Recent advances in biomaterial-boosted adoptive cell therapy.

Adoptive immunotherapies based on the transfer of functional immune cells hold great promise in treating a wide range of malignant diseases, especially cancers, autoimmune diseases, and infectious diseases. However, manufacturing issues and biological barriers lead to the insufficient population of target-selective effector cells at diseased sites after adoptive transfer, hindering effective clinical translation. The convergence of immunology, cellular biology, and materials science lays a foundation for developing biomaterial-based engineering platforms to overcome these challenges. Biomaterials can be rationally designed to improve ex vivo immune cell expansion, expedite functional engineering, facilitate protective delivery of immune cells in situ, and navigate the infused cells in vivo. Herein, this review presents a comprehensive summary of the latest progress in biomaterial-based strategies to enhance the efficacy of adoptive cell therapy, focusing on function-specific biomaterial design, and also discusses the challenges and prospects of this field.

miR-144 suppresses cell proliferation and migration in colorectal cancer by targeting NRAS.

Colorectal cancer (CRC) is a type of malignant cancer that has become particularly prevalent worldwide. It is of crucial importance to CRC treatment that the underlying molecular mechanism of CRC progression is determined. The NRAS gene is an important small G protein that is involved in various biological processes, including cancers. NRAS is an oncogene in many neoplasms but its function and regulation in CRC have seldom been investigated. In this study, it was uncovered that the NRAS protein was significantly upregulated in CRC tissues. According to a bioinformatics prediction, we identified that miR-144 may target NRAS to suppress its expression. In vitro experiments indicated that miR-144 decreased NRAS expression in different CRC cell lines (SW480, LoVo, and Caco2). By inhibiting NRAS, miR-144 repress SW480 cell proliferation and migration. Moreover, miR-144 decelerated the growth of SW480 xenograft tumors in vivo by targeting NRAS. In summary, our results identified a novel miR-144-NRAS axis in CRC that could promote the research and treatment of CRC.

A Novel Specific Anti-CD73 Antibody Inhibits Triple-Negative Breast Cancer Cell Motility by Regulating Autophagy.

Increasing researches have focused on cancer metastasis and development. The ectonucleotidase CD73 is one of the most common cell surface enzymes that are involved in immunosuppression. In this study, the recombinant plasmid pET28a-CD73 was constructed and the CD73 protein was overexpressed in E. coli as an inclusion body that was then subjected to refolding. The anti-CD73 monoclonal antibody (3F7) was obtained by hybridoma technology. The antibody subtype was identified as IgG2a with an affinity constant of 5.75 nM. This antibody could be applied to immunofluorescence and flow cytometry. The results showed that the CD73 protein was not only located in the cytoplasm but also distributed on the surface of triple-negative breast cancer cells MDA-MB-231 and MDA-MB-468. Moreover, the level of CD73 protein was associated with the survival rate. Although the anti-CD73 antibody was not able to inhibit tumor cell growth, it could enhance the cytotoxic effect of Doxorubicin to triple-negative breast cancer cells. In vitro function assay results indicated that anti-CD73 mAb could inhibit cell migration and invasion in both human triple-negative breast cancer and mouse 4T1 cell lines. In this process, both the LC3I/LC3II ratio and p62 protein levels increased, which indicated that the blockage of CD73 could inhibit cell autophagy, and cell migration and invasion were restored by rapamycin. In vivo, anti-CD73 mAb could significantly inhibit lung metastasis of 4T1 cells in a mouse xenograft model. Taken together, this novel anti-CD73 antibody could be developed as an adjuvant drug for triple-negative breast cancer therapy and can be useful in tumor diagnosis.

Optimized Expression and Characterization of a Novel Fully Human Bispecific Single-Chain Diabody Targeting Vascular Endothelial Growth Factor165 and Programmed Death-1 in Pichia pastoris and Evaluation of Antitumor Activity In Vivo.

Bispecific antibodies, which can bind to two different epitopes on the same or different antigens simultaneously, have recently emerged as attractive candidates for study in various diseases. Our present study successfully constructs and expresses a fully human, bispecific, single-chain diabody (BsDb) that can bind to vascular endothelial growth factor 165 (VEGF165) and programmed death-1 (PD-1) in Pichia pastoris. Under the optimal expression conditions (methanol concentration, 1%; pH, 4.0; inoculum density, OD600 = 4, and the induction time, 96 h), the maximum production level of this BsDb is achieved at approximately 20 mg/L. The recombinant BsDb is purified in one step using nickel-nitrilotriacetic acid (Ni-NTA) column chromatography with a purity of more than 95%. Indirect enzyme-linked immune sorbent assay (ELISA) and sandwich ELISA analyses show that purified BsDb can bind specifically to VEGF165 and PD-1 simultaneously with affinities of 124.78 nM and 25.07 nM, respectively. Additionally, the BsDb not only effectively inhibits VEGF165-stimulated proliferation, migration, and tube formation in primary human umbilical vein endothelial cells (HUVECs), but also significantly improves proliferation and INF-γ production of activated T cells by blocking PD-1/PD-L1 co-stimulation. Furthermore, the BsDb displays potent antitumor activity in mice bearing HT29 xenograft tumors by inhibiting tumor angiogenesis and activating immune responses in the tumor microenvironment. Based on these results, we have prepared a potential bispecific antibody drug that can co-target both VEGF165 and PD-1 for the first time. This work provides a stable foundation for the development of new strategies by the combination of an angiogenesis inhibition and immune checkpoint blockade for cancer therapy.

The role of haplotype in 15q25.1 locus in lung cancer risk: results of scanning chromosome 15.

The role of haplotypes and the interaction of haplotypes and smoking in lung cancer risk have not been well characterized. We analyzed data from an Italian population-based, case-control study with 1815 lung cancer patients and 1959 healthy controls in discovery, and performed a validation using a case-control study with 2983 lung cancer patients and 3553 healthy controls of European ancestry for replication. Sliding window haplotype analysis within chromosome 15, evaluating 4722250 haplotypes and pair-wise haplotype analysis identified that CHRNA5 rs588765-rs16969968 was the most significant haplotype associated with lung cancer risk (omnibus P = 8.35×10(-15) in discovery and 7.26×10(-14) in replication), and improved the prediction of case status over that provided by the individual SNPs rs16969968 or rs588765 (likelihood ratio test P = 0.006 for rs16969968 and 3.83×10(-14) for rs588765 in discovery, 0.009 for rs16969968 and 4.62×10(-13) for rs588765 in replication, compared with rs588765-rs16969968). Compared with the wild-type homozygous diplotype, CA/CA homozygote exhibited an approximately 2-fold increase risk for lung cancer (OR = 2.12; 95% CI 1.46-3.07 in discovery, and OR = 2.01; 95% CI 1.51-2.67 in replication). Even among never-smokers, CA/CA homozygote showed an increased risk of lung cancer with borderline significance in discovery (adjusted OR = 1.75, 95% CI 0.96-3.19) and statistical significance in replication (adjusted OR = 2.10, 95% CI 1.12-3.96), compared with combined genotypes (CG/CG + CG/TG). Accordingly, rs588765-rs16969968 may be a genetic marker to lung cancer risk, even among never-smokers.

RNA-seq analysis of lung adenocarcinomas reveals different gene expression profiles between smoking and nonsmoking patients.

Lung adenocarcinoma is caused by the combination of genetic and environmental effects, and smoking plays an important role in the disease development. Exploring the gene expression profile and identifying genes that are shared or vary between smokers and nonsmokers with lung adenocarcinoma will provide insights into the etiology of this complex cancer. We obtained RNA-seq data from paired normal and tumor tissues from 34 nonsmoking and 34 smoking patients with lung adenocarcinoma (GEO: GSE40419). R Bioconductor, edgeR, was adopted to conduct differential gene expression analysis between paired normal and tumor tissues. A generalized linear model was applied to identify genes that were differentially expressed in nonsmoker and smoker patients as well as genes that varied between these two groups. We identified 2273 genes that showed differential expression with FDR < 0.05 and |logFC| >1 in nonsmoker tumor versus normal tissues; 3030 genes in the smoking group; and 1967 genes were common to both groups. Sixty-eight and 70% of the identified genes were downregulated in nonsmoking and smoking groups, respectively. The 20 genes such as SPP1, SPINK1, and FAM83A with largest fold changes in smokers also showed similar large and highly significant fold changes in nonsmokers and vice versa, showing commonalities in expression changes for adenocarcinomas in both smokers and nonsmokers for these genes. We also identified 175 genes that were significantly differently expressed between tumor samples from nonsmoker and smoker patients. Gene expression profile varied substantially between smoker and nonsmoker patients with lung adenocarcinoma. Smoking patients overall showed far more complicated disease mechanism and have more dysregulation in their gene expression profiles. Our study reveals pathogenetic differences in smoking and nonsmoking patients with lung adenocarcinoma from transcriptome analysis. We provided a list of candidate genes for further study for disease detection and treatment in both smoking and nonsmoking patients with lung adenocarcinoma.

Covalently dimerized Camelidae antihuman TNFa single-domain antibodies expressed in yeast Pichia pastoris show superior neutralizing activity.

Antagonists of tumor necrosis factor alpha (TNFa) have revolutionized the treatment of selected inflammatory diseases. Recombination Camelidae variable heavy-chain domain-only TNFa antibodies (anti-TNF-VHH) have been developed to antagonize the action of human and murine TNFa. Here, we describe a strategy to obtain functional covalent dimer anti-TNF-VHH molecules with the C-terminal fusion of human IgG1 Fc domain named anti-TNF-VHH-Fc. The resulting fusion proteins were separately expressed by use of the pET28a vector in Escherichia coli ((Ec)) strain BL21 and the pPICZaA vector in Pichia pastoris ((Pp)) strain GS115, then purified by protein A affinity resin. Fc-engineered anti-(Ec)TNF-VHH-Fc was about 40 kDa and anti-(Pp)TNF-VHH-Fc was about 43 kDa. Monomeric VHH was also cloned and expressed in E. coli strain BL21, with the molecular weight of about 18 kDa. Enzyme-linked immunosorbent assay and L929 cell cytotoxicity assay demonstrated that the fusion protein anti-(Pp)TNF-VHH-Fc blocked TNFa activity more effectively than either anti-(Ec)TNF-VHH-Fc or monomeric anti-(Ec)TNF-VHH protein. We suggest that efficient disulfide bond formation using the P. pastoris expression system improves the covalent dimer anti-TNF-VHH-Fc neutralizing activity.

Control of abscisic acid catabolism and abscisic acid homeostasis is important for reproductive stage stress tolerance in cereals.

Drought stress at the reproductive stage causes pollen sterility and grain loss in wheat (Triticum aestivum). Drought stress induces abscisic acid (ABA) biosynthesis genes in anthers and ABA accumulation in spikes of drought-sensitive wheat varieties. In contrast, drought-tolerant wheat accumulates lower ABA levels, which correlates with lower ABA biosynthesis and higher ABA catabolic gene expression (ABA 8'-hydroxylase). Wheat TaABA8'OH1 deletion lines accumulate higher spike ABA levels and are more drought sensitive. ABA treatment of the spike mimics the effect of drought, causing high levels of sterility. ABA treatment represses the anther cell wall invertase gene TaIVR1, and drought-tolerant lines appeared to be more sensitive to the effect of ABA. Drought-induced sterility shows similarity to cold-induced sterility in rice (Oryza sativa). In cold-stressed rice, the rate of ABA accumulation was similar in cold-sensitive and cold-tolerant lines during the first 8 h of cold treatment, but in the tolerant line, ABA catabolism reduced ABA levels between 8 and 16 h of cold treatment. The ABA biosynthesis gene encoding 9-cis-epoxycarotenoid dioxygenase in anthers is mainly expressed in parenchyma cells surrounding the vascular bundle of the anther. Transgenic rice lines expressing the wheat TaABA8'OH1 gene under the control of the OsG6B tapetum-specific promoter resulted in reduced anther ABA levels under cold conditions. The transgenic lines showed that anther sink strength (OsINV4) was maintained under cold conditions and that this correlated with improved cold stress tolerance. Our data indicate that ABA and ABA 8'-hydroxylase play an important role in controlling anther ABA homeostasis and reproductive stage abiotic stress tolerance in cereals.

EGF-functionalized single-walled carbon nanotubes for targeting delivery of etoposide.

To enhance the therapeutic potential of etoposide (ETO), we devised a targeted drug delivery system (TDDS) of epidermal growth factor-chitosan-carboxyl single-walled carbon nanotubes-ETO (EGF/CHI/SWNT-COOHs/ETO) using modified SWNTs (m-SWNTs) as the carrier, EGF-functionalized SWNTs (f-SWNTs) as the targeted moiety and ETO as the drug. After SWNT-COOHs were conjugated with CHI (CHI/SWNT-COOHs/ETO), they displayed high solubility and stable dispersion in aqueous solution. The drug loading capacity was approximately 25-27%. The m-SWNTs and f-SWNTs had only slight cytotoxicity. ETO was released from EGF/CHI/SWNT-COOHs/ETO at low pH and taken up by tumour cells via adenosine triphosphate (ATP)-dependent endocytosis. The cell death induced by EGF/CHI/SWNT-COOHs/ETO was as much as 2.7 times that due to ETO alone. In summary, these results demonstrated that our TDDS had a greater anticancer effect than free ETO in vitro.

Endothelial-Specific Molecule 1 Inhibition Lessens Productive Angiogenesis and Tumor Metastasis to Overcome Bevacizumab Resistance.

The development of drug resistance in malignant tumors leads to disease progression, creating a bottleneck in treatment. Bevacizumab is widely used clinically, and acts by inhibiting angiogenesis to "starve" tumors. Continuous treatment can readily induce rebound proliferation of tumor blood vessels, leading to drug resistance. Previously, we found that the fragment crystallizable (Fc) region of bevacizumab cooperates with the Toll-like receptor-4 (TLR4) ligand to induce M2b polarization in macrophages and secrete tumor necrosis factor-α (TNFα), which promotes immunosuppression, tumor metastasis, and angiogenesis. However, the downstream mechanism underlying TNFα-mediated bevacizumab resistance requires further investigation. Our RNA-Seq analysis results revealed that the expression of endothelial cell specific molecule-1 (ESM1) increased significantly in drug-resistant tumors and promoted metastasis and angiogenesis in vitro and in vivo. Furthermore, TNFα induced the upregulation of ESM1, which promotes metastasis and angiogenesis and regulates matrix metalloprotease-9 (MMP9), vascular endothelial growth factor (VEGF), and delta-like ligand-4 molecules (DLL4). Accordingly, the curative effect of bevacizumab improved by neutralizing ESM1 with high-affinity anti-ESM1 monoclonal antibody 1-2B7 in bevacizumab-resistant mice. This study provides important insights regarding the molecular mechanism by which TNFα-induced ESM1 expression promotes angiogenesis, which is significant for elucidating the mechanism of bevacizumab drug resistance and possibly identifying appropriate biosimilar molecules.

Nasopharyngeal carcinoma risk by histologic type in central China: impact of smoking, alcohol and family history.

The role of cigarette smoking, alcohol use, family history of cancer and the interaction of cigarettes and family history in the etiology of Nasopharyngeal carcinoma (NPC) in general and within each histologic type are unclear. We conducted a case-control study among 1,044 Han Chinese patients with NPC and 1,095 Han Chinese cancer-free control subjects. Logistic regression was used to analyze the association between histologic type of NPC and cigarette smoking, alcohol drinking and family history. The results indicated that NPC was significantly associated with cigarette smoking [adjusted odds ratio (OR) = 2.97, 95% confidence interval (CI), 2.38-3.70], and the association exhibited a dose-response relationship for intensity, duration, and cumulative consumption of cigarettes (p(trend) < 0.0001 for intensity, duration and cumulative consumption of cigarettes). Positive family history of cancer led to a significant 12-fold elevated risk of NPC (adjusted OR = 12.95, 95% CI, 7.12-23.54) and acted jointly with cigarettes in contributing to NPC risk (adjusted OR = 56.68, 95% CI, 17.25-186.19). The association of NPC risk with cigarettes was stronger for nonkeratinizing carcinoma than for keratinizing squamous cell carcinoma (KSCC), whereas family history was more closely associated with KSCC. NPC risk was not associated with alcohol consumption. Our study demonstrated that cigarette smoking and family history of cancer could serve independently and jointly as risk factors for etiology of NPC and might affect the risk of histology-specific NPC differently. This knowledge may help facilitate comprehension of NPC etiology in general as well as within each histologic type, and thereby improve prevention efforts.

Importance of pre-anthesis anther sink strength for maintenance of grain number during reproductive stage water stress in wheat.

Reproductive stage water stress leads to spikelet sterility in wheat. Whereas drought stress at anthesis affects mainly grain size, stress at the young microspore stage of pollen development is characterized by abortion of pollen development and reduction in grain number. We identified genetic variability for drought tolerance at the reproductive stage. Drought-tolerant wheat germplasm is able to maintain carbohydrate accumulation in the reproductive organs throughout the stress treatment. Starch depletion in the ovary of drought-sensitive wheat is reversible upon re-watering and cross-pollination experiments indicate that the ovary is more resilient than the anther. The effect on anthers and pollen fertility is irreversible, suggesting that pollen sterility is the main cause of grain loss during drought conditions in wheat. The difference in storage carbohydrate accumulation in drought-sensitive and drought-tolerant wheat is correlated with differences in sugar profiles, cell wall invertase gene expression and expression of fructan biosynthesis genes in anther and ovary (sucrose : sucrose 1-fructosyl-transferase, 1-SST; sucrose : fructan 6-fructosyl-transferase, 6-SFT). Our results indicate that the ability to control and maintain sink strength and carbohydrate supply to anthers may be the key to maintaining pollen fertility and grain number in wheat and this mechanism may also provide protection against other abiotic stresses.

Preparation of RGD4C fused anti-TNFα nanobody and inhibitory activity on triple-negative breast cancer in vivo.

AIMS: Triple-negative breast cancer (TNBC) is not sensitive to current endocrine treatments, so new treatment strategies need to be explored. Based on previous antitumour studies on anti-TNFα nanobody, we designed a novel fusion nanobody to enhance antitumour activity of the anti-TNFα nanobody in TNBC. MAIN METHODS: The RGD4C contains RGD sequence, which is the smallest recognition unit binding to the αvß3 receptor on tumour cell membranes and involved in tumour cell adhesion, proliferation, and metastasis. RGD4C was fused to anti-TNFα nanobody to investigate the antitumour activity in vitro and in vivo. KEY FINDINGS: The antitumour effects of fusion nanobody V-L-R-H could effectively bind to αvß3 and inhibit cell migration and proliferation of MDA-MB-231, which had satisfying purification efficiency and approving antigen or receptor binding activity. V-L-R-H could inhibit the TNFα-mediated PI3K/AKT/NF-κB signal pathway and integrin αvß3 correlative FAK focal adhesion signal pathway. Mouse xenograft tumour experiments showed that the V-L-R-H could inhibit tumour proliferation and metastasis; reduce the TNFα, HIFα, Ki67, and CD31 concentrations in tumour; and inhibit the process of epithelial-mesenchymal transition. SIGNIFICANCE: The fusion nanobody enhanced antitumour activity of the anti-TNFα nanobody on TNBC. It provided a reference for the design of dual functional fusion proteins and development of tumour treatment strategies of antagonistic TNFα and αvß3, and a new therapeutic strategy and research direction for the treatment of TNBC.

Tumor necrosis factor α inhibition overcomes immunosuppressive M2b macrophage-induced bevacizumab resistance in triple-negative breast cancer.

Bevacizumab in neoadjuvant therapy provides a new hope of improved survival for patients with triple-negative breast cancer (TNBC) by targeting vascular endothelial growth factor in combination with chemotherapy, but curative effect is limited by bevacizumab's continuous use while mechanisms remain incompletely understood. More and more researches reported that tumor-associated macrophages mediate resistance to chemotherapy and radiotherapy in various tumors. Here we developed a TNBC model resistant to bevacizumab under bevacizumab continuous administration. It was found that proportion of a specific subset of tumor-associated macrophages characterized as M2b (CD11b+ CD86high IL10high) increased and responsible for acquired resistance to bevacizumab. Then, we showed that RAW264.7 macrophages could be polarized to M2b subtype on simultaneous exposure to bevacizumab and TLR4 ligands as occurs in the context of continuous bevacizumab treatment. Concordantly, in TLR4-deleted C57BL/10ScNJNju (TLR4lps-del) mut/mut mice with bevacizumab treatment model, it was verified that the M2b macrophage could be induced by Fc gamma receptor-TLR4 cross-talk. In MDA-MB-231-resistant tumor-bearing mice, the content of TNFα in serum kept going up consistent with CCL1, a chemokine of M2b macrophage. In vitro neutralizing tumor necrosis factor α (TNFα) could inhibit the tumor progression caused by M2b culture medium and tumor IDO1 expression. Therefore, we thought that TNFα is a key tumor-promoting effector molecule secreted by M2b macrophage. Accordingly, the curative effect of bevacizumab was proved to be significantly improved by neutralizing TNFα with anti-TNFα nanobody. This study is expected to provide theoretical and clinical evidence elucidating the drug resistance in patients receiving bevacizumab.

A new efficient method to detect genetic interactions for lung cancer GWAS.

BACKGROUND: Genome-wide association studies (GWAS) have proven successful in predicting genetic risk of disease using single-locus models; however, identifying single nucleotide polymorphism (SNP) interactions at the genome-wide scale is limited due to computational and statistical challenges. We addressed the computational burden encountered when detecting SNP interactions for survival analysis, such as age of disease-onset. To confront this problem, we developed a novel algorithm, called the Efficient Survival Multifactor Dimensionality Reduction (ES-MDR) method, which used Martingale Residuals as the outcome parameter to estimate survival outcomes, and implemented the Quantitative Multifactor Dimensionality Reduction method to identify significant interactions associated with age of disease-onset. METHODS: To demonstrate efficacy, we evaluated this method on two simulation data sets to estimate the type I error rate and power. Simulations showed that ES-MDR identified interactions using less computational workload and allowed for adjustment of covariates. We applied ES-MDR on the OncoArray-TRICL Consortium data with 14,935 cases and 12,787 controls for lung cancer (SNPs = 108,254) to search over all two-way interactions to identify genetic interactions associated with lung cancer age-of-onset. We tested the best model in an independent data set from the OncoArray-TRICL data. RESULTS: Our experiment on the OncoArray-TRICL data identified many one-way and two-way models with a single-base deletion in the noncoding region of BRCA1 (HR 1.24, P = 3.15 × 10-15), as the top marker to predict age of lung cancer onset. CONCLUSIONS: From the results of our extensive simulations and analysis of a large GWAS study, we demonstrated that our method is an efficient algorithm that identified genetic interactions to include in our models to predict survival outcomes.

Protein-altering germline mutations implicate novel genes related to lung cancer development.

Few germline mutations are known to affect lung cancer risk. We performed analyses of rare variants from 39,146 individuals of European ancestry and investigated gene expression levels in 7,773 samples. We find a large-effect association with an ATM L2307F (rs56009889) mutation in adenocarcinoma for discovery (adjusted Odds Ratio = 8.82, P = 1.18 × 10-15) and replication (adjusted OR = 2.93, P = 2.22 × 10-3) that is more pronounced in females (adjusted OR = 6.81 and 3.19 and for discovery and replication). We observe an excess loss of heterozygosity in lung tumors among ATM L2307F allele carriers. L2307F is more frequent (4%) among Ashkenazi Jewish populations. We also observe an association in discovery (adjusted OR = 2.61, P = 7.98 × 10-22) and replication datasets (adjusted OR = 1.55, P = 0.06) with a loss-of-function mutation, Q4X (rs150665432) of an uncharacterized gene, KIAA0930. Our findings implicate germline genetic variants in ATM with lung cancer susceptibility and suggest KIAA0930 as a novel candidate gene for lung cancer risk.

Recombinant expressing angiopep-2 fused anti-VEGF single chain Fab (scFab) could cross blood-brain barrier and target glioma.

In 2009, the FDA approved bevacizumab for the treatment of adult patients diagnosed with recurrent glioblastoma. However, the poor permeability of the macromolecules across the blood-brain barrier, determined by multifactorial anatomical and physiological milieu, restricts the clinical therapeutic effect of bevacizumab. The low-density lipoprotein receptor related protein 1 (LRP1) is highly expressed in the endothelial cells of the brain capillary and the glioma cells. Angiopep-2 (ANG) is a 19-aa oligopeptide that can bind to LRP1 and penetrate the blood-brain barrier by receptor-mediated transport. Therefore, ANG can be used as a dual-targeting drug delivery carrier into the brain and the glioma sites. In this study, ANG gene was fused with the C-terminal domain of single-chain antigen binding fragment (scFab) of the anti-VEGF antibody and recombinant scFab-ANG protein was expressed and purified using Rosatte (DE3) strain. We confirmed that ANG could carry anti-VEGF-scFab, penetrate a three-dimensional model of the brain tumor, and cross the hCMEC/D3 monolayer in the in vitro blood-brain barrier model. The animal experiments demonstrated that 3 h after the tail intravenous protein injection, the fluorescent signals in the brains of the mice in the scFab-ANG group were stronger than that in the scFab group. Furthermore, the study of the in situ rat glioma model shows that scFab-ANG could target glioma while anti-VEGF-scFab could not. These findings indicate that scFab-ANG had stronger transepithelial permeability and glioma targeting capacity. Thus, it can be a potential candidate drug for glioblastoma therapy.

Biological characterisation and application of human MTH1 and monoclonal antibody preparation.

Human MutT homolog 1 (MTH1) hydrolyses oxidised nucleotide triphosphates, thereby preventing them from being incorporated into DNA; MTH1 has been found to be elevated in many types of cancers, including lung, stomach cancer, melanoma and breast cancer. Thus, tumour­targeted hMTH1 may be valuable for developing novel anticancer therapies. In the present study, we prepared human MTH1 protein and its monoclonal antibody (mAb). The hMTH1 gene was cloned into the prokaryotic expression vector pET28a and optimally expressed in the E. coli Transetta (DE3) strain. Using an Ni­NTA column and a G­50 gel filtration column, 20.1 mg of active hMTH1 was obtained from 1,000 ml of bacterial culture, and the purity was over 98%, as detected by high­performance liquid chromatography (HPLC). The half maximal inhibitory concentration (IC50) of TH287 (hMTH1 inhibitor) was determined to be 3.53±0.47 nM using the recombinant hMTH1 protein (rhMTH1). The enzyme activity assay showed the Michaelis constant (Km) and the catalytic constant (kcat) of the protein were 106.13±48.83 µM and 3.64±0.58 sec­1, respectively. The anti­hMTH1 mAb was obtained via the hybridoma technique and validated by western blot analysis. In addition, an immunofluorescence assay (IFA) and ELISA determined that the mAb could efficiently bind to natural hMTH1 expressed on the human breast cancer cell line MCF­7. Taken together, the results showed the rhMTH1 is an active protein and has practical applications for inhibitor selection, and our prepared hMTH1 mAb will provide a valuable tool for the further characterisation of hMTH1 and antitumour medicinal development in future.

Lung Cancer Risk in Never-Smokers of European Descent is Associated With Genetic Variation in the 5p15.33 TERT-CLPTM1Ll Region.

INTRODUCTION: Inherited susceptibility to lung cancer risk in never-smokers is poorly understood. The major reason for this gap in knowledge is that this disease is relatively uncommon (except in Asians), making it difficult to assemble an adequate study sample. In this study we conducted a genome-wide association study on the largest, to date, set of European-descent never-smokers with lung cancer. METHODS: We conducted a two-phase (discovery and replication) genome-wide association study in never-smokers of European descent. We further augmented the sample by performing a meta-analysis with never-smokers from the recent OncoArray study, which resulted in a total of 3636 cases and 6295 controls. We also compare our findings with those in smokers with lung cancer. RESULTS: We detected three genome-wide statistically significant single nucleotide polymorphisms rs31490 (odds ratio [OR]: 0.769, 95% confidence interval [CI]: 0.722-0.820; p value 5.31 × 10-16), rs380286 (OR: 0.770, 95% CI: 0.723-0.820; p value 4.32 × 10-16), and rs4975616 (OR: 0.778, 95% CI: 0.730-0.829; p value 1.04 × 10-14). All three mapped to Chromosome 5 CLPTM1L-TERT region, previously shown to be associated with lung cancer risk in smokers and in never-smoker Asian women, and risk of other cancers including breast, ovarian, colorectal, and prostate. CONCLUSIONS: We found that genetic susceptibility to lung cancer in never-smokers is associated to genetic variants with pan-cancer risk effects. The comparison with smokers shows that top variants previously shown to be associated with lung cancer risk only confer risk in the presence of tobacco exposure, underscoring the importance of gene-environment interactions in the etiology of this disease.

p53 codon 72 polymorphism associated with risk of human papillomavirus-associated squamous cell carcinoma of the oropharynx in never-smokers.

The tumor suppressor p53 protein can be bound, degraded and inactivated by the human papillomavirus (HPV) E6 oncoprotein. The p53 protein's susceptibility to this oncoprotein may be influenced by the p53 codon 72 polymorphism, but the role of such a polymorphism in the development of HPV16-associated squamous cell carcinoma of the oropharynx (SCCOP) has not been established. To investigate the role of the p53 codon 72 polymorphism in the risk of HPV16-associated SCCOP, we conducted a hospital-based case-control study of 188 non-Hispanic white patients with newly diagnosed SCCOP and 342 cancer-free control subjects frequency matched by age (+/-5 years), sex, tobacco smoking status and alcohol drinking status. We found that HPV16 seropositivity was associated with an increased risk of SCCOP [adjusted odds ratio (OR), 5.7; 95% confidence interval (CI), 3.7-8.7], especially among never-smokers (adjusted OR, 14.1; 95% CI, 6.0-32.9) and among subjects with the p53 codon 72 variant genotypes [Arginine (Arg)/Proline (Pro) and Pro/Pro] (adjusted OR, 9.2; 95% CI, 4.7-17.7). A significant multiplicative interaction on the risk of SCCOP was also found between the p53 codon 72 polymorphism and HPV16 seropositivity (P = 0.05). Among never-smokers, the risk of SCCOP for those who had both HPV16 seropositivity and p53 codon 72 variant genotypes (Arg/Pro + Pro/Pro) was particularly high (adjusted OR, 22.5; 95% CI, 4.8-106.2). These findings suggest that p53 codon 72 variant genotypes modify the risk of HPV16-associated SCCOP and may be markers of genetic susceptibility to HPV16-associated SCCOP, especially among never-smokers.