Regulating Tumor-Associated Macrophage Polarization by Cyclodextrin-Modified PLGA Nanoparticles Loaded with R848 for Treating Colon Cancer.

Purpose: This study aimed to develop a novel and feasible modification strategy to improve the solubility and antitumor activity of resiquimod (R848) by utilizing the supramolecular effect of 2-hydroxypropyl-beta-cyclodextrin (2-HP-ß-CD). Methods: R848-loaded PLGA nanoparticles modified with 2-HP-ß-CD (CD@R848@NPs) were synthesized using an enhanced emulsification solvent-evaporation technique. The nanoparticles were then characterized in vitro by several methods, such as scanning electron microscopy (SEM), differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectroscopy, particle size analysis, and zeta potential analysis. Then, the nanoparticles were loaded with IR-780 dye and imaged using an in vivo imaging device to evaluate their biodistribution. Additionally, the antitumor efficacy and underlying mechanism of CD@R848@NPs in combination with an anti-TNFR2 antibody were investigated using an MC-38 colon adenocarcinoma model in vivo. Results: The average size of the CD@R848@NPs was 376 ± 30 nm, and the surface charge was 21 ± 1 mV. Through this design, the targeting ability of 2-HP-ß-CD can be leveraged and R848 is delivered to tumor-supporting M2-like macrophages in an efficient and specific manner. Moreover, we used an anti-TNFR2 antibody to reduce the proportion of Tregs. Compared with plain PLGA nanoparticles or R848, CD@R848@NPs increased penetration in tumor tissues, dramatically reprogrammed M1-like macrophages, removed tumors and prolonged patient survival. Conclusion: The new nanocapsule system is a promising strategy for targeting tumor, reprogramming tumor -associated macrophages, and enhancement immunotherapy.

Combinational delivery of TLR4 and TLR7/8 agonist enhanced the therapeutic efficacy of immune checkpoint inhibitors to colon tumor.

Immunotherapy is regarded as a potent cancer treatment, with DC vaccines playing a crucial role. Although clinical trials have demonstrated the safety and efficacy of DC vaccines, loading antigens in vitro is challenging, and their therapeutic effects remain unpredictable. Moreover, the diverse subtypes and maturity states of DCs in the body could induce both immune responses and immune tolerance, potentially affecting the vaccine's efficacy. Hence, the optimization of DC vaccines remains imperative. Our study discovered a new therapeutic strategy by using CT26 and MC38 mouse colon cancer models, as well as LLC mouse lung cancer models. The strategy involved the synergistic activation of DCs through intertumoral administration of TLR4 agonist high-mobility group nucleosome binding protein 1 (HMGN1) and TLR7/8 agonist (R848/resiquimod), combined with intraperitoneal administration of TNFR2 immunosuppressant antibody. The experimental results indicated that the combined use of HMGN1, R848, and α-TNFR2 had no effect on LLC cold tumors. However, it was effective in eradicating CT26 and MC38 colon cancer and inducing long-term immune memory. The combination of these three drugs altered the TME and promoted an increase in anti-tumor immune components. This may provide a promising new treatment strategy for colon cancer.

Psychological distress influences lung cancer: Advances and perspectives on the immune system and immunotherapy.

Lung cancer has the highest incidence rate and mortality worldwide. Moreover, multiple factors may cause heterogeneity in the efficacy of immunotherapy for lung cancer, and preclinical studies have gradually uncovered the promotive effects of psychological distress (PD) on tumor hallmarks. Therefore, treatment targeted at PD may be a vital factor in adjusting and improving immunotherapy for lung cancer. Here, by focusing on the central nervous system, as well as stress-related crucial neurotransmitters and hormones, we highlight the effects of PD on the lung immune system, the lung tumor microenvironment (TME) and immunotherapy, which brings a practicable means and psychosocial perspective to lung cancer treatment.

Anti-TNFR2 enhanced the antitumor activity of a new HMGN1/3M-052 stimulated dendritic cell vaccine in a mouse model of colon cancer.

Immunotherapy is the new approach for cancer treatment that can be achieved through several strategies, one of which is dendritic cells (DCs) vaccine therapy. However, traditional DC vaccination lacks accurate targeting, so DC vaccine preparation needs to be optimized. Immunosuppressive CD4+Foxp3+ regulatory T cells (Tregs) in the tumor microenvironment can promote tumor immune escape. Therefore, targeting Tregs has become a strategy for tumor immunotherapy. In this study, we found that HMGN1 (N1, a dendritic cell-activating TLR4 agonist) and 3M-052 (a newly synthesized TLR7/8 agonist) synergistically stimulate DCs maturation and increase the production of proinflammatory cytokines TNFα and IL-12. In a colon cancer mice model, vaccination with N1 and 3M-052 stimulated and tumor antigen-loaded DCs combined with anti-TNFR2 inhibited tumor growth in mice, and the antitumor effect was mainly achieved through stimulation of cytotoxic CD8 T cell activation and depletion of Tregs. Overall, the combinating of DC activation by N1 and 3M-052 with inhibition of Tregs by antagonizing TNFR2 as a therapeutic strategy may represent a more effective strategy for cancer treatment.

Ansofaxine hydrochloride inhibits tumor growth and enhances Anti-TNFR2 in murine colon cancer model.

Introduction: As psychoneuroimmunology flourishes, there is compelling evidence that depression suppresses the anti-tumor immune response, promotes the progression of cancer, and inhibits the effectiveness of cancer immunotherapy. Recent studies have reported that antidepressants can not only alleviate the depressant condition of cancer patients, but also strengthen the anti-tumor immunity, thus suppressing tumors. Tumor necrosis factor receptor 2 (TNFR2) antagonistic antibodies (Anti-TNFR2) targeting tumor-infiltrating regulatory T cells (Tregs) has achieved great results in preclinical studies, and with a favorable toxicity profile than existing immunotherapies, and is expected to become a new generation of more effective treatment strategies. Understanding the effects of combination therapy with antidepressants and Anti-TNFR2 may help design new strategies for cancer immunotherapy. Methods: We treated CT26, HCT116, MCA38 and SW620 colon cancer cells with fluoxetine (0-50 µM), ansofaxine hydrochloride (0-50 µM) and amitifadine hydrochloride (0-150 µM) to examine their effects on cell proliferation and apoptosis. We explored the antitumor effects of ansofaxine hydrochloride in combination with or without Anti-TNFR in subcutaneously transplanted CT26 cells in tumor-bearing mouse model. Antitumor effects were evaluated by tumor volume. NK cell, M1 macrophage cell, CD4+ T cell, CD8+ T cell, exhausted CD8+ T and regulatory T cell (Tregs) subtypes were measured by flow cytometry. 5-hydroxytryptamine, dopamine and norepinephrine levels were measured by ELISA. Results: Oral antidepression, ansofaxine hydrochloride, enhanced peripheral dopamine levels, promoted CD8+T cell proliferation, promoted intratumoral infiltration of M1 and NK cells, decreased the proportion of tumor-infiltrating exhausted CD8+T cells, and strengthened anti-tumor immunity, thereby inhibiting colon cancer growth. In combination therapy, oral administration of ansofaxine hydrochloride enhanced the efficacy of Anti-TNFR2, and produced long-term tumor control in with syngeneic colorectal tumor-bearing mice, which was attributable to the reduction in tumor-infiltrating Treg quantity and the recovery of CD8+ T cells function. Discussion: In summary, our data reveal the role of ansofaxine hydrochloride in modulating the anti-tumor immunity. Our results support that exhausted CD8+T is an important potential mechanism by which ansofaxine hydrochloride activates anti-tumor immunity and enhances anti-tumor effects of anti-TNFR2.

Pantothenate Kinase 1 Inhibits the Progression of Hepatocellular Carcinoma by Negatively Regulating Wnt/ß-catenin Signaling.

Hyperactivation of Wnt/ß-catenin signaling has been reported in hepatocellular carcinoma (HCC). However, the mechanisms underlying the hyperactivation of Wnt/ß-catenin signaling are incompletely understood. In this study, Pantothenate kinase 1 (PANK1) is shown to be a negative regulator of Wnt/ß-catenin signaling. Downregulation of PANK1 in HCC correlates with clinical features. Knockdown of PANK1 promotes the proliferation, growth and invasion of HCC cells, while overexpression of PANK1 inhibits the proliferation, growth, invasion and tumorigenicity of HCC cells. Mechanistically, PANK1 binds to CK1α, exerts protein kinase activity and cooperates with CK1α to phosphorylate N-terminal serine and threonine residues in ß-catenin both in vitro and in vivo. Additionally, the expression levels of PANK1 and ß-catenin can be used to predict the prognosis of HCC. Collectively, the results of this study highlight the crucial roles of PANK1 protein kinase activity in inhibiting Wnt/ß-catenin signaling, suggesting that PANK1 is a potential therapeutic target for HCC.

Mesenchymal stromal cells protect hepatocytes from lipotoxicity through alleviation of endoplasmic reticulum stress by restoring SERCA activity.

The aim of this study was to investigate how mesenchymal stromal cells (MSCs) modulate metabolic balance and attenuate hepatic lipotoxicity in the context of non-alcoholic fatty liver disease (NAFLD). In vivo, male SD rats were fed with high-fat diet (HFD) to develop NAFLD; then, they were treated twice by intravenous injections of rat bone marrow MSCs. In vitro, HepG2 cells were cocultured with MSCs by transwell and exposed to palmitic acid (PA) for 24 hours. The endoplasmic reticulum (ER) stressor thapsigargin and sarco/ER Ca2+ -ATPase (SERCA2)-specific siRNA were used to explore the regulation of ER stress by MSCs. We found that MSC administration improved hepatic steatosis, restored systemic hepatic lipid and glucose homeostasis, and inhibited hepatic ER stress in HFD-fed rats. In hepatocytes, MSCs effectively alleviated the cellular lipotoxicity. Particularly, MSCs remarkably ameliorated the ER stress and intracellular calcium homeostasis induced by either PA or thapsigargin in HepG2 cells. Additionally, long-term HFD or PA stimulation would activate pyroptosis in hepatocytes, which may contribute to the cell death and liver dysfunction during the process of NAFLD, and MSC treatment effectively ameliorates these deleterious effects. SERCA2 silencing obviously abolished the ability of MSCs against the PA-induced lipotoxicity. Conclusively, our study demonstrated that MSCs were able to ameliorate liver lipotoxicity and metabolic disturbance in the context of NAFLD, in which the regulation of ER stress and the calcium homeostasis via SERCA has played a key role.

Mesenchymal stem cells alleviate palmitic acid-induced endothelial-to-mesenchymal transition by suppressing endoplasmic reticulum stress.

High levels of plasma free fatty acids (FFAs) lead to endothelial dysfunction (ED), which is involved in the pathogenesis of metabolic syndrome, diabetes, and atherosclerosis. Endoplasmic reticulum (ER) stress and endothelial-to-mesenchymal transition (EndMT) are demonstrated to be mechanistically related to endothelial dysfunction. Mesenchymal stem cells (MSCs) have exhibited an extraordinary cytoprotective effect on cellular lipotoxicity and vasculopathy. However, the underlying mechanisms have not been clearly defined. In the present study, we investigated whether MSCs could ameliorate palmitic acid (PA)-induced endothelial lipotoxicity by reducing ER stress and EndMT. We observed that MSC cocultures substantially alleviated PA-induced lipotoxicity in human umbilical vein endothelial cells (HUVECs). MSCs were able to restore the cell viability, increase tubule formation and migration ability, and decrease inflammation response and lipid deposition. Furthermore, PA caused endothelial-to-mesenchymal transition in HUVECs, which was abrogated by MSCs possibly through inhibiting ER stress. In addition, PA stimulated MSCs to secrete more stanniocalcin-1 (STC-1). Knocking down of STC-1 in MSCs attenuated their effects on PA-induced lipotoxicity in HUVECs. In vivo, MSC transplantation alleviated dyslipidemia and endothelial dysfunction in high-fat diet-fed Sprague-Dawley rats. MSC-treated rats showed reduced expressions of ER stress-related genes in aortas and suppressed expressions of EndMT-related proteins in rat aortic endothelial cells. Overall, our findings indicated that MSCs were able to attenuate endothelial lipotoxicity through inhibiting ER stress and EndMT, in which STC-1 secreted from MSCs may play a critical role.

Oleic acid protects insulin-secreting INS-1E cells against palmitic acid-induced lipotoxicity along with an amelioration of ER stress.

PURPOSE: It is demonstrated that unsaturated fatty acids can counteract saturated fatty acids-induced lipotoxicity, but the molecular mechanisms are unclear. In this study, we investigated the protective effects of monounsaturated oleic acid (OA) against saturated palmitic acid (PA)-induced cytotoxicity in rat ß cells as well as islets, and mechanistically focused on its regulation on endoplasmic reticulum (ER) stress. METHODS: Rat insulinoma cell line INS-1E cells and primary islets were treated with PA with or without OA for 24 h to determine the cell viability, apoptosis, and ER stress. SD rats were fed with high-fat diet (HFD) for 16 w, then, HFD was half replaced by olive oil to observe the protective effects of monounsaturated fatty acids rich diet. RESULTS: We demonstrated that PA impaired cell viability and insulin secretion of INS-1E cells and rat islets, but OA robustly rescued cells from cell death. OA substantially alleviated either PA or chemical ER stressors (thapsigargin or tunicamycin)-induced ER stress. Importantly, OA attenuated the activity of PERK-eIF2α-ATF4-CHOP pathway and regulated the ER Ca2+ homeostasis. In vivo, only olive oil supplementation did not cause significant changes, while high-fat diet (HFD) for 32 w obviously induced islets ER stress and impaired insulin sensitivity in SD rats. Half replacement of HFD with olive oil (a mixed diet) has ameliorated this effect. CONCLUSION: OA alleviated PA-induced lipotoxicity in INS-1E cells and improved insulin sensitivity in HFD rats. The amelioration of PA triggered ER stress may be responsible for its beneficial effects in ß cells.

Oleic acid protects saturated fatty acid mediated lipotoxicity in hepatocytes and rat of non-alcoholic steatohepatitis.

Aim This study aims to demonstrate the protective effects of monounsaturated oleic acid (OA) against saturated palmitic acid (PA) induced cellular lipotoxicity in hepatocytes and rats with non-alcoholic steatohepatitis (NASH). MAIN METHODS: Human hepatoma cell line HepG2 cells and neonatal rat primary hepatocytes were treated with PA or/and OA for 24 h. SD rats were fed with high fat diet (HFD) to induce NASH. From the 16th w, the HFD was full or half replaced by olive oil to observe the protective effects. KEY FINDINGS: In vitro, OA substantially alleviated PA induced cellular apoptosis, oxidative stress, ER stress, mitochondrial dysfunction, as well as inflammation in hepatocytes. In vivo, only olive oil supplementation had no detrimental effects, while HFD developed NASH in normal rats. Full replacement of HFD with olive oil had profoundly reversed NASH. Noteworthily, half replacement of HFD with olive oil (a mixed diet) has ameliorated NASH injury as well. It strikingly changed the hepatic histology from macrovesicular-steatosis into entire microvesicular-steatosis, and significantly reduced inflammation, ballooning and fibrosis. SIGNIFICANCE: Our study has demonstrated in both hepatocytes and NASH rats that oleic acids had great potential to combat the saturated fatty acids induced hepatic lipotoxicity. Only half replacement of HFD by monounsaturated fatty acids rich diet still had significant therapeutic outcome in NASH rats. Redirecting the toxic saturated fatty acids into triglyceride storage and reduction of cholesterol accumulation might be the possible explanation of OA driven protection in this scenario.

Glucocorticoid treatment facilitates development of a metabolic syndrome in ovariectomized Macaca Mulatta fed a high fat diet.

Metabolic syndrome (MetS) is characterized by a cluster of key features, which include abdominal obesity, insulin resistance, hypertension, and dyslipidemia. The aim of this study was to assess the impact of elevated glucocorticoid levels on the development of MetS in middle-aged female rhesus monkeys (Macaca Mulatta) after ovariectomy. Six female ovariectomized rhesus monkeys (9-13years) were randomly assigned to either a control group (normal diet, n=3) or a group in which MetS was facilitated (n=3). The MetS group fed with HFD (15% fat) and received oral prednisone acetate treatment (50mg/day). After 24months, the GCs treatment was withdrawn with continuation of high-fat feeding for a further 12months. After 24months, the MetS group displayed a significant increase in body weight and abdominal circumference. Additionally, the MetS animals displayed abnormal serum lipids, insulin resistance and impaired glucose tolerance. Histology of liver biopsies indicated marked accumulation of lipid droplets in hepatocytes of MetS animals. Withdrawal of GCs treatment led to recovery from above-mentioned metabolic disorders. Whereas GCs treatment increased leptin expression, it lowered expression of adiponectin and other factors in adipose tissue. Expression of Hydroxy-steroid dehydrogenase-1 and glucose transporter type-4 in the livers of MetS animals were reduced. We conclude that in the context of high fat diet, high levels of exogenous GCs contribute to the development of MetS in non-human primates.

High-fat diet combined with low-dose streptozotocin injections induces metabolic syndrome in Macaca mulatta.

Metabolic syndrome (MetS) is associated with abdominal obesity, hyperlipidemia, insulin resistance, and type 2 diabetes mellitus, and increases the risk of cardiovascular disease. Given the complex multifactorial pathogenesis of MetS, qualified animal models are currently seriously limited for researchers. The aim of our study was to develop a MetS model in juvenile rhesus monkeys (Macaca mulatta). Rhesus monkeys (1-year-old) fed a high-fat diet (15 % fat, 2 % cholesterol) were used as the HF group (n = 6), and those on a normal diet (5 % fat) were used as the control group (n = 4). After being fed a high-fat diet for approximately 12 months, 2 monkeys (HF + STZ group) were injected with low-dose streptozotocin (STZ, 25 mg/kg) twice, with a 7 days interval, and were then fed the same diet continuously for another 24 months. After 36 months of treatment, the high-fat diet monkeys, including the HF and HF + STZ groups, had acquired increased body weights, abnormal serum lipids, and impaired glucose tolerance compared to the control group. In addition, much more marked metabolic changes were observed in the two monkeys of the HF + STZ group, particularly in terms of high-blood glucose level and insulin resistance. Morphological observation of biopsies of liver and pancreatic tissues showed decreased islet number and mass and decreased insulin staining in the monkeys of the HF + STZ group. In addition, Oil red O staining suggested remarkable accumulation of lipid droplets in the hepatocytes. Our study suggested that a long-term high-fat diet followed with a low-dose STZ was able to induce MetS in juvenile rhesus monkeys with faster pathophysiological progress compared with high-fat diet induction alone. Our primary data showed that this method may have potentials to develop MetS animal model in non-human primates.