Current progress in chimeric antigen receptor-modified T cells for the treatment of metastatic breast cancer.

Breast cancer is the leading cancer in women. Around 20-30% breast cancer patients undergo invasion or metastasis after radical surgical resection and eventually die. Number of breast cancer patients show poor sensitivity toward treatments despite the advances in chemotherapy, endocrine therapy, and molecular targeted treatments. Therapeutic resistance and tumor recurrence or metastasis develop with the ongoing treatments. Conducive treatment strategies are thus required. Chimeric antigen receptor (CAR)-modified T-cell therapy has progressed as a part of tumor immunotherapy. However, CAR-T treatment has not been effective in solid tumors because of tumor microenvironment complexity, inhibitory effects of extracellular matrix, and lacking ideal tumor antigens. Herein, the prospects of CAR-T cell therapy for metastatic breast cancer are discussed, and the targets for CAR-T therapy in breast cancer (HER-2, C-MET, MSLN, CEA, MUC1, ROR1, EGFR) at clinical level are reviewed. Moreover, solutions are proposed for the challenges of breast cancer CAR-T therapy regarding off-target effects, heterogeneous antigen expression by tumor cells and immunosuppressive tumor microenvironment. Ideas for improving the therapeutics of CAR-T cell therapy in metastatic breast cancer are suggested.

Clinical application status and prospect of the combined anti-tumor strategy of ablation and immunotherapy.

Image-guided tumor ablation eliminates tumor cells by physical or chemical stimulation, which shows less invasive and more precise in local tumor treatment. Tumor ablation provides a treatment option for medically inoperable patients. Currently, clinical ablation techniques are widely used in clinical practice, including cryoablation, radiofrequency ablation (RFA), and microwave ablation (MWA). Previous clinical studies indicated that ablation treatment activated immune responses besides killing tumor cells directly, such as short-term anti-tumor response, immunosuppression reduction, specific and non-specific immune enhancement, and the reduction or disappearance of distant tumor foci. However, tumor ablation transiently induced immune response. The combination of ablation and immunotherapy is expected to achieve better therapeutic results in clinical application. In this paper, we provided a summary of the principle, clinical application status, and immune effects of tumor ablation technologies for tumor treatment. Moreover, we discussed the clinical application of different combination of ablation techniques with immunotherapy and proposed possible solutions for the challenges encountered by combined therapy. It is hoped to provide a new idea and reference for the clinical application of combinate treatment of tumor ablation and immunotherapy.

Rbm14 maintains the integrity of genomic DNA during early mouse embryogenesis via mediating alternative splicing.

OBJECTIVE: In this study, we generated an Rbm14 knockout mouse model to explore its functions during early mouse embryogenesis. MATERIALS AND METHODS: The Rbm14 knockout mouse model was generated by a combination of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and microinjection techniques. The developmental defects of the knockout embryos were characterized by histological analyses. The accumulation of DNA damage in mouse embryonic stem cells (ESCs) was detected by γH2AX staining and comet assay. The altered mRNA splicing of DNA damage response (DDR)-related genes was detected by RNA-Seq analysis and confirmed by semi-quantitative PCR. The interaction of RBM14 with alternative splicing-related genes was detected by immunoprecipitation-mass spectra (IP-MS) and confirmed by co-immunoprecipitation (Co-IP). RESULTS: Rbm14 knockout in mice results in apoptosis and cell proliferation defects in early post-implantation epiblast cells, leading to gastrulation disruption and embryonic lethality. FACS and immunostaining demonstrate accumulation of DNA damage in Rbm14 knockout ES cells. We also identified altered splicing of DDR-related genes in the knockout mouse ESCs by RNA-Seq, indicating that RBM14-mediated alternative splicing is required for the maintenance of genome integrity during early mouse embryogenesis. CONCLUSIONS: Our work reveals that Rbm14 plays an essential role in the maintenance of genome integrity during early mouse embryonic development by regulating alternative splicing of DDR-related genes.

Zika virus infection induces RNAi-mediated antiviral immunity in human neural progenitors and brain organoids.

The re-emergence of Zika virus (ZIKV) in the Western Hemisphere has resulted in global public health crisis since 2015. ZIKV preferentially infects and targets human neural progenitor cells (hNPCs) and causes fetal microcephaly upon maternal infection. hNPCs not only play critical roles during fetal brain development, but also persist in adult brain throughout life. Yet the mechanism of innate antiviral immunity in hNPCs remains largely unknown. Here, we show that ZIKV infection triggers the abundant production of virus-derived small interfering RNAs in hNPCs, but not in the more differentiated progenies or somatic cells. Ablation of key RNAi machinery components significantly enhances ZIKV replication in hNPCs. Furthermore, enoxacin, a broad-spectrum antibiotic that is known as an RNAi enhancer, exerts potent anti-ZIKV activity in hNPCs and other RNAi-competent cells. Strikingly, enoxacin treatment completely prevents ZIKV infection and circumvents ZIKV-induced microcephalic phenotypes in brain organoid models that recapitulate human fetal brain development. Our findings highlight the physiological importance of RNAi-mediated antiviral immunity during the early stage of human brain development, uncovering a novel strategy to combat human congenital viral infections through enhancing RNAi.

Differential antiviral immunity to Japanese encephalitis virus in developing cortical organoids.

Japanese encephalitis (JE) caused by Japanese encephalitis virus (JEV) poses a serious threat to the world's public health yet without a cure. Certain JEV-infected neural cells express a subset of previously identified intrinsic antiviral interferon stimulated genes (ISGs), indicating brain cells retain autonomous antiviral immunity. However, whether this happens in composited brain remains unclear. Human pluripotent stem cell (hPSC)-derived organoids can model disorders caused by human endemic pathogens such as Zika virus, which may potentially address this question and facilitate the discovery of a cure for JE. We thus generated telencephalon organoid and infected them with JEV. We found JEV infection caused significant decline of cell proliferation and increase of cell death in brain organoid, resulting in smaller organoid spheres. JEV tended to infect astrocytes and neural progenitors, especially the population representing outer radial glial cells (oRGCs) of developing human brain. In addition, we revealed variable antiviral immunity in brain organoids of different stages of culture. In organoids of longer culture (older than 8 weeks), but not of early ones (less than 4 weeks), JEV infection caused typical activation of interferon signaling pathway. Preferential infection of oRGCs and differential antiviral response at various stages might explain the much more severe outcomes of JEV infection in the younger, which also provide clues to develop effective therapeutics of such diseases.

RBM14 is indispensable for pluripotency maintenance and mesoderm development of mouse embryonic stem cells.

The pluripotency of embryonic stem cells (ESCs) is maintained by core pluripotency transcription factors, cofactors and several signaling pathways. RBM14 is a component of the para-speckle complex, which has been implicated in multiple important biological processes. The role of RBM14 in ESCs and lineage differentiation remains to be elucidated. In the present study, we provided evidence that RBM14 plays important roles in maintaining pluripotency and in the early differentiation of ESCs. RBM14 was demonstrated to be expressed in mouse embryonic stem cells (mESCs) and localized in the nucleus. RBM14 expression was depleted in mESCs using clustered regularly interspaced short palindromic repeats (CRISPR) technology. Our results also showed that RBM14 depletion altered the gene expression profiles of mESCs. In particular, pluripotency-associated genes and genes involved in the Wnt and TGF-ß signaling pathways were downregulated in RBM14 knockout mESCs. Furthermore, RBM14 was found to be essential for mesoderm development in vitro and in vivo. The specific effects of RBM14 depletion were verified by conducting a rescue experiment. Our findings demonstrated that RBM14 not only plays an important role in maintaining the pluripotency of mESCs but is also indispensable for mesoderm development.

A synthetic dl-nordihydroguaiaretic acid (Nordy), inhibits angiogenesis, invasion and proliferation of glioma stem cells within a zebrafish xenotransplantation model.

The zebrafish (Danio rerio) and their transparent embryos represent a promising model system in cancer research. Compared with other vertebrate model systems, we had previously shown that the zebrafish model provides many advantages over mouse or chicken models to study tumor invasion, angiogenesis, and tumorigenesis. In this study, we systematically investigated the biological features of glioma stem cells (GSCs) in a zebrafish model, such as tumor angiogenesis, invasion, and proliferation. We demonstrated that several verified anti-angiogenic agents inhibited angiogenesis that was induced by xenografted-GSCs. We next evaluated the effects of a synthetic dl-nordihydroguaiaretic acid compound (dl-NDGA or "Nordy"), which revealed anti-tumor activity against human GSCs in vitro by establishing parameters through studying its ability to suppress angiogenesis, tumor invasion, and proliferation. Furthermore, our results indicated that Nordy might inhibit GSCs invasion and proliferation through regulation of the arachidonate 5-lipoxygenase (Alox-5) pathway. Moreover, the combination of Nordy and a VEGF inhibitor exhibited an enhanced ability to suppress angiogenesis that was induced by GSCs. By contrast, even following treatment with 50 µM Nordy, there was no discernible effect on zebrafish embryonic development. Together, these results suggested efficacy and safety of using Nordy in vivo, and further demonstrated that this model should be suitable for studying GSCs and anti-GSC drug evaluation.

Induced pluripotency for translational research.

The advent of induced pluripotent stem cells (iPSCs) has revolutionized the concept of cellular reprogramming and potentially will solve the immunological compatibility issues that have so far hindered the application of human pluripotent stem cells in regenerative medicine. Recent findings showed that pluripotency is defined by a state of balanced lineage potency, which can be artificially instated through various procedures, including the conventional Yamanaka strategy. As a type of pluripotent stem cell, iPSCs are subject to the usual concerns over purity of differentiated derivatives and risks of tumor formation when used for cell-based therapy, though they provide certain advantages in translational research, especially in the areas of personalized medicine, disease modeling and drug screening. iPSC-based technology, human embryonic stem cells (hESCs) and direct lineage conversion each will play distinct roles in specific aspects of translational medicine, and continue yielding surprises for scientists and the public.

TGF-ß1 enhances tumor-induced angiogenesis via JNK pathway and macrophage infiltration in an improved zebrafish embryo/xenograft glioma model.

Angiogenesis plays a crucial role at the early stage of tumorigenesis and tumor progression. A suitable model will be useful not only for the clarification of the underlying molecular mechanisms, but also for high-throughput identification of novel anti-angiogenesis compounds. Here, we established a zebrafish model for the purpose to investigate angiogenesis and screen anti-angiogenic compounds. Glioma U87 cells expressing red fluorescent protein (RFP) were transplanted in fli:GFP transgenic zebrafish embryos where significant angiogenesis was observed. TGF-ß1 enhanced glioma-induced angiogenesis, which was inhibited by JNK inhibitor SP600125 but not p38 MAPK inhibitor SB202190, ERK inhibitor PD98059, or PI3K inhibitor LY294002, indicating the important role of TGF-ß1 and JNK pathways in this process. Moreover, the glioma-induced angiogenesis was associated with macrophage infiltration that was further enhanced by TGF-ß1. Therefore, our zebrafish model provides a powerful in vivo tool for the investigation of tumor-induced angiogenesis, and a cost-effective system for high-throughput screening of anti-angiogenic compounds.