Novel chimeric antigen receptor-expressing T cells targeting the malignant mesothelioma-specific antigen sialylated HEG1.

The selection of highly specific target antigens is critical for the development of clinically efficient and safe chimeric antigen receptors (CARs). In search of diagnostic marker for malignant mesothelioma (MM), we have established SKM9-2 monoclonal antibody (mAb) which recognizes a MM-specific molecule, sialylated Protein HEG homolog 1 (HEG1), with high specificity and sensitivity. In this study, to develop a novel therapeutic approach against MM, we generated SKM9-2 mAb-derived CARs that included the CD28 (SKM-28z) or 4-1BB (SKM-BBz) costimulatory domain. SKM-28z CAR-T cells showed continuous growth and enhanced Tim-3, LAG-3, and PD-1 expression in vitro, which might be induced by tonic signaling caused by self-activation; however, these phenotypes were not observed in SKM-BBz CAR-T cells. In addition, SKM-BBz CAR-T cells exhibited slightly stronger in vitro killing activity against MM cell lines than SKM-28z CAR-T cells. More importantly, only SKM-BBz CAR-T cells, but not SKM-28z CAR-T cells, significantly inhibited tumor growth in vivo in a MM cell line xenograft mouse model. Gene expression profiling and reporter assays revealed differential signaling pathway activation; in particular, SKM-BBz CAR-T cells exhibited enhanced NF-kB signaling and reduced NFAT activation. In addition, SKM-BBz CAR-T cells showed upregulation of early memory markers, such as TCF7 and CCR7, as well as downregulation of pro-apoptotic proteins, such as BAK1 and BID, which may be associated with phenotypical and functional differences between SKM-BBz and SKM-28z CAR-T cells. In conclusion, we developed novel SKM9-2-derived CAR-T cells with the 4-1BB costimulatory domain, which could provide a promising therapeutic approach against refractory MM.

Inhibitory Effect of Zinc on Colorectal Cancer by Granzyme B Transcriptional Regulation in Cytotoxic T Cells.

Zinc is one of the essential trace elements and is involved in various functions in the body. Zinc deficiency is known to cause immune abnormalities, but the mechanism is not fully understood. Therefore, we focused our research on tumor immunity to elucidate the effect of zinc on colorectal cancer and its mechanisms. Mice were treated with azoxymethane (AOM) and dextran sodium sulfate (DSS) to develop colorectal cancer, and the relationship between zinc content in the diet and the number and area of tumors in the colon was observed. The number of tumors in the colon was significantly higher in the no-zinc-added group than in the normal zinc intake group, and about half as many in the high-zinc-intake group as in the normal-zinc-intake group. In T-cell-deficient mice, the number of tumors in the high-zinc-intake group was similar to that in the normal-zinc-intake group, suggesting that the inhibitory effect of zinc was dependent on T cells. Furthermore, we found that the amount of granzyme B transcript released by cytotoxic T cells upon antigen stimulation was significantly increased by the addition of zinc. We also showed that granzyme B transcriptional activation by zinc addition was dependent on calcineurin activity. In this study, we have shown that zinc exerts its tumor-suppressive effect by acting on cytotoxic T cells, the center of cellular immunity, and increases the transcription of granzyme B, one of the key molecules in tumor immunity.

Arginase-1 inhibition reduces migration ability and metastatic colonization of colon cancer cells.

BACKGROUND: Arginase-1 (ARG1), a urea cycle-related enzyme, catalyzes the hydrolysis of arginine to urea and ornithine, which regulates the proliferation, differentiation, and function of various cells. However, it is unclear whether ARG1 controls the progression and malignant alterations of colon cancer. METHODS: We established metastatic colonization mouse model and ARG1 overexpressing murine colon cancer CT26 cells to investigate whether activation of ARG1 was related to malignancy of colon cancer cells in vivo. Living cell numbers and migration ability of CT26 cells were evaluated in the presence of ARG inhibitor in vitro. RESULTS: Inhibition of arginase activity significantly suppressed the proliferation and migration ability of CT26 murine colon cancer cells in vitro. Overexpression of ARG1 in CT26 cells reduced intracellular L-arginine levels, enhanced cell migration, and promoted epithelial-mesenchymal transition. Metastatic colonization of CT26 cells in lung and liver tissues was significantly augmented by ARG1 overexpression in vivo. ARG1 gene expression was higher in the tumor tissues of liver metastasis than those of primary tumor, and arginase inhibition suppressed the migration ability of HCT116 human colon cancer cells. CONCLUSION: Activation of ARG1 is related to the migration ability and metastatic colonization of colon cancer cells, and blockade of this process may be a novel strategy for controlling cancer malignancy.

IFN-γ-STAT1-mediated NK2R expression is involved in the induction of antitumor effector CD8+ T cells in vivo.

The induction of antitumor effector T cells in the tumor microenvironment is a crucial event for cancer immunotherapy. Neurokinin receptor 2 (NK2R), a G protein-coupled receptor for neurokinin A (NKA), regulates diverse physiological functions. However, the precise role of NKA-NK2R signaling in antitumor immunity is unclear. Here, we found that an IFN-γ-STAT1 cascade augmented NK2R expression in CD8+ T cells, and NK2R-mediated NKA signaling was involved in inducing antitumor effector T cells in vivo. The administration of a synthetic analog of double-stranded RNA, polyinosinic-polycytidylic acid (poly I:C), into a liver cancer mouse model induced type I and type II IFNs and significantly suppressed the tumorigenesis of Hepa1-6 liver cancer cells in a STAT1-dependent manner. The reduction in tumor growth was diminished by the depletion of CD8+ T cells. IFN-γ stimulation significantly induced NK2R and tachykinin precursor 1 (encodes NKA) gene expression in CD8+ T cells. NKA stimulation combined with anti-CD3 monoclonal antibody (mAb) treatment significantly augmented IFN-γ and granzyme B production by CD8+ T cells compared with the anti-CD3 mAb alone in vitro. ERK1/2 phosphorylation and IκBα degradation in activated CD8+ T cells were suppressed under NK2R deficiency. Finally, we confirmed that tumor growth was significantly increased in NK2R-deficient mice compared with that in wild-type mice, and the antitumor effects of poly I:C were abolished by NK2R absence. These findings suggest that IFN-γ-STAT1-mediated NK2R expression is involved in the induction of antitumor effector T cells in the tumor microenvironment, which contributes to the suppression of cancer cell tumorigenesis in vivo. In this study, we revealed that IFN-γ-STAT1-mediated NK2R expression is involved in the induction of antitumor effector CD8+ T cells in the tumor microenvironment, which contributes to suppressing the tumorigenesis of liver cancer cells in vivo.

IFN-α/ß-mediated NK2R expression is related to the malignancy of colon cancer cells.

Neurokinin 2 receptor (NK2R), a G protein-coupled receptor for neurokinin A (NKA), a tachykinin family member, regulates various physiological functions including pain response, relaxation of smooth muscle, dilation of blood vessels, and vascular permeability. However, the precise role and regulation of NK2R expression in cancer cells have not been fully elucidated. In this study, we found that high NK2R gene expression was correlated with the poor survival of colorectal cancer patients, and Interferon (IFN-α/ß) stimulation significantly enhanced NK2R gene expression level of colon cancer cells in a Janus kinas 1/2 (JAK 1/2)-dependent manner. NKA stimulation augmented viability/proliferation and phosphorylation of Extracellular-signal-regulated kinase 1/2 (ERK1/2) levels of IFN-α/ß-treated colon cancer cells and NK2R blockade by using a selective antagonist reduced the proliferation in vitro. Administration of an NK2R antagonist alone or combined with polyinosinic-polycytidylic acid, a synthetic analog of double-stranded RNA, to CT26-bearing mice significantly suppressed tumorigenesis. NK2R-overexpressing CT26 cells showed enhanced tumorigenesis and metastatic colonization in both lung and liver after the inoculation into mice. These findings indicate that IFN-α/ß-mediated NK2R expression is related to the malignancy of colon cancer cells, suggesting that NK2R blockade may be a promising strategy for colon cancers.

Clinical significance of plasma-free amino acids and tryptophan metabolites in patients with non-small cell lung cancer receiving PD-1 inhibitor: a pilot cohort study for developing a prognostic multivariate model.

BACKGROUND: Amino acid metabolism is essential for tumor cell proliferation and regulation of immune cell function. However, the clinical significance of free amino acids (plasma-free amino acids (PFAAs)) and tryptophan-related metabolites in plasma has not been fully understood in patients with non-small cell lung cancer (NSCLC) who receive immune checkpoint inhibitors. METHODS: We conducted a single cohort observational study. Peripheral blood samples were collected from 53 patients with NSCLC before treatment with PD-1 (Programmed cell death-1) inhibitors. The plasma concentrations of 21 PFAAs, 14 metabolites, and neopterin were measured by liquid chromatography-mass spectrometry. Using Cox hazard analysis with these variables, a multivariate model was established to stratify patient overall survival (OS). Gene expression in peripheral blood mononuclear cells (PBMCs) was compared between the high-risk and low-risk patients by this multivariate model. RESULTS: On Cox proportional hazard analysis, higher concentrations of seven PFAAs (glycine, histidine, threonine, alanine, citrulline, arginine, and tryptophan) as well as lower concentrations of three metabolites (3h-kynurenine, anthranilic acid, and quinolinic acid) and neopterin in plasma were significantly correlated with better OS (p<0.05). In particular, the multivariate model, composed of a combination of serine, glycine, arginine, and quinolinic acid, could most efficiently stratify patient OS (concordance index=0.775, HR=3.23, 95% CI 2.04 to 5.26). From the transcriptome analysis in PBMCs, this multivariate model was significantly correlated with the gene signatures related to immune responses, such as CD8 T-cell activation/proliferation and proinflammatory immune responses, and 12 amino acid-related genes were differentially expressed between the high-risk and low-risk groups. CONCLUSIONS: The multivariate model with PFAAs and metabolites in plasma might be useful for stratifying patients who will benefit from PD-1 inhibitors.

An ultra-sensitive T 2-weighted MR contrast agent based on Gd3+ ion chelated Fe3O4 nanoparticles.

An ultra-sensitive T 2-weighted MR imaging contrast agent was prepared based on Fe3O4 nanoparticles and Gd3+ ions (Fe3O4@Gd). Amino modified Fe3O4 nanoparticles were conjugated to diethylenetriamine pentaacetic acid, and finally coordinated with Gd3+ ions. The nanoparticles had a uniform morphology with a size of 100 nm and a Gd/Fe mass ratio of 1/110. The r 2 (transverse relaxivity) of the Fe3O4 nanoparticles increased from 131.89 mM-1 s-1 to 202.06 mM-1 s-1 after coordination with Gd3+ ions. MR measurements showed that the aqueous dispersion of Fe3O4@Gd nanoparticles had an obvious concentration-dependent negative contrast enhancement. Hepatoma cells were selected to test the cytotoxicity and MR imaging effect. The application of Fe3O4@Gd nanoparticles as contrast agents was also exploited in vivo for T 2-weighted MR imaging of rat livers. All the results showed the effectiveness of the nanoparticles in MR diagnosis.

IL6 Modulates the Immune Status of the Tumor Microenvironment to Facilitate Metastatic Colonization of Colorectal Cancer Cells.

It is unknown as to how liver metastases are correlated with host immune status in colorectal cancer. In this study, we found that IL6, a proinflammatory cytokine produced in tumor-bearing states, promoted the metastatic colonization of colon cancer cells in association with dysfunctional antitumor immunity. In IL6-deficient mice, metastatic colonization of CT26 cells in the liver was reduced, and the antitumor effector function of CD8+ T cells, as well as IL12 production by CD11c+ dendritic cells, were augmented in vivo IL6-deficient mice exhibited enhanced IFN-AR1-mediated type I interferon signaling, which upregulated PD-L1 and MHC class I expression on CT26 cells. In vivo injection of anti-PD-L1 effectively suppressed the metastatic colonization of CT26 cells in Il6 -/- but not in Il6 +/+ mice. Finally, we confirmed that colorectal cancer patients with low IL6 expression in their primary tumors showed prolonged disease-free survival. These findings suggest that IL6 may be a promising target for the treatment of metastasis in colorectal cancers by improving host immunity.

The antitumour growth and antiangiogenesis effects of xanthatin in murine glioma dynamically evaluated by dynamic contrast-enhanced magnetic resonance imaging.

To investigate the suppressive effects of xanthatin on glioma growth in a nude mouse xenograft model and rat orthotopic implantation model using magnetic resonance imaging (MRI) to dynamically monitor the antitumour growth and antiangiogenesis effects of xanthatin. The nude mouse xenograft tumour model and rat orthotopic implantation model were established to observe the antitumour effects of xanthatin in vivo. In the rat orthotopic implanted tumour model, MRI scanning was used to dynamically monitor the antitumour growth effect and evaluate the antiangiogenesis effect of xanthatin. We found that xanthatin at a dose of 0.4 mg/10 g dramatically decreased the growth of xenograft tumours in nude mice. The antiangiogenesis effect of xanthatin C6 glioma was evaluated by dynamic contrast-enhanced (DCE) MRI via comparison of the volume transfer constant (Ktrans ) value, a parameter that reflects vessel permeability. We found that xanthatin at the doses of 8 and 16 mg/kg significantly decreased the Ktrans value, which suggests that xanthatin has antiangiogenesis effects. These data demonstrate the suppressive effects of xanthatin on C6 glioma occur via antiangiogenesis. Meanwhile, this study also provides evidence for the application of quantitative parameters of DCE-MRI for dynamically evaluating the growth and angiogenesis of intracranial tumours and for experimental and clinical research.

L-Type Calcium Channel-Mediated Zinc Wave Is Involved in the Regulation of IL-6 by Stimulating Non-IgE with LPS and IL-33 in Mast Cells and Dendritic Cells.

The trace element zinc is essential for the immune system, and its dysregulation and deficiency results in impaired immune function. Recent studies have shown that zinc can behave as an intracellular signaling molecule in immune cells. We have previously demonstrated that L-type calcium channel (LTCC) is involved in the regulation of zinc signaling, Zinc wave and cytokine production by stimulating Fc epsilon receptor for immunoglobulin E (IgE) in mast cells. However, it is not known whether LTCC-mediated Zinc wave is required for cytokine production by stimulation of toll-like receptors and cytokine receptors in mast cells. Here we report that stimulation of toll-like receptors and cytokine receptors can induce Zinc wave in mast cells and regulate the expression of cytokine genes. The LTCC antagonist nicardipine inhibited lipopolysaccharide (LPS)- and interleukin-33 (IL-33)-mediated Zinc wave and the induction of cytokine genes such as IL-6. Consistent with these results, the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) also inhibited LPS- and IL-33-induced cytokine gene expression. Furthermore, LPS induced Zinc wave not only in mast cells but also in dendritic cells. Together, these observations show that Zinc wave is activated by various stimuli and is linked to cytokine gene induction in immune cells.

Facile fabrication of water-dispersible nanocomposites based on hexa-peri-hexabenzocoronene and Fe3O4 for dual mode imaging (fluorescent/MR) and drug delivery.

The facile fabrication of multifunctional nanocomposites (Fe3O4/HBC@F127) consisting of superparamagnetic Fe3O4 nanoparticles and fluorescent organic hexa-peri-hexabenzocoronene (HBC) molecules incorporated in block copolymer diacylphospholipid-polyethyleneglycol F127 have been demonstrated for dual mode imaging (fluorescent/MR) and drug delivery. The obtained nanocomposites were water-dispersible, stable and biocompatible, as confirmed by dynamic light scattering (DLS) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Relativity measurements showed a T 2 relaxivity (r 2) of 214.61 mM-1 s-1, which may be used as T 2-weighted MR imaging agents. In vitro imaging studies indicated that the nanocomposites had good MR and fluorescence imaging effects with low cytotoxicity. Besides, the developed nanocomposites could also be applied as drug delivery vehicles. Doxorubicin (DOX) loaded Fe3O4/HBC@F127 nanocomposites significantly inhibited the growth of human hepatoma cells (HepG2). These findings suggested that the facile synthesized multifunctional nanocomposites may be used as a platform for dual mode imaging (both MR and fluorescence) and drug delivery.

Lack of interleukin-6 in the tumor microenvironment augments type-1 immunity and increases the efficacy of cancer immunotherapy.

Conquering immunosuppression in tumor microenvironments is crucial for effective cancer immunotherapy. It is well known that interleukin (IL)-6, a pleiotropic cytokine, is produced in the tumor-bearing state. In the present study, we investigated the precise effects of IL-6 on antitumor immunity and the subsequent tumorigenesis in tumor-bearing hosts. CT26 cells, a murine colon cancer cell line, were intradermally injected into wild-type and IL-6-deficient mice. As a result, we found that tumor growth was decreased significantly in IL-6-deficient mice compared with wild-type mice and the reduction was abrogated by depletion of CD8+ T cells. We further evaluated the immune status of tumor microenvironments and confirmed that mature dendritic cells, helper T cells and cytotoxic T cells were highly accumulated in tumor sites under the IL-6-deficient condition. In addition, higher numbers of interferon (IFN)-γ-producing T cells were present in the tumor tissues of IL-6-deficient mice compared with wild-type mice. Surface expression levels of programmed death-ligand 1 (PD-L1) and MHC class I on CT26 cells were enhanced under the IL-6-deficient condition in vivo and by IFN-γ stimulation in vitro. Finally, we confirmed that in vivo injection of an anti-PD-L1 antibody or a Toll-like receptor 3 ligand, polyinosinic-polycytidylic acid, effectively inhibited tumorigenesis under the IL-6-deficient condition. Based on these findings, we speculate that a lack of IL-6 produced in tumor-bearing host augments induction of antitumor effector T cells and inhibits tumorigenesis in vivo, suggesting that IL-6 signaling may be a promising target for the development of effective cancer immunotherapies.

Lactoferrin modified graphene oxide iron oxide nanocomposite for glioma-targeted drug delivery.

Targeting delivery of drugs in a specific manner represents a potential powerful technology in gliomas. Herein, we prepared a multifunctional targeted delivery system based on graphene oxide (GO) that contains a molecular bio-targeting ligand and superparamagnetic iron oxide nanoparticles on the surface of GO for magnetic targeting. Superparamagnetic Fe3O4 nanoparticles was loaded on the surface of GO via chemical precipitation method to form GO@Fe3O4 nanocomposites. Lactoferrin (Lf), an iron-transporting serum glycoprotein that binds to receptors overexpressed at the surface of glioma cells and vascular endothelial cell of the blood brain barrier, was chosen as the targeted ligand to construct the targeted delivery system Lf@GO@Fe3O4 through EDC/NHS chemistry. With the confirmation of TEM, DLS and VSM, the resulting Lf@GO@Fe3O4 had a size distribution of 200-1000nm and exhibited a superparamagnetic behavior. The nano delivery system had a high loading capacity and exhibited a pH-dependent release behavior. Compared with free DOX and DOX@GO@Fe3O4, Lf@GO@Fe3O4@DOX displayed greater intracellular delivery efficiency and stronger cytotoxicity against C6 glioma cells. The results demonstrated the potential utility of Lf conjugated GO@Fe3O4 nanocomposites for therapeutic application in the treatment of gliomas.

Association of HLA-B*1502 and *1511 allele with antiepileptic drug-induced Stevens-Johnson syndrome in central China.

Previous studies have demonstrated a strong association between carbamazepine (CBZ)-induced Stevens-Johnson syndrome (SJS) and HLA-B*1502 in Han Chinese. Here, we extended the study of HLA-B*1502 susceptibility to two different antiepileptic drugs, oxcarbazepine (OXC) and phenobarbital (PB). In addition, we genotyped HLA-B*1511 in a case of CBZ-induced SJS with genotype negative for HLA-B*1502. The presence of HLA-B*1502 was determined using polymerase chain reaction with sequence-specific primers (PCR-SSP). Moreover, we genotyped HLA-B*1502 in 17 cases of antiepileptic drugs (AEDs)-induced cutaneous adverse drug reactions (cADRs), in comparison with AEDs-tolerant (n=32) and normal controls (n=38) in the central region of China. The data showed that HLA-B*1502 was positive in 5 of 6 cases of AEDs-induced SJS (4 CBZ, 1 OXC and 1 PB), which was significantly more frequent than AEDs-tolerant (2/32, 18 CBZ, 6 PB and 8 OXC) and normal controls (3/38). Compared with AEDs-tolerant and normal controls, the OR for patients carrying the HLA-B*1502 with AEDs-induced SJS was 6.25 (95% CI: 1.06-36.74) and 4.86 (95% CI: 1.01-23.47). The sensitivity and specificity of HLA-B*1502 for prediction of AEDs-induced SJS were 71.4%. The sensitivity and specificity of HLA-B*1502 for prediction of CBZ-induced SJS were 60% and 94%. HLA-B*1502 was not found in 11 children with maculopapular exanthema (MPE) (n=9) and hypersensitivity syndrome (HSS) (n=2). However, we also found one case of CBZ-induced SJS who was negative for HLA-B*1502 but carried HLA-B*1511. It was suggested that the association between the CBZ-induced SJS and HLA-B*1502 allele in Han Chinese children can extend to other aromatic AEDs including OXC and PB related SJS. HLA-B*1511 may be a risk factor for some patients with CBZ-induced SJS negative for HLA-B*1502.