Preventing loss of sirt1 lowers mitochondrial oxidative stress and preserves C2C12 myotube diameter in an in vitro model of cancer cachexia.

Cancer cachexia is a multifactorial syndrome associated with advanced cancer that contributes to mortality. Cachexia is characterized by loss of body weight and muscle atrophy. Increased skeletal muscle mitochondrial reactive oxygen species (ROS) is a contributing factor to loss of muscle mass in cachectic patients. Mice inoculated with Lewis lung carcinoma (LLC) cells lose weight, muscle mass, and have lower muscle sirtuin-1 (sirt1) expression. Nicotinic acid (NA) is a precursor to nicotinamide dinucleotide (NAD+) which is exhausted in cachectic muscle and is a direct activator of sirt1. Mice lost body and muscle weight and exhibited reduced skeletal muscle sirt1 expression after inoculation with LLC cells. C2C12 myotubes treated with LLC-conditioned media (LCM) had lower myotube diameter. We treated C2C12 myotubes with LCM for 24 h with or without NA for 24 h. C2C12 myotubes treated with NA maintained myotube diameter, sirt1 expression, and had lower mitochondrial superoxide. We then used a sirt1-specific small molecule activator SRT1720 to increase sirt1 activity. C2C12 myotubes treated with SRT1720 maintained myotube diameter, prevented loss of sirt1 expression, and attenuated mitochondrial superoxide production. Our data provides evidence that NA may be beneficial in combating cancer cachexia by maintaining sirt1 expression and decreasing mitochondrial superoxide production.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Taken together, PRPS2 plays a crucial role in modulating the antitumor immune response by reprogramming CCL2-mediated TAM and MDSC.

Tumor promoting effect of PDLIM2 downregulation involves mitochondrial ROS, oncometabolite accumulations and HIF-1α activation.

BACKGROUND: Cancer is characterized by dysregulated cellular metabolism. Thus, understanding the mechanisms underlying these metabolic alterations is important for developing targeted therapies. In this study, we investigated the pro-tumoral effect of PDZ and LIM domain 2 (PDLIM2) downregulation in lung cancer growth and its association with the accumulation of mitochondrial ROS, oncometabolites and the activation of hypoxia-inducible factor-1 (HIF-1) α in the process. METHODS: Databases and human cancer tissue samples were analyzed to investigate the roles of PDLIM2 and HIF-1α in cancer growth. DNA microarray and gene ontology enrichment analyses were performed to determine the cellular functions of PDLIM2. Seahorse assay, flow cytometric analysis, and confocal microscopic analysis were employed to study mitochondrial functions. Oncometabolites were analyzed using liquid chromatography-mass spectrometry (LC-MS). A Lewis lung carcinoma (LLC) mouse model was established to assess the in vivo function of PDLIM2 and HIF-1α. RESULTS: The expression of PDLIM2 was downregulated in lung cancer, and this downregulation correlated with poor prognosis in patients. PDLIM2 highly regulated genes associated with mitochondrial functions. Mechanistically, PDLIM2 downregulation resulted in NF-κB activation, impaired expression of tricarboxylic acid (TCA) cycle genes particularly the succinate dehydrogenase (SDH) genes, and mitochondrial dysfunction. This disturbance contributed to the accumulation of succinate and other oncometabolites, as well as the buildup of mitochondrial reactive oxygen species (mtROS), leading to the activation of hypoxia-inducible factor 1α (HIF-1α). Furthermore, the expression of HIF-1α was increased in all stages of lung cancer. The expression of PDLIM2 and HIF-1α was reversely correlated in lung cancer patients. In the animal study, the orally administered HIF-1α inhibitor, PX-478, significantly reduces PDLIM2 knockdown-promoted tumor growth. CONCLUSION: These findings shed light on the complex action of PDLIM2 on mitochondria and HIF-1α activities in lung cancer, emphasizing the role of HIF-1α in the tumor-promoting effect of PDLIM2 downregulation. Additionally, they provide new insights into a strategy for precise targeted treatment by suggesting that HIF-1α inhibitors may serve as therapy for lung cancer patients with PDLIM2 downregulation.

Antitumor Effects of an Anthocyanin-Rich Grain Diet in a Mouse Model of Lewis Lung Carcinoma.

Functional foods enriched with plant polyphenol anthocyanins attract particular attention due to their health-promoting properties, including antitumor activity. We evaluated the effects of a grain diet rich in anthocyanins in a mouse model of Lewis lung carcinoma. Mice of the C57BL/6 strain were fed with wheat of near-isogenic lines differing in the anthocyanin content for four months prior to tumor transplantation. Although a significant decrease in the size of the tumor and the number of metastases in the lungs was revealed in the groups with both types of grain diet, the highest percentage of animals without metastases and with attenuated cell proliferation in the primary tumor were observed in the mice with the anthocyanin-rich diet. Both grain diets reduced the body weight gain and spleen weight index. The antitumor effects of the grain diets were associated with the activation of different mechanisms: immune response of the allergic type with augmented interleukin(IL)-9 and eotaxin serum levels in mice fed with control grain vs. inhibition of the IL-6/LIF system accompanied by a decrease in the tumor-associated M2 macrophage marker arginase 1 gene mRNA levels and enhanced autophagy in the tumor evaluated by the mRNA levels of Beclin 1 gene. Thus, anthocyanin-rich wheat is suggested as a promising source of functional nutrition with confirmed in vivo antitumor activity.

Head-to-head Comparison of Green Fluorescent Protein (GFP) Imaging With Luciferase-luciferin Imaging In Vivo Using Single-nanometer Laser-excitation Tuning and an Ultra-low-light-detection Camera and Optics Demonstrates the Superiority of GFP.

BACKGROUND/AIM: Genetic reporters encoding fluorescent proteins or luciferase have been used in vivo for the last three decades with claims about their superiority or inferiority over each other. In the present report, a head-to-head in vivo comparison of green fluorescent protein (GFP) fluorescence imaging and luciferase-luciferin imaging, using single-nanometer laser-excitation tuning of fluorescence excitation and an ultra-low-light-detection camera and optics was performed. MATERIALS AND METHODS: Mouse Lewis-lung carcinoma cells labeled with GFP (LLC-GFP) or luciferase (LL/2-Luc2) were injected subcutaneously into the flank of nude mice. One week after injection, GFP-fluorescence imaging and luciferase-luciferin imaging was performed using the UVP Biospectrum Advanced system with excitation at 487 nm and peak emission at 513 nm for GFP, and with emission at 560 nm for luciferase-luciferin. GFP fluorescence images were obtained at 0, 10, and 20 min. Luciferase-luciferin images were obtained 10 and 20 min after the injection of D-luciferin. RESULTS: The intensity of GFP images was 55,909 at 0 min, 56,186 at 10 min, and 57,085 at 20 min, and maintained after 20 min. The intensity of luciferase-luciferin images was 28,065 at 10 min after the injection of D-luciferin and 5,199 at 20 min after the injection. The intensity of luciferase-luciferin images decreased by approximately 80% at 20 min compared to 10 min. An exposure time of 30 s for luciferase-luciferin imaging was needed compared to 100 ms for GFP fluorescence imaging in order to detect signals. CONCLUSION: An imaging system with single-nanometer tuning fluorescence excitation and an ultra-low-light detection camera and optics was able to directly visualize both GFP and luciferase-luciferin images in vivo. The intensity and stability of the signals were both greater for GFP than for luciferase-luciferin, and the exposure time for GFP was 300 times faster, demonstrating the superiority of GFP.

Cigarette smoke sustains immunosuppressive microenvironment inducing M2 macrophage polarization and viability in lung cancer settings.

BACKGROUND: It is amply demonstrated that cigarette smoke (CS) has a high impact on lung tumor progression worsening lung cancer patient prognosis and response to therapies. Alteration of immune cell types and functions in smokers' lungs have been strictly related with smoke detrimental effects. However, the role of CS in dictating an inflammatory or immunosuppressive lung microenvironment still needs to be elucidated. Here, we investigated the effect of in vitro exposure to cigarette smoke extract (CSE) focusing on macrophages. METHODS: Immortalized murine macrophages RAW 264.7 cells were cultured in the presence of CS extract and their polarization has been assessed by Real-time PCR and cytofluorimetric analysis, viability has been assessed by SRB assay and 3D-cultures and activation by exposure to Poly(I:C). Moreover, interaction with Lewis lung carcinoma (LLC1) murine cell models in the presence of CS extract were analyzed by confocal microscopy. RESULTS: Obtained results indicate that CS induces macrophages polarization towards the M2 phenotype and M2-phenotype macrophages are resistant to the CS toxic activity. Moreover, CS impairs TLR3-mediated M2-M1 phenotype shift thus contributing to the M2 enrichment in lung smokers. CONCLUSIONS: These findings indicate that, in lung cancer microenvironment of smokers, CS can contribute to the M2-phenotype macrophages prevalence by different mechanisms, ultimately, driving an anti-inflammatory, likely immunosuppressive, microenvironment in lung cancer smokers.

Salvia miltiorrhiza inhibited lung cancer through aerobic glycolysis suppression.

Lung cancer causes the most cancer deaths and needs new treatment strategies urgently. Salvia miltiorrhiza is a classical Chinese herb and a strong candidate for tumor treatment. The study found that the aqueous extract of Salvia miltiorrhiza (DSAE), ethanol extract of Salvia miltiorrhiza (DSEE), and its active components danshensu (DSS) and dihydrotanshinone I (DHI), exhibited antineoplastic effects in vivo and in vitro. Meanwhile, DSAE, DSEE, DSS, and DHI reduced glycolysis metabolites (ATP, lactate, and pyruvate contents) production, decreased aerobic glycolysis enzymes, and inhibited Seahorse indexes (OCR and ECAR) in Lewis lung cancer cells (LLC). Data suggests that aerobic glycolysis could be inhibited by Salvia miltiorrhiza and its components. The administration of DSS and DHI further reduced the level of HKII in lung cancer cell lines that had been inhibited with HK-II antagonists (2-deoxyglucose, 2-DG; 3-bromo-pyruvate, 3-BP) or knocked down with siRNA, thereby exerting an anti-lung cancer effect. Although DSS and DHI decreased the level of HKII in HKII-Knock-In lung cancer cell line, their anti-lung cancer efficacy remained limited due to the persistent overexpression of HKII in these cells. Reiterating the main points, we have discovered that the anti-lung cancer effects of Salvia miltiorrhiza may be attributed to its ability to regulate HKII expression levels, thereby inhibiting aerobic glycolysis. This study not only provides a new research paradigm for the treatment of cancer by Salvia miltiorrhiza, but also highlights the important link between glucose metabolism and the effect of Salvia Miltiorrhiza.

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.

Antitumor progenitor exhausted CD8+ T cells are sustained by TCR engagement.

The durability of an antitumor immune response is mediated in part by the persistence of progenitor exhausted CD8+ T cells (Tpex). Tpex serve as a resource for replenishing effector T cells and preserve their quantity through self-renewal. However, it is unknown how T cell receptor (TCR) engagement affects the self-renewal capacity of Tpex in settings of continued antigen exposure. Here we use a Lewis lung carcinoma model that elicits either optimal or attenuated TCR signaling in CD8+ T cells to show that formation of Tpex in tumor-draining lymph nodes and their intratumoral persistence is dependent on optimal TCR engagement. Notably, attenuated TCR stimulation accelerates the terminal differentiation of optimally primed Tpex. This TCR-reinforced Tpex development and self-renewal is coupled to proximal positioning to dendritic cells and epigenetic imprinting involving increased chromatin accessibility at Egr2 and Tcf1 target loci. Collectively, this study highlights the critical function of TCR engagement in sustaining Tpex during tumor progression.

The Application of 2d Mxene Nanosheet -Based Thermosensitive Gel Delivery System Loaded with Cisplatin and Imiquimod for Lung Cancer.

Introduction: Lung cancer's high incidence and dismal prognosis with traditional treatments like surgery and radiotherapy necessitate innovative approaches. Despite advancements in nanotherapy, the limitations of single-treatment modalities and significant side effects persist. To tackle lung cancer effectively, we devised a temperature-sensitive hydrogel-based local injection system with near-infrared triggered drug release. Utilizing 2D MXene nanosheets as carriers loaded with R837 and cisplatin (DDP), encapsulated within a temperature-sensitive hydrogel-forming PEG-MXene@DDP@R837@SHDS (MDR@SHDS), we administered in situ injections of MDR@SHDS into tumor tissues combined with photothermal therapy (PTT). The immune adjuvant R837 enhances dendritic cell (DC) maturation and tumor cell phagocytosis, while PTT induces tumor cell apoptosis and necrosis by converting light energy into heat energy. Methods: Material characterization employed transmission electron microscopy, X-ray photoelectron spectroscopy, phase transition temperature, and near-infrared thermography. In vitro experiments assessed Lewis cell proliferation and apoptosis using CCK-8, Edu, and TUNEL assays. In vivo experiments on C57 mouse Lewis transplant tumors evaluated the photothermal effect via near-infrared thermography and assessed DC maturation and CD4+/CD8+ T cell ratios using flow cytometry. The in vivo anti-tumor efficacy of MDR@SHDS was confirmed by tumor growth curve recording and HE and TUNEL staining of tumor sections. Results: The hydrogel exhibited excellent temperature sensitivity, controlled release properties, and high biocompatibility. In vitro experiments revealed that MDR@SHDS combined with PTT had a greater inhibitory effect on tumor cell proliferation compared to MDR@SHD alone. Combining local immunotherapy, chemotherapy, and PTT yielded superior anti-tumor effects than individual treatments. Conclusion: MDR@SHDS, with its simplicity, biocompatibility, and enhanced anti-tumor effects in combination with PTT, presents a promising therapeutic approach for lung cancer treatment, offering potential clinical utility.

Effects of reprogrammed splenic CD8+ T-cells in vitro and in mice with spontaneous metastatic Lewis lung carcinoma.

BACKGROUND: Metastatic disease is a major and difficult-to-treat complication of lung cancer. Considering insufficient effectiveness of existing therapies and taking into account the current problem of lung cancer chemoresistance, it is necessary to continue the development of new treatments. METHODS: Previously, we have demonstrated the antitumor effects of reprogrammed CD8+ T-cells (rCD8+ T-cells) from the spleen in mice with orthotopic lung carcinoma. Reprogramming was conducted by inhibiting the MAPK/ERK signalling pathway through MEKi and the immune checkpoint PD-1/PD-L1. Concurrently, CD8+ T-cells were trained in Lewis lung carcinoma (LLC) cells. We suggested that rCD8+ T-cells isolated from the spleen might impede the development of metastatic disease. RESULTS: The present study has indicated that the reprogramming procedure enhances the survival and cytotoxicity of splenic CD8+ T-cells in LLC culture. In an LLC model of spontaneous metastasis, splenic rCD8 + T-cell therapy augmented the numbers of CD8+ T-cells and CD4+ T-cells in the lungs of mice. These changes can account for the partial reduction of tumors in the lungs and the mitigation of metastatic activity. CONCLUSIONS: Our proposed reprogramming method enhances the antitumor activity of CD8+ T-cells isolated from the spleen and could be valuable in formulating an approach to treating metastatic disease in patients with lung cancer.

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

BACKGROUND AND PURPOSE: In 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. METHOD: C57BL/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. RESULTS: This 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. CONCLUSION: Exercise 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.

DPP Inhibition Enhances the Efficacy of PD-1 Blockade by Remodeling the Tumor Microenvironment in Lewis Lung Carcinoma Model.

The remarkable efficacy of cancer immunotherapy has been established in several tumor types. Of the various immunotherapies, PD-1/PD-L1 inhibitors are most extensively used in the treatment of many cancers in clinics. These inhibitors restore the suppressed antitumor immune response and inhibit tumor progression by blocking the PD-1/PD-L1 signaling. However, the low response rate is a major limitation in the clinical application of PD-1/PD-L1 inhibitors. Therefore, combination strategies that enhance the response rate are the need of the hour. In this investigation, PT-100 (also referred to as Talabostat, Val-boroPro, and BXCL701), an orally administered and nonselective dipeptidyl peptidase inhibitor, not only augmented the effectiveness of anti-PD-1 therapy but also significantly improved T immune cell infiltration and reversed the immunosuppressive tumor microenvironment. The combination of PT-100 and anti-PD-1 antibody increased the number of CD4+ and CD8+ T cells. Moreover, the mRNA expression of T cell-associated molecules was elevated in the tumor microenvironment. The results further suggested that PT-100 dramatically reduced the ratio of tumor-associated macrophages. These findings provide a promising combination strategy for immunotherapy in lung cancer.

Tumor cells inhibit the activation of ILC2s through up-regulating PD-1 expression.

OBJECTIVE: Up-regulating programmed cell death ligand-1(PD-L1) expressed on tumor cells and tumor-infiltrating myeloid cells interacting with up-regulated programmed cell death-1 (PD-1) expressed on tumor-infiltrating lymphoid cells greatly hinder their tumor-inhibiting effect. It is necessary to explore the deep mechanism of this negative effect, so as to find the potential methods to improve the immunotherapy efficiency. METHODS AND RESULTS: In this study, we found that the PD-1 expression in lung cancer-infiltrating type II innate lymphoid cells (ILC2s) was highly up-regulated, which greatly restrained the activation and function of ILC2s. Furthermore, anti-PD-1 could restore the inhibition and effective cytokine secretion of ILC2s when co-cultured with tumor cells. In vivo studies proved that anti-PD-1 treatment promoted the activation of tumor-infiltrating ILC2s and inhibited the tumor growth of LLC-bearing nude mice. DISCUSSION: Our studies demonstrate a new PD-1/PD-L1 axis regulating mechanism on innate immune cells, which provide a useful direction to ILC2s-based immunotherapy to cancer diseases.

The ameliorative effect on chemotherapy-induced injury and tumor immunosuppressive microenvironment of the polysaccharide from the rhizome of Menispermum dauricum DC.

Combined medication has attracted increasing attention as an important treatment option for tumors due to the serious adverse effects of chemotherapy. In this study, as a new therapy strategy, a combination treatment of MDP (a polysaccharide from the rhizome of Menispermum dauricum DC.) with cyclophosphamide (CTX) was investigated. The results showed that combination treatment with MDP and CTX exerted a significantly synergistic anti-tumor effect in Lewis tumor-bearing mice, improved CTX-induced emaciation and hair loss, as well as increased the number of leukocytes, erythrocytes, hemoglobin, and platelets in the peripheral blood. In addition, compared with CTX alone, the thymus index and spleen index of the MDP + CTX group were increased, the number of CD3 + T cells, CD8 + T cells, white blood cells and B cells in spleen also increased significantly. MDP could also ameliorate the increase in liver and kidney index caused by CTX. In the Lewis lung cancer model, MDP showed a certain degree of anti-tumor effects, which may be related to its promotion of tumor-associated macrophages (TAMs) to M1 phenotype polarisation, enhancement of the number of T cells in tumor tissues and promotion of Th cells in tumor tissues to Th1 phenotype polarisation, thus alleviating the immunosuppressive microenvironment in tumor tissues. This study laid the foundation for the development of MDP as a polysaccharide drug for the treatment or adjuvant therapy of tumors and has important significance for the further clinical application of polysaccharides.

Fucoidan MF4 from Fucus vesiculosus inhibits Lewis lung cancer via STING-TBK1-IRF3 pathway.

Fucoidan, a sulfated polysaccharide of marine origin found in brown algae and sea cucumbers, has been identified as a neuroprotective compound. In this study, a novel fucoidan MF4 was extracted from Fucus vesiculosus and isolated using Q-Sepharose fast-flow ion-exchange chromatography. The physicochemical properties of MF4 were characterized. MF4 is primarily composed of fucose, xylose, galactose, glucose, and mannose in a molar ratio of 12.3: 4.9: 1.1: 1.0: 1.1, with an average molecular weight of 67.7 kDa. Notably, MF4 demonstrated suppression of LLC tumor growth in vivo. RNA-sequencing analysis revealed that MF4 enhanced the expression of type I interferon-associated downstream genes in macrophages. Furthermore, MF4 increased the levels of phosphorylated TBK1 and IRF3 proteins in vitro. By activating the STING-TBK1-IRF3 signaling pathway, MF4 may enhance the antitumor activity of macrophages. Taken together, MF4 has promising potential as an antitumor and immunomodulatory agent.

Age-Related Features of the Response of Cancer Stem Cells and T Cells in Experimental Lung Cancer.

The responses of tumor stem cells and various populations of CD4 and CD8 T cells of young and aged C57BL/6 mice were studied in a lung cancer model. Using Lewis lung carcinoma cell line, an orthotopic model of lung cancer was modeled. Cancer stem cells, circulating tumor cells, and various populations of CD4 and CD8 T cells in the blood and lung tissue were studied by cytometry. We revealed age-related differences in the content of various populations of CD4 and CD8 T cells in the blood and lungs of intact young and aged mice. Age-related features of the reaction of various populations of cancer stem cells and CD4 and CD8 T cells in the blood and lungs of animals in the Lewis lung carcinoma were shown.

Single-cell RNA sequencing reveals recruitment of the M2-like CCL8high macrophages in Lewis lung carcinoma-bearing mice following hypofractionated radiotherapy.

BACKGROUND: Tumor-associated macrophages (TAMs) play a pivotal role in reshaping the tumor microenvironment following radiotherapy. The mechanisms underlying this reprogramming process remain to be elucidated. METHODS: Subcutaneous Lewis lung carcinoma (LLC) murine model was treated with hypofrationated radiotherapy (8 Gy × 3F). Single-cell RNA sequencing was utilized to identify subclusters and functions of TAMs. Multiplex assay and enzyme-linked immunosorbent assay (ELISA) were employed to measure serum chemokine levels. Bindarit was used to inhibit CCL8, CCL7, and CCL2. The infiltration of TAMs after combination treatment with hypofractionated radiotherapy and Bindarit was quantified with flow cytometry, while the influx of CD206 and CCL8 was assessed by immunostaining. RESULTS: Transcriptome analysis identified a distinct subset of M2-like macrophages characterized by elevated Ccl8 expression level following hypofractionated radiotherapy in LLC-bearing mice. Remarkbly, hypofractionated radiotherapy not only promoted CCL8high macrophages infiltration but also reprogrammed them by upregulating immunosuppressive genes, thereby fostering an immunosuppressive tumor microenvironment. Additioinally, hypofractionated radiotherapy enhanced the CCL signaling pathway, augmenting the pro-tumorigenic functions of CCL8high macrophages and boosting TAMs recruitment. The adjunctive treatment combining hypofractionated radiotherapy with Bindarit effectively reduced M2 macrophages infiltration and prolonged the duration of local tumor control. CONCLUSIONS: Hypofractionated radiotherapy enhances the infiltration of CCL8high macrophages and amplifies their roles in macrophage recruitment through the CCL signaling pathway, leading to an immunosuppressive tumor microenvironment. These findings highlight the potential of targeting TAMs and introduces a novel combination to improve the efficacy of hypofractionated radiotherapy.

MHY1485 potentiates immunogenic cell death induction and anti-cancer immunity following irradiation.

Recent in vitro experiments showed that combined treatment with MHY1485, a low-molecular-weight compound, and X-ray irradiation significantly increased apoptosis and senescence in tumor cells, which was associated with oxidative stress, endoplasmic reticulum (ER) stress and p21 stabilization, compared to radiation treatment alone. However, evidence for MHY1485 treatment-mediated suppression of tumor growth in animals is still lacking. Furthermore, it has been shown that ER stress enhances immunogenic cell death (ICD) in tumor cells, as it can exert a favorable influence on the anti-cancer immune system. In the present study, we examined whether co-treatment of MHY1485 and X-ray irradiation induces ICD and in vivo tumor growth suppression using the CT26 and Lewis lung carcinoma murine tumor cell lines. We found that MHY1485 + X-ray treatment promotes ICD more effectively than X-ray treatment alone. MHY1485 suppresses tumor growth in vivo under co-treatment with X-rays and increases INF-γ, tumor necrosis factor, interleukin-2 and interleukin-12 levels in the spleen as well as the presence of CD8+ cells in the tumor. The results suggest that MHY1485 treatment leads to the conversion of irradiated tumors into effective vaccines. Thus, MHY1485 is a promising lead compound for use in combination with radiotherapy.

18ß-glycyrrhetinic acid suppresses Lewis lung cancer growth through protecting immune cells from ferroptosis.

PURPOSE: 18ß-glycyrrhetinic acid (GA), the main metabolite of glycyrrhizic acid extracted from the root of licorice, has been reported to possess anti-cancer and immunomodulatory activity, but the mechanisms are not well understood. Recent studies have shown that ferroptosis of immune cells is involved in tumor-associated immune suppression. The purpose of this study was to investigate whether the enhanced immune response via inhibiting immune cell ferroptosis contributed to the anticancer effect of 18ß-GA. METHODS: Lewis Lung carcinoma mouse model and Murine CD8 + T cell culture model were used to examine the changes of immune response and ferroptosis of immune cells. RESULTS: We found that 18ß-GA was effective against lung cancer accompanied by enhanced activation of tumor-infiltrating CD8+ T cells in Lewis Lung carcinoma mouse model. Furthermore, we demonstrated that the boosted immune response by GA was attributed to its ability to inhibit arachidonic acid (AA)-mediated CD8+ T ferroptosis via suppressing CD36 expression. CONCLUSION: The findings of the present study unraveled a novel mechanism underlying the anti-cancer and immunomodulatory activity of 18ß-GA and support that 18ß-GA holds potential to be used as an immune enhancer for lung cancer prevention or treatment.