Swine pseudorabies virus attenuated vaccine reprograms the kidney cancer tumor microenvironment and synergizes with PD-1 blockade.

The global incidence rate of kidney cancer (KC) has been steadily increasing over the past 30 years. With the aging global population, kidney cancer has become an escalating concern that necessitates vigilant surveillance. Nowadays, surgical intervention remains the optimal therapeutic approach for kidney cancer, while the availability of efficacious treatments for advanced tumors remains limited. Oncolytic viruses, an emerging form of immunotherapy, have demonstrated encouraging anti-neoplastic properties and are progressively garnering public acceptance. However, research on oncolytic viruses in kidney cancer is relatively limited. Furthermore, given the high complexity and heterogeneity of kidney cancer, it is crucial to identify an optimal oncolytic virus agent that is better suited for its treatment. The present study investigates the oncolytic activity of the Pseudorabies virus live attenuated vaccine (PRV-LAV) against KC. The findings clearly demonstrate that PRV-LAV exhibits robust oncolytic activity targeting KC cell lines. Furthermore, the therapeutic efficacy of PRV-LAV was confirmed in both a subcutaneous tumor-bearing nude mouse model and a syngeneic mouse model of KC. Combined RNA-seq analysis and flow cytometry revealed that PRV-LAV treatment substantially enhances the infiltration of a diverse range of lymphocytes, including T cells, B cells, macrophages, and NK cells. Additionally, PRV-LAV treatment enhances T cell activation and exerts antitumor effects. Importantly, the combination of PRV-LAV with anti-PD-1 antibodies, an approved drug for KC treatment, synergistically enhances the efficacy against KC. Overall, the discovery of PRV-LAV as an effective oncolytic virus holds significant importance for improving the treatment efficacy and survival rates of KC patients.

The potential of swine pseudorabies virus attenuated vaccine for oncolytic therapy against malignant tumors.

BACKGROUND: Oncolytic viruses are now well recognized as potential immunotherapeutic agents against cancer. However, the first FDA-approved oncolytic herpes simplex virus 1 (HSV-1), T-VEC, showed limited benefits in some patients in clinical trials. Thus, the identification of novel oncolytic viruses that can strengthen oncolytic virus therapy is warranted. Here, we identified a live-attenuated swine pseudorabies virus (PRV-LAV) as a promising oncolytic agent with broad-spectrum antitumor activity in vitro and in vivo. METHODS: PRV cytotoxicity against tumor cells and normal cells was tested in vitro using a CCK8 cell viability assay. A cell kinase inhibitor library was used to screen for key targets that affect the proliferation of PRV-LAV. The potential therapeutic efficacy of PRV-LAV was tested against syngeneic tumors in immunocompetent mice, and against subcutaneous xenografts of human cancer cell lines in nude mice. Cytometry by time of flight (CyTOF) and flow cytometry were used to uncover the immunological mechanism of PRV-LAV treatment in regulating the tumor immune microenvironment. RESULTS: Through various tumor-specific analyses, we show that PRV-LAV infects cancer cells via the NRP1/EGFR signaling pathway, which is commonly overexpressed in cancer. Further, we show that PRV-LAV kills cancer cells by inducing endoplasmic reticulum (ER) stress. Moreover, PRV-LAV is responsible for reprogramming the tumor microenvironment from immunologically naïve ("cold") to inflamed ("hot"), thereby increasing immune cell infiltration and restoring CD8+ T cell function against cancer. When delivered in combination with immune checkpoint inhibitors (ICIs), the anti-tumor response is augmented, suggestive of synergistic activity. CONCLUSIONS: PRV-LAV can infect cancer cells via NRP1/EGFR signaling and induce cancer cells apoptosis via ER stress. PRV-LAV treatment also restores CD8+ T cell function against cancer. The combination of PRV-LAV and immune checkpoint inhibitors has a significant synergistic effect. Overall, these findings point to PRV-LAV as a serious potential candidate for the treatment of NRP1/EGFR pathway-associated tumors.

Babao Dan alleviates gut immune and microbiota disorders while impacting the TLR4/MyD88/NF-кB pathway to attenuate 5-Fluorouracil-induced intestinal injury.

Adjuvant chemotherapy based on 5-fluorouracil (5-FU), such as FOLFOX, is suggested as a treatment for gastrointestinal cancer. Yet, intestinal damage continues to be a prevalent side effect for which there are no practical prevention measures. We investigated whether Babao Dan (BBD), a Traditional Chinese Medicine, protects against intestinal damage induced by 5-FU by controlling immune response and gut microbiota. 5-FU was injected intraperitoneally to establish the mice model, then 250 mg/kg BBD was gavaged for five days straight. 5-FU led to marked weight loss, diarrhea, fecal blood, and histopathologic intestinal damage. Administration of BBD reduced these symptoms, inhibited proinflammatory cytokine (IL-6, IL-1ß, IFN-γ, TNF-α) secretion, and upregulated the ratio of CD3(+) T cells and the CD4(+)/CD8(+) ratio. According to 16S rRNA sequencing, BBD dramatically repaired the disruption of the gut microbiota caused in a time-dependent way, and increased the Firmicutes/Bacteroidetes (F/B) ratio. Transcriptomic results showed that the mechanism is mainly concentrated on the NF-κB pathway, and we found that BBD reduced the concentration of LPS in the fecal suspension and serum, and inhibited TLR4/MyD88/NF-κB pathway activation. Furthermore, at the genus level on the fifth day, BBD upregulated the abundance of unidentified_Corynebacteriaceae, Aerococcus, Blautia, Jeotgalicoccus, Odoribacter, Roseburia, Rikenella, Intestinimonas, unidentified_Lachnospiraceae, Enterorhabdus, Ruminiclostridium, and downregulated the abundance of Bacteroides, Parabacteroides, Parasutterella, Erysipelatoclostridium, which were highly correlated with intestinal injury or the TLR4/MyD88/NF-κB pathway. In conclusion, we established a network involving 5-FU, BBD, the immune response, gut microbiota, and key pathways to explain the pharmacology of oral BBD in preventing 5-FU-induced intestinal injury.

Chemotherapeutic Drugs Induce Different Gut Microbiota Disorder Pattern and NOD/RIP2/NF-κB Signaling Pathway Activation That Lead to Different Degrees of Intestinal Injury.

5-Fluorouracil (5-FU), irinotecan (CPT-11), oxaliplatin (L-OHP), and calcium folinate (CF) are widely used chemotherapeutic drugs to treat colorectal cancer. However, chemotherapeutic use is often accompanied by intestinal inflammation and gut microbiota disorder. Changes in gut microbiota may destroy the intestinal barrier, which contributes to the severity of intestinal injury. However, intestinal injury and gut microbiota disorder have yet to be compared among 5-FU, CPT-11, L-OHP, and CF in detail, thereby limiting the development of targeted detoxification therapy after chemotherapy. In this study, a model of chemotherapy-induced intestinal injury in tumor-bearing mice was established by intraperitoneally injecting chemotherapeutic drugs at a clinically equivalent dose. 16S rRNA gene sequencing was used to detect gut microbiota. We found that 5-FU, CPT-11, and l-OHP caused intestinal injury, inflammatory cytokine (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], interleukin-1ß [IL-1ß], and IL-6) secretion, and gut microbiota disorder. We established a complex but clear network between the pattern of changes in gut microbiota and degree of intestinal damage induced by different chemotherapeutic drugs. L-OHP caused the most severe damage in the intestine and disorder of the gut microbiota and showed a considerable overlap of the pattern of changes in microbiota with 5-FU and CPT-11. Analysis by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt v.1.0) showed that the microbiota disorder pattern induced by 5-FU, CPT-11, and L-OHP was related to the NOD-like signaling pathway. Therefore, we detected the protein expression of the NOD/RIP2/NF-κB signaling pathway and found that L-OHP most activated this pathway. Redundancy analysis/canonical correlation analysis (RDA/CCA) revealed that Bifidobacterium, Akkermansia, Allobaculum, Catenibacterium, Mucispirillum, Turicibacter, Helicobacter, Proteus, Escherichia Shigella, Alloprevotealla, Vagococcus, Streptococcus, and "Candidatus Saccharimonas" were highly correlated with the NOD/RIP2/NF-κB signaling pathway and influenced by chemotherapeutic drugs. IMPORTANCE Chemotherapy-induced intestinal injury limits the clinical use of drugs. Intestinal injury involves multiple signaling pathways and gut microbiota disruption. Our results suggested that the degree of intestinal injury caused by different drugs of the first-line colorectal chemotherapy regimen is related to the pattern of changes in microbiota. The activation of the NOD/RIP2/NF-κB signaling pathway was also related to the pattern of changes in microbiota. l-OHP caused the most severe damage to the intestine and showed a considerable overlap of the pattern of changes in microbiota with 5-FU and CPT-11. Thirteen bacterial genera were related to different levels of intestinal injury and correlated with the NOD/RIP2/NF-κB pathway. Here, we established a network of different chemotherapeutic drugs, gut microbiota, and the NOD/RIP2/NF-κB signaling pathway. This study likely provided a new basis for further elucidating the mechanism and clinical treatment of intestinal injury caused by chemotherapy.

Babao Dan Alleviates 5-Fluorouracil-Induced Intestinal Damage via Wnt/ß-Catenin Pathway.

OBJECTIVE: To evaluate the protective function of Babao Dan (BBD) on 5-flurouracil (5-FU)-induced intestinal mucositis (IM) and uncover the underlying mechanism. METHODS: A total of 18 male mice were randomly divided into 3 groups by a random number table, including control, 5-FU and 5-FU combined BBD groups, 6 mice in each group. A single intraperitoneal injection of 5-FU (150 mg/kg) was performed in 5-FU and 5-FU combined BBD groups on day 0. Mice in 5-FU combined BBD group were gavaged with BBD (250 mg/kg) daily from day 1 to 6. Mice in the control group were gavaged with saline solution for 6 days. The body weight and diarrhea index of mice were recorded daily. On the 7th day, the blood from the heart of mice was collected to analyze the proportional changes of immunological cells, and the mice were subsequently euthanized by mild anesthesia with 2% pentobarbital sodium. Colorectal lengths and villus heights were measured. Intestinal-cellular apoptosis and proliferation were evaluated by Tunel assay and immunohistochemical staining of proliferating cell nuclear antigen, respectively. Immunohistochemistry and Western blot were performed to investigate the expressions of components in Wnt/ß-catenin pathway (Wnt3, LRP5, ß-catenin, c-Myc, LRG5 and CD44). RESULTS: BBD obviously alleviated 5-FU-induced body weight loss and diarrhea, and reversed the decrease in the number of white blood cells, including monocyte, granulocyte and lymphocyte, and platelet (P<0.01). The shortening of colon caused by 5-FU was also reversed by BBD (P<0.01). Moreover, BBD inhibited apoptosis and promoted proliferation in jejunum tissues so as to reduce the intestinal mucosal damage and improve the integrity of villus and crypts. Mechanically, the expression levels of Wnt/ß -catenin mediators such as Wnt3, LRP5, ß-catenin were upregulated by BBD, activating the transcription of c-Myc, LRG5 and CD44 (P<0.01). CONCLUSIONS: BBD attenuates the adverse effects induced by 5-FU via Wnt/ß-catenin pathway, suggesting it may act as a potential agent against chemotherapy-induced intestinal mucositis.

Qingjie Fuzheng Granule suppresses lymphangiogenesis in colorectal cancer via the VEGF-C/VEGFR-3 dependent PI3K/AKT pathway.

SCOPE: To investigate the effect of Qingjie Fuzheng Granule (QFG) on lymphangiogenesis and lymphatic metastasis in colorectal cancer. METHODS: The effects of QFG on the expression and secretion of vascular endothelial growth factor-C (VEGF-C) in HCT-116 cells were investigated both in vitro and in vivo. HCT-116 cells were treated with different concentrations (0.2, 0.5, and 1.0 mg/mL) of QFG. The VEGF-C expression level was determined using RT-qPCR and western blotting, and the VEGF-C concentration in supernatant was measured by ELISA. Tumor xenograft models of HCT-116 cells were generated using BALB/c nude mice, and the mice were randomly divided into a control group (gavaged with normal saline) and QFG group (gavaged with 2 g/kg QFG). The effect of QFG on tumor growth was evaluated by comparing the volume and weight of tumors between two groups. Immunohistochemistry (IHC) and RT-qPCR were performed to detect the expression levels of VEGF-C, vascular endothelial growth factor receptor 3 (VEGFR-3), and LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1). ELISA was performed to measure the concentration of serum VEGF-C. TMT proteomics technology and Reactome pathway analysis were used to explore the mechanism of QFG inhibiting lymphangiogenesis in tumor. The VEGF-C (5 ng/mL)-stimulated human lymphatic endothelial cell (HLEC) model was conducted to evaluate the effect of QFG on lymphangiogenesis in vitro. The model cells were treated with different concentrations (0.2, 0.5, and 1.0 mg/mL) of QFG. Cell viability was then determined using an MTT assay. The cell migration, invasion, and tube-formation ability were analyzed using transwell migration, matrigel invasion and tube formation assays, respectively. The underlying mechanism was uncovered, the levels of VEGFR-3, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), p-PI3K/PI3K, p-AKT/AKT and p-mTOR/ mTOR were detected using western blotting. RESULTS: QFG significantly reduced VEGF-C expression and secretion in HCT-116 cells. QFG evidently suppressed in vivo tumor growth and the expression of VEGF-C, VEGFR-3, and LYVE-1. The serum VEGF-C level was also reduced by QFG. Moreover, TMT proteomics technology and Reactome pathway analysis identified 95 differentially expressed protein and multiple enriched pathway about matrix metalloproteinase and extracellular matrix, which is direct associate with lymphangiogenesis. In vitro experiment, QFG inhibited the viability, migration, invasion and tube formation of HLECs. Additionally, QFG reduced the VEGFR-3, MMP-2, MMP-9 expression levels, and the p-PI3K/PI3K, p-AKT/AKT, p-mTOR/ mTOR ratios. CONCLUSION: QFG can exert its effect on both tumor cells and HLECs, exhibiting ani- lymphangiogenesis in colorectal cancer via the VEGF-C/VEGFR-3 dependent PI3K/AKT pathway pathway.

Exploration of the effect of salvianolate on myocardial infarction in rats based on tandem mass tags.

Salvianolate is a compound from traditional Chinese medicine widely used in the treatment of various cardiovascular diseases. This study explored the effects of salvianolate on myocardial infarction and used tandem mass tags (TMT) to discover differentially expressed proteins. Male Sprague Dawley rats were randomly divided into the sham operation group, model group, and salvianolate group. The myocardial infarction model was established by ligating the left anterior descending coronary artery while the sham group had a sham operation. The rats were intraperitoneally injected with 2 ml of 5% glucose once a day, with 48.438 mg/kg/d salvianolate for the rats in the salvianolate group. After 4 weeks, the rats' hemodynamics were measured to evaluate cardiac function, and Masson staining assessed the area of myocardial infarction. TMT analysis was performed and validated by western blot. Salvianolate improved cardiac function after myocardial infarction, reduced the myocardial infarction area, and protected the myocardial tissue. 100 differentially expressed proteins were identified between the sham operation and model groups, salvianolate reversed the expression of 25 of those proteins, that were mainly involved in the metabolism of extracellular collagen matrix and the response to growth factor stimulation. Type I collagen, type V collagen, chymase, ß-myosin heavy chain, and A-Raf differential expression were consistent in western blotting. In conclusion, salvianolate had a protective effect on myocardial tissues of rats with myocardial infarction. Several proteins including type I collagen, type V collagen, chymase, ß-myosin, and A-Raf may be salvianolate targets for treatment of myocardial infarction.