Murine Lung Cancer Research Articles

Detection and analysis of lung microbiota in mice with lung cancer lacking the NLRP3 gene

To explore whether the lung microbiota have changed in the process of NLRP3 inflammasome promoting cancer, we constructed a murine lung cancer model using tracheal instillation of benzo(a)pyrene and an equal volume of tricaprylin, and characterized lung microbiota in bronchoalveolar lavage fluid from 24 SPF wild-type and NLRP3 gene knockout (NLRP3−/−) C57BL/6 mice. 16SrDNA sequencing was used to analyze the changes in the microbiota. The wild-type and the NLRP3−/− lung cancer group had statistically significant differences in tumor formation rate, tumor number, and tumor size. At the phylum and the genus level, the relative abundance of Proteobacteria and Sphingomonas were the highest in each group respectively. Simpson (P = 0.002) and Shannon (P = 0.008) indexes showed that the diversity of microbiota in the lung cancer group was lower than that in the control group under the NLRP3−/− background. According to the ANOSIM and MRPP analysis, there was a difference between the NLRP3−/− lung cancer group and the NLRP3−/− control group (P < 0.05). The knockout of the NLRP3 gene caused changes in the lung microbiota of mice. There may be a regulatory relationship between the NLRP3 inflammasome and the lung microbiota, which affects the occurrence and development of lung cancer.

Lack of SIRP-alpha reduces lung cancer growth in mice by promoting anti-tumour ability of macrophages and neutrophils.

Signal regulatory protein-alpha (SIRPα) is a transmembrane glycoprotein specifically expressed on myeloid cells. Blockade of SIRPα/CD47 interaction is effective in combinational therapy of some cancers. This study aimed to explore into the role and underlying molecular mechanisms of SIRPα in lung cancer growth. A mouse model with lung cancer in wild-type (WT) and SIRPα-knockout mouse (KO) mice was established by subcutaneous injection of Lewis murine lung cancer cells (LLC). Circulating monocytes and neutrophils were depleted in mice by intraperitoneal administration of clodronate liposomes and anti-Ly6G antibody, respectively. Phenotypes and phagocytosis of macrophages and neutrophils were analysed by flow cytometry. Transwell assay was used to analyse LLC cells migration and invasion. Lack of SIRPα inhibited LLC cells growth in KO mice, associated with reduced infiltrating PD-1+ CD8+ T cells and production of IL-6 from infiltrating macrophages and neutrophils in tumour tissues. Depletion of circulating monocytes and neutrophils reduced LLC cells growth in WT mice, which was abolished in KO mice. Studies in vitro showed that lack of SIRPα increased M1/M2 ratio, and reduced LLC cell migration and invasion via attenuated IL-6 secretion. Lack of SIRPα expression in neutrophils effectively increased the cytotoxic activity to LLC cells in vitro. Lack of SIRPα suppressed lung cancer cell growth in mice, dependent on circulating macrophages and neutrophils, in association with improved phagocytosis and reduced IL-6 expression.

Anlotinib Enhances the Antitumor Activity of High-Dose Irradiation Combined with Anti-PD-L1 by Potentiating the Tumor Immune Microenvironment in Murine Lung Cancer

<h3>Purpose/Objective(s)</h3> Radioimmunotherapy has become one of the most promising strategies for cancer treatment. Preclinical and clinical studies have demonstrated that antiangiogenic therapy can improve the efficacy of immunotherapy and sensitize radiotherapy through a variety of mechanisms. However, it is undefined whether angiogenesis inhibitors can enhance the effect of radioimmunotherapy. In this study, we aim to explore the role of anlotinib (AL3818) on the combination of radiotherapy and immune checkpoint inhibitors in Lewis lung carcinoma mouse. <h3>Materials/Methods</h3> C57BL/6 mouse subcutaneous tumor model was used to evaluate the ability of different treatment regimens in tumor growth control. Immune response and immunophenotyping including the quantification and activation were determined by flow cytometry, multiplex immunofluorescence and multiplex immunoassay. <h3>Results</h3> Triple therapy (radiotherapy combined with anti-PD-L1 and anlotinib) increased tumor-infiltrating lymphocytes and reversed the immunosuppressive effect of radiation on the tumor microenvironment in mouse model. Compared with radioimmunotherapy, the addition of anlotinib also boosted the infiltration of CD8⁺ T cells and M1 cells, and caused a decrease in the number of MDSCs and M2 cells in mice. The levels of IFN-gamma, IL-18 were the highest in the triple therapy group, while the levels of IL-23, IL-13, IL-1 beta, IL-2, IL-6, IL-10 and Arg-1 were significantly reduced. NF-κB, MAPK and AKT pathways were down regulated in triple therapy compared with radioimmunotherapy. Thus, the tumor immune microenvironment was significantly improved. As a consequence, triple therapy displayed greater benefit in antitumor efficacy. <h3>Conclusion</h3> Our findings indicate that anlotinib might be a potential synergistic treatment for radioimmunotherapy to achieve better antitumor efficacy in NSCLC patients by potentiating the tumor immune microenvironment.

OR11-2 RNA Sequencing Reveals Unique Transcriptomic Signatures of the Thyroid in a Murine Lung Cancer Model Treated with PD-1 and PD-L1 Antibodies

Abstract Immune checkpoint inhibitors (ICI) have revolutionized cancer therapy, however, are associated with immune-related adverse events (irAEs). Thyroid dysfunction is among the most common irAEs, yet its mechanism has not been fully elucidated. To further explore the molecular mechanism of ICI-induced thyroid dysfunction, we profiled changes in transcription induced by PD-1/PD-L1 antibody treatment in a non-small cell lung cancer murine model using RNA-seq. We used bioinformatic tools to compare transcriptional changes in our model to previously published transcription data sets of drug-induced thyroiditis. C57BL6/129SvJ mice harboring LKR-M (Lung cancer K-ras metastatic tumor) flank tumors were treated with PD-1 antibody, PD-L1 antibody, or vehicle every 3 days over a period of 7 days when mice were sacrificed, and thyroid glands were removed. The tumor volume increased in the control mice (1.38 ± 0.19) and decreased in the anti PD-1 treated (0.64 ± 0.15; P=0.0001) and PD-L1 treated mice (0.74 ± 0.11; P=0.004). RNA-seq was performed in the LKR-M tumor-bearing control mice and in the anti PD-1/PD-L1 treated mice (n=4 per group). A total of 952 differentially expressed genes (DEGs), including 265 upregulated and 688 downregulated, were identified with anti PD-1 treatment (fold-change ≥1.8, FDR ≤0.05). Only 35 genes were differentially expressed with anti PD-L1 treatment, and we therefore chose to focus on the anti PD-1 treated group alone. Using Ingenuity Pathway Analysis (IPA), we determined that of the DEGs in the PD-1 treatment group, 362 were associated with functions of cell death and survival, with a predicated activation of pathways for apoptosis and necrosis (Z=2.89 and 3.21, respectively) and negative activation of pathways for cell viability and cell survival (Z=-6.22 and -6.45, respectively). When compared to previously published datasets of interleukin-1β and interferon gamma-treated human thyroid cells, the apoptosis pathways were similarly activated. To our knowledge this is the first study to evaluate transcriptomic changes in thyroid tissue following ICI therapy. Our data suggest that there are unique changes in gene expression in the thyroid associated with anti PD-1 therapy. ICI-induced thyroid dysfunction may be mediated by increased tissue apoptosis resulting in destructive thyroiditis. Presentation: Sunday, June 12, 2022 11:15 a.m. - 11:30 a.m.

Famitinib enhances the antitumor effect of radioimmunotherapy in murine lung cancer.

Combining antiangiogenic therapy with radioimmunotherapy is believed to further improve antitumor efficacy, but there is still a lack of evidence to support this. This study aimed to investigate the role of the tumor vascular-targeted agent famitinib with a combination of radiotherapy and an immune checkpoint inhibitor in murine lung cancer. The effect of VEGFA and HIF1A on clinical prognosis and the tumor immune microenvironment was analyzed using public databases. Enrichment analyses of post-irradiation gene expression and mRNAs related to immunotherapy efficacy were carried out based on GEO datasets. A C57BL/6 mouse subcutaneous tumor model was used to evaluate the antitumor effects of different treatment schemes. The tumor immunophenotyping was identified by flow cytometry. We demonstrated that high level of VEGFA and HIF1A expression in lung cancer was related to poor prognosis and immunosuppressive tumor microenvironment. In a mouse model, the triple therapy of famitinib, radiotherapy and immunotherapy had the most dramatic antitumor activity. It significantly increased tumor infiltrating lymphocytes and reversed the immunosuppressive state of the tumor microenvironment in mice. Compared with radioimmunotherapy, the addition of famitinib further promoted the infiltration of CD8+ T cells and M1 type tumor associated macrophages, and reduced the number of myeloid suppressor cells. Therefore, triple therapy converted the immunosuppressive tumor microenvironment into an immunostimulatory one. Famitinib can synergize with radioimmunotherapy by regulating the tumor immune microenvironment in murine lung cancer.

Evaluating Therapeutic Effects of In Vivo 15MV Radiodynamic Therapy (RDT) with 5-Aminolevulinic Acid (5-ALA) Using 1.5T MRI and [18F]FDG PET/CT

<h3>Purpose/Objective(s)</h3> A combination of photosensitizer and high-energy photon beam is required to perform radiodynamic therapy (RDT) for tumor treatment. High-energy photon beams activate the photosensitizer, leading to cellular toxicity by producing extra reactive oxygen species to kill malignant cells. The purpose of this study was to investigate the therapeutic effect of RDT with 15 MV photon beams combined with 5-aminolevulinic acid (5-ALA) using a mouse model. <h3>Materials/Methods</h3> All animal studies were approved by our Institutional Animal Care and Use Committee. A subcutaneous murine KP1 small-cell lung cancer model using C57BL/6 mice was used. The tumors (n=126) were randomly assigned into four groups, including control (untreated), radiation treatment (RT) only, 5-ALA only, and RDT to observe individual and synergistic effects of 5-ALA and radiation treatment. Treatment was performed when the tumor reached 5-8 mm in diameter. A single fraction of 4 Gy was delivered to the tumors using 15 MV photons on a clinical linear accelerator for RT only and RDT groups. 5-ALA was injected at 100 mg/kg by tail-vein 4 hours prior to RT for 5-ALA only and RDT groups. Tumor volumes were measured using a 1.5 T MR scanner on the day of treatment and weekly post-treatment. Tumor growth for each tumor was quantified as the ratio of tumor volume one- and two-week post-treatment to the tumor volume on the treatment day. SUV_max and total lesion glycolysis (TLG) were measured using [¹⁸F]FDG PET/CT for control and RDT groups. A 10 min static scan was acquired an hour after an injection of 80 µCi [¹⁸F]FDG. SUV_max was calculated as the maximum measured activity concentration divided by the injected whole bodyweight activity concentration. TLG was quantified as a product of SUV_mean and the metabolic tumor volume. <h3>Results</h3> The tumor growth in the RDT group was delayed by 48.3±11.3%, 46.3±9.2%, and 54.6±12.7% at one-week post-treatment compared to 5-ALA only, RT only, and the control group (P<0.001), respectively. There were no significant differences in tumor growth between 5-ALA only, RT only, and the control group. Moreover, about a 40% additional decrease in tumor growth was observed, showing a synergistic effect of RDT. SUV_max and TLG showed 47.4±8.8% and 66.5±5.3% decrease at one-week post-treatment, respectively. Also, a higher number of distant metastases were observed using [¹⁸F]FDG PET/CT in the control group than in the RDT group. <h3>Conclusion</h3> Assessment of radiodynamic therapy combined with 5-ALA administration and 15 MV high-energy radiation treatment showed improved treatment outcomes compared to the control, RT only, and 5-ALA only groups. Tumor growth delay and tumor activity suppression were significantly improved. Moreover, a synergistic effect of 5-ALA and 15 MV photon beams was observed. The preliminary (hypothesis-generating) results advocate a further investigation into larger sample sizes.

IL-9 stimulates an anti-tumor immune response and facilitates immune checkpoint blockade in the CMT167 mouse model

ObjectivesThere is mounting evidence that interleukin-9 (IL-9) is associated with various cancers although its function in lung cancer remains elusive. This study aimed to elucidate the role(s) of IL-9 in lung cancer and the mechanisms involved. Materials and methodsExpression of IL-9 receptor (IL-9R) in two murine lung cancer cell lines: CMT167 and Lewis lung carcinoma (LLC) were assessed and syngeneic murine lung cancer models were established. Tumor growth, intratumoral immune responses and downstream signaling pathways in tumor-bearing mice were analyzed upon IL-9 treatment. Human lung cancer cell lines A549 and H1975 were included for in vitro validation. Synergistic effects and immune responses of IL-9 in combination with anti-PD-1 were studied. ResultsIL-9R expression was only detected in CMT167 but not LLC cells. IL-9 suppressed CMT167 tumor growth and enhanced anti-tumor T cell responses, both of which were absent in IL-9R-deficient LLC model and lost upon IL-9R knockdown in CMT167 model. In CMT167 tumors, while IL-9 increased CD4+ and CD8+ T cells and dendritic cells, the cytotoxic T subset was the key driver of IL-9-induced tumor suppression. Consistently, in CMT167 and A549 cells, IL-9/IL-9R signaling promoted MHC class I upregulation. Inhibition of ERK signaling abolished IL-9-mediated MHC class I upregulation in CMT167 cells. IL-9 induced expression of PD-1 and PD-L1 on CD8+ T lymphocytes and CMT167 cells respectively. Combined IL-9 treatment with PD-1 blockade further upregulated tumor-infiltrating CD8+ T cell frequencies and synergistically suppressed tumor growth in CMT167 model. ConclusionIL-9 suppresses tumor growth by promoting tumor-derived MHC class I presentation and enhancing cytotoxic T cell immunity. Expression of IL-9R might be used as a biomarker for identification of potential target population susceptible to IL-9 treatment. Our study proposes IL-9 as a promising therapeutic immunomodulatory agent that can be used in combination with PD-1 blockade in lung cancer.

Anti-PD-1 antibody-activated Th17 cells subvert re-invigoration of antitumor cytotoxic T-lymphocytes via myeloid cell-derived COX-2/PGE2.

Anti-PD-1 antibody-mediated activation of type 17T-cells undermines checkpoint inhibitor therapy in the LSL-KrasG12D murine lung cancer model. Herein, we establish that the Th17 subset is the primary driver of resistance to therapy demonstrate that the ontogeny of dysplasia-associated Th17 cells is driven by microbiota-conditioned macrophages; and identify the IL-17-COX-2-PGE2 axis as the mediator of CD8+ cytotoxic T-lymphocyte de-sensitization to checkpoint inhibitor therapy. Specifically, anti-PD-1 treatment of LSL-KrasG12D mice, in which CD4+ T-cells were deficient for RORc, resulted in a 60% increase in CTL cytotoxicity and a 2.5-fold reduction in tumor burden confirming the critical role of Th17 cells in resistance to therapy. Lung-specific depletion of microbiota reduced Th17 cell prevalence and tumor burden by 5- and 2.5-fold, respectively; establishing a link between microbiota and Th17 cell-driven tumorigenesis. Importantly, lung macrophages from microbiota sufficient, but not from microbiota-deficient, mice polarized naïve CD4+ T-cells to a Th17 phenotype, highlighting their role in bridging microbiota and Th17 immunity. Further, treatment with anti-PD-1 enhanced COX-2 and PGE2 levels, whereas neutralization of IL-17 diminished this effect. In contrast, inhibition of COX-2 rescued CTL activity and restored tumor suppression in anti-PD-1-treated mice, revealing the molecular basis of IL-17-mediated resistance to checkpoint blockade. Clinical implications of these findings are discussed.

EP16.01-028 Immunomodulatory Effects of Cryoablation Combined With Immune Checkpoint Inhibitors in a Murine Lung Cancer Model

As part of our research on cryoablation, a combination with immune checkpoint inhibitors (ICIs), such as inhibitors of the programmed cell death 1 (PD-1) receptor and its ligand, programmed cell death 1-ligand 1 (PD-L1), to disrupt the immunosuppressive tumor microenvironment has proven a beneficial effect on enhancing immune response. We specifically addressed myeloid-derived suppressor cells (MDSCs) and T-cell immunity, which are thought to play an important role in the formation of the tumor microenvironment.

EP16.01-024 Differential Efficacy of Combined GITR Co-stimulation with PD-1 Blockade in Preclinical Non-small Cell Lung Cancer Models

Lung cancer remains as the most lethal cancer disease in Hong Kong. Although blockade of inhibitory immune checkpoints achieved remarkable clinical outcomes, continuous efforts are being made to explore novel therapeutic strategies. We have previously found that anti-programmed cell death protein 1 (anti-PD-1) induced systemic and intra-tumoral upregulation of an immunoregulatory molecule, glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) in a subcutaneous murine lung cancer model.

Abstract 361: MicroRNA expression in murine spontaneous non-small cell lung cancer and human cell lines post-treatment of PRIMA-1

Abstract A variety of cancers have been correlated with having a mutation on the TP53 gene, including lung cancer. Lung cancer has been one of the deadliest cancers in the United States. TP53-dependent reactivation and induction of massive apoptosis (PRIMA-1) changes mutant TP53’s conformation, resulting in the restoration of DNA binding and activation of p53 target genes. PRIMA-1 methylated analog, PRIMA-1Met (APR-246, Eprenetapopt) have been used in clinical trials to target cancers containing mutant TP53. It has been reported that PRIMA-1 induced apoptosis through the regulation of microRNAs. Our research group has previously developed a transgenic mice model with a mutant form of TP53 being expressed lung specifically, to closely model human lung cancer development. The aim of the current study is to investigate the effects of PRIMA-1 on miRNA expressions in a murine spontaneous lung cancer mice model, human lung cancer cell lines H211 and H1155. The transgenic FVB/N mice lung tumor model was developed using the lung specific human surfactant protein C (SPC) promoter to control the expression of mutant TP53(273H) following the standard injection method. Tumor bearing mice were identified with a micro-CT and were treated with the PRIMA-1 at a dose of 100mg/kg. Additionally, we treated two human cell lines H211 (containing mutant TP53 R248Q) and H1155 (containing mutant TP53 R273H) with the PRIMA-1. The miRNA expression profile was evaluated with the NanoString nCounter array analysis and qt-PCR array analysis. We found that three miRNAs were up-regulated (&amp;gt;1.5-fold change) in the human lung cancer cells H211, H1155, and murine spontaneous non-small cell lung cancers model post treatment of PRIMA-1. The result of the study showed that miR-200c, miR-181c, and miR-183 was up-regulated in the two human cell lines and the transgenic mice lung tumors after treatment of PRIMA-1. While there was no significant downregulation in miRNA expression in these human lung cancer cells and the murine spontaneous lung cancer model. However, the overall miRNA expression is lower in murine spontaneous lung tumors when compared to the overall miRNA expression of the cell lines post PRIMA-1 treatment. This difference is due to model specific expression. Further investigation is needed to better understand the effects of PRIMA-1 in regulation of micro-RNA expression in the treatment of lung cancers containing mutant p53. Citation Format: Melanie Perez, Shaylyn Grier, Li Gao, Wenrui Duan. MicroRNA expression in murine spontaneous non-small cell lung cancer and human cell lines post-treatment of PRIMA-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 361.

Abstract 874: MSI2 regulates VEGFR2 signaling and tumor progression in NSCLC

Abstract Lung cancer holds second place in incidence and first place in mortality among all cancers, both in men and women, with metastasis contributing to the vast majority of deaths. Recently, expression of vascular endothelial growth factor receptor-2 (VEGFR2) on both endothelial and tumor cells was demonstrated as one of the key mechanisms contributing to growth of non-small cell lung cancer (NSCLC), the most common subtype of lung cancer. VEGFR2 dependent signaling is critical both for tumor autocrine functions related to NSCLC migration, and in paracrine control of tumor angiogenesis through action on tumor-proximal endothelial cells, suggesting a potential mechanism for growth control. Several drugs targeting VEGFR2 signaling pathway demonstrated their clinical efficacy in NSCLC setting, including VEGFR2 and VEGF-A inhibitors, VEGF-A/PIGF trap. Recently our group demonstrated that RNA-binding protein Musashi-2 (MSI2) is driving progression and metastasis of NSCLC. MSI2 regulates mRNA translation of multiple targets, its expression is upregulated in the metastasis-competent murine and human lung cancer cell lines and is progressively elevated in lung cancer samples. The goal of this study is to define the role of MSI2 in sustaining VEGFR2 signaling during NSCLC progression. Using reverse RNA immunoprecipitation (RIP) qPCR analysis, we demonstrate that MSI2 directly binds VEGFR2 mRNA. Reverse protein phase array (RPPA) of several NSCLC cell line models with expression or knockdown of MSI2 and subsequent direct validation showed that MSI2 strongly and positively regulates expression of VEGFR2. Subsequent mechanistical analysis revealed that MSI2 knockdown leads to decreased VEGFR2 protein and mRNA levels expression in several NSCLC cell lines in vitro, resulting in increased apoptosis via inhibition of VEGFR2 signaling pathway. To better characterize MSI2 biology, we’ve crossed the B6/129S4-Msi2tm1.1Cjl/J (M2) mice with B6/129S/Sv-Krastm3Tyj/J;Trp53tm1Brn/J (KP) mice and generated novel B6/129S/Sv-KP;Msi2-/- (KPM2) mouse model. Induction of tumorigenesis in KP mice using Cre adenovirus leads to development of lung adenocarcinoma. Induction of lung-specific Cre resulted in tumorigenesis in both KP and KPM2 mice. Interestingly, we observed significant decrease in both total lung tumor number and total lung tumor burden in KPM2 compared to KP mice. Next, we established three novel KP and KPM2 derived cell lines from mouse lung tumors and found that KPM2 cell lines demonstrate reduced proliferation capacity and VEGFR2 mRNA level relative to KP cell lines. Additional studies to understand an impact of MSI2 deletion in KP cell lines are ongoing. Taken together, MSI2 is a promising target for NSCLC treatment, as this protein regulates VEGFR2 signaling and apoptosis in cell models and contributes to KP tumorigenesis in vivo. Citation Format: Igor Bychkov, Alexander Deneka, Iuliia Topchu, Petr Makhov, Alexander Kudinov, Anna Nikonova, John Karanicolas, Erica Golemis, Christopher Lengner, Hossein Borghaei, Jyoti Patel, Yanis Boumber. MSI2 regulates VEGFR2 signaling and tumor progression in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 874.

Abstract 3286: Development and characterization of an oncolytic adenovirus armed with the immune checkpoint stimulator SA-4-1BBL

Abstract Introduction: 4-1BBL is a co-stimulatory molecule that is particularly important for the expansion of T cells and induction of robust CD8+ T-effector and memory responses, critical for tumor rejection. The natural 4-1BBL is a trimeric transmembrane protein that has no activity in soluble form. To overcome this limitation, a unique soluble form of 4-1BBL was developed by linking core streptavidin (SA) to the extracellular domain of murine 4-1BBL (SA-4-1BBL). This chimeric protein forms oligomers, and in soluble form, has pleiotropic effects on cells of innate, adaptive, and regulatory immunity. However, production and purification of SA-4-1BBL is cost- and time-intensive. Therefore, development of an expression platform that is more efficient, translational, and augments anti-tumor efficacy of SA-4-1BBL will have a significant impact for clinical development of this immune adjuvant. Thus, we developed a novel prototype oncolytic adenoviral system to deliver SA-4-1BBL (OAdSA-4-1BBL) to the solid tumors. Methods: The AdenoQuick 2.0 system was used to construct two OAds, one expressing SA-4-1BBL and the other expressing SA (as control virus). The expression of transgenes was confirmed by western blot and the SA-4-1BBL biological function was evaluated by splenocyte proliferation assay in vitro. The OAdSA-4-1BBL and OAdSA-mediated cancer killing effect was evaluated by crystal violet staining, the activation of autophagy-mediate immunogenic cell death was assessed by detection of autophagy markers (e.g., LC3-I and II as well as p62), the damage-associated molecular patterns (DAMPs) molecules such as adenosine triphosphate (ATP), high mobility group box 1 protein (HMGB1), and calreticulin were evaluated by luminescence, ELISA, and flow cytometry, respectively. Expression of SA, SA-4-1BBL, and Hexon within the tumors were evaluated by immunohistochemistry. The OAdSA-4-1BBL therapeutic efficacy was evaluated in a C57BL/6 mice bearing subcutaneous TC-1 lung tumor. Results: We successfully constructed two OAds expressing SA or SA-4-1BBL, we demonstrated that SA-4-1BBL can produce biologically active oligomers, selectively induces autophagy-mediated immunogenic cell death in murine and human lung cancer cell lines with little cytotoxicity in normal cell lines. The transgene expressions within the subcutaneous tumor were confirmed and OAdSA-4-1BBL shows greater anti-tumor efficacy than control viruses in a TC-1 tumor mouse model. Conclusions: Overall, our data suggests that OAd expressing the immune checkpoint stimulator SA-4-1BBL may represent alternative therapeutic strategy against lung cancer, which could be used alone or in combination with immune checkpoint inhibitors, or other approaches aimed at enhancing the anti-tumor immune response. Citation Format: Jorge G. Gómez-Gutiérrez, Martin Ramos-Gonzalez, Rodolfo Garza-Morales, Mohammad Tarique, Feyza Arguc, Lalit Batra, Haval Shirwan, Esma Yolcu. Development and characterization of an oncolytic adenovirus armed with the immune checkpoint stimulator SA-4-1BBL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3286.

Abstract 3136: Molecular characterization of the effects of cancer-derived exosomes on murine lung cancer tumors

Abstract Lung cancer is the second most diagnosed type of cancer and is the leading cause of cancer-related deaths in both men and women. An estimated 235,760 new cases of lung cancer and 131,880 lung cancer deaths are expected this year accounting for 12% of new cancer cases and 22% of cancer deaths in the United States. Exosomes are secreted vesicles that contain selectively packaged biomaterials (protein, lipids, and RNA) and serve as a form of extracellular communication between cells in the tumor microenvironment. Previous studies have shown the effects of cancer-derived exosomes on specific cell types or gross tumor response in vivo yet, the competitive nature and dynamics of exosomal uptake in the tumor microenvironment remains largely unknown. This work seeks to identify molecular responses induced by cancer-derived exosomes on cells residing in the tumor microenvironment. In order to control tumor cell exposure to cancer-derived exosomes, heterogeneous ex vivo cultures derived from subcutaneous Lewis lung carcinoma (LLC) tumors in C57BL6 mice were exposed to fluorescently dyed LLC exosomes for up to 72 hours. Flow cytometric analysis was performed to identify preferential uptake of cancer exosomes into the stromal subpopulations of tumor cells. Increased uptake was observed throughout the duration of the exposure and preferential uptake was observed into neutrophils, macrophages, and endothelial cells while cancer cells, fibroblasts, and lymphocytes were underrepresented in the exosome uptaken population. Additionally, single cell RNA-sequencing was performed 24 hours post-exosomal exposure to identify transcriptional changes promoted by cancer exosome uptake. We found tumor-derived cancer cells to up-regulate transcripts associated with proliferation and down-regulated genes associated with interferon signaling in response to exosome uptake. These preliminary findings suggest that cancer-derived exosomes have targeted effects on specific cell types in the tumor microenvironment. Specifically in cancer cells, exosomal uptake can elicit protumor (cancer proliferation and immune suppression via decreased interferon signaling) effects that may serve as mediators of disease progression. An understanding of both cancer and host-derived exosomal response to disease progression may identify novel biomarkers and potential therapeutic targets previously undiscovered using canonical discovery methods. This study received funding from LLNL LDRD grant 21-LW-028. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). LLNL-ABS-829271 Citation Format: Nicholas R. Hum, Nicole F. Leon, Aimy Sebastian, Kelly A. Martin, Gabriela G. Loots. Molecular characterization of the effects of cancer-derived exosomes on murine lung cancer tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3136.

Abstract 1980: Cigarette smoke induced oxidative phosphorylation and mitochondrial biogenesis enhance response to immune therapy in NSCLC patients

Abstract Background: The treatment regimen of non-small cell lung cancer (NSCLC) has drastically changed owing to the superior anti-cancer effects generated by the immune-checkpoint blockade (ICB). However, only a subset of patients experience benefit after receiving ICBs. There are several treatments including combination of PD-1 blockade and low dose chemotherapy, but only few robust synergetic effects were reported. Therefore, it is of paramount importance to increase the response rate by elucidating the underlying molecular mechanisms and identifying novel therapeutic targets to enhance the efficacy of IBCs in non-responders. Methods: We analyzed the progression-free survival (PFS) and overall survival (OS) of 295 NSCLC patients who received anti-PD-1 therapy by segregating them with multiple clinical factors including sex, age, race, smoking history, BMI, tumor grade and subtype. Tumor samples from 48 patients were sequenced and their genomic profiles were extracted for analyses. We identified key signaling pathways and mutations that are enriched in those patients with distinct responses to ICB by gene set enrichment analysis (GSEA) and mutational analyses. The efficacy of combination treatment with mitochondrial activator, all-trans retinoic acid (ATRA) and PD-1 antibody is evaluated in LL/2 and CMT167 murine lung cancer model. Results: We found that smoking status was the only parameter that was correlated with the ICB responses compared to sex, age, race, BMI, tumor grade and subtype. Former and current smokers have a higher response rate to anti-PD-1 treatment than non-smokers. GSEA results revealed that oxidative phosphorylation (OXPHOS) and mitochondrial related pathways are significantly enriched in both responders and smokers, suggesting a potential role of cellular metabolism in regulating immune response to ICB. We also demonstrated that ATRA which increased mitochondrial function significantly by MitoSox and MitoTrack enhanced the efficacy of anti-PD-1 treatment in vivo. Our clinical and bioinformatics based analyses revealed a connection between smoking induced metabolic switch and the response to immunotherapy, which can be the basis for developing novel combination therapies that are beneficial to never smoked NSCLC patients. Conclusion: In this study, we discovered that smokers respond better to immune therapy than non-smokers due to elevated OXPHOS capacity and mitochondrial activity, which could be a potential target to boost the response to immune therapy in NSCLC patients who never smoked. Citation Format: Yuezhu Wang, Jimmy Ruiz, Yin Liu, Margaret Smith, Gregory Kucera, Umit Topaloglu, Michael Chan, Stacey O’Neill, Jing Su, Fei Xing. Cigarette smoke induced oxidative phosphorylation and mitochondrial biogenesis enhance response to immune therapy in NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1980.

Abstract 1561: Lnc-NR2F1 upregulated by a ZEB1/NR2F1 axis promotes the migration and invasion of lung cancer cells via TWIST2 regulation

Abstract Long non-coding RNAs (lncRNAs) are RNA transcripts longer than 200 nucleotides lacking protein-coding potential, which are involved in diverse pathophysiologic conditions including cancer progression and metastasis. Metastasis is initiated by a cellular process, epithelial-to-mesenchymal transition (EMT), and many lncRNAs can be affected by and participate in the EMT process. To find out lncRNAs regulated by the EMT signals, we performed an NGS-based RNA sequencing in murine lung cancer cells, and finally selected lnc-NR2F1 that is upregulated by ZEB1, an EMT-inducing transcription factor. lnc-NR2F1 expression levels showed a positive correlation with ZEB1 mRNA levels and EMT scores in 13 mouse lung cancer cells or in lung adenocarcinoma data from TCGA. The migration and invasion of lung cancer cells were decreased after lnc-NR2F1 knockdown and increased after lnc-NR2F1 overexpression. lnc-NR2F1 was transcriptionally up-regulated by NR2F1, a transcription factor transcribed from the antisense strand. Interestingly, NR2F1 was transcriptionally induced by ZEB1, and promoted the migration and invasion of lung cancer cells. Through the analysis of public ChIRP-seq data, we found that lnc-NR2F1 can modulate the expression of TWIST2, and add-back of TWIST2 restored the migration and invasion of lung cancer cells suppressed by lnc-NR2F1 knockdown. In conclusion, lnc-NR2F1 is induced by a ZEB1-NR2F1 axis and stimulates the migration and invasion of lung cancer cells via transcriptional regulation of TWIST2. Citation Format: Eun Ju Kim, Young-Ho Ahn. Lnc-NR2F1 upregulated by a ZEB1/NR2F1 axis promotes the migration and invasion of lung cancer cells via TWIST2 regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1561.

Abstract 5944: Euglena water extract attenuates lung tumorigenesis induced by tobacco-specific carcinogen through modulation of gut microbiota metabolites

Abstract Lung cancer is the leading cause of cancer-related mortality in the world. Our previous studies have shown that the oral administration of Euglena water extract significantly inhibits the growth of grafted lung carcinoma tumors in mice. Euglena gracilis, a single-celled alga used as a nutritional dietary supplement, possesses a broad range of medicinal properties including anticancer activity against a few types of cancers. Most studies describing this anticancer activity have used xenograft cancer mouse models, but not carcinogen-induced cancer models. Since tobacco smoke carcinogen-induced lung carcinoma in mice mimics human lung cancer development, in this study, we investigated the anti-cancer properties and the underlying mechanism of the E. gracilis water extract against lung tumorigenesis induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). A/J mice (n=10 per treatment group) were used and the treatment regimen consisted of: (i) PBS control, (ii) Euglena water extract 2 weeks before NNK injection (pre-NNK) and (iii) this extract 10 weeks after NNK injection (post-NNK). We examined the metabolite landscape of the gut microbiota by a high-throughput metabolomics approach. The Euglena water extract treatment greatly attenuated NNK-induced tumorigenesis in lungs of A/J mice. A Partial Least-Squares Discriminant Analysis demonstrated a separation of the fecal metabolites between the control and treatment groups. Using the untargeted metabolomics, we identified 20 potential metabolites that were differentially expressed in the Euglena extract (pre- and post-NNK) treatment groups as compared to the control group. Specifically, succinate, malate, triethanolamine, acetylserine were increased with the Euglena water extract treatment in comparison to the control. These metabolites are involved in the biosynthesis of short-chain fatty acids (SCFA). Furthermore, using the targeted analysis of SCFA, we observed a significant increase in acetic acid in the feces of mice treated with Euglena water extract both before and after NNK exposure; however, butyric and propionic acids were significantly higher in only post-treated mice. Moreover, in vitro treatment of both human and murine lung cancer cells with SCFA significantly suppressed their proliferation. The present study indicated that treating with the Euglena water extract both pre- and post-NNK exposure greatly inhibits NNK-induced lung tumorigenesis in mice even after early tumorigenesis has been established. This study also found that Euglena water extract increases the levels of SCFA and their precursors in the feces of the treated mice indicating a role of gut microbiota in suppressing the NNK-induced tumorigenesis. This study was supported by 2017 EUGLENA-RC2 (MT and JC), Kansas State University Johnson Cancer Research Center (MT and JC), and NIH grant P20 GM103418 (MT). Citation Format: Deepa Upreti, Susumu Ishiguro, Ayaka Nakashima, Kengo Suzuki, Jeffrey Comer, Masaaki Tamura. Euglena water extract attenuates lung tumorigenesis induced by tobacco-specific carcinogen through modulation of gut microbiota metabolites [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5944.

OX40 enhances T cell immune response to PD-1 blockade therapy in non-small cell lung cancer

Immune-checkpoint blockade is widely studied for cancer therapy. Although the co-inhibitory receptor Programmed death-1(PD-1) blockade benefits some non-small cell lung cancer (NSCLC) patients, a large portion of NSCLC patients still fail to respond to this immunotherapy, and the underlying mechanism is unclear. Thus, a synergistic therapy to enhance the effect of PD-1 is urgently needed to improve the poor outcome of NSCLC patients. Here, we demonstrated that effector memory T cells were increased and T cell response became stronger in PD-1 immunotherapy responders (n = 20) but not in non-responders (n = 10). The expression of co-stimulatory receptor OX40 was upregulated on T cells following PD-1 immunotherapy and was positively associated with the percentage of PD-1+T cells and the responsiveness of T cells. Combination treatment of antagonistic anti-PD-1 and agonistic anti-OX40 antibodies (Abs) promoted the proliferation and cytokines production of T cells from PBMCs of non-responders ex vivo. Consistently, anti-PD-1 and anti-OX40 therapy synergistically augmented T cell response in an in vivo mouse lung cancer model. Our study confirmed the antitumor effects of anti-PD-1/OX40 combination in lung cancer patients as well as in the murine lung cancer model, and the results provide a rationale for clinical trials evaluating the therapeutic effect of this combination of antibodies for NSCLC immunotherapy.

The TLR7/8 agonist R848 optimizes host and tumor immunity to improve therapeutic efficacy in murine lung cancer.

Treatment with the Toll-like receptor 7 (TLR7) agonist, resiquimod (R848), is effective in various types of cancer, such as breast, pancreatic and colorectal cancer. The reported antitumor effect of R848 in lung cancer is considered to be achieved by targeting macrophages. In the present study, it was demonstrated that TLR7 expression on various immune cell types initially rises, then declines in the late stage of lung cancer. Intraperitoneal injection of R848 resulted in a reduction in tumor burden and prolonged survival in both subcutaneous and metastatic lung cancer models in C57BL/6 mice. Initial treatment with R848 at an early stage was found to be the optimal choice. Systemic injection of R848 promoted the activation of innate and adaptive immune responses. Systemic administration of R848 upregulated TLR7 expression in dendritic cells (DCs) and enhanced the activation of DCs and natural killer (NK) cells. Moreover, this treatment also resulted in increased production of T helper cell-associated cytokines in serum, including IFN-γ, TNF-α and IL-2. In addition, continuous treatment with R848 increased the proportion of DCs, NK and CD8+ T cells, and reduced that of Foxp3+ regulatory T cells in the tumor microenvironment. These findings supported the use of R848 treatment for lung cancer via TLR7 targeting and provided insight into the underlying therapeutic mechanism.

Tumor-associated Tregs obstruct antitumor immunity by promoting T cell dysfunction and restricting clonal diversity in tumor-infiltrating CD8+ T cells

BackgroundAccumulation of regulatory T cells (Treg) has been described to often correlate with poor prognosis in many solid tumors. How Treg presence impinges on limited functionality and clonal composition of...