Non-small Cell Lung Cancer Tumors Research Articles

Curcumin Inhibits the Progression of Non-small Cell Lung Cancer by Regulating DMRT3/SLC7A11 Axis.

Non-small cell lung cancer (NSCLC) is a fatal malignancy all over the world. Emerging studies have shownthat curcumin might repress NSCLC progression by regulating ferroptosis, but the underlying mechanism remains unclear. 16HBE, LK-2, and H1650 cell viability was detected using Cell Counting Kit-8 assay. LK-2 and H1650 cell proliferation, apoptosis, and angiopoiesis were measured using 5-ethynyl-2'-deoxyuridine, flow cytometry, and tube formation assay. Superoxide dismutase, Malondialdehyde, Glutathione, and lactate dehydrogenase levels in LK-2 and H1650 cells were examined using special assay kits. Fe+ level was assessed using an iron assay kit. Doublesex and Mab-3 related Transcription Factor 3 (DMRT3) and solutecarrierfamily7member11(SLC7A11) protein levels were detected using western in NSCLC tissues, adjacent matched normal tissues, 16HBE cells, LK-2 cells, H1650 cells, and xenograft tumor tissues. Glutathione peroxidase 4, Acyl-CoA Synthetase Long Chain Family Member 4, and transferrinreceptor1 protein levels in LK-2 and H1650 cells were examined by western blot assay. DMRT3 and SLC7A11 levels were determined using real-time quantitative polymerase chain reaction. After JASPAR prediction, binding between DMRT3 and SLC7A11 promoter was verified using Chromatin immunoprecipitation and dual-luciferase reporter assays in LK-2 and H1650 cells. Role of curcumin on NSCLC tumor growth was assessed using the xenograft tumor model in vivo. Curcumin blocked NSCLC cell proliferation and angiopoiesis, and induced apoptosis and ferroptosis. DMRT3 or SLC7A11 upregulation partly abolished the suppressive role of curcumin on NSCLC development. In mechanism, DMRT3 was a transcription factor of SLC7A11 and increased the transcription of SLC7A11 via binding to its promoter region. Curcumin inhibited NSCLC growth in vivo by modulating DMRT3. Curcumin might constrain NSCLC cell malignant phenotypes partly through the DMRT3/SLC7A11 axis, providing a promising therapeutic strategy for NSCLC.

CircPDK1 competitively binds miR-4731-5p to mediate GIGYF1 expression and increase paclitaxel sensitivity in non-small cell lung cancer

ObjectiveTo investigate the action of circPDK1 in paclitaxel (PTX) resistance in non-small cell lung cancer (NSCLC).MethodscircPDK1, miR-4731-5p, and GIGYF1 levels were determined by RT-qPCR and Western blot. Cell proliferation was detected by CCK-8 and colony formation assay, apoptosis by flow cytometry, invasion by Transwell assay. The targeting relationship between miR-4731-5p and circPDK1 or GIGYF1 was confirmed by dual luciferase reporter gene and RIP assay. A xenograft tumor model was established to determine the role of circPDK1 in PTX resistance.ResultscircPDK1 was overexpressed in PTX-resistant NSCLC, and depleting circPDK1 hampered proliferation and invasion of PTX-resistant cells, activated apoptosis, and improved PTX sensitivity. circPDK1 bound to miR-4731-5p, and increasing miR-4731-5p expression salvaged the effect of circPDK1 depletion on PTX resistance. miR-4731-5p directly targeted GIGYF1, and upregulating GIGYF1 offset the promoting effect of circPDK1 knockdown on PTX sensitivity. NSCLC tumor growth was inhibited and PTX sensitivity improved when circPDK1 was suppressed.ConclusionDepleting circPDK1 promotes PTX sensitivity of NSCLC cells via miR-4731-5p/GIGYF1 axis, thereby inhibiting NSCLC pregnancy.

Detection of Oncogene Hotspot Mutations in Female NSCLC Tumor DNA and Cell-Free DNA.

Non-small-cell lung cancer (NSCLC) is the most prevalent type of lung cancer, with extensively characterized mutational spectra. Several biomarkers (such as EGFR, BRAF, KRAS gene mutations, etc.) have emerged as predictive and prognostic markers for NSCLC. Unfortunately, the quality of the available tumor biopsy and/or cytology material is not always adequate to perform the necessary molecular testing, prompting the search for alternatives. Cell-free DNA (cfDNA) found in plasma is emerging as a highly promising avenue or a supplementary method for assessing the efficacy of cancer treatments. This is especially valuable in instances where conventional biopsy specimens, like formalin-fixed, paraffin-embedded (FFPE), or freshly frozen tumor tissues prove inadequate for conducting molecular pathology analyses subsequent to the initial diagnostic procedures. By leveraging cfDNA from plasma, clinicians gain an additional tool to gauge the effectiveness of cancer therapies, thereby enhancing their ability to optimize tailored treatment strategies. In this study, 51 Lithuanian females with NSCLC were analyzed, with adenocarcinoma being the predominant pathology diagnosis in 40 cases (78%). Target mutations were identified in 38 out of 51 patients (74.5%) in tumor tissue samples, while in plasma samples, they were identified in only 10 patients' samples (19.6%). Even though we did not have enough voluminous plasma samples in our study, gene mutations were detected in plasma from ten women, three of whom were diagnosed with early stages of lung cancer (stages I and II). For these patients, the following mutations were detected: deletion in exon 19 of the EGFR gene and single nucleotide polymorphisms in the TP53 and MET genes. All other women were diagnosed with stages III or IV of lung cancer. This indicates that the later stages of cancer contribute more cfDNA in plasma, making extraction less complicated.

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.

Genomic and Immune Landscape Comparison of MET Exon 14 Skipping and MET-Amplified Non-Small Cell Lung Cancer

Background: Mutation or amplification of the mesenchymal-epithelial transition (MET) tyrosine kinase receptor causes dysregulation of receptor function and stimulates tumor growth in non-small cell lung cancer (NSCLC) with the most common mutation being MET exon 14 (METex14). We sought to compare the genomic and immune landscape of MET-altered NSCLC with MET wild-type NSCLC. Methods: 18,047 NSCLC tumors were sequenced with Tempus xT assay. Tumors were categorized based on MET exon 14 (METex14) mutations; low MET amplification defined as a copy number gain (CNG) 6-9, high MET amplification defined as CNG ≥ 10, and MET other type mutations. Immuno-oncology (IO) biomarkers and the frequency of other somatic gene alterations were compared across MET-altered and MET wild-type groups. Results: 276 (1.53%) METex14, 138 (0.76%) high METamp, 63 (0.35%) low METamp, 27 (0.15%) MET other, and 17,543 (97%) MET wild-type were identified. Patients with any MET mutation including METex14 were older, while patients with METex14 were more frequently female and nonsmokers. MET gene expression was highest in METamp tumors. PD-L1 positivity rates were higher in MET-altered groups than MET wild-type. METex14 exhibited the lowest tumor mutational burden (TMB) and lowest neoantigen tumor burden (NTB). METamp exhibited the lowest proportion of CD4 T cells and the highest proportion of NK cells. There were significant differences in co-alterations between METamp and METex14. Conclusions: METex14 tumors exhibited differences in IO biomarkers and the somatic landscape compared to non-METex14 NSCLC tumors. Variations in immune profiles can affect immunotherapy selection in MET-altered NSCLC and require further exploration. MicroAbstract: The MET oncogene can be mutated or amplified in non-small cell lung cancer. Using a large population-based database we compared the genomic and immune landscape of MET-altered lung cancer. MET-altered tumors exhibited differences in immune biomarkers and somatic mutations compared to non-MET-altered tumors. Variations in immune profiles may affect immunotherapy selection in these patients and require further exploration.

Inhibition of human UDP-glucuronosyltransferase enzyme by entrectinib: Implications for drug-drug interactions

As a new type of oral tyrosine kinase inhibitor, entrectinib can act on multiple targets and exert efficacy and has been approved for the treatment of non-small cell lung cancer (NSCLC) and solid tumors. However, whether entrectinib affects the activities of recombinant human UDP-glucuronosyltransferases (UGTs) remains unclear. Herein, we aimed to investigate the inhibitory effects of entrectinib on human UGTs and to assess the potential risk of causing drug-drug interactions (DDIs) based on the inhibition against UGTs. High-performance liquid chromatography (HPLC) was used to evaluate the inhibitory effects of entrectinib on UGTs according to the product formation rate of UGT substrate with or without entrectinib, and the inhibition kinetics experiment was conducted to assess the inhibitory type of entrectinib on UGTs. Our results showed that entrectinib exhibited extensive inhibitory effects on most human UGTs, and especially inhibited the activities of UGT1A7, UGT1A8, and UGT2B15 with Ki (Inhibition constant) of lower than 5 μM (0.95–4.38 μM). Furthermore, the results from quantitative prediction research suggested that the combination of entrectinib at 600 mg/day with substrates primarily metabolized by hepatic UGT2B15 or intestinal UGT1A7 and UGT1A8 might cause clinical DDIs. Thus, special attention should be paid to avoid adverse reactions induced by DDIs when co-administration of entrectinib and drugs metabolized by UGTs.

Bithiophene-Functionalized Infrared Two-Photon Absorption Metal Complexes as Single-Molecule Platforms for Synergistic Photodynamic, Photothermal, and Chemotherapy.

A planar conjugated ligand functionalized with bithiophene and its Ru(II), Os(II), and Ir(III) complexes have been constructed as single-molecule platform for synergistic photodynamic, photothermal, and chemotherapy. The complexes have significant two-photon absorption at 808 nm and remarkable singlet oxygen and superoxide anion production in aqueous solution and cells when exposed to 808 nm infrared irradiation. The most potent Ru(II) complex Ru7 enters tumor cells via the rare macropinocytosis, locates in both nuclei and mitochondria, and regulates DNA-related chemotherapeutic mechanisms intranuclearly including DNA topoisomerase and RNA polymerase inhibition and their synergistic effects with photoactivated apoptosis, ferroptosis and DNA cleavage. Ru7 exhibits high efficacy in vivo for malignant melanoma and cisplatin-resistant non-small cell lung cancer tumors, with a 100% survival rate of mice, low toxicity to normal cells and low residual rate. Such an infrared two-photon activatable metal complex may contribute to a new generation of single-molecule-based integrated diagnosis and treatment platform to address drug resistance in clinical practice and phototherapy for large, deeply located solid tumors.

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.

Molecular characteristics of non-small cell lung cancer tissue based on quantitative indicators of progesterone receptors expression

Background. Progesterone receptors (PR) are regulators of cell proliferation and therefore can be considered as an aim for targeted medications in the treatment of oncological diseases. At the same time, a quantitative assessment of PR expression in the tissue of non-small cell lung cancer (NSCLC), which has not yet been carried out in other studies, will determine the possibility of using PR modulators for the treatment of this disease and identify the potential category of patients most susceptible to these drugs.Purpose. To characterize NSCLC by quantitative indicators of PR expression and to determine the correlation of clinically significant characteristics of patients and clinical and morphological parameters of a NSCLC tumor with the PR expression to assess the possibility of using PR modulators in the treatment of this disease.Methods. The PR expression in 130 surgical samples of NSCLC was quantified using an immunofluorescence method associated with flow cytometry. Primary antibodies to PR (NBP2-46388) and secondary antibodies conjugated with DyLight650 (ab98729) were used.Results. The expression of progesterone receptors was detected in all the studied tumors; an abnormal distribution of the marker expression level was noted (P=0.01). The mean expression level was 55.3±16.2%, and the median was 57% with a range of 70%, which indicates heterogeneity of PR expression in tumors of different patients. There were no statistically significant differences in the level of PR expression depending on the histotype and stage of NSCLC, as well as on the sex of patients. At the same time, the level of expression and the frequency of overexpression of PR (>67%) in tumors in non-smoking patients are higher than in smokers (P⩽0.02).Conclusion. The high frequency of occurrence and level of PR expression in NSCLC indicate the possible effectiveness of the use of their modulators in the treatment of this disease, especially in non-smoking patients.

Abstract LB365: Generation of a novel severely immunodeficient Sprague Dawley rat model for xenograft studies

Abstract The recombinant-activating gene 2 (Rag2) plays a critical role in the recombination of the immunoglobulin and T cell receptor genes during the maturation of T and B cells. Loss of Rag2 protein leads to a lack of functional T and B cells, which eliminates the adaptive immune response. However, Rag2 deficiency alone may not guarantee the success of xenograft implantation, due to NK cells and other innate cells remaining. Il2rg is a key component of several cytokine receptors involved in differentiation and/or function of numerous adaptive and innate immune cells, including NK cells, mast cells, basophils, and dendritic cells. To generate a more severely immunodeficient rat model, both the Rag2 and Il2rg genes were knocked out in Sprague Dawley (SD) rats; this model was termed B-SDG rats. The immune profile and organ weights of thymus and spleen were investigated in B-SDG rats in comparison with Rag2-/- and wild-type SD rats. The spleen weights of both Rag2-/- and B-SDG rats were similar, but significantly reduced compared to wild-type SD rats. Notably, the thymus weights of B-SDG male rats were significantly smaller than those of Rag2-/- males. The immune cell profiles in thymus, spleen and blood were next evaluated by flow cytometry. A complete lack of B and T cells was observed in both Rag2-/- and B-SDG rats. B-SDG rats lacked CD161+ NK cells in the spleen and blood, which were maintained in Rag2-/- rats. Dendritic cells, monocytes, macrophages, and neutrophils were detected in all strains. In vivo xenograft implantation with human lung, bladder, breast, pancreas, acute monocytic and myelomonocytic leukemia tumor cell lines was next evaluated in B-SDG rats, which demonstrated successful engraftment. Furthermore, several efficacy studies were performed in B-SDG rats, including cisplatin chemotherapy on human breast invasive ductal carcinoma cell (HCC1954) and human large cell lung cancer cell (NCI-H460) xenografts, and a HER2-targeting ADC therapy was tested in B-SDG rats harboring human non-small cell lung cancer tumors (NCI-H1975). Taken together, these data confirm that B-SDG rats provide a powerful immunodeficient model for preclinical in vivo evaluation of novel drug candidates. Citation Format: Yanhui Nie, Meiqi Zhang, Wanling Zhong, Jia Feng, Bahetiyaer Huwatibieke, Zhaoxue Yu. Generation of a novel severely immunodeficient Sprague Dawley rat model for xenograft studies [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 LB365.

Abstract CT292: Phase II study of pembrolizumab in combination with lenvatinib in patients with triple negative breast cancer (TNBC), non-small cell lung cancer (NSCLC), and other tumor types and brain metastases

Abstract Background: Many patients develop brain metastasis during the course of their disease and it represents the cause of death in more than half of them. The prognosis and survival of patients with brain metastases remains poor with limited palliative treatment options. Thus, there is a need to develop new strategies for the therapeutic management of patients with brain metastases. Inflammatory stimuli originating from CNS tumors may increase the permeability of the blood-brain barrier and promote immune cell activation and infiltration into tumors. Several studies have demonstrated now the efficacy of immune checkpoint inhibitors in patients with melanoma with active brain metastases. The modulation of VEGF-mediated immune suppression via angiogenesis inhibition may augment the activity of immune checkpoint inhibitors. Lenvatinib is an oral, potent multiple receptor tyrosine kinase inhibitor that selectively inhibits VEGFRs, VEGFR1 (FLT1), VEGFR2 (KDR), and VEGFR3 (FLT4), in addition to other pro-angiogenic and oncogenic pathway-related receptor tyrosine kinases. The combination of lenvatinib and pembrolizumab is approved for patients with metastatic endometrial carcinoma and renal cancer, and it is investigated in other tumor types with promising preliminary results. We hypothesize that pembrolizumab and lenvatinib will be an effective treatment for TNBC, NSCLC, and other solid tumor types with brain metastases by decreasing angiogenic tumor activity and improving antitumor T-cell activity. Methods: This is a single-center, open-label, multi-cohort Phase II study evaluating the efficacy and safety of pembrolizumab in combination with lenvatinib in patients with solid tumors and brain metastases. The study has 3 cohorts: triple negative breast cancer (TNBC), non-small cell lung cancer (NSCLC), and other solid tumor types with established or preliminary clinical evidence of efficacy of programmed cell death-1 (PD-1) and angiogenesis inhibitors. Eligible patients will have at least 1 unirraditated or progressing brain metastasis of 0.5-2 cm on brain MRI. The study is conducted using a Simon’s optimal two-stage design, and approximately 87 patients will be enrolled concurrently (n=29 per cohort). Pembrolizumab (200 mg intravenously on Day 1 of each cycle) and lenvatinib (20 mg orally once daily) are administered in 21-day cycles for a maximum of 24 months. The primary endpoint is intracranial objective response rate at 4 months as assessed by the modified Response Evaluation Criteria in Solid Tumors (mRECIST). Exploratory analyses will include evaluation of tissue and blood-based immune-related correlates of response to the pembrolizumab and lenvatinib combination. The first patient was enrolled in January 2022 and accrual of patients is ongoing (NCT05064280). Citation Format: Ecaterina E. Dumbrava, Emma J. Montazari, Uyen M. Vu, Tiantian Cai, Mehmet Altan, Nuhad K. Ibrahim, Debra N. Yeboa, Jing Li, Frederick F. Lang, Gisela Sanchez, Isabella C. Glitza, Barbara J. O'Brien, Rashmi K. Murthy, Jianbo Wang, Tanisha T. Darko, Denisse Velazquez, Komal Shah, Funda Meric-Bernstam, Hussein Tawbi, Jordi Rodon. Phase II study of pembrolizumab in combination with lenvatinib in patients with triple negative breast cancer (TNBC), non-small cell lung cancer (NSCLC), and other tumor types and brain metastases [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 CT292.

Abstract LB020: Extracellular vesicle-mediated intrinsic induction of ferroptosis causes reversal of chemoresistance in lung cancer

Abstract Introduction: Resistance to chemotherapeutic drugs is the major cause of treatment failures and high mortality in lung cancer(LC). Thus, strategies to overcome chemoresistance is the major thrust area of cancer research. Ciplatin (CDDP) is the first line of chemotherapeutic interventions in lung cancer however, the treatment benefit diminishes due to develop resistance against the drug. Dysregulation of apoptosis is the major reason for developing chemoresistance. Ferroptosis is a recently discovered form of non-apoptotic regulated cell death (RCD) that can overcome the apoptosis mediated resistance. To exploit this our laboratory has developed a novel Extracellular Vesicle (EV)-iron oxide nanoparticle (IONP) hybrid system- 'ExoFeR,' that can induce ferroptosis. Experiments conducted on LC cells and patient derived tumoroids (PDTs) have shown reversal of CDDP resistance after ExoFeR treatment. Our study demonstrates the utility of ferroptosis as alternate treatment intervention for treating chemoresistant LC tumors. Materials and methods: EVs derived from normal lung fibroblast cells (MRC9) were loaded with IONPs. The complex was characterized by Transmission electron microscopy (TEM), Inductively coupled plasma mass spectrometry (ICP-MS) and through Zeta potential measurements. The induction ferroptosis by ExoFeR was confirmed in Non-Small Cell Lung Cancer (NSCLC) A549 cells and NSCLC PDTs by Prussian blue, FerroOrange, and Mito-FerroGreen staining. Further, ferroptosis markers like GPX4 and SCL7A11/xCT were evaluated to examine the induction of ferroptosis. Finally, A549 cells and resistant NSCLC PDTs were tested for sensitivity to chemo drugs. Results: Physicochemical characterization indicated the successful formulation of ExoFeR. Reduction in the expression level of SLC7A11/xCT- and GPX4 proteins in ExoFeR-treated NSCLC cells and PDTs suggested induction of ferroptosis which also showed sensitization of chemo drug (cisplatin). Summary/Conclusion: We report here the synthesis of a novel EV-IONP-based system that can intrinsically induce ferroptosis and overcome chemoresistance by circumventing the conventional apoptosis-based resistance. Our finding is expected to develop a potent cancer treatment strategy for efficiently treating resistant NSCLC tumors. Citation Format: Akhil Srivastava, Anjugam Paramananantham, Rahmat Asfiya. Extracellular vesicle-mediated intrinsic induction of ferroptosis causes reversal of chemoresistance in lung cancer [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 LB020.

Abstract SY35-02: Spatial mapping of mitochondrial networks in lung cancer

Abstract Mitochondria are critical to the governance of metabolism and bioenergetics in cancer cells. The mitochondria form highly organized networks, in which their outer and inner membrane structures define their bioenergetic capacity. However, in vivo studies delineating the relationship between the structural organization of mitochondrial networks and their bioenergetic activity have been limited. Here we present an in vivo structural and functional profiling of mitochondrial networks and bioenergetic phenotypes in non-small cell lung cancer (NSCLC) using an integrated platform comprised of positron emission tomography (PET) imaging, respirometry and 3-dimensional scanning block-face electron microscopy (3D SBEM). Interestingly, the diverse bioenergetic phenotypes and metabolic dependencies we identified in NSCLC tumors align with distinct structural organization of mitochondrial networks present. Further, we discovered that mitochondrial networks are organized into distinct compartments within tumor cells. In tumors with high rates of oxidative phosphorylation (OXPHOSHI) and fatty acid oxidation we identified peri-droplet mitochondrial networks wherein mitochondria contact and surround lipid droplets. In contrast, we discovered that in tumors with low rates of OXPHOS (OXPHOSLO), high glucose flux regulated i) peri-nuclear localization of mitochondria, ii) structural remodeling of cristae and iii) mitochondrial respiratory capacity. Our findings suggest that in NSCLC, mitochondrial networks are compartmentalized into distinct subpopulations that govern the bioenergetic capacity of tumors. Structure-and-function studies defining the relationship between mitochondrial architecture and metabolic dependencies may hold promise as strategies to profile metabolic liabilities unique to lung cancer subtypes. We anticipate that coupling PET imaging with 3D SBEM will have dynamic applications beyond that of lung cancer and enrich our understanding of how mitochondrial bioenergetics impact human disease. Citation Format: David B. Shackelford. Spatial mapping of mitochondrial networks in lung cancer [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 SY35-02.

ITGAL expression in non-small-cell lung cancer tissue and its association with immune infiltrates.

Integrin subunit alpha L (ITGAL) encodes an integrin component of LFA-1 and is a membrane receptor molecule widely expressed on leukocytes. It plays a key role in the interaction between white blood cells and other cells. There was a significant correlation between the expression of ITGAL and the tumor microenvironment in a number of cancers. However, experimental studies targeting ITGAL and immune cell infiltration in non-small-cell lung cancer (NSCLC) and the response to immune checkpoint inhibitor therapy are lacking. Data were obtained from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and Clinical Proteomic Tumor Analysis Consortium (CPTAC) databases to explore the relationship between ITGAL expression and prognosis, as well as the immune cell infiltration in patients with NSCLC. In addition, immunohistochemical staining for ITGAL and multiplex immunofluorescence (mIF) staining for ITGAL, CD20, CD68, CD4, and CD8 from tissue microarrays containing 118 tumor tissues and paired paracancerous tissues from patients with NSCLC were performed. The correlation between ITGAL expression and clinical factors, as well as the immunophenotypes of tumor-infiltrating immune cells, were also analyzed. In NSCLC tumor tissues, ITGAL was downregulated compared with matched paracancerous tissues, and low ITGAL expression was associated with a poor prognosis of NSCLC patients. Subsequently, immunohistochemistry results for tissue microarray showed that ITGAL expression was mainly elevated in tumor stroma and areas with highly infiltrated immune cells. ITGAL expression was higher in paracancerous tissues than tumor tissues. Furthermore, mIF results indicated that the patients with ITGAL-high expression tend had significantly higher CD8+ T cells, CD68+ macrophages, CD4+ T cells, and CD20+ B cells infiltration in their tumor tissues. Immunophenotypes were classified into three categories, that is deserted, excluded, and inflamed types, according to each kind of immune cell distribution in or around the cancer cell nest. MIF results showed that ITGAL expression level was correlated with the immunophenotypes. Furthermore, ITGAL expression was associated with the prognosis of NSCLC in patients with immune checkpoint inhibitor therapy and the patients with high ITGAL expression tends have better outcomes. ITGAL may be used as a biomarker for assessing the immune microenvironment in patients with NSCLC.

Hedgehog ligand and receptor cooperatively regulate EGFR stability and activity in non-small cell lung cancer.

The hyperactivation of epidermal growth factor receptor (EGFR) plays a crucial role in non-small cell lung cancer (NSCLC). Hedgehog (Hh) signaling has been implicated in the tumorigenesis and progression of various cancers, however, its function in NSCLC cells remains controversial. Herein, we present a novel finding that challenges the current understanding of Hh signaling in tumor growth. Expression of Hh ligands and receptor were assessed using TCGA datasets, immunoblotting and immunohistochemical. Biological function of Hh ligands and receptor in NSCLC were tested using colony formation, cell count kit-8 (CCK-8) and xenograft assays. Biochemical effect of Hh ligands and receptor on regulating EGFR stability and activity were checked via immunoblotting. Expression of Hh ligands and receptor was suppressed in NSCLC tissues, and the lower expression levels of these genes were associated with poor prognosis. Ptch1 binds to EGFR and facilitates its poly-ubiquitylation and degradation independent of downstream transcriptional signaling. Moreover, Hh ligands cooperate with Ptch1 to regulate the protein stability and activity of EGFR. This unique mechanism leads to a suppressive effect on NSCLC tumor growth. Non-canonical Hh signaling pathway, involving cooperation between Hh ligands and their receptor Ptch1, facilitates the degradation of EGFR and attenuates its activity in NSCLC. These findings provide novel insights into the regulation of EGFR protein stability and activity, offer new diagnostic indicators for molecular typing of NSCLC and identify potential targets for targeted therapy of this challenging disease.

Abstract 510: Flow cytometry evaluation of cell cycle arrest in A549 human non-small cell lung cancer cells and tumors following treatment with cell cycle inhibitors: A multifaceted approach to targeting cancer

Abstract The contemporary landscape of cancer treatment has witnessed significant progress with FDA-approved targeted drugs. The FDA's Project Optimus has underscored the growing need for innovative pharmacodynamic endpoints to determine the optimal biological dose in clinical development. Flow cytometry, a potent analytical tool in clinical and preclinical labs, now plays a leading role in cancer research, enabling the evaluation of new drug effects on cell cycle regulation and consideration of rational drug combinations with approved drugs. At TD2, we strive to enhance the translatability of in vitro induced cell cycle arrest studies to in vivo biomarker proof of concept and efficacy. We employ a comprehensive approach, utilizing flow cytometry to assess cell cycle dynamics in cultured cells and in vivo tumor models after treatment with approved and novel drugs. This abstract focuses on the translational relevance of combined in vitro and in vivo testing of well-known drugs that disrupt the cell cycle, laying the foundation for optimized treatment strategies and potentially personalized cancer therapies. Human A549 non-small cell lung carcinoma cells were treated with either thymidine (2.0mM) or the microtubule-depolymerizing drug nocodazole (100ng/ml) for 18 hours, then examined by flow cytometry. Thymidine induced a greater than 85% S-phase arrest, while nocodazole induced a greater than 88% G2/M-phase arrest. A549 cells (5 × 106 cells/mouse) were subcutaneously implanted in SCID mice, followed by treatment with an FDA-approved cell cycle inhibitor, Palbociclib, or cisplatin chemotherapy. Palbociclib, a CDK4/6 inhibitor, and low-dose cisplatin treatment resulted in an increased accumulation of cells in the G0/G1 phase, greater than 80% for each. Both treatments exhibited promising anti-cancer activity, with tumor growth inhibition (TGI) of 69% and 87%, respectively. Our findings highlight flow cytometry's multifaceted utility in assessing cell cycle arrest, providing a comprehensive view of agent effects on cancer cells in culture and within the complex in vivo tumor microenvironment. Determining the impact of any new drug on specific cell cycle checkpoints could allow for a more strategic plan for combination therapy. Integrating flow cytometry could enable early biomarker monitoring of tumor cell cycle changes, offering insights into the therapeutic potential of new agents alone or in combination. This multidimensional evaluation contributes to understanding cancer biology and has the potential to guide the development of novel therapeutic strategies, optimize treatment regimens to identify optimal biological doses, and improve patient outcomes in the era of precision medicine. Citation Format: Erin E. Trachet, Emily Cluff, Ana Lemos de Matos, Damaris Diaz, Chelsea Holmquist, Paul Gonzales. Flow cytometry evaluation of cell cycle arrest in A549 human non-small cell lung cancer cells and tumors following treatment with cell cycle inhibitors: A multifaceted approach to targeting 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 510.

Abstract 2: T cells selected from lymph node acquisition for adoptive cell therapyin NSCLC

Abstract Background: A primary limitation of PD-1 inhibitors in non-small cell lung cancer (NSCLC) arises from their inability to act on 'cold' tumors without tumor-reactive T cells, necessitating alternative approaches. Adoptive cell therapy (ACT) using (CAR)-engineered cells, strives to enhance antitumor immunity but faces several challenges such as identifying safe antigens, tumor heterogeneity, antigen escape, cell trafficking, and T cell persistence. To address these issues, we explored a new source of T cells from the benign tumor draining lymph nodes (tdLNs) of NSCLC patients. Our initial results have shown that tdLNs is a reservoir for of tumor-relevant 'stem-like' T cells. We posit that employing these pluripotent T cells for ACT could achieve significant tumor rejection in NSCLC. Methods: Resected tumors, tdLN, non-draining (ndLN), and blood from NSCLC patients, as well as a syngeneic murine lung cancer model (344SQ), underwent analysis. T cells were profiled using flow cytometry, cytotoxicity and proliferation assays. TCR and single cell (sc)RNA sequencing assessed clonal expansion, diversity, and transcriptional profiles of tumor-relevant T cells. T cells were then transduced with an ICAM-1 targeting CAR, in vivo efficacy was evaluated in an A549 murine lung cancer model. Results: T cell subsets with stem-cell memory characteristics, as indicated by PD-1+, TCF1hi, CXCR5+, and CD8+ expression, which were not significantly found in the tumor or PB. These T cells exhibited progenitor-like transcriptional signatures, enhanced SELL and TCF-1 expression, fewer exhaustion markers, and superior in vitro proliferation compared to TILs from both a murine lung cancer model and patient-derived tissues. scRNA sequencing, coupled with TCR "tumor matching" (TM) techniques, exposed a rich clonal diversity of tumor-relevant clones within tdLNs, which showcased a broader transcriptional memory profile and distinct CD4+ and CD8+ phenotypes. Upon analyzing the top 100 expanded (n>3) TM clones, 47 featured the presence of tdLN-derived T cells, covering progenitor, stem cell-like, and central memory clusters. T cell subsets were then transduced with a CAR targeting ICAM-1—a cell surface protein frequently overexpressed in NSCLC tumors. Manufacturing protocol yielded high transduction efficiency and T cell expansion within two weeks in 6/6 patients, consistent with PB-derived CAR T cells and on par with the optimal dosing requirements of an ICAM-1 CAR Phase I trial (NCT04420754). tdLN-CAR T cells showed potent antitumor efficacy compared to the control in an aggressive NSCLC murine model (Median survival 103d vs 66d; respectively; p=0.006). Conclusions: This represents the first reported use of T cells from tdLN for genetically engineered ACT. The data indicate that modifying antigen-experienced, stem-like T cells from tdLN with CAR is a promising and efficient method in a NSCLC murine model, warranting further research. Citation Format: Tatiana Delgado Cruz, Yanping Yang, Rachel Honigsberg, Geoffrey Markowitz, Nasser K Altorki, Vivek Mittal, Moonsoo M. Jin, Jonathan Villena-Vargas. T cells selected from lymph node acquisition for adoptive cell therapyin 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 2.

Abstract 5101: Comparative genomic analysis reveals distinctive immune profiles in MET exon 14 mutated and MET amplified lung cancer

Abstract Background: In non-small cell lung cancer (NSCLC), the MET tyrosine kinase receptor can be mutated or amplified resulting in dysregulation of receptor function leading to tumor proliferation. MET exon 14 (METEx14) mutated and MET amplified NSCLC tumors have shown varied response to immunotherapy. Therefore, we sought to compare the genomic and immune landscape of MET-altered NSCLC. Methods: The genomic profiles of 3,821 NSCLC tumors sequenced using the Strata Select assay on the Strata Oncology Platform were analyzed. Tumors were sorted based on METex14 mutations, MET amplification (METamp) defined as copy number gain (CNG) ≥6 and MET wild-type (METwt). The RNA expression of 19 immune regulatory genes, tumor mutation burden (TMB), Strata Immunotherapy Response Signature (IRS) score and the frequency of gene co-alterations were compared across groups. Statistical comparisons of medians were done with Kruskal-Wallis testing and categorical comparisons with Chi square using R Studio version 4.3.2. Results: 122 (3.2%) METex14, 70 (1.8%) METamp, and 3,629 (95.0%) METwt were identified. Patients with METex14 were older and more frequently female. MET gene expression was found to be higher in METamp (median 14.4) compared to METex14 (median 12.3) (P<0.01). TMB was lowest in METex14 (median 2.7) group compared to METamp (median 7.1) and METwt (median 5.1) (P<0.001). High PD-L1 expression occurred in 71.4% of METamp, 68.0% of METex14 and 41.3% of METwt (P<0.001). Overall, METamp tumors had a greater proportion of high IRS scores (52.9%) compared to METex14 (35.2%) and METwt (45.4%) (P<0.01). mRNA expression of immune checkpoints CTLA4, PD-1, LAG3, IDO1, TIGIT and HAVCR2 were all higher in METex14 compared to METamp and METwt (P<0.001 for all). METex14 tumors also exhibited higher gene expression of CD4, CD8A and FOXP3 compared to METamp and METwt (p<0.001). METex14 had higher prevalence of MDM2 amplification (34% vs 3% vs 4%) and lower prevalence of alterations in EGFR (0.8% vs 22% vs 21%), KRAS (0.8% vs 10% vs 26%), CDKN2A (17% vs 29% v 28%) and TP53 (34% vs 87% vs 64%) compared with METamp and METwt tumors, respectively (p<0.05). Conclusion: METex14 and METamp tumors harbor distinct immune-gene expression profiles and co-occurring gene alterations that distinguish themselves from each other and METwt tumors. Further analysis must be conducted to determine how these variations impact immunotherapy in MET-altered NSCLC. Citation Format: Saahil Javeri, Rachel Minne, Shrey Ramesh, Andrew Baschnagel, Randy Kimple, Laura Lamb, Scott Tomlins, Bryan Johnson, Nickolay Khazanov. Comparative genomic analysis reveals distinctive immune profiles in MET exon 14 mutated and MET amplified 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 5101.

Abstract 4007: AIC100 CAR-T cells targeting ICAM-1 are efficacious against solid tumors in xenograft mouse models of non-small cell lung cancer (NSCLC) and cervical cancer (CC)

Abstract Purpose: Establish preclinical proof of concept efficacy with CAR T cells (AIC100) in xenograft models of solid tumors (NSCLC and CC) expressing high levels of surface ICAM-1. Experimental: AIC100 is a third generation CAR T cell with micromolar affinity to ICAM-1, tuned lower than most CARs used to date in pre-/clinical studies. Affinity tuned AIC100 drug product is expected to selectively bind and kill tumor cells while safely sparing healthy cells. AIC100 also co-expresses somatostatin receptor 2 (SSTR2), which enables in vivo monitoring of AIC100 distribution and expansion by DOTATATE PET/CT imaging. To test preclinical efficacy of AIC100 in NSCLC and CC, four xenograft mouse models were generated using tumor cell lines known to have ICAM-1 expression (verified by flow cytometry) to test efficacy of AIC100. Reporter cell lines transfected with Luciferase were H226 NSCLC adenocarcinoma, H441 NSCLC squamous cell carcinoma, HeLa CC adenocarcinoma and SW756 CC squamous cell carcinoma. AIC100 efficacy was established by in vitro cytotoxicity prior to in vivo studies. NSG mice (n=10 per cell line) were inoculated via tail vein injection (1.0x106 cells) in cell growth media. Mice were imaged to ensure solid tumor formation which occurred in all mice within 3-5 days in the lung. At D0, mice were randomized into 2 groups (n=5 per group) of vehicle (PBS) and AIC100. At D1, mice were injected with AIC100 (1.0x107 live T cells) or vehicle. Mice were followed by weight and clinical observations 2x weekly and imaging by IVIS weekly. The study continued until vehicle group was euthanized due to tumor related co-morbidities at which time the study concluded. Results: AIC100 showed clear treatment benefit in both models of NSCLC and CC. In CC HeLa model, in mice treated with AIC100, primary tumors were significantly reduced by D8 and eliminated in 5/5 mice by D22. In CC SW756 model, in mice treated with AIC100, tumor growth was reduced in size by D22. In NSCLC H441 model, in mice treated with AIC100, primary tumors were largely eliminated in 5/5 mice by D15. In NSCLC H226 model, in mice treated with AIC100, tumors were significantly reduced by D8 and largely eliminated by D57 (4/5 mice). A clear survival benefit was observed in AIC100 treated mice in NSCLC H226 xenograft model. These results show treatment benefit by AIC100 in 2 tumor models per indication and establish proof of concept for the potential development of AIC100 as a treatment for ICAM-1 expressing NSCLC and CC cancers. Conclusions: AIC100 has significant treatment benefit in NSCLC and CC tumors with ICAM-1 expression in in vivo preclinical studies. Citation Format: Alyssa Birt, Serene Josiah, Kelsey L. Joyce, Srinija Alla, Michael P. Gallagher, Janusz Puc, Hanh Nguyen, Emily Walsh Martin, Sonal Gupta. AIC100 CAR-T cells targeting ICAM-1 are efficacious against solid tumors in xenograft mouse models of non-small cell lung cancer (NSCLC) and cervical cancer (CC) [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 4007.

Abstract 217: Establishment of a spatially defined co-culture model of non-small cell lung cancer organoids and cancer-associated fibroblasts to investigate phenotypic heterogeneity

Abstract Background: Cancer-associated fibroblasts (CAFs) are heterogeneous, with CAF subtypes being localized in spatially defined areas of tumors. However, it remains unclear which CAF subsets specifically play a role in fundamental processes such as tumor growth, invasion, and response to therapy. This study aims to establish a novel in vitro model that recapitulates the in vivo tumor-stroma spatial relationship to investigate CAF heterogeneity and its impact on tumor cell growth in non-small cell lung cancer (NSCLC). Methods: The in vitro model was established using NSCLC patient-derived CAFs and tumor organoids as a 6:1 ratio. Tumor cells originating from xenograft-derived organoids (XDOs) were embedded in a hydrogel containing CAFs (Day 0) and co-cultured for 5 days. Confocal microscopy was utilized to characterize and measure the proliferation of the XDO tumor cells in the co-culture setup, and the proliferation rate was defined as differences in tumor spheroid area between Day 1 and 5. Single-cell RNA-sequencing (scRNAseq) and gene set enrichment analysis (GSEA) were then used to assess the gene expression and signaling pathways dominant in mono- and co-culture to characterize CAF heterogeneity in this model. Results: Initially, the culture media formulations and the matrix for the growth of both CAFs and tumor cells were screened to assess the compatibility of different culture elements. Both CAFs and XDO tumor cells proliferated well in reduced organoid media (M27RD). This reduced medium and the novel matrix composition (50% Matrigel/50% Rat Tail type I collagen) also allowed for maintaining CAF contractility and XDO proliferation. Utilizing these harmonized media and matrix conditions, a spatially defined co-culture model, which incorporated XDOs as a central cluster surrounded by CAFs, was generated. In confocal microscopy, the proliferation rate of tumor cells was significantly higher in co-cultures with CAFs (adjusted p-value < 0.01), while not significant in mono-culture of tumor cells. The GSEA using the Hallmark gene sets on scRNAseq data generated in three co-cultures identified the heterogeneity among CAFs: two CAFs showed significantly higher effect on tumor cell proliferation with the most highly differential expressed genes (DEGs) enriched with inflammation and mitosis and the top high DEGs associated with epithelial-mesenchymal transition, while the other CAF has no effect. Conclusion: The harmonized co-culture conditions for NSCLC organoids and CAFs enabled the characterization of both cell phenotypes and the building of an informed model architecture to study tumor-stroma communication. The presence of CAFs increased tumor cell growth with enrichment in secreted proteins involved in epithelial-mesenchymal transition. Citation Format: Cheol-Kyu Park, Roya Navab, Elisa D'Arcangelo, Quan Li, Hiroyuki Ogawa, Nikolina Radulovich, Nhu-An Pham, Ming-Sound Tsao. Establishment of a spatially defined co-culture model of non-small cell lung cancer organoids and cancer-associated fibroblasts to investigate phenotypic heterogeneity [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 217.