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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

Additional file 2: Supplementary Fig. S2. TEM analysis of left ventricular tissue from control rat. Panel a: low magnification image of a cardiomyocyte (N, nucleus) filled of mitochondria (Mit) and myofibrils (MF). In the interstitial space an endothelial cell (Ec) is lining a capillary lumen (RB, red blood cells). Panel b: image showing mitochondria (Mit) and myofribrils (MF) in a CTRL rat myocardium (N, myocyte nucleus) at higher magnification. Scale Bars: a: 5 μm; b: 2 μm.

Background Nanotoxicology is an increasingly relevant field and sound paradigms on how inhaled nanoparticles (NPs) interact with organs at the cellular level, causing harmful conditions, have yet to be established. This is particularly true in the case of the cardiovascular system, where experimental and clinical evidence shows morphological and functional damage associated with NP exposure. Giving the increasing interest on cobalt oxide (Co3O4) NPs applications in industrial and bio-medical fields, a detailed knowledge of the involved toxicological effects is required, in view of assessing health risk for subjects/workers daily exposed to nanomaterials. Specifically, it is of interest to evaluate whether NPs enter cardiac cells and interact with cell function. We addressed this issue by investigating the effect of acute exposure to Co3O4-NPs on excitation-contraction coupling in freshly isolated rat ventricular myocytes. Results Patch clamp analysis showed instability of resting membrane potential, decrease in membrane electrical capacitance, and dose-dependent decrease in action potential duration in cardiomyocytes acutely exposed to Co3O4-NPs. Motion detection and intracellular calcium fluorescence highlighted a parallel impairment of cell contractility in comparison with controls. Specifically, NP-treated cardiomyocytes exhibited a dose-dependent decrease in the fraction of shortening and in the maximal rate of shortening and re-lengthening, as well as a less efficient cytosolic calcium clearing and an increased tendency to develop spontaneous twitches. In addition, treatment with Co3O4-NPs strongly increased ROS accumulation and induced nuclear DNA damage in a dose dependent manner. Finally, transmission electron microscopy analysis demonstrated that acute exposure did lead to cellular internalization of NPs. Conclusions Taken together, our observations indicate that Co3O4-NPs alter cardiomyocyte electromechanical efficiency and intracellular calcium handling, and induce ROS production resulting in oxidative stress that can be related to DNA damage and adverse effects on cardiomyocyte functionality.

Additional file of Cobalt oxide nanoparticles induce oxidative stress and alter electromechanical function in rat ventricular myocytes

Additional file 1: Supplementary Fig. S1. TEM analysis of left ventricular tissue from cobalt NP inhaled rat. Panel a: low magnification image of a cardiomyocyte (N, nucleus) filled of mitochondria (Mit) and myofibrils (MF). On the left, collagen bundles (Col) are present in the interstitial space. Aggregates of Co3O4-NPs (asterisk) within myofibrils and mitochondria are shown at higher magnification in (b). Panel c: low magnification image of LV myocardium showing the sarcolemma (arrow) lining the surface of a cardiomyocyte with abundant mitochondria (Mit) and myofibrils (MF). Collagen bundles (Col) are present in the interstitial space where an endothelial cell (Ec) is lining a capillary lumen (RB, red blood cells). The black rectangle inscribes an area shown at higher magnification in (d) in which the asterisks indicate NPs located in the mitochondria (Mit). Panel e: image showing a detail of mitochondria (Mit) and myofribrils (MF) in an NP-treated cardiomyocyte. The black rectangle inscribes an area shown at higher magnification in (f) in which NPs are located within the myofibrils (asterisk). Scale Bars: c: 5 μm; a,e: 2 μm; d, f: 1 μm, b: 500 nm

We recently showed that the long-term in vivo administration of green tea catechin extract (GTE) resulted in hyperdynamic cardiomyocyte contractility. The present study investigates the mechanisms underlying GTE action in comparison to its major component, epigallocatechin-3-gallate (EGCG), given at the equivalent amount that would be in the entirety of GTE. Twenty-six male Wistar rats were given 40 mL/day of a tap water solution with either standardized GTE or pure EGCG for 4 weeks. Cardiomyocytes were then isolated for the study. Cellular bioenergetics was found to be significantly improved in both GTE- and EGCG-fed rats compared to that in controls as shown by measuring the maximal mitochondrial respiration rate and the cellular ATP level. Notably, the improvement of mitochondrial function was associated with increased levels of oxidative phosphorylation complexes, whereas the cellular mitochondrial mass was unchanged. However, only the GTE supplement improved cardiomyocyte mechanics and intracellular calcium dynamics, by lowering the expression of total phospholamban (PLB), which led to an increase of both the phosphorylated-PLB/PLB and the sarco-endoplasmic reticulum calcium ATPase/PLB ratios. Our findings suggest that GTE might be a valuable adjuvant tool for counteracting the occurrence and/or the progression of cardiomyopathies in which mitochondrial dysfunction and alteration of intracellular calcium dynamics constitute early pathogenic factors.

The identification of SERCA (sarco/endoplasmic reticulum calcium ATPase) as a target for modulating gain-of-function NOTCH1 mutations in Notch-dependent cancers has spurred the development of this compound class for cancer therapeutics. Despite the innate toxicity challenge associated with SERCA inhibition, we identified CAD204520, a small molecule with better drug-like properties and reduced off-target Ca2+ toxicity compared with the SERCA inhibitor thapsigargin. In this work, we describe the properties and complex structure of CAD204520 and show that CAD204520 preferentially targets mutated over wild-type NOTCH1 proteins in T cell acute lymphoblastic leukemia (T-ALL) and mantle cell lymphoma (MCL). Uniquely among SERCA inhibitors, CAD204520 suppresses NOTCH1-mutated leukemic cells in a T-ALL xenografted model without causing cardiac toxicity. This study supports the development of SERCA inhibitors for Notch-dependent cancers and extends their application to cases with isolated mutations in the PEST degradation domain of NOTCH1, such as MCL or chronic lymphocytic leukemia (CLL). Clinical translation of SERCA inhibitors has been hampered by the risk of adverse cardiac events. In this work, Marchesini and Gherli et al. identified a tolerable oral available SERCA inhibitor, CAD204520, and showed that modulation of clinically relevant NOTCH1 mutations in T cell acute lymphoblastic leukemia and mantle cell lymphoma.

Background/Aims: Dietary polyphenols from green tea have been shown to possess cardio-protective activities in different experimental models of heart diseases and age-related ventricular dysfunction. The present study was aimed at evaluating whether long term in vivo administration of green tea extracts (GTE), can exert positive effects on the normal heart, with focus on the underlying mechanisms. Methods: The study population consisted of 20 male adult Wistar rats. Ten animals were given 40 mL/day tap water solution of GTE (concentration 0.3%) for 4 weeks (GTE group). The same volume of water was administered to the 10 remaining control rats (CTRL). Then, in vivo and ex vivo measurements of cardiac function were performed in the same animal, at the organ (hemodynamics) and cellular (cardiomyocyte mechanical properties and intracellular calcium dynamics) levels. On cardiomyocytes and myocardial tissue samples collected from the same in vivo studied animals, we evaluated: (1) the intracellular content of ATP, (2) the endogenous mitochondrial respiration, (3) the expression levels of the Sarcoplasmic Reticulum Ca2+-dependent ATPase 2a (SERCA2), the Phospholamban (PLB) and the phosphorylated form of PLB, the L-type Ca2+ channel, the Na+-Ca2+ exchanger, and the ryanodine receptor 2. Results: GTE cardiomyocytes exhibited a hyperdynamic contractility compared with CTRL (the rate of shortening and re-lengthening, the fraction of shortening, the amplitude of calcium transient, and the rate of cytosolic calcium removal were significantly increased). A faster isovolumic relaxation was also observed at the organ level. Consistent with functional data, we measured a significant increase in the intracellular ATP content supported by enhanced endogenous mitochondrial respiration in GTE cardiomyocytes, as well as higher values of the ratios phosphorylated-PLB/PLB and SERCA2/PLB. Conclusions: Long-term in vivo administration of GTE improves cell mechanical properties and intracellular calcium dynamics in normal cardiomyocytes, by increasing energy availability and removing the inhibitory effect of PLB on SERCA2.

In early diabetes, hyperglycemia and the associated metabolic dysregulation promote early changes in the functional properties of cardiomyocytes, progressively leading to the appearance of the diabetic cardiomyopathy phenotype. Recently, the interplay between histone acetyltransferases (HAT) and histone deacetylases (HDAC) has emerged as a crucial factor in the development of cardiac disorders. The present study evaluates whether HDAC inhibition can prevent the development of cardiomyocyte contractile dysfunction induced by a short period of hyperglycemia, with focus on the potential underlying mechanisms. Cell contractility and calcium dynamics were measured in unloaded ventricular myocytes isolated from the heart of control and diabetic rats. Cardiomyocytes were either untreated or exposed to the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) for 90 min. Then, a fraction of each group of cells was used to evaluate the expression levels of proteins involved in the excitation&ndash, contraction coupling, and the cardiomyocyte metabolic activity, ATP content, and reactive oxygen species levels. SAHA treatment was able to counteract the initial functional derangement in cardiomyocytes by reducing cell oxidative damage. These findings suggest that early HDAC inhibition could be a promising adjuvant approach for preventing diabetes-induced cardiomyocyte oxidative damage, which triggers the pro-inflammatory signal cascade, mitochondrial damage, and ventricular dysfunction.

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.

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.