Murine Lung Tumors Research Articles

An easy-to-perform protocol for culturing primary murine lung tumor cells as organoids

Cancer research involves significant animal consumption and suffering. Tumor cells can be differentiated in vitro into three-dimensional organoids that resemble the primary tumor. In basic cancer research, however, tumor organoids are usually only used alongside animal experiments. We have established an easy-to-perform protocol that allows to culture KRAS-driven lung tumor cells as organoids for extended periods of time. Like the corresponding tumors in mice, the organoids produce surfactant protein C but no markers of airway epithelial cells (e.g. SCGB1A1, KRT5). The organoids can be passaged as single cell suspensions. Our organoid model contributes to replace animal experiments with cell culture systems and can be used for drug testing or functional studies in cancer research.

Transforming tumor immune microenvironments with a novel systemic enveloped oncolytic virotherapy targeting all tumor sites.

2559 Background: Systemic oncolytic virotherapy offers a promising solution for treating both local and metastatic diseases. However, the rapid inactivation of virotherapeutics by the immune system has resulted in disappointing clinical efficacy. To address this challenge, we have built a new program (ImmunoNova) to develop a cellular-based technology that protects oncolytic virotherapy, allowing for successful targeting of the therapy to tumor sites and effectively overcoming clinical challenges. This approach involves utilizing a newly selected and engineered, tumor-selective strain of vaccinia virus (RT-01). This strain produces high levels of extracellular enveloped virions (EEVs) that contain a second human cell-derived membrane, providing augmented protection against elimination by the immune system when administered systemically. The process requires specific manufacturing methods to preserve this crucial second human cellular membrane. Methods: The resistance of the manufactured enveloped RT-01 (envRT-01) virus against human humoral immunity and its rapid spread were assessed ex-vivo using a plaque assay. EnvRT-01 was administered in various xenograft and syngeneic models to evaluate its specificity in targeting tumors and its therapeutic efficacy, either alone or in combination with cell therapies. The amplification of virus-encoded RFP was monitored using an imaging system. Additionally, flow cytometry and immunohistochemistry (IHC) were employed to analyze immune infiltration in both treated and untreated tumors. Results: EnvRT-01 particles exhibited an approximately 80% survival rate in the presence of active complement. In animal studies, a single systemic dose of envRT-01 selectively targeted three distinct human cancer indications (lung, melanoma, head & neck), leading to the suppression of tumor growth in all three cases. Similarly, in an immunocompetent syngeneic lung tumor model, envRT-01 effectively targeted and reduced multiple murine lung tumors with just a low single dose of treatment. EnvRT-01 was capable of targeting and expressing viral-encoded proteins in all tumor sites and drastically modifying the tumor immune microenvironment, favoring an anti-tumor immune phenotype and facilitating other cell therapies such as innate-based cell therapies. Conclusions: The development of this innovative enveloped oncolytic virotherapeutic, coupled with advancements in its manufacturing methods, opens up new possibilities in the realm of cancer therapy. It addresses the limitations posed by untargetable and untreatable metastatic diseases, presenting a transformative solution with broad implications for the field.

Lung cancer cell-intrinsic IL-15 promotes cell migration and sensitizes murine lung tumors to anti-PD-L1 therapy

BackgroundIL-15 plays a vital role in enhancing NK cell- and T-cell-mediated antitumor immune responses; however, the direct effect of IL-15 on tumor cells has not been fully elucidated. Herein, we investigated the effect of IL-15 on lung adenocarcinoma cells.MethodsSilencing and overexpression techniques were used to modify endogenous IL-15 expression in tumor cells. Transwell assays were used to assess tumor cell migration and invasion; a live-cell analysis system was used to evaluate cell motility; cellular morphological changes were quantified by confocal fluorescence microscopy; the molecular mechanisms underlying the effect of IL-15 on tumor cells were analyzed by western blotting; and RhoA and Cdc42 activities were evaluated by a pulldown assay. NCG and C57BL/6 mouse models were used to evaluate the functions of IL-15 in vivo.ResultsCancer cell-intrinsic IL-15 promoted cell motility and migration in vitro and metastasis in vivo via activation of the AKT-mTORC1 pathway; however, exogenous IL-15 inhibited cell motility and migration via suppression of the RhoA-MLC2 axis. Mechanistic analysis revealed that both the intracellular and extracellular IL-15-mediated effects required the expression of IL-15Rα by tumor cells. Detailed analyses revealed that the IL-2/IL-15Rβ and IL-2Rγ chains were undetected in the complex formed by intracellular IL-15 and IL-15Rα. However, when exogenous IL-15 engaged tumor cells, a complex containing the IL-15Rα, IL-2/IL-15Rβ, and IL-2Rγ chains was formed, indicating that the differential actions of intracellular and extracellular IL-15 on tumor cells might be caused by their distinctive modes of IL-15 receptor engagement. Using a Lewis lung carcinoma (LLC) metastasis model, we showed that although IL-15 overexpression facilitated the lung metastasis of LLC cells, IL-15-overexpressing LLC tumors were more sensitive to anti-PD-L1 therapy than were IL-15-wild-type LLC tumors via an enhanced antitumor immune response, as evidenced by their increased CD8+ T-cell infiltration compared to that of their counterparts.ConclusionsCancer cell-intrinsic IL-15 and exogenous IL-15 differentially regulate cell motility and migration. Thus, cancer cell-intrinsic IL-15 acts as a double-edged sword in tumor progression. Additionally, high levels of IL-15 expressed by tumor cells might improve the responsiveness of tumors to immunotherapies.

Abstract 491: Aerosol delivery of immunotherapy and hesperetin nanoparticles in murine lung cancer model

Abstract Purpose: Radiation is the most common modality for lung cancer treatment method. Radiationinduces apoptosis, causing cancer cells to be fragmented and expose the cancer-associatedantigens to the tumor microenvironment. These can be recognized by antigen-presenting cells(APC) to induce the antineoplastic effect by activating cytotoxic T cells. Our prior studies showadding immunoadjuvant like AntiCD40 with radiation can further activate the APCs in its anti-tumor (M1) format. Recent studies have shown that flavonoids like Hesperetin, an ACE2receptor agonist also can induce apoptosis in cancer cells whereas ACE2 receptors are abundant in lung cancer cells.This project aims to develop a novel treatment for non-small cell lung cancer (NSCLC) using nanotechnology for inhalation drug delivery Hesperetin, a flavonoid with antioxidant and pro-apoptotic activity; bound to PLGA-coated nanoparticles (Hesperetin Nanoparticles, HNPs) andAntiCD40 aiming for localized targeted dosing to reduce the treatment-related systemic toxicity.We plan to test the efficacy and safety of this aerosol spray. Methods: In-vitro studies were performed in human A549 (ATCC) and murine LLC1 (ATCC)lung cancer cell lines for growth (MTT) and clonogenic survival assays to demonstrate the anti-cancer cell activity of Hesperetin (Sigma). Next, HNP was prepared using NanoFabTx™ nano-formulation reagent kits (Millipore-Sigma). A syngeneic orthotopic murine model of Lungadenoma was generated in wild (+/+) C57/BL6 background mice with similar background LLC1cell lines. We developed syngeneic orthotopic murine lung tumors with luciferase genetransfected LL/2-Luc2 (ATCC) Lewis Lung Cancer cell line in wild-type C57BL/6 mice(Techonic). Lung tumor-bearing mice were treated with HNP, antiCD40, or both with aerosolspray with a control group treated with the solvent (dH2O) only. To assess the drug delivery withaerosol treatment ex-vivo lung tissue was analyzed for Fluorescence tagged antiCD40 andnanoparticle uptakes. A survival assay was performed to analyze the efficacy of aerosoltreatment of HNPs with or without AntiCD40. All cohorts were also analyzed for body score, body weight, and liver and kidney functions. Results: We were able to successfully develop an aerosol drug delivery model to administer bothAnti-CD40 and HNP in a murine lung cancer model. In our analysis of an orthotopic murinelung cancer model, we demonstrate a high intake of the HNP and AntiCD40 by the cancer cellssparing the normal lung tissue. Moreover, the highest percentage of survival rate was observed with the combination aerosol treatment with HNP+AntiCD40 (p<0.001), compared to CD40alone (p<0.01) or HNP (p<0.01) alone. Comments: This treatment model will allow us to make the lung cancer treatment method easilyavailable for the mass population without having hazardous radiation treatment in the lungcancer model. Citation Format: Sayeda Yasmin-Karim, Geraud Richard, Amanda Fam, Alina-Marissa Ogurek, G. Mike Makrigiorgos. Aerosol delivery of immunotherapy and hesperetin nanoparticles in murine lung cancer model [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 491.

Applying deep learning to segmentation of murine lung tumors in pre-clinical micro-computed tomography

Lung cancer remains a leading cause of cancer-related death, but scientists have made great strides in developing new treatments recently, partly owing to the use of genetically engineered mouse models (GEMMs). GEMM tumors represent a translational model that recapitulates human disease better than implanted models because tumors develop spontaneously in the lungs. However, detection of these tumors relies on in vivo imaging tools, specifically micro-Computed Tomography (micro-CT or µCT), and image analysis can be laborious with high inter-user variability. Here we present a deep learning model trained to perform fully automated segmentation of lung tumors without the interference of other soft tissues. Trained and tested on 100 3D µCT images (18,662 slices) that were manually segmented, the model demonstrated a high correlation to manual segmentations on the testing data (r2=0.99, DSC=0.78) and on an independent dataset (n = 12 3D scans or 2328 2D slices, r2=0.97, DSC=0.73). In a comparison against manual segmentation performed by multiple analysts, the model (r2=0.98, DSC=0.78) performed within inter-reader variability (r2=0.79, DSC=0.69) and close to intra-reader variability (r2=0.99, DSC=0.82), all while completing 5+ hours of manual segmentations in 1 minute. Finally, when applied to a real-world longitudinal study (n = 55 mice), the model successfully detected tumor progression over time and the differences in tumor burden between groups induced with different virus titers, aligning well with more traditional analysis methods. In conclusion, we have developed a deep learning model which can perform fast, accurate, and fully automated segmentation of µCT scans of murine lung tumors.

Overcoming lung cancer immunotherapy resistance by combining nontoxic variants of IL-12 and IL-2.

Engineered cytokine-based approaches for immunotherapy of cancer are poised to enter the clinic, with IL-12 being at the forefront. However, little is known about potential mechanisms of resistance to cytokine therapies. We found that orthotopic murine lung tumors were resistant to systemically delivered IL-12 fused to murine serum albumin (MSA, IL12-MSA) because of low IL-12 receptor (IL-12R) expression on tumor-reactive CD8+ T cells. IL2-MSA increased binding of IL12-MSA by tumor-reactive CD8+ T cells, and combined administration of IL12-MSA and IL2-MSA led to enhanced tumor-reactive CD8+ T cell effector differentiation, decreased numbers of tumor-infiltrating CD4+ regulatory T cells, and increased survival of lung tumor-bearing mice. Predictably, the combination of IL-2 and IL-12 at therapeutic doses led to significant dose-limiting toxicity. Administering IL-12 and IL-2 analogs with preferential binding to cells expressing Il12rb1 and CD25, respectively, led to a significant extension of survival in mice with lung tumors while abrogating dose-limiting toxicity. These findings suggest that IL-12 and IL-2 represent a rational approach to combination cytokine therapy whose dose-limiting toxicity can be overcome with engineered cytokine variants.

Targeting METTL3 reprograms the tumor microenvironment to improve cancer immunotherapy

The tumor microenvironment (TME) is a heterogeneous ecosystem containing cancer cells, immune cells, stromal cells, cytokines, and chemokines which together govern tumor progression and response to immunotherapies. Methyltransferase-like 3 (METTL3), a core catalytic subunit for RNA N6-methyladenosine (m6A) modification, plays a crucial role in regulating various physiological and pathological processes. Whether and how METTL3 regulates the TME and anti-tumor immunity in non-small-cell lung cancer (NSCLC) remain poorly understood. Here, we report that METTL3 elevates expression of pro-tumorigenic chemokines including CXCL1, CXCL5, and CCL20, and destabilizes PD-L1 mRNA in an m6A-dependent manner, thereby shaping a non-inflamed TME. Thus, inhibiting METTL3 reprograms a more inflamed TME that renders anti-PD-1 therapy more effective in several murine lung tumor models. Clinically, NSCLC patients who exhibit low-METTL3 expression have a better prognosis when receiving anti-PD-1 therapy. Collectively, our study highlights targeting METTL3 as a promising strategy to improve immunotherapy in NSCLC patients.

Clearance of senescent macrophages ameliorates tumorigenesis in KRAS-driven lung cancer.

The accumulation of senescent cells in the tumor microenvironment can drive tumorigenesis in a paracrine manner through the senescence-associated secretory phenotype (SASP). Using a new p16-FDR mouse line, we show that macrophages and endothelial cells are the predominant senescent cell types in murine KRAS-driven lung tumors. Through single cell transcriptomics, we identify a population of tumor-associated macrophages that express a unique array of pro-tumorigenic SASP factors and surface proteins and are also present in normal aged lungs. Genetic or senolytic ablation of senescent cells, or macrophage depletion, result in a significant decrease in tumor burden and increased survival in KRAS-driven lung cancer models. Moreover, we reveal the presence of macrophages with senescent features in human lung pre-malignant lesions, but not in adenocarcinomas. Taken together, our results have uncovered the important role of senescent macrophages in the initiation and progression of lung cancer, highlighting potential therapeutic avenues and cancer preventative strategies.

An analysis of the effect of Oxaliplatin in inducing immunogenetic celldeath and improving the efficacy of checkpoint inhibitor on SGC-7901

Previous studies demonstrated Oxaliplatin, a derivative of Cisplatin, to have anti-cancer properties in a Lewis LungCarcinoma (LLC). It can induce immunogenic cell death (ICD) in tumor cells and co-administrate with checkpointinhibitors to improve the therapeutic efficacy. This study aims to investigate Oxaliplatin’s effect on a different gastriccarcinoma, SGC-7901, and analyze the therapeutic efficacy of its co-administration with the checkpoint inhibitor. Toevaluate the immunogenic cell death (ICD) induced by Oxaliplatin in SGC-7901, flow cytometry, HMGB1, ATP release,and immunoblotting were conducted. The effectiveness of Oxaliplatin was analyzed using a vaccination approach andsubcutaneous tumor models to observe tumour regression. PD-L1 mRNA and protein levels in SGC-7901 were alsoexamined. The therapeutic efficacy of Oxaliplatin in murine lung tumor models will be enhanced by co-administeringwith aPD-L1. Cisplatin will be employed as a positive control, while PBS will be a negative control. The result of thestudy will provide important insight into the experimental effectiveness of Oxaliplatin in SGC-7901, it also sets thebasis for future experimental, preclinical, and clinical studies of the drug. Future studies should focus on practicing theactual experiment on the experimental effectiveness of Oxaliplatin in SGC-7901 and look for the applicability of theeffectiveness of Oxaliplatin on other tumor cells.

Abstract 1856: Pemetrexed-cisplatin-bevacizumab combo in lung cancer models: impact on tumor immunity

Abstract Dual chemotherapy (i.e., Pemetrexed and Cisplatin, CT) plus anti-VEGF Bevacizumab could be associated with anti-PD1 such as Pembrolizumab in NSCLC patients with low PDL1 levels. To support this new combination, to what extent those drugs could reshape tumor immunity remains to be evaluated, prior to establishing optimal scheduling or sequencing with Pembrolizumab. Both Pemetrexed and Cisplatin are expected to trigger immunogenic cell death, whereas Bevacizumab could help T lymphocytes to extravasate and better infiltrate tumor micro-environment. Here, we studied the impact of Cisplatin and Pemetrexed at two dose levels (i.e., low dose and Maximal Tolerated Dose, MTD) and fixed flat-dose Bevacizumab, alone or in combination (i.e., Cisplatin, Pemetrexed, Cisplatin + Pemetrexed, with our without Beva), on the immune repertoire of BalbC mice ectopically grafted with KLN-205 murine lung tumors. CD45, CD4, CD8, FoxP3-CD4 (Tregs), and MDSCs were monitored by FACS analysis in blood, spleen, and tumors on D2, D7, D14, and D21 after treatment. Results were normalized by CD45 counts. All treatments led to an increase in CD4+ T cells in blood as compared with control (i.e., untreated) animals. Little increase was observed in tumors, regardless of the treatments but mice treated with the Beva+CT combo (both low dose and MTD CT) showed an increase in CD4+ in the spleen on D14. Regarding CD8+ T cells, all treatments led to an increase from D2 to D7. Beva + CT low dose led to a continuous increase in CD8+ cells, whereas in Beva+CT MTD mice, after an initial increase, a reduction in CD8+ was observed after D7 eventually. In the spleen, CD8+ proved to be stable apart from the Beva + CT low dose group which showed an increase up to D14. In tumors, an increase in CD8+ cells was observed in all the groups. No change in MDSCs levels was observed, regardless of the drugs, the dosing and the organ. Conversely, Tregs decreased in the blood in the Beva + CT low-dose group on D14, and decreased in spleen and tumors as compared with Control mice, regardless of the drugs and the dosing. Overall, our data suggest that the triple combination between Beva, Pemetrexed, and Cisplatin has a stronger impact on immune cells than each drug used as a single agent. MDSCs were the only cells to be unaffected by the treatments. Dosing could be relevant as well since the reduction in CD8+ T cells observed with MTD CT could come from drug-related lymphopenia. The Beva followed by low dose Pemetrexed + Cisplatin group is thus the more likely to harness tumor immunity, with peaks in CD4+ and CD8+ T cells and drop in Tregs observed on D14. This pilot study suggests therefore that sequencing Beva + CT low dose followed by a two-week lag time prior to administrating anti-PD1 could lead to synergism in lung cancer models. Citation Format: Guillaume Sicard, Sarah Giacometti, Fabrice Barlesi, Raphaelle Fanciullino, Joseph Ciccolini. Pemetrexed-cisplatin-bevacizumab combo in lung cancer models: impact on tumor immunity [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 1856.

Abstract 6192: MSI2 regulates DNA damage response signaling and tumor progression in murine NSCLC

Abstract Lung cancer is one of the most frequently diagnosed cancers and is the leading cause of cancer-related death worldwide, accounting for >2.2 million of new cancer diagnoses and 1.8 million cancer-related deaths in 2020. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, including adenocarcinomas and squamous cell carcinomas. Many lung adenocarcinomas have activating driver mutations in KRAS (~32% tumors), often co-occurring with mutations inactivating the TP53 tumor suppressor. Expression of the RNA-binding protein Musashi-2 (MSI2) MSI2 regulates target mRNA translation, and its expression is progressively elevated along with tumor stage in lung cancers. We previously showed that MSI2 actively promotes lung cancer growth, invasion, and metastasis, in part through promoting expression and activity of the TGFβR1/SMAD3 and EGFR signaling cascades. We now find that genetic MSI2 deficiency in a murine lung tumor model, Krasmut/Trp53KO/Msi2KO (KPM2) decreases both the total tumor number and tumor burden in comparison to control Krasmut/Trp53KO (KP) mice. Using a panel of KPM2 and KP cell lines we established from tumors from these mice, we found KPM2 cell lines demonstrate a significant decrease in proliferation compared to KP cell lines. Interestingly, reverse phase protein array (RPPA) comparison analysis of KPM2 and KP cell lines showed that MSI2 strongly and positively regulates expression of Ataxia-Telangiectasia Mutated (ATM) protein, with lower levels observed in KPM2 cells. The ATM serine/threonine protein kinase is a key transducer of DDR signaling in cases of double-strand breaks; ATM activation causes G1/S cell cycle arrest, contributes to efficiency of DNA repair, and triggers apoptosis in cases of repair failure. Notably, KPM2 cells showed significantly higher levels of γH2AX, suggesting inefficient DDR. Finally, untreated or cisplatin-treated KPM2 cells demonstrated G2/M arrest and had increased level of apoptosis as demonstrated by increase in caspase 3 cleavage, consistent with ATM loss and compensatory activation of Ataxia-telangiectasia and Rad3-related protein (ATR). Taken together, our data suggests that one of the consequences of MSI2 upregulation in NSCLC includes maintenance of an efficient DDR. Targeting MSI2 and its downstream signaling would be of therapeutic interest. Citation Format: Igor Bychkov, Alexander Deneka, Iuliia Topchu, Rajendra P. Pangeni, Christopher Lengner, Evgeny Izumchenko, Jyoti Patel, John Karanicolas, Petr Makhov, Yanis Boumber. MSI2 regulates DNA damage response signaling and tumor progression in murine NSCLC [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 6192.

Precision immunointerception of EGFR-driven tumorigenesis for lung cancer prevention.

Epidermal growth factor receptor (EGFR) mutations occur in about 50% of lung adenocarcinomas in Asia and about 15% in the US. EGFR mutation-specific inhibitors have been developed and made significant contributions to controlling EGFR mutated non-small cell lung cancer. However, resistance frequently develops within 1 to 2 years due to acquired mutations. No effective approaches that target mutant EGFR have been developed to treat relapse following tyrosine kinase inhibitor (TKI) treatment. Vaccination against mutant EGFR is one area of active exploration. In this study, we identified immunogenic epitopes for the common EGFR mutations in humans and formulated a multi-peptide vaccine (Emut Vax) targeting the EGFR L858R, T790M, and Del19 mutations. The efficacy of the Emut Vax was evaluated in both syngeneic and genetic engineered EGFR mutation-driven murine lung tumor models with prophylactic settings, where the vaccinations were given before the onset of the tumor induction. The multi-peptide Emut Vax effectively prevented the onset of EGFR mutation-driven lung tumorigenesis in both syngeneic and genetically engineered mouse models (GEMMs). Flow cytometry and single-cell RNA sequencing were conducted to investigate the impact of Emut Vax on immune modulation. Emut Vax significantly enhanced Th1 responses in the tumor microenvironment and decreased suppressive Tregs to enhance anti-tumor efficacy. Our results show that multi-peptide Emut Vax is effective in preventing common EGFR mutation-driven lung tumorigenesis, and the vaccine elicits broad immune responses that are not limited to anti-tumor Th1 response.

Inhaled dry powder cisplatin increases antitumour response to anti-PD1 in a murine lung cancer model.

Despite advances in targeted therapies and immunotherapy in lung cancer, chemotherapy remains the backbone of treatment in most patients at different stages of the disease. Inhaled chemotherapy is a promising strategy to target lung tumours and to limit the induced severe systemic toxicities. Cisplatin dry powder for inhalation (CIS-DPI) was tested as an innovative way to deliver cisplatin locally via the pulmonary route with minimal systemic toxicities. In vivo, CIS-DPI demonstrated a dose-dependent antiproliferative activity in the M109 orthotopic murine lung tumour model and upregulated the immune checkpoint PD-L1 on lung tumour cells. Combination of CIS-DPI with the immune checkpoint inhibitor anti-PD1 showed significantly reduced tumour size, increased the number of responders and prolonged median survival over time in comparison to the anti-PD1 monotherapy. Furthermore, the CIS-DPI and anti-PD1 combination induced an intra-tumour recruitment of conventional dendritic cells and tumour infiltrating lymphocytes, highlighting an anti-tumour immune response. This study demonstrates that combining CIS-DPI with anti-PD1 is a promising strategy to improve lung cancer therapy.

Miz1 promotes KRAS-driven lung tumorigenesis by repressing the protocadherin Pcdh10

Targeting KRAS-mutated non-small-cell lung cancer (NSCLC) remains clinically challenging. Here we show that loss of function of Miz1 inhibits lung tumorigenesis in a mouse model of oncogenic KRAS-driven lung cancer. In vitro, knockout or silencing of Miz1 decreases cell proliferation, clonogenicity, migration, invasion, or anchorage-independent growth in mutant (MT) KRAS murine or human NSCLC cells but has unremarkable impact on non-tumorigenic cells or wild-type (WT) KRAS human NSCLC cells. RNA-sequencing reveals Protocadherin-10 (Pcdh10) as the top upregulated gene by Miz1 knockout in MT KRAS murine lung tumor cells. Chromatin immunoprecipitation shows Miz1 binding on the Pcdh10 promoter in MT KRAS lung tumor cells but not non-tumorigenic cells. Importantly, silencing of Pcdh10 rescues cell proliferation and clonogenicity in Miz1 knockout/knockdown MT KRAS murine or human tumor cells, and rescues allograft tumor growth of Miz1 knockout tumor cells in vivo. Miz1 is upregulated in MT KRAS lung tumor tissues compared with adjacent non-involved tissues in mice. Consistent with this, Miz1 is upregulated while Pcdh10 is downregulated in human lung adenocarcinomas (LUAD) compared with normal tissues, and high Miz1 levels or low Pcdh10 levels are associated with poor survival in lung cancer patients. Furthermore, the Miz1 signature is associated with worse survival in MT but not WT KRAS LUAD, and Pcdh10 is downregulated in MT compared to WT KRAS LUAD. Taken together, our studies implicate the Miz1/Pcdh10 axis in oncogenic KRAS-driven lung tumorigenesis.

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 (>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.

TSAFinder: exhaustive tumor-specific antigen detection with RNAseq.

Tumor-specific antigen (TSA) identification in human cancer predicts response to immunotherapy and provides targets for cancer vaccine and adoptive T-cell therapies with curative potential, and TSAs that are highly expressed at the RNA level are more likely to be presented on major histocompatibility complex (MHC)-I. Direct measurements of the RNA expression of peptides would allow for generalized prediction of TSAs. Human leukocyte antigen (HLA)-I genotypes were predicted with seq2HLA. RNA sequencing (RNAseq) fastq files were translated into all possible peptides of length 8-11, and peptides with high and low expressions in the tumor and control samples, respectively, were tested for their MHC-I binding potential with netMHCpan-4.0. A novel pipeline for TSA prediction from RNAseq was used to predict all possible unique peptides size 8-11 on previously published murine and human lung and lymphoma tumors and validated on matched tumor and control lung adenocarcinoma (LUAD) samples. We show that neoantigens predicted by exomeSeq are typically poorly expressed at the RNA level, and a fraction is expressed in matched normal samples. TSAs presented in the proteomics data have higher RNA abundance and lower MHC-I binding percentile, and these attributes are used to discover high confidence TSAs within the validation cohort. Finally, a subset of these high confidence TSAs is expressed in a majority of LUAD tumors and represents attractive vaccine targets. The datasets were derived from sources in the public domain as follows: TSAFinder is open-source software written in python and R. It is licensed under CC-BY-NC-SA and can be downloaded at https://github.com/RNAseqTSA. Supplementary data are available at Bioinformatics online.

Picturing of the Lung Tumor Cellular Composition by Multispectral Flow Cytometry.

The lung tumor microenvironment plays a critical role in the tumorigenesis and metastasis of lung cancer, resulting from the crosstalk between cancer cells and microenvironmental cells. Therefore, comprehensive identification and characterization of cell populations in the complex lung structure is crucial for development of novel targeted anti-cancer therapies. Here, a hierarchical clustering approach with multispectral flow cytometry was established to delineate the cellular landscape of murine lungs under steady-state and cancer conditions. Fluorochromes were used multiple times to be able to measure 24 cell surface markers with only 13 detectors, yielding a broad picture for whole-lung phenotyping. Primary and metastatic murine lung tumor models were included to detect major cell populations in the lung, and to identify alterations to the distribution patterns in these models. In the primary tumor models, major altered populations included CD324+ epithelial cells, alveolar macrophages, dendritic cells, and blood and lymph endothelial cells. The number of fibroblasts, vascular smooth muscle cells, monocytes (Ly6C+ and Ly6C–) and neutrophils were elevated in metastatic models of lung cancer. Thus, the proposed clustering approach is a promising method to resolve cell populations from complex organs in detail even with basic flow cytometers.

Novel 3D Lung Tumor Spheroids for Oncoimmunological Assays

Lung cancer thrives in a complex multicellular tumor microenvironment (TME) that impacts tumor growth, metastasis, response, and resistance to therapy. While orthotopic murine lung cancer models can partly recapitulate this complexity, they do not resonate with high‐throughput immunotherapeutic drug screening assays. To address the current need for relevant and easy‐to‐use lung tumor models, a protocol is established to generate and evaluate fully histocompatible murine and human lung tumor spheroids, generated by coculturing lung fibroblasts with tumor cells in ultralow adherence 96‐well plates. A spheroid generation protocol with the murine KrasG12Dp53−/−(KP) and Lewis Lung Carcinoma (LLC) cell lines is delivered next to the human lung H1650 adenocarcinoma line. In addition, their application potential to study tumor‐stroma organization, T‐cell motility, and infiltration as well as distinct macrophage subsets’ behavior using confocal microscopy is described. Finally, a 3D target‐specific T‐cell killing assay that allows spatiotemporal assessment of different tumor to T‐cell ratios and immune checkpoint blockade regimens using flow cytometry and live cell imaging is described. This 3D lung tumor spheroid platform can serve as a blueprint for other solid cancer types to comply with the need for straightforward murine and human oncoimmunology assays.

Abstract PO-124: EZH2 blockade overcomes suppression of the proinflammatory senescence-associated secretory phenotype in the pancreas and drives NK cell-mediated pancreatic tumor responses

Abstract T cell-targeting immunotherapies that produce durable and sometimes curative responses in other malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to poor T cell infiltration and tumor immunogenicity. We and others have demonstrated that cellular senescence and its associated secretory phenotype (SASP), which leads to production of proinflammatory cytokines, chemokines, and growth factors, can be a powerful way to reactivate a different type of Natural Killer (NK) cell immunity. Recently, we found that RAS pathway targeting MEK and CDK4/6 inhibitors can induce senescence in KRAS mutant lung tumor and PDAC models; however, therapy-induced senescence (TIS) led to NK cell-mediated tumor regressions only in the lungs. Here we set out to understand how the pancreas tumor microenvironment (TME) suppresses NK immunity and develop strategies to harness NK cell surveillance for PDAC immunotherapy. Syngeneic murine KRAS-driven lung tumor and PDAC cells were transplanted into lungs, pancreas, or liver of C57BL/6 mice. Following 2-week treatment with the MEK inhibitor trametinib and CDK4/6 inhibitor palbociclib (T/P) to induce senescence, immune responses were assessed by flow cytometry, and the SASP profile assessed by RNA- and ATAC-seq analysis. An NK1.1-targeted antibody was also used to deplete NK cells and evaluate treatment efficacy. shRNA-mediated EZH2 knockdown and treatment with EZH2 methyltransferase inhibitors in murine and human PDAC cells and mouse models was used to determine the role of EZH2 in SASP regulation by qPCR and cytokine array. The effects of EZH2 blockade on NK cell immunity were determined in co-culture migration and cytotoxicity assays in vitro and in PDAC transplant models in vivo by flow cytometry. The efficacy of T/P treatment in combination with EZH2-targeting shRNAs or inhibitors was measured by ultrasound tumor measurements and survival in PDAC-bearing animals. Tumors propagated in the pancreas TME display transcriptional repression and reduced chromatin accessibility of SASP transcriptional activators (NF-KB, IRFs) and factors necessary for NK cell activity (IL-15,-18) and chemotaxis (CCL2,-7,-8; CXCL9,-10) following TIS, and do not undergo anti-tumor NK immune surveillance as compared to tumors grown in the lungs and liver. We identified induction of EZH2 and repression of its targets, including key SASP factors and regulators, specifically in tumor cells grown in the pancreas TME. Genetic or pharmacological inhibition of EZH2 enhanced proinflammatory SASP signaling and resulted in NK cell infiltration and anti-tumor cytotoxicity in PDAC models. Remarkably, EZH2 knockdown in combination with T/P treatment led to complete tumor regressions in PDAC-bearing mice that were reversed following NK cell depletion. These results demonstrate that EZH2 mediates transcriptional repression of the proinflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with TIS could be a powerful means to reactivate absent NK cell surveillance in PDAC to achieve immune-mediated tumor control. Citation Format: Loretah Chibaya, Yvette Lopez-Diaz, Haibo Liu, Katherine C. Murphy, John P. Morris IV, Yu-jui Ho, Janelle Simon, Wei Luan, Amanda Kulick, Lakhena Leang, Elisa de Stanchina, Lihua J. Zhu, Scott W. Lowe, Marcus Ruscetti. EZH2 blockade overcomes suppression of the proinflammatory senescence-associated secretory phenotype in the pancreas and drives NK cell-mediated pancreatic tumor responses [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-124.

USP28 deletion and small-molecule inhibition destabilizes c-MYC and elicits regression of squamous cell lung carcinoma.

Lung squamous cell carcinoma (LSCC) is a considerable global health burden, with an incidence of over 600,000 cases per year. Treatment options are limited, and patient's 5-year survival rate is less than 5%. The ubiquitin-specific protease 28 (USP28) has been implicated in tumourigenesis through its stabilization of the oncoproteins c-MYC, c-JUN, and Δp63. Here, we show that genetic inactivation of Usp28-induced regression of established murine LSCC lung tumours. We developed a small molecule that inhibits USP28 activity in the low nanomole range. While displaying cross-reactivity against the closest homologue USP25, this inhibitor showed a high degree of selectivity over other deubiquitinases. USP28 inhibitor treatment resulted in a dramatic decrease in c-MYC, c-JUN, and Δp63 proteins levels and consequently induced substantial regression of autochthonous murine LSCC tumours and human LSCC xenografts, thereby phenocopying the effect observed by genetic deletion. Thus, USP28 may represent a promising therapeutic target for the treatment of squamous cell lung carcinoma.