Microenvironment In Non-small Cell Lung Cancer Research Articles

C5a induces A549 cell proliferation of non-small cell lung cancer via GDF15 gene activation mediated by GCN5-dependent KLF5 acetylation

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, and multiple evidence has confirmed that C5a production is elevated in NSCLC microenvironment. Although NSCLC cell proliferation induced by C5a has been reported, the involved mechanism has not been elucidated. In this study, we examined the proliferation-related genes (i.e., KLF5, GCN5, and GDF15) and C5a receptor (C5aR) expression in tumor tissues as well as C5a concentration in plasma of NSCLC patients, and then determined the roles of KLF5, GCN5, and GDF15 in C5a-triggered NSCLC cell proliferation and the related mechanism both in vitro and in vivo. Our results found that the expression of KLF5, GCN5, GDF15, C5aR, and C5a was significantly upregulated in NSCLC patients. Mechanistic exploration in vitro revealed that C5a could facilitate A549 cell proliferation through increasing KLF5, GCN5, and GDF15 expression. Besides, KLF5 and GCN5 could form a complex, binding to GDF15 promoter in a KLF5-dependent manner and leading to GDF15 gene transcription. More importantly, GCN5-mediated KLF5 acetylation contributing to GDF15 gene transcription and cell proliferation upon C5a stimulation, the region (−103 to +58 nt) of GDF15 promoter which KLF5 could bind to, and two new KLF5 lysine sites (K335 and K391) acetylated by GCN5 were identified for the first time. Furthermore, our experiment in vivo demonstrated that the growth of xenograft tumors in BALB/c nude mice was greatly suppressed by the silence of KLF5, GCN5, or GDF15. Collectively, these findings disclose that C5a-driven KLF5–GCN5–GDF15 axis had a critical role in NSCLC proliferation and might serve as targets for NSCLC therapy.

The effect of chemotherapy on the tumor immune microenvironment in non-small cell lung cancer (NSCLC): A single-institution retrospective analysis.

155 Background: Platinum-doublet chemotherapy (chemo) is routinely used for treatment of early stage and metastatic NSCLC. Immunotherapy is a proven treatment in these patients as well. The presence of tumor infiltrating lymphocytes (TILs) has been shown to be prognostic of this disease; however, the effect of chemo on TILs and the tumor immune microenvironment in NSCLC has not been well studied. We tested the hypothesis that chemo results in a decrease in the presence of TILs in NSCLC. Methods: We retrospectively identified patients treated with neo-adjuvant platinum-doublet chemo who underwent surgical resection from 2007-2015 at the University of Virginia. The specimens were sectioned and IHC stained for CD8, FoxP3, and PD-L1. Two independent investigators assessed a central and a peripheral high power field of tumor for number of CD8 and FoxP3 positive TILs. An independent pathologist assessed tumor cell PD-L1 expression. The nonparametric Wilcoxon Mann Whitney test was used to compare measurements of TILs in chemo-exposed tissue to a control cohort from a database of NSCLC surgical specimens that had not received pre-operative therapy. Results: Of the 17 patients who met eligibility criteria, 11 had sufficient tissue for evaluation. Results are shown in Table 1. There was a significant decrease in CD8 and FoxP3 TILs in both the center and periphery in patients exposed to chemo. There was no difference in PD-L1 expression. Conclusions: These data suggest that platinum-doublet chemo significantly decreases the presence of cytotoxic and regulatory subsets of TILs without effecting PD-L1 expression. This study is limited by the lack of pre-treatment tumor samples to compare pre- and post-treatment TILs in individual patients. Further studies are warranted to evaluate the impact of decreased TILs in the setting of concurrent or sequential immunotherapy treatment with chemo in NSCLC. [Table: see text]

Low PD-1 Expression in Cytotoxic CD8+ Tumor-Infiltrating Lymphocytes Confers an Immune-Privileged Tissue Microenvironment in NSCLC with a Prognostic and Predictive Value.

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.

Determination of poor prognostic immune features of tumour microenvironment in non-smoking patients with lung adenocarcinoma

We have previously demonstrated that the prognostic significance of tumour-infiltrating CD8+ T cells significantly differs according to histological type and patient smoking habits in non-small cell lung cancer (NSCLC). This work suggested that infiltrating CD8+ T cells may not be activated sufficiently in the immunosuppressive microenvironment in non-smokers with adenocarcinoma. To understand the immunogenic microenvironment in NSCLC, we characterised immune cells comprehensively by performing an immunohistochemical evaluation using an alternative counting method and multicolour staining method (n = 234), and assessed immune-related gene expression by using genetic analytical approaches (n = 58). We found that high infiltration of activated CD8+ T cells expressing interferon gamma (IFN-γ) and granzyme was correlated with postoperative survival in patients with non-adenocarcinoma. On the contrary, CD8+ T-cell accumulation was identified as a worse prognostic factor in patients with adenocarcinoma, particularly in non-smokers. Infiltrating CD8+ T cells were significantly less activated in this microenvironment with high expression of various immunoregulation genes. Potentially immunoregulatory CD8+ FOXP3+ T cells and immunodysfunctional CD8+ GATA3+ T cells were increased in adenocarcinoma of non-smokers. CD4+ FOXP3+ regulatory T cells expressing chemokine receptor-4 (CCR4)- and chemokine ligand (CCL17)-expressing CD163+ M2-like macrophages also accumulated correlatively and significantly in adenocarcinoma of non-smokers. These characteristic immune cells may promote tumour progression possibly by creating an immunosuppressive microenvironment in non-smoking patients with lung adenocarcinoma. Our findings may be helpful for refining the current strategy of personalised immunotherapy including immune-checkpoint blockade therapy for NSCLC.

Analysis of non-small cell lung cancer microenvironment indicates preponderance of T cell exhaustion marker expression

Lung cancer metastasis causes 70% of an estimated 1.4 million deaths per annum. The major shortcoming in lung cancer is the tendency to have inherent or develop acquired resistance to chemotherapy. It is now evolving that such resistance might develop due to differential contribution and interaction with tumor microenvironment, stromal cells, and the extracellular matrix. The objective of the current study was to define the lung cancer tumor microenvironment. We have identified multiple tumor-infiltrating T lymphocyte subsets in patients with lung cancer, which were independent of disease stage. Functional analysis indicated high expression of the inhibitory receptors, cytotoxic T-lymphocyte-associated protein 4 (CTLA4), lymphocyte activated gene 3 (LAG3) and programmed cell death protein 1 (PD-1) in both CD4 and CD8 subsets, compared to non-malignant controls. Inhibitory receptors expressed by the tumor infiltrating T cells might mediate tolerance to tumor antigens with co-expression of these receptors exacerbating lung carcinogenesis and metastatic progression.

Periostin and tumor-stroma interactions in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-associated mortality globally. Interactions of the cancer cells with the tumor microenvironment are essential carcinogenic features for the majority of solid tumors, such as pancreatic cancer. The present study investigated the role of stromal activation in NSCLC and analyzed the surgical specimens of 93 patients by immunohistochemistry with regard to periostin (an extracellular matrix protein), α-smooth muscle actin (α-SMA; a marker of myofibroblasts) and cluster of differentiation 31 (CD31; a marker of endothelial cells), and the activated stroma index. There was a trend towards reduced overall survival for patients with high periostin expression (hazard ratio, 1.80; 95% confidence interval, 0.99–3.27; P=0.050). No significant correlations with overall survival were identified for α-SMA (P=0.930), CD31 (P=0.923), collagen (P=0.441) or the activated stroma index (P=0.706). In a multivariable analysis, the histological tumor subtype, tumor stage, lymph node involvement and resection status were independent prognostic factors in NSCLC, but none of the investigated immunohistochemical markers were prognostic factors. Thus, the tumor microenvironment and stroma activation did not prove to be of prognostic relevance for lung cancer, as it has been previously described for pancreatic cancer. Other markers of the microenvironment of NSCLC may be of higher prognostic value, pointing towards tumor-type specific effects.

Long noncoding RNA HOTAIR, a hypoxia-inducible factor-1α activated driver of malignancy, enhances hypoxic cancer cell proliferation, migration, and invasion in non-small cell lung cancer.

Despite the fact that great advances have been made in the management of non-small cell lung cancer (NSCLC), the prognosis of advanced NSCLC remains very poor. HOX transcript antisense intergenic RNA (HOTAIR) has been identified as an oncogenic long noncoding RNA (lncRNA) that is involved in the progression of a variety of carcinomas and acts as a negative prognostic biomarker. Yet, little is known about the effect of HOTAIR in the hypoxic microenvironment of NSCLC. The expression and promoter activity of HOTAIR were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and luciferase reporter assay. The function of the hypoxia-inducible factor-1α (HIF-1α) binding site to hypoxia-responsive elements (HREs) in the HOTAIR promoter region was tested by luciferase reporter assay with nucleotide substitutions. The binding of HIF-1α to the HOTAIR promoter in vivo was confirmed by chromatin immunoprecipitation assay (CHIP) and electrophoretic mobility shift assay (EMSA). The effect of HIF-1α suppression by small interference RNA or YC-1 on HOTAIR expression was also determined. In the present study, we demonstrated that HOTAIR was upregulated by hypoxia in NSCLC cells. HOTAIR is a direct target of HIF-1α through interaction with putative HREs in the upstream region of HOTAIR in NSCLC cells. Furthermore, HIF-1α knockdown or inhibition could prevent HOTAIR upregulation under hypoxic conditions. Under hypoxic conditions, HOTAIR enhanced cancer cell proliferation, migration, and invasion. These data suggested that suppression of HOTAIR upon hypoxia of NSCLC could be a novel therapeutic strategy.

Reciprocal activation between IL-6/STAT3 and NOX4/Akt signalings promotes proliferation and survival of non-small cell lung cancer cells.

Inflammatory cytokines and oxidative stress are two critical mediators in inflammation-associated cancer. Interleukin-6 (IL-6) is one of the most critical tumor-promoting cytokines in non-small cell lung cancer (NSCLC). In our recent study, we confirmed that NADPH oxidase 4 (NOX4), an important source of reactive oxygen species (ROS) production in NSCLC cells, promotes malignant progression of NSCLC. However, whether the crosstalk of NOX4 and IL-6 signalings exists in NSCLC remains undentified. In this study, we show that NOX4 expression is positively correlated with IL-6 expression in NSCLC tissues. Exogenous IL-6 treatment significantly enhances NOX4/ROS/Akt signaling in NSCLC cells. NOX4 also enhances IL-6 production and activates IL-6/STAT3 signaling in NSCLC cells. Specifically, NOX4 is confirmed to functionally interplay with IL-6 to promote NSCLC cell proliferation and survival. The in vivo results were similar to those obtained in vitro. These data indicate a novel NOX4-dependent link among IL-6 in the NSCLC microenvironment, oxidative stress in NSCLC cells and autocrined IL-6 in NSCLC cells. NOX4/Akt and IL-6/STAT3 signalings can reciprocally and positively regulate each other, leading to enhanced NSCLC cell proliferation and survival. Therefore, NOX4 may serve as a promising target against NSCLC alone with IL-6 signaling.

The tumor immune microenvironment in octogenarians with stage I non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality and has increasingly become a disease of elderly patients. Elderly patients are underrepresented in clinical trials that evaluate treatments for NSCLC. It has been suggested that patients >65 years of age have less robust immune responses to infections, immunizations, and tumors compared with younger patients. With increasing focus and number of immunotherapy clinical trials for NSCLC, we investigated the relationship between patient age and the tumor immune microenvironment in NSCLC. Using tissue microarrays from 1,278 patients with surgically resected Stage I NSCLC (≤65 years [33%], 66–79 years [55%], and ≥80 years [12%]), we determined whether quantitative and qualitative immune cell infiltration in the tumor differed between younger and older patients. Furthermore, we investigated the prognostic value of immune cell infiltration with respect to recurrence in octogenarians. We found that there were no statistically significant differences between older and younger patients in tumoral immune infiltration or effector regulatory immune response ratios (FoxP3/CD3, FoxP3/CD4, and FoxP3/CD8 ratios). In octogenarians, presence of low tumoral CD68+ immune cells was an independent predictor of recurrence. In the uniform cohort of surgically selected and resected Stage I NSCLC patients, tumor immune cell infiltration among the older age group resembled other age groups. Our study provides information that supports inclusion of older age patients selected for surgical resection in neoadjuvant or adjuvant immunotherapy clinical trials for lung cancer.

Treatment with EGCG in NSCLC leads to decreasing interstitial fluid pressure and hypoxia to improve chemotherapy efficacy through rebalance of Ang-1 and Ang-2

AimMicrovasculature and microenvironment play important roles in proliferation, invasion, metastasis and prognosis in non-small cell lung cancer (NSCLC), which might be altered by many anti-angiogenic drugs. Epigallocatechin-3-gallate (EGCG), a natural anti-angiogenesis agent refined from green tea, was defined to have multiple effects on angiogenesis factors, such as endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and angiopoietins (ANGs). Hypothesizing that EGCG might regulate microvasculature and microenvironment in NSCLC, the effects of EGCG on microvessel density (MVD), expression of Ang-1 and Ang-2, interstitial fluid pressure (IFP), tumor hypoxia, and chemotherapy sensitivity were examined. Methods and ResultsEGCG treatment of A549 cells in mice bearing xenografts in vivo led to a significant decrease of MVD detected by CD31, and of Ang-2 expression detected by quantum dots double-label immunofluorescence assessment, while Ang-1 decreased with no significance. Decreased IFP was measured by the Wink-in-needle method, while hypoxia was assessed by polarographic electrode and pimonidazole (PIMO) immunohistochemistry. Assuming that these changes would increase response to chemotherapy, tumor growth studies were p[erformed in nude mice with xenografts, which were then treated with EGCG and the chemotherapeutic agent cisplatin. EGCG therapy combined with cisplatin led to synergistic inhibition of tumor growth, compared with administration of each treatment separately (P < 0.001). According to linear regression analysis, IFP was positively correlated with PIMO staining (R2 = 0.618, P = 0.002), Ang-2 was correlated with MVD (R2 = 0.423, P = 0.022), IFP (R2 = 0.663, P = 0.01) and PIMO staining (R2 = 0.694, P = 0.01). ConclusionIFP and delivery of oxygen might be improved by rebalance of Ang-1/Ang-2 under the treatment of EGCG in NSCLC, which also acts as a sensitizer of chemotherapy. These studies established a new mechanism for using EGCG as an adjuvant chemotherapy agent through modifying microvasculature and microenvironment.

Abstract 2439: Molecular imaging of spatial and temporal heterogeneity of tumor micro-environment in mouse models of non-small cell lung cancer macroscopic xenografts and micro-metastases

Abstract 18F-fluoro-2-deoxyglucose (18F-FDG, imaging glucose metabolism), 18F-fluorothymidine (18F-FLT, cell proliferation) and 18F-misonidazole (18F-FMISO, hypoxia) PET scans have emerged as important clinical tools for management of cancer. The objective of this study was to spatially and temporally visualize tumor microenvironment of tumor hypoxia, proliferation and glucose metabolism using 18F-FDG, 18F-FLT and 18F-FMISO digital autoradiography and microPET comparing with histological findings. Methods: We used human non-small cell lung cancer (NSCLC) A549 and HTB177 cells to generate subcutaneous and peritoneal metastases in nude mice. Animals were coinjected with the mixture of one PET radiotracer, pimonidazole (hypoxia marker) and bromodeoxyuridine (proliferation marker) intravenously 1 hour before animal euthanasia. The intratumoral distributions of radiotracers were visualized by digital autoradiography (DAR) and related to microscopic visualization of cellular proliferation, tumor hypoxia, stroma and necrosis. Serial microPET scans (day 1to day5) were also performed in the same animals to investigate change in glucose metabolism (using 18F-FDG), proliferation (18F-FLT) and hypoxia (18F-FMISO). Results: NSCLC microenvironment was complex and highly heterogeneous: xenografts had complex structures with intermingled regions of well oxygenated (negative pimonidazole) and highly proliferative (positive bromodeoxyuridine) cancer cells, hypoxic (positive pimonidazole) and low proliferation (little bromodeoxyuridine) cancer cells stroma and necrosis. Hypoxic cancer cells had high18F-FDG and 18F-FMISO but low 18F-FLT accumulation, indicating increased glucose metabolism is not a common feature of cancer cells but only hypoxic ones. Well oxygenated cancer cells with high proliferation rate accumulated high level of 18F-FLT, but low 18F-FDG and18F-FMISO. Stroma and necrotic zones always associated with low activity in all radiotracers we tested. MicroPET scans revealed apparent change in intratumor distribution of 18F-FLT, 18F-FDG as well as 18F-FMISO in as short as ∼48 hrs interval, indicating temporal heterogeneity of tumor microenvironment in term of proliferation, glucose metabolism and hypoxia. Conclusions: Both macroscopic xenografts and micrometastases of NSCLC have spatial heterogeneity of tumor microenvironment. Temporal change of tumor microenvironment did occur in a very short of interval of natural growth process in NSCLC. We have investigated to spatial and temporal behavior of heterogeneity of tumor microenvironment which is important for better understanding cancer biology and cancer management, if our findings in mice models are clinically applicable. Acknowledgement: This study was supported by Kentucky Lung Cancer Research Program Award to Dr. Xiao-Feng Li. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2439. doi:1538-7445.AM2012-2439