Lewis Lung Cancer Cells Research Articles

Morphine promotes non-small cell lung cancer progression by downregulating E-cadherin via the PI3K/AKT/mTOR pathway

Morphine has been suggested to affect cancer cell dynamics and decrease survival rates in lung cancer patients at specific doses, but the precise mechanisms poorly understood. In this study, we aimed to investigate the molecular mechanisms by which morphine modulates the malignant characteristics of non-small cell lung cancer. Cell proliferation was assessed via the Cell Counting Kit-8 assay, and cell migration and invasion were examined via wound healing and Transwell assays. We employed immunofluorescence staining to evaluate E-cadherin expression in A549 and Lewis lung cancer (LLC) cell lines and immunohistochemistry to evaluate E-cadherin expression in nude mice tumours. Additionally, the in vivo effects of morphine on lung cancer progression were explored in a xenograft tumour experiments, in which naloxone was used as a morphine antagonist. Western blot analysis was performed to detect E-cadherin, phosphorylated mTOR (p-mTOR), mTOR, phosphorylated AKT (p-AKT), AKT, phosphorylated PI3K (p-PI3K), and PI3K protein levels in A549 and LLC cells as well as in tumour samples. Morphine (10 µM) significantly increased the proliferation of A549 and LLC cells in vitro (p < 0.05). It also enhanced the migratory and invasive capacities of these cell lines (p < 0.01). Mechanistically, morphine treatment (10 µM) led to a reduction in the expression of E-cadherin, and an increase in the phosphorylation of PI3K, AKT, and mTOR in A549 and LLC cells (p < 0.01). Morphine treatment (1.5 mg/kg) also reduced E-cadherin expression in xenograft tumours and promoted tumour growth in vivo (p < 0.05). This effect was reversed by naloxone (0.1 mg/kg). The results demonstrated that morphine stimulates the malignant proliferation of A549 and LLC cell lines and promotes xenograft tumour growth. Perhaps by specifically targeting MOR, morphine triggers a signalling cascade that activates the PI3K/AKT/mTOR pathway while inhibiting the EMT marker E-cadherin, which may consequently promote the progression of lung cancer.

Batroxase promotes the effect of NK cell adoptive therapy on lung cancer by enhancing immune cell infiltration.

Batroxobin, isolated from Bothrops moojeni, is a defibrinogenating agent used as a thrombin-like serine protease against fibrinogen for improving microcirculation. Here, we investigated whether, and if so, how batroxobin acts in concert with NK cells in terms of anti-tumor effects. CD3+/CD56+ NK cells were isolated and cultured from C57BL/6 mouse spleen. NK cells' viability was tested via Lactate dehydrogenase (LDH) assay. Lewis lung cancer cell (1*107 cell/ml) was used to build animal models. All animals were divided into five groups and treated with Batroxobin and NK cells respectively. HE staining was used to detect the pathological morphology of tumor tissue. The contents of fibrinogen and TNF-α in serum were determined by ELISA. The protein expression levels of MMP2, MMP9, VEGF and CD44 in tumor tissues were detected by Western Blot or immunohistochemistry. Compared with Control group, Tumor growth was not significantly affected in the group treated with Batroxobin or NK cells alone, However, tumor growth was significantly inhibited in the NK cell combined with the Batroxobin group. Serum levels of Fbg and TNF-αin mice treated with Batroxobin combined with NK cells dropped significantly, bringing them closer to normal levels. WB results showed that the expression levels of MMP2/9, VEGF and CD44 in Batroxobin combined with NK cell group also significantly decreased. Batroxobin combined with adoptive immunotherapy with NK cells significantly inhibited the growth of Lewis lung cancer in mice.

Molecular mechanism of basolateral amygdala involved in electroacupuncture-induced amelioration of cancer pain and concomitant depression based on transcriptomics techniques.

To observe the effect of electroacupuncture (EA) of "Zusanli" (ST36) and "Sanyinjiao" (SP6) on cancer pain and concomitant negative emotion in cancer pain model mice, and to explore its molecular mechanisms in the basolateral amygdala (BLA) by using transcriptomics techniques. C57BL/6 mice were randomized into sham operation, model and EA groups, with 10 mice in each group. The cancer pain model was established by injecting PBS suspension containing Lewis lung cancer cells into the femur. The mice in the EA group received EA stimulation(1 mA, 2 Hz) on ST36 and SP6 from the 10th day after modeling, 20 min per day for 12 successive days. The bone damage of the distal femur was observed with X-ray and H.E. staining, respectively. The mechanical pain threshold (MPT) was detected by using von Frey. The depression-like behavior was detected by using sucrose-preference test (sucrose preference index in 12 h), and the immobility (feeling of despair) duration of forced swimming within 4 min. The BLA tissue was extracted for RNA sequencing (RNA library construction, and screening differential gene profiling by transcriptomic sequencing) and bioinformatics analysis. The real-time PCR was used to validate the mRNA expression of differentially expressed genes：tumor necrosis factor superfamily 8 (Tnfsf8), bone marrow stromal cell antigen 1 (Bst1), prodynorphin (Pdyn) and voltage-gated sodium channelβ4 (Scn4b). H.E. staining and X-ray showed significant bone damage in the distal femur in cancer pain mice. In contrast to the sham operation group, the MPT on the 1st , 4th, 7th , 10th, 14th and 21st day after modeling and sucrose preference index were significantly decreased (P<0.001, P<0.000 1), and the immobility time of the forced swimming was considerably increased in the model group (P<0.001). In contrast to the model group, the MPT values on the 14th and 21st day and sucrose preference index were obviously increased (P<0.000 1, P<0.05), and the immobility time was strikingly decreased in the EA group (P<0.01). RNA sequencing showed that a total of 404 differentially expressed genes (205 up-regulated, 199 down-regulated) were screened in the model group compared with the sham operation group, and a total of 329 differentially expressed genes (206 up-regulated and 123 down-regulated) were screened in the EA group compared with the model group. Venn diagram analysis of the differentially expressed genes showed that 45 up-regulated and 28 down-regulated genes in the model group were completely reversed by EA. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the screened differentially expressed genes revealed that the above differential genes were mainly enriched in the ligand receptor activity, cytokine receptor binding, and cytokine activity related to neuro-inflammation, as well as in neuropeptide signaling pathways related to neuronal excitability, and calcium ion mediated signal transduction. The analysis of KEGG pathway showed that the differentially expressed genes were mainly enriched in the inflammation-related pathways, such as interleukin-17 pathway. Validation analysis of the differentially expressed genes showed that the expression levels of Tnfsf8 and Bst1 were significantly up-regulated in the model group compared with the sham operation group (P<0.01, P<0.05), and down-regulated by EA (P<0.01, P<0.05), while the expression levels of Pdyn and Scn4b were down-regulated in the model group in comparison with the sham operation group (P<0.01), and up-regulated by EA (P<0.05, P<0.01), which was consistent with the changing trend of the gene sequencing results. Acupuncture of ST36 and SP6 can significantly relieve cancer pain and concomitant negative emotion in cancer pain mice, which may be related to its functions in alleviating neuro-inflammation and relieving the abnormal activities of specific neurons in the BLA.

Parthenolide enhances the metronomic chemotherapy effect of cyclophosphamide in lung cancer by inhibiting the NF-kB signaling pathway.

Parthenolide (PTL), a sesquiterpene lactone derived from the medicinal herb Chrysanthemum parthenium, exhibits various biological effects by targeting NF-kB, STAT3, and other pathways. It has emerged as a promising adjunct therapy for multiple malignancies. To evaluate the in vitro and in vivo effect of PTL on cyclophosphamide (CTX) metronomic chemotherapy. The cytotoxicity of PTL and CTX on Lewis lung cancer cells (LLC cells) was assessed by measuring cell activity and apoptosis. The anti-tumor efficiency was evaluated using a tumor xenograft mice model, and the survival of mice and tumor volume were monitored. Additionally, the collected tumor tissues were analyzed for tumor microenvironment indicators and inflammatory factors. In vitro, PTL demonstrated a synergistic effect with CTX in inhibiting the growth of LLC cells and promoting apoptosis. In vivo, metronomic chemotherapy combined with PTL and CTX improved the survival rate of tumor-bearing mice and reduced tumor growth rate. Furthermore, metronomic chemotherapy combined with PTL and CTX reduced NF-κB activation and improved the tumor immune microenvironment by decreasing tumor angiogenesis, reducing Transforming growth factor β, and α-SMA positive cells. PTL is an efficient compound that enhances the metronomic chemotherapy effects of CTX both in vitro and in vivo, suggesting its potential as a supplementary therapeutic strategy in metronomic chemotherapy to improve the chemotherapy effects.

PRPS2-mediated modulation of the antitumor immune response in lung cancer through CCL2-mediated tumor-associated macrophages and myeloid-derived suppressor cells.

Phosphoribosyl pyrophosphate synthetase 2 (PRPS2) is known as an oncogene in many types of cancers, including lung cancer. However, its role in regulating tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) remains unclear. Our study aimed to explore the involvement of PRPS2 in TAM and MDSC regulation. Stable Lewis lung cancer (LLC) cell lines were established using a lentivirus system. These LLC lines were then used to establish tumor model in mice. The levels of target genes were determined using qPCR, western blotting, and ELISA assays. The percentage of different immune cell types was analyzed using fluorescence-activated cell sorting. The chemotaxis ability of TAM and MDSC was evaluated using an in vitro transwell chemotaxis assay. Notably, PRPS2 was found to regulate the chemotaxis of TAM and MDSC in tumor cells, as evidenced by the positive correlation of PRPS2 expression levels and abundance of TAM and MDSC populations. In addition, the expression of CCL2, mediated by PRPS2, was identified as a key factor in the chemotaxis of TAM and MDSC, as evidenced by a significant reduction in macrophages and MDSC numbers in the presence of the CCL2 antibody. Furthermore, in vivo experiments confirmed the involvement of PRPS2 in mediating CCL2 expression. PRPS2 was also found to regulate immune cell infiltration into tumors, whereas knockdown of CCL2 reversed the phenotype induced by PRPS2 overexpression. In tumor tissues from mice implanted with LLC-PRPS2-shCCL2 cells, a notable increase in CD4+ and CD8+ T cell percentages, alongside a marked decrease in TAMs, M-MDSC, and PMN-MDSC, was observed. Taken together, PRPS2 plays a crucial role in modulating the antitumor immune response by reprogramming CCL2-mediated TAM and MDSC.

High-fat-diet-induced obesity promotes simultaneous progression of lung cancer and atherosclerosis in apolipoprotein E-knockout mice.

Clinical studies have shown that atherosclerotic cardiovascular disease and cancer often co-exist in the same individual. The present study aimed to investigate the role of high-fat-diet (HFD)-induced obesity in the coexistence of the two diseases and the underlying mechanism in apolipoprotein E-knockout (ApoE-/-) mice. Male ApoE-/- mice were fed with a HFD or a normal diet (ND) for 15 weeks. On the first day of Week 13, the mice were inoculated subcutaneously in the right axilla with Lewis lung cancer cells. At Weeks 12 and 15, serumlectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and vascular endothelial growth factor levels were measured by enzyme-linked immunosorbent assay, and blood monocytes and macrophages were measured by fluorescence-activated cell sorting. At Week 15, the volume and weight of the local subcutaneous lung cancer and metastatic lung cancer and the amount of aortic atherosclerosis were measured. At Week 15, compared with mice in the ND group, those in the HFD group had a larger volume of local subcutaneous cancer (p = 0.0004), heavier tumors (p = 0.0235), more metastatic cancer in the lungs (p < 0.0001), a larger area of lung involved in metastatic cancer (p = 0.0031), and larger areas of atherosclerosis in the aorta (p < 0.0001). At Week 12, serum LOX-1, serum vascular endothelial growth factor, and proportions of blood monocytes and macrophages were significantly higher in the HFD group than those in the ND group (p = 0.0002, p = 0.0029, p = 0.0480, and p = 0.0106, respectively); this trend persisted until Week 15 (p = 0.0014, p = 0.0012, p = 0.0001, and p = 0.0204). In this study, HFD-induced obesity could simultaneously promote progression of lung cancer and atherosclerosis in the same mouse. HFD-induced upregulation of LOX-1 may play an important role in the simultaneous progression of these two conditions via the inflammatory response and VEGF.

Exercise accelerates recruitment of CD8+ T cell to promotes anti-tumor immunity in lung cancer via epinephrine

Background and purposeIn recent years, there has been extensive research on the role of exercise as an adjunctive therapy for cancer. However, the potential mechanisms underlying the anti-tumor therapy of exercise in lung cancer remain to be fully elucidated. As such, our study aims to confirm whether exercise-induced elevation of epinephrine can accelerate CD8+ T cell recruitment through modulation of chemokines and thus ultimately inhibit tumor progression.MethodC57BL/6 mice were subcutaneously inoculated with Lewis lung cancer cells (LLCs) to establish a subcutaneous tumor model. The tumor mice were randomly divided into different groups to performed a moderate-intensity exercise program on a treadmill for 5 consecutive days a week, 45 min a day. The blood samples and tumor tissues were collected after exercise for IHC, RT-qPCR, ELISA and Western blot. In addition, another group of mice received daily epinephrine treatment for two weeks (0.05 mg/mL, 200 µL i.p.) (EPI, n = 8) to replicate the effects of exercise on tumors in vivo. Lewis lung cancer cells were treated with different concentrations of epinephrine (0, 5, 10, 20 µM) to detect the effect of epinephrine on chemokine levels via ELISA and RT-qPCR.ResultsThis study reveals that both pre- and post-cancer exercise effectively impede the tumor progression. Exercise led to an increase in EPI levels and the infiltration of CD8+ T cell into the lung tumor. Exercise-induced elevation of EPI is involved in the regulation of Ccl5 and Cxcl10 levels further leading to enhanced CD8+ T cell infiltration and ultimately inhibiting tumor progression.ConclusionExercise training enhance the anti-tumor immunity of lung cancer individuals. These findings will provide valuable insights for the future application of exercise therapy in clinical practice.

Abstract 172: Targeting the poliovirus receptor to activate T cells and induce myeloid-derived suppressor cells to differentiate to M1-like macrophages via the IFNγ-pSTAT1-IRF8 axis in cancer therapy

Abstract T cell immunoglobulin and ITIM domain (TIGIT) is one of the most important immune checkpoints expressed on lymphocytes, and poliovirus receptor (PVR, also CD155) serves as the most crucial ligand for TIGIT, harboring an important function in cancer cells and influencing the tumor microenvironment (TME). While it's well-established that TIGIT blockade could reverse immunosuppression, the question of whether direct inhibition of PVR yields comparable results remains to be fully elucidated. In this study, we generated a mouse gene Pvr knock out (KO) Lewis lung cancer (LLC) cell line and found that the Pvr-KO tumors tended to develop a pro-inflammatory TME, as reflected by a downregulation of monocytic-myeloid derived suppressor cells (M-MDSCs) and an upregulation of CD8+ central memory T cells. Through bulk RNA-seq and multi-cytokines measurement, we quantified the transcription and protein levels of immunostimulatory cytokines and chemokines and found they were significantly upregulated in Pvr-KO TME, including GM-CSF, IFN-γ, TNF-α, IL-2/4/6/10, CCLs (including CCL2/3/4/5) and CXCLs (including CXCL1/2/5). We further employed single-cell RNA sequencing (scRNA-seq) on 20,026 intra-tumoral CD45+ immune cells. Using a conserved MDSCs gene set and the 'AddModuleScore' function of Seurat package, we identified the clusters of MDSCs and found the differentiation ability of M-MDSCs in Pvr-KO TME was enhanced. Through pseudo-temporal ordering, we found the M-MDSCs in the Pvr-KO TME have a trend to differentiate towards M1-like macrophages, which could play an essential positive role in anti-tumor immunity. We further focused on the scRNA-seq of T cell subsets and found the enhanced function of cytotoxic CD8+ T cells, leading to increased production of IFN-γ. In the intercellular communications analysis, a significant upregulation of the IFNG-IFNR signal between T cells: MDSCs in the Pvr-KO group was observed. Moreover, we found that Irf8, a key factor in myeloid cell differentiation, was significantly upregulated in the M-MDSCs of Pvr-KO tumor. We then employed CUT &amp; Tag sequencing and confirmed that IFN-γ induces phosphate Stat1, which then binds on the promoter of Irf8. These findings suggest that the IFN-γ-Stat1-Irf8 axis plays a crucial role in MDSCs differentiation and may be a potential target for therapeutic intervention in cancer. Next, an anti-PVR nanobody was synthesized and its efficacy was evaluated in the context of LLC and 4T1 tumor grafts. When in combination with anti-PD-1 therapy, anti-PVR exhibited a remarkable capacity to reduce tumor size in both tumor models with no notable indications of toxicity observed. Our findings provide new insights into the role of PVR in modulating the TME and offer potential strategies for enhancing the effectiveness of immunotherapy. Citation Format: Mingyang Feng, Sirui Tan, Qizhi Ma, Benxia Zhang, Yue Chen, Qiu Li, Yongsheng Wang. Targeting the poliovirus receptor to activate T cells and induce myeloid-derived suppressor cells to differentiate to M1-like macrophages via the IFNγ-pSTAT1-IRF8 axis in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 172.

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

Abstract Purpose: Radiation is the most common modality for lung cancer treatment method. Radiationinduces apoptosis, causing cancer cells to be fragmented and expose the cancer-associatedantigens to the tumor microenvironment. These can be recognized by antigen-presenting cells(APC) to induce the antineoplastic effect by activating cytotoxic T cells. Our prior studies showadding immunoadjuvant like AntiCD40 with radiation can further activate the APCs in its anti-tumor (M1) format. Recent studies have shown that flavonoids like Hesperetin, an ACE2receptor agonist also can induce apoptosis in cancer cells whereas ACE2 receptors are abundant in lung cancer cells.This project aims to develop a novel treatment for non-small cell lung cancer (NSCLC) using nanotechnology for inhalation drug delivery Hesperetin, a flavonoid with antioxidant and pro-apoptotic activity; bound to PLGA-coated nanoparticles (Hesperetin Nanoparticles, HNPs) andAntiCD40 aiming for localized targeted dosing to reduce the treatment-related systemic toxicity.We plan to test the efficacy and safety of this aerosol spray. Methods: In-vitro studies were performed in human A549 (ATCC) and murine LLC1 (ATCC)lung cancer cell lines for growth (MTT) and clonogenic survival assays to demonstrate the anti-cancer cell activity of Hesperetin (Sigma). Next, HNP was prepared using NanoFabTx™ nano-formulation reagent kits (Millipore-Sigma). A syngeneic orthotopic murine model of Lungadenoma was generated in wild (+/+) C57/BL6 background mice with similar background LLC1cell lines. We developed syngeneic orthotopic murine lung tumors with luciferase genetransfected LL/2-Luc2 (ATCC) Lewis Lung Cancer cell line in wild-type C57BL/6 mice(Techonic). Lung tumor-bearing mice were treated with HNP, antiCD40, or both with aerosolspray with a control group treated with the solvent (dH2O) only. To assess the drug delivery withaerosol treatment ex-vivo lung tissue was analyzed for Fluorescence tagged antiCD40 andnanoparticle uptakes. A survival assay was performed to analyze the efficacy of aerosoltreatment of HNPs with or without AntiCD40. All cohorts were also analyzed for body score, body weight, and liver and kidney functions. Results: We were able to successfully develop an aerosol drug delivery model to administer bothAnti-CD40 and HNP in a murine lung cancer model. In our analysis of an orthotopic murinelung cancer model, we demonstrate a high intake of the HNP and AntiCD40 by the cancer cellssparing the normal lung tissue. Moreover, the highest percentage of survival rate was observed with the combination aerosol treatment with HNP+AntiCD40 (p&amp;lt;0.001), compared to CD40alone (p&amp;lt;0.01) or HNP (p&amp;lt;0.01) alone. Comments: This treatment model will allow us to make the lung cancer treatment method easilyavailable for the mass population without having hazardous radiation treatment in the lungcancer model. Citation Format: Sayeda Yasmin-Karim, Geraud Richard, Amanda Fam, Alina-Marissa Ogurek, G. Mike Makrigiorgos. Aerosol delivery of immunotherapy and hesperetin nanoparticles in murine lung cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 491.

Bifunctional molecular probe targeting tumor PD-L1 enhances anti-tumor efficacy by promoting ferroptosis in lung cancer mouse model

PurposeImmune checkpoint inhibitors (ICIs) targeting tumor-specific PD-1/PD-L1 significantly improve the overall survival rate of patients with advanced cancer by reactivating the immune system to attack cancer cells. To explore their tumor killing effect, we used the radionuclide iodine-131 (131I) to label the anti-PD-L1 antibody Atezolizumab (131I-PD-L1 mAb). MethodWe prepared the radioimmunoassay molecular probe 131I-PD-L1 mAb by the chloramine-T method and evaluated its affinity using Lewis lung cancer (LLC) cells. The uptake of 131I-PD-L1 mAb by transplanted tumors was examined through SPECT and its in vivo distribution. We then compared the in vitro and in vivo anti-tumor efficacy of groups treated with control, PD-L1 mAb, 131I-PD-L1 mAb, and 131I-PD-L1 mAb + PD-L1 mAb combined treatment. We performed H&E staining to examine the changes in tumor, as well as the damage in major tissues and organs caused by potential side effects. The anti-tumor mechanism of 131I-PD-L1 mAb was analyzed by Western blot, RT-qPCR and immunohistochemistry (IHC). Result131I-PD-L1 mAb was highly stable and specific, and easily penetrated into tumor. 131I-PD-L1 mAb suppressed cancer cell proliferation in vitro, and inhibited tumor growth in vivo by inducing ferroptosis, thus prolonging the survival of experimental animals while demonstrating biological safety. ConclusionTherefore, our study suggested that 131I-PD-L1 mAb affected the expression of tumor-related factors through β-rays and thus promoted ferroptosis in tumor. Combined treatment showed better anti-tumor effect compared to single ICI treatment.

A novel neoadjuvant therapy for early-stage non-small cell lung cancer in a mouse model.

Lung cancer is the common malignancy with high mortality rate in the world. Even with curative resection for early-stage lung cancer patients, the rate of postoperative recurrence and metastasis is still high. Neoadjuvant nivolumab combined with chemotherapy leads to improved pathological complete response rate and event-free survival in resectable non-small cell lung cancer (NSCLC) patients. However, the neoadjuvant therapy is not only accompanied by grade 3 or above adverse events which resulting in the potential missing out on the window for curative surgery for the patients, but also has low efficacy especially in patients with low programmed death ligand 1 (PD-L1) expression. Hence, it is particularly important to explore innovative ways to inhibit tumour recurrence and metastasis. In the present study, we investigated whether neoadjuvant therapy with intralesional Rose Bengal (RB) elicited specific immune responses compared with control group, and then the lung cancer mouse model was used to evaluated the immunological mechanism. The secondary Lewis lung cancer cells (LLCs) tumour growth was significantly suppressed by RB intralesional injection into subcutaneous tumour; the formation rate of secondary tumours induced by the B16 melanoma cell injection was 100%. Intralesional RB neoadjuvant therapy before surgical resection exhibited effectively enhanced T central memory cells (Tcm) and T memory stem cells (Tscm) + naïve T cells (Tn) infiltration, elicited stronger cytotoxic T lymphocyte (CTL) responses against LLCs, and displayed markedly higher proportions of splenic lymphocytes that produce tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) upon restimulation in a lung cancer mouse model. Based on our preclinical data, neoadjuvant therapy with intralesional RB injection generated immune memory and prevented the recurrence and metastasis of tumour in a lung cancer mouse model, which provides a new strategy for neoadjuvant treatment of early-stage NSCLC.

A Novel Translational PET Imaging Approach to Quantifying Distal Tumor Immune Activation After Targeted Radiation Therapy and Checkpoint Blockade

ObjectivesThis study aimed to provide a novel non-invasive method to quantify abscopal immune activation and predict combinational treatment response using [68Ga]-NOTA-GZP PET imaging. Materials/Methods4T1 breast cancer cells were implanted bilaterally in the mammary fat pad of Balb/c mice and Lewis's lung cancer cells (LLC) were implanted bilaterally on the shoulders of C57/Bl6 mice. One of the tumors received a single fraction of 12Gy irradiation followed by combination of concurrent PD-1 and CTLA-4 inhibitors or controls. Tumor growth of the irradiated and non-irradiated tumors was measured and compared to 12Gy irradiation only, checkpoint inhibitor only, and no treatment control group. Changes in granzyme B activity were assessed with [68Ga]-NOTA-GZP PET imaging from baseline and every three days until day 9. ResultsIn the 4T1 model, concurrent treatment with dual checkpoint inhibitors (CPI) and radiation resulted in reduction of the irradiated tumor volume at day 30. At this same time point, the non-irradiated tumor volume for combination treatment decreased significantly, consistent with abscopal immune activation. Similarly, in the LLC model, concurrent treatment inhibited tumor growth on the non-irradiated tumor at day 15. Importantly, on day 9, granzyme B PET signal in both 4T1 and LLC models was significantly higher in the non-irradiated tumors that responded to concurrent treatment compared to subsequent non-responding tumors. A similar lack of granzyme B signal was observed in the non-irradiated tumors from mice that received radiation or CPI only and control tumors. ROC analysis identified a PET threshold of 1.505 and 1.233 on day 9 that predicted treatment response in 4T1 and LLC models, respectively. Conclusion[68Ga]-NOTA-GZP PET imaging was able to non-invasively predict abscopal immune activation prior to subsequent tumor volume changes after combination treatment. It provides a potential translational paradigm for investigating distal immune activation post-radiation in a clinical setting.

Combination of radiotherapy and Anlotinib enhances benefit from immunotherapy to liver metastasis and abscopal tumor from lung cancer

Many studies have shown that liver metastasis can weaken the efficacy of immunotherapy. Immunotherapy combined with radiotherapy or anti-angiogenic therapy has been proven to have synergistic anti-tumor effects. So we devote to explore whether the combination of the three therapies can exert effective anti-tumor effects on liver metastasis. The clinical information of 118 patients with liver metastasis were collected to compare the intrahepatic progression-free survival between immunotherapy and immunotherapy combined with other treatments. We used Lewis lung cancer (LLC) cell to establish a mouse liver metastasis tumor model and record tumor burden and survival. Tumor-infiltrating immune cells detected by flow cytometry. RNA sequencing was performed and the proportion of immune cells were analyzed by TIMER2.0 database. Compared with immunotherapy group, the combination therapy group showed a trend for longer median intrahepatic progression-free survival. Radiotherapy combined with PD-1 inhibitor and Anlotinib can inhibit liver metastasis and subcutaneous tumor growth and prolong the survival compared with other groups in vivo. Compared with the anti-PD-1 treatment group, triple therapy can increase CD4+T, CD8+T, and IFN-γ+CD8+T cells and decrease infiltration of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) in tumors. PPAR signaling pathway were significantly activated and CD8+T and dendritic cells (DC) were increased in the triple therapy group compared to the PD-1 inhibitor combined with Anlotinib group. Radiotherapy combined with PD-1 inhibitor and Anlotinib can effectively exert anti-tumor efficacy and reshape the tumor immune microenvironment by increasing the infiltration of anti-tumor immune cells and reducing the infiltration of immunosuppressive immune cells.

Juglone triggers apoptosis of non-small cell lung cancer through the reactive oxygen species -mediated PI3K/Akt pathway.

Non-small cell lung cancer (NSCLC) is one of the most common malignancies worldwide, and oxidative stress plays a crucial role in its development. Juglone, a naturally occurring naphthoquinone in J. mandshurica, exhibits significant cytotoxic activity against various cancer cell lines. However, whether the anticancer activity of juglone is associated with oxidative stress remains unexplored. In this study, mouse Lewis lung cancer (LLC) and human non-small cell lung cancer A549 cells were used to explore the anticancer mechanisms of juglone. Juglone inhibited LLC and A549 cells viability, with IC50 values of 10.78 μM and 9.47 μM, respectively, for 24 h, and substantially suppressed the migration and invasion of these two lung cancer cells. Additionally, juglone arrested the cell cycle, induced apoptosis, increased the cleavage of caspase 3 and the protein expression of Bax and Cyt c, and decreased the protein expression of Bcl-2 and caspase-3. Furthermore, juglone treatment considerably increased intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) levels, but suppressed glutathione peroxidase 4 (GPX4) and superoxide dismutase (SOD) activities. It also inhibited the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, which was attenuated by 1,3-diCQA (an activator of PI3K/Akt). Moreover, N-acetylcysteine (a ROS scavenger) partially reversed the positive effects of juglone in terms of migration, invasion, ROS production, apoptosis, and PI3K/Akt pathway-associated protein expression. Finally, in tumor-bearing nude mouse models, juglone inhibited tumor growth without any apparent toxicity and significantly induced apoptosis in NSCLC cells. Collectively, our findings suggest that juglone triggers apoptosis via the ROS-mediated PI3K/Akt pathway. Therefore, juglone may serve as a potential therapeutic agent for the treatment of NSCLC.

One new prenylated flavonoid with cytotoxic activity from Epimedium brevicornu maxim

One previously undescribed prenylated flavonoid (1) and three known ones (2-4) were isolated from leaves of Epimedium brevicornu maxim. Their structures were determined through extensive spectroscopic analysis and comparison with the NMR data in the literature. Compound 1 showed a moderate cytotoxicity with an IC50 value of 18.7 μM, while known compounds 2 and 3 elicited weak cytotoxicities with IC50 values of 29.2 and 32.8 μM against Lewis Lung cancer cells (LLC cells), respectively.

1,25(OH)2 D3 inhibits Lewis lung cancer cell migration via NHE1-sensitive metabolic reprograming.

High prevalence and metastasis rates are characteristics of lung cancer. Glycolysis provides energy for the development and metastasis of cancer cells. The 1,25-dihydroxy vitamin D3 (1,25(OH)2 D3 ) has been linked to reducing cancer risk and regulates various physiological functions. We hypothesized that 1,25(OH)2 D3 could be associated with the expression and activity of Na+ /H+ exchanger isoform 1 (NHE1) of Lewis lung cancer cells, thus regulating glycolysis as well as migration by actin reorganization. Followed by online public data analysis, Vitamin D3 receptor, the receptor of 1,25(OH)2 D3 has been proved to be abundant in lung cancers. We demonstrated that 1,25(OH)2 D3 treatment suppressed transcript levels, protein levels, and activity of NHE1 in LLC cells. Furthermore, 1,25(OH)2 D3 treatment resets the metabolic balance between glycolysis and OXPHOS, mainly including reducing glycolytic enzymes expression and lactate production. In vivo experiments showed the inhibition effects on tumor growth as well. Therefore, we concluded that 1,25(OH)2 D3 could amend the NHE1 function, which leads to metabolic reprogramming and cytoskeleton reconstruction, finally inhibits the cell migration.

Phyllanthi Tannin Loaded Solid Lipid Nanoparticles for Lung Cancer Therapy: Preparation, Characterization, Pharmacodynamics and Safety Evaluation

The objective of the present study was to develop PTF-loaded solid lipid nanoparticles (PTF-SLNs) and investigate their efficacy in treating lung cancer. The PTF-SLNs were prepared by the thin film hydration method and verified by FTIR and TEM. Their physicochemical properties were characterized by particle size, polydispersity index (PDI), zeta potential, entrapment efficiency (EE), drug loading (DL), etc. Then, the pharmacodynamic studies of PTF-SLNs were performed on Lewis lung cancer cells and tumor-bearing mice. Finally, the safety studies were assessed by organ index, serum biochemical indicators, and histopathological changes. The PTF-SLNs were characterized by around 50 nm sphere nanoparticles, sustained ideal stability, and controlled drug release effects. The pharmacodynamic evaluation results showed that PTF-SLNs had stronger anti-tumor efficacy than PTF. An in vitro study revealed a more obvious cytotoxicity and apoptosis effect. The IC 50 values of PTF and PTF-SLNs were 67.43 μg/mL and 20.74 μg/mL, respectively. An in vivo study showed that the tumor inhibition rates of 2 g/kg PTF and 0.4 g/kg PTF-SLNs were 59.97% and 64.55%, respectively. The safety preliminary study indicated that PTF-SLNs improve the damage of PTF to normal organs to a certain extent. This study provides a nanoparticle delivery system with phenolic herbal extract to improve anti-tumor efficacy in lung cancer.

Lapachol inhibits the growth of lung cancer by reversing M2-like macrophage polarization via activating NF-κB signaling pathway

Resetting tumor-associated macrophages (TAMs) is a promising strategy to ameliorate the immunosuppressive tumor microenvironment (TME) and improve innate and adaptive antitumor immunity. Lapachol, a naturally occurring 1,4-naphthoquinone, exhibits various pharmacological activities including antitumor, anti-leishmanial, antimalarial and antiseptic. In this study, we investigated the relevance of macrophage polarization and the antitumor effect of lapachol in Lewis lung cancer (LLC) both in vitro and in vivo. This study demonstrated that lapachol significantly reversed the polarization of M2-like macrophages thus that were endowed with the ability to kill LLC cells by activating NF-κB signaling pathway. Furthermore, lapachol effectively suppressed tumor growth in C57BL/6 mice bearing lung tumors by reducing the proportion of M2-like macrophages. Overall, our findings clearly illustrated that lapachol could reverse the polarization of M2-like macrophages to improve the immunosuppressive tumor microenvironment, and had the potential to be developed as an immunomodulatory antitumor agent.

Exosomal lncRNA Mir100hg derived from cancer stem cells enhance glycolysis and promote metastasis of lung adenocarcinoma through mircroRNA-15a-5p/31-5p

BackgroundExosomes are a new class of molecular entities in the metastatic microenvironment, which can mediate bidirectional communication between cells. While exosomes-mediated interactions between tumor cells and other cell populations in the tumor microenvironment have attracted most attention, little is known about the significance of exosomes in mediating the interaction between non-stemness cancer cells and cancer stem cells during cancer progression.MethodsThe structure, sequence and downstream target miRNAs of lncRNA Mir100hg were predicted by online web resources. The bioinformatics prediction results were validated with experimental verification: exosome tracing, electron microscopy, Luciferase assay, metabolomics sequencing and mouse tail vein model of pulmonary metastasis. A complex regulatory network of "cancer stem cells-exosomal lncRNA-non-stem cancer cells" was constructed.ResultsThis study demonstrates firstly that lncRNA Mir100hg is upregulated in lung cancer stem cell LLC-SD (Lung cancer stem cells) and can be delivered to non-stemness cancer cells LLC (Lewis lung cancer cells) via exosomes. In LLC, Mir100hg targets miR-15a-5p and miR-31-5p which leads to the increase of the global glycolytic activity of lung cancer cells and consequently, the enhancement of their metastatic capability.ConclusionWe delineated a complex regulatory network that utilized by cancer stem cells to transfer their high metastatic activity to the low-metastatic non-stemness cancer cells through exosomal Mir100hg, thereby providing new mechanistic insights into the communication between two heterogeneous tumor cells.EX9vzfLURWcPee8x9snm8iVideo

Nicaraven Exerts a Limited Effect on Radiation-Induced Inhibition of Tumor Growth in a Subcutaneous Murine Tumor Model.

Nicaraven selectively protects normal tissue from radiation-induced injury. To further develop the clinical application of nicaraven for mitigating the side effects of cancer radiotherapy, we investigated the potential effect of nicaraven administration in radiation-induced inhibition of tumor growth. A subcutaneous tumor model was established in mice by the injection of Lewis lung cancer cells at the back of the chest. X-ray radiation was delivered to the thoracic area and different doses of nicaraven (0, 20, 50, 100 mg/kg) were administrated intraperitoneally pre- or post-irradiation. The tumor size was measured every other day. Mice were euthanized on day 30, and the tumor weight and the levels of cytokines in tumor tissue were measured. Pre- or post-irradiation administration of nicaraven up to a dose of 100 mg/kg did not significantly diminish the radiation-induced inhibition of tumor growth, but post-irradiation administration of 20 and 50 mg/kg nicaraven resulted in relatively lower tumor weight. The levels of IL-1β, IL-6, IL-10, MCP-1, MIP-2a, TGF-β1, VEGF, p53, p21, cyclin D1 and caspase-3 in tumor tissue did not change by nicaraven administration and were not significantly associated with the tumor weights. According to our experimental data, nicaraven will not significantly diminish the radiation-induced inhibition of tumor growth, even with pre-irradiation administration at a high dose.