Mouse Model Of Lung Adenocarcinoma Research Articles

Trametinib sensitizes KRAS-mutant lung adenocarcinoma tumors to PD-1/PD-L1 axis blockade via Id1 downregulation

BackgroundThe identification of novel therapeutic strategies to overcome resistance to the MEK inhibitor trametinib in mutant KRAS lung adenocarcinoma (LUAD) is a challenge. This study analyzes the effects of trametinib on Id1 protein, a key factor involved in the KRAS oncogenic pathway, and investigates the role of Id1 in the acquired resistance to trametinib as well as the synergistic anticancer effect of trametinib combined with immunotherapy in KRAS-mutant LUAD.MethodsWe evaluated the effects of trametinib on KRAS-mutant LUAD by Western blot, RNA-seq and different syngeneic mouse models. Genetic modulation of Id1 expression was performed in KRAS-mutant LUAD cells by lentiviral or retroviral transductions of specific vectors. Cell viability was assessed by cell proliferation and colony formation assays. PD-L1 expression and apoptosis were measured by flow cytometry. The anti-tumor efficacy of the combined treatment with trametinib and PD-1 blockade was investigated in KRAS-mutant LUAD mouse models, and the effects on the tumor immune infiltrate were analyzed by flow cytometry and immunohistochemistry.ResultsWe found that trametinib activates the proteasome-ubiquitin system to downregulate Id1 in KRAS-mutant LUAD tumors. Moreover, we found that Id1 plays a major role in the acquired resistance to trametinib treatment in KRAS-mutant LUAD cells. Using two preclinical syngeneic KRAS-mutant LUAD mouse models, we found that trametinib synergizes with PD-1/PD-L1 blockade to hamper lung cancer progression and increase survival. This anti-tumor activity depended on trametinib-mediated Id1 reduction and was associated with a less immunosuppressive tumor microenvironment and increased PD-L1 expression on tumor cells.ConclusionsOur data demonstrate that Id1 expression is involved in the resistance to trametinib and in the synergistic effect of trametinib with anti-PD-1 therapy in KRAS-mutant LUAD tumors. These findings suggest a potential therapeutic approach for immunotherapy-refractory KRAS-mutant lung cancers.Graphical

Abstract LT06: Understanding the Role of Mutant TP53 in Lung Cancer Using Novel Genetically Engineered Mouse Models

Abstract The tumour suppressor gene TP53 (Trp53 in mouse) is mutated in ~50% of human cancers and this is associated with poor therapeutic responses. Mutant TP53 protein was reported to promote tumour development in three ways: (1) loss of function (LOF) effects which abrogate normal TP53-mediated cellular processes; (2) dominant negative effects (DNE); and (3) gain of function effects (GOF) whereby mutant TP53 proteins bind to and modulate the functions of proteins that are not impacted by wildtype TP53. Despite extensive research efforts, the relative contributions of the LOF, DNE and GOF effects of mutant TP53 in cancer are largely unknown. This project aims to understand the relative importance of these three effects of TP53 mutant proteins in the initiation and sustained expansion of lung adenocarcinoma (LUAD). We will do this by using novel LUAD mouse models with switchable Trp53 status to trigger tumorigenesis through expression of mutant Trp53 and then conversion of mutant Trp53 either back to wildtype Trp53 or into a Trp53 knock-out state when desired. In vitro and in vivo studies were performed to investigate the immediate and long-term effects of different Trp53 status on tumour growth. Our data thus far show that conversion of mutant Trp53 back to wildtype Trp53 induced cellular senescence and cell cycle arrest in LUAD cells in vitro and substantially delayed tumour progression in vivo, which improved the overall survival of mice. In contrast, conversion of mutant Trp53 into a Trp53 knock-out state did not impair the sustained growth of these tumour cells either in vitro or in vivo. This suggest that the LOF but not GOF of mutant TRP53 is critical to sustain the expansion of lung cancer. These findings have important implications for the design of novel therapeutic approaches for cancers expressing mutant TP53. Citation Format: Tianwei Chen, Elizabeth Lieschke, Leanne Scott, Danielle Boyd, Kate Sutherland, Andreas Strasser, Gemma Kelly. Understanding the Role of Mutant TP53 in Lung Cancer Using Novel Genetically Engineered Mouse Models [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr LT06.

KRAS/PI3K axis driven GTF3C6 expression and promotes LUAD via FAK pathway

IntroductionEffective targeting drugs for KRAS mutation-mediated Lung Adenocarcinoma (LUAD) are currently are limited. ObjectivesInvestigating and intervening in the downstream key target genes of KRAS is crucial for clinically managing KRAS mutant-driven LUAD. GTF3C6, a newly identified member of the general transcription factor III (GTF3) family, plays a role in the transcription of RNA polymerase III (pol III)-dependent genes. However, its involvement in cancer remains unexplored. MethodsThis study examined the expression, roles, and potential molecular mechanisms of GTF3C6 in LUAD tissues, LSL-KrasG12D/+;LSL-p53−/− LUAD mouse models, and LUAD patients-derived organoid using Western blot, qRT-PCR, immunofluorescence, immunohistochemistry, and gene manipulation assays. ResultsWe present the first evidence that GTF3C6 is highly expressed in LUAD tissues, LSL-KrasG12D/+;LSL-p53−/− LUAD mouse models, and LUAD organoids, correlating with poor clinical prognosis. Furthermore, GTF3C6 was found to promote anchorage-independent proliferation, migration, and invasion of LUAD cells. Mechanistically, KRAS mutation drives GTF3C6 expression through the PI3K pathway, and GTF3C6 knockdown reverses the malignant phenotype of KRAS mutation-driven LUAD cells. Additionally, the FAK pathway emerged as a crucial downstream signaling pathway through which GTF3C6 mediates the malignant phenotype of LUAD. Finally, GTF3C6 knockdown suppresses LUAD organoid formation and inhibits tumor growth in vivo. ConclusionOur findings demonstrate that GTF3C6, driven by KRAS mutation, promotes LUAD development by regulating FAK phosphorylation, suggesting its potential as a biomarker and therapeutic target in KRAS mutant-driven LUAD.

Abstract 2762: Spatial proteomics of lung adenocarcinoma mouse models with divergent propensities for metastasis

Abstract Introduction: Lung cancer is the leading cause of cancer deaths worldwide with most deaths attributed to metastasis. Metastases in existing mouse models of lung adenocarcinoma (LUAD) is sporadic and often requires several months of aging. These factors limit these models for basic and pre-clinical research aimed at identifying mechanisms of metastatic disease. We have recently developed two novel mouse models of LUAD that feature constitutively active Kras, loss of Trp53 function and truncated Dicer1 in different cell types. The observed survival and metastatic phenotypes of the models are dependent on the cell type in which the genetic alterations are made. We applied spatial proteomics and metabolomics to investigate molecular drivers underlying the phenotypic differences in our models. Methods and Results: Mouse models were generated by adding mutations in Dicer1, an RNAse III enzyme within the microRNA (miRNA) biosynthesis pathway, to a mouse model of Kras-driven LUAD. For both models, tumorigenesis was induced by conditional expression of an oncogenic allele of Kras, deletion of both alleles of Trp53 and deletion of one allele of Dicer1 in one cell type and the expression of a truncated Dicer1 allele in a different cell type. Mice expressing KrasG12D and deleted Trp53 in club cells without Dicer1 truncation have a reported median survival of 28.6 weeks after tumor induction. We observed accelerated development of LUAD and lymph node metastasis (12.1 weeks) only when we induced tumorigenesis in club cells and truncate Dicer1 in alveolar type II (ATII) cells. Induction of tumorigenesis in ATII cells and truncation of Dicer1 in club cells did not accelerate tumorigenesis or lead to detectable metastasis. Prelminary spatial proteomic analysis revealed significant upregulation of proteins associated with mitochondrial respiratory chain complex I in the tumors of both models which is consistent with processes of bioenergetic adaptation of tumor cells to hypoxic conditions. Gene Ontology annotation term enrichment revealed that the tumors of the two models have distinctive molecular function profiles. Both models show significant disruption of amino acid metabolism but only the model with increased metastatic potential showed apparent disruption in metabolites associated with steroidogenesis. Conclusions: Through cell type specific truncation/deletion of Dicer1 we have generated a mouse model that rapidly develops Kras/Trp53 driven LUAD and metastatic disease. Our findings suggest that tumorigenesis and metastasis are influenced by miRNA-mediated communication between different cell types. Additional studies are underway to link miRNA expression in these models to the results reported here for spatial proteomics and metabolomics. We will continue to develop this model to investigate the molecular mechanisms underlying metastasis. Citation Format: Paige Ramkissoon, Naomi Mitchell-Hutchinson, Julie Wells, Rick Maser, Tim Stodola, Brian Hoffmann, Anne Marchini, Elaine Bechtel, Rosalinda Doty, Carol J. Bult. Spatial proteomics of lung adenocarcinoma mouse models with divergent propensities for metastasis [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 2762.

Abstract 6592: Deciphering the effects of age on T cell immunity and immunotherapy

Abstract Aging induces profound alterations in host biology and correlates with unfavorable clinical outcomes in age-related diseases such as cancer. While the pivotal role of the immune system in regulating tumor progression is well-acknowledged, the precise mechanisms through which aging influences anti-tumor T cell immunity and responsiveness to immune checkpoint blockade (ICB) remain elusive. In this study, we employed syngeneic murine tumor models (B16, MC38, and E0771) in young (2-4 months) and aged (20-24 months) cohorts to elucidate the early impacts of aging on tumor development and T cell responses. Our findings demonstrate that aged mice exhibit accelerated tumor growth across various models. In-depth analysis of tumor-infiltrating lymphocytes (TILs) uncovered distinct alterations in the CD8+ T cell population, characterized by decreased activation status and diminished tumor specificity in aged mice. Correspondingly, aged mice exhibited resistance to anti-PD-1 immunotherapy. To unravel the underlying environmental factors contributing to compromised anti-tumor T cell responses and ICB resistance, we characterized the immune and metabolic landscapes of the aged tumor-draining lymph nodes and tumors. Furthermore, we established a genetically engineered mouse model of lung adenocarcinoma in both young and aged mice, unveiling novel functions and mechanisms associated with age-related T cells actively impeding effective tumor control. In the context of increasing average life expectancy, comprehending the intricate interplay between age-related T cell dysfunction and cancer assumes paramount importance. Such insights hold the key to the development of innovative therapeutic strategies tailored for the growing population of older patients. Citation Format: SeongJun Han, Peter Georgiev, Jaime Schneider, Amy Huang, Zhanel Nugmanova, Thao H. Nguyen, Conghui Yao, Sheila Johnson, Shakchhi Joshi, Ishita Dhiman, Jefte M. Drijvers, Megan Fung, Jared H. Rowe, Brian C. Miller, Gordon J. Freeman, Alison E. Ringel, Marcia C. Haigis, Arlene H. Sharpe. Deciphering the effects of age on T cell immunity and immunotherapy [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 6592.

Abstract 5352: SOX9-dependent immune microenvironment remodeling drives KRAS-induced lung adenocarcinoma progression

Abstract Interactions between tumor intrinsic factors and tumor microenvironment play a vital role in disease progression and treatment response. SOX9, a transcription factor crucial for tissue development and homeostasis, has been linked to the advancement of tumor growth. However, its specific role as a driver in lung adenocarcinoma (LUAD) is not well-defined. Additionally, the impact of SOX9 on the tumor microenvironment has not been previously explored. We here identify SOX9 transcription factor to play a critical role in defining tumor immune microenvironment and driving disease progression. We employed CRISPR/Cas9 and Cre-LoxP gene knockout methods within the Kras G12D-induced mouse model of lung adenocarcinoma (LUAD). Our objective was to investigate the mechanisms through which SOX9 contributes to the development and advancement of lung adenocarcinoma. To substantiate our findings, we conducted immune profiling, gene expression analyses, RT-qPCR, and immunohistochemistry assessments in the Kras G12D-driven murine LUAD. These results were subsequently validated by examining bulk and single-cell gene expression profiles, as well as immunohistochemistry, in human lung adenocarcinoma. Our studies indicated that SOX9 played a very important role by enhancing lung tumor development, burden, and progression, leading to lower overall survival. SOX9. SOX9 consistently facilitated the growth of organoids in vitro. However, the promotion of tumor growth by SOX9 was notably reduced in immunocompromised mice compared to syngeneic mice. SOX9 demonstrated a suppressive effect on immune cell infiltration, particularly affecting the functionality of tumor-associated CD8+ T cells, natural killer cells, and dendritic cells. CD8+ T cells in the SOX9 overexpressing tumors showed upregulation of exhaustion markers. SOX9-mediated tumor growth advantage could not be observed in the CD8 knockout mice model, suggesting a CD8+ T cell-dependent role. In addition, SOX9 significantly upregulated collagen-related gene expression, leading to a substantial increase in collagen fibers possibly affecting the migration of immune cells into the tumor microenvironment. Together, our studies indicate that SOX9 plays a very critical role in modulating tumor microenvironment by controlling immune cell infiltration and CD8+ T cell exhaustion driving Kras G12D- driven lung tumor progression. Citation Format: Dinoop Ravindran Menon, Hua Zhong, Shridar Ganesan, Hatim Sabaawy, Sharon Pine. SOX9-dependent immune microenvironment remodeling drives KRAS-induced lung adenocarcinoma progression [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 5352.

Abstract 3938: A novel mouse model reveals potential therapeutic strategies for RIT1-driven lung adenocarcinomas

Abstract Gain-of-function oncogenic mutations in RIT1 have been identified in lung adenocarcinoma and are mutually exclusive with other reported driver mutations, highlighting the role of RIT1 as a cancer driver. RIT1 is a poorly-studied non-classical RAS GTPase and, to date, there are no reported genetically-engineered mouse models of RIT1-driven lung adenocarcinoma. Here, we have generated a conditional knock-in allele within the endogenous mouse Rit1 locus, introducing the most frequent mutation observed in lung adenocarcinomas (RIT1M90I). Upon intranasal administration of adenovirus encoding the Cre recombinase to induce somatic recombination in the mouse lungs, we observed the formation of discrete adenomas. Although Rit1M90I mutation can promote lung tumor initiation in mice, we found that this phenotype is less penetrant than the common KrasG12D mutation. Immunohistochemistry analysis of Rit1M90I and KrasG12D tumors showed expression of typical histological markers associated with lung adenocarcinoma (i.e., TTF1 and SPC), as well as increased levels of Ki67 and pERK expression. Next, to develop potential pharmacological strategies for RIT1-driven lung tumors, we first explored drugs that inhibit the RAS/MAPK pathway and are either FDA-approved or under clinical development. In vitro experiments with cell lines carrying the RIT1M90I mutation demonstrated high sensitivity to SHP2, MEK, RAF, and RAS inhibitors. In vivo, SHP2 and RAS, but not MEK, inhibitors slowed down tumor progression in both cell line and patient-derived xenografts. Our results indicate that, although RAS signaling might be needed for RIT1-dependent growth, these tumors are not sensitive to MAPK inhibition as a monotherapy and, hence, other pathways could contribute to tumor growth. Therefore, we decided to explore whether targeting RIT1 directly, could be a more beneficial strategy for these tumors. In order to inhibit oncogenic RIT1 directly, we have undertaken two complementary approaches. First, because RIT1 mutations were shown to impair its proteolysis, we explored whether restoring RIT1 proteasomal degradation could be a promising strategy. Recently, the development of Proteolysis Targeting Chimeras (PROTAC) has demonstrated that targeted degradation can be used as a pharmacological approach for the treatment of cancer. Therefore, we have tested successfully several proof-of-concept PROTAC approaches that caused degradation of oncogenic RIT1 in different cellular models. Second, we used a chemical biology approach to assess whether covalent inhibitors directed against the closely-related GTPase KRAS could cross-react and bind to oncogenic RIT1. We have identified a molecule that binds RIT1 and we are currently characterizing it in our preclinical models. Overall, our work provides comprehensive insights into the oncogenic role of RIT1 in lung cancer emphasizing its potential as a therapeutic target. Citation Format: Alessandro Mauro Mozzarelli, Antonio Cuevas-Navarro, Anatoly Urisman, Pau Castel. A novel mouse model reveals potential therapeutic strategies for RIT1-driven lung adenocarcinomas [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 3938.

Abstract 2667: Mucin 5AC inhibition via intratracheal RNAi delivery as an effective alternative strategy for prevention and treatment of Kras mutant lung cancer

Abstract Lung cancer, including lung adenocarcinoma (LUAD), is the leading cause of cancer-related deaths in the United States. KRAS mutations are the most prevalent oncogenic driver alteration in human LUAD. We have previously shown that high expression of Mucin 5 AC (MUC5AC), a main airway secretory mucin, is significantly associated with poor prognosis. Mucins are essential for mucociliary clearance and the maintenance of airway homeostasis, however, their overproduction could negatively affect the lung immune microenvironment and has been shown to promote airway diseases such as chronic obstructive pulmonary disease (COPD) and lung cancer. Previously, we have shown that genetic deletion of MUC5AC in a murine model of K-ras mutant lung adenocarcinoma (KM-LUAD) impedes tumor development and reduces pro-tumor inflammatory and immune phenotypes. These findings indicate that MUC5AC is a potential druggable target against KM-LUAD. In this study, we sought a translational approach and inhibited MUC5AC in our K-rasG12D mutant mouse model of LUAD via RNA interference (RNAi). Six-week-old mice were given RNAi (5mg/kg) or vehicle (saline) via biweekly intratracheal administration for 8 weeks. This approach led to a significant decrease in tumor burden (40%) and a trending increase in total white blood cell counts, predominantly macrophages in the lung tumor microenvironment which was associated with phenotypic changes in macrophages characterized by reduced expression of Fizz1, and M2 pro-tumor macrophage marker. Additionally, RNAi-treated mice had reduced expression of Siglec-E, a mucin receptor and known inhibitor of the TLR4 endocytosis and promoter of NF-ĸB signaling, as well as a significant decrease in IL-6, a product of the NF-ĸB pathway and promoter of STAT3 signaling which we have previously shown to have an essential role in promotion of KM-LUAD. In summary, these findings elucidate a potential mechanism by which MUC5AC protects tumor cells from apoptosis and immune-mediated cytotoxic activity but also highlights how MUC5AC RNAi is an effective indirect preventive and therapeutic strategy in high-risk individuals (smokers with COPD) and patients with early stage KM-LUAD, respectively. Citation Format: Melvin Zarghooni, Maria T. Grimaldo, Avantika Krishna, Michael J. Clowers, Linda Phan, Yasmina H. Rezai, Arnav Gaitonde, Carlos Rodriguez Reyna, Walter Velasco Torrez, Erik Bush, Seyed Javad Moghaddam. Mucin 5AC inhibition via intratracheal RNAi delivery as an effective alternative strategy for prevention and treatment of Kras mutant 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 2667.

Abstract 668: Enhancing chemosensitivity of lung adenocarcinoma via Y-box binding protein 1 (YB-1) inhibition

Abstract Background: Y-box binding protein 1 (YB-1, YBX1) is a multifunctional nucleic acid binding protein that regulates the expression of various genes by binding to their mRNA and stabilizing them. In over 20 cancer types, including lung adenocarcinoma (LUAD), YBX1 expression is significantly elevated in tumor samples compared to normal tissue. Within cancer cells, YB-1 promotes the expression of genes involved in tumorigenesis and chemoresistance. Consequently, elevated YBX1 expression in tumor is associated with poor clinical outcomes and significantly reduced overall survival in LUAD patients. Therefore, YB-1 emerges as a promising therapeutic target for LUAD treatment. Utilizing a small molecule YB-1 inhibitor, CET056, we performed a series of in vitro and in vivo analyses to assess the therapeutic efficacy of YB-1 inhibition and unravel YB-1’s role in tumorigenesis and disease progression in LUAD. Methods: Four human and one mouse LUAD cell lines were treated with CET056 and/or chemotherapeutic drugs and analyzed for the in vitro therapeutic activity of CET056. Using xenograft and syngeneic LUAD mouse models, the preclinical therapeutic efficacy of CET056 on disease progression of LUAD was also tested. Results: CET056 was shown to effectively decrease YB-1 expression in both human and mouse LUAD cell lines and exert inhibitory effects on LUAD in vitro and in vivo. This treatment delayed tumor progression and metastasis in CMT-64 syngeneic mouse model. Proteomics analysis revealed that translational machinery-associated pathways were down-regulated in LUAD cells after CET056 treatment whereas apoptosis and cell death-associated pathways were up-regulated. In alignment with this finding, CET056 treatment in combination with cisplatin showed chemo-sensitizing effect on LUAD in vitro and in vivo. Conclusion: Our studies suggest that YB-1 could be a promising target for combating chemoresistance in LUAD. Inhibition of YB-1 with a small molecule drug as a monotherapy or combination therapy could an effective strategy to treat LUAD. Citation Format: Jee Min Lee, Dhanir Tailor, Fernando J. Garcia-Marques, Abel Bermudez, Sharon Pitteri, Sanjay V. Malhotra. Enhancing chemosensitivity of lung adenocarcinoma via Y-box binding protein 1 (YB-1) inhibition [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 668.

FAM86A methylation of eEF2 links mRNA translation elongation to tumorigenesis

eEF2 post-translational modifications (PTMs) can profoundly affect mRNA translation dynamics. However, the physiologic function of eEF2K525 trimethylation (eEF2K525me3), a PTM catalyzed by the enzyme FAM86A, is unknown. Here, we find that FAM86A methylation of eEF2 regulates nascent elongation to promote protein synthesis and lung adenocarcinoma (LUAD) pathogenesis. The principal physiologic substrate of FAM86A is eEF2, with K525me3 modeled to facilitate productive eEF2-ribosome engagement during translocation. FAM86A depletion in LUAD cells causes 80S monosome accumulation and mRNA translation inhibition. FAM86A is overexpressed in LUAD and eEF2K525me3 levels increase through advancing LUAD disease stages. FAM86A knockdown attenuates LUAD cell proliferation and suppression of the FAM86A-eEF2K525me3 axis inhibits cancer cell and patient-derived LUAD xenograft growth invivo. Finally, FAM86A ablation strongly attenuates tumor growth and extends survival in KRASG12C-driven LUAD mouse models. Thus, our work uncovers an eEF2 methylation-mediated mRNA translation elongation regulatory node and nominates FAM86A as an etiologic agent in LUAD.

Urinary VOCs as biomarkers of early stage lung tumour development in mice.

Lung cancer is the primary cause of cancer-induced death. In addition to prevention and improved treatment, it has increasingly been established that early detection is critical to successful remission. The aim of this study was to identify volatile organic compounds (VOCs) in urine that could help diagnose mouse lung cancer at an early stage of its development. We analysed the VOC composition of urine in a genetically engineered lung adenocarcinoma mouse model with oncogenic EGFR doxycycline-inducible lung-specific expression. We compared the urinary VOCs of 10 cancerous mice and 10 healthy mice (controls) before and after doxycycline induction, every two weeks for 12 weeks, until full-blown carcinomas appeared. We used SPME fibres and gas chromatography- mass spectrometry to detect variations in cancer-related urinary VOCs over time. This study allowed us to identify eight diagnostic biomarkers that help discriminate early stages of cancer tumour development (i.e., before MRI imaging techniques could identify it). The analysis of mice urinary VOCs have shown that cancer can induce changes in odour profiles at an early stage of cancer development, opening a promising avenue for early diagnosis of lung cancer in other models.

Advancing the Boundaries of Immunotherapy in Lung Adenocarcinoma with Idiopathic Pulmonary Fibrosis by a Biomimetic Proteinoid Enabling Selective Endocytosis.

The coexistence of lung adenocarcinoma (LUAD) with idiopathic pulmonary fibrosis (IPF), which has been extensively documented as a prominent risk factor for checkpoint inhibitor-related pneumonitis (CIP) in patients undergoing immunotherapy, has long been considered a restricted domain for the use of immune checkpoint inhibitors (ICIs). To overcome it, an approach was employed herein to specifically target PD-L1 within the cellular interior, surpassing the conventional focus solely on the cytomembrane, thereby facilitating the development of ICIs capable of distinguishing between LUAD cells and noncancerous cells based on their distinctive endocytic propensities. By exploiting the aurophilicity-driven self-assembly of a PD-L1 binding peptide (PDBP) and subsequently encapsulating it within erythrocyte membranes (EM), the resulting biomimetic ICIs protein EMS-PDBP exhibited extraordinary selectivity in internalizing LUAD cells, effectively targeting PD-L1 within cancer cells while hindering its membrane translocation. The EMS-PDBP treatment not only reactivated the antitumor immune response in the LUAD orthotopic allograft mouse model but also demonstrated a favorable safety profile by effectively eliminating any immune-related adverse events (irAEs). Most significantly, EMS-PDBP successfully and safely restored the antitumor immune response in a mouse model of LUAD with coexistent IPF, thus shattering the confines of ICIs immunotherapy. The reported EMS-PDBP collectively offers a potential strategy for immune reactivation to overcome the limitations of immunotherapy in LUAD coexisting with IPF.

Non-targeted metabolomics analysis of indoleamine 2,3-dioxygenase inhibitor treatment in a mouse model of early-stage lung adenocarcinoma.

Lung adenocarcinoma is a common malignant tumor, and its early diagnosis and treatment are key to improving patient survival rates. However, due to the non-specific early symptoms, many patients are already at an advanced stage when diagnosed. Non-targeted metabolomics analysis, as a method for comprehensive analysis of metabolites in the body, has been shown to have potential in the early diagnosis of cancer. This study aims to identify early-stage lung adenocarcinoma-specific biomarkers using non-targeted metabolomics analysis in an established mouse model. The intervention mechanism of indoleamine 2,3-dioxygenase (IDO) inhibitor in early-stage lung adenocarcinoma is explored to provide evidence for clinical disease treatment. Twenty specific-pathogen-free-grade female Kunming mice were divided into control group, experimental group, Epacadostatlow group, and Epacadostathigh group. After modeling, immune therapy intervention (epacadostat) was administered to the mice, and plasma and urine samples were collected from all mice on day 7 and day 28. Ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) analysis was performed to identify potential biomarkers for diagnosing early-stage lung adenocarcinoma. Cluster analysis and correlation analysis were used to explore the differential expression patterns of metabolites in different samples. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was used to identify enriched pathways of differentially expressed metabolites. A total of 348 metabolites were identified after merging the positive and negative ion modes. Among them, organic acids and derivatives (16.954%) and lipids and lipid-like molecules (15.517%) were the two major classes of metabolites in the early-stage lung adenocarcinoma mice. Anthranilic acid (vitamin L1), 1-methylhistidine, 12(R)-HETE, and hippuric acid were the major differentially expressed metabolites on both day 7 and day 28, and they showed correlations with each other. Metabolic pathway analysis revealed multiple dysregulated pathways in lung adenocarcinoma mice. UPLC-QTOF-MS analysis is a feasible method for identifying biomarkers of lung adenocarcinoma. Epacadostat, a novel and promising IDO inhibitor, may exert its therapeutic effect by modulating 1-methylhistidine and anthranilic acid (vitamin L1).

SRCAP complex promotes lung cancer progression by reprograming the oncogenic transcription of Hippo-YAP/TAZ signaling pathway

The activation of YAP/TAZ, a pair of paralogs of transcriptional coactivators, initiates a dysregulated transcription program, which is a key feature of human cancer cells. However, it is not fully understood how YAP/TAZ promote dysregulated transcription for tumor progression. In this study, we employed the BioID method to identify the interactome of YAP/TAZ and discovered that YAP/TAZ interact with multiple components of SRCAP complex, a finding that was further validated through endogenous and exogenous co-immunoprecipitation, as well as immunofluorescence experiments. CUT&Tag analysis revealed that SRCAP complex facilitates the deposition of histone variant H2A.Z at target promoters. The depletion of SRCAP complex resulted in a decrease in H2A.Z occupancy and the oncogenic transcription of YAP/TAZ target genes. Additionally, the blockade of SRCAP complex suppressed YAP-driven tumor growth. In a genetically engineered lung adenocarcinoma mouse model and non-small cell lung cancer patients, SRCAP complex and H2A.Z deposition were found to be upregulated. This upregulation was statistically correlated with YAP expression, pathological stages, and poor survival in lung cancer patients. Together, our study uncovers that SRCAP complex plays a critical role in YAP/TAZ oncogenic transcription by coordinating H2A.Z deposition during cancer progression, providing potential targets for cancer diagnosis and prevention.

Inhalable point-of-care urinary diagnostic platform.

Although low-dose computed tomography screening improves lung cancer survival in at-risk groups, inequality remains in lung cancer diagnosis due to limited access to and high costs of medical imaging infrastructure. We designed a needleless and imaging-free platform, termed PATROL (point-of-care aerosolizable nanosensors with tumor-responsive oligonucleotide barcodes), to reduce resource disparities for early detection of lung cancer. PATROL formulates a set of DNA-barcoded, activity-based nanosensors (ABNs) into an inhalable format. Lung cancer-associated proteases selectively cleave the ABNs, releasing synthetic DNA reporters that are eventually excreted via the urine. The urinary signatures of barcoded nanosensors are quantified within 20 min at room temperature using a multiplexable paper-based lateral flow assay. PATROL detects early-stage tumors in an autochthonous lung adenocarcinoma mouse model with high sensitivity and specificity. Tailoring the library of ABNs may enable not only the modular PATROL platform to lower the resource threshold for lung cancer early detection tools but also the rapid detection of chronic pulmonary disorders and infections.

Bcl-xL targeting eliminates ageing tumor-promoting neutrophils and inhibits lung tumor growth

Elevated peripheral blood and tumor-infiltrating neutrophils are often associated with a poor patient prognosis. However, therapeutic strategies to target these cells are difficult to implement due to the life-threatening risk of neutropenia. In a genetically engineered mouse model of lung adenocarcinoma, tumor-associated neutrophils (TAN) demonstrate tumor-supportive capacities and have a prolonged lifespan compared to circulating neutrophils. Here, we show that tumor cell-derived GM-CSF triggers the expression of the anti-apoptotic Bcl-xL protein and enhances neutrophil survival through JAK/STAT signaling. Targeting Bcl-xL activity with a specific BH3 mimetic, A-1331852, blocked the induced neutrophil survival without impacting their normal lifespan. Specifically, oral administration with A-1331852 decreased TAN survival and abundance, and reduced tumor growth without causing neutropenia. We also show that G-CSF, a drug used to combat neutropenia in patients receiving chemotherapy, increased the proportion of young TANs and augmented the anti-tumor effect resulting from Bcl-xL blockade. Finally, our human tumor data indicate the same role for Bcl-xL on pro-tumoral neutrophil survival. These results altogether provide preclinical evidence for safe neutrophil targeting based on their aberrant intra-tumor longevity.

An optimized protocol for the generation and monitoring of conditional orthotopic lung cancer in the KP mouse model using an adeno-associated virus vector compatible with biosafety level 1

BackgroundThe inducible Kras/p53 lung adenocarcinoma mouse model, which faithfully recapitulates human disease, is routinely initiated by the intratracheal instillation of a virus-based Cre recombinase delivery system. Handling virus-based delivery systems requires elevated biosafety levels, e.g., biosafety level 2 (BSL-2). However, in experimental animal research facilities, following exposure to viral vectors in a BSL-2 environment, rodents may not be reclassified to BSL-1 according to standard practice, preventing access to small animal micro-computed tomography (micro-CT) scanners that are typically housed in general access areas such as BSL-1 rooms. Therefore, our goal was to adapt the protocol so that the Cre-induced KP mouse model could be handled under BSL-1 conditions during the entire procedure.ResultsThe Kras-Lox-STOP-Lox-G12D/p53 flox/flox (KP)-based lung adenocarcinoma mouse model was activated by intratracheal instillation of either an adenoviral-based or a gutless, adeno-associated viral-based Cre delivery system. Tumor growth was monitored over time by micro-CT. We have successfully substituted the virus-based Cre delivery system with a commercially available, gutless, adeno-associated, Cre-expressing vector that allows the KP mouse model to be handled and imaged in a BSL-1 facility. By optimizing the anesthesia protocol and switching to a microscope-guided vector instillation procedure, productivity was increased and procedure-related complications were significantly reduced. In addition, repeated micro-CT analysis of individual animals allowed us to monitor tumor growth longitudinally, dramatically reducing the number of animals required per experiment. Finally, we documented the evolution of tumor volume for different doses, which revealed that individual tumor nodules induced by low-titer AAV-Cre transductions can be monitored over time by micro-CT.ConclusionModifications to the anesthesia and instillation protocols increased the productivity of the original KP protocol. In addition, the switch to a gutless, adeno-associated, Cre-expressing vector allowed longitudinal monitoring of tumor growth under BSL-1 conditions, significantly reducing the number of animals required for an experiment, in line with the 3R principles.

Blocking LTB4 signaling-mediated TAMs recruitment by Rhizoma Coptidis sensitizes lung cancer to immunotherapy

BackgroundImmune checkpoint blockade (ICB) induces durable immune responses across a spectrum of advanced cancers and revolutionizes the oncology field. However, only a subset of patients achieves long-lasting clinical benefits. Tumor-associated macrophages (TAMs) usually secrete immunosuppressive cytokines and contribute to the failure of ICB therapy. Therefore, it is crucial to mechanically manipulate the abundance and function of TAMs in the tumor microenvironment (TME), which can offer a promising molecular basis to improve the clinical response efficacy of ICB in cancer patients. PurposeThis study aims to investigate TAMs in the immunosuppressive microenvironment to identify new therapeutic targets, improve the ability to predict and guide responses to clinical immunotherapy, and develop new strategies for immunotherapy of lung tumors. MethodsLewis lung carcinoma (LLC) xenograft-bearing mouse models were established to analyze the antitumor activity of Rhizoma Coptidis (RC) in vivo. A systems pharmacology strategy was used to predict the correlation between RC and M2 macrophages. The effect of RC on the abundance of M2 macrophages was analyzed by flow cytometry of murine samples. Western blot was performed to analyze the expression of Leukotriene A4 hydrolase (LTA4H) and LTB4 receptor 1 (BLT1) in harvested lung cancer tissues. The impact of blocking leukotriene B4 (LTB4) signaling by RC on the recruitment of M2 macrophages was assessed in vitro and in vivo. Transwell migration assays were conducted to clarify the inhibition of macrophage migration by blocking LTB4. Lta4h−/− mice were used to investigate the sensitivity of immunotherapy to lung cancer by blocking the LTB4 signaling. ResultsHere, we report that RC, an herbal medicine from the family Ranunculaceae, suppresses the recruitment and immunosuppressive function of TAMs, which in turn sensitizes lung cancer to ICB therapy. Firstly, a systems pharmacology strategy was proposed to identify combinatorial drugs for ICB therapy with a systems biology perspective of drug-target-pathway-TME phenotype. We predicted and verified that RC significantly inhibits tumor growth and the infiltration of M2-TAMs into TME of LLC tumor-bearing mice. Then, RC inhibits the recruitment of macrophages to the tumor TME via blocking LTB4 signaling, and suppresses the expression of immunosuppressive factors (IL-10, TGF-β and VEGF). As a result, RC enables CD8+ T cells to retain their proliferative and infiltrative abilities within the TME. Ultimately, these events promote cytotoxic T-cell-mediated clearance of tumor cells, which is further enhanced by the addition of anti-PD-L1 therapy. Furthermore, we employed LTA4H deficient mice (Lta4h−/− mice) to evaluate the antitumor efficiency, the results showed that the efficacy of immunotherapy was enhanced due to the synergistic effect of LTB4 signaling blockage and ICB inhibition, leading to remarkable inhibition of tumor growth in a mouse model of lung adenocarcinoma. ConclusionsTaken together, these findings suggest that RC enhances antitumor immunity, providing a rationale for combining RC with immunotherapies as a potential anti-cancer treatment strategy.

Grading of lung adenocarcinomas with simultaneous segmentation by artificial intelligence (GLASS-AI)

Preclinical genetically engineered mouse models (GEMMs) of lung adenocarcinoma are invaluable for investigating molecular drivers of tumor formation, progression, and therapeutic resistance. However, histological analysis of these GEMMs requires significant time and training to ensure accuracy and consistency. To achieve a more objective and standardized analysis, we used machine learning to create GLASS-AI, a histological image analysis tool that the broader cancer research community can utilize to grade, segment, and analyze tumors in preclinical models of lung adenocarcinoma. GLASS-AI demonstrates strong agreement with expert human raters while uncovering a significant degree of unreported intratumor heterogeneity. Integrating immunohistochemical staining with high-resolution grade analysis by GLASS-AI identified dysregulation of Mapk/Erk signaling in high-grade lung adenocarcinomas and locally advanced tumor regions. Our work demonstrates the benefit of employing GLASS-AI in preclinical lung adenocarcinoma models and the power of integrating machine learning and molecular biology techniques for studying the molecular pathways that underlie cancer progression.

Lung Adenocarcinoma Expression of the Aryl Hydrocarbon Receptor Promotes PD-L1 and Immune Suppression

Abstract Immunotherapy has shown dramatic results in treating cancer, but only a minority of patients benefit. Therefore, there is an unmet medical need to understand the regulation of immune suppression in tumors. We show that the aryl hydrocarbon receptor (AhR) is a central player in regulating immune checkpoints in a mouse model of lung adenocarcinoma (LUAD). Within the murine malignant cell, AhR regulates expression of several genes important to suppressive immune signaling, including PD-L1. We find similar immune-related regulatory mechanisms in human LUAD cells. Notably, the presence of IFNγ boosts the expression of these AhR-regulated suppressive genes, suggesting a deleterious role for IFNγ in LUAD. Transplantation of mouse AhR-knockout CMT167 (CMT167 AhR-KO) LUAD cells leads to partial or complete tumor rejection and protection from wild type (WT) LUAD challenge. CMT167 AhR-KOtumors that do form have decreased PD-L1 expression and increased T cell infiltration relative to AhR +control tumors. To identify specific immune cell subsets involved in AhR-related tumor immunity, we employed an imaging mass cytometry (IMC) platform coupled with single cell RNA sequencing of tumor infiltrating CD45 +immune cells. Understanding the phenotypes and spatial orientation of the immune cells interacting with malignant cells will provide evidence of the key immune populations responsible for anti-tumor immunity and thus help pave the way for future therapies and biomarkers relevant to improving treatments for LUAD patients. Find the Cause Consortium Gifts