Non-small Cell Lung Cancer Tissue Samples Research Articles

Downregulation of lncRNA MIR17HG reduced tumorigenicity and Treg-mediated immune escape of non-small-cell lung cancer cells through targeting the miR-17-5p/RUNX3 axis.

Long noncoding RNA MIR17HG was involved with the progression of non-small-cell lung cancer (NSCLC), but specific mechanisms of MIR17HG-mediated immune escape of NSCLC cells were still unknown. The present study investigated the function of MIR17HG on regulatory T cell (Treg)-mediated immune escape and the underlying mechanisms in NSCLC. Expression of MIR17HG and miR-17-5p in NSCLC tissue samples were detected using quantitative real-time PCR (qRT-PCR). A549 and H1299 cells were transfected with sh-MIR17HG, miR-17-5p inhibitor, or sh-MIR17HG + miR-17-5p inhibitor, followed by cocultured with Tregs. Cell proliferation was measured using 5-ethynyl-20-deoxyuridine (Edu) staining assay and cell counting kit-8 (CCK-8) assay. Flow cytometry was used for determining positive numbers of FOXP3+CD4+/CD25+/CD8+ Tregs. Through subcutaneous injection with transfected A549 cells, a xenograft nude mouse model was established. Weights and volumes of xenograft tumors were evaluated. Additionally, the expressions of immune-related factors includingtransforming growth factor beta (TGF-β), vascular endothelial growth factor A (VEGF-A), interleukin-10 (IL-10), IL-4, and interferon-gamma (IFN-γ) in cultured cells, were evaluated by enzyme-linked immunosorbent assay and western blot analysis. Then, miR-17-5p was decreased and MIR17HG was enhanced in both NSCLC tissues and cell lines. MIR17HG knockdown significantly suppressed cell proliferation, tumorigenicity, and immune capacity of Tregs in A549 and H1299 cells, whereas sh-MIR17HG significantly reduced expression levels of VEGF-A, TGF-β, IL-4, and IL-10 but promoted the IFN-γ level in vitro and in vivo. Moreover, downregulation of miR-17-5p significantly reversed the effects of sh-MIR17HG. Additionally, we identified that runt- related transcription factor 3 (RUNX3) was a target of miR-17-5p, and sh-MIR17HG and miR-17-5p mimics downregulated RUNX3 expression. In conclusion, downregulation of MIR17HG suppresses tumorigenicity and Treg-mediated immune escape in NSCLC through downregulating the miR-17-5p/RUNX3 axis, indicating that this axis contains potential biomarkers for NSCLC.

Abstract 4977: The prevalence of key oncogenic driver mutations in stage I-III non-small cell lung cancer: Results from the AACR GENIE BPC dataset

Abstract Background: While there is growing interest in targeted therapy for non-small cell lung cancer (NSCLC) in perioperative settings, data on key oncogenic driver mutations is limited in early-stage NSCLC. This real-world database study aims to evaluate the prevalence of select genomic alterations in stage I-III NSCLC. Methods: From the American Association for Cancer Research (AACR) Project Genomics Evidence Neoplasia Information Exchange (GENIE) Biopharma Collaborative (BPC) V2.1-consortium dataset, we included adult patients with a confirmed diagnosis of NSCLC who had genomic testing using stage I-III NSCLC tissue samples. Descriptive statistics were used to summarize baseline characteristics and the prevalence of select genomic alterations. Results: A total of 465 patients were included in this study, of whom 61% were female, 85% were white, 19% were never smokers, and 77% had adenocarcinoma. Less than 2% of the study population had BRAF V600E, HER2 activating mutations, HER2 amplification, MET amplification, HER3 amplification, RET fusion, ALK fusion, or ROS1 fusion. Among genomic alterations with a prevalence of 2% or higher, such as EGFR sensitizing mutations, KRAS G12C, MET exon 14 skipping, STK11, KEAP1, and TP53, some variations in prevalence were observed across baseline patient and disease characteristics such as race, smoking status, and histology (Table 1). Conclusion: The prevalence of key oncogenic drivers and its relationship to select baseline disease characteristics among patients with early-stage NSCLC are similar to those of advanced NSCLC observed in other real-world clinico-genomic databases. Table 1. Prevalence of Select Genomic Alterations in Stage I-III NSCLC (N=465) Prevalence of Positive Biomarker Status (%) EGFR KRAS G12C MET Exon 14 Skipping STK11* KEAP1* TP53 Overall 17.6 12.7 4.5 16.0 11.0 52.7 Gender Male 7.1 9.3 5.5 18.5 18.5 59.6 Female 24.5 15.9 3.9 14.3 6.3 48.2 Race White 15.6 14.1 4.5 16.5 12.0 53.7 Black 15.0 5.0 5.0 22.2 11.1 60.0 Asian 44.0 4.0 8.0 8.3 4.2 44.0 Smoking Status Never smoker 41.4 1.2 6.9 4.7 1.2 36.8 Former smoker 13.7 16.0 4.6 17.4 12.9 52.4 Current smoker 5.7 12.9 1.4 23.9 15.4 72.9 Histology Adenocarcinoma 20.1 14.3 4.6 18.1 10.4 46.0 Squamous cell carcinoma 0 2.9 4.4 4.6 14.1 86.8 *The numbers of patients tested for STK11 and KEAP1 were 457 and 445, respectively. Citation Format: Ke Zu, Jiemin Ma, Cai Chen, Christine Pierce. The prevalence of key oncogenic driver mutations in stage I-III non-small cell lung cancer: Results from the AACR GENIE BPC dataset [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4977.

Abstract 3759: First clinical evidence of the tissue nanomechanical signature as a novel diagnostic and prognostic tool in patients with non-small-cell lung cancer

Abstract Introduction: Complex structural remodelling of cells and extracellular matrix during cancer initiation and progression elicit substantial biomechanical alterations, which can be measured by Atomic Force Microscopy (AFM) at the nanoscale level. Clinical and translational evidence indicates that tissue nanomechanical signature (NS) can be a useful tool for rapid cancer diagnosis, and assessment of cancer aggressiveness. We demonstrated that the AFM-based Automated and Reliable Tissue Diagnostics (ARTIDIS) platform represents a valid method for rapid diagnosis, prognosis, and treatment response assessment in patients with breast cancer. Here we aimed to use this platform to examine tumour biopsies from non-small cell lung cancer (NSCLC) patients for the first time. Methods: This is an ongoing, single center study at the Hospital Clinic of Barcelona and University of Barcelona (UHB) Spain, aiming to enroll 75 patients by January 2024 in the first phase, followed by 200 patients in the expansion phase. To date, 54 matching tumor and non-tumor fresh tissue biopsies were taken by experienced pathologists from fresh surgical resection specimens from 27 patients with previously diagnosed NSCLC. Tissue biopsies were measured with the ARTIDIS device in a blinded manner, without knowing the malignancy status of the biopsy. Routine clinico-pathologic parameters have been collected. The multiparametric nanomechanical signature has been analyzed in an automated manner using the proprietary AI-based ARTIDISNET software platform together with available clinico-pathologic parameters. Results: In total, the NSCLC tissue samples included 16 squamous cell carcinoma (SCC), 6 Adenocarcinoma (ADC), 1 large cell carcinoma (LCC) and 4 other histological subtypes. Non-tumor tissues exhibited significantly varied composition, including normal lung parenchyma, bronchial tissues, fibrosis and inflammatory changes. Among the 27 patients, 5 patients developed distant progression within 12 months after ARTIDIS measurements. Results analysis showed that the ARTIDIS NS can reliably differentiate malignant NSCLC lesions from non-tumor lung tissue, with a sensitivity of 0.84, specificity of 0.81, positive predictive value (PPV) of 0.79, negative predictive value (NPV) of 0.86 and accuracy of 0.82. In addition, the ARTIDIS NS can differentiate patients with and without distant progression with a sensitivity of 0.62, specificity of 0.81, PPV of 0.42, NPV of 0.90 and accuracy of 0.77. Conclusions: Here we demonstrated first clinical evidence of the ARTIDIS tissue NS as a novel diagnostic and prognostic tool in patients with NSCLC. This study aims to establish the diagnostic, prognostic, and predictive NS in patients NSCLC. In addition to local expansion at the UHB, study results will be further validated in ARTIDIS clinical program at MD Anderson Cancer Centre, USA. Citation Format: Jordi Alcaraz, Héctor Sanz-Fraile, Elba Marin, Paula Gausa Busquets, Kate Neal, Marselina Arshakyan, Marc Rico, Marc Boada, David Sanchez, Reinier Oropesa Nunez, Gitika Srivastava, Ahmed Jizawi, Sara Nizzero, Tobias Appenzeller, Mariam Gachechiladze, Marko Loparic, Marija Plodinec, Noemi Reguart. First clinical evidence of the tissue nanomechanical signature as a novel diagnostic and prognostic tool in patients with non-small-cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3759.

Establishment of a 21-color Panel for the Detection of Immune Cell Subsets in Human Non-small Cell Lung Cancer Tumor Tissues with Flow Cytometry

With the rise of multicolor flow cytometry, flow cytometry has become an important means to detect the immune microenvironment of lung cancer, but most of them are used to detect the proportion of cell subsets or the function of major cell subsets, and they cannot be detected at the same time. Therefore, a reliable 21-color flow cytometry protocol was established to detect the immune cell subsets in human non-small cell lung cancer (NSCLC) tumor tissues. Cell membrane surface antibodies cluster of differentiation (CD)45, CD3, CD19, CD4, CD8, programmed cell death 1 (PD-1), CD39, CD103, CD25, CD127, chemokine receptor 8 (CCR8), CD56, CD11c, human leukocyte antigen (HLA)-DR, CD38, CD27, CD69, CD62L, CD45RA, CCR7 and nucleic acid dye L/D were used to develop the protocol. Firstly, antibody titration experiments, voltage optimization, subtraction of one color staining and single color staining experiments were carried out for each antibody, and after the experimental conditions and detection schemes were determined, the feasibility of the scheme was verified by using peripheral blood mononuclear cells (PBMCs) specimens of six healthy adult volunteers. Tumor tissue samples from 6 NSCLC patients were tested and analyzed. The established 21-color flow cytometry protocol was used to detect the tumor tissue samples of 6 NSCLC patients, and the proportion of each cell subset in lung cancer tissue, as well as the immunophenotype and differentiation of the main cell population, were analyzed. The successfully established 21-color flow cytometry protocol is suitable for the detection of PBMCs and NSCLC tissue samples, which provides an effective new idea for monitoring the immune microenvironment status in lung cancer.

IGF2BP1 facilitates non-small cell lung cancer progression by regulating the KIF2A-mediated Wnt/β-catenin pathway.

Insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs) are crucially implicated in the cancer progression. The current study intends to excavate and clarify the mechanisms of the key IGF2BPs in non-small cell lung cancer (NSCLC). The expression of IGF2BPs and kinesin family member 2A (KIF2A) was examined using immunohistochemistry, real-time quantitative polymerase chain reaction, and western blot in NSCLC tissue samples or cell lines. NSCLC cell viability was examined using a cell counting kit-8 assay. Cell apoptotic rate was assessed using flow cytometry analysis. The migration and invasion of H1299 cells were subject to scratch test and Transwell assays, respectively. Starbase 2.0 was used to detect the downstream factors of the IGF2BP1 protein. The binding of IGF2BP with KIF2A was detected using RNA binding protein immunoprecipitation assays. Ki-67 immunohistochemistry assay and TUNEL assays were applied for the evaluation of proliferation and apoptosis in vivo, respectively. IGF2BP1 was upregulated in NSCLC tissue samples and cells. Functionally, IGF2BP1 overexpression promoted the proliferative ability, migration, and invasiveness of H1299 cells, while inhibiting cell apoptosis in vitro. In vivo studies revealed that overexpression of IGF2BP1 promoted tumor growth of NSCLC. Mechanistically, IGF2BP1 was involved in KIF2A mRNA stabilization. KIF2A exerted the same functions as IGF2BP1 via the Wnt/β-catenin signaling. In conclusion, IGF2BP1 enhances NSCLC malignant progression by stabilizing KIF2A to modulate the Wnt/β-catenin pathway.

Thymidine kinase 1 as a target is regulated by the hsa-let-7b-5p/LINC00665 axis and affects NSCLC prognosis

BackgroundIn the past, multiple studies have offered incremental evidence that indicates that competitive endogenous RNA (ceRNA) regulatory networks are involved in tumor growth and present novel therapeutic targets. Herein, we investigated the impact of thymidine kinase 1 (TK1)-related ceRNA networks on the prognosis of non-small cell lung cancer (NSCLC). MethodsTK1 expression data in NSCLC and normal tissue samples were retrieved from the Cancer Genome Atlas (TCGA) database and were then compared. Thereafter, the findings of the immunohistochemical staining experiments and clinical follow-up data derived from patients with NSCLC were used for conducting prognostic analysis. The starBase database was searched to determine TK1-targeted microRNAs and long non-coding RNAs, and clinical data from TCGA were used for survival analysis to construct a ceRNA network associated with TK1 expression and prognosis. Finally, the roles played by methylation and immunity in the prognosis and treatment of NSCLC were analyzed. ResultsOur findings revealed that the cancer tissues expressed significantly higher TK1 levels than normal tissues, and the follow-up clinical data revealed that the prognosis was generally worse in the high-expression patients than in the low-expression patients. In addition, clinical data collected from the starBase and TCGA databases showed that the LINC00665/has-let-7b-5p/TK1 network could influence the growth and prognosis of NSCLC. It was also noted that the TK1 methylation site was correlated with the prognosis of NSCLC, and immunoprognostic analysis further indicated that patients with higher TK1 expression levels displayed a worse prognosis. ConclusionWhen the regulatory network of LINC00665/has-let-7b-5p/TK1 was assessed, it was observed that elevated TK1 levels may affect the prognosis of NSCLC. Therefore, it could be considered a prognostic biomarker and a probable therapeutic target for predicting NSCLC prognosis.

Zinc finger protein 263 upregulates interleukin 33 and suppresses autophagy to accelerate the malignant progression of non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a complex disease that remains a major public health concern worldwide. One promising avenue for NSCLC treatment is the targeting of transcription factors that regulate key pathways involved in cancer progression. In this study, we investigated the role of the transcription factor ZNF263 in NSCLC and its impact on the regulation of IL33, apoptosis, and autophagy. Levels of ZNF263 in tissues and cell lines were identified, after which the effects of its knockdown on cellular malignant behaviors, apoptosis and autophagy were assessed. Based on bioinformatics analysis, ZNF263 was found to bind to IL33 promoter, their mutual relationship was confirmed, as well as the role of IL33 in the regulation of ZNF263. The involvement of ZNF263 in the growth of xenograft tumors was assessed using tumor-bearing nude mouse models. Experimental results revealed that ZNF263 was upregulated in NSCLC tissue samples and cell lines. Its expression level is positively correlated with cellular malignant behaviors. We further demonstrated that ZNF263 upregulated IL33 expression, which, in turn, promoted the proliferation and migration, inhibited apoptosis and autophagy in NSCLC cells. Furthermore, ZNF263 knockdown reduced the growth of xenograft tumors in nude mice. This finding suggests that the inhibition of ZNF263 or IL33 may represent a novel therapeutic strategy for NSCLC. Importantly, our results highlight the crucial role of transcription factors in NSCLC and their potential as therapeutic targets.

Utility of multigene panel next-generation sequencing in routine clinical practice for identifying genomic alterations in newly diagnosed metastatic nonsmall cell lung cancer.

The standard of care in newly diagnosed metastatic non-small cell lung cancer (NSCLC) is to test for aberrations in three genes for driver mutations - ALK, ROS1 and epidermal growth factor receptor (EGFR) - and also for immunohistochemistry to be performed for programmed death-ligand 1 expression level. Next-generation sequencing (NGS), with or without RNA fusion testing, is increasingly used in standard clinical practice to identify patients with potentially actionable mutations. Stratification of NGS mutation tiers is currently based on the European Society of Medical Oncology Scale for Clinical Actionability of Molecular Targets (ESCAT) Tiers I-V and X. Our aim was to analyse NSCLC tumour samples for the prevalence of Tiers I-V mutations to establish guidance for current and novel treatments in patients with metastatic disease. NGS was performed employing the Oncomine Precision Assay (without RNA fusion testing) that interrogates DNA hotspot variants across 45 genes to screen 210 NSCLC tissue samples obtained across six Sydney hospitals between June 2021 and March 2022. In our cohort, 161 of 210 (77%) had at least one gene mutation identified, with 41 of 210 (20%) having two or more concurrent mutations. Tier I mutations included 42 of 210 (20%) EGFR mutations (EIA) and five of 210 (3%) MET exon 14 skipping mutations (EIB). Non-Tier I variants included 22 of 210 (11%) KRAS G12C hotspot mutations (EIIB), with a further 47 of 210 (22%) having non-G12C KRAS (EX) mutations. NGS testing revealed an additional 15% of cases with Tier II ESCAT mutations in NSCLC. Forty-six percent of patients also demonstrated potential Tier III and IV mutations that are currently under investigation in early-phase clinical trials. In addition to identifying patients with genomic alterations suitable for clinically proven standard-of-care therapeutic options, the 45-gene NGS panel has significant potential in identifying potentially actionable non-Tier 1 mutations that may become future standard clinical practice in NSCLC.

Potential of miR-181a-5p and miR-630 as clinical biomarkers in NSCLC

BackgroundThe development of drug resistance and high mortality rates are the major problems observed in non-small cell lung cancer (NSCLC). Biomarkers indicating and predicting disease development towards these unfavorable directions are therefore on high demand. Many studies have demonstrated that changes in miRNAs expression may be associated with a response to treatment and disease prognosis, thus suggesting its potential biomarker value for a broad spectrum of clinical applications. The aim of the present study was to investigate the expression level of miR-181a-5p, miR-630, and its targets in NSCLC tumor tissue and plasma samples; and to analyze its association with NSCLC patient’s response to treatment and disease prognosis.MethodsThe study was performed in 89 paired tissue specimens and plasma samples obtained from NSCLC patients who underwent surgical treatment at the Department of Thoracic Surgery and Oncology of the National Cancer Institute. Analysis of miR-181a-5p and miR-630 expression was performed by qRT-PCR using TaqMan miRNA specific primers. Whereas BCL2, LMO3, PTEN, SNAI2, WIF1 expression levels were identified with KAPA SYBR FAST qPCR Kit. Each sample was examined in triplicate and calculated following the 2-ΔΔCt method. When the p-value was less than 0.05, the differences were considered statistically significant.ResultsIt was found that miR-181a-5p and miR-630 expression levels in NSCLC tissue and plasma samples were significantly decreased compared with control samples. Moreover, patients with low miR-181a-5p expression in tumor tissue and plasma had longer PFS rates than those with high miRNA expression. Decreased miR-630 expression in tumor was statistically significantly associated with better NSCLC patients’ OS. In addition, the expression of miR-181a-5p, as well as miR-630 in tumor tissue, are the statistically significant variables for NSCLC patients’ OS. Moreover, in NSCLC patient plasma samples circulating miR-181a-5p can be evaluated as significant independent prognostic factors for OS and PFS.ConclusionsOur findings indicate the miR-181a-5p and miR-630 expression levels have the potential to prognose and predict and therefore improve the treatment individualization and the outcome of NSCLC patients. Circulating miR-181a-5p has the potential clinical value as a non-invasive biomarker for NSCLC.

Inhibition of inflammation and infiltration of M2 macrophages in NSCLC through the ATF3/CSF1 axis: Role of miR-27a-3p.

Non-small cell lung cancer (NSCLC) imposes a significant economic burden on patients and society due to its low overall cure and survival rates. Tumour-associated macrophages (TAM) affect tumour development and may be a novel therapeutic target for cancer. We collected NSCLC and tumour-adjacent tissue samples. Compared with the tumour-adjacent tissues, the Activation Transcription Factor 3 (ATF3) and Colony Stimulating Factor 1 (CSF-1) were increased in NSCLC tissues. Levels of ATF3 and CSF-1 were identified in different cell lines (HBE, A549, SPC-A-1, NCI-H1299 and NCI-H1795). Overexpression of ATF3 in A549 cells increased the expression of CD68, CD206 and CSF-1. Moreover, levels of CD206, CD163, IL-10 and TGF-β increased when A549 cells were co-cultured with M0 macrophages under the stimulation of CSF-1. Using the starbase online software prediction and dual-luciferase assays, we identified the targeting between miR-27a-3p and ATF3. Levels of ATF3, CSF-1, CD206, CD163, IL-10 and TGF-β decreased in the miR-27a mimics, and the tumour growth was slowed in the miR-27a mimics compared with the mimics NC group. Overall, the study suggested that miR-27a-3p might inhibit the ATF3/CFS1 axis, regulate the M2 polarization of macrophages and ultimately hinder the progress of NSCLC. This research might provide a new therapeutic strategy for NSCLC.

MiR-196b-5p activates NF-κB signaling in non-small cell lung cancer by directly targeting NFKBIA

BackgroundOur recent study found that QKI-5 regulated miRNA, miR-196b-5p, promotes non-small cell lung cancer (NSCLC) progression by directly targeting GATA6, TSPAN12 and FAS. However, the biological functions of miR-196b-5p in NSCLC progression and metastasis still remain elusive. MethodsCell proliferation, migration, colony formation, cell cycle assays were used to investigate cellular phenotypic changes. Quantitative real-time PCR (qRT-PCR) and western blot analyses were used to measure expressions of relative gene and protein. Interaction between QKI-5 and miR-196b-5p was determined by RNA immunoprecipitation (RIP) assay. Luciferase reporter assay was used to determine direct binding between miR-196b-5p and NFKBIA 3′-UTR. ELISA assay was used to measure secreted IL6 proteins. Mice xenograft model was used to assess the functions of NFKBIA on in vivo tumor growth. ResultsWe demonstrated that the miR-196b-5p facilitates lung cancer cell proliferation, migration, colony formation, and cell cycle by directly targeting NFKBIA, a negative regulator of NFκB signaling. Knocking down NFKBIA increases IL6 mediated phosphorylation of signal transducer and activator of transcription 3 (STAT3) to promote lung cancer cell growth by activating NFκB signaling. The expression of NFKBIA was significantly downregulated in NSCLC tissue samples, and was negatively correlated with the expression miR-196b-5p. In addition, we found that downregulated QKI-5 expression was associated with the elevated miR-224 expression in NSCLC. ConclusionsOur findings indicated that the miR-224/QKI-5/miR-196b-5p/NFKBIA signaling pathway might play important functions in the progression of NSCLC, and suggested that targeting this pathway might be an effective therapeutic strategy in treating NSCLC.

Homeobox D9 Promotes Malignant Progression of Non-Small Cell Lung Cancer Through Negatively Regulating Kallikrein Related Peptidase 9

We investigated the association between HOXD9 expression and prognosis in non-small cell lung cancer (NSCLC) and explored its underlying mechanisms in tumor progression. HOXD9 expression was examined in 42 pairs of NSCLC tissue samples using qRT-PCR, and its effects were evaluated in NSCLC cell lines through lentiviral vector transfection, assessing cell growth and migration using CCK-8 and Transwell assays. Bioinformatics analysis identified KLK9 as a target gene of HOXD9, and their interaction was investigated in vitro and in vivo. HOXD9 mRNA expression was significantly higher in NSCLC tissues compared to adjacent tissues, positively correlating with metastasis incidence and poor prognosis. HOXD9 knockdown reduced NSCLC cell migration and proliferation, while overexpression had the opposite effect. Luciferase assays confirmed HOXD9′s binding to KLK9, leading to negative regulation of KLK9 expression. Knockdown of KLK9 reversed the inhibitory effect of HOXD9 on NSCLC cell migration and proliferation, while co-overexpression of KLK9 and HOXD9 had the opposite effect. Our findings demonstrate the close association of high HOXD9 expression in NSCLC tissues with metastasis incidence and poor prognosis. HOXD9 likely exerts its biological function in NSCLC by negatively regulating KLK9. These results provide valuable insights into the molecular mechanisms driving NSCLC progression, highlighting HOXD9 as a potential prognostic biomarker and therapeutic target.

Quantitative 3D assessment of the lung cancer microenvironment using multi-resolution open-top light-sheet microscopy.

2616 Background: Non-small cell lung cancer (NSCLC) tissue is a valuable resource for diagnosis, treatment planning, and drug development. Current 2D histopathological techniques introduce under-sampling error (i.e., a single 5 µm section represents 0.5% of a 1 mm thick biopsy), interobserver variability, and failure to capture the biology contained within the entire tissue sample. We have developed a suite of technologies to stain, chemically clarify, image, visualize, and analyze entire, intact lung tissue samples. Herein, we present analysis methods for comprehensive 3D assessment of intact NSCLC tissue. Methods: Human NSCLC tissue, stored frozen in optimal cutting temperature (OCT) compound, was fixed in 4% paraformaldehyde, stained with nuclear (TOPRO-3) and general protein (Eosin) fluorescent dyes, and optically cleared using a modified iDISCO protocol with ethyl cinnamate as the refractive index matching solution. Twenty tissue samples measuring 1-5 mm3 in volume were imaged at 2 µm/pixel resolution with a hybrid open-top light-sheet microscope. Smaller regions of interest (ROIs) with interesting pathologic features were re-imaged at higher resolution, 0.17 µm/pixel. Visualization and analysis was performed using Aivia software, custom spatial analysis software and python scripts. Results: All NSCLC tissue samples were successfully imaged in 3D at both low and high resolution. Computational approaches, such as machine learning, were used to segment and classify tumor cells and lymphocytes within the high resolution ROIs. In one example we quantified a 0.0045 mm3 ROI containing 1x109 voxels. We segmented 1,474 unique objects, and classified 683 (46%) of the objects as lymphocytes and 309 (20%) of the cells as tumor cells. We found that on average the lymphocytes were within 12 µm of the nearest tumor cell. Additionally the tissue was broken into separate lymphocyte dense (58% of volume) and tumor dense regions (42% of volume). Further analysis is in progress to quantify more features, such as tertiary lymphoid structures, stromal cells, and fibrosis over the full 20 sample dataset. Conclusions: We successfully implemented 3D machine learning analysis pipelines using images from intact NSCLC tissue samples measuring up to 5 mm3. The imaging was performed using our custom hybrid open-top light-sheet microscope. This novel method enables us to identify and quantify key histological features such as tumor cells and lymphocytes within the tumor microenvironment. In addition, machine learning driven algorithms clustered cells into distinct tissue regions in 3D which enables a more precise assessment of disease state and heterogeneity. Further investigation is ongoing to link these results to patient outcomes.

A differential analysis of TCR repertoire between patients with EGFR-mutant and KRAS-mutant non–small-cell lung cancer.

e21012 Background: T-cell receptor (TCR) repertoire could partially represent tumor micro-environment. EGFR and KRAS are common driver genes mutated and often mutually exclusive in non-small cell lung cancer (NSCLC). There’s a hypothesis that the immune system responds differently according to different mutations. Here we investigated the TCR features, including diversity, clonality, evenness and sequence similarity in EGFR and KRAS mutated NSCLC patients, to better understand the immune response in carcinogenesis. Methods: A total of 71 EGFR-mutant and 31 KRAS-mutant NSCLC tissue samples underwent multiplex PCR based TCR-β sequencing. TCR-β cdr3 amino acid sequences were obtained from the sequencing data using MiXCR and VDJtools. To compare TCR repertoires in EGFR-mutant and KRAS-mutant groups, each assemble CDR3 amino acid sequence was taken as a word, and each repertoire was taken as a document. Each repertoire was compared with every other one as a sample pair. TF-IDF (term frequency-inverse document frequency) document vectors was built for each sample and cosine similarity scores were computed for sample pairs. Results: The total cosine similarity score within EGFR-mutant group is 11.07 and average cosine similarity score is 0.0044±0.01, while in the KRAS-group, the total cosine similarity score is 13.41 and average cosine similarity score is 0.029±0.04. Between these two groups, the total cosine similarity score is 8.76 and average cosine similarity score is 0.0019±0.0009. See table for summary. The intergroup similarity score is significantly less than that within each group respectively (p < 1E10). Conclusions: Our results demonstrated that the TCR repertoire was clearly associated with cancer mutational profile. The mechanism of how mutations affect the immune system and shape the TCR repertoire is yet to be investigated. [Table: see text]

Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis

F-box/LRR-repeat protein 7 (FBXL7) was predicted as a differentially expressed E3 ubiquitin ligase in non-small cell lung cancer (NSCLC), which has been suggested to influence cancer growth and metastasis. In this study, we aimed to decipher the function of FBXL7 in NSCLC and delineate the upstream and downstream mechanisms. FBXL7 expression was verified in NSCLC cell lines and GEPIA-based tissue samples, after which the upstream transcription factor of FBXL7 was bioinformatically identified. The substrate PFKFB4 of the FBXL7 was screened out by tandem affinity purification coupled with mass-spectrometry (TAP/MS). FBXL7 was downregulated in NSCLC cell lines and tissue samples. FBXL7 ubiquitinated and degraded PFKFB4, thus suppressing glucose metabolism and malignant phenotypes of NSCLC cells. Hypoxia-induced HIF-1α upregulation elevated EZH2 and inhibited FBXL7 transcription and reduced its expression, thus promoting PFKFB4 protein stability. By this mechanism, glucose metabolism and the malignant phenotype were enhanced. In addition, knockdown of EZH2 impeded tumor growth through the FBXL7/PFKFB4 axis. In conclusion, our work reveals that the EZH2/FBXL7/PFKFB4 axis plays a regulatory role in glucose metabolism and tumor growth of NSCLC, which is expected to be potential biomarkers for NSCLC.

Hsa_circ_0088036 promotes nonsmall cell lung cancer progression by regulating miR-1343-3p/Bcl-3 axis through TGFβ/Smad3/EMT signaling.

Nonsmall cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Circular RNAs (circRNAs) have been the focus of numerous studies, and some circRNAs have been linked to the development of multiple malignant tumors, including NSCLC. Nevertheless, the functional role and mechanisms of circRNAs in NSCLC remain largely unknown. The primary objective of this study was to screen the associated circRNA in NSCLC and investigate its mechanism. CircRNA microarray was used to identify circRNAs that were abnormally expressed in NSCLC tissue samples. Expression of hsa_circRNA_0088036 was validated in NSCLC tissues and cell lines after the correlation between hsa_circRNA_0088036 and prognosis was determined. We then used a series of function gain-and-loss assays to determine the role of hsa_circ_0088036 in NSCLC progression. RNA-binding protein immunoprecipitation (RIP), RNA pull-down, and RNA interference assays were used to assess the interaction between hsa_circ_0088036 and miR-1343-3p/Bcl-3 axis. Moreover, mechanistic assays were applied to investigate the involved signaling pathway regulated by the hsa_circ_0088036/miR-1343-3p/Bcl-3 axis. Microarray analysis and reverse transcription polymerase chain reaction confirmed the presence of a circRNA termed hsa_circ 0088036 that was upregulated in NSCLC tissue samples and cell lines and indicated a positive association with patient prognosis. Functionally, hsa_circ_0088036 silencing inhibited proliferative, invasive, and migrative potential of NSCLC cells as well as epithelial-mesenchymal transition (EMT)-related proteins by sponging miR-1343-3p to inhibit Bcl-3. Furthermore, mechanistic experiments demonstrated that hsa_circ_0088036 promoted NSCLC progression by activating the TGFβ/Smad3/EMT signaling pathway via miR-1343-3p/Bcl-3 axis. In conclusion, hsa_circ_0088036 functions as an oncogene by targeting the miR-1343-3p/Bcl-3 axis via TGFβ/Smad3/EMT signaling pathway.

PLCG2 can exist in eccDNA and contribute to the metastasis of non-small cell lung cancer by regulating mitochondrial respiration

Extrachromosomal circular DNAs (eccDNAs) participate in tumorigenesis and tumor progression. However, the role and mechanism of eccDNAs have yet to be elucidated in non-small cell lung cancer (NSCLC). In our research, three surgically matched NSCLC tissue samples, NSCLC cell lines (H1299, A549, and H460), and a normal lung cell line (MRC-5) were used as study objects. High-throughput eccDNA sequencing and bioinformatics analysis were performed to study the distribution pattern and level of eccDNA expression. The upregulated candidate eccDNA-encoding PLCG2 was validated by routine PCR. Plasmid transfection, RNA interference, qRT‒PCR and western blotting experiments were used to verify the expression level of PLCG2. Our results showed that the chromosome distribution, length distribution, and genomic annotation of the eccDNAs were comparable between the NSCLC and normal groups. Nevertheless, there were no significant differences in eccDNAs between NSCLC tissues and matched normal lung tissues. The eccDNA derived from PLCG2 was upregulated in NSCLC cells. TCGA analysis and immunohistochemistry showed that PLCG2 was highly expressed in lung cancer tissues and tended to be associated with poor outcome. We also demonstrated that PLCG2 can promote metastasis through the regulation of mitochondrial respiration. These results suggested that PLCG2 identified by eccDNA sequencing acts as an oncogene and might be a new biomarker for NSCLC diagnosis and prognosis evaluation.

Abstract 6624: 3D assessment of the lung cancer microenvironment using multi-resolution open-top light-sheet microscopy

Abstract Background: Non-small cell lung cancer (NSCLC) tissue is a valuable resource for diagnosis, treatment planning, and drug development. Current 2D histopathological techniques introduce under-sampling error (i.e., a single 5 um section represents 0.5% of a 1 mm thick biopsy), interobserver variability, and fail to capture the biology contained within the entire tissue sample. We have developed a suite of technologies to stain, chemically clarify, image, visualize, and analyze entire intact NSCLC tissue samples. Methods: Human NSCLC tissue, stored frozen in OCT, was fixed in 4% paraformaldehyde, stained with nuclear (TOPRO-3) and general protein (Eosin) fluorescent dyes, and optically cleared using a modified iDISCO protocol with ethyl cinnamate as the refractive index matching solution. Whole, intact tissue samples, roughly 1-5mm3 in volume, were imaged at 2 microns/pixel resolution with an open-top light-sheet microscope (3Di, Alpenglow Biosciences). Smaller regions of interest (ROIs) with key pathologic features were reimaged at higher resolution, 0.17 microns/pixel, to reveal subnuclear features and for cell typing. Visualization was performed using Aivia software. Results: NSCLC tissue samples were successfully imaged in 3D. Low resolution images (2 microns/pixel) were obtained within 4-31 minutes, depending on the tissue volume. The 3D distribution of cancer cells, immune cells, vessels, and fibrosis varied substantially throughout the volume of the tissue. Recognizable histologic features, including nests of tumor cells surrounded by vasculature and immune cells, were readily visualized. Squamous and adenocarcinoma with its subtypes (solid, acinar, lepidic, and micropapillary) morphologies were recognizable in 2D optical sections of the 3D datasets. Imaging quality degraded in tissue deeper than 1 mm due to light scattering. Conclusion: We assessed intact NSCLC tissue samples measuring up to 5 mm3 using our custom light-sheet microscope and tissue clearing techniques. This novel method enables us to visualize key features of NSCLC such as the tumor interfaces, tertiary lymphoid structures, vessels and fibrosis in the entire tissue sample, preventing under sampling error, and potentially enabling new biologic insights. Next steps include segmentation and quantification of key tissue structures such as tumor volume, immune cell distribution, and fibrosis/immune cell exclusion. This proof-of-concept study provides motivation for further investigation into the significance of 3D tissue features in NSCLC tissue samples. Citation Format: Alexandra Alvarsson, Carl Storey, Brandy Olin Pope, Caleb Stoltzfus, Robert Vierkant, Jessica Tufariello, Aaron Bungum, Julia Naso, Cheuk Ki Chan, Eric Edell, Christopher Hartley, Janani Reisenauer, Nicholas Reder. 3D assessment of the lung cancer microenvironment using multi-resolution open-top light-sheet microscopy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6624.

Long non-coding RNA ATB expedites non-small cell lung cancer progression by the miR-200b/fibronectin 1 axis.

Long non-coding RNA (lncRNA) ATB belongs to an active modulator in multiple cancers, but its expression along with potential underlying non-small cell lung cancer (NSCLC) is obscure. Our study aimed to investigate the role and potential mechanism of LncRNA ATB in NSCLC. LncRNA ATB expression in NSCLC tissues and cell lines was detected by qRT-PCR. Effects of LncRNA ATB on NSCLC cell proliferation, migration and invasion were assessed by MTS, colony formation and transwell assays. The connection among LncRNA ATB, miR-200b and fibronectin 1 (FN1) was determined by bioformatics prediction and luciferase reporter assay. In this research, upregulation of LncRNA ATB was discovered in NSCLC tissue samples and cell lines. LncRNA ATB was positively related to advanced tumor phase as well as lymph node metastasis. Cell function assays reflected LncRNA ATB expedited NSCLC cells proliferation, migration and invasion. LncRNA ATB promoted fibronectin 1 (FN1) expression via inhibiting miR-200b. Furthermore, LncRNA ATB depletion suppressed NSCLC cells proliferation, migration and invasion, while miR-200b inhibitor or pcDNA-FN1 rescued these effects. In summary, our outcomes elucidated that LncRNA ATB/miR-200b axis expedited NSCLC cells proliferation, migration and invasion by up-regulating FN1.

Tastin promotes non-small-cell lung cancer progression through the ErbB4, PI3K/AKT, and ERK1/2 pathways.

Tastin might be involved in tumorigenesis, but its role in non-small-cell lung cancer (NSCLC) has not been adequately explored. This work aimed to examine tastin's role in NSCLC and to explore the underlying mechanism. The Gene Expression Omnibus (GEO), Gene Expression Database of Normal and Tumor tissues (GENT), and Cancer Genome Atlas (TCGA) databases were used. Four GEO datasets (GSE81089, GSE40419, GSE74706, and GSE19188) containing gene expression data for NSCLC and normal tissue samples were analyzed for tastin mRNA expression. Tastin expression levels in different tissues were compared using the GENT website. TCGA biolinks were used to download gene expression quantification (n = 594) and overall survival data (n = 535). In total, 30 lung adenocarcinoma and 25 lung squamous cell carcinoma cases were enrolled. In addition, four-week-old male BALB/c nude mice (n = 9/group) were used to establish xenograft mouse models. Furthermore, cultured HEK293T, A549, and NCI-H226 cells assessed. Immunoblot, hematoxylin and eosin (H&E) staining, immunohistochemistry, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), fluorescence microscopy, flow cytometry, lentiviral transduction, and MTT, colony formation, wound healing, and Transwell assays were carried out. Tastin expression levels were markedly increased in NSCLC tumor tissue specimens and correlated with a poorer prognosis. Silencing of tastin inhibited the proliferative and migratory abilities of NSCLC cells. Bioinformatic analysis suggested that tastin interacts with ErbB4. The PI3K/AKT and ERK1/2 downstream pathways were suppressed in tastin-deficient cells. In conclusion, tastin might be involved in NSCLC growth and invasion and is a potential therapeutic target in NSCLC.