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Background: Accumulating evidence has demonstrated the immunomodulatory effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in rheumatoid arthritis and the tumor microenvironment, besides its known capacity of specifically inducing the apoptosis of cancer cells. Mice are common available animal models for studying the roles of TRAIL. However, mice express only a single TRAIL receptor (mTRAILR) with an intracellular death domain, in contrast to the two TRAIL receptors (TRAILR1 and TRAILR2) in humans. Moreover, human TRAIL binds weakly to mTRAILR, whereas mouse TRAIL has high affinity for the human TRAIL-Rs. Therefore, we considered that murine TRAIL would be more suitable than human TRAIL for exploring the immunoregulatory effect of TRAIL in immunocompetent mice or when using mouse cells as the target. To our knowledge, the detailed method for production of recombinant murine TRAIL has not been reported. Objective: In this study, we aimed to design and express two soluble forms of murine TRAIL and verify the properties of the protein. Methods: Recombinant murine TRAILs were expressed in Escherichia coli BL21 (DE3, and Ni-chelating affinity chromatography was used for protein purification. SDS-PAGE, GDS-PAGE and HPLC were applied to analyze the protein structure. The cytotoxicity of our purified murine TRAILs was evaluated in the TRAIL-sensitive human breast cancer ZR-75-30 cells and murine breast cancer 4T1 cells. Finally, validation of the tumor-killing ability of the murine protein in vivo. Results: Two soluble forms of murine TRAILs (mT_N99 and mT_N188) were purified and demonstrated with high purity and trimeric structure. In addition, Zn2+ supplement was essential to produce soluble murine TRAILs in E.coli BL21 (DE3). The two purified soluble mTRAILs showed similar cytotoxicity to cancer cells, moreover, mT_N99 also showed a good anti-tumor effect in vivo and is more suitable for the treatment of murine tumor models. Conclusion: A production approach for recombinant murine TRAIL was determined, which covered the design of shortened forms, expression, purification and characterization.

Traditional anticancer treatments directly target tumor cells. In contrast, cancer immunotherapy fortifies host immunity. Nanoparticles that incorporate both immunomodulatory and chemotherapeutic agents regulate the tumor microenvironment by activating immune cells and enhancing antitumor immunity. Nanoparticle-based cancer immunotherapy has received considerable attention and has been extensively studied in recent years. In this study, we developed a targeted drug delivery system to enhance immunotherapeutic efficacy and overcome drug resistance by inducing tumor apoptosis and immunogenic cell death (ICD), and activating immune cells. Periodic mesoporous organosilica nanoparticles (PMOs) bore tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on their surfaces, and their inner cores were loaded with doxorubicin (DOX). TRAIL enhanced the nanoparticle-targeting capacity and worked synergistically with DOX against breast cancer cells in vitro and in vivo. Furthermore, we revealed for the first time the ability of PMOs to activate dendritic cells (DCs) and elevate ICD levels of DOX in vitro, and TRAIL further enhances the immunomodulatory function of PMOs. Systemic exposure to DOX@PMO-hT induced an immune response, activated DCs and CD4

Accumulating evidence has demonstrated the immunomodulatory effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in rheumatoid arthritis and the tumor microenvironment, besides its known capacity of specifically inducing the apoptosis of cancer cells. Mice are common available animal models for studying the roles of TRAIL. However, mice express only a single TRAIL receptor (mTRAILR) with an intracellular death domain, in contrast to the two TRAIL receptors (TRAILR1 and TRAILR2) in humans. Moreover, human TRAIL binds weakly to mTRAILR, whereas mouse TRAIL has a high affinity for human TRAIL-Rs. Therefore, we considered that murine TRAIL would be more suitable than human TRAIL for exploring the immunoregulatory effect of TRAIL in immunocompetent mice or when using mouse cells as the target. To our knowledge, the detailed method for the production of recombinant murine TRAIL has not been reported.In this study, we aimed to design and express two soluble forms of murine TRAIL and verify the properties of the protein.Recombinant murine TRAILs were expressed in Escherichia coli BL21 (DE3, and Nichelating affinity chromatography was used for protein purification. SDS-PAGE, GDS-PAGE and HPLC were applied to analyze the protein structure. The cytotoxicity of our purified murine TRAILs was evaluated in the TRAIL-sensitive human breast cancer ZR-75-30 cells and murine breast cancer 4T1 cells. Finally, validation of the tumor-killing ability of the murine protein in vivo.Two soluble forms of murine TRAILs (mT_N99 and mT_N188) were purified and demonstrated with high purity and trimeric structure. In addition, ZnA production approach for recombinant murine TRAIL was determined, which covered the design of shortened forms, expression, purification and characterization.

Glioblastoma, the most common human brain tumor, is highly invasive and difficult to cure using conventional cancer therapies. As an alternative, adenovirus-mediated virotherapies represent a popular and maturing technology. However, the cell surface coxsackievirus and adenovirus receptor (CAR)-dependent infection mechanism limits the infectivity and oncolytic effects of Adenovirus type 5. To address this limitation, in this study we aimed to develop a novel oncolytic adenovirus for enhanced infectivity and therapeutic efficacy toward glioblastoma. We developed a novel genetically modified oncolytic adenovirus vector with dual capsid modifications to facilitate infection and specific cytotoxicity toward glioma cells. Modification of the adenoviral capsid proteins involved the incorporation of a synthetic leucine zipper-like dimerization domain into the capsid protein IX (pIX) of human adenovirus serotype 5 (Ad5) and the exchange of the fiber knob from Ad37. The virus infection mechanism and anti-tumor efficacy of modified vectors were evaluated in both in vitro (cell) and in vivo (mouse) models. Ad37-knob exchange efficiently promoted the virus infection and replication-induced glioma cell lysis by oncolytic Ad5. We also found that gene therapy mediated by the dual-modified oncolytic Ad5 vector coupled with the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exhibited significantly enhanced anti-tumor efficacy in vitro and in vivo. This genetically modified oncolytic adenovirus provides a promising vector for future use in glioblastoma gene-viral-based therapies.

The immune status of the tumor microenvironment is a key indicator determining the antitumor effect of immunotherapy. Oncolytic viruses directly target tumor cells or indirectly modulate the tumor microenvironment (TME) especially when properly armed. It was previously demonstrated that conditionally replicating adenovirus serotype 5 (CRAd5) encoding tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) had outstanding antitumor effects in different human cancer cells xenograft models; however, its antitumor immune mechanism has not been evaluated in immunocompetent preclinical mouse models. We first explored the antitumor activity of CRAd5-TRAIL in several murine tumor models and found that the expression of TRAIL induced increases or activation in tumor-infiltrating T cells. To further improve the antitumor effects, mouse CD40 ligand (mCD40L) as an immune activator expressed by recombinant Ad5 vector was firstly used in combination with CRAd5-TRAIL for tumor immunotherapy. Both in vitro and in vivo studies demonstrated that mCD40L effectively activated dendritic cells (DCs), B cells, and tumor-infiltrating T cells, and also promoted tumor cell apoptosis by increasing the expression of TRAIL receptors, thereby significantly enhancing the antitumor activity of oncolytic adenoviruses in CT26 and B16 tumor-bearing models. Although affected by the restriction of oncolytic adenovirus replication in mouse cells, the combination treatment failed to completely eliminate tumor cells, our research still provided a promising strategy for oncolytic adenovirus-mediated solid tumor immunotherapy.