Murine Lung Cancer Model Research Articles

A Novel In Situ Dendritic Cell Vaccine Triggered by Rose Bengal Enhances Adaptive Antitumour Immunity.

Dendritic cell- (DC-) based vaccination has emerged as a promising antitumour immunotherapy. However, overcoming immune tolerance and immunosuppression in the tumour microenvironment (TME) is still a great challenge. Recent studies have shown that Rose Bengal (RB) can effectively induce immunogenic cell death (ICD) in cancer cells, presenting whole tumour antigens for DC processing and presentation. However, the synergistic antitumour effect of combining intralesional RB with immature DCs (RB-iDCs) remains unclear. In the present study, we investigated whether RB-iDCs have superior antitumour effects compared with either single agent and evaluated the immunological mechanism of RB-iDCs in a murine lung cancer model. The results showed that intralesional RB-iDCs suppressed subcutaneous tumour growth and lung metastasis, which resulted in 100% mouse survival and significantly increased TNF-α production by CD8+ T cells. These effects were closely related to the induction of the expression of distinct ICD hallmarks by RB in both bulk cancer cells and cancer stem cells (CSCs), especially calreticulin (CRT), thus enhancing immune effector cell (i.e., CD4+, CD8+, and memory T cells) infiltration and attenuating the accumulation of immunosuppressive cells (i.e., Tregs, macrophages, and myeloid-derived suppressor cells (MDSCs)) in the TME. This study reveals that the RB-iDC vaccine can synergistically destroy the primary tumour, inhibit distant metastasis, and prevent tumour relapse in a lung cancer mouse model, which provides important preclinical data for the development of a novel combinatorial immunotherapy.

Preclinical Pharmacokinetics and Dosimetry of an 89Zr Labelled Anti-PDL1 in an Orthotopic Lung Cancer Murine Model.

Anti-PDL1 is a monoclonal antibody targeting the programmed death-cell ligand (PD-L1) by blocking the programmed death-cell (PD-1)/PD-L1 axis. It restores the immune system response in several tumours, such as non-small cell lung cancer (NSCLC). Anti-PDL1 or anti-PD1 treatments rely on PD-L1 tumoural expression assessed by immunohistochemistry on biopsy tissue. However, depending on the biopsy extraction site, PD-L1 expression can vary greatly. Non-invasive imaging enables whole-body mapping of PD-L1 sites and could improve the assessment of tumoural PD-L1 expression.MethodsPharmacokinetics (PK), biodistribution and dosimetry of a murine anti-PDL1 radiolabelled with zirconium-89, were evaluated in both healthy mice and immunocompetent mice with lung cancer. Preclinical PET (μPET) imaging was used to analyse [89Zr]DFO-Anti-PDL1 distribution in both groups of mice. Non-compartmental (NCA) and compartmental (CA) PK analyses were performed in order to describe PK parameters and assess area under the concentration-time curve (AUC) for dosimetry evaluation in humans.ResultsOrgan distribution was correctly estimated using PK modelling in both healthy mice and mice with lung cancer. Tumoural uptake occurred within 24 h post-injection of [89Zr]DFO-Anti-PDL1, and the best imaging time was at 48 h according to the signal-to-noise ratio (SNR) and image quality. An in vivo blocking study confirmed that [89Zr]DFO-anti-PDL1 specifically targeted PD-L1 in CMT167 lung tumours in mice. AUC in organs was estimated using a 1-compartment PK model and extrapolated to human (using allometric scaling) in order to estimate the radiation exposure in human. Human-estimated effective dose was 131 μSv/MBq.ConclusionThe predicted dosimetry was similar or lower than other antibodies radiolabelled with zirconium-89 for immunoPET imaging.

The BCL-2 family member BOK promotes KRAS-driven lung cancer progression in a p53-dependent manner

A variety of cancer entities are driven by KRAS mutations, which remain difficult to target clinically. Survival pathways, such as resistance to cell death, may represent a promising treatment approach in KRAS mutated cancers. Based on the frequently observed genomic deletions of BCL-2-related ovarian killer (BOK) in cancer patients, we explored the function of BOK in a mutant KrasG12D-driven murine model of lung cancer. Using KrasG12D/+Bok−/− mice, we observed an overall tumor-promoting function of BOK in vivo. Specifically, loss of BOK reduced proliferation both in cell lines in vitro as well as in KrasG12D-driven tumor lesions in vivo. During tumor development in vivo, loss of BOK resulted in a lower tumor burden, with fewer, smaller, and less advanced tumors. Using KrasG12D/+Tp53Δ/ΔBok−/− mice, we identified that this phenotype was entirely dependent on the presence of functional p53. Furthermore, analysis of a human dataset of untreated early-stage lung tumors did not identify any common deletion of the BOK locus, independently of the TP53 status or the histopathological classification. Taken together our data indicate that BOK supports tumor progression in Kras-driven lung cancer.

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.

Fast DNA Vaccination Strategy Elicits a Stronger Immune Response Dependent on CD8+CD11c+ Cell Accumulation.

Conventional DNA vaccine strategies usually employ a regimen of immunizations at 2-week or longer intervals to induce effective memory cell-dependent immune responses. Clinical cancer treatment requires a faster immunization strategy to contend with tumor progression. In this study, a novel fast immunization strategy was established, wherein a DNA vaccine was intramuscularly administered on days 0, 2, and 5 in a murine lung cancer model. Effector cells peaked 7 to 10 days after the last vaccination. Compared with traditional 2-week-interval immunization strategies, antigen-specific cytolysis and INF-γ secretion were significantly enhanced under the fast vaccination approach. As a result, the rapidly administered DNA vaccine elicited stronger and more prompt antitumor effects. The probable underlying mechanism of fast immunization was the accumulation of CD8+CD11c+ antigen-presenting cells at the injection site, which enhanced subsequent antigen presentation. In conclusion, the fast DNA vaccination strategy shortened vaccination time to 5 days and elicited a stronger antitumor immune response.

A radioenhancing nanoparticle mediated immunoradiation improves survival and generates long-term antitumor immune memory in an anti-PD1-resistant murine lung cancer model

BackgroundCombining radiotherapy with PD1 blockade has had impressive antitumor effects in preclinical models of metastatic lung cancer, although anti-PD1 resistance remains problematic. Here, we report results from a triple-combination therapy in which NBTXR3, a clinically approved nanoparticle radioenhancer, is combined with high-dose radiation (HDXRT) to a primary tumor plus low-dose radiation (LDXRT) to a secondary tumor along with checkpoint blockade in a mouse model of anti-PD1-resistant metastatic lung cancer.MethodsMice were inoculated with 344SQR cells in the right legs on day 0 (primary tumor) and the left legs on day 3 (secondary tumor). Immune checkpoint inhibitors (ICIs), including anti-PD1 (200 μg) and anti-CTLA4 (100 μg) were given intraperitoneally. Primary tumors were injected with NBTXR3 on day 6 and irradiated with 12-Gy (HDXRT) on days 7, 8, and 9; secondary tumors were irradiated with 1-Gy (LDXRT) on days 12 and 13. The survivor mice at day 178 were rechallenged with 344SQR cells and tumor growth monitored thereafter.ResultsNBTXR3 + HDXRT + LDXRT + ICIs had significant antitumor effects against both primary and secondary tumors, improving the survival rate from 0 to 50%. Immune profiling of the secondary tumors revealed that NBTXR3 + HDXRT + LDXRT increased CD8 T-cell infiltration and decreased the number of regulatory T (Treg) cells. Finally, none of the re-challenged mice developed tumors, and they had higher percentages of CD4 memory T cells and CD4 and CD8 T cells in both blood and spleen relative to untreated mice.ConclusionsNBTXR3 nanoparticle in combination with radioimmunotherapy significantly improves anti-PD1 resistant lung tumor control via promoting antitumor immune response.Graphical

NBTXR3 Nanoparticle With Immunoradiation Might Reshape Metastatic Tumor-Infiltrating T Cell Repertoire in Murine Lung Cancer Model

Radiation therapy (XRT) induces lymphocyte infiltration of primary tumors that is increased when using NBTXR3 radioenhancer nanoparticle, leading to improved response to combination immunotherapy (IT). However, treatment effects on the metastatic tumor microenvironment are poorly understood. We studied the effects of NBTXR3 injection into primary tumors plus XRT plus dual-agent immunotherapy on secondary tumor-infiltrating T cells in a mouse model of lung cancer. The hypothesis is that intratumoral primary tumor treatment with NBTXR3 nanoparticle enriches conserved T cell receptor repertoires among metastatic tumor-infiltrating lymphocytes.Four groups of 4 mice each were inoculated subcutaneously with anti-PD1-resistant 344SQR murine lung cancer cells in each hind leg, 3 days apart, to establish 'primary' (right) and 'secondary' (left, which represented metastasis) tumors. All mice received intraperitoneal anti-PD1 and anti-CTLA-4 (IT) on days 4, 7, 10, and 13, and 12-Gy high-dose XRT (HD) to the primary tumors on days 7, 8 and 9. Primary tumors in groups 2 and 4 also received intratumoral NBTXR3 on day 6. Secondary tumors in groups 3 and 4 were irradiated with 1-Gy low-dose XRT (LD) on days 12 and 13. Treatment groups were designated as 1 = HD+IT, 2 = NBTXR3+HD+IT, 3 = HD+LD+IT, and 4 = NBTXR3+HD+LD+IT. All secondary tumors were harvested on day 19 for tumor-infiltrating T-cell RNA extraction and sequencing on an Illumina MiSeq. T-cell receptor (TCR) beta complementarity-determining region 3 (CDR3b) repertoires were analyzed with MiXCR v3.0.12 software, and CDR3b analyses and Circos plots were generated using software. P < 0.05 from Mann-Whitney U tests were considered statistically significant.NBTXR3 significantly increased the proportion of conserved CDR3b clonotypes within the secondary tumors among mice that received only HD XRT to the primary tumors (group 2 vs 1, P = 0.0476). When the secondary tumors were exposed to LD XRT, NBTXR3 still increased the frequency of overlapping clonotypes although not significantly (group 4 vs 3, P = 0.3095). Most interestingly, there were significantly more conserved CDR3b repertoires from the secondary tumors that were shared between groups 2 and 4 that received NBTXR3 compared to those shared between groups 1 and 3 (P < 0.0001). Comparisons of CDR3b normalized Shannon clonality indices and Circos plots did not reveal any difference in repertoire diversity between NBTXR3-treated and non-NBTXR3-treated groups.NBTXR3 radioenhancer nanoparticle, given with immunoradiation, might reshape the TCR repertoire of metastatic lung cancer tumor-infiltrating T cells by driving the selection of conserved CDR3b clonotypes.

P12.01 A Novel 89Zr-α-PD-1 Immuno-PET-CT May Improve Pseudoprogression Detection in a Lung Cancer Murine Model Receiving Immunotherapy

Immune checkpoint inhibitors (ICIs) have revolutionized lung cancer treatment improving survival of non-small cell lung cancer (NSCLC) patients. Response assessment of patients on ICIs represents a challenge since an increase in tumor size or the appearance of new lesions might not reflect true disease progression (P) but pseudoprogression (PP). Pseudoprogression has been reported in a range of 3-6% in NSCLC. Conventional 18F-FDG-PET scans do not accurately discriminate P from PP. We have recently reported the efficacy of a combined blockade of PD-1 and Id1 in a lung cancer mouse model (Baraibar et al.

Monocarboxylate Transporter-2 Expression Restricts Tumor Growth in a Murine Model of Lung Cancer: A Multi-Omic Analysis.

Monocarboxylate transporter 2 (MCT2) is a major high-affinity pyruvate transporter encoded by the SLC16A7 gene, and is associated with glucose metabolism and cancer. Changes in the gut microbiota and host immune system are associated with many diseases, including cancer. Using conditionally expressed MCT2 in mice and the TC1 lung carcinoma model, we examined the effects of MCT2 on lung cancer tumor growth and local invasion, while also evaluating potential effects on fecal microbiome, plasma metabolome, and bulk RNA-sequencing of tumor macrophages. Conditional MCT2 mice were generated in our laboratory using MCT2loxP mouse intercrossed with mCre-Tg mouse to generate MCT2loxP/loxP; Cre+ mouse (MCT2 KO). Male MCT2 KO mice (8 weeks old) were treated with tamoxifen (0.18 mg/g BW) KO or vehicle (CO), and then injected with mouse lung carcinoma TC1 cells (10 × 105/mouse) in the left flank. Body weight, tumor size and weight, and local tumor invasion were assessed. Fecal DNA samples were extracted using PowerFecal kits and bacterial 16S rRNA amplicons were also performed. Fecal and plasma samples were used for GC−MS Polar, as well as non-targeted UHPLC-MS/MS, and tumor-associated macrophages (TAMs) were subjected to bulk RNAseq. Tamoxifen-treated MCT2 KO mice showed significantly higher tumor weight and size, as well as evidence of local invasion beyond the capsule compared with the controls. PCoA and hierarchical clustering analyses of the fecal and plasma metabolomics, as well as microbiota, revealed a distinct separation between the two groups. KO TAMs showed distinct metabolic pathways including the Acetyl-coA metabolic process, activation of immune response, b-cell activation and differentiation, cAMP-mediated signaling, glucose and glutamate processes, and T-cell differentiation and response to oxidative stress. Multi-Omic approaches reveal a substantial role for MCT2 in the host response to TC1 lung carcinoma that may involve alterations in the gut and systemic metabolome, along with TAM-related metabolic pathway. These findings provide initial opportunities for potential delineation of oncometabolic immunomodulatory therapeutic approaches.

A novel EGFR inhibitor suppresses survivin expression and tumor growth in human gefitinib-resistant EGFR-wild type and -T790M non-small cell lung cancer

Tyrosine kinase inhibitors of epidermal growth factor receptor (EGFR-TKIs) are currently used therapy for non-small cell lung cancer (NSCLC) patients; however, drug resistance during cancer treatment is a critical problem. Survivin is an anti-apoptosis protein, which promotes cell proliferation and tumor growth that highly expressed in various human cancers. Here, we show a novel synthetic compound derived from gefitinib, do-decyl-4-(4-(3-(4-(3-chloro-4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)propyl) piper-azin-1-yl)-4-oxobutanoate, which is named as SP101 that inhibits survivin expression and tumor growth in both the EGFR-wild type and -T790M of NSCLC. SP101 blocked EGFR kinase activity and induced apoptosis in the A549 (EGFR-wild type) and H1975 (EGFR-T790M) lung cancer cells. SP101 reduced survivin proteins and increased active caspase 3 for inducing apoptosis. Ectopic expression of survivin by a survivin-expressed vector attenuated the SP101-induced cell death in lung cancer cells. Moreover, SP101 inhibited the gefitinib-resistant tumor growth in the xenograft human H1975 lung tumors of nude mice. SP101 substantially reduced survivin proteins but conversely elicited active caspase 3 proteins in tumor tissues. Besides, SP101 exerted anticancer abilities in the gefitinib resistant cancer cells separated from pleural effusion of a clinical lung cancer patient. Consistently, SP101 decreased the survivin proteins and the patient-derived xenografted lung tumor growth in nude mice. Anti-tumor ability of SP101 was also confirmed in the murine lung cancer model harboring EGFR T790M-L858R. Together, SP101 is a new EGFR inhibitor with inhibiting survivin that can be developed for treating EGFR wild-type and EGFR-mutational gefitinib-resistance in human lung cancers.

Lung Cancer Stem Cells and Their Clinical Implications.

It is now widely accepted that stem cells exist in various cancers, including lung cancer, which are referred to as cancer stem cells (CSCs). CSCs are defined in this context as the subset of tumor cells with the ability to form tumors in serial transplantation and cloning assays and form tumors at metastatic sites. Mouse models of lung cancer have shown that lung CSCs reside in niches that are essential for the maintenance of stemness, plasticity, enable antitumor immune evasion, and provide metastatic potential. Similar to normal lung stem cells, Notch, Wnt, and the Hedgehog signaling cascades have been recruited by the CSCs to regulate stemness and also provide therapy-driven resistance in lung cancer. Compounds targeting β-catenin and Sonic hedgehog (Shh) activity have shown promising anti-CSC activity in preclinical murine models of lung cancer. Understanding CSCs and their niches in lung cancer can answer fundamental questions pertaining to tumor maintenance and associated immune regulation and escape that appear important in the quest to develop novel lung cancer therapies and enhance sensitivity to currently approved chemo-, targeted-, and immune therapeutics.

Neutralizing interleukin-6 in tumor-bearing mice does not abrogate behavioral fatigue induced by Lewis lung carcinoma

Tumor growth is associated with metabolic reprogramming of various organs including the liver. This metabolic reprogramming is responsible for the development of behavioral fatigue represented by decreased voluntary wheel running in a murine model of lung cancer. To determine whether interleukin (IL-)6 induced by the tumor is responsible for the metabolic reprogramming, mice injected with Lewis lung carcinoma cells in the flank were treated with an anti-mouse IL-6 monoclonal neutralizing antibody using a 2 × 2 factorial design (+/- tumor and +/- anti-IL-6 antibody). Endpoints were represented by behavioral, metabolic and immune phenotypes. Despite its ability to abrogate the increase in plasma levels of IL-6 that was apparent in tumor-bearing mice and decrease inflammatory signaling in the liver, immunoneutralization of IL-6 had no effect on voluntary wheel running and did not modify the tumor-induced alterations in hepatic gene expression of inflammatory cytokines and metabolic factors. These negative results indicate that IL-6 does not mediate the communication between tumor and host in mice implanted with Lewis lung carcinoma

EVT-701 is a novel selective and safe mitochondrial complex 1 inhibitor with potent anti-tumor activity in models of solid cancers.

Targeting the first protein complex of the mitochondrial electron transport chain (MC1) in cancer has become an attractive therapeutic approach in the recent years, given the metabolic vulnerabilities of cancer cells. The anticancer effect exerted by the pleiotropic drug metformin and the associated reduction in hypoxia‐inducible factor 1α (HIF‐1α) levels putatively mediated by MC1 inhibition led to the development of HIF‐1α inhibitors, such as BAY87‐2243, with a more specific MC1 targeting. However, the development of BAY87‐2243 was stopped early in phase 1 due to dose‐independent emesis and thus there is still no clinical proof of concept for the approach. Given the importance of mitochondrial metabolism during cancer progression, there is still a strong therapeutic need to develop specific and safe MC1 inhibitors. We recently reported the synthesis of compounds with a novel chemotype and potent action on HIF‐1α degradation and MC1 inhibition. We describe here the selectivity, safety profile and anti‐cancer activity in solid tumors of lead compound EVT‐701. In addition, using murine models of lung cancer and of Non‐Hodgkin's B cell lymphoma we demonstrated that EVT‐701 reduced tumor growth and lymph node invasion when used as a single agent therapy. LKB1 deficiency in lung cancer was identified as a potential indicator of accrued sensitivity to EVT‐701, allowing stratification and selection of patients in clinical trials. Altogether these results support further evaluation of EVT‐701 alone or in combination in preclinical models and eventually in patients.

543P Neoadjuvant MRx0518 treatment is associated with significant gene and metagene signature changes in solid tumours

MRx0518 is an oral live biotherapeutic with potent immunostimulatory activity and anti-tumorigenic efficacy in murine models of lung (LLC1), kidney (Renca) and breast (EMT6) cancer. Previous reports have demonstrated a favourable safety profile in neoadjuvant and metastatic clinical settings, with emerging evidence of immune modulation. We performed a comprehensive analysis of the gene and metagene signature in cancer patients treated with MRx0518 monotherapy.

Abstract 440: Potentiation of EGFR peptide cancer vaccine by an orally bioavailable glutamine antagonist prodrug JHU-083

Abstract Lung cancer is the leading cause of cancer mortality worldwide. More than 85% of lung cancers are non-small cell lung cancer (NSCLC). EGFR mutations occur in 47.9% of Asia-Pacific patients with NSCLC and 19.2% of Western patients. The most common EGFR mutations (&amp;gt;90%) are deletions in exon 19 and/or point mutations in exon 21 (L858R). Th1 helper cellular immunity is critical for immunotherapy-mediated tumor regression. We have previously characterized an MHC-II-restricted EGFR multi-peptide vaccine (two peptides: “SCVRACGADSYEMEEDGVRK” and “VWSYGVTVWELMTFGSKPY”) (EGFR-V) that targets the EGFR protein and decreases EGFR-driven lung tumorigenesis by ~80% in EGFRL858R transgenic mice that were vaccinated before doxycycline-induction of the EGFR protein [1]. However, diminished efficacy was observed when this MHC-II-restricted EGFR multi-peptide vaccine was given two weeks after doxycycline induction of the EGFR protein, suggesting that expression of the EGFR oncoprotein significantly increased the immunosuppressive microenvironment. JHU083, an orally bioavailable glutamine antagonist, has recently been shown to not only inhibit tumor growth but also boost anti-cancer immunity [2]. To determine if JHU083 could potentiate the efficacy of EGFR vaccine in a post-initiation setting, we evaluated in vivo antitumor efficacy of EGFR vaccine combined with JHU083 using an EGFRL858R transgenic mouse model. JHU083 inhibited tumor burden by 31% and EGFR vaccine suppressed tumor burden by 33%, whereas combining JHU083 with anti-EGFR peptide vaccine had an additive effect (54% tumor inhibition). Mechanistically, JHU083 by itself, markedly reduced the immunosuppressive monocytic myeloid-derived suppressor cells (MDSCs) in lung tissues. The combination of JHU083 and the vaccine significantly reduced Tregs in lung tissues. The anti-EGFR vaccine primarily induced expansion of antigen specific antitumor CD4+ effector T cells. Long term administration of JHU-083 did not decrease bodyweight in mice. Together with previous results, these data suggest that JHU083 could be used to reshape the tumor microenvironment toward one that enhances antitumor T cell responses and could further enhance the efficacy of the EGFR anticancer vaccine.

Fish Oil, Se Yeast, and Micronutrient-Enriched Nutrition as Adjuvant Treatment during Target Therapy in a Murine Model of Lung Cancer.

Despite the effectiveness of primary treatment modalities for cancer, the side effects of treatments, medication resistance, and the deterioration of cachexia after disease progression lead to poor prognosis. A supportive treatment modality to overcome these limitations would be considered a major breakthrough. Here, we used two different target drugs to demonstrate whether a nutraceutical formula (fish oil, Se yeast, and micronutrient-enriched nutrition; NuF) can interfere with cancer cachexia and improve drug efficacy. After Lewis lung cancer (LLC) tumor injection, the C57BL/6 mice were orally administered targeted therapy drugs Iressa and Sutent alone or combined with NuF for 27 days. Sutent administration effectively inhibited tumor size but increased the number of lung metastases in the long term. Sutent combined with NuF had no significant difference in tumor weight and metastasis compare with Sutent alone. However, NuF slightly attenuated metastases number in lung may via mesenchymal marker N-cadherin suppression. NuF otherwise increased epithelial-like marker E-cadherin expression and induce NO-mediated intrinsic apoptotic pathway in tumor cells, thereby strengthening the ability of the targeted therapy drug Iressa for inhibiting tumor progression. Our results demonstrate that NuF can promote the anticancer effect of lung cancer to targeted therapy, especially in Iressa, by inhibiting HIF-1α and epithelial-mesenchymal transition (EMT) and inducing the apoptosis of lung cancer cells. Furthermore, NuF attenuates cancer-related cachectic symptoms by inhibiting systemic oxidative stress.

Tumour draining lymph node-generated CD8 T cells play a role in controlling lung metastases after a primary tumour is removed but not when adjuvant immunotherapy is used

Surgical resection of cancer remains the frontline therapy for millions of patients annually, but post-operative recurrence is common, with a relapse rate of around 45% for non-small cell lung cancer. The tumour draining lymph nodes (dLN) are resected at the time of surgery for staging purposes, and this cannot be a null event for patient survival and future response to immune checkpoint blockade treatment. This project investigates cancer surgery, lymphadenectomy, onset of metastatic disease, and response to immunotherapy in a novel model that closely reflects the clinical setting. In a murine metastatic lung cancer model, primary subcutaneous tumours were resected with associated dLNs remaining intact, completely resected or partially resected. Median survival after surgery was significantly shorter with complete dLN resection at the time of surgery (49 days (95%CI)) compared to when lymph nodes remained intact (> 88 days; p < 0.05). Survival was partially restored with incomplete lymph node resection and CD8 T cell dependent. Treatment with aCTLA4 whilst effective against the primary tumour was ineffective for metastatic lung disease. Conversely, aPD-1/aCD40 treatment was effective in both the primary and metastatic disease settings and restored the detrimental effects of complete dLN resection on survival. In this pre-clinical lung metastatic disease model that closely reflects the clinical setting, we observe decreased frequency of survival after complete lymphadenectomy, which was ameliorated with partial lymph node removal or with early administration of aPD-1/aCD40 therapy. These findings have direct relevance to surgical lymph node resection and adjuvant immunotherapy in lung cancer, and perhaps other cancer, patients.

Encapsulation of a CpG oligonucleotide in cationic liposomes enhances its local antitumor activity following pulmonary delivery in a murine model of metastatic lung cancer

Immunotherapy brings new hope to the fight against lung cancer. General immunostimulatory agents represent an immunotherapy strategy that has demonstrated efficacy with limited toxicity when delivered intratumorally. The goal of this study was to enhance the antitumor efficacy of unmethylated oligodeoxynucleotides containing CpG motifs (CpG) and polyinosinic-polycytidylic acid (poly I:C) double-stranded RNA following their local delivery in lung cancer by encapsulating them in liposomes. Liposomes encapsulation of nucleic acids could increase their uptake by lung phagocytes and thereby the activation of toll-like receptors within endosomes. Liposomes were prepared using a cationic lipid, dioleoyltrimethylammoniumpropane (DOTAP), and dipalmitoylphosphatidylcholine (DPPC), the main phospholipid in lung surfactant. The liposomes permanently entrapped CpG but could not efficiently withhold poly I:C. Both poly I:C and CpG delayed tumor growth in the murine B16F10 model of metastatic lung cancer. However, only CpG increased IFN-γ levels in the lungs. Pulmonary administration of CpG was superior to its intraperitoneal injection to slow the growth of lung metastases and to induce the production of granzyme B, a pro-apoptotic protein, and IFNγ, MIG and RANTES, T helper type 1 cytokines and chemokines, in the lungs. These antitumor activities of CpG were strongly enhanced by CpG encapsulation in DOTAP/DPPC liposomes. Delivery of low CpG doses to the lungs induced increased inflammation markers in the airspaces but the inflammation did not reach the systemic compartment in a significant manner. These data support the use of a delivery carrier to strengthen CpG antitumor activity following its pulmonary delivery in lung cancer.

Implementation of CRISPR/Cas9 Genome Editing to Generate Murine Lung Cancer Models That Depict the Mutational Landscape of Human Disease.

Lung cancer is the most common cancer worldwide and the leading cause of cancer-related deaths in both men and women. Despite the development of novel therapeutic interventions, the 5-year survival rate for non-small cell lung cancer (NSCLC) patients remains low, demonstrating the necessity for novel treatments. One strategy to improve translational research is the development of surrogate models reflecting somatic mutations identified in lung cancer patients as these impact treatment responses. With the advent of CRISPR-mediated genome editing, gene deletion as well as site-directed integration of point mutations enabled us to model human malignancies in more detail than ever before. Here, we report that by using CRISPR/Cas9-mediated targeting of Trp53 and KRas, we recapitulated the classic murine NSCLC model Trp53fl/fl:lsl-KRasG12D/wt. Developing tumors were indistinguishable from Trp53fl/fl:lsl-KRasG12D/wt-derived tumors with regard to morphology, marker expression, and transcriptional profiles. We demonstrate the applicability of CRISPR for tumor modeling in vivo and ameliorating the need to use conventional genetically engineered mouse models. Furthermore, tumor onset was not only achieved in constitutive Cas9 expression but also in wild-type animals via infection of lung epithelial cells with two discrete AAVs encoding different parts of the CRISPR machinery. While conventional mouse models require extensive husbandry to integrate new genetic features allowing for gene targeting, basic molecular methods suffice to inflict the desired genetic alterations in vivo. Utilizing the CRISPR toolbox, in vivo cancer research and modeling is rapidly evolving and enables researchers to swiftly develop new, clinically relevant surrogate models for translational research.

Dual Inhibition of MEK and AXL Targets Tumor Cell Heterogeneity and Prevents Resistant Outgrowth Mediated by the Epithelial-to-Mesenchymal Transition in NSCLC.

The epithelial-to-mesenchymal transition (EMT) is a dynamic epigenetic reprogramming event that occurs in a subset of tumor cells and is an initiating step toward invasion and distant metastasis. The process is reversible and gives plasticity to cancer cells to survive under variable conditions, with the acquisition of cancer stem cell-like characteristics and features such as drug resistance. Therefore, understanding survival dependencies of cells along the phenotypic spectrum of EMT will provide better strategies to target the spatial and temporal heterogeneity of tumors and prevent their ability to bypass single-inhibitor treatment strategies. To address this, we integrated the data from a selective drug screen in epithelial and mesenchymal KRAS/p53 (KP)-mutant lung tumor cells with separate datasets including reverse-phase protein array and an in vivo shRNA dropout screen. These orthogonal approaches identified AXL and MEK as potential mesenchymal and epithelial cell survival dependencies, respectively. To capture the dynamicity of EMT, incorporation of a dual fluorescence EMT sensor system into murine KP lung cancer models enabled real-time analysis of the epigenetic state of tumor cells and assessment of the efficacy of single agent or combination treatment with AXL and MEK inhibitors. Both two- and three-dimensional culture systems and in vivo models revealed that this combination treatment strategy of MEK plus AXL inhibition synergistically killed lung cancer cells by specifically targeting each phenotypic subpopulation. In conclusion, these results indicate that cotargeting the specific vulnerabilities of EMT subpopulations can prevent EMT-mediated drug resistance, effectively controlling tumor cell growth and metastasis. SIGNIFICANCE: This study shows that a novel combination of MEK and AXL inhibitors effectively bypasses EMT-mediated drug resistance in KRAS/p53-mutant non-small cell lung cancer by targeting EMT subpopulations, thereby preventing tumor cell survival.