Mouse Lung Adenocarcinoma Research Articles

Immune profiling of mouse lung adenocarcinoma paraffin tissues using multiplex immunofluorescence panel: a pilot study

BackgroundImmune profiling has become an important tool for identifying predictive, prognostic and response biomarkers for immune checkpoint inhibitors from tumor microenvironment (TME). We aimed to build a multiplex immunofluorescence (mIF) panel to apply to formalin-fixed and paraffin-embedded tissues in mice tumors and to explore the programmed cell death protein 1/ programmed cell death 1 ligand 1 (PD-1/PD-L1) axis.ResultsAn automated eight-color mIF panel was evaluated to study the TME using seven antibodies, including cytokeratin 19, CD3e, CD8a, CD4, PD-1, PD-L1, F4-80 and DAPI, then was applied in six mice lung adenocarcinoma samples. Cell phenotypes were quantified by software to explore the co-localization and spatial distribution between immune cells within the TME. This mice panel was successfully optimized and applied to a small cohort of mice lung adenocarcinoma cases. Image analysis showed a sparse degree of immune cell expression pattern in this cohort. From the spatial analysis we found that T cells and macrophages expressing PD-L1 were close to the malignant cells and other immune cells.ConclusionsComprehensive immune profiling using mIF in translational studies improves our ability to correlate the PD-1/PD-L1 axis and spatial distribution of lymphocytes and macrophages in mouse lung cancer cells to provide new cues for immunotherapy, that can be translated to human tumors for cancer intervention.

Combined cryoablation and PD-1 inhibitor synergistically enhance antitumor immune responses in Lewis lung adenocarcinoma mice via the PI3K/AKT/mTOR pathway

Cryoablation is a therapeutic modality for lung adenocarcinoma that destroys target tumors using lethal levels of cold, resulting in the release of large amounts of specific antigens that activate immune responses. However, tumor immune checkpoint escape mechanisms prevent these released self-antigens from inducing effective anti-tumor immune responses. To overcome this challenge, we propose the use of immune checkpoint inhibitors to relieve T cell inhibition by immune checkpoints and enhance the anti-tumor immune response mediated by cryoablation. We used bilateral tumor-bearing mouse models and a specific cryoablation instrument to study the efficacy of cryoablation combined with PD-1 inhibitors in Lewis lung adenocarcinoma model mice. We found that cryoablation combined with PD-1 inhibitors significantly inhibited the growth of mouse lung adenocarcinoma, prolonged mouse survival, and enhanced the anti-tumor immune response. Moreover, this combined regimen could synergistically promote the activation and proliferation of T cells via the PI3K/AKT/mTOR pathway. The present study provides a strong theoretical basis for the clinical combination of cryoablation and PD-1 inhibitors.

MEN1 deficiency stabilizes PD-L1 and promotes tumor immune evasion of lung cancer.

Multiple Endocrine Neoplasia 1 gene (MEN1), which is known to be a tumor suppressor gene in lung tissues, encodes a 610 amino acid protein menin. Previous research has proven that MEN1 deficiency promotes the malignant progression of lung cancer. However, the biological role of this gene in the immune microenvironment of lung cancer remains unclear. In this study, we found that programmed cell death-ligand 1 (PD-L1) is upregulated in lung-specific KrasG12D mutation-induced lung adenocarcinoma in mice, after Men1 deficiency. Simultaneously, CD8+ and CD3+ T cells are depleted, and their cytotoxic effects are suppressed. Invitro, PD-L1 is inhibited by the overexpression of menin. Mechanistically, we found that MEN1 inactivation promotes the deubiquitinating activity of COP9 signalosome subunit 5 (CSN5) and subsequently increases the level of PD-L1.

Role of COX-2/PGE2/EP4 Axis-induced Macrophage Functional Activation in NSCLC Development

Tumor microenvironment (TME) is one of the important factors in tumorigenesis and progression, in which tumor-associated macrophages (TAMs) play an important role in non-small cell lung cancer (NSCLC) progression. However, the mechanism of TAMs in NSCLC progression remains unclear, so this study aimed to investigate the role of TAMs in NSCLC progression and to find potential therapeutic targets. Gene Expression Profiling Interactive Analysis (GEPIA) database was used to analyze the expression of prostaglandin E2 receptor 4 (EP4) mRNA in NSCLC and normal lung tissues; the protein expression levels of cyclooxygenase-2 (COX-2), EP4, cluster of differentiation 86 (CD86), CD163 and CD31 were detected by immunohistochemistry (IHC) in 120 NSCLC tissues and 24 paracancerous tissues specimens. The nude mouse lung adenocarcinoma cell A549 and macrophage RAW264.7 co-transplanted tumor model was established. And the samples were collected by gavage with EP4 inhibitor E7046, and then stained with hematoxylin-eosin (HE), IHC, and immunofluorescence (IF), and then detected by Western blot for the epithelial mesenchymal transformation (EMT) of the tumor tissues of the nude mice in each group. Western blot was used to detect the expressions of EMT related protiens in each group of nude mice; full-length transcriptome sequencing was used to screen the key genes causing liver metastasis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed. EP4 mRNA expression level in NSCLC tissues was generally lower than that in normal lung tissues (P<0.05); COX-2, EP4, CD163, CD31 proteins were differentially expressed in NSCLC tissues and adjacent tissues, and differences were observed in many clinicopathological parameters of NSCLC patients; RAW264.7 shortened the latency period of tumorigenesis of A549 and promoted the proliferation of tumors and liver metastasis of tumors, and E7046 could reduce tumor cell proliferation activity, tumor tissue vascular density and M2-type macrophage infiltration in nude mice; IF staining showed that macrophages were mainly distributed around the metastatic foci of tumors; Western blot results showed that compared with A549 alone transplantation group, the relative expression of E-cadherin protein in tumor tissues of mice in A549 and RAW264.7 co-transplantation group was significantly decreased, and the difference was statistically significant (P<0.05), while the relative expression of N-cadherin protein was up-regulated, but the difference was not statistically significant (P>0.05); the main pathways enriched in the differential genes of the full-length transcriptome were the PI3K-AKT and MAPK signaling pathways. During NSCLC development, the COX-2/PGE2/EP4 axis may promote tumor progression by inducing macrophage functional activation, and EP4 may be a potential new target for tumor immunotherapy. This study provides new perspectives and ideas for in-depth exploration of the mechanisms of NSCLC development, as well as a theoretical basis for the development of new therapeutic strategies for NSCLC.

Cytosolic DNA sensor AIM2 promotes KRAS-driven lung cancer independent of inflammasomes.

Constitutively active KRAS mutations are among the major drivers of lung cancer, yet the identity of molecular co-operators of oncogenic KRAS in the lung remains ill-defined. The innate immune cytosolic DNA sensor and pattern recognition receptor (PRR) Absent-in-melanoma 2 (AIM2) is best known for its assembly of multiprotein inflammasome complexes and promoting an inflammatory response. Here, we define a role for AIM2, independent of inflammasomes, in KRAS-addicted lung adenocarcinoma (LAC). In genetically defined and experimentally induced (nicotine-derived nitrosamine ketone; NNK) LAC mouse models harboring the KrasG12D driver mutation, AIM2 was highly upregulated compared with other cytosolic DNA sensors and inflammasome-associated PRRs. Genetic ablation of AIM2 in KrasG12D and NNK-induced LAC mouse models significantly reduced tumor growth, coincident with reduced cellular proliferation in the lung. Bone marrow chimeras suggest a requirement for AIM2 in KrasG12D-driven LAC in both hematopoietic (immune) and non-hematopoietic (epithelial) cellular compartments, which is supported by upregulated AIM2 expression in immune and epithelial cells of mutant KRAS lung tissues. Notably, protection against LAC in AIM2-deficient mice is associated with unaltered protein levels of mature Caspase-1 and IL-1β inflammasome effectors. Moreover, genetic ablation of the key inflammasome adapter, ASC, did not suppress KrasG12D-driven LAC. In support of these invivo findings, AIM2, but not mature Caspase-1, was upregulated in human LAC patient tumor biopsies. Collectively, our findings reveal that endogenous AIM2 plays a tumor-promoting role, independent of inflammasomes, in mutant KRAS-addicted LAC, and suggest innate immune DNA sensing may provide an avenue to explore new therapeutic strategies in lung cancer.

Abstract CT252: Nicotinamide in combination with EGFR-TKIs for the treatment of stage IV lung adenocarcinoma with EGFR mutations: A randomized double-blind (Phase IIb) trial

Abstract Background: RUNX3 is a tumor suppressor gene, which is inactivated in approximately 70% of lung adenocarcinomas. Nicotinamide, a sirtuin inhibitor, has demonstrated potential in re-activating epigenetically silenced RUNX3 in cancer cells. This study assessed the therapeutic benefits of combining nicotinamide with first-generation EGFR-TKIs for patients with stage IV lung cancer carrying EGFR mutations. Patients and Methods: We assessed the impact of nicotinamide on carcinogen-induced lung adenocarcinomas in mice and observed that nicotinamide increased RUNX3 levels and inhibited lung cancer growth. Subsequently, 110 consecutive patients with stage IV lung cancer who had EGFR mutations were recruited: 70 females (63.6%) and 84 never-smokers (76.4%). The patients were randomly assigned to receive either nicotinamide (1 g/day, n = 55) or placebo (n = 55). The primary and secondary endpoints were progression-free survival (PFS) and overall survival (OS), respectively. Results: After a median follow-up of 54.3 months, the nicotinamide group exhibited a median PFS of 12.7 months (95% CI, 10.4-18.3), while the placebo group had a PFS of 10.9 months (9.0-13.2; p = 0.2). The median OS was similar in the two groups (31.0 months with nicotinamide vs. 29.4 months with placebo; p = 0.2). Notably, subgroup analyses revealed a significant reduction in mortality risk for females (p = 0.01) and never-smokers (p = 0.03) treated with nicotinamide. Conclusion: The addition of nicotinamide with EGFR-TKIs demonstrated potential improvements in PFS and OS, with notable survival benefits for female patients and those who had never smoked. (ClinicalTrials.gov Identifier: NCT02416739) Citation Format: Cheol-Kyu Park, Hyung-Joo Oh, Suk-Chul Bae, In-Jae Oh, Kyoung-Mi Jung, Da-Mi Kim, Jung-Won Lee, Chang Kyun Kang, Il Yeong Park, Young-Chul Kim. Nicotinamide in combination with EGFR-TKIs for the treatment of stage IV lung adenocarcinoma with EGFR mutations: A randomized double-blind (Phase IIb) trial [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 CT252.

Abstract 7062: CIP2A enhances mitochondrial respiratory electron transport chain complexes and induces lung cancer in vivo

Abstract Identification of oncogenes that can induce cancer in vivo is critical to elucidation of tumorigenesis and development of targeted therapies. Here, we reported for the first time that forced ectopic expression of CIP2A, the cancerous inhibitor of protein phosphatase 2A (PP2A), in lung tissues induced lung adenocarcinoma in mice. CIP2A upregulated the transcription and translation of mitochondrial DNA (mtDNA), and increases the enzymatic activity of mitochondrial electron transport chain (ETC) complex IV and V, thus enhancing the function of mitochondria and promoting the level of oxidative phosphorylation in tumors. CIP2A inhibited the binding of PP2A-B56α to FOXO3a, leading to the enhancement of the phosphorylation level of FOXO3a. The phosphorylated FOXO3a then translocated into the mitochondrion, and acted as a transcription factor to regulate CIP2A-mediated metabolic reprogramming in cancer cells. We found that oxidative phosphorylation inhibitors effectively inhibited CIP2A-induced cell proliferation. In non-small cell lung cancer, CIP2A level in tumor tissues was positively associated with the level of mtDNA. Our results indicated that CIP2A is able to induce lung adenocarcinoma in mice and has a critical role in mitochondrial oxidative phosphorylation, and represents a promising therapeutic target for non-small cell lung cancer. Competing interests: The authors declare no competing interests. Citation Format: Fu-Ying Yang, Di Wang, Li-Jun liang, Gui-Zhen Wang, Guang-Biao Zhou. CIP2A enhances mitochondrial respiratory electron transport chain complexes and induces lung cancer in vivo [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 7062.

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.

Low-dose radiotherapy promotes the formation of tertiary lymphoid structures in lung adenocarcinoma.

A tertiary lymphoid structure (TLS) refers to an organized infiltration of immune cells that is linked to a positive prognosis and improved response to immunotherapy. However, methods that promote TLS formation are limited and challenging to implement in clinical settings. In this study, we aimed to promote the formation and maturation of TLSs in lung adenocarcinoma (LUAD) by combining low-dose radiotherapy (LDRT) with immunotherapy. Tissue sections from 198 patients who had undergone surgery were examined. Risk factors for patient survival were assessed, and the relationship between TLSs and five-year survival was analyzed. The Kras-LSL-G12D spontaneous lung cancer mouse model was used to screen the optimal irradiation dose (0/1/2 Gy whole lung irradiation) for promoting TLS formation. LDRT combined with anti-PD-1 was used to promote the formation and maturation of TLSs. TLS+, TLSHigh, TLS+GC+ and CD8High within TLS+ were associated with a favorable prognosis. LDRT increased the formation of early TLSs in the Kras-LSL-G12D lung cancer mouse model. In addition, LDRT combined with anti-PD-1 treatment can significantly improve the maturity of TLSs in mouse LUAD, resulting in greater antitumor effects. This antitumor effect was strongly associated with the number of CD8+ T cells within the TLSs. We successfully applied LDRT combined with PD-1 inhibitor therapy for the first time, which increased both the quantity and maturity of TLSs in lung cancer. This approach achieved a promising antitumor effect.

Prebiotics Modulate Gut Microbiota-mediated T-cell Immunity to Enhance the Inhibitory Effect of Sintilimab in Lewis Lung Adenocarcinoma Model Mice.

Sintilimab (Sin) helps the body to restore the anti-tumor response of T lymphocytes. However, in clinical use, the treatment process is more complicated due to adverse effects and different dosing regimens. It is not clear whether prebiotics (PREB) have a potentiating effect on Sin for lung adenocarcinoma, and this study intends to investigate the inhibitory effect, safety and possible mechanism of Sin combined with PREB on lung adenocarcinoma from animal experiments. Lewis lung adenocarcinoma cells were inoculated into the right axilla of mice subcutaneously to prepare the Lewis lung cancer mouse model and treated in groups. The volume of transplanted tumors was measured, the histopathology of the liver and kidney of mice was observed by H&E staining, the levels of ALT, AST, UREA, CREA, WBC, RBC, and HGB in blood were analyzed biochemically; the ratio of T-cell subpopulations in blood, spleen, and bone marrow was detected by flow cytometry, the expression of PD-L1 in tumor tissue was detected by immunofluorescence staining, and 16S rRNA to analyze the diversity of fecal flora. Sin inhibited tumor growth and regulated immune cell homeostasis in lung adenocarcinoma mice, but liver and kidney histopathology showed different degrees of damage after Sin treatment, while the addition of PREB reduced liver and kidney damage in lung adenocarcinoma mice and promoted Sin's regulation of immune cells. In addition, the beneficial effects of Sin were associated with changes in intestinal flora diversity. The mechanism by which Sintilimab combined with prebiotics inhibits tumor volume and regulates immune cell subpopulation balance in lung adenocarcinoma mice may be related to gut microbes.

Thymol targeting interleukin 4 induced 1 expression reshapes the immune microenvironment to sensitize the immunotherapy in lung adenocarcinoma.

Immune checkpoint blockades are the most promising therapy in lung adenocarcinoma (LUAD). However, the response rate remains limited, underscoring the urgent need for effective sensitizers. Interleukin 4 induced 1 (IL4I1) is reported to have immunoinhibitory and tumor-promoting effects in several cancers. However, the targetable value of IL4I1 in sensitizing the immunotherapy is not clear, and there is a lack of effective small molecules that specifically target IL4I1. Here, we show that silencing IL4I1 significantly remodels the immune microenvironment via inhibiting aryl hydrocarbon receptor (AHR) signaling, thereby enhancing the efficacy of anti-PD-1 antibody in LUAD, which suggests that IL4I1 is a potential drug target for the combination immunotherapy. We then identify thymol as the first small molecule targeting IL4I1 transcription through a drug screening. Thymol inhibits the IL4I1 expression and blocks AHR signaling in LUAD cells. Thymol treatment restores the antitumor immune response and suppresses the progression of LUAD in an orthotopic mouse model. Strikingly, the combination treatment of thymol with anti-PD-1 antibody shows significant tumor regression in LUAD mice. Thus, we demonstrate that thymol is an effective small molecule to sensitize the PD-1 blockade in LUAD via targeting IL4I1, which provides a novel strategy for the immunotherapy of LUAD.

Tumor microenvironment-derived monoacylglycerol lipase provokes tumor-specific immune responses and lipid profiles

We recently described that monoacylglycerol lipase (MGL) is present in the tumor microenvironment (TME), increasing tumor growth. In this study we compare the implications of MGL deficiency in the TME in different tumor types.We show that subcutaneous injection of KP (KrasLSL-G12D/p53fl/fl, mouse lung adenocarcinoma) or B16-F10 cells (mouse melanoma) induced tumor growth in MGL wild type (WT) and knockout (KO) mice. MGL deficiency in the TME attenuated the growth of KP cell tumors whereas tumors from B16-F10 cells increased in size. Opposite immune cell profiles were detected between the two tumor types in MGL KO mice. In line with their anti-tumorigenic function, the number of CD8+ effector T cells and eosinophils increased in KP cell tumors of MGL KO vs. WT mice whereas their presence was reduced in B16-F10 cell tumors of MGL KO mice. Differences were seen in lipid profiles between the investigated tumor types. 2-arachidonoylglycerol (2-AG) content significantly increased in KP, but not B16-F10 cell tumors of MGL KO vs. WT mice while other endocannabinoid-related lipids remained unchanged. However, profiles of phospho- and lysophospholipids, sphingomyelins and fatty acids in KP cell tumors were clearly distinct to those measured in B16-F10 cell tumors.Our data indicate that TME-localized MGL impacts tumor growth, as well as levels of 2-AG and other lipids in a tumor specific manner.

Tumor-suppressive action of miR-30a-5p in lung adenocarcinoma correlates with ABL2 inhibition and PI3K/AKT pathway inactivation.

ABL2 contributes to the oncogenic potential of cancers, pointing to its inhibition as a possible strategy against malignant diseases. Bioinformatics prediction of upstream effector miR-30a-5p for ABL2 allowed us to hypothesize and then validate mechanistic actions of miR-30a-5p in lung adenocarcinoma (LUAD). The ABL2 expression in LUAD was analyzed in the TCGA data, clinical samples, and cell lines. The shRNA-mediated silencing of ABL2 was introduced to illustrate its effect on malignant phenotypes of LUAD cells. The binding affinity between ABL2 and miR-30a-5p was verified by luciferase activity and RNA pull-down assay. Ectopic expression, knockdown methods, and PI3K inhibitor LY294002 were used to investigate their effects on in vitro biological characteristics and in vivo tumor growth of LUAD cells. Using nude mouse lung adenocarcinoma in situ and brain metastasis models to validate the inhibitory effect of miR-30a-5p on LUAD by regulating the ABL2/PI3K/AKT signaling axis. High expression of ABL2 and poor expression of miR-30a-5p were noticed in LUAD tissues and cell lines. Importantly, miR-30a-5p was demonstrated to target and downregulate ABL2, subsequently inactivating the PI3K/AKT pathway. miR-30a-5p inhibited the malignant phenotypes of LUAD cells by inhibiting ABL2 expression and inactivating the PI3K/AKT pathway. For in vivo experiments, miR-30a-5p was substantiated to thwart tumor tumorigenesis by regulating the ABL2/PI3K/AKT axis. In addition, miR-30a-5p suppresses the occurrence and development of in situ lung cancer and brain metastasis via the ABL2/PI3K/AKT signaling pathway. This study underscores the inhibitory role of miR-30a-5p in LUAD through the ABL2/PI3K/AKT axis, which may be a viable target for LUAD treatment.

Veillonella parvula promotes the proliferation of lung adenocarcinoma through the nucleotide oligomerization domain 2/cellular communication network factor 4/nuclear factor kappa B pathway

Enrichment of Veillonella parvula in the lung microbiota is strongly associated with non-small cell lung cancer (NSCLC) and induces the progression of lung adenocarcinoma in vivo, but its actual role and mechanism remain unexplored. This study analyzed the correlation between NSCLC and V. parvula abundance based on 16 s rRNA sequencing results. The effects of V. parvula on the progression of lung adenocarcinoma were observed in vivo and in vitro using a C57 bl/6j mouse tumor-bearing model, a bacterial cell co-culture model, combined with transcriptome sequencing, and a TCGA database to explore and validate the growth promotion of lung adenocarcinoma by V. parvula and its molecular mechanism. 16 s rRNA sequencing revealed that V. parvula was significantly enriched in lung adenocarcinoma. In vivo, V. parvula promoted the growth of lung adenocarcinoma in mice by suppressing the infiltration of tumor-associated T lymphocytes and peripheral T lymphocytes. It showed a higher affinity for lung adenocarcinoma in vitro and promoted lung adenocarcinoma cell proliferation through adhesion or intracellular invasion. Further analysis of differential gene expression and KEGG enrichment by transcriptome sequencing revealed that V. parvula induced CCN4 expression and activated NOD-like receptor and NF-κB signaling pathway in lung adenocarcinoma cells. Further analysis clarified that V. parvula promoted activation of the NF-κB pathway via Nod2/CCN4 signaling, which promoted lung adenocarcinoma cell proliferation. Thus, V. parvula mediates activation of the Nod2/CCN4/NF-κB signaling pathway to promote non-small cell lung adenocarcinoma progression, thereby providing a potential target for diagnosing and treating lung adenocarcinoma.Graphical

A2aR on lung adenocarcinoma cells: A novel target for cancer therapy via recruiting and regulating tumor-associated macrophages

Adenosine 2a receptor (A2aR), a typical GPCR with a high affinity for adenosine, is widely expressed on immune cells, inhibiting anti-tumor immune response accordingly. Here, we identify that A2aR is specifically expressed on tumor cells from lung adenocarcinoma (LUAD) patients, closely related to their prognosis and positively correlated with tumor-associated macrophages (TAMs) infiltration. We hypothesize that blocking A2aR on LUAD cells will inhibit the role of TAMs and control tumor growth. Constructing models of TAMs and LUAD mice, we find that A2aR highly expressed on LUAD cells promotes the secretion of chemokines and polarizing factors through activating PI3K/AKT/NF-κB pathway, thereby promoting the migration and invasion of TAMs. Functionally, blocking A2aR significantly suppresses TAMs infiltration and attenuates tumor burden in LUAD mice. Notably, the M2 polarization of TAMs can also be prevented by inhibiting A2aR in vitro. Together, our studies demonstrate that A2aR on LUAD cells drives TAMs migration and polarization, and blockade of A2aR may support a novel and potent therapeutic option for LUAD.

Abstract IA01: Targeting KRAS: Light at the end of the tunnel

Abstract KRAS, an oncogene responsible for at least one fourth of all human cancers, was discovered four decades ago. Yet, the first KRAS selective inhibitor could not be approved until 2021. In spite of this long-awaited breakthrough, approved KRAS inhibitors only block one of the multiple oncogenic isoforms, KRASG12C, an isoform primarily present in lung adenocarcinomas. Moreover, tumor resistance to these KRAS inhibitors is becoming a significant issue in the clinic (Awad et al., N Engl J Med, 2021; Zhao et al., Nature 2021). To provide experimental information that might help to improve current KRAS targeting strategies, we have generated two experimental mouse tumor models of Kras-driven lung adenocarcinoma to compare the therapeutic consequences of eliminating, instead of inhibiting, KRAS oncoproteins. In addition, we have interrogated the molecular mechanisms responsible for the induction of resistance in sotorasib treated KrasG12C/p53 mouse lung adenocarcinomas. We have also investigated the potential role of tampering with KRAS signaling pathways to develop alternative therapeutic strategies against KRAS mutant lung and pancreatic cancers. Previous studies have shown that ablation of RAF1, either by itself, or in combination with the expression of a CDK4 kinase dead isoform, induce significant levels of tumor regression in Kras/p53 driven-lung adenocarcinomas without inducing significant toxicities (Sanclemente et al. Cancer Cell, 2018; Esteban-Burgos et al., PNAS 2020). However, Kras/p53 driven-pancreatic ductal adenocarcinomas (PDAC) are completely resistant to RAF1 ablation, unless RAF1 is deleted in combination with EGFR, an upstream KRAS effector (Blasco et al, Cancer Cell 2019). Yet, ablation of RAF1 and EGFR only resulted in the complete regression of a fraction of PDAC tumors. We have now identified activation of STAT3 as the main mechanism responsible for the resistance of PDAC tumors to RAF1/EGFR ablation. Indeed, combined ablation of RAF1/EGFR/STAT3 completely blocked proliferation of all tested mouse tumor cell lines and organoids. Moreover, all tumors tested in orthotopic allograft models regressed completely with no evidence of tumor regression for at least two months of TMX exposure. Finally, I will discuss potential strategies to pharmacologically validate this triple therapeutic strategy as well as plans to combine it with current KRAS inhibitors. Citation Format: Mariano Barbacid. Targeting KRAS: Light at the end of the tunnel [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA01.

Abstract LB104: Preclinical evaluation of the role of Galectin-1 in tumor immune evasion associated mechanisms using a highly selective Galectin-1 small molecule inhibitor

Abstract Galectin-1 (Gal-1) is a β-galactoside-binding lectin that is highly expressed within the tumor microenvironment (TME) of some aggressive cancers and whose high expression correlates strongly with poor survival. Following a data mining exercise of external publication data and additional clinical biomarker datasets, we found strong evidence concluding the potential role of Gal-1 in the establishment of a fibrotic tumor stroma and mechanisms associated with tumor invasion, metastasis and angiogenesis. However, there was limited data regarding the role of Gal-1 in immune escape by tumors to make any strong conclusions/pathway reconstruction around this specific TME mechanism of action (MOA). To further strengthen the potential role of Gal-1 in tumor immune evasion we completed a series of preclinical in vivo and in vitro activities, using a highly selective Gal-1 small molecule inhibitor developed at Galecto Biotech. To examine the role of Gal-1 inhibition in vivo, we investigated the effects on tumor volume of the highly selective Gal-1 inhibitor, in a syngeneic mouse model of lung adenocarcinoma. RNA profiling of vehicle and compound treated tumors was also implemented following completion of the in-life phase. Oral administration of the Gal-1 inhibitor reduced mouse lung adenocarcinoma growth and increased the immune gene expression within the tumors, showing a strong association to that of exacerbated NK cell activity. No additional cytotoxic T cell gene changes were uncovered in the Gal-1 inhibitor treated tumors. We further evaluated the overall T cell compartment, relevant to the clinical TME setting, by stimulating human peripheral blood mononuclear cells (hPBMC’s) incubated with and without the Gal-1 inhibitor to observe the potential blockade of any known immune checkpoint inhibitor (ICI) resistance mechanisms. Interestingly, we found a variety of immune suppressive proteins to be inhibited, including IL-17, when hPBMC’s were incubated with the Gal-1 inhibitor. In summary, the data presented suggests highly selective Gal-1 inhibition could provide an effective, monotherapy (or be used in combination with ICI's) to boost immune infiltration and/or activation in the TME of lung adenocarcinoma and potentially other aggressive cancers. Citation Format: Ian Holyer, Rob Slack, Kristoffer Petersen, Fredrik Zetterberg, Alison Mackinnon. Preclinical evaluation of the role of Galectin-1 in tumor immune evasion associated mechanisms using a highly selective Galectin-1 small molecule inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB104.

Antibodies against endogenous retroviruses promote lung cancer immunotherapy

B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS)1,2. Although TLS have been found to correlate with improved patient survival and response to immune checkpoint blockade (ICB), the underlying mechanisms of this association remain elusive1,2. Here we investigate lung-resident B cell responses in patients from the TRACERx 421 (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy) and other lung cancer cohorts, and in a recently established immunogenic mouse model for lung adenocarcinoma3. We find that both human and mouse lung adenocarcinomas elicit local germinal centre responses and tumour-binding antibodies, and further identify endogenous retrovirus (ERV) envelope glycoproteins as a dominant anti-tumour antibody target. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted inhibition of KRAS(G12C) in the mouse model. ERV-reactive antibodies exert anti-tumour activity that extends survival in the mouse model, and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Finally, we find that effective immunotherapy in the mouse model requires CXCL13-dependent TLS formation. Conversely, therapeutic CXCL13 treatment potentiates anti-tumour immunity and synergizes with ICB. Our findings provide a possible mechanistic basis for the association of TLS with immunotherapy response.

Abstract 16: The role of innate and adaptive immunity in the sex bias of LKB1-mutant lung adenocarcinoma

Abstract The tumor suppressor gene, LKB1/STK11, is one of the most frequently mutated genes in lung adenocarcinoma (LUAD). A multidimensional analysis of TCGA-LUAD patient data indicates that LKB1 mutation is one of the top sex-biased mutations, being more frequent in males than in females. A similar pattern was observed in other large LUAD cohorts. However, the underlying mechanism was unknown, partially due to a lack of an appropriate mouse model to represent this clinical phenomenon. We previously demonstrated a similar sex bias in a Lenti-Cre-induced KRAS/LKB1-mutant genetically engineered mouse model (GEMM) and a syngeneic tail-vein injection model. Here, we demonstrated that intravenous injection of the same cell line in male and female NOD scid gamma (NSG) mice does not exhibit a sex-bias pattern of lung metastasis formation (P=0.9746). However, sex bias was observed when we used CB17-SCID mice. This data suggest that innate immunity is partly responsible for the sex bias of LKB1-mutant lung cancer formation. We also characterized immune cells from the lung with LKB1-mutant tumors. Monocytes/macrophages decreased from 23% to 19%, and neutrophils increased from 15% to 19%. Lymphoid cells remained the same at 58%, and T-cells account for 34% of all CD45+ cells. Interestingly, T-cells doubled to account for 81% of all CD45+ cells from the lung in female mice resistant to the formation of lung metastasis. Our data indicated that both the innate and the adaptive immunity are responsible for eliminating LKB1-mutant lung adenocarcinoma in female mice. Citation Format: Yijian Fan, Lenore Monterroza, Rui Jin, Xiuju Liu, Periasamy Selvaraj, Rabindra Tirouvanziam, Wei Zhou. The role of innate and adaptive immunity in the sex bias of LKB1-mutant lung adenocarcinoma [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 16.

Abstract 3234: OCT-598, a novel EP2/EP4 dual antagonist, promotes anti-tumor immune responses in syngeneic mouse tumor models in combination with standard-of-care chemo- and immunotherapies

Abstract Prostaglandin E2 (PGE2) is widely recognized as one of the major bioactive lipids that, with the striking regenerative potential, promote drug-resistance in cancer cells as well as immune evasion in the tumor microenvironment (TME). Primarily driven by apoptotic cell death, PGE2 is thought to elicit wound-healing responses to help provide an immunosuppressive and proliferative niche that supports cancer stem cell repopulation and thereby therapy-resistance. While COX1/2 inhibitors that attenuate PGE2 production have shown promising anti-cancer effects in various (pre-)clinical settings, the gastrointestinal- and cardiotoxicities precluded their development as anti-cancer agents. It is anticipated that specific targeting of PGE2 signaling via its cognate receptors constitutes a safer and potentially more effective approach. Of the receptor subtypes EP1-4, Gα,s-coupled EP2 and EP4 are believed to be directly involved in immunosuppressive effects of PGE2.OCT-598 is a novel, highly potent and selective EP2/EP4 dual antagonist with Ki values of 23 nM and 0.2 nM vs EP2 and EP4, respectively. PGE2 inhibited normal differentiation of human monocytes into CD1a+CD16- dendritic cells under the presence of GM-CSF and IL-4 and promoted differentiation towards CD1a-CD16+ macrophages in vitro. However, EP2/EP4 dual inhibition by OCT-598 reversed this phenomenon to a greater extent than either EP2- or EP4-specific inhibitor alone. In vivo, OCT-598 effected tumor growth inhibition in multiple syngeneic mouse models as a single agent as well as in combination with an immune checkpoint blocker (ICB). Furthermore, the addition of OCT-598 to the lung cancer standard-of-care regimen (anti-PD-1 plus chemotherapy) in TC-1 mouse lung adenocarcinoma model gave rise to complete tumor regression. In conclusion, dual blockade of EP2 and EP4 by OCT-598 is shown to be a compelling strategy to reinforce antitumor effects by thwarting PGE2-mediated therapy resistance and immune evasion.Findings from this study provide a rationale for clinical development of OCT-598 as a therapeutic option for human malignant cancers. Citation Format: Youngrae Lee, Sujeong Baek, Dong Kwon Kim, Yeri Lee, Donggeon Kim, Seongin Jo, Sang Kyun Lim, Young Sook Shin, Soonsang Kwon, Seung Min Yang, Young Taek Kim, Seong-San Kang, Chun-Bong Synn, Kwangmin Na, Mi Hyun Kim, Heekyung Han, Yu Jin Han, Sungwoo Lee, Jae Hwan Kim, Mi Ran Yun, Youngseon Byeon, Young Seob Kim, Ji Yun Lee, Jii Bum Lee, Chang Gon Kim, Min Hee Hong, Sun Min Lim, Kyoung-Ho Pyo, Byoung Chul Cho, Taeyoung Yoon. OCT-598, a novel EP2/EP4 dual antagonist, promotes anti-tumor immune responses in syngeneic mouse tumor models in combination with standard-of-care chemo- and immunotherapies [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 3234.