exerted a long-lasting activity after stopping therapy. In vitro studies demonstrated the stronger efficacy of the combination over the single treatments in inhibiting cell proliferation and inducing cell death in PC9T790M cells as well as in T790M negative PC9 and HCC827 cell lines, suggesting the potential role of this strategy also as first-line treatment. Finally, we demonstrated that osimertinib resistant clones, either derived from resistant tumors or generated in vitro, were less sensitive to pemetrexed prompting to use a chemotherapy regimen non-containing pemetrexed in patients after progression to osimertinib treatment. Conclusions Our results identify a combination between osimertinib and pemetrexed or cisplatin potentially useful in the treatment of EGFR-mutated NSCLC patients, which might delay the appearance of osimertinib resistance with long-lasting effects. Electronic supplementary material The online version of this article (10.1186/s13046-019-1240-x) contains supplementary material, which is available to authorized users.

Background: Malignant Pleural Mesothelioma (MPM) is an aggressive tumor that has a significant incidence related to asbestos exposure with no effective therapy and poor prognosis. The role of mesenchymal stromal cells (MSCs) in cancer is controversial due to their opposite effects on tumor growth and in particular, only a few data are reported on MSCs and MPM. Methods: We investigated the in vitro efficacy of adipose tissue-derived MSCs, their lysates and secretome against different MPM cell lines. After large-scale production of MSCs in a bioreactor, their efficacy was also evaluated on a human MPM xenograft in mice. Results: MSCs, their lysate and secretome inhibited MPM cell proliferation in vitro with S or G0/G1 arrest of the cell cycle, respectively. MSC lysate induced cell death by apoptosis. The efficacy of MSC was confirmed in vivo by a significant inhibition of tumor growth, similar to that produced by systemic administration of paclitaxel. Interestingly, no tumor progression was observed after the last MSC treatment, while tumors started to grow again after stopping chemotherapeutic treatment. Conclusions: These data demonstrated for the first time that MSCs, both through paracrine and cell-to-cell interaction mechanisms, induced a significant inhibition of human mesothelioma growth. Since the prognosis for MPM patients is poor and the options of care are limited to chemotherapy, MSCs could provide a potential new therapeutic approach for this malignancy.

Radiomics has emerged as a noninvasive tool endowed with the potential to intercept tumor characteristics thereby predicting clinical outcome. In a recent study on resected non-small cell lung cancer (NSCLC), we identified highly prognostic computed tomography (CT) -derived radiomic features (RFs), which in turn were able to discriminate hot from cold tumor immune microenvironment (TIME). We aimed at validating a radiomic model capable of dissecting specific TIME profiles bearing prognostic power in resected NSCLC. The validation cohort included 31 radically resected NSCLCs clinicopathologically matched with the training set (n = 69). TIME was classified in hot and cold according to a multiparametric immunohistochemical analysis involving PD-L1 score and incidence of immune effector phenotypes among tumor infiltrating lymphocytes (TILs). High- throughput radiomic features (n = 841) extracted from CT images were correlated to TIME parameters to ultimately define prognostic classes. We confirmed PD-1 to CD8 ratio as best predictor of clinical outcome among TIME characteristics. Significantly prolonged overall survival (OS) was observed in patients carrying hot (median OS not reached) vs cold (median OS 22 months; hazard ratio 0.28, 95% confidence interval 0.09 -0.82; p = 0.015) immune background, thus validating the prognostic impact of these two TIME categories in resected NSCLC. Importantly, in the validation setting, three out of eight previously identified RFs sharply distinguishing hot from cold TIME were endorsed. Among signature-related RFs, Wavelet-HHH_gldm_HighGrayLevelEmphasis highly performed as descriptor of hot immune contexture (area under the receiver operating characteristic curve 0.94, 95% confidence interval 0.81 -1.00; p = 0.01). Based on our findings, Radiomics may decipher specific TIME profiles providing a noninvasive prognostic approach in resected NSCLC and an exploitable predictive strategy in advanced cases.

Background: Evaluation of fibrosis and its severity is the commonest parameter to screen potential new medicines in rodents treated with bleomycin (BLM). Results are mainly reported by histological analysis and indirect measurement of collagen through hydroxyproline (HYP) quantification. Objectives: In the present study we performed a comparative analysis of different methods to assess collagens induced by BLM in the mouse lung. Methods: BLM (0.02 U/kg) was administered by oropharyngeal aspiration to male C57BL/6 mice on day 0 and day 4. Oral Nintedanib (60 mg/kg/day) started from day 7. Three and four weeks after the first BLM administration, histopathological examination and collagen genes expression analysis were applied. Moreover, HYP and collagen isoforms (LC-MS) quantification in lung homogenates were assessed. Results: Ashcroft score and automated measure of lung fibrosis revealed significant fibrotic lesions in BLM-treated animals. Specific collagen genes appeared up-regulated in BLM group. LC-MS quantification of collagen isoforms correlated with HYP levels although, interestingly, showed a higher therapeutic window between control and BLM group (1.5 vs. 0.5 fold, respectively). Nintedanib significantly affected all the evaluated readouts. Conclusions: Our study provides a comprehensive analysis of changes in collagen genes and proteins following BLM treatment in mice and the effect of Nintedanib. Importantly, our data indicate for the first time LC-MS as a sensitive and reliable method for collagen quantification in lung homogenate. This approach might implement the most employed HYP assay to achieve reliable predictions of efficacy for therapeutics in lung fibrosis.

Heart failure (HF) remains one of the leading causes of death worldwide; most commonly developing after myocardial infarction (MI). Since adult cardiomyocytes characteristically do not proliferate, cells lost during MI are not replaced. As a result, the heart has a limited regenerative capacity. There is, therefore, a need to develop novel cell-based therapies to promote the regeneration of the heart after MI. The delivery and retention of cells at the injury site remains a significant challenge. In this context, we explored the potential of using an injectable, RGDSP-functionalised self-assembling peptide - FEFEFKFK - hydrogel as scaffold for the delivery and retention of rat cardiac progenitor cells (CPCs) into the heart. Our results show that culturing CPCs in vitro within the hydrogel for one-week promoted their spontaneous differentiation towards adult cardiac phenotypes. Injection of the hydrogel on its own, or loaded with CPCs, into the rat after injury resulted in a significant reduction in myocardial damage and left ventricular dilation.

The immune regulation of cancer growth and regression has been underscored by the recent success of immunotherapy. The possibility that immune microenvironmental factors may impact on clinical outcome and treatment response still requires intense investigations. Hereby, supporting data of the research article “Integrated CT Imaging and Tissue Immune Features Disclose a Radio-Immune Signature with High Prognostic Impact on Surgically Resected NSCLC” [1], are presented. With the ultimate aim to provide non-invasive prognostic scores, we report on our approach to correlate different Tumor Immune Microenvironment (TIME) profiles with CT imaging-derived qualitative (semantic, CT-SFs) and quantitative (radiomic, CT-RFs) features in a cohort of 60 surgically resected NSCLC. The renowned characterization of TIME, essentially based on the score evaluation of Programme Death Ligand-1 (PD-L1) and Tumor Infiltrating Lymphocytes (TILs), was implemented here by the assessment of effector and suppressor phenotypes including the analysis of Programme Death receptor 1 (PD-1). Thus, we defined two main TIME categories: hot inflamed (PD-L1high, CD8/CD3high and PD-1/CD8low) as opposed to cold inactive (PD-L1low, CD8/CD3lowand PD-1/CD8high). Importantly, as reported in the extended publication [1], these distinctive immune contextures identified different prognostic classes and were decoded by radiomics. To corroborate our radiomic approach, a comparative estimation of CT-RFs extracted from 60 NSCLC and 13 non neoplastic tissues was undertaken, documenting high discrimination ability. Moreover, we tested the potential association of qualitative radiologic features with clinico-pathological and TIME parameters. Taken together, our findings suggest that CT-SFs and CT-RFs may underlay specific patterns of lung cancer.

The use of injectable scaffolds to repair the infarcted heart is receiving great interest. Thermosensitive polymers, in situ polymerization, in situ cross-linking, and self-assembling peptides are the most investigated approaches to obtain injectability.Aim of the present work was the preparation and characterization of a novel bioactive scaffold, in form of injectable microspheres, for cardiac repair. Gellan/gelatin microspheres were prepared by a water-in-oil emulsion and loaded by adsorption with Insulin-like growth factor 1 to promote tissue regeneration. Obtained microspheres underwent morphological, physicochemical and biological characterization, including cell culture tests in static and dynamic conditions and in vivo tests. Morphological analysis of the microspheres showed a spherical shape, a microporous surface and an average diameter of 66 ± 17µm (under dry conditions) and 123 ± 24 µm (under wet conditions). Chemical Imaging analysis pointed out a homogeneous distribution of gellan, gelatin and Insulin-like growth factor-1 within the microsphere matrix. In vitro cell culture tests showed that the microspheres promoted rat cardiac progenitor cells adhesion, and cluster formation. After dynamic suspension culture within an impeller-free bioreactor, cells still adhered to microspheres, spreading their cytoplasm over microsphere surface. Intramyocardial administration of microspheres in a cryoinjury rat model attenuated chamber dilatation, myocardial damage and fibrosis and improved cell homing.Overall, the findings of this study confirm that the produced microspheres display morphological, physicochemical, functional and biological properties potentially adequate for future applications as injectable scaffold for cardiac tissue engineering.

In recent years, there has been an increasing interest toward the covalent binding of bioactive peptides from extracellular matrix proteins on scaffolds as a promising functionalization strategy in the development of biomimetic matrices for tissue engineering. A totally new approach for scaffold functionalization with peptides is based on Molecular Imprinting technology. In this work, imprinted particles with recognition properties toward laminin and fibronectin bioactive moieties were synthetized and used for the functionalization of biomimetic sponges, which were based on a blend of alginate, gelatin, and elastin. Functionalized sponges underwent a complete morphological, physicochemical, mechanical, functional, and biological characterization. Micrographs of functionalized sponges showed a highly porous structure and a quite homogeneous distribution of imprinted particles on their surface. Infrared and thermal analyses pointed out the presence of interactions between blend components. Biodegradation and mechanical properties appeared adequate for the aimed application. The results of recognition tests showed that the deposition on sponges did not alter the specific recognition and binding behavior of imprinted particles. In vitro biological characterization with cardiac progenitor cells showed that early cell adherence was promoted. In vivo analysis showed that developed scaffolds improved cardiac progenitor cell adhesion and differentiation toward myocardial phenotypes.

Adequate mechanical properties to withstand the surgical procedure and decoration with bioactive molecules promoting tissue regeneration are crucial aspects in the development of successful matrice...

Introduction: Injectable scaffolds are emerging as a promising strategy in the field of myocardial tissue engineering. Among injectable scaffolds, microparticles have been poorly investigated. The goal of this study was the development of novel gelatin/gellan microparticles that could be used as an injectable scaffold to repair the infarcted myocardium. In particular, the effect of particle size on cardiac progenitor cell response was investigated. Methods: Particles were produced by a water-in-oil emulsion method. Phosphatidylcholine was used as a surfactant. Particles with different diameter ranges (125–300 µm and 350–450 µm) were fabricated using two different surfactant concentrations. Morphological, physicochemical, and functional characterizations were carried out. Cardiac progenitor cell adhesion and growth on microparticles were tested both in static and dynamic suspension culture conditions. Results: Morphological analysis of the produced particles showed a spherical shape and porous surface. The hydrophilicity of particle matrix and the presence of intermolecular interactions between gellan and gelatin were pointed out by the physicochemical characterization. A weight loss of 75 ± 5 % after 90 days of hydrolytic degradation was observed. Injectability through a narrow needle (26 G) and persistence of the microparticles at the injection site were preliminarily verified by ex vivo test. In vitro cell culture tests showed a preservation of rat cardiac progenitor biologic properties and indicated a preferential cell adherence to microparticles with a smaller size. Conclusion: Overall, the obtained results indicate that the produced gelatin/gellan microparticles could be potentially employed as injectable scaffolds for myocardial regeneration.

Malignant pleural mesothelioma (MPM) is a progressive malignancy associated to the exposure of asbestos fibers. The most frequently inactivated tumor suppressor gene in MPM is CDKN2A/ARF , encoding for the cell cycle inhibitors p16 INK4a and p14 ARF , deleted in about 70% of MPM cases. Considering the high frequency of alterations of this gene, we tested in MPM cells the efficacy of palbociclib (PD-0332991), a highly selective inhibitor of cyclin-dependent kinase (CDK) 4/6. The analyses were performed on a panel of MPM cell lines and on two primary culture cells from pleural effusion of patients with MPM. All the MPM cell lines, as well as the primary cultures, were sensitive to palbociclib with a significant blockade in G0/G1 phase of the cell cycle and with the acquisition of a senescent phenotype. Palbociclib reduced the phosphorylation levels of CDK6 and Rb, the expression of myc with a concomitant increased phosphorylation of AKT. Based on these results, we tested the efficacy of the combination of palbociclib with the PI3K inhibitors NVP-BEZ235 or NVP-BYL719. After palbociclib treatment, the sequential association with PI3K inhibitors synergistically hampered cell proliferation and strongly increased the percentage of senescent cells. In addition, AKT activation was repressed while p53 and p21 were up-regulated. Interestingly, two cycles of sequential drug administration produced irreversible growth arrest and senescent phenotype that were maintained even after drug withdrawal. These findings suggest that the sequential association of palbociclib with PI3K inhibitors may represent a valuable therapeutic option for the treatment of MPM.

OBJECTIVES Tumour-infiltrating lymphocytes (TILs) are critically implicated in the clinical outcome and response to immunotherapy in non-small-cell lung cancer (NSCLC) patients. The functional competence of lymphocyte subpopulations is strongly conditioned by their spatial arrangement within the tumour immune microenvironment. The aim of this study was to determine whether the tissue localization of specific TIL subpopulations might have an impact on the risk of recurrence in surgically resected NSCLC. METHODS High-speed scanning of whole slide images was performed on immunohistochemically stained tissue sections from 97 NSCLC patients to assess the number and ratio of CD3+, CD8+ and PD-1+ T-lymphocytes. TIL distribution was computed considering the intratumoural (proximal or distal) and peripheral (invasive margin) localization as well as their location within the fibrotic tissue (immune excluded). The tumour proliferative index was assessed by Ki67 labelling. The impact of TILs number and distribution on clinical-pathological characteristics and outcomes were statistically analysed. RESULTS High density and percentage of proximal CD8+ TILs and low PD-1-to-CD8 ratio had a positive impact on disease-free-survival (P = 0.03) and overall survival (P = 0.003). An inverse correlation was observed between the abundance of intratumoural CD8+ TILs carrying PD-1 inhibitory receptor and cancer cell proliferation. Cases with high compared to low fraction of immune excluded CD8+ TILs had significantly reduced 5-year overall survival (n events: 22 vs 12; P = 0.04) and disease-free survival (n events: 24 vs 16; P = 0.03) rates while the amount of CD3+ and CD8+ TILs located at the invasive margin had a favourable effect on the clinical course. CONCLUSIONS Mapping TIL subpopulations may implement the definition of prognostic parameters in surgically resected NSCLC.

Introduction: A prospective investigation of the circulating immune profile in NSCLC patients receiving nivolumab was performed to identify potentially predictive parameters. Methods: Flow Cytometry of peripheral blood (PB) CD3+, CD8+, CD4+, NK, Treg and MDSCs was prospectively performed in 31 consecutive advanced NSCLC patients at baseline (T0) and after 2 (T1) and 4 (T2) cycles of bi-weekly nivolumab. Functional molecules (PD-1, CD3ζ Granzyme B, Perforin), cell proliferation (Ki67) and NK receptors (NKG2 A, NKG2D, NKp30) were also explored. The immunohistochemical evaluation of PD-L1 and TILs was restricted to available tumor biopsies. Tissue and circulating parameters were correlated to clinico-pathological features and treatment outcomes. Results: KRAS mutations, active smoking, COPD and steroid treatment conditioned a different distribution of circulating phenotypes. At baseline, clinical benefit (CB, n = 19) group displayed higher number of phenotypically active NK and PD-1+CD8+ cells (p < 0.01) compared to non-responders (NR, n = 12). Prolonged survival outcomes (p < 0.01) were recorded in cases with high baseline circulating NK and PD-1+CD8+ cells. At tissue level, low PD-1 expression in CD8 + TILs was a positive prognostic feature (p < 0.001). Strikingly, high circulating NK and PD-1+CD8+ cells combined with low PD-1/CD8+ ratio in TILs characterized a privileged context able to provide a significantly prolonged (p < 0.01) progression-free survival (PFS). During PD-1 blockade, NKs progressively raised in CB while declined in NR (p < 0.05) and this phenomenon was counterbalanced by parallel changes in Treg. Conclusion: The functional pool of circulating NKs associated with a divergent PD-1 expression in blood and tissue CD8+ lymphocytes portrays an immune profile predictive of anti-PD1 treatment efficacy.

OBJECTIVES: Lymphangiogenesis plays a critical role in the immune response, tumour progression and therapy effectiveness. The aim of this study was to determine whether the interplay between the lymphatic and the blood microvasculature, tumour-infiltrating lymphocytes and the programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) immune checkpoint constitutes an immune microenvironment affecting the clinical outcome of patients with non-small-cell lung cancer. METHODS: Samples from 50 squamous cell carcinomas and 42 adenocarcinomas were subjected to immunofluorescence to detect blood and lymphatic vessels. CD3pos, CD8pos and PD-1pos tumour-infiltrating lymphocytes and tumour PD-L1 expression were assessed by immunohistochemical analysis. RESULTS: Quantification of vascular structures documented a peak of lymphatics at the invasive margin together with a decreasing gradient of blood and lymphatic vessels from the peritumour area throughout the neoplastic core. Nodal involvement and pathological stage were strongly associated with vascularization, and an increased density of vessels was detected in samples with a higher incidence of tumour-infiltrating lymphocytes and a lower expression of PD-L1. Patients with a high PD-L1 to PD-1 ratio and vascular rarefaction had a gain of 10 months in overall survival compared to those with a low ratio and prominent vascularity. CONCLUSIONS: Microvessels are an essential component of the cancer immune microenvironment. The clinical impact of the PD-1/PD-L1-based immune contexture may be implemented by the assessment of microvascular density to potentially identify patients with non-small-cell lung cancer who could benefit from immunotherapy and antiangiogenic treatment.

Cardiovascular complications are included among the systemic effects of tyrosine kinase inhibitor (TKI)-based therapeutic strategies. To test the hypothesis that inhibition of Kit tyrosine kinase that promotes cardiac progenitor cell (CPC) survival and function may be one of the triggering mechanisms of imatinib mesylate (IM)-related cardiovascular effects, the anatomical, structural and ultrastructural changes in the heart of IM-treated rats were evaluated. Cardiac anatomy in IM-exposed rats showed a dose-dependent, restrictive type of remodeling and depressed hemodynamic performance in the absence of remarkable myocardial fibrosis. The effects of IM on rat and human CPCs were also assessed. IM induced rat CPC depletion, reduced growth and increased cell death. Similar effects were observed in CPCs isolated from human hearts. These results extend the notion that cardiovascular side effects are driven by multiple actions of IM. The identification of cellular mechanisms responsible for cardiovascular complications due to TKIs will enable future strategies aimed at preserving concomitantly cardiac integrity and anti-tumor activity of advanced cancer treatment.

Purpose: The success of immune checkpoint inhibitors strengthens the notion that tumor growth and regression are immune regulated. To determine whether distinct tissue immune microenvironments differentially affect clinical outcome in non–small cell lung cancer (NSCLC), an extended analysis of PD-L1 and tumor-infiltrating lymphocytes (TIL) was performed. Experimental Design: Samples from resected adenocarcinoma (ADC 42), squamous cell carcinoma (SCC 58), and 26 advanced diseases (13 ADC and 13 SCC) treated with nivolumab were analyzed. PD-L1 expression and the incidence of CD3, CD8, CD4, PD-1, CD57, FOXP3, CD25, and Granzyme B TILs were immunohistochemically assessed. Results: PD-L1 levels inversely correlated with N involvement, although they did not show a statistically significant prognostic value in resected patients. The incidence and phenotype of TILs differed in SCC versus ADC, in which EGFR and KRAS mutations conditioned a different frequency and tissue localization of lymphocytes. NSCLC resected patients with high CD8pos lymphocytes lacking PD-1 inhibitory receptor had a longer overall survival (OS: HR = 2.268; 95% CI, 1.056–4.871, P = 0.03). PD-1-to-CD8 ratio resulted in a prognostic factor both on univariate (HR = 1.952; 95% CI, 1.34–3.12, P = 0.001) and multivariate (HR = 1.943; 95% CI, 1.38–2.86, P = 0.009) analysis. Moreover, low PD-1 incidence among CD8pos cells was a distinctive feature of nivolumab-treated patients, showing clinical benefit with a prolonged progression-free survival (PFS: HR = 4.51; 95% CI, 1.45–13.94, P = 0.004). Conclusions: In the presence of intrinsic variability in PD-L1 expression, the reservoir of PD-1–negative effector T lymphocytes provides an immune-privileged microenvironment with a positive impact on survival of patients with resected disease and response to immunotherapy in advanced NSCLC. Clin Cancer Res; 24(2); 407–19. ©2017 AACR.

Squamous cell lung carcinoma (SCC) accounts for 30% of patients with NSCLC and to date, no molecular targeted agents are approved for this type of tumor. However, recent studies have revealed several oncogenic mutations in SCC patients, including an alteration of the PI3K/AKT pathway, i.e. PI3K point mutations and amplification, AKT mutations and loss or reduced PTEN expression. Prompted by our observation of a correlation between PTEN loss and FAK phosphorylation in a cohort of patients with stage IV SCC, we evaluated the relevance of PTEN loss in cancer progression as well as the efficacy of a new combined treatment with the pan PI3K inhibitor buparlisip and the FAK inhibitor defactinib. An increase in AKT and FAK phosphorylation, associated with increased proliferation and invasiveness, paralleled by the acquisition of mesenchymal markers, and overexpression of the oncomir miR-21 were observed in SKMES-1-derived cell clones with a stable reduction of PTEN. Notably, the combined treatment induced a synergistic inhibition of cell proliferation, and a significant reduction in cell migration and invasion only in cells with reduced PTEN. The molecular mechanisms underlying these findings were unraveled using a specific RTK array that showed a reduction in phosphorylation of key kinases such as JNK, GSK-3 α/β, and AMPK-α2, due to the concomitant decrease in AKT and FAK activation. In conclusion, the combination of buparlisib and defactinib was effective against cells with reduced PTEN and warrants further studies as a novel therapeutic strategy for stage IV SCC patients with loss of PTEN expression.

// Claudia Fumarola 1 , Daniele Cretella 1 , Silvia La Monica 1 , Mara A. Bonelli 1 , Roberta Alfieri 1 , Cristina Caffarra 1 , Federico Quaini 1 , Denise Madeddu 1 , Angela Falco 1 , Andrea Cavazzoni 1 , Graziana Digiacomo 1 , Giulia Mazzaschi 1 , Valentina Vivo 2 , Elisabetta Barocelli 2 , Marcello Tiseo 3 , Pier Giorgio Petronini 1, * and Andrea Ardizzoni 4, * 1 Department of Medicine and Surgery, University of Parma, Parma, Italy 2 Food and Drug Department, University of Parma, Parma, Italy 3 Medical Oncology Unit, University Hospital of Parma, Parma, Italy 4 Division of Medical Oncology, Sant’Orsola-Malpighi University Hospital, Bologna, Italy * Joint last authors Correspondence to: Claudia Fumarola, email: claudia.fumarola@unipr.it Mara A. Bonelli, email: mara.bonelli@unipr.it Keywords: FGFR1, glucose metabolism, dovitinib, NVP-BGJ398, SQCLC Received: March 03, 2017 Accepted: June 18, 2017 Published: July 17, 2017 ABSTRACT Fibroblast Growth Factor Receptor (FGFR) signaling is a complex pathway which controls several processes, including cell proliferation, survival, migration, and metabolism. FGFR1 signaling is frequently deregulated via amplification/over-expression in NSCLC of squamous histotype (SQCLC), however its inhibition has not been successfully translated in clinical setting. We determined whether targeting downstream signaling implicated in FGFR1 effects on glucose metabolism potentiates the anti-tumor activity of FGFR1 inhibition in SQCLC. In FGFR1 amplified/over-expressing SQCLC cell lines, FGF2-mediated stimulation of FGFR1 under serum-deprivation activated both MAPK and AKT/mTOR pathways and increased glucose uptake, glycolysis, and lactate production, through AKT/mTOR-dependent HIF-1α accumulation and up-regulation of GLUT-1 glucose transporter. These effects were hindered by PD173074 and NVP-BGJ398, selective FGFR inhibitors, as well as by dovitinib, a multi-kinase inhibitor. Glucose metabolism was hampered by the FGFR inhibitors also under hypoxic conditions, with consequent inhibition of cell proliferation and viability. In presence of serum, glucose metabolism was impaired only in cell models in which FGFR1 inhibition was associated with AKT/mTOR down-regulation. When the activation of the AKT/mTOR pathway persisted despite FGFR1 down-regulation, the efficacy of NVP-BGJ398 could be significantly improved by the combination with NVP-BEZ235 or other inhibitors of this signaling cascade, both in vitro and in xenotransplanted nude mice. Collectively our results indicate that inhibition of FGFR1 signaling impacts on cancer cell growth also by affecting glucose energy metabolism. In addition, this study strongly suggests that the therapeutic efficacy of FGFR1 targeting molecules in SQCLC may be implemented by combined treatments tackling on glucose metabolism.

Background: Notch pathway is involved in regulating colon cancer stem cells (CSCs), which play an important role in angiogenesis and resistance to conventional therapies. CD44 and CD133 are transmembrane glycoproteins reported as putative markers for isolating CSCs. High expression of Notch Intracellular Cleaved Domain (NICD) has been associated with resistance to anti-vascular endothelial growth factor therapy. Based on these reports, we evaluated NICD, CD44 and CD133 expression in a series of consecutive metastatic colon cancer patients treated with bevacizumab-based chemotherapy within first-line clinical trials. Methods: Histological samples obtained from 45 primary adenocarcinomas were tested by immunohistochemistry for NICD, CD44 and CD133. A scoring system based on staining intensity and percentage of stained cells was used. Vessels density was measured in hot-spot areas by CD31 antibody. Results: CD44 levels were higher in high NICD cases than in low NICD cases (63% vs. 16%, respectively, p=0.014). High NICD and CD44 levels predicted shorter progression-free (p

OBJECTIVES: The aim of the present study was to characterize the biological properties and in vivo tumourigenic potential of mesenchymal cells (MCs) obtained from non-small-cell lung cancer (NSCLC) samples. METHODS: NSCLC samples (53 adenocarcinomas and 24 squamous-cell carcinomas) surgically removed from 46 males and 31 females were processed to identify mesenchymal cells from human lung cancer (hLc-MCs). hLc-MCs were separated from neoplastic epithelial cells, expanded and extensively characterized in vitro. Subsequently, female BALB/c nude mice were subcutaneously injected with either 10 6 or 2.5 × 10 6 Calu-3 (human adenocarcinoma cell line able to reproducibly induce xenografted tumours) alone or in combination with equal doses of hLc-MCs. Control animals were injected with the two doses of hLc-MCs only. RESULTS: Primary cultures of hLc-MCs were obtained from >80% of NSCLC specimens. The typical MCs immunophenotype was documented by the expression of CD90, CD105, CD73, CD13 and CD44 at fluorescence-activated cell sorting analysis. CD45, CD14, CD34 and epithelial antigens were negative while CD117 (c-kit) and CD133 (prominin) were partially expressed. Interestingly, nuclear transcription factors octamer-binding transcription factor 3/4 and sex determining region Y-box 2 involved in stemness, thyroid transcription factor 1 in bronchoalveolar commitment, and ETS1 in carcinogenesis, were expressed in hLc-MCs isolated from NSCLC. Specific conditioned media and cocultures confirmed the supportive role of hLc-MCs for cancer cells. In vivo experiments showed that at both doses Calu-3 xenografts doubled in size when hLc-MCs were coinjected. Cell tracking in xenografted tumours, by immunofluorescence combined with fluorescence in situ hybridization analysis, documented hX-chromosome-labelled, Calu-3-derived cytokeratin-positive adenocarcinoma structures surrounded by hLc-MCs. CONCLUSIONS: Tumour-propagating cells require the inductive interaction of resident mesenchymal cells to foster lung cancer development.

In humans, there is a documented association between anxiety disorders and cardiovascular disease. Putative underlying mechanisms may include an impairment of the autonomic nervous system control of cardiac function. The primary objective of the present study was to characterize cardiac autonomic modulation and susceptibility to arrhythmias in genetic lines of rats that differ largely in their anxiety level. To reach this goal, electrocardiographic recordings were performed in high-anxiety behavior (HAB, n=10) and low-anxiety behavior (LAB, n=10) rats at rest, during stressful stimuli and under autonomic pharmacological manipulations, and analyzed by means of time- and frequency-domain indexes of heart rate variability. During resting conditions, HAB rats displayed a reduced heart rate variability, mostly in terms of lower parasympathetic (vagal) modulation compared to LAB rats. In HAB rats, this relatively low cardiac vagal control was associated with smaller heart rate responsiveness to acute stressors compared to LAB counterparts. In addition, beta-adrenergic pharmacological stimulation induced a larger incidence of ventricular tachyarrhythmias in HABs compared to LABs. At sacrifice, a moderate increase in heart-body weight ratio was observed in HAB rats. We conclude that high levels of anxiety-related behavior in rats are associated with signs of i) impaired autonomic modulation of heart rate (low vagally-mediated heart rate variability), ii) poor adaptive heart rate responsiveness to stressful stimuli, iii) increased arrhythmia susceptibility, and iv) cardiac hypertrophy. These results highlight the utility of the HAB/LAB model for investigating the mechanistic basis of the comorbidity between anxiety disorders and cardiovascular disease.