PD-L1 siRNA hitched polyethyleneimine-elastase constituting nanovesicle induces tumor immunogenicity and PD-L1 silencing for synergistic antitumor immunotherapy.

BACKGROUND: PD-1/PD-L1 blockade has become a powerful method to treat malignant tumors. However, a large proportion of patients still do not benefit from this treatment, due to low tumor immunogenicity and low tumor penetration of the agents. Recently, neutrophil elastase has been shown to induce robust tumor immunogenicity, while the insufficient enzyme activity at the tumor site restricted its anti-tumor application. Here, we designed polyethyleneimine-modified neutrophil elastase (PEI-elastase) loaded with PD-L1small interfering RNA (PD-L1 siRNA) for improving enzymatic activity and delivering siRNA to tumor, which was expected to solve the above-mentioned problems. RESULTS: We first demonstrated that PEI-elastase possessed high enzymatic activity, which was also identified as an excellent gene-delivery material. Then, we synthesized anti-tumor lipopolymer (P-E/S Lip) by encapsulating PEI-elastase and PD-L1siRNA with pH-responsive anionic liposomes. The P-E/S Lip could be rapidly cleaved in tumor acidic environment, leading to exposure of the PEI-elastase/PD-L1 siRNA. Consequently, PEI-elastase induced powerful tumor immunogenicity upon direct tumor killing with minimal toxicity to normal cells. In parallel, PEI-elastase delivered PD-L1siRNA into the tumor and reduced PD-L1 expression. Orthotopic tumor administration of P-E/S Lip not only attenuated primary tumor growth, but also produced systemic anti-tumor immune response to inhibit growth of distant tumors and metastasis. Moreover, intravenous administration of P-E/S Lip into mice bearing subcutaneous tumors leaded to an effective inhibition of established B16-F10 tumor and 4T1 tumor, with histological analyses indicating an absence of detectable toxicity. CONCLUSIONS: In our study, a protease-based nanoplatform was used to cooperatively provoke robust tumor immunogenicity and down-regulate PD-L1 expression, which exhibited great potential as a combination therapy for precisely treating solid tumors.

Lobar lung transplantation in the mouse.

Background: As an important supplementary approach to clinical in orthotopic lung transplantation (LTx), lobar LTx currently lacks a stable animal model and in the orthotopic left LTx model, the right lung of the donor mouse is completely removed and discarded. We introduce a novel mouse lobar LTx model that potentially provides a mouse model for clinical lobar LTx and increase the utilization rate of the experimental donor. Methods: Lobar and orthotopic left LTx were performed in syngeneic strain combinations. We performed micro-computed tomography and tested arterial blood gases to assess the graft function 28 days after transplantation. Hematoxylin-eosin and Masson's trichrome staining were used to evaluate pathological changes. Results: We performed ten lobar LTx with an operation success rate of 90%, accompanied by ten orthotopic left LTx from the same donors with an operation success rate of 100%. The graft preparation for lobar LTx was longer than that of the orthotopic left LTx (42.11±3.79 vs. 30.10±3.14 minutes, P<0.001). The recipient procedure for lobar LTx was nearly equivalent to the orthotopic left LTx. The graft function and histopathological changes for lobar LTx were comparable to those of orthotopic left LTx 28 days after transplantation. Conclusions: We describe a lobar LTx model in the mouse, which potentially provides a model for clinical lobar LTx and effectively addresses the issue of resource wastage in the orthotopic left LTx model.

Multiple RNA Rapid In Situ Imaging Based on Cas9 Code Key System.

Existing RNA in situ imaging strategies mostly utilize parallel repetitive nucleic acid self-assembly to achieve multiple analysis, with limitations of complicated systems and cumbersome steps. Here, a Cas9 code key system with key probe (KP) encoder and CRISPR/Cas9 signal exporter is developed. This system triggers T-protospacer adjacent motif (T-PAM structural transitions of multiple KP encoders to form coding products with uniform single-guide RNA (sgRNA) target sequences as tandem nodes. Only single sgRNA/Cas9 complex is required to cleave multiple coding products, enabling efficient "many-to-one" tandem signaling, and non-collateral cleavage activity-dependent automatic signaling output through active introduction of mismatched bases. Compared with conventional parallel multiple signaling analysis model, the proposed system greatly simplifies reaction process and enhances detection efficiency. Further, a rapid multiple RNA in situ imaging system is developed by combining the Cas9 code key system with a T-strand displacement amplification (T-SDA) signal amplifier. The constructed system is applied to tumor cells and clinicopathology slices, generating clear multi-mRNA imaging profiles in less than an hour with just one step. Therefore, this work provides reliable technical support for clinical tumor typing and molecular mechanism investigation.