Mouse Lung Carcinoma Research Articles

Single-cell RNA sequencing reveals enhanced antitumor immunity after combined application of PD-1 inhibitor and Shenmai injection in non-small cell lung cancer

BackgroundImmune checkpoint inhibitors (ICIs) have altered the clinical management of non-small cell lung cancer (NSCLC). However, the low response rate, severe immune-related adverse events (irAEs), and hyperprogressive disease following ICIs monotherapy require attention. Combination therapy may overcome these limitations and traditional Chinese medicine with immunomodulatory effects provides a promising approach. Shenmai injection (SMI) is a clinically effective adjuvant treatment for cancer with chemotherapy and radiotherapy. Therefore, the combined effects and mechanisms of SMI and programmed death-1 (PD-1) inhibitor against NSCLC was focused on this study.MethodsA Lewis lung carcinoma mouse model and a lung squamous cell carcinoma humanized mouse model were used to investigate the combined efficacy and safety of SMI and PD-1 inhibitor. The synergistic mechanisms of the combination therapy against NSCLC were explored using single-cell RNA sequencing. Validation experiments were performed using immunofluorescence analysis, in vitro experiment, and bulk transcriptomic datasets.ResultsIn both models, combination therapy alleviated tumor growth and prolonged survival without increasing irAEs. The GZMAhigh and XCL1high natural killer (NK) cell subclusters with cytotoxic and chemokine signatures increased in the combination therapy, while malignant cells from combination therapy were mainly in the apoptotic state, suggesting that mediating tumor cell apoptosis through NK cells is the main synergistic mechanisms of combination therapy. In vitro experiment confirmed that combination therapy increased secretion of Granzyme A by NK cells. Moreover, we discovered that PD-1 inhibitor and SMI combination blocked inhibitory receptors on NK and T cells and restores their antitumoral activity in NSCLC better than PD-1 inhibitor monotherapy, and immune and stromal cells exhibited a decrease of angiogenic features and attenuated cancer metabolism reprogramming in microenvironment of combination therapy.ConclusionsThis study demonstrated that SMI reprograms tumor immune microenvironment mainly by inducing NK cells infiltration and synergizes with PD-1 inhibitor against NSCLC, suggested that targeting NK cells may be an important strategy for combining with ICIs.4EoQVoCxPUSr8SKnFMutzeVideo

Dual inhibition of IDO1/TDO2 enhances anti-tumor immunity in platinum-resistant non-small cell lung cancer

BackgroundThe impact of non-small cell lung cancer (NSCLC) metabolism on the immune microenvironment is not well understood within platinum resistance. We have identified crucial metabolic differences between cisplatin-resistant (CR) and cisplatin-sensitive (CS) NSCLC cells with elevated indoleamine 2,3-dioxygenase-1 (IDO1) activity in CR, recognized by increased kynurenine (KYN) production.MethodsCo-culture, syngeneic, and humanize mice models were utilized. C57BL/6 mice were inoculated with either Lewis lung carcinoma mouse cells (LLC) or their platinum-resistant counterpart (LLC-CR) cells. Humanized mice were inoculated with either A (human CS cells) or ALC (human CR cells). Mice were treated with either IDO1 inhibitor or TDO2 (tryptophan 2,3-dioxygenase-2) inhibitor at 200 mg/kg P.O. once a day for 15 days; or with a new-in-class, IDO1/TDO2 dual inhibitor AT-0174 at 170 mg/kg P.O. once a day for 15 days with and without anti-PD1 antibody (10 mg/kg, every 3 days). Immune profiles and KYN and tryptophan (TRP) production were evaluated.ResultsCR tumors exhibited a more highly immunosuppressive environment that debilitated robust anti-tumor immune responses. IDO1-mediated KYN production from CR cells suppressed NKG2D on immune effector natural killer (NK) and CD8+ T cells and enhanced immunosuppressive populations of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Importantly, while selective IDO1 inhibition attenuated CR tumor growth, it concomitantly upregulated the TDO2 enzyme. To overcome the compensatory induction of TDO2 activity, we employed the IDO1/TDO2 dual inhibitor, AT-0174. Dual inhibition of IDO1/TDO2 in CR mice suppressed tumor growth to a greater degree than IDO1 inhibition alone. Significant enhancement in NKG2D frequency on NK and CD8+ T cells and a reduction in Tregs and MDSCs were observed following AT-1074 treatment. PD-L1 (programmed death-ligand-1) expression was increased in CR cells; therefore, we assessed dual inhibition + PD1 (programmed cell death protein-1) blocking and report profound anti-tumor growth and improved immunity in CR tumors which in turn extended overall survival in mice.ConclusionOur study reports the presence of platinum-resistant lung tumors that utilize both IDO1/TDO2 enzymes for survival, and to escape immune surveillance as a consequence of KYN metabolites. We also report early in vivo data in support of the potential therapeutic efficacy of the dual IDO1/TDO2 inhibitor AT-0174 as a part of immuno-therapeutic treatment that disrupts tumor metabolism and enhances anti-tumor immunity.

Eicosapentaenoic Acid-Enriched Phospholipids Alleviate Skeletal Muscle Atrophy in Lewis Lung Carcinoma Mouse Model.

Skeletal muscle atrophy is a critical feature of cancer-associated cachexia (CAC) and it is responsible for poor quality of life and high mortality in cancer patients. The previous study demonstrates that eicosapentaenoic acid-enriched phospholipids (EPA-PL) prevent body weight loss in a mouse model of CAC. However, the role of EPA-PL on cancer-induced skeletal muscle atrophy remains unclear. In the present study, a Lewis lung carcinoma (LLC) mouse model is established, then the effect and underlying mechanism of EPA-PL on skeletal muscle atrophy in LLC-bearing mice are investigated. The results reveal that EPA-PL treatment significantly attenuates skeletal muscle atrophy in LLC-bearing mice, as evidenced by suppressing the reductions of skeletal muscle mass, myofiber cross-sectional area, and grip strength. Besides, the study finds that EPA-PL alleviated cancer-induced skeletal muscle atrophy via balancing muscle protein degradation and synthesis, inhibiting type I oxidative muscle fibers atrophy, and promoting mitochondrial function. Furthermore, the results also indicate that EPA-PL may counteract skeletal muscle atrophy in LLC mouse model via a sirtuin 1-dependent mechanism. These findings provide evidence that EPA-PL may be beneficial as a nutritional supplement for prevention and treatment of cancer-induced skeletal muscle atrophy.

Macrophage CD5L is a target for cancer immunotherapy

Reprogramming of immunosuppressive tumor-associated macrophages (TAMs) presents an attractive therapeutic strategy in cancer. The aim of this study was to explore the role of macrophage CD5L protein in TAM activity and assess its potential as a therapeutic target. Monoclonal antibodies (mAbs) against recombinant CD5L were raised by subcutaneous immunization of BALB/c mice. Peripheral blood monocytes were isolated from healthy donors and stimulated with IFN/LPS, IL4, IL10, and conditioned medium (CM) from different cancer cell lines in the presence of anti-CD5L mAb or controls. Subsequently, phenotypic markers, including CD5L, were quantified by flow cytometry, IF and RT-qPCR. Macrophage CD5L protein expression was studied in 55 human papillary lung adenocarcinoma (PAC) samples by IHC and IF. Anti-CD5L mAb and isotype control were administered intraperitoneally into a syngeneic Lewis Lung Carcinoma mouse model and tumor growth was measured. Tumor microenvironment (TME) changes were determined by flow cytometry, IHC, IF, Luminex, RNAseq and RT-qPCR. Cancer cell lines CM induced an immunosuppressive phenotype (increase in CD163, CD206, MERTK, VEGF and CD5L) in cultured macrophages. Accordingly, high TAM expression of CD5L in PAC was associated with poor patient outcome (Log-rank (Mantel-Cox) test p=0.02). We raised a new anti-CD5L mAb that blocked the immunosuppressive phenotype of macrophages invitro. Its administration invivo inhibited tumor progression of lung cancer by altering the intratumoral myeloid cell population profile and CD4+ T-cell exhaustion phenotype, thereby significantly modifying the TME and increasing the inflammatory milieu. CD5L protein plays a key function in modulating the activity of macrophages and their interactions within the TME, which supports its role as a therapeutic target in cancer immunotherapy. For a full list of funding bodies, please see the Acknowledgements.

PCSK9 regulates the efficacy of immune checkpoint therapy in lung cancer.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) secreted by tumors was reported as a deleterious factor that led to the reduction of lymphocyte infiltration and the poorer efficacy of ICIs in vivo. This study aimed to explore whether PCSK9 expression in tumor tissue could predict the response of advanced non-small cell lung cancer (NSCLC) to anti-PD-1 immunotherapy and the synergistic antitumor effect of the combination of the PCSK9 inhibitor with the anti-CD137 agonist. One hundred fifteen advanced NSCLC patients who received anti-PD-1 immunotherapy were retrospectively studied with PCSK9 expression in baseline NSCLC tissues detected by immunohistochemistry (IHC). The mPFS of the PCSK9lo group was significantly longer than that of the PCSK9hi group [8.1 vs. 3.6 months, hazard ratio (HR): 3.450; 95% confidence interval (CI), 2.166-5.496]. A higher objective response rate (ORR) and a higher disease control rate (DCR) were observed in the PCSK9lo group than in the PCSK9hi group (54.4% vs. 34.5%, 94.7% vs. 65.5%). Reduction and marginal distribution of CD8+ T cells were observed in PCSK9hi NSCLC tissues. Tumor growth was retarded by the PCSK9 inhibitor and the anti-CD137 agonist alone in the Lewis lung carcinoma (LLC) mice model and further retarded by the PCSK9 inhibitor in combination with the CD137 agonist with long-term survival of the host mice with noticeable increases of CD8+ and GzmB+ CD8+ T cells and reduction of Tregs. Together, these results suggested that high PCSK9 expression in baseline tumor tissue was a deleterious factor for the efficacy of anti-PD-1 immunotherapy in advanced NSCLC patients. The PCSK9 inhibitor in combination with the anti-CD137 agonist could not only enhance the recruitment of CD8+ and GzmB+ CD8+ T cells but also deplete Tregs, which may be a novel therapeutic strategy for future research and clinical practice.

Highlights from Recent Cancer Literature.

Highlights from Recent Cancer Literature.

Synergistic effects of radiotherapy with JNK inhibitor-incorporated nanoparticle in an intracranial Lewis Lung Carcinoma Mouse Models.

Radiosurgery has been recognized as a reasonable treatment for metastatic brain tumors. Increasing the radiosensitivity and synergistic effects are possible ways to improve the therapeutic efficacy of specific regions of tumors. c-Jun-N-terminal kinase (JNK) signaling regulates H2AX phosphorylation to repair radiation-induced DNA breakage. We previously showed that blocking JNK signaling influenced radiosensitivity in vitro and in an in vivo mouse tumor model. Drugs can be incorporated into nanoparticles to produce a slow-release effect. This study assessed JNK radiosensitivity following the slow release of the JNK inhibitor SP600125 from a poly (DL-lactide-co-glycolide) (LGEsese) block copolymer in a brain tumor model. A LGEsese block copolymer was synthesized to fabricate SP600125-incorporated nanoparticles by nanoprecipitation and dialysis methods. The chemical structure of the LGEsese block copolymer was confirmed by 1H nuclear magnetic resonance (NMR) spectroscopy. The physicochemical and morphological properties were observed by transmission electron microscopy (TEM) imaging and measured with particle size analyzer. The blood-brain barrier (BBB) permeability to the JNK inhibitor was estimated by BBBflammaTM 440-dye-labeled SP600125. The effects of the JNK inhibitor were investigated using SP600125-incorporated nanoparticles and by optical bioluminescence, magnetic resonance imaging (MRI), and a survival assay in a mouse brain tumor model for Lewis lung cancer (LLC)-Fluc cells. DNA damage was estimated by histone γH2AX expression and apoptosis was assessed by the immunohistochemical examination of cleaved caspase 3. The SP600125-incorporated nanoparticles of the LGEsese block copolymer were spherical and released SP600125 continuously for 24h. The use of BBBflammaTM 440-dye-labeled SP600125 demonstrated the ability of SP600125 to cross the BBB. The blockade of JNK signaling with SP600125-incorporated nanoparticles significantly delayed mouse brain tumor growth and prolonged mouse survival after radiotherapy. γH2AX, which mediates DNA repair protein, was reduced and the apoptotic protein cleaved-caspase 3 was increased by the combination of radiation and SP600125-incorporated nanoparticles.

Arsenic trioxide promotes ERK1/2-mediated phosphorylation and degradation of BIMEL to attenuate apoptosis in BEAS-2B cells

Inorganic arsenic is highly toxic, widely distributed in the human environment and may result in multisystem diseases and several types of cancers. The BCL-2-interacting mediator of cell death protein (BIM) is a key modulator of the intrinsic apoptosis pathway. Interestingly, in the present study, we found that arsenic trioxide (As2O3) decreased BIMEL levels in human bronchial epithelial cell line BEAS-2B and increased BIMEL levels in human lung carcinoma cell line A549 and mouse Sertoli cell line TM4. Mechanismly, the 26S proteasome inhibitors MG132 and bortezomib could effectively inhibit BIMEL degradation induced by As2O3 in BEAS-2B cells. As2O3 activated extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways, but only the ERK1/2 MAPK inhibitor PD98059 blocked BIMEL degradation induced by As2O3. Furthermore, As2O3 induced-phosphorylation of BIMEL at multiple sites was inhibited by ERK1/2 MAPK inhibitor PD98059. Inhibition of As2O3-induced ERK1/2 MAPK phosphorylation increased the levels of BIMEL and cleaved-caspase-3 proteins and decreased BEAS-2B cell viability. As2O3 also markedly mitigated tunicamycin-induced apoptosis of BEAS-2B cells by increasing ERK1/2 phosphorylation and BIMEL degradation. Our results suggest that As2O3-induced activation of the ERK1/2 MAPK pathway increases phosphorylation of BIMEL and promotes BIMEL degradation, thereby alleviating the role of apoptosis in As2O3-induced cell death. This study provides new insights into how to maintain the survival of BEAS-2B cells before malignant transformation induced by high doses of As2O3.

The atypical β-blocker S-oxprenolol reduces cachexia and improves survival in a rat cancer cachexia model.

Beta-blockers and selected stereoisomers of beta-blockers, like bisoprolol and S-pindolol (ACM-001), have been shown to be effective in preclinical cancer cachexia models. Here, we tested the efficacy of stereoisomers of oxprenolol in two preclinical models of cancer cachexia-the Yoshida AH-130 rat model and the Lewis lung carcinoma (LLC) mouse model. In the Yoshida AH130 hepatoma rat cancer cachexia model and compared with placebo, 50mg/kg/d S-oxprenolol (HR: 0.49, 95% CI: 0.28-0.85, P=0.012) was superior to 50mg/kg/d R-oxprenolol (HR: 0.83, 95% CI 0.38-1.45, P=0.51) in reducing mortality (=reaching ethical endpoints). Combination of the three doses (12.5, 25 and 50mg/kg/d) that had a significant effect on body weight loss in the S-oxprenolol groups vs the same combination of the R-oxprenolol groups lead to a significantly improved survival of S-oxprenolol vs R-oxprenolol (HR: 1.61, 95% CI: 1.08-2.39, P=0.0185). Interestingly, there is a clear dose dependency in S-oxprenolol-treated (5, 12.5, 25 and 50mg/kg/d) groups, which was not observed in groups treated with R-oxprenolol. A dose-dependent attenuation of weight and lean mass loss by S-oxprenolol was seen in the Yoshida rat model, whereas R-oxprenolol had only had a significant effect on fat mass. S-oxprenolol also non-significantly reduced weight loss in the LLC model and also improved muscle function (grip strength 428±25 and 539±37g/100g body weight for placebo and S-oxprenolol, respectively). However, there was only a minor effect on quality of life indicators food intake and spontaneous activity in the Yoshida model (25mg/kg/S-oxprenolol: 11.9±2.5g vs placebo: 4.9±0.8g, P=0.013 and also vs 25mg/kg/d R-oxprenolol: 7.5±2.6g, P=0.025). Both enantiomers had no effects on cardiac dimensions and function at the doses used in this study. Western blotting of proteins involved in the anabolic/catabolic homoeostasis suggest that anabolic signalling is persevered (IGF-1 receptor, Akt) and catabolic signalling is inhibited (FXBO-10, TRAF-6) by S-pindolol, but not he R-enantiomer. Expression of glucose transporters Glut1 and Glut 4 was similar in all groups, as was AMPK. S-oxprenolol is superior to R-oxprenolol in cancer cachexia animal models and shows promise for a human application in cancer cachexia.

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.

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.

Experimental photodynamic therapy in the treatment of lung tumor.

e20515 Background: Study of the effect of photodynamic therapy (PDT) with Photoditazine using a high-energy laser at various periods after tumor transplantation on the dynamics of growth of experimental Lewis lung carcinoma in mice. Methods: The study included 176 male C57Bl6 mice weighing 18-20 g with Lewis lung carcinoma (LLC) transplanted subcutaneously. The animals received experimental PDT sequential overlapping of fields of influence (662 nm, 3 W, Photoditazine 25 mg/kg, 300 J/cm2). Animals received PDT on day 11 after the transplantation (experimental series I) and on day 21 (experimental series II). Experimental groups were divided in dependence on the exposure area (to the primary tumor -1gr, thorax (area of metastases)-2 gr and to both zones-3 gr). Animals with tumors without PDT were used as controls. Results: PDT with Photoditazine using the 3W laser stimulated the growth of primary tumor node. The degree of stimulation depended on the time of PDT. Increase in tumor sizes differed already on day 7 after PDT at various periods of the LCC growth. During PDT on the 11th day from the moment of tumor inoculation, the percentage of growth on the 7th day after the end of treatment was 60.7; 69.6 and 86.7% in groups 1, 2 and 3, respectively. In the groups of animals that underwent PDT on the 21st day of the growth of the grafted tumor on the 7th day after the end of treatment, the percentage of tumor growth was 20; 35.6 and 24.4% in groups 1, 2 and 3, respectively. Increase in the median survival was registered in experimental animals with PDT on 22.6% compared to control. A smaller increase in tumor volume was observed in animals receiving PDT to the primary tumor, and the group receiving therapy to both areas was in between. Conclusions: The study was shown that with an earlier initiation of PDT, a greater stimulation of tumor growth was revealed than with therapy at a later date. The revealed characteristics of the development of experimental LLC at PDT exposure allows further selection of parameters for photodynamic therapy regimens.

The effect of the iron chelator Deferasirox in combination with Cisplatin chemotherapy against lung carcinoma

The preferred treatment for patients with lung carcinoma (LC) is platinum‐based regimens, eg., Cisplatin (CisPt). Unfortunately, despite improvements in LC outcomes, many of these tumors metastasize and develop resistance against such Pt‐drugs, inducing abysmally low survival prognosis. Due to this, there is an urgent need to develop alternative therapies for LC mostly focused on overcome or diminish chemoresistance and inhibit the metastatic spread. FDA‐approved drugs which target cancer metabolic pathways are considered as repurposing drugs that could diminish these adverse effects. Deferasirox (Def), an FDA‐approved iron chelation therapy, is a drug under study for cancer therapy due to iron plays a key role in cell growth and energy production in cancer. Herein, we determined the effect of deferasirox (Def) alone and in combination with CisPt on the genes related to chemoresistance and metastasis in LC cells. Viability assay results showed that Def has synergistic cytotoxic effects in combination with CisPt after 24 h of incubation in A549 LC cells. qPCR gene expression studies showed that Def induced a significant downregulation of EGFR, VEGF, MMP9, Pgp, P53, NDRG1, CHD4 genes in A549 cells. In addition, we determined that Def induce the production of reactive oxygen species in LC. Furthermore, in in vivo studies performed in Lewis Lung Carcinoma mouse model, Def co‐administered with CisPt showed tumor size reduction. The results obtained from this study will impact the field of lung cancer therapy adding knowledge on the Def cellular mechanisms and genetic regulation against chemoresistance and metastasis processes.

Air pollution particles hijack peroxidasin to disrupt immunosurveillance and promote lung cancer.

Although fine particulate matter (FPM) in air pollutants and tobacco smoke is recognized as a strong carcinogen and global threat to public health, its biological mechanism for inducing lung cancer remains unclear. Here, by investigating FPM's bioactivities in lung carcinoma mice models, we discover that these particles promote lung tumor progression by inducing aberrant thickening of tissue matrix and hampering migration of antitumor immunocytes. Upon inhalation into lung tissue, these FPM particles abundantly adsorb peroxidasin (PXDN) - an enzyme mediating type IV collagen (Col IV) crosslinking - onto their surface. The adsorbed PXDN exerts abnormally high activity to crosslink Col IV via increasing the formation of sulfilimine bonds at the NC1 domain, leading to an overly dense matrix in the lung tissue. This disordered structure decreases the mobility of cytotoxic CD8+ T lymphocytes into the lung and consequently impairs the local immune surveillance, enabling the flourishing of nascent tumor cells. Meanwhile, inhibiting the activity of PXDN abolishes the tumor-promoting effect of FPM, indicating the key impact of aberrant PXDN activity on the tumorigenic process. In summary, our finding elucidates a new mechanism for FPM-induced lung tumorigenesis and identifies PXDN as a potential target for treatment or prevention of the FPM-relevant biological risks.

181P Anlotinib enhances the antitumor activity of high-dose irradiation combined with anti-PD-L1 by potentiating the tumor immune microenvironment in murine lung cancer

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.

32P Anlotinib enhances the antitumor activity of high-dose irradiation combined with anti-PD-L1 by potentiating the tumor immune microenvironment in murine lung cancer

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.

Folate-Decorated Cross-Linked Cytochrome c Nanoparticles for Active Targeting of Non-Small Cell Lung Carcinoma (NSCLC).

The folate receptor alpha (FR), which is overexpressed in solid tumors including NSCLC, can be utilized for active tumor targeting to afford more effective cancer therapies. In this context, cytochrome c (Cyt c) has drawn attention to cancer research because it is non-toxic, yet, when delivered to the cytoplasm of cancer cells, can kill them by inducing apoptosis. Cyt c nanoparticles (NPs, 169 ± 9 nm) were obtained by solvent precipitation with acetonitrile, and stabilized by reversible homo-bifunctional crosslinking to accomplish a Cyt-c-based drug delivery system that combines stimulus-responsive release and active targeting. Cyt c was released under intracellular redox conditions, due to an S–S bond in the NPs linker, while NPs remained intact without any release under extracellular conditions. The NP surface was decorated with a hydrophilic folic acid–polyethylene glycol (FA–PEG) polymer for active targeting. The FA-decorated NPs specifically recognized and killed cancer cells (IC50 = 47.46 µg/mL) that overexpressed FR, but showed no toxicity against FR-negative cells. Confocal microscopy confirmed the preferential uptake and apoptosis induction of our NPs by FR-positive cancer cells. In vivo experiments using a Lewis lung carcinoma (LLC) mouse model showed visible NP accumulation within the tumor and inhibited the growth of LLC tumors.

Atractylenolide II inhibits tumor-associated macrophages (TAMs)-induced lung cancer cell metastasis

Objective M2-like tumor-associated macrophages (TAMs) play a crucial role in promoting tumor proliferation, angiogenesis, and metastasis. In the current study, we investigated the relationship between macrophage polarization and the antitumor effect of Atractylenolide II (AT-II) in lung cancer cells. Materials and methods Cell viability, migration, and invasion were determined by MTT assay, wound healing assay, and transwell assay, respectively. Flow cytometry analysis showed the percentage of CD206+ cells. Gene expression was determined by real-time PCR, western blotting, and immunofluorescence staining. Lewis lung carcinoma mouse xenograft and metastasis models were used to examine the effects of AT-II on lung cancer in vivo. Results AT-II (2.5 and 5 µM) did not cause significant inhibition of A549 cell viability but markedly inhibited IL-4/IL-13-induced M2-like polarization, evidenced by the decreased expression of the M2 surface marker CD206, down-regulation of specific M2-marker genes (Arg-1, IL-10 and TGF-β) as well as inhibition of M2 macrophages-mediated invasion and migration of A549 cells. In addition, AT-II inhibited IL-4/IL-13-induced activation of the STAT6 signaling pathway that is vital in the M2-like polarization of macrophages. In animal models, administration of AT-II (50 mg kg−1, i.g., QD for 21 days) significantly inhibited tumor growth, reduced pulmonary metastatic nodules, and down-regulated the percentages of M2 macrophages (F4/80+ and CD206+) in total macrophages (F4/80+) in tumor tissues and pulmonary metastatic nodules. Conclusions AT-II effectively inhibits M2-like polarization, thereby inhibiting lung cancer cell metastasis both in vivo and in vitro, revealing a novel potential strategy for the antitumor effect of AT-II.

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

Background 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. Methods 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. Results 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 and 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 downregulated 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. Conclusion 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.

Network Pharmacology and Experimental Verification to Explore the Potential Mechanism of Yin-Huo-Tang for Lung Adenocarcinoma Recurrence.

PurposeYin-Huo-Tang (YHT) is a classic traditional Chinese prescription, used to prevent lung adenocarcinoma (LUAD) relapse by “nourishing yin and clearing heat”. In this study, the mechanism of YHT in LUAD recurrence was investigated.MethodsFirstly, the bioactive compounds and targets of YHT, as well as related targets of LUAD recurrence, were collected from public databases. The protein–protein interaction network, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed to find the pivotal compounds, hub genes, functional annotation and main pathways. Subsequently, RNA sequencing of recurrent tumor tissues from Lewis lung carcinoma mice treated with YHT was used to explore the main pathways. At the same time, pathways screened by network pharmacology and RNA sequencing analysis were considered the most important pathways. Finally, liquid chromatography mass spectrometry was used to validate the pivotal active ingredients. Molecular docking technology was performed to validate the binding association between the hub genes and the pivotal active ingredients. PCR and WB analysis were used to validate the main pathways.ResultsThere were 128 active compounds and 419 targets interacting with YHT and LUAD recurrence. Network analysis identified 4 pivotal compounds, 28 hub genes and 30 main pathways. Sphingolipid signaling pathway was the common main pathway in network pharmacology and RNA sequencing results. The hub gene related to the sphingolipid signaling pathway was S1PR5. Qualitative phytochemical analysis confirmed the presence of 3 pivotal compounds, namely stigmasterol, nootkatone and ergotamine. The molecular docking verified that the pivotal compounds could good affinity with S1PR5. The PCR and WB analysis verified YHT suppressed Lewis lung cancer cells proliferation and migration by inhibiting the sphingolipid signaling pathway.ConclusionThe potential mechanism and therapeutic effect of YHT against the recurrence of LUAD may be ascribed to inhibition of the sphingolipid signaling pathway.