Biomarker For Non-small Cell Lung Cancer Research Articles

A novel prognostic signature based on smoking-associated genes for predicting prognosis and immune microenvironment in NSCLC smokers

BackgroundAs a highly heterogeneous tumor, non-small cell lung cancer (NSCLC) is famous for its high incidence and mortality worldwide. Smoking can cause genetic changes, which leading to the occurrence and progress of NSCLC. Nevertheless, the function of smoking-related genes in NSCLC needs more research.MethodsWe downloaded transcriptome data and clinicopathological parameters from Gene Expression Omnibus (GEO) databases, and screened smoking-related genes. Lasso regression were applied to establish the 7-gene signature. The associations between the 7-gene signature and immune microenvironment analysis, survival analysis, drug sensitivity analysis and enriched molecular pathways were studied. Ultimately, cell function experiments were conducted to research the function of FCGBP in NSCLC.ResultsThrough 7-gene signature, NSCLC samples were classified into high-risk group (HRG) and low-risk group (LRG). Significant difference in overall survival (OS) between HRG and LRG was found. Nomograms and ROC curves indicated that the 7-gene signature has a stable ability in predicting prognosis. Through the analysis of immune microenvironment, we found that LRG patients had better tumor immune activation. FCGBP showed the highest mutation frequency among the seven prognostic smoking related genes (LRRC31, HPGD, FCGBP, SPINK5, CYP24A1, S100P and FGG), and was notable down-regulated in NSCLC smokers compared with non-smoking NSCLC patients. The cell experiments confirmed that FCGBP knockdown promoting proliferation, migration, and invasion in NSCLC cells.ConclusionThis smoking-related prognostic signature represents a promising tool for assessing prognosis and tumor microenvironment in smokers with NSCLC. The role of FCGBP in NSCLC was found by cell experiments, which can be served as diagnostic biomarker and immunotherapy target for NSCLC.

Serum IL-24 combined with CA125 as screening and prognostic biomarkers for NSCLC.

Noninvasive and effective methods for early screening of non-small cell lung cancer(NSCLC) still need to be developed. At present, a reasonable conclusion is that a combination of tumor markers is a superior predictor of screening. Cytokines, as important regulators of cancer development, have great potential for the screening and prognosis of NSCLC. This study screened novel biomarkers related to the early screening and prognosis of NSCLC. In the present study, the biological significance and immunoregulation of interleukin-24(IL-24) were analyzed based on The Cancer Genome Atlas data. Next, 150 serum samples from initially treated patients with NSCLC and 70 controls were collected, and we obtained pathological sections from 60 patients with NSCLC. The ELISA and immunohistochemistryresults showed the differential expression of IL-24 and carbohydrate antigen 125(CA125). The results show that IL-24 is an important tumor suppressor in NSCLCthat helps to improve the poor prognosis of these patients. A significantly negative correlation between IL-24 and CA125 levels was also found. Notably, serum IL-24 levels were significantly negatively correlated with the TNM stage of patients with NSCLC, consistent with an important role for tumor suppressors in NSCLC. The receiver operating characteristic curve analysis showed that a combination of IL-24 and CA125 was an effective panel for discriminating patients with NSCLC from HD, and individuals with other lung diseases. Serum IL-24 and CA125 levels were identified as independent prognostic markers for NSCLC. The IL-24 and CA125 panel exhibited good performance in the screening of NSCLC.

Exosome-transported circ_0061407 and circ_0008103 play a tumour-repressive role and show diagnostic value in non-small-cell lung cancer

BackgroundCircular RNAs (circRNAs), one of the major contents of exosomes, have been shown to participate in the occurrence and progression of cancers. The role and the diagnostic potential of exosome-transported circRNAs in non-small-cell lung cancer (NSCLC) remain largely unknown.MethodsThe NSCLC-associated exosomal circ_0061407 and circ_0008103 were screened by circRNA microarray. The role of circ_0061407 and circ_0008103 in NSCLC was examined in vitro and in vivo. The encapsulation of the two circRNAs into exosomes and the transport to recipient cells were observed by confocal microscopy. The effects of exosome-transported circ_0061407 and circ_0008103 on recipient cells were investigated using a co-culture device. Bioinformatics analyses were performed to predict the mechanisms by which circ_0061407 and circ_0008103 affected NSCLC. The quantitative polymerase chain reaction was used to quantify the exosome-containing circ_0061407 and circ_0008103 in the serum samples of healthy, pneumonia, benign lung tumours, and NSCLC. The diagnostic efficacy was evaluated using receiver operating characteristic curves.ResultsThe levels of circ_0061407 and circ_0008103 within exosomes were down-regulated in the serum of patients with NSCLC. The up-regulation of circ_0061407 and circ_0008103 inhibited the proliferation, migration/invasion, cloning formation of NSCLC cells in vitro and inhibited lung tumour growth in vivo. Circ_0061407 and circ_0008103 were observed to be packaged in exosomes and transported to recipient cells, where they inhibited the proliferation, migration/invasion, and cloning formation abilities of the recipient cells. Moreover, circ_0061407 and circ_0008103 might be involved in the progression of NSCLC by interacting with microRNAs and proteins. Additionally, lower serum exosomal circ_0061407 and circ_0008103 levels were associated with advanced pathological staging and distant metastasis.ConclusionsThis study identified two novel exosome-transported circRNAs (circ_0061407 and circ_0008103) associated with NSCLC. These findings may provide additional insights into the development of NSCLC and potential diagnostic biomarkers for NSCLC.

Cuproptosis-related gene LIPT1 as a prognostic indicator in non-small cell lung cancer: Functional involvement and regulation of ATOX1 expression.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths, necessitating a deeper understanding of novel cell death pathways like cuproptosis. This study explored the relevance of cuproptosis-related genes in NSCLC and their potential prognostic significance. We analyzed the expression of 16 cuproptosis-related genes in 1017 NSCLC tumors and 578 Genotype-Tissue Expression (GTEx) normal samples from The Cancer Genome Atlas (TCGA) to identify significant genes. A risk model and prognostic nomogram were employed to identify the pivotal prognostic gene. Further in vitro experiments were conducted to investigate the functions of the identified genes in NSCLC cell lines. LIPT1, a gene for lipoate-protein ligase 1 enzyme, emerged as the central prognostic gene with decreased expression in NSCLC. Importantly, elevated LIPT1 levels were associated with a favorable prognosis for NSCLC patients. Overexpression of LIPT1 inhibited cell growth and enhanced apoptosis in NSCLC. We confirmed that LIPT1 downregulates the copper chaperone gene antioxidant 1 (ATOX1), thereby impeding NSCLC progression. Our study identified LIPT1 as a valuable prognostic biomarker in NSCLC as it elucidates its tumor-inhibitory role through the modulation of ATOX1. These findings offered insights into the potential therapeutic targeting of LIPT1 in NSCLC, contributing to a deeper understanding of this deadly disease.

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.

Identifying lncRNAs and mRNAs related to survival of NSCLC based on bioinformatic analysis and machine learning.

Non-small cell lung cancer (NSCLC) is the most common histopathological type, and it is purposeful for screening potential prognostic biomarkers for NSCLC. This study aims to identify the lncRNAs and mRNAs related to survival of non-small cell lung cancer (NSCLC). The expression profile data of lung adenocarcinoma and lung squamous cell carcinoma were downloaded in The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) dataset. A total of eight survival related long non-coding RNAs (lncRNAs) and 262 survival related mRNAs were filtered. By gene set enrichment analysis, 17 significantly correlated Gene Ontology signal pathways and 14 Kyoto Encyclopedia of Genes and Genomes signal pathways were screened. Based on the clinical survival and prognosis information of the samples, we screened eight lncRNAs and 193 mRNAs by single factor Cox regression analysis. Further single and multifactor Cox regression analysis were performed, 30 independent prognostication-related mRNAs were obtained. The PPI network was further constructed. We then performed the machine learning algorithms (Least absolute shrinkage and selection operator, Recursive feature elimination, and Random forest) to screen the optimized DEGs combination, and a total of 17 overlapping mRNAs were obtained. Based on the 17 characteristic mRNAs obtained, we firstly built a Nomogram prediction model, and the ROC values of training set and testing set were 0.835 and 0.767, respectively. By overlapping the 17 characteristic mRNAs and PPI network hub genes, three genes were obtained: CDC6, CEP55, TYMS, which were considered as key factors associated with survival of NSCLC. The in vitro experiments were performed to examine the effect of CDC6, CEP55, and TYMS on NSCLC cells. Finally, the lncRNAs-mRNAs networks were constructed.

Tumor-suppressive miR-4732-3p is sorted into fucosylated exosome by hnRNPK to avoid the inhibition of lung cancer progression

BackgroundAberrant fucosylation observed in cancer cells contributes to an augmented release of fucosylated exosomes into the bloodstream, where miRNAs including miR-4732-3p hold promise as potential tumor biomarkers in our pilot study. However, the mechanisms underlying the sorting of miR-4732-3p into fucosylated exosomes during lung cancer progression remain poorly understood.MethodsA fucose-captured strategy based on lentil lectin-magnetic beads was utilized to isolate fucosylated exosomes and evaluate the efficiency for capturing tumor-derived exosomes using nanoparticle tracking analysis (NTA). Fluorescence in situ hybridization (FISH) and qRT-PCR were performed to determine the levels of miR-4732-3p in non-small cell lung cancer (NSCLC) tissue samples. A co-culture system was established to assess the release of miRNA via exosomes from NSCLC cells. RNA immunoprecipitation (RIP) and miRNA pull-down were applied to validate the interaction between miR-4732-3p and heterogeneous nuclear ribonucleoprotein K (hnRNPK) protein. Cell functional assays, cell derived xenograft, dual-luciferase reporter experiments, and western blot were applied to examine the effects of miR-4732-3p on MFSD12 and its downstream signaling pathways, and the impact of hnRNPK in NSCLC.ResultsWe enriched exosomes derived from NSCLC cells using the fucose-captured strategy and detected a significant upregulation of miR-4732-3p in fucosylated exosomes present in the serum, while its expression declined in NSCLC tissues. miR-4732-3p functioned as a tumor suppressor in NSCLC by targeting 3'UTR of MFSD12, thereby inhibiting AKT/p21 signaling pathway to induce cell cycle arrest in G2/M phase. NSCLC cells preferentially released miR-4732-3p via exosomes instead of retaining them intracellularly, which was facilitated by the interaction of miR-4732-3p with hnRNPK protein for selective sorting into fucosylated exosomes. Moreover, knockdown of hnRNPK suppressed NSCLC cell proliferation, with the elevated levels of miR-4732-3p in NSCLC tissues but the decreased expression in serum fucosylated exosomes.ConclusionsNSCLC cells escape suppressive effects of miR-4732-3p through hnRNPK-mediated sorting of them into fucosylated exosomes, thus supporting cell malignant properties and promoting NSCLC progression. Our study provides a promising biomarker for NSCLC and opens a novel avenue for NSCLC therapy by targeting hnRNPK to prevent the "exosome escape" of tumor-suppressive miR-4732-3p from NSCLC cells.

Pirin Promotes the Progression of Non-Small-Cell Lung Cancer by Increasing ODC1 to Suppress Autophagy.

Non-small-cell lung cancer (NSCLC), a common malignant tumor, requires deeper pathogenesis investigation. Autophagy is an evolutionarily conserved lysosomal degradation process that is frequently blocked during cancer progression. It is an urgent need to determine the novel autophagy-associated regulators in NSCLC. Here, we found that pirin was upregulated in NSCLC, and its expression was positively correlated with poor prognosis. Overexpression of pirin inhibited autophagy and promoted NSCLC proliferation. We then performed data-independent acquisition-based quantitative proteomics to identify the differentially expressed proteins (DEPs) in pirin-overexpression (OE) or pirin-knockdown (KD) cells. Among the pirin-regulated DEPs, ornithine decarboxylase 1 (ODC1) was downregulated in pirin-KD cells while upregulated along with pirin overexpression. ODC1 depletion reversed the pirin-induced autophagy inhibition and pro-proliferation effect in A549 and H460 cells. Immunohistochemistry showed that ODC1 was highly expressed in NSCLC cancer tissues and positively related with pirin. Notably, NSCLC patients with pirinhigh/ODC1high had a higher risk in terms of overall survival. In summary, we identified pirin and ODC1 as a novel cluster of prognostic biomarkers for NSCLC and highlighted the potential oncogenic role of the pirin/ODC1/autophagy axis in this cancer type. Targeting this pathway represents a possible therapeutic approach to treat NSCLC.

Advancing Nonsmall Cell Lung Cancer Diagnosis Accuracy via Dual Detection Fluorescent Nanoprobes.

Among the primary threats to human health worldwide, nonsmall cell lung cancer (NSCLC) remains a significant factor and is a leading cause of cancer-related deaths. Due to subtle early symptoms, NSCLC patients are diagnosed at advanced stages, resulting in low survival rates. Herein, novel Au-Se bond nanoprobes (NPs) designed for the specific detection of Calpain-2 (CAPN2) and Human Neutrophil Elastase (HNE), pivotal biomarkers in NSCLC, were developed. The NPs demonstrated exceptional specificity and sensitivity toward CAPN2 and HNE, enabling dual-color fluorescence imaging to distinguish between NSCLC cells and normal lung cells effectively. The NPs' performance was consistent across a wide pH range (6.2 to 8.0), and it exhibited remarkable resistance to biological thiol interference, indicating its robustness in complex physiological environments. These findings suggest the nanoprobe is a promising tool for early NSCLC diagnosis, offering a novel approach for enhancing the accuracy of cancer detection.

BIRC3-HSP90B1 Interaction Inhibits Non-Small Cell Lung Cancer Progression through the Extracellular Signal-Regulated Kinase Pathway.

The long-term prognosis of nonsmall cell lung cancer (NSCLC) remains unsatisfactory, which is a major challenge in lung cancer treatment. BIRC3 is an inhibitor of apoptosis (IAP) protein that contributes to tumor regulation. However, the underlying regulatory mechanisms of BIRC3 in NSCLC remains unknown. We initiated an analysis of BIRC3 expression data in NSCLC tumors and adjacent tissues using the TCGA and GEO databases and examined the variations in prognosis. Further, we conducted overexpression (OE) and knockdown (KD) studies on BIRC3 to evaluate its effects on NSCLC cell proliferation, migration, and invasion. Additionally, through utilization of a nude mouse model, the regulatory effects of BIRC3 on NSCLC were verified in vivo. Co-immunoprecipitation (Co-IP) assay served to pinpoint the proteins with which BIRC3 interacts. The results indicated that BIRC3 is down-regulated in NSCLC tissues and that patients with high BIRC3 expression demonstrate a better prognosis. BIRC3 is a tumor suppressor, inhibiting the proliferation and metastasis of NSCLC. Co-IP results revealed that BIRC3 interacts with HSP90B1, leading to a decrease in HSP90B1 expression and subsequent negative regulation of the ERK signaling pathway. BIRC3 may serve as a prognostic biomarker for NSCLC. It directly interacts with HSP90B1 to negatively regulate the ERK signaling pathway, thereby hindering the progression of NSCLC.

CircMYBL1 suppressed acquired resistance to osimertinib in non-small-cell lung cancer

Circular RNAs (circRNAs) play an important role in the development of acquired resistance to many anticancer drugs. We developed the Non-Small-Cell Lung Cancer (NSCLC) cell lines with acquired resistance to osimertinib, a third-generation of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), and evaluated the different expression profiles of circRNAs in osimertinib-sensitive and -resistant NSCLC cell lines using RNA sequencing (RNA-Seq). The expression of selected differentially expressed circRNAs was verified using quantitative real-time PCR (qRT-PCR) in paired osimertinib-sensitive and -resistant NSCLC cell lines, and in plasma samples of osimertinib-sensitive and -resistant NSCLC patients. We found that circMYBL1(has_circ_0136924) was downregulated after acquired resistance to osimertinib, inhibiting circMYBL1 expression facilitated the proliferation, migration, and invasion in osimertinib-sensitive NSCLC cells. CircMYBL1 may be a novel molecular biomarker and therapeutic target for osimertinib-resistant NSCLC.

Isolation and Characterization of a Triterpenoid Compound from Sodhita <i>Semecarpus anacardium</i> L. Drupe Ethanolic Extract and its Anticancer Potential <i>In Vitro</i>

Semecarpus anacardium is listed in Schedule E1 of the Drugs and Cosmetics Act, of 1940. It is purified (sodhana) and used in traditional medicine to treat various diseases, including gulma (hard mass/cystic growth or lump). Studies on Semecarpus anacardium in Non-Small Cell Lung Cancer (NSCLC) cell lines are hitherto unavailable, hence this study was conducted. The drupes of sodhita Semecarpus anacardium were extracted with solvents of different polarity, and the half-maximal inhibitory concentration (IC50) of each extract was determined in the NSCLC cell line (A549). The ethanolic extract with an IC50 of 47.09±0.032 μg/ml was found to be better than the other extracts and, therefore subjected to compound isolation by wet-pack column chromatography. The fractions (6-9 and 15-19) with a single spot identified by thin-layer chromatography were crystallized (56 mg) and used for spectral techniques. The probable compound structure of C30H50O2, elucidated by FTIR, 1H and 13C NMR revealed it as a triterpenoid derivative. The IC50 of the isolated compound was 15.73±0.019 μg/ml which was further used to investigate anti-cancer properties The treated cells showed apoptotic features like cell blebbing, loss of cell adhesion and membrane integrity. A semi-fragmented piece of unresolved DNA and G2/M phase arrest was observed in the treated cells. RT-qPCR analysis revealed that the expression of the NSCLC biomarkers c-ROS oncogene-1 (ROS1) and proinflammatory factor nuclear factor kappa B (NF-κB) was significantly (p<0.05) downregulated in the treated cells. Based on these observations, it was concluded that the isolated compound could be a potential candidate for lung cancer therapy.

LncRNA RGMB-AS1 inhibits HMOX1 ubiquitination and NAA10 activation to induce ferroptosis in non-small cell lung cancer

Ferroptosis, an iron-dependent regulated cell death caused by excessive lipid peroxide accumulation, has emerged as a promising therapeutic target in various cancers, including non-small cell lung cancer (NSCLC). In this study, we identified the long non-coding RNA RGMB-AS1 as a key regulator of ferroptosis in NSCLC. Mechanistically, RGMB-AS1 interacted with heme oxygenase 1 (HMOX1) and prevented its ubiquitination by the E3 ligase TRC8, leading to increased HMOX1 stability and enhanced ferroptosis. Additionally, RGMB-AS1 bound to the 82–87 amino acid region of N-alpha-acetyltransferase 10 (NAA10), stimulating its acetyltransferase activity and promoting the conversion of acetyl-CoA to HMG-CoA, further contributing to ferroptosis. The RGMB-AS1-HMOX1 and RGMB-AS1-NAA10 axes synergistically inhibited NSCLC growth both in vitro and in vivo. Clinically, low RGMB-AS1 expression was associated with advanced tumor stage and poor overall survival in NSCLC patients. Furthermore, adeno-associated virus-mediated RGMB-AS1 overexpression significantly suppressed tumor growth in mouse xenograft models. Our findings uncover a novel lncRNA-mediated regulatory mechanism of ferroptosis and highlight the potential of RGMB-AS1 as a prognostic biomarker and therapeutic target in NSCLC.

Can hypoxia marker carbonic anhydrase IX serve as a potential new diagnostic marker and therapeutic target of non-small cell lung cancer?

Lung cancer represents the leading cause of cancer-related deaths. Non-small cell lung cancer (NSCLC), the most common form of lung cancer, is a molecularly heterogeneous disease with intratumoral heterogeneity and a significant mutational burden associated with clinical outcome. Tumor microenvironment (TME) plays a fundamental role in the initiation and progression of primary de novo lung cancer and significantly influences the response of tumor cells to therapy. Hypoxia, an integral part of the tumor microenvironment and a serious clinical phenomenon, is associated with increased genetic instability and a more aggressive phenotype of NSCLC, which correlates with the risk of metastasis. Low oxygen concentration influences all components of TME including the immune microenvironment. Hypoxia-inducible pathway activated in response to low oxygen supply mediates the expression of genes important for the adaptation of tumor cells to microenvironmental changes. A highly active transmembrane hypoxia-induced metalloenzyme - carbonic anhydrase IX (CAIX), as a part of transport metabolon, contributes to the maintenance of intracellular pH within physiological values and to the acidification of the extracellular space. CAIX supports cell migration and invasion and plays an important role in NSCLC tumor tissue and pleural effusion. Due to its high expression, it also represents a potential diagnostic differential biomarker and therapeutic target in NSCLC. To test new potential targeted therapeutic compounds, suitable models are required that more faithfully simulate tumor tissue, TME components, and spatial architecture.

CircFTO from M2 macrophage-derived small extracellular vesicles (sEV) enhances NSCLC malignancy by regulation miR-148a-3pPDK4 axis

BackgroundAccumulation studies found that tumor-associated macrophages (TAMs) are a predominant cell in tumor microenvironment (TME), which function essentially during tumor progression. By releasing bioactive molecules, including circRNA, small extracellular vesicles (sEV) modulate immune cell functions in the TME, thereby affecting non-small cell lung cancer (NSCLC) progression. Nevertheless, biology functions and molecular mechanisms of M2 macrophage-derived sEV circRNAs in NSCLC are unclear.MethodsCellular experiments were conducted to verify the M2 macrophage-derived sEV (M2-EV) roles in NSCLC. Differential circRNA expression in M0 and M2-EV was validated by RNA sequencing. circFTO expression in NSCLC patients and cells was investigated via real-time PCR and FISH. The biological mechanism of circFTO in NSCLC was validated by experiments. Our team isolated sEV from M2 macrophages (M2Ms) and found that M2-EV treatment promoted NSCLC CP, migration, and glycolysis.ResultsHigh-throughput sequencing found that circFTO was highly enriched in M2-EV. FISH and RT-qPCR confirmed that circFTO expression incremented in NSCLC tissues and cell lines. Clinical studies confirmed that high circFTO expression correlated negatively with NSCLC patient survival. Luciferase reporter analysis confirmed that miR-148a-3p and PDK4 were downstream targets of circFTO. circFTO knockdown inhibited NSCLC cell growth and metastasis in in vivo experiments. Downregulating miR-148a-3p or overexpressing PDK4 restored the malignancy of NSCLC, including proliferation, migration, and aerobic glycolysis after circFTO silencing.ConclusionThe study found that circFTO from M2-EV promoted NSCLC cell progression and glycolysis through miR-148a-3p/PDK4 axis. circFTO is a promising prognostic and diagnostic NSCLC biomarker and has the potential to be a candidate NSCLC therapy target.

Abstract 4286: Proteolytic degradation of BIGH3 can be quantified non-invasively in serum with biomarker potential for patients with non-small cell lung cancer

Abstract Introduction: Transforming growth factor beta induced protein ig-h3 (BIGH3/TGFBI) is expressed widely by multiple cell types, including macrophages and fibroblasts, and is participating in various biological processes including adhesion, migration, angiogenesis and wound healing. BIGH3 is known to bind multiple collagens and are often embedded in the matrix where it seems to function as a linker between ECM and cell surfaces. The increased proteolytic activity found in non-small cell lung cancer (NSCLC) may degrade BIGH3 that affect this interaction as well as generate small peptide fragments that can serve as novel non-invasive biomarker targets if released to circulation. The aim of this study is to develop a tool to quantify degraded BIGH3 non-invasively and explore its potential as a biomarker in NSCLC. Methods: An ELISA, named nordicBIGH3M-N targeting a cleaved fragment of BIGH3 was developed to reflect BIGH3 degradation and enable serological quantification. We incubated recombinant BIGH3 with and without collagenase to confirm that nordicBIGH3M-N only measured degraded BIGH3. In addition, we generated matrixes from normal fibroblasts and cancer associated fibroblasts (CAFs) grown with or without tgf-b. These matrixes were cleaved with collagenase to confirm that NordicBIGH3M-N could be generated from degradation of matrix. nordicBIGH3M-N was also measured in serum from 39 patients with NSCLC (18 patients with adenocarcinoma and 21 patients with squamous cell carcinoma) and 35 healthy controls. BIGH3M-N levels in serum from patients with the two subtypes of NSCLC and healthy controls was compared using Dunn’s multiple comparison test and area under receiver operation characteristic curves (AUROC). Results: BIGH3M-N was only detectable after incubating recombinant BIGH3 and fibroblasts matrices with collagenase. A 7-fold increase of BIGH3M-N levels was seen between the collagenase degraded matrix from tgf-b treated fibroblasts compared to the degraded matrix from untreated fibroblasts. No difference in BIGH3M-N levels was observed between degraded matrix from tgf-b treated or untreated CAFs. BIGH3M-N was significantly elevated in patients with squamous cell carcinoma compared to healthy controls (p = 0.006, AUROC = 0.73) and patients with adenocarcinoma (p = 0.022, AUROC = 0.78). However, there was no significant difference between BIGH3M-N levels in serum from patients with adenocarcinoma and healthy controls (p>0.99, AUROC = 0.51). Conclusion: Degradation of BIGH3 can be reflected by non-invasive quantification of the cleaved fragment of BIGH3, BIGH3M-N, in serum. BIGH3M-N is a promising biomarker in NSCLC with potential for discriminating between subtypes. Moreover, BIGH3M-N is connected to fibroblast matrix biology so the optimal use for this biomarker might be in combination with other ECM biomarkers. Citation Format: Rasmus S. Pedersen, Morten Karsdal, Nicholas Willumsen. Proteolytic degradation of BIGH3 can be quantified non-invasively in serum with biomarker potential for 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 4286.

Abstract 4287: Serological quantification of fibroblast activation protein (FAP) cleaved type III collagen: A biomarker for FAP activity

Abstract Introduction: Fibroblast activation protein (FAP) has unique proteolytic activity with very low expression in healthy tissues, while being upregulated in diseased tissues including many types of cancer for example non-small cell lung cancer (NSCLC). The disease specific expression and unique proteolytic activity have made FAP an interesting protein to be utilized for drug targeting purposes. Therefore, it is important to identify the patients with FAP activity. The aim of the study was to develop a tool to non-invasively quantify FAP activity and investigate its potential as a biomarker for patients with NSCLC. Methods: We developed an ELISA (named C3F), with monoclonal antibodies raised against a FAP generated fragment of type III collagen that was identified by MS, to quantify FAP-mediated cleavage of type III collagen in serum to reflect proteolytic activity of FAP. We confirmed that the biomarker reflects proteolytic activity of FAP by incubating type III collagen with or without FAP and measured C3F. In addition, we confirmed elevated FAP expression in NSCLC (adenocarcinoma and squamous cell carcinoma) compared to healthy tissue and correlation between FAP and COL3A1 expression using data from The Cancer Genome Atlas (TCGA). The comparisons between FAP expression in healthy and cancer tissue was done using one-way ANOVA and correlation between FAP and COL3A1 was done using Spearman correlation. Lastly, C3F was measured in serum samples from patients with NSCLC (n = 109) and healthy controls (n = 42) and the two groups was compared using Mann Whitney test and area under receiver operating characteristic curves (AUROCs). Results: A C3F signal only appeared after type III collagen incubation with FAP, supporting that C3F reflects FAP proteolytic activity. C3F was significantly elevated in serum from patients with NSCLC compared to healthy controls (p < 0.0001, AUROC = 0.78). In support, FAP expression (TCGA) was significantly elevated in NSCLC tissue compared to normal lung tissue (p < 0.0001) and FAP expression correlated with expression of COL3A1 (persons r = 0.83). Conclusion: The FAP-cleavage mediated type III collagen fragment C3F reflects FAP activity and can be quantified non-invasively in serum. In this study we show C3F as promising diagnostic biomarker in NSCLC, however as the biomarker reflects FAP activity the optimal use is more is potentially as a predicter of treatment response from FAP-based drug delivery systems. Citation Format: Rasmus Sund Pedersen, Morten Karsdal, Nicholas Willumsen. Serological quantification of fibroblast activation protein (FAP) cleaved type III collagen: A biomarker for FAP activity [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 4287.

Abstract 4912: Unsupervised detection of stromal phenotypes with distinct fibrogenic and inflamed properties in NSCLC

Abstract Background: Understanding the composition of cancer-associated stroma (CAS) is vital, as the number and location of immune cells and fibroblasts, as well as the degree of extracellular matrix deposition, have implications for cancer progression and response to treatment, including in non-small cell lung cancer (NSCLC). Manual analysis of CAS does not fully describe the stromal milieu, especially from a spatial perspective, and is highly subjective. To this end, we have developed an unsupervised machine learning (ML) model to characterize the CAS in NSCLC from hematoxylin and eosin (H&E) stained whole slide images (WSI) at scale. Methods: PathExploreTM models were deployed to predict stromal tissue and cell types, while another ML model was used to detect collagen fibers from H&E stained WSIs from the TCGA LUAD (N=536) and LUSC (N=464) datasets. Stroma was divided into small regions (median = 0.02 mm2), and 88 features characterizing cell distribution, tissue composition and fiber density were extracted from each region. Graphs were generated connecting neighboring regions (nodes), and an unsupervised variational graph auto-encoder (VGAE) model was trained to learn 8 latent features through dimensionality reduction. Stromal phenotypes were then derived from the latent features using k-means clustering. The fraction of each phenotype in the stroma was correlated against immune- and stroma-related gene expression signatures (GES) and overall survival (OS). Results: Deployment of VGAE on LUAD and LUSC WSIs revealed three distinct stromal phenotypes - P0, P1 and P2. Fibroblast density was elevated in P0 and P1 regions (p<0.001), immune cell density was elevated in P2 regions (p<0.001), and collagen fiber intensity was highest in P1 regions (p<0.001). P2 enrichment was correlated with elevated expression of the T cell-inflamed gene expression profile (TGEP; Spearman ρ = 0.43 in LUAD; ρ = 0.27 in LUSC) and with improved OS (HR = 0.696; 95% CIs: 0.571-0.847 in LUSC). Conversely, P1 enrichment was positively associated with a transforming growth factor-β-induced cancer associated fibroblast GES (TGFβ-CAF: ρ = 0.19 in LUAD and ρ = 0.12 in LUSC) and poor OS (HR = 1.358; 95% CIs: 1.149-1.603 in LUSC). These phenotypes are consistent with fibroblast-enriched, collagen-depleted stroma (P0), collagen-rich, fibroblast-enriched tumor-promoting stroma (P1), and immune cell-enriched, tumor-suppressive stroma (P2). Conclusions: We describe an unsupervised, data-driven method of predicting stromal regions with discrete patterns of cell composition and collagen deposition in NSCLC. This approach identified three phenotypes of NSCLC stroma. These results highlight the ability of ML models to characterize and find meaningful patterns within the cell, tissue, and matrix components of a tumor. This work provides further evidence of the potential of ML to discover novel precision medicine biomarkers in NSCLC. Citation Format: Neel Patel, Nhat Le, Tan Nguyen, Fedaa Najdawi, Sandhya Srinivasan, Adam Stanford-Moore, Deeksha Kartik, Jun Zhang, Jacqueline Brosnan-Cashman, Robert Egger, Justin Lee, Matthew Bronnimann. Unsupervised detection of stromal phenotypes with distinct fibrogenic and inflamed properties in NSCLC [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 4912.

Abstract 7501: Gene expression profiling of rare cells captured from liquid biopsy using the genesis cell isolation system

Abstract Background: Targeting rare cell types such as circulating tumor cells (CTCs), considered to be metastatic precursors in cancer progression, may provide long-term clinical benefits by enhancing patient outcomes. The study of CTCs comes with technical challenges due to their heterogeneity and low cell numbers, leading to difficulties in genomic characterization. This study aims to overcome these technical challenges by developing a comprehensive gene expression profiling workflow using qPCR and RNA sequencing (RNA-Seq) on rare cells enriched with Genesis System with Celselect SlidesTM. Methods: To mimic CTCs in blood, we created a rare cell model sample by spiking A549 non-small cell lung cancer cells at concentrations of 50, 100, 500 and 1000 cells/ml in whole blood from healthy donors. The Genesis System, which can capture rare cells of 8µm-30µm, isolated 83.73% of spiked A549 cells in whole blood. Using the same blood samples without A549 spiked in, we enriched cells using the Genesis and it was used as a baseline for gene expression controls. High quality RNA was extracted from each of the enriched samples and used for both qPCR and RNA-Seq to detect the gene expression signature of the non-small cell lung cancer cells. Results: Our analyses revealed significant differential expression of cancer-related genes (p < 0.05) in all A549-spiked samples when compared to the blood-only controls. Notably, in the 50 cells/ml dilution, RNA-Seq analysis identified over 3,000 differentially expressed genes, encompassing protein-coding genes, long non-coding RNAs, and small non-coding RNAs. Among these, 759 genes were consistently present across all enriched samples, suggesting a common expression pattern. To investigate the potential utility of these genes as non-small cell lung cancer biomarkers, we constructed a custom qPCR array comprising 38 of these genes and pre-amplified their targets using 50pg of RNA from the enriched cell samples. Notably, six genes from this panel (MET, TOP2A, NF1, SPP1, GAPDH, and ANXA5) exhibited significant differential expression (+/- 4-fold, p<0.05) in the enriched samples. MET and TOP2A, in particular, are well-known biomarkers for non-small cell lung cancer. Conclusion: Our liquid biopsy workflow demonstrates the recovery of high-quality RNA from enriched rare cells, including CTCs. It enables comprehensive profiling of the tumor transcriptome, encompassing RNAs of various sizes, including miRNAs. Our approach has the potential to facilitate the identification of patient-specific biomarkers, enabling swift monitoring using preamplification and qPCR in advancement of targeted oncology therapy and research. Citation Format: Srikanth Perike, Angelica Olcott, Cissy Jiang, Nish Kumar, Nathan Knapp, Jennifer Placek, Candice Cox, Yoon-Tae Kang, Linda Lingelbach, Elizabeth Dreskin. Gene expression profiling of rare cells captured from liquid biopsy using the genesis cell isolation system [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 7501.

Abstract 383: Mic19 regulates mitophagy in non-small cell lung cancer under hypoxic microenvironment

Abstract Background: Mitophagy is a pivotal cellular process crucial for maintaining homeostasis in hypoxic conditions, selectively eliminating dysfunctional mitochondria through autophagy. This process is implicated in various diseases, including cancer. Non-Small Cell Lung Cancer (NSCLC), known for its prevalence and aggressiveness, often encounters hypoxic microenvironments. Mic19, a core protein of the mitochondrial cristae membrane, plays vital roles in maintaining mitochondrial architecture and function. However, its involvement in mitophagy in hypoxic NSCLC and the underlying molecular mechanisms remain inadequately explored. Methods: NSCLC cells were cultured under conditions with 1% O2 oxygen concentration to simulate a hypoxic microenvironment. Protein expression levels of Mic19 were analyzed using Western Blot and immunofluorescence (IF). Cellular proliferation and metastasis capability were assessed through CCK8 and transwell experiments. Immunoprecipitation experiments were conducted to validate the interaction between Mic19 and key mitophagy regulatory proteins. IF, confocal microscopy, ATP measurement, oxygen consumption rate (OCR), mitochondrial membrane potential, and transmission electron microscopy were employed to assess the formation of mitochondrial cristae and the level of mitophagy. Results: In hypoxic conditions, Mic19 exhibited up-regulation in NSCLC cells. Subsequent in vitro experiments confirmed Mic19's pivotal role in promoting the growth and metastasis of lung cancer cell lines. Through clinical data analysis, Mic19 emerged as an independent prognostic indicator for NSCLC. Inhibition of Mic19 expression exacerbated the disruption of mitochondrial morphology and structure in lung cancer cells, accompanied by the loss of mitochondrial cristae. Notably, Mic19 expression closely correlated with ATP and OCR production in tumor cells. Assessment of mitochondrial membrane potential and transmission electron microscopy revealed Mic19 as a critical downstream target of hypoxia, mediating mitophagy. Overall, these findings not only underscore the significance of Mic19 in regulating mitochondrial dynamics but also highlight its multifaceted role in tumor progression, positioning Mic19 as a promising therapeutic target and prognostic biomarker for NSCLC. Conclusion: This study provides novel insights into the mechanistic understanding of hypoxia-induced mitophagy in NSCLC, emphasizing the regulatory role of Mic19. Mic19 emerges as a potential therapeutic target for future interventions in NSCLC patients. Citation Format: Hua Huang, Chen Ding, Zhanrui Zhang, Di Wu, Chen Chen, Yongwen Li, Hongyu Liu, Jun Chen. Mic19 regulates mitophagy in non-small cell lung cancer under hypoxic microenvironment [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 383.