Journal: Journal of Oncology

Article Title: Cancer-Associated Fibroblast-Derived Exosomal miRNA-320a Promotes Macrophage M2 Polarization In Vitro by Regulating PTEN/PI3K γ Signaling in Pancreatic Cancer

doi: 10.1155/2022/9514697

Figure Lengend Snippet: CAFs-derived conditioned medium (CAFs-CM) facilitated macrophage M2 polarization. (a) qPCR analysis of mRNA expression of classic M2 signature markers (CD163, CD206, and IL-10) and classic M1 signature markers (HLA-DR and iNOS) in U937 cells cultured with HPDE6-C7-CM, PANC-CM, NF3-CM, NF6-CM, CAF3-CM, or CAF6-CM. (b) qPCR analysis of mRNA expression of CD163, CD206, and IL-10 in U937 cells cultured with NFs-CM, CAFs-CM, or CAFs/GW4869-CM. (c) Western blot analysis of CD163, CD206, and IL-10 protein expression in U937 cells cultured with NFs-CM, CAFs-CM, or CAFs/GW4869-CM. (d) Quantitative analysis of CD163, CD206, and IL-10 protein expression in U937 cells cultured with NFs-CM, CAFs-CM, or CAFs/GW4869-CM. (e) The effect of NFs-CM, CAFs-CM, or CAFs/GW4869-CM on proliferation of pancreatic cancer cells by CCK-8 assay. (f) The effect of NFs-CM, CAFs-CM, or CAFs/GW4869-CM on pancreatic cancer cells invasion was determined by the Transwell assay. Representative photographs (magnification, 100) and the number of invaded cells are displayed. ∗∗ p < 0.01. # vs CAFs-CM. qPCR, quantitative real-time PCR; CM, conditioned medium; NFs, normal fibroblasts; CAFs, cancer-associated fibroblasts.

Article Snippet: Human monocytic cell line U937 was purchased from Merck Millipore (Temecula, California, USA) and cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 10 nM 12-o-tetradecanoyl-phorbol-13-acetate (TPA).

Techniques: Derivative Assay, Expressing, Cell Culture, Western Blot, CCK-8 Assay, Transwell Assay, Real-time Polymerase Chain Reaction

Journal: Journal of Oncology

Article Title: Cancer-Associated Fibroblast-Derived Exosomal miRNA-320a Promotes Macrophage M2 Polarization In Vitro by Regulating PTEN/PI3K γ Signaling in Pancreatic Cancer

doi: 10.1155/2022/9514697

Figure Lengend Snippet: CAFs-derived exosomes (CAFs-Exo) facilitated macrophage M2 polarization. (a) Electron micrograph analysis of exosomes collected from CAFs-CM (bar, 500 nm). (b) Size distribution and concentration range characterization of exosomes collected from CAFs-CM assayed with qNano. (c) Western blot analysis of protein expression of CD63, CD81, and HSP70 (markers of exosomes) in exosomes collected from CAFs-CM assayed. (d) qPCR analysis of CD163, CD206, and IL-10 mRNA expression in U937 cells cultured with NFs-Exo or CAFs-Exo. (e) Western blot analysis of protein expression of CD163, CD206, and IL-10 in U937 cells cultured with NFs-Exo or CAFs-Exo. (f) Quantitative analysis of CD163, CD206, and IL-10 protein expression in U937 cells cultured with NFs-Exo or CAFs-Exo. ∗∗ p < 0.01. qPCR, quantitative real-time PCR; CM, conditioned medium; NFs, normal fibroblasts; CAFs, cancer-associated fibroblasts; Exo, exosome.

Article Snippet: Human monocytic cell line U937 was purchased from Merck Millipore (Temecula, California, USA) and cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 10 nM 12-o-tetradecanoyl-phorbol-13-acetate (TPA).

Techniques: Derivative Assay, Concentration Assay, Western Blot, Expressing, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Journal of Oncology

Article Title: Cancer-Associated Fibroblast-Derived Exosomal miRNA-320a Promotes Macrophage M2 Polarization In Vitro by Regulating PTEN/PI3K γ Signaling in Pancreatic Cancer

doi: 10.1155/2022/9514697

Figure Lengend Snippet: Exosomes mediated the transfer of miRNA-320a from CAFs to macrophages. qPCR analysis of miR-106b, miR-148a, miR-125b, miR-320c, miR-320a, miR-1285, miR-422a, miR-29a, and miR-378d mRNA expression in NFs and CAFs (a) or NFs-Exo and CAFs-Exo (b). (c, d) qPCR analysis of miR-320a mRNA expression in 7 pancreatic cancer tissues-derived CAFs and 7 pancreatic cancer tissues-derived NFs or 7 pancreatic cancer tissues-derived CAFs-Exo and 7 pancreatic cancer tissues-derived NFs-Exo. (e–g) qPCR analysis of miR-320a mRNA expression in miRNA-320a-overexpressed CAFs (e), corresponding CAFs-Exo (f), and U937 cells after treatment with miRNA-320a-overexpressed CAFs-Exo (g). (h) Fluorescence microscope analysis of FAM-tagged miRNA-320a in U937cells treated with CAFs-Exo/FAM-miRNA-320a. ∗∗ p < 0.01. qPCR, quantitative real-time PCR; CM, conditioned medium; NFs, normal fibroblasts; CAFs, cancer-associated fibroblasts; Exo, exosome; CAFs-Exo/FAM-miRNA-320a, FAM-miRNA-320a-overexpressed CAFs-Exo.

Article Snippet: Human monocytic cell line U937 was purchased from Merck Millipore (Temecula, California, USA) and cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 10 nM 12-o-tetradecanoyl-phorbol-13-acetate (TPA).

Techniques: Expressing, Derivative Assay, Fluorescence, Microscopy, Real-time Polymerase Chain Reaction

Journal: Journal of Oncology

Article Title: Cancer-Associated Fibroblast-Derived Exosomal miRNA-320a Promotes Macrophage M2 Polarization In Vitro by Regulating PTEN/PI3K γ Signaling in Pancreatic Cancer

doi: 10.1155/2022/9514697

Figure Lengend Snippet: miRNA-320a facilitated macrophage M2 polarization. qPCR analysis of classic M2 signature markers (CD163, CD206, and IL-10) and classic M1 signature markers (HLA-DR and iNOS) mRNA expression in U937 cells cultured with miRNA-320a mimics (a) or inhibitor (b). Western blot (c) and quantitative analysis (d) of CD163, CD206, and IL-10 protein expression in U937 cells cultured with miRNA-320a mimics. (e) The effect of U937/miR-320a-CM on pancreatic cancer cell proliferation by CCK-8 assay. (f, g) The effect of U937/miR-320a-CM on pancreatic cancer cells invasion was determined by the Transwell assay. Representative photographs (magnification, 100) and the number of invaded cells are displayed. ∗∗ p < 0.01. qPCR, quantitative real-time PCR; CM, conditioned medium; NFs, normal fibroblasts; CAFs, cancer-associated fibroblasts; U937/miR-320a-CM, miRNA-320a-overexpressed U937-derived CM.

Article Snippet: Human monocytic cell line U937 was purchased from Merck Millipore (Temecula, California, USA) and cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 10 nM 12-o-tetradecanoyl-phorbol-13-acetate (TPA).

Techniques: Expressing, Cell Culture, Western Blot, CCK-8 Assay, Transwell Assay, Real-time Polymerase Chain Reaction, Derivative Assay

Journal: Journal of Oncology

Article Title: Cancer-Associated Fibroblast-Derived Exosomal miRNA-320a Promotes Macrophage M2 Polarization In Vitro by Regulating PTEN/PI3K γ Signaling in Pancreatic Cancer

doi: 10.1155/2022/9514697

Figure Lengend Snippet: miRNA-320a functions by targeting PTEN/PI3K γ signaling. (a) Schematic representation of the miR-320a site in PTEN-3′UTR. (b, c) Luciferase activity was assayed in PCa cells co-transfected with miR-320a and luciferase reporters containing PTEN-3′UTR. Data are presented as the relative ratio of firefly luciferase activity to renilla luciferase activity. Western blot (d) and quantitative analysis (e) of PTEN, PI3K γ , AKT, and p-AKT protein expression in miRNA-320a-overexpressed U937 cells. Western blot (f) and quantitative analysis (g) of CD163 and CD206 protein expression in U937 cells overexpressed with miRNA-320a in the presence or absence of PI3K γ siRNA. ∗∗ p < 0.01. # vs miR-320a mimic group. qPCR, quantitative real-time PCR.

Article Snippet: Human monocytic cell line U937 was purchased from Merck Millipore (Temecula, California, USA) and cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 10 nM 12-o-tetradecanoyl-phorbol-13-acetate (TPA).

Techniques: Luciferase, Activity Assay, Transfection, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: Blood Cancer Journal

Article Title: Bispecific antibody releasing-mesenchymal stromal cell machinery for retargeting T cells towards acute myeloid leukemia blasts

doi: 10.1038/bcj.2015.73

Figure Lengend Snippet: Efficient T-cell mediated killing of target cell lines with varying CD33 expression levels is induced in the presence of bsAb-releasing hMSCs. ( a ) HEK293T, OCI-AML3, U937 and MOLM-13 were analyzed for CD33 surface expression levels by staining with anti-CD33/PE mAb (in black) or matched isotype control Ab (in gray), respectively. Numbers represent mean fluorescence intensity (MFI) of total cells. ( b ) In a standard chromium release assay 51 Cr labeled CD33 + MOLM-13 cells and CD33 - HEK293T cells were incubated with freshly isolated T cells at effector-to-target (e:t) cell ratio of 5:1 for 20 h with decreasing concentrations of the purified bsAb CD33–CD3. Mean±s.d. of two independent donors is shown. ( c ) Specific cell lysis of AML cell lines U937 (upper) and MOLM-13 (lower) measured with standard chromium release assay. Freshly isolated CD3 + T cells were co-cultured for 10 and 20 h with 51 Cr labeled CD33 + target cells at an e:t cell ratio of 5:1 in the presence of hMSC lines seeded at different concentrations 48 h before adding effector T cells and target cells. Data are presented as means±s.d. from two or three different donors, respectively. ( d ) Decreasing densities of 51 Cr labeled gene-modified hMSCs were co-cultured with PBMCs in the presence or absence of CD33 + MOLM-13 cells at an e:t ratio of 5:1. After 20 h of co-incubation the specific hMSCs lysis was examined via chromium release assay. Data shown as mean±s.d. from two independent donors.

Article Snippet: The human AML cell lines U937 (ACC 5) and MOLM-13 (ACC 554) were cultured in complete RPMI 1640 medium (Biochrom AG, Berlin, Germany).

Techniques: Expressing, Staining, Control, Fluorescence, Release Assay, Labeling, Incubation, Isolation, Purification, Lysis, Cell Culture, Modification