Journal: Journal for Immunotherapy of Cancer

Article Title: Intratumoral combination therapy with poly(I:C) and resiquimod synergistically triggers tumor-associated macrophages for effective systemic antitumoral immunity

doi: 10.1136/jitc-2021-002408

Figure Lengend Snippet: Toxicological and immunomodulatory evaluation in vitro of intracellular TLR agonists alone or combined using primary human macrophages. Macrophages were in vitro differentiated from purified monocytes stimulated with 25 ng/mL of recombinant human macrophage colony-stimulating factor for 6 days (M0); M1 and M2 polarized macrophages were obtained by stimulation with 100 ng/mL of lipopolysaccharide+50 ng/mL of interferon-γ or 20 ng/mL of IL-4, respectively, for 24 hours. (A) Cell viability (Alamar blue) of M0 macrophages exposed 24 hours to TLR agonists alone or in different combinations. concentrations used were 5, 10, 20 and 50 µg/mL, represented by color gradient from left to right. (B–D) cytokine secretion (ELISA) of (B) CXCL10, (C) CCL5, and (D) IL-10 by macrophages exposed for 24 hours to 5 µg/mL of TLR agonists or untreated M0 macrophages. In each panel, M1 and M2 polarized macrophages from the same individuals are shown as reference populations. Each dot corresponds to macrophages from each blood donor. (E) THP-1-Lucia cells for monitoring the NF-kB signal transduction pathway were exposed for 16 hours to pIC and/or R848 at indicated concentrations. Bars represent mean±SD, n=3. (F, G) Cytotoxic activity of TLR-treated macrophages toward human Panc1 cancer cells stained with CellTrace. Each dot corresponds to macrophages from each blood donor. Bars represent mean±SEM. Statistical comparison was performed using one-way analysis of variance followed by Tukey’s multiple comparison test. Statistically significant differences are represented as follows: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.001. IL, interleukin; NF-kB, nuclear factor-kappa B; ns, non-significant; pIC, poly(I:C); R837, imiquimod; R848, resiquimod; TLR, toll-like receptor.

Article Snippet: Human pancreatic carcinoma cell line PANC1 (ATCC), murine fibrosarcoma cell line MN/MCA1, and murine lung adenocarcinoma cell line CMT167 (ECACC) were cultured in DMEM (Lonza) with 10% FBS and 0.5% penicillin/streptomycin (Gibco) at 37°C and 5% CO 2 .

Techniques: In Vitro, Purification, Recombinant, Enzyme-linked Immunosorbent Assay, Transduction, Activity Assay, Staining, Comparison

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblasts promote progression and gemcitabine resistance via the SDF-1/SATB-1 pathway in pancreatic cancer

doi: 10.1038/s41419-018-1104-x

Figure Lengend Snippet: a The expression of α-SMA in tumor stroma was assayed by immunohistochemical staining, indicating that CAFs were abundant in pancreatic tumor stroma. b The morphological images of NAFs and CAFs. c Immunofluorescence staining showed the subcellular localization and the expression of α-SMA and FAP in NAFs and CAFs. Scale bar = 50 μm, magnification, ×400. d The mRNA expression levels of α-SMA, FAP, and FSP1 in NAFs and CAFs (passage 4) isolated from three patients were detected by qRT-PCR analysis. n = 3 (replicating from three patients), *** p < 0.001. e Western blot analysis shows the expression of α-SMA and FAP in NAFs and CAFs derived from four pairs of non-neoplastic pancreatic tissues and tumor tissues. f , g qRT-PCR and western blot analysis show the different expression levels of α-SMA and FAP in CAFs at different passages. Compared with the fourth-passage CAFs (CAFs-P4), the expression of α-SMA and FAP in CAFs-P8 had no significant difference, but the expression in CAFs-P12 significantly decreased. n = 3 (replicating from three patients), ns: not significantly different, * p < 0.05

Article Snippet: Human pancreatic cancer cell lines (CFPAC-1, HPAC-1, PANC-1, Capan-2, MIAPaCa-2, SW1990, and BxPC-3) were purchased from the American Type Culture Collection (ATCC, Manassas, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Immunofluorescence, Isolation, Quantitative RT-PCR, Western Blot, Derivative Assay

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblasts promote progression and gemcitabine resistance via the SDF-1/SATB-1 pathway in pancreatic cancer

doi: 10.1038/s41419-018-1104-x

Figure Lengend Snippet: a , b The qRT-PCR and western blot analysis show the mRNA and protein levels of SATB-1 in SW1990 and PANC-1 cells cultured with (co-culture) or without CAFs (monoculture). n = 3, *** p < 0.001. c The qRT-PCR analysis shows the mRNA expression of SATB-1 in 32 pancreatic tumor tissues and matched non-neoplastic pancreatic tissues. Dots represent each patient, and error bars indicate standard deviation (SD). n = 3, *** p < 0.001. d Western blot analysis shows the protein levels of SATB-1 in 12 pancreatic cancer tissues (PC) and matched non-neoplastic pancreatic tissues (NP). e , f The qRT-PCR and western blot analyses show the mRNA and protein levels of SATB-1 in various pancreatic cancer cell lines. n = 3

Article Snippet: Human pancreatic cancer cell lines (CFPAC-1, HPAC-1, PANC-1, Capan-2, MIAPaCa-2, SW1990, and BxPC-3) were purchased from the American Type Culture Collection (ATCC, Manassas, USA).

Techniques: Quantitative RT-PCR, Western Blot, Cell Culture, Co-Culture Assay, Expressing, Standard Deviation

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblasts promote progression and gemcitabine resistance via the SDF-1/SATB-1 pathway in pancreatic cancer

doi: 10.1038/s41419-018-1104-x

Figure Lengend Snippet: a Wound-healing assay shows the abilities of SW1990 and PANC-1 cells with SATB-1 silenced or co-cultured. b The transwell assay shows the fractions of migrated and invaded SW1990 and PANC-1 cells with SATB-1 silenced or co-cultured. Knockdown of SATB-1 inhibited the migration and invasion abilities of PANC-1 and SW1990 cells. Coculturing with CAFs enhanced the migration and invasion abilities of PANC-1 and SW1990 cells, but neutralization with anti-SDF-1 antibody reduced these abilities. Compared with control pancreatic cancer cells, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001. c Wound-healing and transwell assays show the upregulated migration and invasion abilities of Capan-2 and BXPC-3 cells transfected with pcDNA3.1-SATB-1. Compared with control pancreatic cancer cells, n = 3, ** p < 0.01, *** p < 0.001

Article Snippet: Human pancreatic cancer cell lines (CFPAC-1, HPAC-1, PANC-1, Capan-2, MIAPaCa-2, SW1990, and BxPC-3) were purchased from the American Type Culture Collection (ATCC, Manassas, USA).

Techniques: Wound Healing Assay, Cell Culture, Transwell Assay, Knockdown, Migration, Neutralization, Control, Transfection

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblasts promote progression and gemcitabine resistance via the SDF-1/SATB-1 pathway in pancreatic cancer

doi: 10.1038/s41419-018-1104-x

Figure Lengend Snippet: The relationship between SDF-1 expression and SATB-1 expression in pancreatic cancer tissues

Article Snippet: Human pancreatic cancer cell lines (CFPAC-1, HPAC-1, PANC-1, Capan-2, MIAPaCa-2, SW1990, and BxPC-3) were purchased from the American Type Culture Collection (ATCC, Manassas, USA).

Techniques: Expressing

Journal: Scientific Reports

Article Title: α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in human, and transgenic (Kras G12D , and Kras G12D /tp53R270H) mice

doi: 10.1038/srep32743

Figure Lengend Snippet: Pancreatic CSCs, and cancer cell lines (AsPC-1 and PANC-1) were treated with coumarin-6 containing Mang-NPs for 2 h. Cells were incubated with Hoechst 33342 for nuclear staining. The fluorescence microscope was used to observe the uptake of Mang-NPs. Green color = Mang-NP. Blue color = Nucleus.

Article Snippet: Pancreatic cancer cell lines (AsPC-1, MIA PaCa-2, and PANC-1) were purchased from ATCC.

Techniques: Incubation, Staining, Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in human, and transgenic (Kras G12D , and Kras G12D /tp53R270H) mice

doi: 10.1038/srep32743

Figure Lengend Snippet: ( A ) Pancreatic CSCs were treated with PLGA-NPs (NPs), Mangostin or Mang-NPs (0–10 μM) for 48 hrs, and cell proliferation was measured. Data represent mean (n = 4) ± SD. *, #, &, % and $ = significantly different from control (NPs group), and each other, P < 0.05. ( B ) and ( C ) AsPC-1 and PANC-1 cells were treated with NPs, Mangostin or Mang-NPs (0–10 μM) for 48 hrs, and cell proliferation was measured. Data represent mean (n = 4) ± SD. *, #, &, % and $ = significantly different from control (NPs group), and each other, P < 0.05. ( D ) Human pancreatic normal ductal epithelial (HPNE) cells were treated with NPs, Mangostin or Mang-NPs (0–10 μM) for 48 hrs, and cell proliferation was measured. Data represent mean (n = 4) ± SD. Mango-NPs = Mang-NPs

Article Snippet: Pancreatic cancer cell lines (AsPC-1, MIA PaCa-2, and PANC-1) were purchased from ATCC.

Techniques: Control

Journal: Scientific Reports

Article Title: α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in human, and transgenic (Kras G12D , and Kras G12D /tp53R270H) mice

doi: 10.1038/srep32743

Figure Lengend Snippet: ( A ) Pancreatic CSCs (Pan CSCs), and cancer cell lines (PANC-1, AsPC-1, and MIA PaCa-2) were treated with Mang-NPs (0–10 μM) for 21 days. Number of colonies were counted. Data represent mean ± SD. *, # and % = significantly different from control, and each other, P < 0.05. ( B ) Pan CSCs, and cancer cell lines (PANC-1, AsPC-1, and MIA PaCa-2) were treated with Mang-NPs (0–10 μM) for 48 hrs. Apoptosis was measured by TUNEL assay. Data represent mean ± SD. *, # and % = significantly different from control, and each other, P < 0.05.

Article Snippet: Pancreatic cancer cell lines (AsPC-1, MIA PaCa-2, and PANC-1) were purchased from ATCC.

Techniques: Control, TUNEL Assay

Journal: Scientific Reports

Article Title: α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in human, and transgenic (Kras G12D , and Kras G12D /tp53R270H) mice

doi: 10.1038/srep32743

Figure Lengend Snippet: ( A ) Human pancreatic CSCs were treated with Mang-NPs (0–10 μM) for 7 days to obtain primary spheroids. At the end of incubation period, spheroids were collected, reseeded and treated with Mang-NPs for another week to obtain secondary spheroids. Secondary spheroids were collected, reseeded and treated with Mang-NPs for another week to obtain tertiary spheroids. Cell viability in spheroids was measured by trypan blue assay at the end of 7, 14 and 21 days. Data represent mean ± SD. *, & and # = significantly different from control, P < 0.05. ( B ) Mouse pancreatic CSCs were treated with Mang-NPs (0–10 μM) for 7 days to obtain primary spheroids. At the end of incubation period, spheroids were collected, reseeded and treated with Mang-NPs for another week to obtain secondary spheroids. Secondary spheroids were collected, reseeded and treated with Mang-NPs for another week to obtain tertiary spheroids. Cell viability in spheroids was measured by trypan blue assay at the end of 7, 14 and 21 days. Data represent mean ± SD. *, & and # = significantly different from control, P < 0.05.

Article Snippet: Pancreatic cancer cell lines (AsPC-1, MIA PaCa-2, and PANC-1) were purchased from ATCC.

Techniques: Incubation, Control

Journal: Scientific Reports

Article Title: α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in human, and transgenic (Kras G12D , and Kras G12D /tp53R270H) mice

doi: 10.1038/srep32743

Figure Lengend Snippet: ( A ) Pancreatic CSCs were treated with Mang-NPs (0–10 μM) for 48 h. The expression of Nanog, and c-Myc was measured by the Western blot analysis. β-actin was used as a loading control. ( B ) Pancreatic CSCs were treated with Mang-NPs (0–10 μM) for 36 h. The expression of c-Myc, Oct-4 and Nanog was measured by q-RT-PCR. Data represent mean ± SD. * and # = significantly different from control, P < 0.05. ( C ) Nanog shRNA enhances the inhibitory effects of Mang-NPs on colony formation. Pan CSCs/Scrambled and CSCs/Nanog shRNA were seeded and treated with Mang-NPs (0–10 μM) for 21 days. At the end of incubation period, number of colonies were counted. Data represent mean ± SD. *, #, %, and @ = significantly different from control, P < 0.05. ( D ) Pancreatic CSCs were treated with Mang-NPs (0–10 μM) for 48 h. The expression of Gli1, Gli2, Patched-1, and Patched-2 was measured by the Western blot analysis. β-actin was used as a loading control. ( E ), Pancreatic CSCs were treated with Mang-NPs (0–10 μM) for 36 h. The expression of Bcl-2 was measured by q-RT-PCR. Data represent mean ± SD. * = significantly different from control, P < 0.05.

Article Snippet: Pancreatic cancer cell lines (AsPC-1, MIA PaCa-2, and PANC-1) were purchased from ATCC.

Techniques: Expressing, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, shRNA, Incubation

Journal: Scientific Reports

Article Title: α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in human, and transgenic (Kras G12D , and Kras G12D /tp53R270H) mice

doi: 10.1038/srep32743

Figure Lengend Snippet: ( A – D ) Pancreatic CSCs, and cancer cell lines (PANC-1, AsPC-1, and MIA PaCa-2) were stably transduced with Gli-responsive GFP/firefly luciferase viral particles (pGreen Fire1-Gli with EF1, System Biosciences). Transduced CSCs and cell lines were treated with Mang-NPs (0–10 μM) for 24 h. Gli reporter activity was measured as we described . Data represent mean ± SD. *, # and % = significantly different from control, P < 0.05.

Article Snippet: Pancreatic cancer cell lines (AsPC-1, MIA PaCa-2, and PANC-1) were purchased from ATCC.

Techniques: Stable Transfection, Transduction, Luciferase, Activity Assay, Control

Journal: Scientific Reports

Article Title: α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in human, and transgenic (Kras G12D , and Kras G12D /tp53R270H) mice

doi: 10.1038/srep32743

Figure Lengend Snippet: ( A ) Inhibition of pancreatic cancer growth and development in KPC mice. KPC mice were treated with Mang-NPs (20 mg/kg) for about 10 weeks. At the end of experiment, mice were sacrificed and pancreas weight was recorded. Data represent mean ± SD. * and # = significantly different from untreated control group, P < 0.05. ( B ) Pancreatic tissues from control and Mang-NPs-treated mice were fixed and stained with H & E. Tissue sections were visualized for the presence of PanIN (PanIN-1A, PanIN-1B, PanIN-2, and PanIN-3) lesions and PDAC. nd = not detected. ( C ) Liver metastasis. Liver tissues from control and Mang-NPs-treated mice were visualized for the presence of nodules. ( D ) Expression of stem cell marker, and pluripotency maintain factors in tumor tissues. Pancreatic cancer tissues from control and Mang-NPs treated mice were subjected to the Western blot analysis, and the expression of CD24, CD133, c-Myc, Nanog and Oct4 was measured. β-actin was used as a loading control. ( E ) Expression of components of Shh pathway in tumor tissues. Pancreatic cancer tissues from control and Mang-NPs treated mice were subjected to the Western blot analysis, and the expression of Gli1, Gli2, Patched1, Patched2, and Smoothened was measured. ( F ) Expression of Bcl2, XIAP and Cyclin D1 in tumor tissues. Pancreatic cancer tissues from control and Mang-NPs treated mice were subjected to the Western blot analysis, and the expression of Bcl2, XIAP and Cyclin D1 was measured. ( G ) Expression of E-cadherin, N-Cadherin, Slug, Snail, and Zeb1 in tumor tissues. Pancreatic cancer tissues from control and Mang-NPs treated mice were subjected to the Western blot analysis, and the expression of E-cadherin, N-Cadherin, Slug, Snail, and Zeb1 was measured.

Article Snippet: Pancreatic cancer cell lines (AsPC-1, MIA PaCa-2, and PANC-1) were purchased from ATCC.

Techniques: Inhibition, Control, Staining, Expressing, Marker, Western Blot