Fig 1: Anti-cath-D antibody-based therapy prevents M2-like macrophage and MDSC recruitment, and triggers antitumor response via NK cell activation in MDA-MB-231 xenografts. a Tumor growth. Nude mice bearing MDA-MB-231 tumors of 50 mm3 were treated with F1 (n = 9), F1Fc (n = 8), or rituximab (CTRL; n = 9) (15 mg/kg) for 35 days. At day 54, mice were sacrificed. *, P < 0.001 for F1 versus CTRL; P = 0.077 for F1Fc versus CTRL; P = 0.069 for F1 versus F1Fc (mixed-effects ML regression test). b Mean tumor volumes at day 54. Mean ± SEM; *, P = 0.011 for F1 versus CTRL; P = 0.231 for F1Fc versus CTRL, P = 0.189 for F1 versus F1Fc (Student’s t-test). c TAM recruitment. The percentage of F4/80+ CD11b+ TAMs was quantified by FACS and expressed relative to all CD45+ immune cells (n = 9 for CTRL; n = 9 for F1; n = 8 for F1Fc); *, P = 0.044 for F1 versus CTRL; P = 0.3 for F1Fc versus CTRL (Student’s t-test). d Linear regression analysis of TAM and tumor volumes. R2 = 0.5425; ***, P < 0.0001; n = 26. e Quantification of CD206 mRNA expression. Total RNA was extracted from MDA-MB-231 tumor xenografts at the end of treatment, and CD206 expression analyzed by RT-qPCR and shown relative to F4/80 (n = 9 for CTRL; n = 9 for F1; n = 8 for F1Fc); P = 0.05 for F1 versus CTRL; P = 0.04 for F1Fc versus CTRL (Student’s t-test). f MDSC recruitment. The percentage of Gr1+ CD11b+ MDSCs was quantified by FACS analysis and expressed relative to all CD45+ cells (n = 9 for CTRL; n = 9 for F1; n = 8 for F1Fc); **, P = 0.008 for F1 versus CTRL; P = 0.079 for F1Fc versus CTRL (Student’s t-test). g Linear regression analysis of MDSC and tumor volumes. R2 = 0.23315; *, P = 0.0125; n = 26. h Quantification of TGFß mRNA expression. Total RNA was extracted from MDA-MB-231 tumor cell xenografts at the end of treatment and TGFß expression analyzed by RT-qPCR. Data are relative to RPS9 expression (n = 9 for CTRL; n = 9 for F1; n = 8 for F1Fc); **, P = 0.009 for F1 versus CTRL; P = 0.1 for F1Fc versus CTRL (Student’s t-test). i NK recruitment. The percentage of CD49b+ CD11b+ NK cells was quantified by FACS and expressed relative to all CD45+ cells (mean ± SEM; n = 9 for rituximab (CTRL); n = 9 for F1; n = 8 for F1Fc); P = 0.7 for F1 versus CTRL; P = 0.8 for F1Fc versus CTRL; P = 0.8 for F1 versus F1Fc (Student’s t-test). j Quantification of IL-15 mRNA expression. Total RNA was extracted from MDA-MB-231 tumor cell xenografts at the end of treatment and IL-15 analyzed by RT-qPCR. Data are the mean ± SEM expression level relative to RPS9 expression (n = 9 for rituximab (CTRL); n = 9 for F1; n = 8 for F1Fc); **, P = 0.0013 for F1 versus CTRL; P = 0.365 for F1Fc versus CTRL; *, P = 0.0127 for F1 versus F1Fc (Student’s t-test). k Linear regression analysis of IL-15 mRNA level and tumor volumes. R2 = 0.3693; **, P = 0.0013; n = 26. l Quantification of granzyme B mRNA expression as in (j). ***, P = 0.0002 for F1 versus CTRL; **, P = 0.0011 for F1Fc versus CTRL; **, P = 0.0076 for F1 versus F1Fc (Student’s t-test). m Quantification of perforin mRNA expression as in (j). *, P = 0.033 for F1 versus CTRL; *, P = 0.0294 for F1Fc versus CTRL; P = 0.386 for F1 versus F1Fc (Student’s t-test). n Quantification of IFN? mRNA expression as in (j). ***, P < 0.0001 for F1 versus CTRL; P = 0.0513 for F1Fc versus CTRL; **, P = 0.0078 for F1 versus F1Fc (Student’s t-test)
Fig 2: Ichthyosis and inflammation are absent in K14-CAP1/Prss8:PAR2-/- mice.(a) Macroscopic appearance of 2-week-old animals. H&E: haematoxylin and eosin staining of skin. Immunofluorescence (green) shows transgenic CAP1/Prss8 expression at the basal layer in K14-CAP1/Prss8:PAR2+/+, K14-CAP1/Prss8:PAR2+/- and K14-CAP1/Prss8:PAR2-/- transgenic mice. Below: negative control (omission of primary antibody, no 1Ab). Nuclei were counterstained with DAPI (blue), n=3 mice per group. Bar represents 20 µm. (b) RT–PCR analysis demonstrates expression of the CAP1/Prss8 transgene in K14-CAP1/Prss8:PAR2+/+, K14-CAP1/Prss8:PAR2+/- and K14-CAP1/Prss8:PAR2-/- transgenic mice, and absence of PAR2 gene expression in PAR2-/- animals. ß-actin was used to control cDNA, and H2O provided negative control. The blot is representative of three animals analysed per group. (c) Transepidermal water loss and body weight analyses (d) in experimental and control animals, n=8 mice per group. *P<0.05, **P<0.01, ***P<0.001. (e) Numbers (nb.) of F4/80- and (f) S100-positive cells evident in the skin of 2-week-old animals; (e, f) n=3 mice per genotype. **P<0.01, ***P<0.001. All data are presented as mean±s.e.m.
Fig 3: Combining ROCK Inhibitors with BRAF Inhibitors In Vivo(A) Top, schematic of experiment. Left, growth of A375/PLX/R xenografts in nude mice after treatment. Middle, Kaplan-Meier survival plot. Right, tumor volume at endpoint (n = 4–6 mice/group).(B) Left, volume of patient no. 2 xenografts in NSG mice after a 21-day treatment (n = 7 mice/group). Right, tumor growth at endpoint versus baseline.(C) p-MLC2 staining in patient no. 2 xenografts. Scale bar, 100 µm.(D) Survival of patient no. 2 cells in the mouse lung 24 h post-injection (n = 8–9 mice from 2 experiments). Scale bar, 100 µm.(E) Left, volume of patient no. 35 xenografts in NSG mice after a 10-day treatment (n = 6 mice/group). Right, p-MLC2 staining. Scale bar, 100 µm.(F and G) Images and quantification of p-MLC2 (F), CD206+ (G) in A375/PLX/R xenografts from (A). Scale bars, 100 µm. Ratio of CD206+/F4/80+ shown. (F and G) Pooled data from 2 experiments.(A–G) ROCKi GSK269962A, BRAFi PLX4720.Boxplots show median (center line); interquartile range (box); min-max (whiskers); and individual mice (circles). p values by ANOVA with Tukey's: (A) right graph, (B) left graph; Benjamini, Krieger, and Yekutieli (C, D, F, G, and E, right) or Dunnett's correction (E, left), Mantel-Cox (A, survival plot), chi-square test: percentage regressions in (A, left) and (B, right). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; n.s., not significant. See also Figure S7.
Fig 4: T-cell therapy induces tumor stroma formation. A and B, Untreated or T-cell–treated intramuscular RH30_19 tumors were excised and stained with H&E, with central and right showing enlargements of the indicated boxed areas (A) or TriChrome stained to highlight collagen deposition (blue; B). Yellow arrows indicate regions of stroma/collagen deposition as opposed to the tumor–stromal interface in untreated mice (black arrows). C, Untreated or T-cell–treated RH30_19 tumors were stained with a-Luciferase (tumor), mouse a-CD11b, and a-F4/80. A phenotype map was created to indicate the identity of each cell within the field of view. Luciferase staining was used to determine tumor cells and create a mask of tumor and nontumor regions. Myeloid cells were counted within both regions (phenotype + mask) as demonstrated. The proportion of myeloid cells in untreated, T-cell–treated, or anti-FGFR4 CAR–treated mice is shown in the representative images and plotted to the right. D, The presence of tumor-associated macrophages was determined with immunofluorescence staining of CD206 along with CD11b and F4/80 in RH30_19 tumors excised from untreated, untransduced T-cell, or FGFR4 CAR T-cell–treated mice. E, CD4 and CD8 T cells localized to myeloid-rich regions as shown by immunofluorescent staining with CD4+CD8, CD11b, and F4/80 in RH30_19 tumor samples with the indicated treatments.
Fig 5: Cdc42-overexpressing mammary glands display characteristics associated with stromal activation. (A) Representative images of H&E-stained longitudinally sectioned TEBs from 1 week dox-treated Cdc42-overexpressing and control mammary glands. Scale bar = 100 µm. Graph depicts average stromal thickness (± SEM) at neck region of TEBs (n = 7,5; *P = 0.008). (B) Representative images of F4/80-immunostained TEBs from 1 week dox-treated Cdc42-overexpressing and control mammary glands (n = 4 to 5 animals per genotype, with at least three TEBs analyzed per mouse). Scale bar = 50 µm. Graph shows percentage of F4/80-positive stromal cells (± SEM). (C) Representative images of Masson’s trichrome-stained TEBs (blue) (n = 4 animals per genotype, with an average of three TEBs analyzed per mouse). Scale bar = 50 µm. Graph depicts quantification of collagen deposition (*P= 0.009). (D) Table of qRT-PCR Super Array results showing genes that were upregulated at least 1.5 fold in stromal cells isolated from Cdc42-overexpressing compared to control mammary glands. Data are representative of three animals per genotype. (E) qRT-PCR and new primer sets were used to validate several genes identified in the Super Array. Graph shows fold change in mRNA expression levels (*P = 0.0003, **P = 0.04, ***P = 0.003, #P = 0.001, ##P = 0.005). Data are representative of three animals per group. Cdc42, cell division cycle 42; TEB, terminal end bud.
Supplier Page from Thermo Fisher Scientific for F4/80 Antibody