Healthy Lung Research Articles

TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation.

Epithelium-derived cytokines or alarmins, such as interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP), are major players in type 2 immunity and asthma. Here, we demonstrate that TNF-like ligand 1A (TL1A) is an epithelial alarmin, constitutively expressed in alveolar epithelium at steady state in both mice and humans, which cooperates with IL-33 for early induction of IL-9high ILC2s during the initiation of allergic airway inflammation. Upon synergistic activation by IL-33 and TL1A, lung ILC2s acquire a transient IL-9highGATA3low "ILC9" phenotype and produce prodigious amounts of IL-9. A combination of large-scale proteomic analyses, lung intravital microscopy, and adoptive transfer of ILC9 cells revealed that high IL-9 expression distinguishes a multicytokine-producing state-of-activated ILC2s with an increased capacity to initiate IL-5-dependent allergic airway inflammation. Similar to IL-33 and TSLP, TL1A is expressed in airway basal cells in healthy and asthmatic human lungs. Together, these results indicate that TL1A is an epithelium-derived cytokine and an important cofactor of IL-33 in the airways.

Effect of Silver-Graphene Oxide-Cobalt Oxide Nanocomposite on Cytotoxic Levels in MRC-5 and HepG2 Cell Lines and Molecular Docking Studies

The cytotoxic properties of cobalt oxide (Co3O4) nanoparticles (NPs), in addition to graphene oxide (GO)-Co3O4 and silver (Ag)-GO-Co3O4 nanocomposites (NCs), were evaluated against both human healthy lung fibroblast (MRC-5) and hepatocellular carcinoma (HepG2) cell lines utilizing the XTT assay. The investigation revealed that synthesized Co3O4 NPs and NCs (GO-Co3O4 and Ag-GO-Co3O4) elicited significant cytotoxic responses in MRC-5 and HepG2 cell lines in a concentration-dependent manner. Through molecular docking analyses, it was observed that all fabricated nanomaterials exhibited DNA recognition via minor groove binding, with molecular affinities ranging from − 4.82 to -11.66 kcal/mol. Furthermore, the docking outcomes illustrated that the angular conformations of GO-Co3O4 and Ag-GO-Co3O4 conferred ‘shape-selective’ characteristics as DNA minor groove binders, leading to heightened cytotoxicity, particularly in the HepG2 cell line compared to the normal MRC-5 cell line.

Abstract LB058: N-glycan deglycosylation ameliorates anti-HER2 antibody drug conjugate-induced interstitial pneumonia in preclinical models

Abstract Background: Current clinical application of trastuzumab deruxtecan (T-DXd), an anti-HER2 antibody-drug conjugate (ADC) has been severely limited by interstitial lung disease (ILD). Recent studies suggest a correlation between ILD and the uptake of T-DXd by alveolar macrophages (AMs), which are major innate immune cells in lung tissues. This underlying mechanism of AMs phagocytosing T-DXd was predicted to be the binding interaction between the crystallizable fragment (Fc) of trastuzumab and the Fcγ receptors on the cell surface of AMs, exerting effector functions. Since monoclonal antibodies typically undergo N-glycosylation in the Fc fragment, here we investigate the impacts of antibody N-glycan deglycosylation on T-DXd-induced ILD in preclinical models. Methods: N-glycan removal was performed on trastuzumab and T-DXd using PNGase F. Using immunofluorescence imaging combined with flow cytometry, we conducted a comparative analysis of the binding affinity and internalization efficiency of wild type and fully N-glycan deglycosylated HER2 antibodies. Next, we determined the in vitro cytotoxicity of WT and N-glycan deglycosylated T-DXd against both macrophages and HER2-positive cancer cells using CCK-8 assay. Furthermore, the deposition of N-glycan deglycosylated T-DXd in healthy lungs and their effects in ILD incidence and severity was determined in immunocompetent BALB/c mice carrying HER2-positive tumors. Results: First, we obtained complete N-glycan deglycosylated HER2 antibodies confirmed by SDS-PAGE and mass spectrometry. Next, we showed that N-glycan removal by PNGase F did not affect the Fab fragments of HER2 antibodies binding to the HER2 antigens on HER2-expressing cancer cells (OE19, SK-OV-3 and CT26-hHER2). Importantly, we did observe an approximately 30-50% reduction in the N-glycan deglycosylated HER2 antibodies binding with a panel of three macrophages (MH-S, RAW264.7, and PMA-induced THP-1) in comparison with WT antibodies, indicating a significant impact of N-glycan removal on innate immune cell-mediated effector functions. Moreover, in vitro cytotoxicity studies further support these observations that N-glycan deglycosylated T-DXd has significantly lower cytotoxicities against MH-S (p=0.046) and RAW264.7(p=0.020) in comparison with WT T-DXd. Correlating with our in vitro findings, in vivo biodistribution data showed a reduced lung deposition of HER2 antibodies and T-DXd after N-glycan removal. Immunofluorescence staining confirms the co-localization of HER2 antibodies with CD68-positive macrophages, indicating that AMs are the major contributor to the lung deposition of HER2 antibodies and T-DXd and N-glycan removal could effectively avoid ADC being phagocytosed by AMs. We also demonstrated chronic administration of N-glycan deglycosylated T-DXd could reduce ADC-associated ILD incidence and ameliorate ILD severity. Conclusions: Collectively, our study suggests that antibody N-glycan deglycosylation has important roles in regulating interactions between ADC and innate immune cells. This finding provides valuable clues for further optimizing ADC design and treatment strategies. Citation Format: Yu-Xuan Yang, Le-Tao Ma, Peng Guo. N-glycan deglycosylation ameliorates anti-HER2 antibody drug conjugate-induced interstitial pneumonia in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB058.

Abstract LB344: A novel approach to limit off-tumor toxicity of CAR-T cells targeting solid cancers

Abstract T cells genetically engineered to express a chimeric antigen receptor (CAR) specific for the molecule CD19 have achieved significant gains in the treatment of chemotherapy-resistant leukemia and lymphoma. Despite evidence of long-lived cures in patients with metastatic solid cancer after receiving tumor-specific T cells, no CAR-T cell treatment is currently approved for use against any solid cancer. A large majority of solid cancers express one of many tumor-associated antigens (TAAs) that are vulnerable to CAR T cell recognition. However, in nearly all cases those same TAAs are present in healthy tissues. Since CAR T cells cannot differentiate between cancer and normal cells, CAR T cell recognition of TAAs on healthy tissues provokes dose-limiting ‘on-target off-tumor’ toxicities at sites of endogenous expression (lung, intestine). Thus, there remains a pressing need to develop strategies to mitigate off-tumor toxicity of CAR T cells. To this end, we develop two immunocompetent models of CAR T cell transfer targeting TAAs abundant in metastatic colorectal cancer and pancreatic cancer. TAA-specific CAR T cells affected tumor shrinkage along with infiltrating and damaging sites of endogenous expression (primarily lung, pancreas, & colon). In both of our models, off-tumor toxicities were antigen-dependent and resulted in tissue damage, systemic inflammation, and host death that was titratable to CAR-T cell dose. TAA-specific CAR T cells infiltrated sites of endogenous expression such as the intestine, pancreas, and lungs. Notably, the respiratory system was the site of greatest pathology in both of our models, with massive CAR T cell accumulation and tissue destruction that resulted in systemic wasting and rapid weight loss. Remarkably, we found that the presence of an antigen-bearing tumor in the liver, but not other sites, greatly exacerbated CAR-T cell pulmonary infiltration, tissue destruction, and host mortality.The liver is the most common site of metastasis in patients with colorectal and pancreatic adenocarcinoma and the only site of metastasis in one-third of colorectal cancers. Our models indicated that TAA-specific CAR-T cells given for the treatment of liver metastasis result in dose-limiting pulmonary toxicity due to the presence of the TAA in the healthy lung. To address this translational barrier, we designed and validated a multiparameter genetic reprogramming targeting T cell trafficking and tissue residency introduced with the CAR to imbue CAR-T cells with liver tropism. In multiple models, CAR T cells programmed with liver-specific tropism augmented CAR T cell liver trafficking, decreased lung infiltration and toxicity, and effectively treated TAA+ liver metastasis. We aim to initiate a pilot clinical trial using tissue resident-programmed CAR T cells to treat colorectal liver metastasis. Citation Format: Alex Bartlett, Matthew Mcwhorter, Camille Collier, Chelsea Jenkins, Robert Eil. A novel approach to limit off-tumor toxicity of CAR-T cells targeting solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB344.

Low-dose and standard-dose whole-body [18F]FDG-PET/CT imaging: implications for healthy controls and lung cancer patients

Aim: High-sensitivity hybrid positron emission tomography (PET) imaging using advanced whole-body (WB) or total-body PET/computed tomography (CT) systems permits reducing injected tracer activity while preserving diagnostic quality. Such approaches are promising for healthy control studies or exploring inter-organ communication in systemic diseases. This study assessed test/retest variations in the fluoro-2-deoxy-D-glucose (FDG) uptake in key organs from low-dose (LD) and standard-dose (STD) [18F]FDG-PET/CT imaging protocols in healthy controls and lung cancer patients.Methods: A total of 19 healthy controls (19–62 years, 46–104 kg, 10 M/9 F) and 7 lung cancer patients (47–77 years, 50–88 kg, 4 M/3 F) underwent [18F]FDG-PET/CT imaging. All subjects were first injected (“test,” LD) with 28 ± 2 MBq FDG and underwent a dynamic (0–67 min post-injection) WB imaging protocol with LD-CT. Then, 90 min post-LD injection, the subjects were repositioned and injected with 275 ± 16 MBq FDG (“retest,” STD). Second LD-CT and STD-CT scans were acquired for healthy controls and patients, respectively. Static images (55–67 min post-injection) were considered for subsequent analysis. The CT images were used to automatically segment the target volumes of interest. Standardized uptake values normalized to the body weight (SUVBW) were extracted for each volume of interest. The mean SUVBW were compared for both LD/STD conditions with paired t-tests. In patients, FDG-avid lesions were manually delineated on LD and STD static images. Effective dose levels were estimated from both the CT and PET acquisitions.Results: Organ-based mean SUVBW were similar between the LD and STD (mean %difference ≤5%) in both healthy controls and cancer patients, except in the heart. Intra-control test/retest variability was significant in the brain, heart, and skeletal muscle (p < 0.05). While 17 lesions were delineated on the STD images of the patients, only 10/17 lesions were identified on the LD images due to increased image noise. Lesion-based mean SUVBW were similar between LD and STD acquisitions (p = 0.49, %difference = 10%). In patients, the effective doses were (1.9 ± 0.2) mSv (LD-CT), (16.6 ± 5.4) mSv (STD-CT), (0.5 ± 0.1) mSv (LD-PET), and (4.6 ± 0.3) mSv (STD-PET).Conclusion: LD and STD [18F]FDG injections in healthy controls and lung cancer patients yielded comparable mean SUVBW, except in the heart. Dose levels may be reduced for [18F]FDG-PET imaging without a loss in mean SUVBW accuracy, promoting LD-PET/CT protocols for studying multi-organ metabolic patterns. In oncology patients, this approach may be hindered by a lower diagnostic quality in the presence of significant noise.

Effect of diaphragmatic stretch technique on thoracic excursion and pulmonary function in COPD patients: Study protocol for randomized controlled trial.

Limited airflow is a defining feature of COPD, a respiratory disease that frequently results in reduced thoracic movement and compromised lung function. The diaphragm, which is the main breathing muscle, is essential for healthy lung expansion and ventilation. The diaphragm muscle’s flexibility and mobility are intended to be enhanced via diaphragmatic stretching. This method can enhance respiratory health and assist in returning the diaphragm to its original state. Patients with COPD may benefit from improved lung expansion and ventilation because of this. The usefulness of integrating the diaphragmatic stretch approach with traditional chest physical therapy is not well-established. Thus, research is needed to ascertain how the diaphragmatic stretch technique affects lung function and thoracic excursion in COPD patients. This study aims to ascertain how the diaphragmatic stretch technique affects thoracic excursion and pulmonary function (FEV1, FVC, FEV1/FVC, FEF25%-75%, PEFR) in individuals with COPD. There will be 58 COPD patients enrolled in total for this trial; 29 will receive traditional chest physiotherapy along with the diaphragmatic stretch technique, and 29 will receive traditional chest physiotherapy exclusively. Two weeks will pass throughout the intervention. Prior to the intervention, pre-outcome measures will be evaluated, and two weeks later, post-outcome measures will be evaluated once more. The anticipated outcome of this study is that the diaphragmatic stretch technique can enhance thoracic excursion and improve pulmonary function just as well as traditional chest physiotherapy when used in conjunction with it.

Design of a flow modulation device to facilitate individualized ventilation in a shared ventilator setup

This study aims to resolve the unmet need for ventilator surge capacity by developing a prototype device that can alter patient-specific flow in a shared ventilator setup. The device is designed to deliver a predictable tidal volume (VT), requiring minimal additional monitoring and workload. The prototyped device was tested in an in vitro bench setup for its performance against the intended use and design criteria. The ventilation parameters: VT and airway pressures, and ventilation profiles: pressure, flow and volume were measured for different ventilator and device settings for a healthy and ARDS simulated lung pathology. We obtained VTs with a linear correlation with valve openings from 10 to 100% across set inspiratory pressures (IPs) of 20 to 30 cmH2O. Airway pressure varied with valve opening and lung elastance but did not exceed set IPs. Performance was consistent in both healthy and ARDS-simulated lung conditions. The ventilation profile diverged from traditional pressure-controlled profiles. We present the design a flow modulator to titrate VTs in a shared ventilator setup. Application of the flow modulator resulted in a characteristic flow profile that differs from pressure- or volume controlled ventilation. The development of the flow modulator enables further validation of the Individualized Shared Ventilation (ISV) technology with individualization of delivered VTs and the development of a clinical protocol facilitating its clinical use during a ventilator surge capacity problem.

(364) - Quantitative CT Scan Analysis of Healthy Lung Transplant Recipients Correlates with Lung Function in a Multicenter Prospective Cohort

(364) - Quantitative CT Scan Analysis of Healthy Lung Transplant Recipients Correlates with Lung Function in a Multicenter Prospective Cohort

Utilizing Magnetic Levitation to Detect Lung Cancer-Associated Exosomes.

Extracellular vesicles, especially exosomes, have attracted attention in the last few decades as novel cancer biomarkers. Exosomal membrane proteins provide easy-to-reach targets and can be utilized as information sources of their parent cells. In this study, a MagLev-based, highly sensitive, and versatile biosensor platform for detecting minor differences in the density of suspended objects is proposed for exosome detection. The developed platform utilizes antibody-functionalized microspheres to capture exosomal membrane proteins (ExoMPs) EpCAM, CD81, and CD151 as markers for cancerous exosomes, exosomes, and non-small cell lung cancer (NSCLC)-derived exosomes, respectively. Initially, the platform was utilized for protein detection and quantification by targeting solubilized ExoMPs, and a dynamic range of 1-100 nM, with LoD values of 1.324, 0.638, and 0.722 nM for EpCAM, CD81, and CD151, were observed, respectively. Then, the sensor platform was tested using exosome isolates derived from NSCLC cell line A549 and MRC5 healthy lung fibroblast cell line. It was shown that the sensor platform is able to detect and differentiate exosomal biomarkers derived from cancerous and non-cancerous cell lines. Overall, this innovative, simple, and rapid method shows great potential for the early diagnosis of lung cancer through exosomal biomarker detection.

Abstract 6217: Differential genome-wide gene regulatory networks for lung cancer

Abstract Background: Lung cancer remains the leading cause of cancer death worldwide due to the fact that most cases are diagnosed at distant stages, and treatment is less effective. During the past two decades, much effort has been devoted to understanding tumor biology and developing targeted therapeutics, which have significantly improved patient survival. With the increasingly available multi-omics data from healthy controls and lung cancer patients, we constructed gene regulatory networks for the two main subtypes of non-small-cell lung cancer (NSCLC), the most common type of lung cancer. In addition, differential networks were constructed by integrative analysis of data from lung cancer tissue and tissues from healthy lungs. Methods: Transcriptomics and genomic data were obtained from the patients with lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) tissue available in The Cancer Genome Atlas (TCGA) project, and both types of omics data of normal lung tissue were downloaded from The Genotype-Tissue Expression (GTEx) consortium. The networks for each subtype and the differential networks were constructed by utilizing our newly developed machine learning tools, two-stage penalized least square (2SPLS) approach, and analysis of variance of directed networks (NetANOVA), respectively. Results: For the top three identified subnetworks of LUAD, we identified some well-known genes associated with lung cancer, such as EGFR, IRF1, KRAS, ERBB2, PIK3CA, MYC, at bootstrap frequency >90%. Based on the top three subnetworks with a bootstrap frequency of 100%, results from the Ingenuity Pathway Analysis (IPA) indicated several significant pathways, primary immunodeficiency signaling and communication between innate and adaptive immune cells. GO enrichment analysis showed statistically significant biological processes, including immune response, extracellular matrix organization, and significant KEGG pathways such as Hematopoietic cell lineage. The differential networks between LUAD, LUSC, and healthy lungs showed the same regulations across all three groups, such as the regulation between EEF2 and RACK1, the perturbations between LUAD and healthy (two-way regulations between HIGD2A and ARL10), and LUSC and healthy (GRB14 regulates EIF3E) respectively. In addition, we identified the differences between LUAD and LUSC, such as bi-directional regulations between CLTB and ARL10. Conclusion: Although all groups share most regulations, we identified differential gene regulations between healthy lung tissue and lung cancer tissue and between LUAD and LUSC of NSCLC. Using multi-omics data generated from lung tissues, coupled with advanced machine learning methods, the causal relationships between genome-wide genes from the analysis will help us understand the molecular mechanism of lung cancer and facilitate the development of personalized treatment strategies. Citation Format: Min Zhang, Zhongli Jiang, Dabao Zhang. Differential genome-wide gene regulatory networks for 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 6217.

Abstract 1281: Antibiotics aerosolization shrinks intratumoral Tregs and impairs lung tumor growth by perturbing the taxonomic structure of tumor-associated microbiota

Abstract The lung has been traditionally regarded as a sterile environment. Emerging evidence has overturned this belief unveiling the existence of a specific lung microbiota that plays a pivotal role in modulating lung immune tolerance. Recent research also sheds light on how the tissue microbiota is significantly altered by the presence of a growing tumor. Here, we investigated the compositional changes in lung-colonizing bacterial populations associated with the growth of LLC1 lung carcinoma cells in C57BL/6 mice, by analyzing the lung 16S rRNA profiling. While overall microbial diversity and structure were similar in tumor-bearing and healthy lungs, abundance analysis of amplicon sequence variants (ASVs) revealed that 48 ASVs were differentially modulated at significant level (FDR ≤ 0.05). Notably, local treatment with aerosolized vancomycin/neomycin antibiotics starting 4 days after tumor injection caused a different representation of 47 ASVs (FDR ≤ 0.05), mostly reducing those ASVs specifically emerging in the lung by the tumor growth, belonging to Bacterioidales, Clostridiales, Lactobacillales and Enterobacteriales. A different microbial composition was also suggested by the results of Partial Least Squares Discriminant Analysis (PLS-DA) that clustered lung tumor samples as saline or antibiotic-aerosolized. Based on the reported ability of some bacteria belonging to these orders to induce immunosuppressive cells in the gut, we examined the impact of antibiotic aerosol on tumor-associated Tregs in the lung. The presence of the tumor promoted the appearance of Tregs expressing CD69 and PD-1 activation markers, but vancomycin/neomycin aerosol treatment strongly dropped Treg frequency in tumor-bearing lungs, compared to saline. In a therapeutic setting, aerosolization of antibiotics starting 4 days after LLC1 tumor intravenous injection significantly reduced the number of lung metastases (p=0.0116). This effect was associated with an increased cytotoxic activity of immune infiltrating effector cells suggesting that the modulation of the lung microbiota impairs tumor growth and enhances antitumor immune response. Some cultivable bacterial strains, that were in agreement with metataxonomic profile results, were isolated from tumor-bearing lungs, but not from healthy lungs. Ongoing experiments suggest an effect of their in vivo transfer in the lung by aerosol on tumor growth and the ability to induce Treg expansion in vitro. Overall, this study unveils that the growth of the murine LLC1 tumor shapes the lung microbial composition by favoring the expansion of microbes with potential immunosuppressive activity eventually affecting the tumor progression. Moreover, our findings unravel the therapeutic potential of localized antibiotics to revert the immunosuppressive microenvironment by targeting tumor-associated bacteria. Citation Format: Giancarla Bernardo, Valentino Le Noci, Elena Montanari, Emerenziana Ottaviano, Giorgio Gargari, Elisa Borghi, Simone Guglielmetti, Federico Ambrogi, Tiziana Triulzi, Elda Tagliabue, Serenella M. Pupa, Michele Sommariva, Lucia Sfondrini. Antibiotics aerosolization shrinks intratumoral Tregs and impairs lung tumor growth by perturbing the taxonomic structure of tumor-associated microbiota [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 1281.

Abstract 3093: Combining lab automation with data analysis automation to enable high-throughput screening of patient-derived organoids

Abstract Although patient-derived tumor organoids (PDOs) better-represent patient tumors compared to traditional in vitro cell cultures, limitations of PDO analysis remain a major hurdle for high-throughput screening (HTS) applications. In particular, current PDO analysis fall short in at least 1 of 2 major criteria: 1) accurate recapitulation of patient tumor or 2) scalability. To address this, we have developed an automation-compatible protocol to satisfy these 2 major criteria to facilitate the adoption of PDOs for HTS applications1. Our novel protocol leverages live-cell imaging techniques to capture the dynamic interactions and intricate complexities of PDOs, which allowed for more precise and comprehensive analysis. To further enhance the accuracy and efficiency of our approach, we developed a fully automated image and data analysis pipeline (Orbits). Using label-free detection of organoids from brightfield images, Orbits integrates growth-rate-based drug response metrics with drug synergy metrics to significantly improve the identification of synergistic drug interactions. Here, present the results of our HTS protocol on 10 distinct drug combination strategies, specifically focusing on the repurposed drug candidate Auranofin, a Thioredoxin reductase inhibitor. We screened on 10 PDOs sourced from healthy lung, non-small cell lung cancer, and pancreatic ductal adenocarcinoma. Not only were we able to distinguishing cytostatic from cytotoxic drug responses at a single-organoid level, we identified drug candidates that can synergistically enhanced the efficacy of Auranofin in a tumor selective manner. Our study presents a major step forward in the field of PDO-based drug discovery, offering a robust protocol for HTS and automated data analysis. The implementation of our method will accelerate the discovery of effective therapeutic strategies, ultimately translating to improved patient outcomes. 1 Le Compte, M., et al JoVE 190 (2022) Citation Format: Abraham Lin, Maxim Le Compte, Tyler Gilcrest, Edgar Cardenas De La Hoz, Geert Roeyen, Jeroen Hendriks, Filip Lardon, Christophe Deben. Combining lab automation with data analysis automation to enable high-throughput screening of patient-derived organoids [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 3093.

Abstract 5018: Evaluation of whole blood fractionation conditions for optimal liquid biopsy NGS applications

Abstract “For research use only. Not for diagnostic procedures.” Introduction: Liquid biopsy is a non-invasive alternative to tissue biopsies for cancer profiling using cell-free total nucleic acids (cfTNA; including cell free DNA (cfDNA) and cell free RNA (cfRNA)), the biological analytes derived from tumor cells floating in blood. However, it is crucial to optimize the whole blood fractionation workflows, since limited access to a suitable centrifuge at blood collection sites poses a challenge to this critical fractionation step, causing delays in processing the blood, leading to decreased sensitivity of variant detection and increased occurrence of false positives. Current study shows a systematic comparison of the effects of 8 different blood fractionation conditions for spin speeds, temperatures and duration on the levels of cfTNA derived from K2-EDTA-derived blood from multiple healthy and non-small cell lung cancer (NSCLC) donors, using next generation sequencing (NGS). Methods: CfTNA from K2-EDTA derived plasma was isolated using the MagMAXTM Cell-Free Total Nucleic Acid kit and the Genexus™ Cell free total nucleic acid kit, utilizing the KingFisher TM Flex and GenexusTMpurification systems. Yield and integrity of cfTNA isolated was evaluated by Qubit™, Agilent Tapestation and quantitative real time PCR (qRT-PCR). Furthermore, the impact of these test conditions on quality control specifications of DNA and RNA was assessed by performing sequencing on an Ion TorrentTM GenexusTM sequencing instrument using the AmpliSeqTM HD target amplification assay, that reports sequence variations in DNA for 42 cancer related genes and RNA in 7 cancer related genes. Results: Results showed comparable performance for all the 8 blood fractionation conditions tested, for yield and cfDNA profiles compared to control condition, indicating that there is no significant impact of these conditions on either yield or integrity of the cfDNA isolated. In addition, NGS data also showed that on target reads and median molecular coverage for DNA are comparable for all the conditions tested. However, sequencing data demonstrated that one test condition showed significantly better performance for RNA quality control specifications in terms of RNA mapped reads and molecular coverage for process controls used in the assay, compared to all other conditions tested. Conclusion: Data suggests that one of the whole blood fractionation conditions tested impact the cfRNA recovery from plasma without negatively affecting the yield, and integrity of isolated cfDNA and shows increased sensitivity for RNA process controls in NGS. Citation Format: MADHU JASTI, Sergio Hernandez, Nick Siepert, Lokesh Kodandaramaswamy, Thilanka Jayaweera, Jeoffrey Schageman, Angie Cheng, Susanne Lopez, Miao Der Chen. Evaluation of whole blood fractionation conditions for optimal liquid biopsy NGS applications [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 5018.

Abstract 710: Post-exposure suppression of radiation pneumonitis suggests non-redundant independent effects of TGF-beta and TRAIL/DR5

Abstract Radiation pneumonitis is one of the most common but challenging toxicities seen in patients that are receiving ionizing radiotherapy for thoracic malignancies. While radiation effectively eliminates cancer cells, the damage it causes to surrounding healthy lung tissue leads to an immune response that can impact treatment regimen and lead to long term fibrotic tissue formation. The mechanism of radiation pneumonitis and the inflammatory response in the lungs is poorly understood. However, it is known that the TRAIL pathway plays an important role in inflammatory and fibrotic response. Through our previous work, it was discovered that modulating the TRAIL pathway via pegylated recombinant long-acting TRAIL (TLY012) in both WT and TRAIL-/- C57Bl/6 could suppress radiation pneumonitis in mice that were exposed to 20 Gy of thoracic radiation with shielding of other organs as early two weeks post exposure. In addition to the TRAIL pathway, transforming growth factor-beta (TGF-β) is a cytokine that is known to play a key role in fibrosis. Broad spectrum integrin inhibitor GLPG0187 is known to inhibit the activation of TGF-β via binding to specific integrin receptors. In order to determine if the inhibition of TGF-β could suppress radiation pneumonitis without stimulation of the TRAIL pathway, DR5 null mice were utilized for the experiment. Male DR5-/- C57Bl/6 mice received a single 20 Gy thoracic radiation dose with shielding of other organs and were treated with 100 mg/kg of GLPG-0187 or control twice a week via IP injection with the first dose being administered 1 hour before radiation (n = 4/treatment/group). 13 days post-irradiation, lungs, sternal bone marrow, and peripheral serum were collected. Upon histological analysis, it was observed that there were thinner alveolar borders and lessened inflammation compared to the control mice. Mice were weighed every three days, and it was discovered that mice treated with GLPG-0187 maintained steady weight compared to the control group. Additional analysis including immunohistochemistry, cytokine profiling, and quantification needs to be conducted. As the TRAIL pathway could not be modulated in the DR5 null mice, these findings suggest that GLPG-0187 was able to suppress radiation pneumonitis via inhibition of TGF-β. While some rescue from radiation pneumonitis was observed via GLPG0187, it was to a lesser extent compared to treatment of WT and TRAIL-/- C57Bl/6 mice with TLY012. Future directions include investigating the synergistic effects between TLY012 and GLPG0187 in suppressing radiation pneumonitis and decreasing fibrotic response to radiation. Citation Format: Jillian Strandberg, Anna Louie, William MacDonald, Praveen Srinivasan, Leiqing Zhang, Lanlan Zhou, Seulki Lee, Wafik El-Deiry. Post-exposure suppression of radiation pneumonitis suggests non-redundant independent effects of TGF-beta and TRAIL/DR5 [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 710.

Abstract 5537: How aging promotes immune mediated reactivation from metastatic melanoma dormancy

Abstract Disseminated cancer cells that escape the primary tumor can seed distal tissues but may take several years to grow into clinically detectable metastases, a phenomenon termed tumor dormancy. Despite its importance in metastasis and residual disease, few studies have been able to successfully model melanoma dormancy. We have previously shown that while tumors grow more slowly in aged mouse skin, they form more lung metastases in aged mice compared to young mice. We queried whether the lung microenvironment undergoes age-related changes that establish a permissive niche for metastatic outgrowth. We identified that Wnt5A and AXL are not only required for efficient dissemination away from the primary tumor but also activate dormancy of melanoma cells within the lung. The young lung microenvironment maintains melanoma cells in a dormant state; however, age-associated reprograming of lung fibroblasts increases secretion of the soluble Wnt antagonist sFRP1, which inhibits both Wnt5A and AXL to enable reactivation from dormancy and the formation of larger metastatic colonies in the aged lung. We found that downregulation of AXL in melanoma cells leads to an increase in the related TAM family receptor MERTK, which promotes a proliferative melanoma phenotype and enables reactivation from dormancy. We have now found that both the aged-pre and post metastatic niche regulate immune cells in the lung to create a permissive niche for reactivation of metastatic melanoma. NK cells are significantly decreased in the healthy lungs of aged mice, are significantly less active, and have increased exhaustion markers. NK cell infiltration remains low in early and late-stage metastasis in aged mice. Depletion of NK cells in young mice is sufficient to induce reactivation from dormancy. Further, MERTK induced reactivation of melanoma cells results in paracrine secretion of its own ligand PROS1 to facilitate a growth permissive lung microenvironment. Melanoma-secreted PROS1 increases infiltration of PMN-MDSCs and decreases macrophage infiltration to maintain an immunosuppressive lung niche for efficient outgrowth. Selective inhibition of PMN-MDSCs using a TRIAL-R2 agonistic antibody significantly decreases metastatic outgrowth and promotes smaller, non-proliferative dormant colonies. Our findings indicate a significant role that aging plays in altering NK-cell infiltration and function to create a growth permissive pre-metastatic niche and how reactivation from dormancy can further contribute to create an immunosuppressive microenvironment for efficient outgrowth via PROS1 induced infiltration of PMN-MDSCs. Importantly, the clear differences found in our young and aged mouse modelling systems for outgrowth in metastatic tissues necessitates the need for age-appropriate pre-clinical modelling for better patient and disease representation. Citation Format: Pulkit Datt, Jhon Passamonte, Christopher Price, Daniel Zabransky, Yash Chhabra, Ashani Weeraratna, Mitchell E. Fane. How aging promotes immune mediated reactivation from metastatic melanoma dormancy [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 5537.

Abstract 5376: Quantitative profiling of HLA class I and class II antigens and neoantigens in tissue biopsy and PBMC samples using an optimized mass spectrometry-based workflow

Abstract Major histocompatibility complex (MHC) molecules play a central role in orchestrating immune responses by presenting antigenic peptides derived from both self and foreign proteins. In the context of cancer, understanding the repertoire of tumor-associated antigens (TAAs) presented by MHC molecules (or HLA molecules in human) is crucial for deciphering how the immune system recognizes and responds to malignant cells. The identification of neoantigens, unique to individual tumors due to somatic mutations, has become a focal point in immunopeptidomic studies. One significant hurdle in systematic immunopeptidomics analysis is the high input material requirement. Here, we present a semi-automated workflow to robustly identify and quantify immunopeptides from reduced amounts of clinical tissue biopsy and peripheral blood mononuclear cell samples. At the core of immunopeptidomics is the enrichment of HLA-associated peptides, followed by identification using mass spectrometry and bioinformatics tools. We optimized the native lysis and a sequential immunoprecipitation workflow for both class I and class II immunopeptides while ensuring scalability and reproducibility. Leveraging the magnetic properties of the beads, 1,000 samples can be processed within a week by a single operator. For both tissue and PBMC samples, we performed a systematic ramping experiment starting with as little as 2.5mg tissue or 5 million PBMCs. In all experiments, The established sample preparation offers high reproducibility and identifications of good quality: 1) Class-I immunopeptides: >60% of the peptides identified are 9-mers, >80% predicted strong binders, and the expected amino acids are enriched at the anchor positions; 2) Class-II immunopeptides, >50% of the peptides identified are 14-to-16-mers, and >50% are predicted strong binders. Furthermore, the pipeline is highly sensitive as we could still identify over 2,800 class-I immunopeptides when processing 2.5 mg fresh frozen tissue and 2,000 - 3,000 class-I immunopeptides when starting from 5 million PBMCs. Furthermore, the developed immunopeptidomics workflow was deployed to profile a cohort of 12 cancerous and matched healthy lung tissue samples. Their Class-I immunopeptidomes clearly displayed a pattern where matched tissues from each subject cluster together, further underlining the fact that the intricate immune-tumor interface is highly personalized. Overall, we established a robust pipeline for for class-I and II immunopeptidome profiling from clinically relevant sample types. Taking advantage of the nature of mass spectrometry-based methods, customized targeted assays can be developed without the need for affinity reagent, allowing specific and absolute quantification of any immunopeptide of interest. Citation Format: Ilja E. Shapiro, Marco Tognetti, Tikira Temu, Oliver M. Bernhardt, Daniel Redfern, Yuehan Feng, Roland Bruderer, Lukas Reiter. Quantitative profiling of HLA class I and class II antigens and neoantigens in tissue biopsy and PBMC samples using an optimized mass spectrometry-based workflow [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 5376.

Abstract 2091: Pharmacological inhibition of chaperone-mediated autophagy via NCoR1/RARα targeting is effective against non-small cell lung cancer

Abstract Dysregulation of autophagy, the lysosomal degradation of intracellular components, has been universally described in cancer. While activity of macroautophagy changes in a context-dependent manner in cancers, the upregulation of chaperone-mediated autophagy (CMA) is well-described in all solid tumors studied to date. Upregulated CMA directly contributes to both tumor growth and protection against anti-oncogenic interventions in cancers. Selective degradation of anti-oncogenic and pro-apoptotic proteins, such as mutant p53 and PUMA, misfolded proteins, and glycolytic enzymes by CMA allows for increased survival, reduced cell death, increased stress resistance, and enhancement of the Warburg effect in cancer cells, respectively. As such, inhibition of CMA is an attractive target for anti-cancer therapeutics, but the field currently lacks protein targets specific to CMA and selective CMA inhibitors. We have previously demonstrated the regulatory effect of retinoic acid receptor alpha (RARα) and its coregulator, nuclear receptor corepressor 1 (NCoR1), on CMA through transcriptional upregulation of the CMA effector proteins (Bourdenx et al. Cell 2021, Gomez-Sintes et al, Nat. Commun. 2022). Here, we identified a high NCoR1/RARα expression ratio in human non-small cell lung cancer (NSCLC) tumors compared to healthy lung tissue. We further illustrated a dependency on NCoR1 expression and its interaction with RARα to maintain high CMA activity in NSCLC cell lines. Provided the reliance on NCoR1 repression of RARα for high CMA activity in NSCLC, we hypothesized that small molecule targeting of the druggable NCoR1/RARα interaction to disrupt the interaction may result in suppression of CMA effector proteins and CMA inhibition. Through a series of in-silico screens for predicted NCoR1/RARα targeting, and functional screens for CMA inhibition, we identified CIM7, a first-in-class potent and selective CMA inhibitor. We validated CIM7 function through binding to RARα and subsequent disruption of NCoR1 binding, resulting in alterations to CMA-related gene expression. CIM7 inhibits CMA in vivo with no observed toxicity in normal tissues. We additionally demonstrated the therapeutic potential of CIM7 through its ability to reduce tumor growth in vivo in mouse NSCLC xenografts. Our findings support the NCoR1/RARα interaction as a major regulator of CMA in NSCLC and demonstrate the potential of pharmacologic CMA inhibition as an anti-cancer therapeutic strategy. Citation Format: Mericka McCabe, Rabia R. Khawaja, Rajanya Bhattacharyya, Antonio Diaz, Thomas P. Garner, Kristen Lindenau, Rebecca Sereda, Olaya Santiago-Fernández, Ana Maria Cuervo, Evripidis Gavathiotis. Pharmacological inhibition of chaperone-mediated autophagy via NCoR1/RARα targeting is effective against 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 2091.

Quantitative profiling and baseline intervals of trace elements in healthy lung tissues

BackgroundHuman lung tissue, as an interface with the environment, is susceptible to various environmental pollutants, including trace metals. However, quantitative data on trace metals in human lung tissues remain poorly described. MethodsThis study aimed to characterize the elemental composition of histologically healthy, unaffected parts of human lung tissues, associated with non-infective, non-infiltrative, and non-malignant diseases (n = 60) for essential (Cr, Mn, Fe, Co, Cu, Zn, and Se) and toxic trace elements (Sr, Ni, As, Cd, and Pb). Additionally, we investigated the influence of personal factors (sex, age, and smoking habits) on the examined trace element profiles, as well as between the trace elements correlations in the healthy human lungs. ResultsAmong the analyzed trace elements, Fe was the most prevalent, while As was the least prevalent in healthy lung tissues. Stratifying by age revealed significantly higher Cr and Co (less Sr, Ni, and Pb) and lower Se levels in older individuals (above 65 years) compared to their younger counterparts. Sex-based differences were also notable, with Cu and Co 1.2- and 2.3-fold higher levels in females than in males. Exploring the impact of smoking habits revealed a striking 10-fold increase in Cd levels in the lung tissues of smokers compared to non-smokers. Correlation analyses showed significant positive associations between concentrations of certain toxic and essential trace elements in healthy lung tissues. ConclusionsThis study could contribute to the establishment of baseline intervals for essential and toxic trace elements, valuable for toxicological and clinical assessment, in healthy, unaffected human lungs, and indicates the influence of sex, age, and smoking. However, further larger-scale studies are needed to make more stable conclusions.

Determination of Tumor Marker Screening for Lung Cancer Using ROC Curves.

Lung cancer ranks first among malignant tumors worldwide and is a leading cause of cancer-related mortality in both men and women. Combining tumor marker testing is a strategy to screen individuals at high risk of pulmonary cancer and minimize pulmonary cancer mortality. Therefore, tumor marker screening is crucial. In this study, we analyzed combinations of tumor markers for lung cancer screening using receiver operating characteristic (ROC) curve analysis. A retrospective descriptive study was conducted on patients diagnosed with lung cancer, as well as healthy and benign lung diseases, using data from the China Huludao Central Hospital database between January 2016 and July 2022. The t-test and ROC curve were utilized to assess the effectiveness of individual tumor marker and the combination of multiple tumor markers. Tumor markers are molecular products metabolized and secreted by tumor tissues, characterized by cells or body fluids. They serve as indicators of tumor stage and grading, monitor treatment response, and predict recurrence. In this study, 267 healthy participants, 385 patients with benign lesions, and 296 patients with lung cancer underwent tumor marker screening. The sensitivity of five tumor markers-CEA, CYFRA21-1, NSE, pro-GRP, and CA125-was found to be <55%. This study revealed that a single tumor marker had limited value in lung cancer screening. However, combining two or more markers yielded varying area under the curves (AUC), with no significant impact on screening accuracy. The combination of CEA + CA125 demonstrated the highest accuracy for lung cancer screening in healthy participants. At a cutoff of 0.447 for CEA + CA125, the combination showed a sensitivity of 0.676 and specificity of 0.846 for lung cancer screening. Conversely, for patients with benign lung lesions, the optimal combination was CEA + NSE, with a cutoff of 0.393, yielding a sensitivity of 0.645 and specificity of 0.766 for lung cancer screening. The five tumor markers-CEA, CA125, CY211, NSE, GRP-show promising results in screening healthy individuals and patients with lung cancer. However, only CEA, NSE, and GRP effectively differentiate patients with benign lung lesions from those with lung cancer. A single tumor marker has limited utility in detecting and screening for lung cancer and should be combined with other tumor markers. CEA + CA125 emerges as a superior tumor marker for distinguishing healthy individuals from those with lung cancer, whereas the CEA + NSE combination is more effective in identifying tumor markers in patients with benign lung lesions and lung cancer.

Novel air-liquid interface culture model to investigate stiffness-dependent behaviors of alveolar epithelial cells

Pulmonary alveoli are functional units in gas exchange in the lung, and their dysfunctions in lung diseases such as interstitial pneumonia are accompanied by fibrotic changes in structure, elevating the stiffness of extracellular matrix components. The present study aimed to test the hypothesis that such changes in alveoli stiffness induce functional alteration of epithelial cell functions, exacerbating lung diseases. For this, we have developed a novel method of culturing alveolar epithelial cells on polyacrylamide gel with different elastic modulus at an air-liquid interface. It was demonstrated that A549 cells on soft gels, mimicking the modulus of a healthy lung, upregulated mRNA expression and protein synthesis of surfactant protein C (SFTPC). By contrast, the cells on stiff gels, mimicking the modulus of the fibrotic lung, exhibited upregulation of SFTPC gene expression but not at the protein level. Cell morphology, as well as cell nucleus volume, were also different between the two types of gels.