Reactive oxygen species (ROS)-induced aberrant oncogenic signalling has been proposed to mediate the progression and development of pleural mesothelioma (PM). In this study, we demonstrate how ROS promote oncogenic signalling, especially in the context of cell migration and immune evasion via YB-1 phosphorylation in mesothelial and PM cell models. Xanthine (X)- and xanthine oxidase (XO)-generated ROS exposure led to increased migration and a more elongated cell shape in mesothelial and PM cells in live-cell videomicroscopy analyses. These effects were associated with the enhanced phosphorylation of ERK, AKT, and YB-1 and the elevated gene expression of PD-L1 and PD-L2, which were analysed with immunoblotting and quantitative real-time RT-PCR, respectively. The pharmacological inhibition of AKT (ipatasertib), MEK (trametinib), and RSK (BI-D1870) resulted in the reversal of ROS-induced effects, with the strongest effects observed upon the inhibition of YB-1 phosphorylation by BI-D1870. The results suggest that ROS exposure has a strong impact on cell migration and immune evasion not only in PM cells but also in mesothelial cells, from which PM arises. Interfering with ROS-responsive kinase pathways, particularly YB-1 phosphorylation, could counteract pro-migratory and immune-evasive effects in PM.

The enrichment of immunosuppressive M2 macrophages, combined with diminished CD8+ T cell infiltration, represents a key mechanism driving tumor progression and limiting immunotherapy efficacy in non-small cell lung cancer (NSCLC). Here, we provide evidence that the purinergic P2Y2 receptor (P2RY2) is a key regulator of M2 macrophage enrichment and contributes to the exclusion of CD8+ T cells from the tumor microenvironment (TME). P2RY2 expression is significantly elevated in human NSCLC compared to non-malignant tissues and M2-like macrophages expressing P2RY2 are more prevalent in tumors with an advanced TNM (Tumor, Node, Metastasis) stage. Elevated P2RY2 mRNA levels are significantly associated with poorer overall survival in a NSCLC patients. Furthermore, we selectively inhibited P2RY2 in syngeneic or autochthonous mouse models of NSCLC driven by Kras or Egfr mutations. This resulted in a significant reduction of M2-like macrophages, enhanced CD8+ T cell migration and tumor infiltration and a marked decrease in tumor burden. Similar results were evident following the genetic deletion of P2ry2, validating the impact on the TME. Importantly, macrophages are the predominant P2RY2-expressing cells within the TME. Moreover, tumor-educated macrophages (TEMs) isolated from P2ry2-/- tumor-bearing mice exhibited reduced proliferation compared with wild-type macrophages when co-cultured with LLC1 cells, revealing a potential mechanism underlying P2RY2-mediated pro-tumorigenic activity. Our study underscores the clinical significance of P2RY2 in NSCLC and provides evidence of its pivotal role in the regulation of M2 macrophage enrichment and the exclusion of CD8+ T cells from the TME. Targeting P2RY2 may offer a novel immunotherapeutic intervention for NSCLC.

BACKGROUND:Characterizing the immune landscape of thymic epithelial tumors (TETs) is essential for patient stratification for emerging immunotherapeutic approaches and for optimizing follow-up strategies. In this study, we examined the expression patterns and clinical significance of five immune-related markers in a large cohort of TETs. MATERIAL AND METHODS:Expression of TIM3, OX40L, GTF2I, XPO1, and GITR was assessed by immunohistochemistry (IHC) in the epithelial and lymphocytic compartments of 137 surgically resected TETs and correlated with clinicopathological parameters and patient outcomes. Tumors were grouped according to their immune profile using cluster analysis. RESULTS:TIM3, GTF2I, and XPO1 showed high and consistent expression in the epithelial compartment of TETs across histological subgroups, whereas GITR expression was mostly absent. In the lymphocytic compartment, only XPO1 was highly expressed, with no significant differences between TET subtypes. Epithelial and lymphocytic OX40L expressions were significantly higher in patients with myasthenia gravis (vs. those without this autoimmune disorder; p = 0.004 and p = 0.045, respectively). Importantly, hierarchical clustering identified four distinct TET subgroups based on their combined immune marker expression profiles, which displayed widely divergent survival outcomes (p < 0.0001). Among these, the subgroup characterized by elevated epithelial expression of GTF2I, XPO1, and TIM3 showed worse survival in our multivariate model (HR 6.37; p = 0.025). CONCLUSIONS:TIM3, GTF2I, and XPO1 are highly expressed in the epithelial compartment of TETs, providing a rationale for therapeutic targeting in future immunotherapy trials. Differential IHC expression of TIM3, OX40L, GTF2I, XPO1, and GITR defines distinct TET subtypes with independent prognostic relevance, enabling more accurate risk assessment in these highly heterogeneous thoracic malignancies.

The current paradigm of inflammatory disease etiology is that it is caused by an aberrant increase in either gene activation or cell death. Studying the linear ubiquitin (linUb) chain assembly complex (LUBAC) was instrumental in proving that immune-receptor-dependent cell death can cause inflammatory disease. As LUBAC regulates both, gene activation and cell death, it can, however, also serve to evaluate the inflammatory-disease-initiating role of gene activation. Investigating mice with two different gene-activation-enhancing point mutations in LUBAC components showed that aberrantly increased gene activation in Hoil 1C458A/C458A mice did not cause pathology. In contrast, in HoipN101A/N101A mice it led to untoward cell death which, unexpectedly, was required for pathological inflammation, culminating in lethal lung fibrosis resembling human idiopathic pulmonary fibrosis (IPF). Hence, we here uncover a new etiology of inflammation, whereby aberrantly enhanced gene activation can be root cause of pathological, fibrogenic inflammation, yet intriguingly via inducing untoward cell death.

Abstract Introduction: Small cell lung cancer (SCLC) represents an aggressive malignancy that initially responds well to frontline platinum-based chemotherapy. However, most patients relapse quickly due to rapid development of chemoresistance, emphasizing the urgent need for novel therapeutic strategies to achieve durable clinical benefit. Methods: To elucidate the mechanisms driving treatment failure, we established in vitro SCLC models that acquired cisplatin resistance across distinct molecular subtypes. These models were characterized by proliferation, cell cycle, and proteomic assays. Drug screens were conducted to identify therapeutic vulnerabilities, including responses to unconjugated antibody-drug conjugate (ADC) payloads and targeted pathway inhibitors. Results: Cisplatin-resistant (CR) SCLC models exhibited broad cross-resistance to clinically relevant cytotoxic drugs such as carboplatin, lurbinectedin, and topoisomerase I and II (TOP1 and TOP2) inhibitors. Resistance was associated with diminished intracellular platinum accumulation, reduced proliferation, and altered cell cycle distribution with G2/M arrest or senescent-like features compared to the corresponding sensitive parental lines. Proteomic analysis of CR models, together with patient-derived samples at treatment relapse, revealed extensive metabolic reprogramming and downregulation of DNA repair and checkpoint control pathways, highlighting shared adaptive signatures. Screening of unconjugated ADC payloads uncovered mechanism-specific vulnerabilities. While CR models were resistant to DNA-damaging payloads such as calicheamicin, microtubule-disrupting ADC payloads (MMAE and DM1) retained sensitivity across all CR models. Responses to TOP1-directed ADC payloads varied and were associated with SLFN11 expression levels. Importantly, inhibition of ATR with ceralasertib synergized with DNA-damaging ADC payloads, re-sensitizing SLFN11-low resistant models by increasing DNA damage and inducing apoptosis. Conclusion: Our findings demonstrate shared adaptive mechanisms of acquired cisplatin resistance in SCLC, including checkpoint rewiring and metabolic flexibility. SLFN11 was identified as a key determinant of TOP1-directed ADC efficacy. Combining ATR inhibition with DNA-damaging ADC payloads represents a promising strategy to restore DNA damage-induced cell death in resistant SCLC. These insights provide a translational framework for developing rational ADC-based combination therapies to overcome chemoresistance in relapsed SCLC. Citation Format: Buesra Ernhofer, Lisa Glatt, Benjamin Morris, Beata Szeitz, Zsolt Megyesfalvi, Abigail Deloria, Diana Piesel, Laura Schnell, Kristiina Boettiger, Amirali Karimi, Monique Nilsson, Melinda Rezeli, Simon Heeke, John Victor Heymach, Clemens Aigner, Balazs Dome, Karin Schelch. In-depth profiling of SCLC models of acquired chemoresistance reveals SLFN11-dependent antibody-drug conjugate payload sensitivity and ATR inhibitor synergy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2971.

Small cell lung cancer (SCLC) is an aggressive thoracic disease characterized by rapid proliferation and early metastatic spread. The survival outcomes for patients with SCLC remain notoriously poor, underlining that only modest improvements have been achieved in clinical settings to date. However, insights gained from human tumors and preclinical models in recent years have shed light on the heterogeneous molecular profile of SCLC. Numerous research groups have, therefore, begun to stratify SCLC into subgroups based on differential transcription factor expression, the tumor immune microenvironment, and other criteria. As SCLC subtypes show major differences in their molecular landscape and biological behavior, they may offer unique therapeutic vulnerabilities and serve as a framework for future personalized clinical trials. Here, we summarize impactful classification attempts from the past 10 years, highlighting discrepancies and connections between the nomenclature of each study, and expound upon relevant factors of SCLC biology influencing subtype composition and plasticity. This review delves into the implications of subgrouping for understanding and treating SCLC, as well as potential future directions for SCLC research.

Ex vivo lung perfusion (EVLP) is a unique platform for lung assessment and preservation. While EVLP has been extensively investigated in donor lungs, its application to translational research is less well-defined. We aimed to evaluate and characterise the EVLP methodology using resected human cancerous lung. Between January 2021 and March 2023, 216 patients at our Institute underwent eligibility screening for experimental inclusion. Following pneumonectomy or lobectomy, 10 cases were included. The lung specimens were inflated, flushed with Perfadex®, and connected to an acellular perfusate circuit primed with Steen® solution. Physiological parameters were monitored at regular intervals with a blood gas analyser. The median duration of the experiments performed was 159 min, during which the functional parameters remained stable despite the progressive development of oedema. Importantly, elevated lactate levels did not correlate with functional deterioration defined by partial O2 and CO2 pressure levels despite a temporal non-significant decline in these parameters. We managed to perfuse pneumonectomy and lobectomy specimens with comparable results regarding the investigated physiological parameters. However, pneumonectomies were associated with shorter median warm ischemic times compared to lobectomies (29.5 min vs. 75 min, respectively). In this study, we have characterised proof-of-concept EVLP in cancerous human lungs regarding physiological parameters. Oedema management and clearly defined endpoints are essential in its translational application. These preliminary observations are intended to guide future investigators for improved protocols and establish standardised criteria for patient selection.

Abstract Small cell lung cancer (SCLC) is one of the most aggressive solid malignancies with limited treatment options. Access to clinically relevant SCLC tissue is restricted because surgical resection is rare in metastatic disease, making this cancer type difficult to study. Through our multicenter rapid research autopsy (RRA) program, however, we are able to collect not only primary tumors but also multiple metastatic lesions, providing a unique and rare collection of SCLC specimens. In this program, rapid autopsies are performed within four hours after death to ensure optimal tissue quality for downstream analyses. In the current project, we analyzed samples from seven patients with histologically confirmed SCLC, including clinical and metastatic lesions, as well as their corresponding patient-derived tumor xenografts (PDTXs) serially passaged through three generations. To assess whether the original tissue architecture and intratumoral heterogeneity are preserved across PDTX generations, we performed immunohistochemical and proteomic analyses, comparing molecular profiles between the original tumors and their corresponding PDTX models. A subset of SCLC clinical specimens, including matched PDTX-clinical pairs, confirmed that the histologic landscape of the tumors of origin is preserved in the derivative PDTX models. Samples were grouped by patient and anatomical site, and pairwise Pearson correlations of global protein abundance profiles were computed within each group. The mean within-lineage correlation was high (typically >0.8), and correlation heatmaps showed tight clustering across PDTX generations from the same lineage. Across all patients, the only pathways consistently and significantly downregulated in PDTX models compared with the original tumors were the ECM-receptor interaction and Complement and Coagulation Cascade pathways. When analyzing metastatic PDTX samples, we compared liver and lymph node metastases with their corresponding primary patient tumors across PDTX generations. In lymph node metastases, proteins involved in ECM-receptor interaction and EMT pathways were upregulated. In contrast, liver metastases showed upregulation of fatty acid metabolism and peroxisome proliferator-activated receptor signaling pathways, with downregulation of EMT, ECM-receptor interaction, E2F targets, and G2M checkpoint. These findings demonstrate that the proteomic identity of SCLC tumors is largely preserved during serial passaging in PDTX models, while site-specific adaptations emerge in distinct metastatic microenvironments. Citation Format: Sára Surguta, Laura Svajda, Zsolt Megyesfalvi, Bence Ferencz, Ildikó Kovács, Vivien Téglás, Lilla Horváth, Szilvia Török, Balázs Döme, Melinda Rezeli, Jozsef Tovari. Proteomic insights into metastatic small cell lung cancer using patient derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2108.

Background/Objective: The identification of novel non-invasive diagnostic and prognostic biomarkers is urgently needed in pleural mesothelioma (PM). While soluble mesothelin-related peptides (SMRP) are the most established circulating biomarker, their prognostic value is limited. A wide range of microRNAs (miRs) play diverse roles in regulating gene expression in PM. MiR-21 has been shown to be upregulated in mesothelioma tissue; nevertheless, the diagnostic and prognostic utility of miR-21 in the circulation and its association with survival in PM have not been extensively investigated to date. The objective of the current study was to evaluate miR-21 as a potential blood-based diagnostic and prognostic biomarker in PM. Methods: Plasma samples from PM patients (n = 94) were collected at the time of diagnosis, prior to treatment. Sex- and age-matched healthy individuals (n = 30) served as controls. MiR-21 levels were measured using quantitative RT-PCR and normalized to miR-16, and potential correlations with clinicopathological data were analyzed. Serum SMRP levels were measured in matched patients (n = 84), and a direct comparative analysis of miR-21 and SMRP was conducted. In situ hybridization (ISH) was used to confirm the presence of miR-21 in tumor cells. Results: Plasma miR-21 levels were significantly elevated in PM patients compared to healthy controls (p < 0.001), demonstrating good diagnostic performance (AUC 0.81). The localization of miR-21 in PM cells was confirmed by ISH. High miR-21 levels were associated with significantly shorter median overall survival (12.4 vs. 24.3 months, p < 0.001). Elevated SMRP levels were also associated with reduced survival (12.4 vs. 19.5 months, p = 0.032); however, SMRP did not retain independent prognostic significance in multivariable analysis. In contrast, high-circulating miR-21 was confirmed as an independent predictor for poor survival (HR 3.12, p < 0.001). Conclusions: Our findings highlight that circulating miR-21 is a potential non-invasive biomarker with both diagnostic and independent prognostic value in pleural mesothelioma and outperforms SMRP in multivariable survival analysis. Further research is warranted to validate its role in the biology of this disease and to assess its correlation with outcome and treatment responses.

Patients with lung metastases frequently receive anti-angiogenic (AA) therapies based on the histological characteristics of the primary tumor. However, clinical benefit remains limited, suggesting the presence of therapeutic resistance. The contribution of intratumoral drug distribution to this limited therapeutic benefit remains insufficiently understood.We aimed to determine whether differences in therapeutic response are associated with differences in intratumoral drug distribution in matched primary and lung metastasis models of breast and renal cell carcinoma.Matched primary and lung metastasis models were established using 4T1, MDA-MB-231, and RENCA cells. Mice received sunitinib or vehicle, and therapeutic response was evaluated by complementary histopathological analyses of tumor growth, microvessel area (MVA), hypoxia, and necrosis, together with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) assessment of intratumoral sunitinib distribution.Sunitinib significantly inhibited tumor growth and reduced MVA in all primary tumor models. In metastasis models, MVA was also significantly reduced, whereas therapeutic response varied between tumor types. MALDI-MSI demonstrated higher normalized intratumoral sunitinib signal in metastases than in corresponding primary tumors, particularly in the breast cancer models. Drug signal also differed between viable, necrotic, and hypoxic tumor regions, demonstrating substantial intratumoral heterogeneity.These findings demonstrate that integrating MALDI-MSI with histopathological analyses enables comparative assessment of intratumoral drug distribution in matched primary and metastatic tumor models. Despite consistent anti-vascular effects and detectable intratumoral drug signal, additional mechanisms beyond inadequate drug delivery are likely to contribute to the limited response observed in breast cancer lung metastases.

Small cell lung cancer (SCLC) is a highly aggressive malignancy with poor survival outcomes. The CD70-CD27 axis has been implicated in immune regulation and tumor progression across cancers, but its role in SCLC has not yet been elucidated. This research explores the expression patterns and prognostic significance of CD70 and CD27 in early-stage SCLC. In this retrospective study, we analyzed 190 surgically resected SCLC tumor samples using immunohistochemistry (IHC) for CD70 and CD27 expression and RNAscope for CD70 RNA detection. Immune infiltration was assessed using CD45, CD8, and CD20 staining. Quantification of RNAscope signals was performed using QPath software. Kaplan–Meier survival analysis and multivariate Cox regression were used to assess the prognostic impact of CD70, CD27, and immune cell infiltrates on overall survival (OS). CD70 was expressed in 46

Proteomic analysis resulting from the comparison of SCLC cell lines showing synergistic or additive responses to entinostat and cisplatin therapy

Background/Objectives: Malignant pleural mesothelioma (MPM) remains challenging to treat, with a poor prognosis. As controversy about clinical management continues, predictive biomarkers for patient selection to indicate the benefit of treatment modalities are urgently needed. Methods: In a retrospective analysis of 195 patients between 1994 and 2020 at the Department of Thoracic Surgery, Medical University of Vienna, Austria, the Mesothelioma Systemic Inflammation Score (MSIS)—consisting of pretreatment neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), C-reactive protein (CRP), and fibrinogen—was tested for its prognostic and predictive significance. The prognostic impact of MSIS was subsequently validated in an independent cohort of 80 patients treated at the Department of Thoracic Surgery, Karl Landsteiner Institute for Clinical and Translational Thoracic Surgery Research, Clinic Floridsdorf, Vienna, Austria. Results: Median overall survival (OS) was 14 months for the entire cohort (95% CI: 11.4–16.6). Patients undergoing multimodality treatment including macroscopic complete resection had a longer OS (22.3 months, 95% CI: 18.6–26.0; p < 0.001). In multivariable analysis, MSIS (p < 0.001), disease stage (p = 0.001), and the type of treatment (p = 0.004) were confirmed as independent predictors for OS. Higher MSIS was associated with shorter OS (p < 0.001). Significant survival benefit of multimodality regimens including surgery was limited to patients with low MSIS. Among patients with low (≤ 2) MSIS, multimodality therapy was associated with significantly prolonged OS when compared with chemo- and/or radiotherapy alone (25.8 months [95% CI: 16.4–35.3] vs. 14.4 months [95% CI: 10.4–18.4], p < 0.001). In contrast, among patients with elevated MSIS, no survival benefit was achieved by surgery over conservative treatment (11.8 months [95% CI: 8.3–15.3] vs. 8.2 months [95% CI: 5.2–11.3], p = 0.233). The ability of MSIS to predict survival was equivalent between the baseline and the independent validation cohort (p < 0.001). Conclusions: The Mesothelioma Systemic Inflammation Score was found to be an independent prognostic score in pleural mesothelioma, predicting benefit from macroscopic complete resection as part of multimodality treatment in distinct patients.

Human tumors are diverse in their natural history and response to treatment, which in part results from genetic and transcriptomic heterogeneity. In clinical practice, single-site needle biopsies are used to sample this diversity, but cancer biomarkers may be confounded by spatiogenomic heterogeneity within individual tumors. Here we investigate clonally expressed genes as a solution to the sampling bias problem by analyzing multiregion whole-exome and RNA sequencing data for 450 tumor regions from 184 patients with lung adenocarcinoma in the TRACERx study. We prospectively validate the survival association of a clonal expression biomarker, Outcome Risk Associated Clonal Lung Expression (ORACLE), in combination with clinicopathological risk factors, and in stage I disease. We expand our mechanistic understanding, discovering that clonal transcriptional signals are detectable before tissue invasion, act as a molecular fingerprint for lethal metastatic clones and predict chemotherapy sensitivity. Lastly, we find that ORACLE summarizes the prognostic information encoded by genetic evolutionary measures, including chromosomal instability, as a concise 23-transcript assay.

Quantifying solute transport across epithelial cell layers grown on transwell inserts is a common approach in early-stage drug development to estimate pharmacokinetic properties such as absorption and bioavailability. To increase throughput and reduce variability, these assays are increasingly automated, including the use of robotic or microfluidic systems for time-resolved sampling. However, both automated and manual sampling can introduce systematic artifacts, such as residual volume retention and surface adsorption, that distort concentration time series and affect downstream analysis. To fully realize the potential precision of automated measurements, we propose a mathematical correction to account for sampling artifacts; then, to fit the corrected data to a three-compartment model that captures membrane diffusion, cellular sequestration, and metabolic loss. The method is demonstrated on datasets from transwell epithelial barrier transport assays. We suggest that the considered three-compartment model yields mechanistically more meaningful parameters than the conventional apparent permeability (Papp) measure. The proposed approach thus enables more accurate characterization of analyte interactions with the barrier cell layer, supporting better-informed assessments of compound behavior in vitro transport systems. Quantifying solute transport across epithelial cell layers grown on transwell inserts is a common approach in early-stage drug development to estimate pharmacokinetic properties such as absorption and bioavailability. To increase throughput and reduce variability, these assays are increasingly automated, including the use of robotic or microfluidic systems for time-resolved sampling. However, both automated and manual sampling can introduce systematic artifacts, such as residual volume retention and surface adsorption, that distort concentration time series and affect downstream analysis. To fully realize the potential precision of automated measurements, we propose a mathematical correction to account for sampling artifacts; then, to fit the corrected data to a three-compartment model that captures membrane diffusion, cellular sequestration, and metabolic loss. The method is demonstrated on datasets from transwell epithelial barrier transport assays. We suggest that the considered three-compartment model yields mechanistically more meaningful parameters than the conventional apparent permeability (Papp) measure. The proposed approach thus enables more accurate characterization of analyte interactions with the barrier cell layer, supporting better-informed assessments of compound behavior in vitro transport systems.

Objectives: Lung squamous cell carcinoma (LUSC) is associated with a poor prognosis and a lack of specific treatment options. The dysregulation of activin A (ActA) has been reported in various malignancies. Herein, we investigated the diagnostic and prognostic significance of ActA in LUSC. Materials and methods: ActA concentrations were measured using ELISA in plasma samples of 128 LUSC patients (stage I-IV) and 73 controls, and correlated those values with clinicopathological parameters and survival. Results: ActA plasma levels were significantly higher in therapy-naive LUSC patients compared to controls (444.1 ± 310.9 pg/mL vs 338.9 ± 145.5 pg/mL, p = 0.010). ActA levels significantly correlated with advanced stage as well as with T and N factors. High circulating ActA levels were significantly increased in metastatic disease patients compared to M0 disease. Further, patients with ActA levels above a computationally established optimal cut-off value of 443.0 pg/mL had a significantly worse median overall (OS, 17.63 vs 64.77 months, HR 0.391, 95 % CI 0.200–0.762, p < 0.001) and median disease-/progression-free survival (DFS/PFS; 11.57 vs 30.20 months, HR 0.502, 95 % CI 0.248–1.019, p = 0.020). Multivariate analysis revealed that high ActA levels were an independent prognostic factor for shorter OS (p = 0.001) and DFS/PFS (p = 0.018). A newly developed score combining CRP and ActA levels was also an independent prognostic factor for OS and DFS/PFS. Conclusion: Measurement of circulating ActA levels may help identify advanced-stage LUSC patients, and this value could serve as a prognostic parameter in LUSC. Thus, ActA may be a novel blood-based biomarker for identifying LUSC patients with distant metastasis.

Proteomic analysis resulting from the comparison of entinostat sensitive versus resistant cell lines

Small cell lung cancer (SCLC) is an aggressive malignancy with distinct molecular subtypes defined by transcription factors and inflammatory characteristics. This follow-up study aimed to validate the unique metabolic phenotype in achaete-scute homologue 1 (ASCL1)-driven SCLC cell lines and human tumor tissue. Metabolic alterations were analyzed using proteomic data. Structural and functional differences of mitochondria were investigated using qPCR, flow cytometry, confocal imaging, and transmission electron microscopy and seahorse assays. Several metabolic inhibitors were tested using MTT-based and clonogenic assays. Single-cell enzyme activity assays were conducted on cell lines and tumor tissue samples of SCLC patients. We found increased mitochondrial numbers correlating with higher oxidative phosphorylation activity in ASCL1-dominant cells compared to other SCLC subtypes. Metabolic inhibitors targeting mitochondrial respiratory complex-I or carnitine palmitoyltransferase 1 revealed higher responsiveness in SCLC-A. Conversely, we demonstrated that non-ASCL1-driven SCLCs with lower oxidative signatures show dependence on glutaminolysis as evidenced by the enhanced susceptibility to glutaminase inhibition. Accordingly, we detected increased glutamate-dehydrogenase activity in non-ASCL1-dominant cell lines as well as in human SCLC tissue samples. Distinct SCLC subtypes exhibit unique metabolic vulnerabilities, suggesting potential for subtype-specific therapies targeting the respiratory chain, fatty acid transport, or glutaminolysis.