Regulation of CTLs/Tregs via Highly Stable and Ultrasound-Responsive Cerasomal Nano-Modulators for Enhanced Colorectal Cancer Immunotherapy.

Immunotherapy is showing good potential for colorectal cancer therapy, however, low responsive rates and severe immune-related drug side effects still hamper its therapeutic effectiveness. Herein, a highly stable cerasomal nano-modulator (DMC@P-Cs) with ultrasound (US)-controlled drug delivery capability for selective sonodynamic-immunotherapy is fabricated. DMC@P-Cs' lipid bilayer is self-assembled from cerasome-forming lipid (CFL), pyrophaeophorbid conjugated lipid (PL), and phospholipids containing unsaturated chemical bonds (DOPC), resulting in US-responsive lipid shell. Demethylcantharidin (DMC) as an immunotherapy adjuvant is loaded in the hydrophilic core of DMC@P-Cs. With US irradiation, reactive oxygen species (ROS) can be effectively generated from DMC@P-Cs, which can not only kill tumor cells for inducing immunogenic cell death (ICD), but also oxidize unsaturated phospholipids-DOPC to change the permeability of the lipid bilayers and facilitate controlled release of DMC, thus resulting in down-regulation of regulatory T cells (Tregs) and amplification of anti-tumor immune responses. After intravenous injection, DMC@P-Cs can efficiently accumulate at the tumor site, and local US treatment resulted in 94.73% tumor inhibition rate. In addition, there is no detectable systemic toxicity. Therefore, this study provides a highly stable and US-controllable smart delivery system to achieve synergistical sonodynamic-immunotherapy for enhanced colorectal cancer therapy.

Metformin-Mediated Fast Charge-Reversal Nanohybrid for Deep Penetration Piezocatalysis-Augmented Chemodynamic Immunotherapy of Cancer.

Immune checkpoint blockade (ICB) therapy still suffers from insufficient immune response and adverse effect of ICB antibodies. Chemodynamic therapy (CDT) has been demonstrated to be an effective way to synergize with ICB therapy. However, a low generation rate of reactive oxygen species and poor tumor penetration of CDT platforms still decline the immune effects. Herein, a charge-reversal nanohybrid Met@BF containing both Fe3O4 and BaTiO3 nanoparticles in the core and Metformin (Met) on the surface was fabricated for tumor microenvironment (TME)- and ultrasound (US)-activated piezocatalysis-chemodynamic immunotherapy of cancer. Interestingly, Met@BF had a negative charge in blood circulation, which was rapidly changed into positive when exposed to acidic TME attributed to quaternization of tertiary amine in Met, facilitating deep tumor penetration. Subsequently, with US irradiation, Met@BF produced H2O2 based on piezocatalysis of BaTiO3, which greatly enhanced the Fenton reaction of Fe3O4, thus boosting robust antitumor immune response. Furthermore, PD-L1 expression was inhibited by the local released Met to further augment the antitumor immune effect, achieving effective inhibitions for both primary and metastatic tumors. Such a combination of piezocatalysis-enhanced chemodynamic therapy and Met-mediated deep tumor penetration and downregulation of PD-L1 provides a promising strategy to augment cancer immunotherapy.

Nanomaterials augmented bioeffects of ultrasound in cancer immunotherapy.

Immunotherapy as a milestone in cancer treatment has made great strides in the past decade, but it is still limited by low immune response rates and immune-related adverse events. Utilizing bioeffects of ultrasound to enhance tumor immunotherapy has attracted more and more attention, including sonothermal, sonomechanical, sonodynamic and sonopiezoelectric immunotherapy. Moreover, the emergence of nanomaterials has further improved the efficacy of ultrasound mediated immunotherapy. However, most of the summaries in this field are about a single aspect of the biological effects of ultrasound, which is not comprehensive and complete currently. This review proposes the recent progress of nanomaterials augmented bioeffects of ultrasound in cancer immunotherapy. The concept of immunotherapy and the application of bioeffects of ultrasound in cancer immunotherapy are initially introduced. Then, according to different bioeffects of ultrasound, the representative paradigms of nanomaterial augmented sono-immunotherapy are described, and their mechanisms are discussed. Finally, the challenges and application prospects of nanomaterial augmented ultrasound mediated cancer immunotherapy are discussed in depth, hoping to pave the way for cancer immunotherapy and promote the clinical translation of ultrasound mediated cancer immunotherapy through the reasonable combination of nanomaterials augmented ultrasonic bioeffects.

Ultrasound-Induced Piezocatalysis Triggered NO Generation for Enhanced Hypoxic Tumor Therapy.

Conventional NO gas generation based on l-arginine (l-Arg) is usually dependent on H2O2 and O2, both of which are very limited within the tumor microenvironment, thus greatly limiting l-Arg's therapeutic effect. Herein, a novel nanoplatform for efficiently triggering NO production based on ultrasound-induced piezocatalysis was developed, which was fabricated by coating amphiphilic poly-l-arginine (DSPE-PEG2000-Arg, DPA) on the piezoelectric material of barium titanate (BTO). The resulting BTO@DPA nanoparticles can efficiently generate H2O2, 1O2, and O2 via ultrasound-induced piezocatalysis based on BTO and oxidize the surface arginine to produce NO, which can even further interact with the reactive oxygen species (ROS) to produce more reactive peroxynitrite, thus inducing serious tumor cell apoptosis both in hypoxia and normoxia. After intravenous injection, BTO@DPA accumulated well at the tumor tissue at 4 h postinjection; later, ultrasound irradiation on the tumor not only achieved the best tumor inhibition rate of ∼70% but also completely inhibited tumor metastasis to the lungs via the alleviation of tumor hypoxia. Such a strategy was not dependent on the tumor microenvironment and can be well controlled by ultrasound irradiation, providing a simple and efficient therapy paradigm for hypoxic tumor.

Ultrasound-Triggered Piezocatalysis for Selectively Controlled NO Gas and Chemodrug Release to Enhance Drug Penetration in Pancreatic Cancer.

Nitric oxide (NO) is drawing widespread attention in treating pancreatic ductal adenocarcinoma (PDAC) as a safe and therapeutically efficient technique through modulating the dense fibrotic stroma in the tumor microenvironment to enhance drug penetration. Considerable NO nanogenerators and NO releasing molecules have been developed to shield the systemic toxicity caused by free diffusion of NO gas. However, on-demand controlled release of NO and chemotherapy drugs at tumor sites remains a problem limited by the complex and dynamic tumor microenvironment. Herein, we present an ultrasound-responsive nanoprodrug of CPT-t-R-PEG2000@BaTiO3 (CRB) which encapsulates piezoelectric nanomaterials barium titanate nanoparticle (BaTiO3) with amphiphilic prodrug molecules that consisted of thioketal bond (t) linked chemotherapy drug camptothecin (CPT) and NO-donor l-arginine (R). Based on ultrasound-triggered piezocatalysis, BaTiO3 can continuously generate ROS in the hypoxic tumor environment, which induces a cascade of reaction processes to break the thioketal bond to release CPT and oxidize R to release NO, simultaneously delivering CPT and NO to the tumor site. It is revealed that CRB shows a uniform size distribution, prolonged blood circulation time, and excellent tumor targeting ability. Moreover, controlled release of CPT and NO were observed both in vitro and in vivo under the stimulation of ultrasound, which is beneficial to the depletion of dense stroma and subsequently enhanced delivery and efficacy of CPT. Taken together, CRB significantly increased the antitumor efficacy against highly malignant Panc02 tumors in mice through inhibiting chemoresistance, representing a feasible approach for targeted therapies against Panc02 and other PDAC.

Genetically Engineering Cell Membrane-Coated BTO Nanoparticles for MMP2-Activated Piezocatalysis-Immunotherapy.

Tumor immunotherapy based on immune checkpoint blockade (ICB) still suffers from low host response rate and non-specific distribution of immune checkpoint inhibitors, greatly compromising the therapeutic efficiency. Herein, cellular membrane stably expressing matrix metallopeptidase 2 (MMP2)-activated PD-L1 blockades is engineered to coat ultrasmall barium titanate (BTO) nanoparticle for overcoming the immunosuppressive microenvironment of tumors. The resulting M@BTO NPs can significantly promote the BTO's tumor accumulation, while the masking domains on membrane PD-L1 antibodies are cleaved when exposure to MMP2 highly expressed in tumor. With ultrasound (US) irradiation, M@BTO NPs can simultaneously generate reactive oxygen species (ROS) and O2 based on BTO mediated piezocatalysis and water splitting, significantly promoting the intratumoral infiltration of cytotoxic T lymphocytes (CTLs) and improving the PD-L1 blockade therapy to the tumor, resulting in effective tumor growth inhibition and lung metastasis suppression in a melanoma mouse model. This nanoplatform combines MMP2-activated genetic editing cell membrane with US responsive BTO for both immune stimulation and specific PD-L1 inhibition, providing a safe and robust strategy in enhancing immune response against tumor.

In situ synthesized nanozyme for photoacoustic-imaging-guided photothermal therapy and tumor hypoxia relief.

Nanozymes have attracted extensive research interest due to their ideal enzymatic catalytic performance; however, uncontrollable activities and nonspecific accumulation limit their further clinical application. To overcome these obstacles, we proposed in situ synthesized nanozyme, and realized the concept through an intelligent nanosystem (ISSzyme) based on Prussian blue (PB) precursor. PB nanozyme was synthesized at the tumor sites through the interaction of ISSzyme with glutathione, which was demonstrated by comparing with conventional PB nanozyme. ISSzyme is capable of tumor-specific photoacoustic imaging (PAI) and photothermal therapy (PTT), reducing the false-positive signals of PAI and the treatment side effects of PTT. ISSzyme has catalase-like activities, resulting in tumor hypoxia relief and metastasis inhibition. More importantly, the in situ synthesized PB nanozyme has the favorable property of minimal liver accumulation. Considering the above advantages, ISSzyme is expected to shed light on the design of the next-generation artificial enzymes, with many new biomedical applications.

Dual-targeting nanozyme for tumor activatable photo-chemodynamic theranostics.

Tumor phototheranostics holds a great promise on account of its high spatiotemporal resolution, tumor-specificity, and noninvasiveness. However, physical limitation of light penetration and "always on" properties of conventional photothermal-conversion agents usually cause difficulty in accurate diagnosis and completely elimination of tumor. Meanwhile, nanozymes mediated Fenton reactions can well utilize the tumor microenvironment (TME) to generate hydroxyl radicals for chemodynamic therapy (CDT), but limited by the concentration of H2O2 in TME and the delivery efficiency of nanozymes. To overcome these problems, a dual-targeting nanozyme (FTRNPs) is developed for tumor-specific in situ theranostics, based upon the assembling of ultrasmall Fe3O4 nanoparticles, 3,3',5,5'-tetrameth-ylbenzidine (TMB) and the RGD peptide. The FTRNPs after H2O2 treatment exhibits superior photothermal stability and high photothermal conversion efficiency (η = 50.9%). FTRNPs shows extraordinary accumulation and retention in the tumor site by biological/physical dual-targeting, which is 3.54-fold higher than that without active targeting. Cascade-dual-response to TME for nanozymes mediated Fenton reactions and TMB oxidation further improves the accuracy of both photoacoustic imaging and photothermal therapy (PTT). The tumor inhibition rate of photo-chemodynamic therapy is ~ 97.76%, which is ~ 4-fold higher than that of PTT or CDT only. Thus, the combination of CDT and PTT to construct "turn on" nanoplatform is of great significance to overcome their respective limitations. Considering its optimized "all-in-one" performance, this new nanoplatform is expected to provide an advanced theranostic strategy for the future treatment of cancers.

In Situ Micro-Nano Conversion Augmented Tumor-Localized Immunochemotherapy.

The immune checkpoint blockade (ICB) therapy based on monoclonal antibodies still suffers from a lower immune response rate and severe immune-related side effects, which greatly compromise its therapeutic benefits. Herein, ultrasound (US) microbubbles (MBs) that locally delivered the camptothecin-floxuridine (CF) drug combination and anti-PD-L1 blocking antibody (αPD-L1) to tumors were developed to improve ICB therapy. The resulting αPCF MBs exhibited good stability, allowing their use as US imaging contrast agents to trace the drug delivery in vivo. Furthermore, the combination of αPCF MBs treatment and disrupted US irradiation triggered tumor in situ conversion of αPCF MBs to αPCF NPs while promoting higher tumor cell uptake and deeper tumor penetration as confirmed by the US/fluorescence bimodal imaging. Camptothecin (CPT) and floxuridine (FUDR) were further released at a fixed 1:1 molar ratio within the tumor microenvironment (TME) to synergistically elicit an immunogenic tumor phenotype and sensitize tumors to αPD-L1-mediated ICB therapy, while the local simultaneous delivery of immunotherapeutic αPD-L1 further reversed the immunosuppressive tumor microenvironment and promoted the infiltration of cytotoxic T lymphocytes (CTLs), thus achieving a synergistic therapeutic effect of chemotherapy and immunotherapy in the CT26 tumor-bearing mice. Thus, αPCF MBs + US mediated local co-delivering of the drug combination and αPD-L1 well augmented the ICB therapy while effectively minimizing the off-target side effects, providing a safe and universal therapeutic strategy for tumor immunotherapy.

Cascade Drug Delivery through Tumor Barriers of Pancreatic Cancer via Ultrasound in Combination with Functional Microbubbles.

The abundant desmoplastic stroma and the lack of sufficient targets on pancreatic cancer cells render poor drug penetration and cellular uptake, which significantly compromise the chemotherapy efficacy. Herein, we reported a three-step cascade delivery strategy for selective delivery of paclitaxel (PTX) to achieve a targeted therapy for pancreatic cancer. cRGD and cCLT1 peptides, which could target the integrin and fibronectin, respectively, overexpressed in pancreatic cancer cells and stroma, were decorated on PTX-loaded microbubbles, resulting in the formation of dual-targeting PTX-RCMBs. In this strategy, ultrasound in combination with PTX-RCMBs first enhanced the permeability of tumor vessels via cavitation effects and simultaneously helped the generated PTX-RCNPs penetrate into the stroma. Then, the cCLT1 peptide modified on PTX-RCNPs selectively bound the fibronectin highly expressed in the stroma and later targeted the integrin (α5ß1) on the cell surface. Finally, another targeting cRGD peptide modified on PTX-RCNPs would further promote PTX uptake via targeting the integrin (αvß3) on the cell surface. This strategy significantly increased the delivery of PTX into tumor tissues. Moreover, the in vivo effective accumulation of PTX was monitored by ultrasound and fluorescence bimodal imaging. The tumor growth inhibition was investigated on subcutaneous tumor mouse models with 89.8% growth inhibition rate during 21 days of treatment, showing great potential for improving pancreatic cancer therapy.

Charge Reversal Polypyrrole Nanocomplex-Mediated Gene Delivery and Photothermal Therapy for Effectively Treating Papillary Thyroid Cancer and Inhibiting Lymphatic Metastasis.

As a traditional treatment for papillary thyroid cancer (PTC), surgical resection of diseased tissues often brings lots of inconveniences to patients, and the tumor recurrence and metastasis are difficult to avoid. Herein, we developed a gene and photothermal combined therapy nanosystem based on a polypyrrole (Ppy)-poly(ethylene imine)-siILK nanocomplex (PPRILK) to achieve minimally invasive ablation and lymphatic metastasis inhibition in PTC simultaneously. In this system, gelatin-stabilized Ppy mainly acted as a photothermal- and photoacoustic (PA)-responsive nanomaterial and contributed to its well-behaved photosensitivity in the near-infrared region. Moreover, gelatin-stabilized Ppy possessed a charge reversal function, facilitating the tight conjunction of siILK gene at physiological pH (7.35-7.45) and its automatic release into acidic lysosomes (pH 4.0-5.5); the proton sponge effect generated during this process further facilitated the escape of siILK from lysosomes to the cytoplasm and played its role in inhibiting PTC proliferation and lymphatic metastasis. With the guidance of fluorescence and PA bimodal imaging, gene delivery and Ppy location in tumor regions could be clearly observed. As a result, tumors were completely eradicated by photothermal therapy, and the recurrences and metastases were obviously restrained by siILK.

Ultrasound Microbubbles Mediated Sonosensitizer and Antibody Co-delivery for Highly Efficient Synergistic Therapy on HER2-Positive Gastric Cancer.

Trastuzumab combined with chemotherapy is the first-line treatment for advanced HER2-positive gastric cancer, but it still suffers from limited therapeutic efficiency and serious side effects, which are usually due to the poor delivery efficiency and the drug resistance of tumor cells to the chemotherapeutic drugs. Herein, a type of ultrasound microbubble for simultaneous delivery of sonosensitizers and therapeutic antibodies to achieve targeting combination of sonodynamic therapy and antibody therapy of HER2-positive gastric cancer was constructed from pyropheophorbide-lipid followed by trastuzumab conjugation (TP MBs). In vitro and in vivo studies showed that TP MBs had good biological safety, and their in vivo delivery can be monitored by ultrasound/fluorescence bimodal imaging. With ultrasound (US) located at the tumor area, TP MBs can be converted into nanoparticles (TP NPs) in situ by US-targeted microbubble destruction; plus the enhanced permeability and retention effects and the targeting effects of trastuzumab, the enrichment of sonosensitizers and antibodies in the tumor tissue can be greatly enhanced (∼2.1 times). When combined with ultrasound, TP MBs can not only increase the uptake of sonosensitizers in HER2-positive gastric cancer NCI-N87 cells but also efficiently generate singlet oxygen to greatly increase the killing effect on cells, obviously inhibiting the tumor growth in HER2-positive gastric cancer NCI-N87 cell models with a tumor inhibition rate up to 79.3%. Overall, TP MBs combined with US provided an efficient way for co-delivery of sonosensitizers and antibodies, greatly enhancing the synergistic therapeutic effect on HER2-positive gastric cancer while effectively reducing the side effects.

Ultrasmall Barium Titanate Nanoparticles for Highly Efficient Hypoxic Tumor Therapy via Ultrasound Triggered Piezocatalysis and Water Splitting.

Hypoxia in a solid tumor microenvironment (TME) can lead to the overexpression of hypoxia-inducible factor-1α (HIF-1α), which correlates to tumor metastasis. Reactive oxygen species (ROS) induced tumor cell apoptosis is becoming a promising method in tumor treatment. Currently, the ROS generating systems, e.g., photodynamic treatment and sonodynamic treatment, highly depend on oxygen (O2) in the tumor microenvironment (TME). However, the level of O2 in TME is too low to produce enough ROS. Herein, we developed an ultrasmall DSPE-PEG2000 coated barium titanate nanoparticle (P-BTO) for tumor treatment based on ultrasound triggered piezocatalysis and water splitting. Interestingly, irradiated by ultrasound, the surface of ultasmall P-BTO nanoparticles produced imbalance charges, which induced a cascade of redox reaction processes to simultaneously generate ROS and O2, the latter one was hardly generated in large-sized barium titanate nanoparticles. The as-synthesized P-BTO reached the highest accumulation in the tumor site at 4 h after intravenous injection. The results showed that the produced O2 significantly alleviated the hypoxia of TME to down-regulate the expression of HIF-1α, and the produced ROS can efficiently kill tumor cells. Moreover, the tumor metastasis was also inhibited, providing a different way to treat triple-negative breast cancer, which was easily metastatic and lacked effective treatments in the clinic.

CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway.

With its high efficiency for site-specific genome editing and easy manipulation, the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (CAS9) system has become the most widely used gene editing technology in biomedical research. In addition, significant progress has been made for the clinical development of CRISPR/CAS9 based gene therapies of human diseases, several of which are entering clinical trials. Here we report that CAS9 protein can function as a genome mutator independent of any exogenous guide RNA (gRNA) in human cells, promoting genomic DNA double-stranded break (DSB) damage and genomic instability. CAS9 interacts with the KU86 subunit of the DNA-dependent protein kinase (DNA-PK) complex and disrupts the interaction between KU86 and its kinase subunit, leading to defective DNA-PK-dependent repair of DNA DSB damage via non-homologous end-joining (NHEJ) pathway. XCAS9 is a CAS9 variant with potentially higher fidelity and broader compatibility, and dCAS9 is a CAS9 variant without nuclease activity. We show that XCAS9 and dCAS9 also interact with KU86 and disrupt DNA DSB repair. Considering the critical roles of DNA-PK in maintaining genomic stability and the pleiotropic impact of DNA DSB damage responses on cellular proliferation and survival, our findings caution the interpretation of data involving CRISPR/CAS9-based gene editing and raise serious safety concerns of CRISPR/CAS9 system in clinical application.

MTR4 drives liver tumorigenesis by promoting cancer metabolic switch through alternative splicing.

The metabolic switch from oxidative phosphorylation to glycolysis is required for tumorigenesis in order to provide cancer cells with energy and substrates of biosynthesis. Therefore, it is important to elucidate mechanisms controlling the cancer metabolic switch. MTR4 is a RNA helicase associated with a nuclear exosome that plays key roles in RNA processing and surveillance. We demonstrate that MTR4 is frequently overexpressed in hepatocellular carcinoma (HCC) and is an independent diagnostic marker predicting the poor prognosis of HCC patients. MTR4 drives cancer metabolism by ensuring correct alternative splicing of pre-mRNAs of critical glycolytic genes such as GLUT1 and PKM2. c-Myc binds to the promoter of the MTR4 gene and is important for MTR4 expression in HCC cells, indicating that MTR4 is a mediator of the functions of c-Myc in cancer metabolism. These findings reveal important roles of MTR4 in the cancer metabolic switch and present MTR4 as a promising therapeutic target for treating HCC.

Micheliolide Inhibits Liver Cancer Cell Growth Via Inducing Apoptosis And Perturbing Actin Cytoskeleton.

PURPOSE: Micheliolide (MCL) is an effector compound of the flower which has been traditionally used to treat inflammation and cancer patients in oriental medicine. MCL has killing effects on several cancer and immune cells by modulating apoptosis, cell cycle, and metabolism. However, the detail of the mechanisms of anti-cancer activity remains to be elucidated and the effect on liver cancer cells is unknown. METHODS: Cell proliferation was determined by CCK8 and clone formation assay. The xenograft liver cancer model formed by injecting Huh7 cells into NUDE mice was used to evaluate the effects of MCL on liver cancer cells in vivo. We evaluated the stemness of cells with spheroid formation assay and flow cytometry assay. The apoptosis was determined by Annexin V assay. F-actin staining and ROS were performed to detect the impairment of the F-actin cytoskeleton and mitochondria. RESULTS: Here, we first show that MCL inhibits liver cancer cells both in vivo and in vitro by triggering apoptosis which was reduced by anti-oxidant, but not cell-cycle arrest. In addition, MCL induces mitochondrial ROS and caspase-3 activation. Also, we found that the aggregation of mitochondria and the perturbation of F-actin fibers in the MCL-treated liver cancer cells coincidently occurred before the induction of apoptosis and mitochondrial ROS. CONCLUSION: These results suggest that F-actin perturbation is involved in impaired mitochondria and apoptosis. Therefore, MCL can be a potent therapeutic reagent for liver cancer, primarily targeting the actin cytoskeleton.

Wild-Type p53 Promotes Cancer Metabolic Switch by Inducing PUMA-Dependent Suppression of Oxidative Phosphorylation.

The tumor suppressor p53 is somatically mutated in half of all human cancers. Paradoxically, the wild-type p53 (WTp53) is often retained in certain human cancers, such as hepatocarcinoma (HCC). We discovered a physiological and oncogenic role of WTp53 in suppressing pyruvate-driven oxidative phosphorylation by inducing PUMA. PUMA inhibits mitochondrial pyruvate uptake by disrupting the oligomerization and function of mitochondrial pyruvate carrier (MPC) through PUMA-MPC interaction, which depends on IκB kinase-mediated phosphorylation of PUMA at Ser96/106. High expression levels of PUMA are correlated with decreased mitochondrial pyruvate uptake and increased glycolysis in HCCs and poor prognosis of HCC patients. These findings are instrumental for cancer drug discovery aiming at activating WTp53 or restoring WTp53 activity to p53 mutants.

[Generation of a new strain of NOD/SCID/IL2Rγ-/- mice with targeted disruption of Prkdc and IL2Rγ genes using CRISPR/Cas9 system].

OBJECTIVE: The NOD/SCID/IL2Rγ- /- (NSG) mouse strain is the most widely used immunodeficient strain for xenograft transplantation. However, the existing SCID mutation is a spontaneous mutation of the Prkdc gene, which leads to leaky T cell developmental block and difficulty in genotyping. It is therefore important to develop a new strain of NSG mice with targeted disruption of Prkdc and IL2Rγ genes. METHODS: Targeted disruption of Prkdc and IL2Rγ genes was achieved using the CRISPR/Cas9 system. By intercrossing the knockout and NOD mice, we obtained a novel strain of NOD/SCID/IL2Rγ- /-(NSG) mice, denoted as cNSG (Chinese NSG) mice. RESULTS: In addition to the NOD mutation, cNSG mice exhibited a complete absence of T cells, B cells and NK cells. cNSG mice allowed more efficient engraftment of human cancer cells than the commonly used immunodeficient nude mice. CONCLUSION: cNSG mice will provide an important xenotransplantation model for biomedical research.