Lung Squamous Cell Carcinoma Tumor Research Articles

TNIK inhibition sensitizes TNIK-overexpressing lung squamous cell carcinoma to radiotherapy.

Most patients with lung squamous cell carcinoma (LSCC) undergo chemotherapy, radiotherapy, and adjuvant immunotherapy for locally advanced disease. The efficacy of these treatments is still limited due to dose-limiting toxicity or locoregional recurrence. New combination approaches and targets such as actionable oncogenic drivers are needed to advance treatment options for LSCC patients. Moreover, other options for chemotherapy-ineligible patients are also limited. As such there is a critical need for the development of selective and potent chemoradiosensitizers for locally advanced LSCC. Here, we investigated inhibiting TRAF2 and NCK-interacting protein kinase (TNIK), which is amplified in 40% of LSCC patients, as a strategy to sensitize LSCC tumors to chemo- and radiotherapy. Employing a range of human LSCC cell lines and the TNIK inhibitor NCB-0846, we investigated the potential of TNIK as a chemo- and radiosensitizing target with in vitro and in vivo preclinical models. The combination of NCB-0846 with cisplatin or etoposide was at best additive. Interestingly, pre-treating LSCC cells with NCB-0846 prior to ionizing radiation (IR) potentiated the cytotoxicity of IR in a TNIK-specific fashion. Characterization of the radiosensitization mechanism suggested that TNIK inhibition may impair the DNA damage response and promote mitotic catastrophe in irradiated cells. In a subcutaneous xenograft in vivo model, pretreatment with NCB-0846 significantly enhanced the efficacy of IR and caused elevated necrosis in TNIKhigh LK2 tumors but not TNIKlow KNS62 tumors. Overall, these results indicate that TNIK inhibition may be a promising strategy to increase the efficacy of radiotherapy in LSCC patients with high TNIK expression.

Abstract 3261: Improving targeted therapy in female patients with lung squamous cell carcinoma

Abstract Non-small cell lung cancer (NSCLC) will claim the lives of ~60,000 female patients in the US in 2023. Lung squamous cell carcinoma (LUSC) is a subtype of Non-Small Cell Lung Cancer (NSCLC) that comprises ~30% of NSCLC and is characterized by high metabolic activity. Specifically, LUSC typically exhibits elevated rates of both glucose and glutamine metabolism driven by the PI3K-AKT-mTOR and KEAP1/NRF2 pathways. Further, a paucity of identifiable genetic drivers in LUSC has resulted in a lack of effective targeted therapies for patients with this devastating disease. Despite recent advances in therapy, the 5-year survival rate for patients with advanced NSCLC is 6%, underscoring the need for better treatment options. Importantly, no significant sex bias is seen in the newly diagnosed cases of lung cancer (~50%M/~50%F) (American Cancer Society, 2023), yet there are no sex-specific therapeutic approaches in clinical use. We demonstrated that the catalytic mTOR kinase inhibitor TAK228 can be utilized to effectively treat LUSC tumors via inhibition of glycolysis, an approach that is currently being evaluated on two phase I/II clinical trials (NCT02417701, NCT04250545), launched as a direct result of our pre-clinical work. However, our pre-clinical data suggests that despite the potential benefit of TAK228 inhibition in LUSC, a subset of tumors are resistant to TAK228 despite on-target activity of mTOR inhibitor, leading to the rationale of the current hypothesis, namely the possibility that sex-specific differential effects of TAK228 may exist. To evaluate this hypothesis, we have generated preliminary data demonstrating that sex is a determinant of TAK228 response in LUSCs, whereby male mice are significantly more sensitive to mTOR inhibition than females. Follow-on experiments have demonstrated that the anti-estrogen therapy (Letrozole) sensitized previously resistant female mice bearing LUSCs to TAK228, suggesting an estrogen-related link to TAK228 resistance in females. Additionally, RNASeq and biochemical analyses identified pathways that are upregulated in TAK228 resistant female mice with LUSC tumors. These results suggest that the estrogen may play a key role in the observed pattern of female resistance to mTOR inhibition. As such, the goal of the current proposal is to evaluate the catalytic mTOR kinase inhibitor TAK228, in combination with anti-estrogen therapy as a therapeutic strategy to treat LUSC in females. Citation Format: Milica Momcilovic, David Shackelford. Improving targeted therapy in female patients with lung squamous cell carcinoma [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 3261.

Abstract C049: Genomic correlates with ancestry in lung squamous cell carcinoma

Abstract Outcome data from lung squamous cell carcinoma (SCC) show significantly lower overall and progression-free survival in Black patients. However, whether any genomic factors correlate with race and ancestry in this lung SCC, and whether this may contribute to disparities, has not been elucidated. We analyzed clinically annotated genomic data from 501 lung SCC tumor samples in the Cancer Genome Atlas (TCGA), with available ancestry admixture data. Here, we analyzed clinical data, gene expression profiles, mutations, and copy number changes to identify significantly differential events between tumors from patients of European (EUR) or African (AFR) ancestry. In tumors from AFR patients, we identified more frequent gain of chromosome arm 8q and/or the chr8q24 locus, where the oncogene MYC and its enhancers are located. 8q24 gain is associated both with global AFR ancestry and local AFR ancestry. Furthermore, expression of MYC targets is significantly increased in AFR samples relative to EUR samples, all of which suggest a role for the MYC pathway in differential cancer progression. MYC activity correlates with decreased immune infiltration and gene signaling, and patients with AFR ancestry are more likely to have decreased immune infiltrate and pathway activity. Our findings are consistent with observations in prostate cancer; this is the first study to find MYC enhancer amplification correlating with African ancestry in lung SCC. We are currently testing additional cohorts, including AACR GENIE, to validate these findings. Future studies will elucidate patient-specific treatment modalities. Citation Format: Sejal Jain, Alexandria Yao, Fatemeh Momen-Heravi, Diane Allen-Gipson, Xiaoyang Zhang, Brian S. Henick, Jian Carrot-Zhang, Alison M. Taylor. Genomic correlates with ancestry in lung squamous cell carcinoma [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C049.

SLC22A3 that encodes organic cation transporter-3 is associated with prognosis and immunogenicity of human lung squamous cell carcinoma.

SLC22A3, the gene which encodes organic cation transporter (OCT)-3, has been linked to the prognosis of several types of cancer. However, its role in lung squamous cell carcinoma (LSCC) has not been addressed elsewhere. We analyzed gene expression, DNA methylation, and clinicopathological data from The Cancer Genome Atlas - Lung Squamous Cell Carcinoma (TCGA-LUSC) (n=501), a publicly available database exclusively consisting of LSCC patients. Using a 5 FPKM (fragments per kilobase of exon per million mapped fragments) cut-off, we divided LSCC patients into two groups: patients with tumors possessing high and low SLC22A3 expression (SLC22A3-high and SLC22A3-low, respectively). Prognostic significance was determined through Cox analyses and Kaplan-Meier curves for overall survival (OS) and disease-free survival (DFS). Differential methylation position (DMP), differentially gene expression, and pathway analyses were performed. Validation was carried out in GSE74777 (n=107), GSE37745 (n=66), GSE162520 (n=45) and GSE161537 (n=17). SLC22A3-high LSCC patients had lower OS and DFS rates than SLC22A3-low LSCC patients. The different expression levels of SLC22A3 in LSCC were correlated with the methylation status of the SLC22A3 gene. Pathway analysis indicated that SLC22A3 expression levels were positively correlated with immune-related pathways such as inflammatory response and abundance of infiltrating immune cells in the tumor microenvironment (TME). Notably, in the SLC22A3-high group, many genes encoding immunological checkpoint inhibitory molecules were upregulated. In addition, SLC22A3 expression positively correlated with the Hot Oral Tumor (HOT) score, indicating high tumor immunogenicity. These findings suggest that high expression of SLC22A3 is associated with poor prognosis and high immunogenicity in LSCC tumors.

Identification of prognostic immune subtypes of lung squamous cell carcinoma by unsupervised consistent clustering.

We performed UCC on the expression data of lung squamous cell carcinoma tumor samples to identify the classification of lung squamous cell carcinoma (LUSC) tumor samples, and calculated the levels of different classified immune cells by single-sample gene enrichment analysis (ssGSEA) to obtain a set of immune-related subtype gene tags, which can be used for subtype classification of lung squamous cell carcinoma. TCGA-LUSC and GSE30219 data of lung squamous cell carcinoma were obtained from TCGA and GEO databases. Prognostic-associated subtypes were identified by unsupervised consensus clustering (UCC). Using ssGSEA analysis to calculate the level of immune cells of different subtypes, obtain the connection between subtypes and immunity, identify the gene signatures recognized by subtypes, and verify this group of gene signatures through GSE30219. We effectively identified 2 subtypes that were significantly associated with prognostic survival by UCC, and calculated according to ssGSEA, the 2 subtypes were significantly different at the level of immune cells, followed by introducing a This weighted thinking computes a set of gene signatures that are significantly associated with subtype 1. During validation, this set of gene signatures could efficiently and robustly identify distinct prognostic immune subtypes, demonstrated the validity of this set of gene signatures, as well as 2 subtypes of lung squamous cell carcinoma. We used lung squamous cell carcinoma data from public databases and identified 2 prognostic immunosubtypes of lung squamous cell carcinoma and a set of gene tags that can be used to classify immune subtypes of lung squamous cell carcinoma, which may provide effective evidence for accurate clinical treatment of lung squamous cell carcinoma.

Immune-active tumor-adjacent tissues are associated with favorable prognosis in stage I lung squamous cell carcinoma

The immunogenomic features of tumor-adjacent lungs (TALs) in stage I lung squamous cell carcinoma (LUSC) are not clear. Multiomics analyses of tumor tissues and paired TALs from 59 stage I LUSC patients were performed. Compared to tumors, TALs exhibited a better-preserved immune contexture indicated by upregulation of immune pathways, increased immune infiltration, and higher expression of immune effector molecules. Notably, TALs had no mutations in PTEN and KEAP1, a lower incidence of human leukocyte antigen (HLA) loss and higher expression of HLA class I genes, major histocompatibility complex (MHC) I chaperones, and interferon (IFN)-γ-associated genes. Digital spatial profiling validated the generally higher immune infiltration in TALs and revealed a higher level of immune heterogeneity in LUSC tumors. Importantly, patients with higher immune infiltration in TALs had significantly longer survival, while high immune heterogeneity was associated with inferior patient survival. Our work can be considered in the selection of patients for adjuvant therapy, especially immunotherapy.

Protein Exaptation by Endogenous Retroviral Elements Shapes Tumor Cell Senescence and Downstream Immune Signaling.

Cancer cell senescence in lung squamous cell carcinoma (LUSC) is associated with a poor response to chemotherapies and immunotherapies due to promotion of an immunosuppressive tumor microenvironment. This environment is shaped by the senescence-associated secretory pathway, which recruits suppressive immune cell populations. In a recent study, Attig and colleagues identified a transcription factor-activated molecular switch that circumvents cellular senescence through increased expression of the calbindin protein. A human endogenous retrovirus (HERV) sequence upstream of the calbindin gene, CALB1, promotes the transcription of an HERVH-CALB1 transcript through a splice event at the third CALB1 exon in a process known as protein exaptation. The KLF5 transcription factor mediates this transcriptional activity by binding at the HERVH sequence, subsequently initiating the chimeric HERVH-CALB1 transcription. This increased expression of calbindin reduces CXCL8 chemokine production and downstream neutrophil recruitment in LUSC tumor cells. CALB1 exaptation by HERVH is one example by which endogenous retroelements (ERE) regulate immunity in human cancers, highlighting the emerging role of EREs in tumor immunity.

Abstract 422: Cell intrinsic determinants of response to mTOR based therapy

Abstract Lung cancer is a leading cause of cancer death with estimated 135,000 deaths in 2021. Recent developments in the immunotherapy field have expanded treatment options available to lung cancer patients, with most patients now receiving checkpoint inhibitors as part of the first line therapy. However, while durable responses have been achieved in 20-30% of patients, the majority of patients inevitably progress on immunotherapy, chemo-immunotherapy or targeted therapy regiments. With five year survival at ~10% there is an urgent need for novel strategies for the treatment of lung cancer. Lung squamous cell carcinoma (LUSC) is an aggressive subtype of non-small cell lung cancer characterized by poor prognosis. LUSC are highly metabolically active, with uptake of high levels of glucose and glutamine supporting their growth and proliferation. In addition to fueling tumor cell growth, high levels of glycolysis and glutaminolysis help create unfavorable tumor microenvironment, contributing to the poor response to immunotherapy. The Cancer Genome Atlas (TCGA) revealed that LUSC have a high mutational burden but few mutations in oncogenes that can be targeted with selective inhibitors. This lack of clear genetic drivers in LUSC has resulted in a lack of targeted therapies for these patients. We have demonstrated that catalytic mTOR kinase inhibitor TAK228 inhibits glycolysis in LUSC cell lines, in xenografts, in patient derived xenografts and genetically engineered mouse models. However, despite inhibiting active mTOR pathway and lowering glucose uptake, LUSC tumors were able to maintain high levels of proliferation. Our work showed that tumors were able to adapt to prolonged treatment with TAK228 by relying on glutaminolysis. Inhibiting both mTOR and GLS, a key enzyme in glutaminolysis, resulted in reduced proliferation index and lower tumor volume. However, while combination therapy halted tumor growth, there was a lack of tumor regression. Therefore, tumors are poised to further adapt to combination therapy. Here we show that upregulation of lysosomal function supports adaptation to TAK228 therapy in LUSC by supporting macropinocytosis, a form of endocytosis that allows cancer cells to uptake extracellular fluids and further process solutes and macromolecules in lysosomes, leading to the release of amino acids inside the cytoplasm. This suggests that macropinocytosis might be a mechanism that is utilized by LUSC in order to escape mTORi therapy, where upon treatment with mTOR inhibitors, lysosomal catabolism of macropinocytosed proteins increases, allowing tumor cells to increase their proliferation.Resistance to targeted therapies is a major hurdle to achieving effective therapies in multiple cancer types. Identifying ways to overcome resistance to targeted therapies would allow for longer and more efficient therapeutic response for larger number of patients, ultimately leading to longer survival and better quality of life. Citation Format: Milica Momcilovic, David Shackelford. Cell intrinsic determinants of response to mTOR based therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 422.

Abstract 4622: Tumor microenvironment differences between lung cancer subtypes revealed by spatial transcriptomics

Abstract Lung cancer is a leading cause of cancer deaths worldwide and has complex underlying genetic drivers, subtypes and immune cell types. Molecular analysis of bulk tumors has repeatedly identified key somatic driver genes and subtypes in lung cancer. However, these key molecular strata of bulk lung tumors still contain significant heterogeneity which if characterized in finer detail may reveal new tumor microenvironment factors and lead to improved patient prognostication and therapy options. Here, we sought to compare the tumor microenvironments of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) through spatial transcriptomics. Using four frozen lung tumors and three with replicated sections (n = 7), we sequenced spatial transcriptomes using the 10X Visium platform and illumina sequencing to measure up to 5,000 latticed-spots throughout 6.5 mm2 of the tumor surface area. Data analysis revealed a large number of latticed-spots per tumor (median 3,486) with a large number of genes detected per median spot (tumor median 4,427). Through unsupervised clustering of spots in each tumor, we found between 8 and 10 clusters per tumor with distinct pathway activities, including multiple immune-enriched clusters per tumor (range: 3-5). Immune-enriched clusters in one LUAD tumor displayed a spatial shape consistent with tertiary lymphoid structures (TLS). Concordantly, this cluster overexpressed both B cell and T cell pathways and as well as a TLS signature from liver cancer. Interestingly by bulk tumor RNA analysis, this TLS+ tumor was classified to be in the terminal respiratory unit expression subtype, which is an immune-mild bulk subtype. This supports that the TLS signal can be a unique property of spatial expression in lung cancer that may be unobservable by bulk tumor RNA sequencing. Then to compare global spatial heterogeneity among tumors, we calculated an index of expression spatial continuity and found LUSC tumors to have more contiguous expression patterns than LUAD tumors (mean 0.60 vs 0.54). We also quantified expression diversity across all tumor latticed-spots and found that LUSC tumors had greater values compared to LUAD tumors (mean 0.37 vs 0.27). Together, our results suggest that LUSC has a more contiguous and heterogeneous tumor expression microenvironment than LUAD. TLS are predictive of immune checkpoint inhibitor response in many other tumor types, and our results suggest that spatial transcriptomics may also identify this responsiveness in lung cancer. Future, larger cohorts of lung tumors are needed to determine recurrent spatial properties associated with patient outcome and treatment response. The views expressed in this abstract are solely of the authors and do not reflect the official policy of the Departments of Army/Navy/Air Force, Department of Defense, USUHS, HJF, or U.S. Government. Citation Format: Matthew D. Wilkerson, Savannah Kounelis-Wuillaume, Camille Alba, Teri J. Franks, Martin L. Doughty, Robert L. Kortum, Robert F. Browning, Clifton L. Dalgard, Craig D. Shriver. Tumor microenvironment differences between lung cancer subtypes revealed by spatial transcriptomics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4622.

Resveratrol improves the cytotoxic effect of CD8 +T cells in the tumor microenvironment by regulating HMMR/Ferroptosis in lung squamous cell carcinoma

Ferroptosis, an iron-dependent cell death process, is a potential therapeutic strategy for Lung squamous cell carcinoma (LUSC). Resveratrol (RES) is an anti-tumor polyphenol. However, whether and how RES treats LUSC is not yet known. This study aimed to investigate the effect of RES on LUSC and to explore its potential mechanism. This study used a combination of proteomics, bioinformatics, clinical samples, and cell experiments to study the interaction between HMMR and the ferroptosis signaling pathway and investigate the role of RES in regulating tumor immune microenvironment and anti-tumor by cytotoxic CD8 +T cells in LUSC. Ferroptosis signaling pathway and HMMR were involved in the LUSC tumor immune microenvironment and correlated with worse prognosis of LUSC patients. RES+H520 cells induced a higher level of ferroptosis and MDA, mainly by reducing the expression of GPX4 and SLC7A11, inducing the expression of ACSL4 and TFRC. HMMR, GSH, and SOD contents were lower observed than in H520 cells. When HMMR was expressed, SLC7A11 was also highly expressed in LUSC, and there was an interaction between HMMR expression and SLC7A11. In addition, RES increased the TNF-α, IFN-γ, IL-12, and IL-2 expression and increased the cytotoxic effects of CD8 +T cells expressions in LUSC. Resveratrol regulates SLC7A11-HMMR interaction, activates ferroptosis, enhances the cytotoxic effect of CD8 +T cells, and regulates the tumor immune microenvironment. Based on the pathogenesis of LUSC and the clinical efficacy of RES, this study explored the influence of RES on LUSC, clarified its biological effects, and further provided cell biological basis for the clinical application of RES, which could guide clinical combination and personalized medicine, improve the response rate of immunotherapy and benefit more patients with LUSC.

Identification of Clinical Prognostic Regulators and Analysis of Ferroptosis-Related Signatures in the Tumor Immune Microenvironment in Lung Squamous Cell Carcinoma.

Lung squamous cell carcinoma (LUSC) is a common respiratory malignancy and presents an increasing prevalence. Ferroptosis is a newly identified controlled cell death that has captured clinical attention worldwide. However, the ferroptosis-related lncRNA expression in LUSC and its relevance to prognosis remain elusive. The research measured predictive ferroptosis-related lncRNAs in LUSC samples from the TCGA datasets. Data on the stemness indices (mRNAsi) and corresponding clinical characteristics were obtained from TCGA. A prognosis model was established using the LASSO regression. Changes within the neoplasm microenvironment (TME) and medicine association were examined to grasp higher immune cell infiltration in numerous risk teams. In line with coexpression studies, the expression of lncRNAs is closely associated with that of ferroptosis. They were overexpressed in unsound people in the absence of alternative clinical symptoms. The low-risk and speculative teams were considered to have substantial differences in CCR and inflammation-promoting genes. C10orf55, AC016924.1, AL161431.1, LUCAT1, AC104248.1, and MIR3945HG were highly expressed in the high-risk group, suggesting their involvement in the oncology process of LUSC. Moreover, AP006545.2 and AL122125.1 were considerably higher in the low-risk group, implying the potential of these genes as LUSC tumor suppressor genes. The biomarkers listed above may serve as therapeutic targets for LUSC. lncRNAs were also linked to patient outcomes in the LUSC trial. lncRNAs of ferroptosis were overexpressed in the high-risk cohort without other clinical signs, implying their potential to predict BLCA prognosis. GSEA highlighted immunological and tumor-related pathways in the high-risk group. LUSC occurrence and progression are linked to lncRNAs of ferroptosis. Corresponding prognostic models help forecast the prognosis of LUSC patients. lncRNAs of ferroptosis and associated immune cell infiltration in the tumor microenvironment (TME) may serve as potential therapeutic targets in LUSC, which requires further trials. In addition, the lncRNAs of ferroptosis signature offer a viable alternative to predict LUSC, and these ferroptosis-lncRNAs show a prospective research area for LUSC-targeted treatment in the future.

Abstract A009: Survival disparities in lung squamous cell carcinoma: Clinical and genomic correlates with genetic ancestry

Abstract Many studies show racial and ethnic survival disparities in lung squamous cell carcinoma (LSCC), and self-identified African-Americans have significantly lower overall survival and progression-free survival than self-identified Caucasians. However, survival models based on genetic ancestry, rather than self-identified race, are needed to assess whether genetic ancestry influences this survival disparity or whether the cause of the disparity is largely socioeconomic. Using clinically annotated genomic data from 501 LSCC tumor samples in the Cancer Genome Atlas (TCGA), we compared clinical data, gene expression profiles, mutations, and copy number changes of tumors across ancestral groups to identify significantly differential events. We then applied the Cox Proportional Hazards model to predict overall survival as a function of genetic ancestry alongside other genetic, molecular, and clinical variables. We found a significant difference in metastatic stage between tumors from patients of African (AFR) and European (EUR) ancestry, which was significant in predicting survival. We identified increased copy number of chromosome arm 8q and locus chr8q24, as well as increased expression of MYC targets in AFR samples relative to EUR samples, all of which suggest a role for the MYC pathway in differential cancer progression. In addition, we found enrichment of amplifications at chr22q11 and chr16p11 in AFR samples compared to EUR samples. We identified top differentially mutated genes between groups, of which mutant SPRR3, PDGFA, FRS3, and C21orf62 correlate with survival. Notably, when survival was modeled as a function of both genetic ancestry and SPRR3 mutation, genetic ancestry was no longer significant as a predictor of survival. The PDGFRA receptor is also significantly differentially expressed between groups, suggesting a possible role of the PDGF axis in differential tumor growth. These results suggest potential genetic and clinical factors implicated in racial survival disparities. Further studies and more diverse sampling of cancer patient populations would allow validation of these differences and future elucidation of patient-specific treatment modalities. Citation Format: Sejal Jain, Alexandria Yao, Diane Allen-Gipson, Fatemeh Momen-Heravi, Alison M. Taylor. Survival disparities in lung squamous cell carcinoma: Clinical and genomic correlates with genetic ancestry [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr A009.

Biomedical Profiling of Lung Tumor via Ventilation-Induced Tumor Deformation: Implications on the Prognosis of Lung Cancer

<h3>Purpose/Objective(s)</h3> To quantitatively characterize tumor biomechanical rigidity via ventilation-induced tumor deformation (VITD) and assess its prognostic values. <h3>Materials/Methods</h3> This study utilized 10-bin 4D-CT and PET scans for 16 lung cancer patients (7 squamous cell carcinoma (SCC) and 9 adenocarcinoma (AC)). First, tumor surface models were generated based on binary segmentation for 10 respiratory phases and regionally aligned using surface-based registration. The resultant shape descriptors () across respiration phases were assembled as an intra-subject VITD. Subsequently, tumor strain was approximate as the magnitude of the averaged shape changes (<i>VITD_mag</i>) between end-of-exhale (EOE) and end-of-inhale (EOI) phases in the relevant regions - areas showing significant morphological correlations with the respective lung volume trajectory. Afterward, para-tumor stress (PTS) was approximated as the averaged displacements in the dilated para-tumor region, by deforming EOE to EOI 4D-CTs. Finally, <i>VITD_rigi</i> was calculated as PTS over <i>VITD_mag</i>. To assess the prognostic value of the <i>VITD</i> model, <i>VITD_mag</i> and <i>VITD_rigi</i> were correlated with PET-derived <i>SUV_max</i> for AC and SCC tumors, respectively. <h3>Results</h3> On lung SCC tumors, <i>VITD_mag</i>, the extent of maximal shape changes in respiration, was negatively associated with <i>SUV_max,</i> indictive of elevated tumor metabolism (r= -0.88, p=0.01). After controlling for PTS, an increase in the derived tumor rigidity (<i>VITD_rigi</i>) was found to be significantly associated with increased <i>SUV_max</i>, with r=0.76 and p=0.04. In contrast, on AC tumors, featuring surfactant overproduction and thus complex tissue composition, neither <i>VITD_mag</i> nor <i>VITD_rigi</i> presented a significant association with <i>SUV_max</i> (p = 0.98 and 0.88). <h3>Conclusion</h3> We introduced a novel noninvasive pipeline to characterize lung tumor rigidity based on VITD in 4D-CT. The derived <i>VITD_rigi</i> in lung SCC significantly correlated with tumor metabolism, indicating tumor aggressiveness. The proposed VITD model provides new biomechanical evidence in lung SCC prognosis. For AC tumors with complex tissue composition, deeper radiographic profiling besides tumor deformation is needed to effectively characterize its biomechanical properties.

Prediction of lung squamous cell carcinoma immune microenvironment and immunotherapy efficiency with pyroptosis-derived genes.

Lung squamous cell carcinoma (LUSC) is a common subtype of lung cancer that exhibits diverse pyroptosis regulatory patterns. Studies have highlighted the significance of pyroptosis in cancer invasion and immune responses. We aimed to explore the signatures of pyroptosis-related genes and their immune relevance in LUSC. Using The Cancer Genome Atlas (TCGA)-LUSC cohort and 5 gene expression omnibus (GEO) datasets, we performed consensus clustering based on 41 pyroptosis-related genes, and single sample gene set enrichment analysis (ssGSEA) was employed to calculate the infiltration levels of distinct clusters. A pyroptosis scoring scheme using the principal component analysis (PCA) method was used to quantify pyroptosis regulation in patients with LUSC and predict their prognosis. Four pyroptosis clusters were identified among 833 LUSC samples, which were associated with different Kyoto encyclopedia of genes and genome (KEGG) signaling pathways and tumor microenvironment infiltration features, and were highly consistent with 4 reported immune phenotypes: immune-responsive, immune-non-functional, immune-exclusion, and immune-ignorance. We then divided the patients into high- and low-pyroptosis score subgroups, and patients with higher scores were characterized by prolonged survival and attenuated immune infiltration. Moreover, higher scores were correlated with male patients, higher microsatellite instability, lower immune checkpoint inhibitor expression (such as CTLA-4 and GAL-9), and high mutation rates of typical mutated genes (e.g., TP53 and TTN). In particular, patients with lower pyroptosis scores showed better immune response to immune checkpoint inhibitor treatment. Pyroptosis regulatory patterns in the immune microenvironment can predict the clinical outcomes of patients with LUSC. Accurately quantifying the pyroptosis of individual patients will strengthen the understanding of heterogeneity within the LUSC tumor microenvironment infiltration areas.

Identification of tumor antigens and immune subtypes in lung squamous cell carcinoma for mRNA vaccine development.

BackgroundCancer vaccines are therapies that activate the patient’s own immune system by inoculating the patient with cancer-specific antigens to identify and clear cancer cells. Messenger ribonucleic acid (mRNA) vaccines have received much attention because of their ease of synthesis, relative affordability, and long-term safety, but have not been studied in lung squamous cell carcinoma (LUSC). Thus, the identification of tumor antigens is necessary to facilitate the development of mRNA vaccines for LUSC.MethodsGenes with significant copy number amplification, the presence of gene mutations in LUSC, and genes associated with survival were identified as potential tumor antigens. Subsequently, the genes significantly correlated with the level of infiltration of 3 antigen-presenting cells in LUSC were identified as LUSC tumor antigens. Unsupervised clustering and immune profiling analysis were used to identify potential suitable patients for mRNA vaccines. Lastly, drug sensitivity analysis was used to explore individualized treatment options for mRNA vaccines suitable and unsuitable patients.ResultsIn this study, among the highly mutated genes in LUSC, we found that bone morphogenetic protein 5 (BMP5) and claudin 5 (CLDN5) expression were positively correlated with antigen-presenting cell infiltration, which indicates that these 2 genes have potential for development as mRNA cancer vaccines. Further, the immune landscape analysis identified different immune subtypes. Our findings provide a reference for the development of treatment strategies and the prediction of treatment responsiveness.ConclusionsOverall, our study provides a new perspective on the development of mRNA vaccines for LUSC and highlights the importance of individualized therapy.

Identification of a dysregulated ceRNA network modulated by copy number variation-driven lncRNAs in lung squamous cell carcinoma.

Lung cancer is primarily responsive for cancer death, and its progression is aggressively affected by copy number variation (CNV). Through bioinformatics approach, a ceRNA network of CNV-driven lncRNAs in lung squamous cell carcinoma (LUSC) patients was constructed. Data on normal and LUSC tumor tissue from The Cancer Genome Atlas (TCGA)-LUSC dataset were subjected to differential analysis, and differentially expressed lncRNAs (DElncRNAs), DEmiRNAs, and DEmRNAs were obtained. Based on TCGA-LUSC, CNVs of normal and tumor tissue samples were then compared using a Chi-square test, and lncRNAs were intersected based on their CNVs and expression alternation. In combination with the Kruskal-Wallis test, CNV-driven lncRNAs were acquired. Afterwards, miRNAs and mRNAs that interacted with CNV-driven lncRNAs were obtained based on databases (LncBase, starBase, miRDB, mirDIP and TargetScan), DElncRNAs, DEmiRNAs and DEmRNAs, and correlation analysis. The acquired lncRNAs, miRNAs and mRNAs were subjected to Cytoscape software to construct a CNV-driven ceRNA network, which involved 5 lncRNAs (MIR143HG, LINC00702, MIR22HG, RP11-180 N14.1, RP11-473 M20.9), 6 miRNAs (miR-3200-3p, miR-1301-3p, miR-93-3p, miR-96-5p, miR-96-5p, miR-130b-5p, miR-205-5p) and 80 mRNAs. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analyses indicated that downstream mRNAs were mainly correlated with blood vessel development and T cell-mediated immunity. In summary, we devoted to analyzing CNV-related lncRNAs, mRNAs, and miRNAs in LUSC, thus clarifying 5 lncRNAs that may influence the malignant progression of LUSC. The ceRNA network regulated by these lncRNAs may be the novel pathogenesis of LUSC.

MiR-6124: A potential new prognosis biomarker for lung squamous cell carcinoma through m5C regulating mechanism.

e20568 Background: RNA methylation is an important epigenetic modification that can act as tumor prognosis biomarkers, and which is regulated by methyltransferase, RNA binding protein, and demethylase. 5-Methylcytosine (m5C) is a well-known post-transcriptional modification that plays an important role in cell differentiation and apoptosis. Although gene mutations in those regulator genes are associated with various cancers, the m5C regulating mechanism and its roles in cancer prognosis largely remains unknown. This study aimed to find new microRNA biomarkers associated with prognosis of clinical outcomes through m5C regulating mechanism in lung squamous cell carcinoma (LUSC). Methods: The RNA-seq expression data of LUSC were collected from the The Cancer Genome Atlas (TCGA). In total, we obtained 501 LUSC tumor samples and 49 normal samples from 502 LUSC patients. We firstly identified differential expressed genes of the 13 previously reported m5C regulators between the tumor tissues and adjacent normal tissues. Then miRNAs were identified using Pearson correlation analysis between the microRNA expression and the differential expressed m5C regulator genes. Pathway enrichment analysis of the miRNAs was performed using clusterProfiler to examine their cancer development roles. Furthermore, cox regression analysis was used to evaluate the risk factor for overall survival. Targeted relationship between miRNA and genes were confirmed by miRTarBase. Results: Differential analysis revealed 4 significantly up-regulated (NOP2, NSUN2, DNMT3B and ALYREF) genes in tumor among 13 interested m5C regulators. Pearson correlation analysis identified 39 correlated miRNAs. After cox regression, we found out one micro-RNA miR-6124, which is highly correlated with DNMT3B gene and is significantly associated with worse overall survival (OS, p &lt; 0.01). We then identified the regulatory targets of miR-6124 using miRTarBase database. In total we identified 181 target genes. Among them, H3F3A which encode histone H3 synergistically work with DNMT3B gene as showed in the previous studies and promote DNA methylation of the oncogenes. Such preliminary results showed miR-6124 may be involved in cancer pathway regulating and was a potential prognosis biomarker candidate in LUSC. Conclusions: We identified a potential new prognosis biomarker in lung squamous cell carcinoma. The identified miR-6124 is involved in DNMT3B and m5C regulating mechanism. Further evidences are needed to study its’ role in clinical applications.

Ubiquitin specific peptidase 47 promotes proliferation of lung squamous cell carcinoma

Ubiquitin specific peptidase 47 (USP47) is a kind of deubiquitinase, which has been reported to play oncogenic roles in several malignancies including colorectal cancer and breast cancer. Here we aimed to investigate the clinical significance of USP47 in lung squamous cell carcinoma (LUSC). We retrospectively enrolled a cohort of LUSC patients who underwent surgical resection in our hospital (n = 280) and conducted immunohistochemistry staining for their tumor tissues targeting USP47. The correlations between USP47 expression and clinicopathological characteristics were evaluated by Chi-square test. Univariate and multivariate analyses were conducted to assess the prognostic predictive role of USP47 in LUSC. Cell lines and mice models were utilized to explore the tumor-related functions of USP47 in vitro and in vivo, respectively. Among the 280 cases, there were 127 cases classified as high-USP47 expression and 153 cases with low-USP47 expression. Statistical analyses revealed that higher USP47 expression was independently correlated with larger tumor size, advanced T stage, and unfavorable prognosis. Knockdown of USP47 by shRNA resulted in impaired proliferation of LUSC cell lines and reduced nucleus beta-catenin level. Furthermore, xenograft assays demonstrated that silencing USP47 can inhibit LUSC tumor growth in vivo. Our research established a novel tumor-promoting effect and prognostic predictive role of USP47 in LUSC, thereby providing evidence for further therapeutic development.

Multimodal analysis suggests differential immuno-metabolic crosstalk in lung squamous cell carcinoma and adenocarcinoma

Immunometabolism within the tumor microenvironment is an appealing target for precision therapy approaches in lung cancer. Interestingly, obesity confers an improved response to immune checkpoint inhibition in non-small cell lung cancer (NSCLC), suggesting intriguing relationships between systemic metabolism and the immunometabolic environment in lung tumors. We hypothesized that visceral fat and 18F-Fluorodeoxyglucose uptake influenced the tumor immunometabolic environment and that these bidirectional relationships differ in NSCLC subtypes, lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). By integrating 18F-FDG PET/CT imaging, bulk and single-cell RNA-sequencing, and histology, we observed that LUSC had a greater dependence on glucose than LUAD. In LUAD tumors with high glucose uptake, glutaminase was downregulated, suggesting a tradeoff between glucose and glutamine metabolism, while in LUSC tumors with high glucose uptake, genes related to fatty acid and amino acid metabolism were also increased. We found that tumor-infiltrating T cells had the highest expression of glutaminase, ribosomal protein 37, and cystathionine gamma-lyase in NSCLC, highlighting the metabolic flexibility of this cell type. Further, we demonstrate that visceral adiposity, but not body mass index (BMI), was positively associated with tumor glucose uptake in LUAD and that patients with high BMI had favorable prognostic transcriptional profiles, while tumors of patients with high visceral fat had poor prognostic gene expression. We posit that metabolic adjunct therapy may be more successful in LUSC rather than LUAD due to LUAD’s metabolic flexibility and that visceral adiposity, not BMI alone, should be considered when developing precision medicine approaches for the treatment of NSCLC.

FP16.01 Development and Characterization of Two Novel Squamous Cell Lung Cancer Mouse Cell Lines

Lung cancer is the leading cause of cancer-related death worldwide. The incidence and mortality associated with lung cancer is a major Public Health challenge in advanced societies. About 20-30% of all lung cancers are classified as lung squamous cell carcinoma (LUSC), also known as squamous cell carcinoma (SSC or SqCC) or epidermoid carcinoma. The absence of preclinical models makes LUSC disease analysis and research more challenging. The development of well-characterized cellular and molecular tools is essential for the advance in lung cancer research. In the present work, we have performed an exhaustive molecular and functional characterization of a new LUSC research model, recently developed in our laboratory. This model consists of two novel A/J mouse-derived syngeneic cell lines from NTCU-induced LUSC.In our opinion, these cell lines may become a robust tool for the study of squamous cell lung cancer in a reliable and reproducible manner. Carcinogenesis and cell line generation: LUSC tumors were induced by N-nitroso-tris-chloroethylurea (NTCU) (Toronto Research Chemicals) treatment applying 0.04 M NTCU by skin painting twice a week for 20 weeks to 8-week-old A/J mice. Immunohistochemical staining: Dissected tumors were fixed in 4% paraformaldehyde and embedded in paraffin. Three-micrometer-thick sections were used for tumors immunohistochemical phenotypic characterization. Exome sequencing: Whole exome sequencing was performed on extracted DNA from the tumor-derived cell lines. RNA sequencing analysis: Samples were prepared with the Illumina TruSeq Stranded mRNA kit and sequenced as reverse paired-end (50 bp) runs on the Nextseq sequencer. Tumor immunotherapy experiments: 2x106 cells were subcutaneously inoculated in one flank of 8-week-old female A/J mice. When the tumors reached a volume of 75 mm3, mice were randomized and treated intraperitoneally with 3 doses of 100 μg of anti-PD1 or anti-CTLA4. Tumor immune landscape analysis: 2x106 cells were subcutaneously inoculated in one flank of 6 8-week-old female A/J mice . Thirteen days after cell inoculation, tumors were collected and processed for flow cytometry analysis. Metastasis in vivo model: Cells were transduced with a triple modality construct containing a triple fusion protein GFP, luciferase and thymidine kinase. Eight-week old female mice were inoculated in the left cardiac ventricle with 1 × 105 cells. Cells were followed by bioluminescence. We have generated two transplantable LUSC cell lines (UN-SCC679 and UN-SCC680) derived from an N-nitroso-tris-chloroethylurea (NTCU) chemically-induced mouse model in A/J mice. Both cells lines expressed cytokeratins and p40, and lacked thyroid transcription factor 1 (TTF1) expression. Furthermore, we genetically characterized the LUSC cell lines by performing whole exome and RNA sequencing. In addition, we characterized the immune landscape of both tumors in vivo and assessed their response to immunotherapy studies combining the gold-standard checkpoint inhibitors in clinic. Finally, we studied the metastatic potential of each of these two models and confirmed that they reflect the human LUSC organotropism to the brain, bones, liver and adrenal glands. We have generated a very valuable tool for LUSC research that recapitulates the complexity of the human disease.