Journal: bioRxiv

Article Title: TMEM Doorway Mediated Metastasis in Pancreatic Ductal Adenocarcinoma by Tie2 Signaling

doi: 10.64898/2025.12.05.692608

Figure Lengend Snippet: (A) Immunofluorescence imaging of Tie2Hi/VEGFAHi macrophages in TMEM doorways in sequential sections. Endothelial cells (EC), macrophage (M) and tumor tissue (TC). In the merge panel, a mask for IBA1 macrophage was created in ImageJ ® for a better visualization of Tie2. Scale bar,10μm. (B and C) Quantification of Tie2+ macrophages in tumor area and withim TMEM doorway area of KPCY T-low (B) and KPC (C) tumors. -total macrophages. -Tie2 positive macrophages. (D) Western blot analysis of phospho-Tie2 immunoprecipitated from extracts of KPC, KPCY T-low, RAW 264.7 or 3B-11 cells treated with Rebastinib +/- Angiopoietin 2. (E) Experimental design of the subluminal to luminal trans-endothelial migration (iTEM) assay. Macrophages = red. Tumor cells = green. The tumor cells (green) are considered transendothelial migrating cells when they are crossing the endothelial layer (pink). (F) Representative image of iTEM assay from confocal microscope. Macrophages = red. Tumor cells = green. Endothelial layer = pink. Quantitation of trans-endothelial migration of KPCY T-low (G) and KPC (H). In KPCY T-low and KPC, we observed an increase of transendothelial migration from 3 cells/field to 12.65 cells/field and 6 cells/field to 16.65 cells/field respectively, in the presence of macrophages (p<0.05), following by a decrease to 8.46 cells/field and 8.46 cells/field after rebastinib treatment (p<0.05). Co-culture with macrophages significantly increased migration, which was inhibited by the Tie2 inhibitor rebastinib. Data are presented as mean ± SEM; (n ≥ 10 per group). *P < 0.05, by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: KPC ( Cat.#: 153600: Cancer tools.org ) , KPCY T-low (6419c5) , or KPCY C-EMT 3077 (provided by Ben Z. Stanger) , murine macrophage cell line RAW264.7 (ATCC-provided by Dianne Cox), and murine endothelial cells 3B11 (ATCC - provided by Camille Duran) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (cat# SH30243, Hyclone, GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (cat# S11550, Atlanta Biologicals, Flowery Branch, GA, USA).

Techniques: Immunofluorescence, Imaging, Western Blot, Immunoprecipitation, Migration, Microscopy, Quantitation Assay, Co-Culture Assay

Journal: bioRxiv

Article Title: TMEM Doorway Mediated Metastasis in Pancreatic Ductal Adenocarcinoma by Tie2 Signaling

doi: 10.64898/2025.12.05.692608

Figure Lengend Snippet: (A) panel 1 shows TMEM doorways, visualized by immunohistochemistry (IHC) staining for Mena, Iba-1, and endomucin. The three cells of the TMEM doorway (contained in black circle, with the three cells forming the TMEM doorway indicated with the yellow triangle) are the TMEM doorway endothelial cell (TEC, endomucin stained in blue), TMEM doorway macrophage (TM, Iba1 stained in brown), and TMEM doorway tumor cell (TTC, Mena stained in pink). Panels 2-4 show different staining channels of the sequential tissue section 2 aligned with the IHC stained section (panel 1) with immunofluorescence (IF) with antibodies against endomucin (panel 3 yellow), dextran (panel 4 pink) and nuclear stain DAPI (blue). Active versus inactive TMEM doorways were distinguished by the presence of extravascular dextran staining (panel), which indicates that the vessel had a TMEM doorway-associated vascular opening (TAVO). Scale bars=20μm. (B-H) Mice were treated with control chow or ∼0.44 mg/day of rebastinib for 3 weeks. (B and D) Quantification of TMEM doorway density in 10 high-power fields (HPFs; no significant difference) in KPCY T-low (B) and KPC tumors (D). (C and E) Immunofluorescence measurement of extravascular dextran levels in Tie2 + TMEM doorways indicate significant inhibition of TMEM doorways by rebastinib inKPCY T – low tumors (C) and KPC tumors (E). TMEM doorways were identified in the IF-stained sections as described above. Active TMEM doorways were identified by the presence of extravascular dextran which is not present in inactive TMEM doorways. *p<0.05. (F) Disseminated tumor cells in the liver stained with p53 (red). Increased magnification of field of the p53 positive cells is shown in the yellow box. The left bottom is a representative image of control and right bottom of rebastinib treated group in magnified fields. (G-H) Quantification of DTCs in the liver of animals as compared to the control with rebastinib treated groups showing significant inhibition of DTC levels. (G) KPCY T-low (H) KPC where each dot is a separate mouse. *p<0.05, analyzed by Student’s t-test. N=8.

Article Snippet: KPC ( Cat.#: 153600: Cancer tools.org ) , KPCY T-low (6419c5) , or KPCY C-EMT 3077 (provided by Ben Z. Stanger) , murine macrophage cell line RAW264.7 (ATCC-provided by Dianne Cox), and murine endothelial cells 3B11 (ATCC - provided by Camille Duran) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (cat# SH30243, Hyclone, GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (cat# S11550, Atlanta Biologicals, Flowery Branch, GA, USA).

Techniques: Immunohistochemistry, Staining, Immunofluorescence, Control, Inhibition

Journal: Signal Transduction and Targeted Therapy

Article Title: Pancreatic cancer cells render tumor-associated macrophages metabolically reprogrammed by a GARP and DNA methylation-mediated mechanism

doi: 10.1038/s41392-021-00769-z

Figure Lengend Snippet: PDA cells reprogram macrophages in TME through DNA methylation. a Tumor and macrophage co-culture experimental schema. b Nqo-1 and Aldh1a3 methylation was examined by methylation-specific PCR (MSP) in mouse BMDMs after co-culturing with KPC PDA cells. * P < 0.05 (paired t test). c The schema of the candidate gene selection process. d Expression of the key genes in glucose metabolism and OXPHOS pathway in mouse BMDMs after co-culturing with KPC cells. The mRNA expression of these genes was measured by RT-PCR and β-actin was used for normalization. e , f Nqo - 1 and Aldh1a3 methylation after pretreating BMDMs with DAC. * P < 0.05 ( d , e , f , Mann–Whitney U test) . g Methylation of Nqo - 1 and Aldh1a3 in TAMs, CD4 + and CD8 + T cells from primary PDA and BMDMs (consider as M0 macrophages) of the same KPC mice, and BMDMs after co-culturing with KPC cells. * P < 0.05 (ANOVA). Data are means ± SEM from technical duplicates and representative of two experiments

Article Snippet: The KPC PDA tumor cell line was developed from a KPC mouse as described previously., Human pancreatic cancer cell line Panc10.05 cell line was established in 1998 in accordance with the Johns Hopkins Medical Institution Institutional Review Board (JHMI IRB)-approved protocols and authenticated by DNA and gene expression profiling as previously described.

Techniques: DNA Methylation Assay, Co-Culture Assay, Methylation, Selection, Expressing, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: Signal Transduction and Targeted Therapy

Article Title: Pancreatic cancer cells render tumor-associated macrophages metabolically reprogrammed by a GARP and DNA methylation-mediated mechanism

doi: 10.1038/s41392-021-00769-z

Figure Lengend Snippet: DNA methylation of the metabolism genes in macrophages is induced by direct interaction with PDA cells through GARP/TGF-βRII-integrin αV/β8. a , b Nqo-1 and Aldh1a3 methylation in mouse BMDMs in a transwell system separated from KPC cells by an 8-μm or 1-μm pore membrane that, respectively, allows or not allows tumor cells to migrate through and direct contract with macrophages and in BMDMs cultured with TCM. Nqo-1 and Aldh1a3 methylation quantified as described in Supplementary Methods. * P < 0.05 (Mann–Whitney U test). c Lucifer Yellow labeled-KPC cells were co-cultured with unlabeled BMDMs. Thick arrows indicate macrophages that contain Lucifer Yellow spread from KPC cells (thin arrow) around them. Scale bar: 20 μm. d GARP expression on M0, M1-like, and M2-like macrophages measured by immunofluorescent staining with FITC-conjugated anti-GARP antibody. Arrow indicates macrophages that have the highest fluorescence within each image. Scale bar: 20 μm. * P < 0.05 (ANOVA). Histogram (right panel) shows quantification of fluorescence intensity. e Multiplex immunohistochemistry (IHC) was performed on a single slide of human PDA tissues for GARP (in green), CD68 (in red) and CD163 (in purple). A representative among 20 human PDAs tested is shown. Arrows (both panels) indicate GARP-expressing CD68 + CD163 + (M2-like) macrophages ; and arrowheads (left panel) indicate GARP-expressing CD68 + CD163 - (M1-like) macrophages . Notched arrowheads (right panel) indicate CD68 + CD163 + (M2-like) macrophages with little GARP expression. Scale bar: 50 μm. f Multiplex IHC staining of GARP (in green) on F4/80 + (in red) macrophages in PDAs from KPC mice. Scale bar: 50 μm. g TGF-βRII and GARP on cell surface of M0, M1-like, and M2-like macrophages co-stained and analyzed by flow cytometry. h Quantification of the percentages of TGF-βRII on cell surface of M0, M1-like, and M2-like macrophages by flow cytometry. * P < 0.05 (ANOVA). i Integrin subunits ɑV and β8 cell-surface expression was measured by flow cytometry. j IHC staining of PDA and normal pancreas tissues from KPC mice with anti-integrin ɑV and β8 antibodies. Scale bar: 100 μm. Data were from technical triplicates and representative of two experiments

Article Snippet: The KPC PDA tumor cell line was developed from a KPC mouse as described previously., Human pancreatic cancer cell line Panc10.05 cell line was established in 1998 in accordance with the Johns Hopkins Medical Institution Institutional Review Board (JHMI IRB)-approved protocols and authenticated by DNA and gene expression profiling as previously described.

Techniques: DNA Methylation Assay, Methylation, Membrane, Cell Culture, MANN-WHITNEY, Labeling, Expressing, Staining, Fluorescence, Multiplex Assay, Immunohistochemistry, Flow Cytometry

Journal: Signal Transduction and Targeted Therapy

Article Title: Pancreatic cancer cells render tumor-associated macrophages metabolically reprogrammed by a GARP and DNA methylation-mediated mechanism

doi: 10.1038/s41392-021-00769-z

Figure Lengend Snippet: GARP mediates Nqo-1 methylation and M2-like phenotypical changes in M1-like macrophages after co-culturing with PDA cells. a Nqo-1 methylation in mouse WT M1-like macrophages compared to GARP KO M1-like macrophages. * P < 0.05 (paired t test). b RT-PCR of M2 marker genes in WT vs. GARP KO M1-like macrophages after co-cultured with KPC cells. Fold changes of these marker genes in co-cultured vs. monocultured M1-like macrophages were shown. Fold change >1: upregulation; fold change <1: downregulation. All results were first normalized by respective β-actin and then respective monocultured BMDMs. * P < 0.05 (Mann–Whitney U test). c Expression of the M2 cytokine IL-10 in WT vs. GARP KO M1-like macrophages after co-culturing with KPC cells, measured by flow cytometry analysis of percentages of IL-10-positive cells with intracellular staining of IL-10. * P < 0.05 (Mann–Whitney U test). d Fold changes of MSP results of the Nqo-1 gene in co - cultured vs. monocultured M1 - like macrophages treated with RGD or TGF-βRII blocking antibody. * P < 0.05 (ANOVA). e Fold changes of RT-PCR results of M2 marker genes in co-cultur e d vs. monocultured M1-like macrophages treated with RGD or TGF-βRII blocking antibody. Data were first normalized by respective β-actin and then respective monocultured M0 macrophages. * P < 0.05 (ANOVA). f Mitochondrial membrane potentials in mouse M0, M1-like and M2-like macrophages after co-culturing with KPC cells by measuring mean fluorescence intensity of TMRM signals on the PE channel of flow cytometry, comparing mono- vs. co-cultured macrophages. * P < 0.05 (ANOVA). g Glucose uptake activities in M0, M1-like, and M2-like macrophages by measuring mean fluorescence intensity of 2-NBDG signals, comparing mono- vs. co-cultured macrophages. * P < 0.05 (ANOVA). h KPC cells were co-cultured with mouse BMDMs or DAC pretreated BMDMs in upper chamber of a transwell system with 8-μm pore membrane that allows them migrating to the lower chamber. Migrated KPC cells were examined by immunofluorescent staining with FITC-conjugated anti-Pan-CK antibody and counted. Fold changes of migrated KPC cell number in co-cultured vs. monocultured group (normalized as 1) were shown. * P < 0.05 (Mann–Whitney U test). i KPC cells were co-cultured with BMDMs pretreated with DAC, glucose uptake inhibitor WZB-117, or DAC + WZB-117, respectively, in the transwell system. Numbers of migrated KPC cells were counted as described in ( h ) and shown. * P < 0.05 (ANOVA). j Il-10 expression per RT-PCR in untreated, DAC, or WZB-117 pretreated BMDMs before (normalized as 1) and after co-culturing with KPC cells. * P < 0.05 (Mann–Whitney U test). Data are means ± SEM from technical duplicates and representative of two experiments

Article Snippet: The KPC PDA tumor cell line was developed from a KPC mouse as described previously., Human pancreatic cancer cell line Panc10.05 cell line was established in 1998 in accordance with the Johns Hopkins Medical Institution Institutional Review Board (JHMI IRB)-approved protocols and authenticated by DNA and gene expression profiling as previously described.

Techniques: Methylation, Reverse Transcription Polymerase Chain Reaction, Marker, Cell Culture, MANN-WHITNEY, Expressing, Flow Cytometry, Staining, Blocking Assay, Membrane, Fluorescence

Journal: Signal Transduction and Targeted Therapy

Article Title: Pancreatic cancer cells render tumor-associated macrophages metabolically reprogrammed by a GARP and DNA methylation-mediated mechanism

doi: 10.1038/s41392-021-00769-z

Figure Lengend Snippet: Downregulation of genes in the metabolic pathway in TAMs from murine PDA. a mRNA expression of metabolism genes as indicated were measured by RT-PCR in TAMs, CD4 + , and CD8 + T cells from primary pancreatic tumors and BMDMs of the same KPC mice. Tumors were identified by ultrasound before sacrifice. β-actin used for normalization. Data are means ± SEM from triplicates and representative of two experiments. * P < 0.05 (ANOVA). b The schematic model of the GARP/integrin-mediated interaction between tumor cells and macrophages in the TME of PDAC and the mechanisms of metabolic, phenotypical, and functional reprogramming of macrophages from M1-like to M2-like macrophages in a DNA methylation-dependent manner

Article Snippet: The KPC PDA tumor cell line was developed from a KPC mouse as described previously., Human pancreatic cancer cell line Panc10.05 cell line was established in 1998 in accordance with the Johns Hopkins Medical Institution Institutional Review Board (JHMI IRB)-approved protocols and authenticated by DNA and gene expression profiling as previously described.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Functional Assay, DNA Methylation Assay

Journal: JCI Insight

Article Title: Schlafen 5 is an intracellular immune checkpoint and controls IFN responses in pancreatic ductal adenocarcinoma

doi: 10.1172/jci.insight.190031

Figure Lengend Snippet: ( A – C ) RT-qPCR analysis to monitor ( A ) efficacy of CRISPR/Cas9-mediated Slfn5 disruption, ( B ) expression of Slfn5 in response to IFN-β treatment (5,000 IU for 6 hours) in Slfn5 WT cells, and ( C ) IFN-β–mediated (5,000 IU for 6 hours) induction of indicated murine ISGs in Slfn5 -WT and Slfn5 -KO cells. The expression levels of the indicated genes were determined using Gapdh for normalization and as an internal control. The data are expressed as fold change over the corresponding controls, and the graphs represent mean ± SEM of 3 independent experiments. * P < 0.05, *** P < 0.001 by 2-tailed unpaired t test with Welch’s correction ( A and B ) or 1-tailed unpaired t test with Mann-Whitney test ( C ). ( D ) Slfn5 -WT and Slfn5 -KO KPC1199 cells were plated in 6-well plates and counted on days 1, 2, and 3 after seeding. Data are mean of number of cells ± SEM of 3 independent experiments, each done in duplicate. Two-way repeated-measures ANOVA with Šidák’s multiple-comparison test; ** P < 0.01, **** P < 0.0001. ( E ) Slfn5 -WT and Slfn5 -KO KPC1199 cells were plated into round-bottom 96-well plates under stem cell–permissive conditions to form 3D spheroids. After 7 days, spheres were imaged using a Cytation 3 cell imaging multi-mode reader to determine cross-sectional area. Data are expressed as percentages of WT parental spheres and represent mean ± SEM of 3 independent experiments, each done in triplicate. Two-tailed unpaired t test with Welch’s correction; * P ≤ 0.05.

Article Snippet: Murine KPC1199 cells were a gift from David Tuveson (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA) and propagated in DMEM supplemented with 10% FBS and antibiotics.

Techniques: Quantitative RT-PCR, CRISPR, Disruption, Expressing, Control, MANN-WHITNEY, Comparison, Imaging, Two Tailed Test

Journal: JCI Insight

Article Title: Schlafen 5 is an intracellular immune checkpoint and controls IFN responses in pancreatic ductal adenocarcinoma

doi: 10.1172/jci.insight.190031

Figure Lengend Snippet: ( A ) KPC1199 luciferase-expressing Slfn5 -WT or Slfn5 -KO cells (5 × 10 4 cells/mouse) were injected into the pancreatic tails of male and female C57BL/6J mice. Mice were randomized into the following treatment groups: Slfn5 WT PBS ( n = 7), Slfn5 WT IFN-α ( n = 7), Slfn5 KO PBS ( n = 7), and Slfn5 KO IFN-α ( n = 8). Where indicated, mice were injected subcutaneously with 600 ng murine IFN-α per mouse once per week for 2 weeks. Tumor growth was monitored at least once per week by bioluminescence imaging (BLI). Normalized BLI values are shown. On day 17, model-based estimate of the mean difference in BLI signal between WT IFN-α and Slfn5 -KO IFN-α was 3.91 × 10 9 photons/s/cm 2 /sr (95% CI 1.72 × 10 9 to 6.09 × 10 9 ). Two-way ANOVA with Šidák’s multiple-comparison test for day 17. Comparison of BLI signals on day 17 is based on mixed-effects model up to day 17. Data are expressed as mean ± SEM of tumor BLI signals for each genotypic treatment group; **** P < 0.0001. ( B ) Survival curves of mice bearing Slfn5 -WT and Slfn5 -KO pancreatic tumors. Survival was estimated using the method of Kaplan-Meier and groups were compared using the log-rank test; ** P < 0.01; *** P < 0.001; **** P < 0.0001. ( C ) Median survival time (days) for indicated genotypic treatment groups was estimated using Simple Survival Analysis (Kaplan-Meier) in GraphPad.

Article Snippet: Murine KPC1199 cells were a gift from David Tuveson (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA) and propagated in DMEM supplemented with 10% FBS and antibiotics.

Techniques: Luciferase, Expressing, Injection, Imaging, Comparison

Journal: JCI Insight

Article Title: Schlafen 5 is an intracellular immune checkpoint and controls IFN responses in pancreatic ductal adenocarcinoma

doi: 10.1172/jci.insight.190031

Figure Lengend Snippet: KPC1199 luciferase-expressing CTRL or Slfn5 -KO cells (5 × 10 4 cells/mouse) were injected into the pancreatic tails of 6- to 8-week-old male and female mice that were grouped as Rag1 WT + CTRL ( n = 9), Rag1 WT + Slfn5 KO ( n = 11), Rag1 KO + CTRL ( n = 11), and Rag1 KO + Slfn5 KO ( n = 11). In the Rag1 WT + Slfn5 KO group, 5 mice were still alive on day 60. Survival curves of indicated mice are shown. Survival was estimated using the method of Kaplan-Meier and groups were compared using the log-rank test; *** P < 0.001; **** P < 0.0001.

Article Snippet: Murine KPC1199 cells were a gift from David Tuveson (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA) and propagated in DMEM supplemented with 10% FBS and antibiotics.

Techniques: Luciferase, Expressing, Injection

Journal: JCI Insight

Article Title: Schlafen 5 is an intracellular immune checkpoint and controls IFN responses in pancreatic ductal adenocarcinoma

doi: 10.1172/jci.insight.190031

Figure Lengend Snippet: ( A – J ) Immunophenotypic analysis of tumor-bearing pancreases by multicolor flow cytometry. ( A , C , E , G , and I ) CTRL ( n = 10) and Slfn5 -KO ( n = 9) KPC1199 luciferase-expressing cells (5 × 10 4 cells/mouse) were injected into the pancreatic tails of C57BL/6J mice and 7 days after cell implantation CTRL and Slfn5 -KO tumor–bearing pancreases were harvested and processed for immunophenotypic analysis by multicolor flow cytometry. ( B , D , F , H , and J ) CTRL ( n = 10) and Slfn5 -KO ( n = 11) KPC1199 luciferase-expressing cells (5 × 10 4 cells/mouse) were injected into the pancreatic tails of C57BL/6J mice and 21 days after cell implantation CTRL and Slfn5 -KO tumor–bearing pancreases were harvested and processed for immunophenotypic analysis by multicolor flow cytometry. Scatter dot plots show the percentage of tumor-infiltrating cells. ( A and B ) Immunosuppressive cells, i.e., TAMs (left panel), Tregs (middle panel), and TAMCs (right panel). ( C and D ) Innate myeloid immune cells, i.e., M1 macrophages (left panel) and M2 macrophages (right panel). ( E and F ) Effector cells, i.e., NK cells (left panel), CD8 + T cells (middle panel), and CD4 + T cells (right panel). ( G and H ) Activation markers (IFN-γ, GZMB, CD69) of NK cells, and ( I and J ) of CD8 + cells. Data are expressed as mean ± SEM of percentages of indicated immune infiltrates as detailed in . Two-tailed unpaired t test with Mann-Whitney test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Murine KPC1199 cells were a gift from David Tuveson (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA) and propagated in DMEM supplemented with 10% FBS and antibiotics.

Techniques: Flow Cytometry, Luciferase, Expressing, Injection, Activation Assay, Two Tailed Test, MANN-WHITNEY