Generation, ex vivo expansion and safety of engineered PD1-knockout primary T cells from cynomolgus macaques.

Programmed cell death protein 1 (PD1) is a cell-surface receptor that plays a vital regulatory role in suppressing inflammatory T cell activity; therefore, it is an ideal target for T cell-redirecting therapies. Here, we describe a cynomolgus macaque model for studying the transfer of PD1-modified T cells. We developed the first T cell engager targeting the disruption of PD1 by electroporation of plasmids encoding sgRNA and Cas9. There were no significant differences between mock T cells and PD1-knockout (PD1-KO) T cells in terms of cell viability, T cell signature marker expression, cell apoptosis, or cell cycling during prolonged in vitro culture. However, in a mixed lymphocyte reaction, PD1-KO T cells exhibited increased proliferation for both CD4+ and CD8+T cells and enhanced IFNγ release. We adoptively transferred autologous PD1-KO T cells into three cynomolgus monkeys. The PD1-KO T cells did not cause overt toxicity as measured by evaluating body weight, hematological parameters, and blood chemistry parameters. Histopathological analyses of tissues showed no lesions related to the infused PD1-KO T cells. Our findings demonstrate the utility of cynomolgus monkeys in expanding PD1-KO T cells and evaluating the safety of this immunotherapy and provide a new strategy for T cell-based adoptive cell therapies.

PD-1 inhibitors dependent CD8+ T cells inhibit mouse colon cancer cell metastasis.

BACKGROUND: Colon cancer is a common digestive tract malignancy which ranks as the third leading cause of cancer death worldwide. A current focus of anti-cancer research is harnessing the patient's own immune system for therapy. Programmed cell death protein 1 (PD-1), an immune suppressor, is upregulated in various activated immune cells, such as T cells, and in viral infections and tumors. PURPOSE: The objective of this study was to investigate the function of PD-1 inhibitor on the metastasisi of mouse colon cancer cells. PATIENTS AND METHODS: In the present study, we established an in situ colon cancer mouse model using the CT26 cell line. Hematoxylin-eosin (HE) staining was performed to detect colon cancer cell metastasis. The levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-12 (IL-12) in serum and mesenteric lymph nodes (MLNs) were detected by Enzyme-linked immunosorbent assay (ELISA). CD44high CD62Llow memory T cells, CD4+ FoxP3+ regulatory T cells, and IFN-γ and TNF-α levels in MLNs and spleen were detected by flow cytometry (FCM). RESULTS: We found that anti-PD-1 therapy inhibited colon cancer cells metastasis to the small intestine, liver, and lung, and lengthened the survival time of mice. However, the depletion of CD8 suppressed the activity of anti-PD-1 antibodies. In response to anti-PD-1 immunotherapy, the levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-12 (IL-12) in serum and mesenteric lymph nodes (MLNs) were significantly increased, while IL-6, IL-17, and transforming growth factor-ß (TGF-ß) were decreased. CD8 depletion had the opposite effect. In addition, anti-PD-1 treatment significantly increased CD44high CD62Llow memory T cells, decreased CD4+ FoxP3+ regulatory T cells, and increased IFN-γ and TNF-α levels in MLNs and spleen. Furthermore, anti-PD-1 treatment cannot exert these roles when CD8 is depleted. CONCLUSION: These results suggest that PD-1 inhibitors rely on CD8+ T cells to exert anti-tumor immunity in colon cancer.

Opposite response to hypoxia by breast cancer cells between cell proliferation and cell migration: A clue from microRNA expression profile.

The majority of tumors possess the features of hypoxia. It is generally accepted that hypoxia is a negative prognostic factor for cancer. Low levels of oxygen are able to modify basic cell metabolism status. Elucidating the basic response, including cell proliferation and migration, to hypoxia by cancer cells is important for understanding the role of hypoxia in the development of cancer. In the present study, CoCl2 stimulation was used to simulate hypoxia. A microRNA (miRNA/miR) array was used to systematically detect the changes in miRNA expression profiles. Following treatment with CoCl2 for 12 h, 15 miRNAs were markedly upregulated and 10 miRNAs were markedly decreased compared with the control. After 24 h CoCl2 incubation, 15 miRNAs were increased and 3 miRNAs were decreased compared with the control. Among them, 7 miRNAs were upregulated and 2 miRNAs were downregulated at 12 and 24 h following CoCl2 stimulation. The potential roles of these miRNA were reviewed and it was identified that the majority of them are associated with cell proliferation and migration. Additional experiments demonstrated that CoCl2 incubation inhibited the proliferation of MCF-7 cells but promoted cell migration. miR-491 may be a key miRNA for hypoxia-inhibited cell proliferation, as it was identified that hypoxia induced the downregulation of B-cell lymphoma-extra large in a miR-491-dependent manner. As the target of miR-302a, CXCR4 may be a key protein for hypoxia-promoted cell migration. In the present study, it was identified that in the early stage of hypoxia, cell proliferation was inhibited but cell migration was promoted. These results support the hypothesis that hypoxia may be a driving force for tumor cell escape from the primary tumor site to other organs, or other sites of the same organ.

MiR-424 Promotes Non-Small Cell Lung Cancer Progression and Metastasis through Regulating the Tumor Suppressor Gene TNFAIP1.

BACKGROUND/AIMS: This study aimed to investigate the potential roles of miR-424 expression in non-small cell lung cancer (NSCLC) metastasis and growth and its underlying mechanism. METHODS: The expression of miR-424 in two NSCLC cell lines (A549 and H1975) was altered by transfection with miR-424 mimic and inhibitor. Effects of miR-424 overexpression and suppression on cells migration, invasion and colony formation were analyzed. Target genes for miR-424 were predicted using bioinformatics method and then verified using luciferase assay. Effects of miR-424 expression on cell migration, invasion and proliferation were reanalyzed on the condition of TNFAIP1 was silenced. Moreover, TNFAIP1 silencing and miR-424 modified A549 cells were subcutaneous injected into node BALB/c mice to confirm the regulation of miR-424 on TNFAIP1 in regulating tumor growth. RESULTS: Compared with the control, miR-424 overexpression significantly increased the migrated and invaded cells, as well as the proliferated colonies. TNFAIP1 was a predicted target gene for miR-424, and was negatively regulated by miR-424. TNFAIP1 silence significantly increased the migrated and invaded cells compared to that in control, while these increases were abolished by miR-424 suppression. Animal experiment further evidenced miR-424 affected tumor growth by regulating TNFAIP1. CONCLUSIONS: These data demonstrate that miR-424 may be a contributor for NSCLC progression and metastasis through involving in cell migration, invasion and proliferation via inhibiting TNFAIP1. This study may provide theoretical basis for miR-424 in NSCLC target therapeutic treatment.

Microarray based analysis of gene regulation by mesenchymal stem cells in breast cancer.

Breast cancer is one of the most common malignant tumors with a high case-fatality rate among women. The present study aimed to investigate the effects of mesenchymal stem cells (MSCs) on breast cancer by exploring the potential underlying molecular mechanisms. The expression profile of GSE43306, which refers to MDA-MB-231 cells with or without a 1:1 ratio of MSCs, was downloaded from Gene Expression Omnibus database for differentially expressed gene (DEG) screening. The Database for Annotation, Visualization and Integrated Discovery was used for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for DEGs. The protein-protein interactional (PPI) network of DEGs was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins. The data was subsequently analyzed using molecular complex detection for sub-network mining of modules. Finally, DEGs in modules were analyzed using GO and KEGG pathway enrichment analyses. A total of 291 DEGs including 193 upregulated and 98 downregulated DEGs were obtained. Upregulated DEGs were primarily enriched in pathways including response to wounding (P=5.92×10-7), inflammatory response (P=5.92×10-4) and defense response (P=1.20×10-2), whereas downregulated DEGs were enriched in pathways including the cell cycle (P=7.13×10-4), mitotic cell cycle (P=6.81×10-3) and M phase (P=1.72 ×10-2). The PPI network, which contained 156 nodes and 289 edges, was constructed, and Fos was the hub node with the degree of 29. A total of 3 modules were mined from the PPI network. In total, 14 DEGs in module A were primarily enriched in GO terms, including response to wounding (P=4.77×10-6), wounding healing (P=6.25×10-7) and coagulation (P=1.13 ×10-7), and these DEGs were also enriched in 1 KEGG pathway (complement and coagulation cascades; P=0.0036). Therefore, MSCs were demonstrated to exhibit potentially beneficial effects for breast cancer therapy. In addition, the screened DEGs, particularly in PPI network modules, including FN1, CD44, NGF, SERPINE1 and CCNA2, may be the potential target genes of MSC therapy for breast cancer.

Identification of featured biomarkers in breast cancer with microRNA microarray.

PURPOSE: We aimed to screen possible biomarkers associated with the molecular mechanism of breast cancer using microRNA (miRNA) microarray. METHODS: The miRNAs expression profile GSE45666 was downloaded from Gene Expression Omnibus database, which included 101 genechips from breast tumor samples and 15 from adjacent breast normal tissue samples. Limma package in R language was used to screen and identify differentially expressed miRNAs (DE-miRNAs) which were classified as up-regulated and down-regulated groups. Then, target genes regulated by the two groups of DE-miRNA were predicted, followed by the functional and pathway enrichment analysis using the DAVID system. RESULTS: Totally, 130 DE-miRNAs were screened out, including 59 up-regulated DE-miRNAs and 71 down-regulated DE-miRNAs. The functional enrichment indicated that target genes of up- and down-regulated DE-miRNA may be most highly associated with positive regulation of gene expression and regulation of cellular metabolic process, respectively. Target genes regulated by the up- and down-regulated DE-miRNAs were mainly enriched in 13 and 14 pathways, respectively, and both were most significant in subcategories in cancer. In addition, we identified three important miRNAs (miR-142-3p, miR-483-5p and miR-483-3p) pivotal for the initiation and progression of this malignant tumor. CONCLUSIONS: MiR-142-3p, miR-483-5p and miR-483-3p are potential key factors for further understanding the molecular mechanism of breast cancer by affecting the normal physiological function of cell.

A potential peptide vector that allows targeted delivery of a desired fusion protein into the human breast cancer cell line MDA-MB-231.

Effective control of breast cancer has been primarily hampered by a lack of tumor specificity in treatments. One potential way to improve targeting specificity is to develop novel vectors that specifically bind to and are internalized by tumor cells. Through a phage display library, an 11-L-amino acid peptide, PI (sequence, CASPSGALRSC), was selected. PI was labeled with fluorescein isothiocyanate (FITC) and named PI-FITC. Subsequently, the specific affinity of PI-FITC to MDA-MB-231 human breast cancer cells and other cancer cell lines was observed by confocal microscopy. Our previous study established that PI-FITC also shows affinity to Calu-1 human lung carcinoma cells and major histocompatibility complex class I antigen molecules; therefore, the cytomembrane proteins of the cell lines were analyzed to determine those that were common to the two cell lines and may be associated with transmembrane transduction. To further test the delivery ability of PI to MDA-MB-231 cells, PI-glutathione-S-transferase (GST) was constructed and the internalization of this fusion protein was visualized by immunofluorescence microscopy. The results revealed that PI exhibited specific affinity to MDA-MB-231 cells. Use of membrane transport inhibitors indicated that macropinocytosis and caveolin-mediated endocytosis may be involved in the endocytosis of PI. In addition, 11 membrane proteins common to MDA-MB-231 and Calu-1 may be associated with transmembrane transduction. In summary, PI was able to deliver PI-GST into MDA-MB-231 cells. Thus, PI could be modified to be a potential vector, and may contribute to the development of targeted therapeutic strategies for breast cancer.

MDK Protein Overexpression Correlates with the Malignant Status and Prognosis of Non-small Cell Lung Cancer.

BACKGROUND AND AIMS: Midkine (MDK) is a heparin-binding growth factor and is overexpressed in various types of human cancer. However, little is known about the clinical significance of MDK in non-small cell lung cancer (NSCLC). The aim of this study was to measure MDK protein levels in patients with NSCLC and to explore its clinical significance. METHODS: The expression status of MDK in NSCLC at Gene Expression Omnibus (GEO accession number: GSE19804) was observed. The expression of MDK mRNA and protein was examined in NSCLC tissues and normal lung tissues through real-time PCR and Western blot. Meanwhile, the relationship of MDK protein expression levels with clinical characteristics of 186 NSCLC patients was analyzed by immunohistochemistry. RESULTS: MDK expression was increased in NSCLC tissues compared with paired normal lung tissues in microarray data (GSE19804). MDK mRNA and protein expression were obviously increased in NSCLC tissues than in paired adjacent normal lung tissues. Using immunohistochemistry, MDK protein overexpression was positively correlated with status of clinical stage, T classification, N classification, and M classification in NSCLC patients. In survival analysis, patients with higher MDK protein expression had a significantly shorter overall survival time than did patients with lower MDK protein expression. Multivariate analysis indicated that the MDK protein overexpression was an independent poor prognostic indicator for patients with NSCLC. CONCLUSIONS: MDK plays an important role in NSCLC progression and prognosis and may act as a convincing prognostic indicator for NSCLC patients.