Journal: Science Advances

Article Title: A stromal PAI1-tPA axis orchestrates immunosuppression in pancreatic cancer

doi: 10.1126/sciadv.aea6734

Figure Lengend Snippet: ( A to I ) Serpine1 +/+ and Serpine1 −/− mice were orthotopically implanted with pancreatic cancer cells (7940B), and tumors were collected at day 21. [(A) and (B)] Flow cytometry quantification of frequencies of total T cells (CD45 + CD3 + ) (A) and conventional dendritic cells (CD45 + F4/80 − CD11c + MHCII + ) (B) among live cells in orthotopic tumors. (C) Co-IF staining for Arg1 (green), F4/80 (red), and DAPI (blue) in orthotopic tumors, and corresponding quantification ( n = 3 FOV per animal). (D) Quantitative RT-PCR analysis of Arg1 in BMDMs treated with vehicle or recombinant PAI1 (rPAI1) for 20 hours. Each dot represents individual primary BMDM lines. (E) Flow cytometry quantification of frequency of CD8 + T cells (CD45 + CD3 + CD8 + ) among live cells in orthotopic tumors. [(F) and (G)] Flow cytometry quantification of frequencies and absolute numbers of GZMB + CD8 + T cells (F) and Ki67 + CD8 + T cells (G) following a 5-hour ex vivo PMA/ionomycin stimulation of tumor digests. (H) Co-IF staining for GZMB (green), CD8 (red), and DAPI (blue) in orthotopic tumors, and corresponding quantification ( n = 5 FOV per animal). (I) Co-IF staining for Ki67 (red), CD8 (green), and DAPI (blue) in orthotopic tumors, and corresponding quantification ( n = 5 FOV per animal). ( J ) Experimental design for CD8 + T cell depletion. Mice received control immunoglobulin G (IgG) or anti-CD8 antibody twice per week, starting 5 days postimplantation (7940B). Tumors were harvested at day 21. ( K ) Tumor weight of orthotopic tumors with CD8⁺ T cell antibody-mediated depletion. Symbols in (A) to (I) and (K) represent individual mice. Data are means ± SEM. P values were determined by two-tailed unpaired t test [(A), (B), right of (C), (D), (E) to (G), and right of (H)], Mann-Whitney test [left of (C), left of (H), and (I)], and Mann-Whitney test with Holm-Sidak post hoc (K).

Article Snippet: The following antibodies were used: Arg1 (D4E3M, 1:400, no. 93668S, Cell Signaling), cleaved caspase-3 (polyclonal, 1:100, no. 9661S, Cell Signaling), CD8 (D4W2Z, 1:200, no. 98941S, Cell Signaling), ECAD (36, 1:1000, no. 610181, BD Biosciences), F4/80 (D2S9R, 1:200, no. 70076S, Cell Signaling), GZMB (D6E9W, 1:100, no. 46890S, Cell Signaling), Ki67 (SP6, 1:200, no. MA5-14520, Invitrogen), PAI1 (polyclonal, 1:1000, no. IRBAHUPAI1AP100UG, Innovative Research), PanCK (monoclonal antibody cocktail, 1:400, no. NBP2-44368-0.1 mg, Novus), PDGFRα/β (Y92, 1:100, no. AB32570, Abcam), and tPA (polyclonal, 1:250, no. 10147-1-AP, Proteintech).

Techniques: Flow Cytometry, Staining, Quantitative RT-PCR, Recombinant, Ex Vivo, Control, Two Tailed Test, MANN-WHITNEY

Journal: Science Advances

Article Title: A stromal PAI1-tPA axis orchestrates immunosuppression in pancreatic cancer

doi: 10.1126/sciadv.aea6734

Figure Lengend Snippet: ( A ) Experimental outline of murine PDAC model. Plat +/+ and Plat −/− mice were orthotopically implanted with pancreatic cancer cells (7940B), and tumors were collected 21 days postimplantation. ( B ) Tumor weight of orthotopic tumors harvested from Plat +/+ and Plat −/− mice. ( C ) ELISA of tPA in tissue supernatants from pancreata of Plat +/+ mice (no tumor) and orthotopic tumors from Plat +/+ and Plat −/− mice. ( D ) Picrosirius red staining of orthotopic tumors, and corresponding quantification ( n = 5 FOV per animal). ( E ) Co-IF staining for cCasp3 (red), ECAD (green), and DAPI (blue) in orthotopic tumors, and corresponding quantification ( n = 4 FOV per animal). ( F to H ) Flow cytometry quantification of frequencies of total T cells (F), CD8 + T cells (G), and conventional dendritic cells (H) among live cells in orthotopic tumors. ( I ) Co-IF staining for Arg1 (green), F4/80 (red), and DAPI (blue) in orthotopic tumors, and corresponding quantification ( n = 3 FOV per animal). Symbols in (B) to (I) represent individual mice. Data are means ± SEM. P values were determined by Mann-Whitney test [(B) and (F)], two-way ANOVA with Holm-Sidak post hoc (C), and two-tailed unpaired t test [(D), (E), and (G) to (I)].

Article Snippet: The following antibodies were used: Arg1 (D4E3M, 1:400, no. 93668S, Cell Signaling), cleaved caspase-3 (polyclonal, 1:100, no. 9661S, Cell Signaling), CD8 (D4W2Z, 1:200, no. 98941S, Cell Signaling), ECAD (36, 1:1000, no. 610181, BD Biosciences), F4/80 (D2S9R, 1:200, no. 70076S, Cell Signaling), GZMB (D6E9W, 1:100, no. 46890S, Cell Signaling), Ki67 (SP6, 1:200, no. MA5-14520, Invitrogen), PAI1 (polyclonal, 1:1000, no. IRBAHUPAI1AP100UG, Innovative Research), PanCK (monoclonal antibody cocktail, 1:400, no. NBP2-44368-0.1 mg, Novus), PDGFRα/β (Y92, 1:100, no. AB32570, Abcam), and tPA (polyclonal, 1:250, no. 10147-1-AP, Proteintech).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, MANN-WHITNEY, Two Tailed Test

Journal: Science Advances

Article Title: A stromal PAI1-tPA axis orchestrates immunosuppression in pancreatic cancer

doi: 10.1126/sciadv.aea6734

Figure Lengend Snippet: ( A ) Experimental outline of murine PDAC model. WT, Serpine1 −/− , and Serpine1 −/− ; Plat −/− mice were orthotopically implanted with pancreatic cancer cells (7940B), and tumors were collected 21 days postimplantation. ( B ) Tumor weight of orthotopic tumors harvested from WT, Serpine1 −/− , and Serpine1 −/− ; Plat −/− mice. ( C ) IHC staining for CD8 in orthotopic tumors, and corresponding quantification ( n = 3 FOV per animal). ( D ) Co-IF staining for Arg1 (green), F4/80 (red), and DAPI (blue) in orthotopic tumors, and corresponding quantification ( n = 3 FOV per animal). Symbols in (B) to (D) represent individual mice. Data are means ± SEM. P values were determined by one-way ANOVA with Holm-Sidak post hoc [(B) to (D)]. ( E ) Working model. Left: Hypoxia stabilizes HIF proteins, inducing PAI1 expression in pancreatic CAFs. Although tPA levels are elevated in PDAC, PAI1 predominantly inhibits its activity, thereby suppressing antitumor immunity. Middle: Elimination of stromal PAI1 restores tPA activity, enhancing antitumor CD8 + T cell responses, accompanied by alleviation of immunosuppressive TAM phenotypes and increased dendritic cell (DC) infiltration. Stromal PAI1-driven tumor growth depends on CD8 + T cells. Right: Removal of stromal tPA abolishes residual tPA activity, further promoting an immunosuppressive TME and tumor growth.

Article Snippet: The following antibodies were used: Arg1 (D4E3M, 1:400, no. 93668S, Cell Signaling), cleaved caspase-3 (polyclonal, 1:100, no. 9661S, Cell Signaling), CD8 (D4W2Z, 1:200, no. 98941S, Cell Signaling), ECAD (36, 1:1000, no. 610181, BD Biosciences), F4/80 (D2S9R, 1:200, no. 70076S, Cell Signaling), GZMB (D6E9W, 1:100, no. 46890S, Cell Signaling), Ki67 (SP6, 1:200, no. MA5-14520, Invitrogen), PAI1 (polyclonal, 1:1000, no. IRBAHUPAI1AP100UG, Innovative Research), PanCK (monoclonal antibody cocktail, 1:400, no. NBP2-44368-0.1 mg, Novus), PDGFRα/β (Y92, 1:100, no. AB32570, Abcam), and tPA (polyclonal, 1:250, no. 10147-1-AP, Proteintech).

Techniques: Immunohistochemistry, Staining, Expressing, Activity Assay

Journal: Toxicological Sciences

Article Title: Ovarian hormone deficiency enhances wood smoke-induced immune dysfunction via transcriptomic and metabolic alterations

doi: 10.1093/toxsci/kfag023

Figure Lengend Snippet: Protein level validation confirms downregulation of ARG1 and PPARγ in OVX-WS BMDMs. A) Representative Western blot images for ARG1, PPARγ, and Vinculin (loading control) in bone marrow-derived macrophage (BMDM) lysates from Sham and ovariectomized (OVX) mice exposed to filtered air (FA) or wood smoke (WS) ( n = 3 biological replicates shown). B) Quantification of relative ARG1 protein levels normalized to Vinculin. C) Quantification of relative PPARγ protein levels normalized to Vinculin. Data in B and C are presented as scatter plots showing individual biological replicates ( n = 3), with bars representing mean ± SD. Statistical significance was determined by 2-way ANOVA followed by Tukey’s multiple comparisons test (* P < 0.05, ** P < 0.01 compared with OVX-FA, mark specific comparisons based on post-hoc results). (D) Summary table of 2-way ANOVA results showing the P values for the main effects of Surgery, Treatment, and their Interaction on ARG1 and PPARγ protein levels. The significant interaction effects confirm the specific downregulation in the OVX-WS group.

Article Snippet: Membranes were blocked with 5% BSA in TBST for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies against ARG1 (#93668, Cell Signaling Technology, 1:1,000), PPARγ (#2443, Cell Signaling Technology, 1:1,000), or Vinculin (#13901, Cell Signaling Technology, 1:1,000) diluted in 5% BSA/TBST.

Techniques: Biomarker Discovery, Western Blot, Control, Derivative Assay

Journal: Toxicological Sciences

Article Title: Ovarian hormone deficiency enhances wood smoke-induced immune dysfunction via transcriptomic and metabolic alterations

doi: 10.1093/toxsci/kfag023

Figure Lengend Snippet: Ovarian hormone deficiency and WS exposure modulate M2-associated gene expression in BMDMs. A to G) Relative mRNA expression of M2-associated genes in bone marrow-derived macrophages (BMDMs) from Sham and ovariectomized (OVX) mice exposed to filtered air (FA) or wood smoke (WS). BMDMs were differentiated from bone marrow of each group and gene expression was measured by quantitative RT-PCR. Panels show expression of (A) Arg1 , (B) Mrc1 , (C) Pparg , (D) Tgfb1 , (E) Ccl2 , (F) Ccl5 , and (G) Il10 . For visualization, gene expression levels were normalized to 18S rRNA and relative quantification was calculated using the 2−ΔΔCt method, with the mean ΔCt of the Sham-FA group serving as the calibrator for all samples. Bars represent mean ± SEM for each group, and each data point represents an individual mouse ( n = 5 biological replicates per group, with 3 technical replicate measurements averaged per mouse). H) Summary of P -values for the main effects and their interaction from the 2-way mixed-effects model. Statistical significance for all panels was determined using a linear mixed-effects model (2 fixed factors: Surgery, Treatment; random intercept: Biological replicate) on the ΔCt values (prior to 2−ΔΔCt transformation) to appropriately account for inter-replicate variability. “Surgery” and “Treatment” were modeled as fixed effects, and “Biological Replicate” as a random effect. Asterisks denote significant differences from post-hoc multiple comparisons tests. ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant. See also for the response patterns of each individual biological replicate. See also for data showing that expression of representative M1-associated genes (e.g. Cxcl1 , Il1b , Tnfa ) was not significantly altered by WS under these conditions.

Article Snippet: Membranes were blocked with 5% BSA in TBST for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies against ARG1 (#93668, Cell Signaling Technology, 1:1,000), PPARγ (#2443, Cell Signaling Technology, 1:1,000), or Vinculin (#13901, Cell Signaling Technology, 1:1,000) diluted in 5% BSA/TBST.

Techniques: Gene Expression, Expressing, Derivative Assay, Quantitative RT-PCR, Quantitative Proteomics, Transformation Assay