Mouse Lewis Lung Carcinoma Research Articles

Opaganib (ABC294640) Induces Immunogenic Tumor Cell Death and Enhances Checkpoint Antibody Therapy.

Antibody-based cancer drugs that target the checkpoint proteins CTLA-4, PD-1 and PD-L1 provide marked improvement in some patients with deadly diseases such as lung cancer and melanoma. However, most patients are either unresponsive or relapse following an initial response, underscoring the need for further improvement in immunotherapy. Certain drugs induce immunogenic cell death (ICD) in tumor cells in which the dying cells promote immunologic responses in the host that may enhance the in vivo activity of checkpoint antibodies. Sphingolipid metabolism is a key pathway in cancer biology, in which ceramides and sphingosine 1-phosphate (S1P) regulate tumor cell death, proliferation and drug resistance, as well as host inflammation and immunity. In particular, sphingosine kinases are key sites for manipulation of the ceramide/S1P balance that regulates tumor cell proliferation and sensitivity to radiation and chemotherapy. We and others have demonstrated that inhibition of sphingosine kinase-2 by the small-molecule investigational drug opaganib (formerly ABC294640) kills tumor cells and increases their sensitivities to other drugs and radiation. Because sphingolipids have been shown to regulate ICD, opaganib may induce ICD and improve the efficacy of checkpoint antibodies for cancer therapy. This was demonstrated by showing that in vitro treatment with opaganib increases the surface expression of the ICD marker calreticulin on a variety of tumor cell types. In vivo confirmation was achieved using the gold standard immunization assay in which B16 melanoma, Lewis lung carcinoma (LLC) or Neuro-2a neuroblastoma cells were treated with opaganib in vitro and then injected subcutaneously into syngeneic mice, followed by implantation of untreated tumor cells 7 days later. In all cases, immunization with opaganib-treated cells strongly suppressed the growth of subsequently injected tumor cells. Interestingly, opaganib treatment induced crossover immunity in that opaganib-treated B16 cells suppressed the growth of both untreated B16 and LLC cells and opaganib-treated LLC cells inhibited the growth of both untreated LLC and B16 cells. Next, the effects of opaganib in combination with a checkpoint antibody on tumor growth in vivo were assessed. Opaganib and anti-PD-1 antibody each slowed the growth of B16 tumors and improved mouse survival, while the combination of opaganib plus anti-PD-1 strongly suppressed tumor growth and improved survival (p < 0.0001). Individually, opaganib and anti-CTLA-4 antibody had modest effects on the growth of LLC tumors and mouse survival, whereas the combination of opaganib with anti-CTLA-4 substantially inhibited tumor growth and increased survival (p < 0.001). Finally, the survival of mice bearing B16 tumors was only marginally improved by opaganib or anti-PD-L1 antibody alone but was nearly doubled by the drugs in combination (p < 0.005). Overall, these studies demonstrate the ability of opaganib to induce ICD in tumor cells, which improves the antitumor activity of checkpoint antibodies.

ALKBH5 promotes the development of lung adenocarcinoma by regulating the polarization of M2 macrophages through CDCA4

ObjectiveLung adenocarcinoma (LUAD) is the most common subtype of lung cancer, with high morbidity and mortality. N6-methyladenosine (m6A) is an important regulator of LUAD progression. Here, we investigated the potential biological functions of ALKBH5 (a m6A demethylated enzyme) and cell division cycle associated protein 4 (CDCA4) in the progression of LUAD. MethodsThe expressions of CDCA4, METTL3, ALKBH5, FTO, YTHDC2 and YTHDC1 mRNA and proteins in LUAD and adjacent tissues, as well as NCI-H1299 and NCI-H157 cells were detected by RT-qPCR and western blot. Meanwhile, the role of ALKBH5 and CDCA4 in macrophage polarization was explored through tumor formation in Lewis lung carcinoma (LLC) mice and the co-culture system of NCI-H1299 and NCI-H157/THP-1 cells. Cell characterization was further analyzed. The expression of Ki-67 in tumor tissue was tested by immunohistochemistry. The scale of M1 and M2 macrophages was determined by flow cytometry. ResultsCDCA4 was significantly overexpressed in NCI-H1299 and NCI-H157 cell lines compared with BEAS-2B cells. The fold enrichment of CDCA4 m6A level in the overexpression (oe)-METTL3 or short hairpin (sh)-ALKBH5 cells was enhanced. Overexpression of CDCA4 promoted the cell viability, proliferation and migration, and inhibited apoptosis, which was reversed by sh-ALKBH5 intervention. Overexpression of YTHDC2 (not YTHDC1) inhibited the effect of CDCA4 on sh-ALKBH5 cells. sh-CDCA4 inhibited tumor growth and weight of LLC cells in mice, and promoted M1/M2 ratio in LLC mice and NCI-H1299/THP-1 and NCI-H157/THP-1 co-culture systems. Oe-CDCA4 promoted the volume and weight of tumor and inhibited the M1/M2 ratio of tumor tissue in LLC mice, but was reversed by sh-ALKBH5 intervention. Conclusionm6A demethylase ALKBH5 promotes the development of LUAD through CDCA4 regulation of malignant characterization and M1/M2 macrophage polarization.

Single Cell Analysis of Macrophage Heterogeneity and NK-Cell Exhaustion in Lewis Lung Cancer Xenograft Tumor

Stereotactic body radiation therapy (SBRT) has attracted much attention because of its ability to stimulate anti-tumor immune response. However, the mechanism of SBRT reprogramming the tumor microenvironment remains to be elucidated. Using Lewis lung carcinoma (LLC) xenograft mice model treated with SBRT (8Gy x 3F), multiplex assay was performed to measure serum chemokine levels, and single-cell RNA sequencing was performed to assess tumor microenvironment. The differential expression genes of each cell subcluster were identified by the "Find-All markers" function with default parameters provided by Seurat. Intercellular communication analysis was explored by using CellPhone DB package. The majority of serum chemokines involved macrophage recruitment, including CCL3, CCL4, CCL8, and CCL20, were highly secreted at 7 days after SBRT. Single-cell RNA sequencing of 108,741 cells were contained from 6 mouse Lewis lung carcinoma samples (n = 3 tumors for SBRT, n = 3 tumors pooled for SHAM). Besides Lewis cancer cells, myeloid cells were 57.61% ,70.82% in Sham-irradiation (SHAM) and SBRT while NT and T cells were 20.50%, 7.81% in SHAM and SBRT, respectively. When compared with SHAM group, upregulation of Ccl3, Ccl4, Ccl8 chemokine genes were observed in cancer cells of SBRT group. Differential expression genes analysis showed high expression level of Ccl8 (Log2FC 2.54, p<0.01) in cluster of Mrc1+macrophage. The SBRT group consisted of more Ccl8+Mrc1+macrophages (proportion 36.28% for SBRT, 27.44% for SHAM) and exhausted NK cells (proportion 22.56% for SBRT, 13.70% for SHAM). More importantly, intercellular communication analysis revealed a potential communication network between Ccl8+Mrc1+macrophages and exhausted NK cells. Our results provide a potential therapeutic strategy by disrupting Ccl8+ Mrc1+macrophages and NK-cell interaction to facilitate the stimulation of the anti-tumor immune response by SBRT.

Pharmacokinetic study of Tdp1 inhibitor resulted in a significant increase in antitumor effect in the treatment of Lewis lung carcinoma in mice by its combination with topotecan

We have previously shown that the Tdp1 inhibitor, enamine derivative of usnic acid, the agent OL9–116, enhances the antitumor activity of topotecan. In the present study, we developed and validated LC-MS/MS method for the quantification of OL9–116 in mouse whole blood and studied pharmacokinetics of the agent. The substance OL9–116 was shown to be stable in the whole blood in vitro. Sample preparation included two steps: mixing 10 µL of a blood sample with 10 µL of 0.2 M ZnSO4 aqueous solution, followed by protein precipitation with 100 µL of acetonitrile containing internal standard. Quantification of the compound was performed using SCIEX 6500 QTRAP mass spectrometer in MRM mode following chromatographic separation on a C8 reversed-phase column. The method was validated in terms of selectivity, linearity, accuracy, precision, recovery, and stability of the prepared sample. When the agent OL9–116 was administered intragastrically at a dose of 150 mg/kg, the maximum concentration in the blood (about 5000 ng/mL) was reached after 2–4 h followed by the distribution and elimination of the compound. A study of the antitumor activity of a combination of OL9–116 and topotecan against Lewis lung carcinoma revealed that administration of topotecan 3 h after OL9–116 resulted in the most pronounced antitumor effect compared to simultaneous or individual administration of both compounds.

Dendritic cells infected with recombinant adenoviral vector encoding mouse fibroblast activation protein-α and human livin α exert an antitumor effect against Lewis lung carcinoma in mice.

Fibroblast activation protein-α (FAP) and livin α are considered as cancer-associated fibroblasts (CAFs) and tumor-specific targets, respectively, for immunogenic tumor vaccines. This study is designed to decipher the antitumor effect of double-gene modified dendritic cells (DCs) on Lewis lung carcinoma (LLC). By encoding mouse FAP cDNA and human livin α (i.e., hlivin α) cDNA into recombinant adenoviral vector (rAd), rAd-FAP, rAd-hlivin α, and rAd-FAP/hlivin α were constructed, which were then transduced into mouse DCs. LLC-bearinig mice were immunized with the infected DCs (5 × 105 cells/mouse), followed by calculation of tumor volume and survival rate. The identification of CAFs from mouse LLC as well as the determination on expressions of FAP and livin α, was accomplished by western blot. Cytotoxic T lymphocyte assay was harnessed to assess the effect of the infected DCs on inducing splenic lymphocytes to lyse CAFs. DCs were successfully transduced with rAd-FAP/hlivin α in vitro. FAP was highly expressed in CAFs. CAFs were positive for α-SMA and negative for CD45 and CD31. Livin α level was upregulated in mouse LLC. Immunization with rAd-FAP/hlivin α-transduced DCs suppressed LLC volume and improved the survival of tumor-bearing mice. Immunization with rAd-FAP/hlivin α-transduced DCs enhanced the cytotoxic effect of splenic lymphocytes on LLC tumor-derived CAFs. Injection with rAd-FAP/hlivin α-transduced DCs promotes immune-enhanced tumor microenvironment by decreasing CAFs and suppresses tumor growth in LLC mouse models.

Muscle p62 stimulates the expression of antioxidant proteins alleviating cancer cachexia.

Oxidative stress plays an important role in skeletal muscle atrophy during cancer cachexia, and more glycolytic muscles are preferentially affected. Sequestosome1/SQSTM1 (i.e., p62), particularly when phosphorylated at Ser 349 (Ser 351 in mice), competitively binds to the Kelch-like ECH-associated protein 1 (Keap1) activating Nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 then stimulates the transcription of antioxidant/electrophile-responsive elements in target genes. However, a potential role for p62 in the protection of muscle wasting in cachexia remains to be determined. Here, using the well-established cachexia-inducing model of Lewis Lung Carcinoma (LLC) in mice we demonstrate higher expression of antioxidant proteins (i.e., NQO1, HO-1, GSTM1, CuZnSOD, MnSOD, and EcSOD) in the more oxidative and cachexia resistant soleus muscle than in the more glycolytic and cachexia prone extensor digitorum longus muscle. This was accompanied by higher p62 (total and phosphorylated) and nuclear Nrf2 levels in the soleus, which were paralleled by higher expression of proteins known to either phosphorylate or promote p62 phosphorylation (i.e., NBR1, CK1, PKCδ, and TAK1). Muscle-specific p62 gain-of-function (i.e., in p62 mTg mice) activated Nrf2 nuclear translocation and increased the expression of multiple antioxidant proteins (i.e., CuZnSOD, MnSOD, EcSOD, NQO1, and GSTM1) in glycolytic muscles. Interestingly, skeletal muscle Nrf2 haplodeficiency blunted the increases of most of these proteins (i.e., CuZnSOD, EcSOD, and NQO1) suggesting that muscle p62 stimulates antioxidant protein expression also via additional, yet to be determined mechanisms. Of note, p62 gain-of-function mitigated glycolytic muscle wasting in LLC-affected mice. Collectively, our findings identify skeletal muscle p62 as a potential therapeutic target for cancer cachexia.

Brief Report: Acetaminophen Reduces Neoadjuvant Chemoimmunotherapy Efficacy in Patients With NSCLC by Promoting Neutrophil Extracellular Trap Formation: Analysis From a Phase 2 Clinical Trial

IntroductionNeoadjuvant chemoimmunotherapy has recently been the standard of care for resectable locally advanced NSCLC. Factors affecting the neoadjuvant immunotherapy efficacy, however, remain elusive. Metabolites have been found to modulate immunity and associate with immunotherapeutic efficacy in advanced tumors. Therefore, we aimed to investigate the impact of plasma metabolites on the pathologic response after neoadjuvant chemoimmunotherapy. MethodsPatients with stage IIIA (N2) NSCLC who underwent neoadjuvant chemoimmunotherapy in a prospective phase 2 clinical trial (NCT04422392) were enrolled. Metabolomic profiling of the plasma before treatment was performed using liquid chromatography–mass spectrometry. A Lewis lung carcinoma mouse model was further used to investigate the underlying mechanisms. Proteomics and multiplexed immunofluorescence of the mice tumor were performed. ResultsA total of 39 patients who underwent three cycles of anti–programmed cell death-protein 1 (anti–PD-1) (sintilimab) and chemotherapy were included. The level of acetaminophen (APAP) was found to be significantly elevated in patients who did not achieve major pathologic response. The level of APAP remained an independent predictor for major pathologic response in multivariate logistic analysis. In the Lewis lung carcinoma mouse model, combination of APAP and anti–PD-1 treatment significantly reduced the treatment efficacy compared with anti–PD-1 treatment alone. Proteomics of the tumor revealed that myeloid leukocyte activation and neutrophil activation pathways were enriched after APAP treatment. Tumor microenvironment featuring analysis also revealed that the combination treatment group was characterized with more abundant neutrophil signature. Further multiplexed immunofluorescence confirmed that more neutrophil extracellular trap formation was observed in the combination treatment group. ConclusionsAPAP could impair neoadjuvant chemoimmunotherapy efficacy in patients with NSCLC by promoting neutrophil activation and neutrophil extracellular trap formation.

Amorphous Calcium Carbonate Shows Anti-Cancer Properties That are Attributed to Its Buffering Capacity.

Amorphous calcium carbonate (ACC) is a non-crystalline form of calcium carbonate, and it is composed of aggregated nano-size primary particles. Here, we evaluated its anti-cancer effect postulated relative to its buffering capabilities in lung cancer. Tumors were evaluated in vivo using the Lewis lung carcinoma (LLC) mouse cell line and A549 human lung cancer carcinoma cell line. LLC and A549 cells were injected subcutaneously into the right hind leg of mice. Treatments (ACC, cisplatin, vehicle, and ACC with cisplatin, all given via daily IP injections) started once tumors reached a measurable size. Treatments were carried out for 14 days in the LLC model and for 22 and 24 days in the xenograft model (two experiments). LLC tumors were resected from ACC at the end of the study, and vehicle groups were evaluated for cathepsin B activity. Differential gene expression was carried out on A549 cells following 8 weeks of in vitro culture in the presence or absence of ACC in a culture medium. The ACC treatment decelerated tumor growth rates in both models. When tumor volumes were compared on the last day of each study, the ACC-treated animal tumor volume was reduced by 44.83% compared to vehicle-treated animals in the LLC model. In the xenograft model, the tumor volume was reduced by 51.6% in ACC-treated animals compared to vehicle-treated animals. A more substantial reduction of 74.75% occurred in the combined treatment of ACC and cisplatin compared to the vehicle (carried out only in the LLC model). Cathepsin B activity was significantly reduced in ACC-treated LLC tumors compared to control tumors. Differential gene expression results showed a shift towards anti-tumorigenic pathways in the ACC-treated A549 cells. This study supports the ACC anti-malignant buffering hypothesis by demonstrating decelerated tumor growth, reduced cathepsin B activity, and altered gene expressions to produce anti-cancerous effects.

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.

Exosomes secreted from cardiomyocytes suppress the sensitivity of tumor ferroptosis in ischemic heart failure

Heart failure (HF) patients in general have a higher risk of developing cancer. Several animal studies have indicated that cardiac remodeling and HF remarkably accelerate tumor progression, highlighting a cause-and-effect relationship between these two disease entities. Targeting ferroptosis, a prevailing form of non-apoptotic cell death, has been considered a promising therapeutic strategy for human cancers. Exosomes critically contribute to proximal and distant organ-organ communications and play crucial roles in regulating diseases in a paracrine manner. However, whether exosomes control the sensitivity of cancer to ferroptosis via regulating the cardiomyocyte-tumor cell crosstalk in ischemic HF has not yet been explored. Here, we demonstrate that myocardial infarction (MI) decreased the sensitivity of cancer cells to the canonical ferroptosis activator erastin or imidazole ketone erastin in a mouse model of xenograft tumor. Post-MI plasma exosomes potently blunted the sensitivity of tumor cells to ferroptosis inducers both in vitro in mouse Lewis lung carcinoma cell line LLC and osteosarcoma cell line K7M2 and in vivo with xenograft tumorigenesis model. The expression of miR-22-3p in cardiomyocytes and plasma-exosomes was significantly upregulated in the failing hearts of mice with chronic MI and of HF patients as well. Incubation of tumor cells with the exosomes isolated from post-MI mouse plasma or overexpression of miR-22-3p alone abrogated erastin-induced ferroptotic cell death in vitro. Cardiomyocyte-enriched miR-22-3p was packaged in exosomes and transferred into tumor cells. Inhibition of cardiomyocyte-specific miR-22-3p by AAV9 sponge increased the sensitivity of cancer cells to ferroptosis. ACSL4, a pro-ferroptotic gene, was experimentally established as a target of miR-22-3p in tumor cells. Taken together, our findings uncovered for the first time that MI suppresses erastin-induced ferroptosis through releasing miR-22-3p-enriched exosomes derived from cardiomyocytes. Therefore, targeting exosome-mediated cardiomyocyte/tumor pathological communication may offer a novel approach for the ferroptosis-based antitumor therapy.

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.

Feiyiliu Mixture sensitizes EGFRDel19/T790M/C797S mutant non-small cell lung cancer to osimertinib by attenuating the PRC1/Wnt/EGFR pathway.

Introduction: Osimertinib is a potent epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) for the treatment of patients with EGFR-mutant non-small cell lung cancer (NSCLC). However, the emergence of acquired resistance due to the EGFR-Del19/T790M/C797S mutation limits the clinical application of osimertinib. Feiyiliu Mixture (FYLM), a clinical experience formula of Chinese medicine, was used to treat lung cancer with good clinical efficacy. In this study, we aimed to investigate the mechanism by which Feiyiliu Mixture delays osimertinib resistance in EGFR-mutant cell lines and EGFR-mutant cell tumor-bearing mice. Methods: The osimertinib-resistant cell models were established in mouse Lewis lung carcinoma (LLC) cells transfected with EGFR-Del19/T790M/C797S mutant lentivirus. In cell experiments, after 48h of treatment with Feiyiliu Mixture-containing serum, MTT assay was used to detect the relative cell viability, and western blotting was used to detect EGFR protein phosphorylation expression. In animal experiments, C57BL/6J mice were subcutaneously injected with Lewis lung carcinoma cells stably expressing EGFR-Del19/T790M/C797S mutations to construct a xenograft model. After 2weeks of Feiyiliu Mixture and/or osimertinib treatment, the expression of proliferation-related, apoptosis-related and PRC1/Wnt/EGFR pathway markers was detected by real-time qPCR, western blotting and immunohistochemistry. Results: The results showed that when combined with osimertinib, Feiyiliu Mixture synergistically reduces proliferation and increases apoptosis to improve drug resistance. In vitro, Feiyiliu Mixture-containing serum reduced the EGFR phosphorylation. In vivo, Feiyiliu Mixture downregulated the expression of cyclin B1 and Bcl-2 while upregulating the level of cleaved Caspase-3 protein, indicating that Feiyiliu Mixture promotes apoptosis. Furthermore, Feiyiliu Mixture reduced the expression of p-EGFR, p-Akt, PRC1 and Wnt pathway-related proteins such as β-catenin, c-Myc and c-Jun. Conclusion: The present study identified that Feiyiliu Mixture inhibited PRC1/Wnt/EGFR pathway activation, reduced proliferation, and promoted apoptosis, thereby increasing the sensitivity of EGFR-mutant non-small cell lung cancer to osimertinib. Our study provided a new idea for Chinese medicine to play a role in enhancing efficacy and reducing toxicity in the treatment of non-small cell lung cancer.

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.

Exosomal IL-8 derived from Lung Cancer and Colon Cancer cells induced adipocyte atrophy via NF-κB signaling pathway

BackgroundCytokines secreted in the tumor microenvironment function in cancer cachexia (CC), a common clinicopathological syndrome associated with adipocyte wasting and skeletal muscle atrophy. Extracellular vesicles (EVs) secreted by cancer cells actively engage in inter-tissue communication; EVs and enclosed cytokines are largely undefined in CC adipocytes wasting.MethodsEVs derived from Lewis lung carcinoma (LLC) and colorectal cancer C26 cells were extracted and characterized. Conditioned medium and EVs from cancer cells were applied to 3 T3-L1 adipocytes. Recombinant IL-8, IL-8 neutralizing antibody, CXCR2 and NF-κB inhibitor were examined in functional assays. Lipolysis of adipocytes was monitored by Western blots, Oil red O staining and glycerol assays. Furthermore, LLC and C26 cell lines were established as cachexia model to explore the relevance of IL-8 and NF-κB signaling in CC adipose wasting. Adipose tissues were collected for histology analyses.ResultsLLC and C26 cell-derived EVs induced lipolysis of 3 T3-L1 adipocytes. Specially, Dil-labeled EVs were effectively taken up by 3 T3-L1 adipocytes, which were motivated by the delivered IL-8 to elicit the NF-κB pathway. In comparison, special IL-8 neutralizing antibody relieved that lipolysis of 3 T3-L1 adipocytes induced by EVs together with conditioned medium of LLC and C26 cells, respectively. Consistently, both CXCR2 and NF-κB inhibitors would lessen the phenotype of lipolysis in 3 T3-L1 adipocytes. In the in vivo settings, both LLC and C26-tumor bearing mice had higher serum IL-8 levels as compared to the control groups. Two typical lipolysis markers, PGC1α and UCP1, were also up-regulated in the adipose tissues of LLC and C26-tumor mice groups, respectively.ConclusionsEVs secreted by LLC and C26 tumor cells would induce adipocyte wasting via extracellular IL-8-mediated NF-κB signaling. Our study pointed out the physiological and therapeutic values of exosomal IL-8 in CC lipolysis.

Gaseous nitric oxide tumor ablation induces an anti-tumor abscopal effect

BackgroundIn-situ tumor ablation provides the immune system with the appropriate antigens to induce anti-tumor immunity. Here, we present an innovative technique for generating anti-tumor immunity by delivering exogenous ultra-high concentration (> 10,000 ppm) gaseous nitric oxide (UHCgNO) intratumorally.MethodsThe capability of UHCgNO to induce apoptosis was tested in vitro in mouse colon (CT26), breast (4T1) and Lewis lung carcinoma (LLC-1) cancer cell lines. In vivo, UHCgNO was studied by treating CT26 tumor-bearing mice in-situ and assessing the immune response using a Challenge assay.ResultsExposing CT26, 4T1 and LLC-1 cell lines to UHCgNO for 10 s–2.5 min induced cellular apoptosis 24 h after exposure. Treating CT26 tumors in-situ with UHCgNO followed by surgical resection 14 days later resulted in a significant secondary anti-tumor effect in vivo. 100% of tumor-bearing mice treated with 50,000 ppm UHCgNO and 64% of mice treated with 20,000 ppm UHCgNO rejected a second tumor inoculation, compared to 0% in the naive control for 70 days. Additionally, more dendrocytes infiltrated the tumor 14 days post UHCgNO treatment versus the nitrogen control. Moreover, T-cell penetration into the primary tumor was observed in a dose-dependent manner. Systemic increases in T- and B-cells were seen in UHCgNO-treated mice compared to nitrogen control. Furthermore, polymorphonuclear-myeloid-derived suppressor cells were downregulated in the spleen in the UHCgNO-treated groups.ConclusionsTaken together, our data demonstrate that UHCgNO followed by the surgical removal of the primary tumor 14 days later induces a strong and potent anti-tumor response.

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.