Journal: Cancer cell

Article Title: Complement C5a Fosters Squamous Carcinogenesis and Limits T Cell Response to Chemotherapy

doi: 10.1016/j.ccell.2018.09.003

Figure Lengend Snippet: (A) Immunofluorescent (IF) detection of C5a (green, DAPI [blue]; top row), and immunohistochemistry (IHC) of C5aR1 (brown; middle row), and quantification (bottom) of ear skin from nontransgenic (NT), and 4-month old C3+/+ versus C3−/− HPV16 mice. Representative images are shown. (B) IF detection of C5a (green, DAPI [blue]; top row), and IHC of C5aR1 (brown middle row and side), and quantitation (bottom) from NT mice and canonical timepoints from HPV16 mice. Representative images are shown. (C) Co-IF of C5aR1 (green) with indicated lineage markers in dysplastic ear skin from HPV16 mice. Representative images for each cell type are shown. Boxed areas on top are shown at higher magnification on bottom. (D) PDSC5 tumor growth kinetics in C5aR1+/− and C5aR1−/− mice (n≥8 mice/group). (E) Quantitation of VEGF protein in lysates by ELISA from SCCs in (D) (F) Manual IHC analysis of CD31+ vessels from SCCs in (D). Please define FOV. (G and H) Growth kinetics of PDSC5 cells admixed with C5aR1+/− or C5aR1−/− BMMCs (G) or BMMFs (H) from donor (d) mice, implanted into syngeneic recipient (r) mice of indicated genotypes. Mice from two independent experiments depicted (BMMCs, n=9–16; BMMΦs n=11–15 mice/group). Data are represented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Significance determined by one-way (A-B) or two-way (D, G-H) ANOVA with Bonferroni post-test for multiple comparisons or by unpaired Student’s t test with Welch’s correction (E-F). Each data point reflects an individual mouse. In (A-C), epidermal (e) and dermal (d) regions are indicated, with dotted lines reflecting epidermal-dermal interface. See also Figures S1 and S2.

Article Snippet: Primary antibodies including rat anti-mouse PECAM1/CD31 (MEC 13.3; BioLegend; 1:50), CD45 (30-F11; BD Pharmingen; 1:100), F4/80, C5aR1 (M19; Santa Cruz; 1:100), C5a (L-14; Santa Cruz; 1:50), CD3 (17A2; BD Pharmingen; 1:100), PDGFRa (APA5; eBioscience; 1:100), CD117 (ACK45; BD Pharmingen; 1:50), CD11c (NB110–97871; BD Pharmingen; 1:100) were diluted in 0.5x blocking buffer and incubated overnight at 4 o C. Slides were washed and incubated with appropriate fluorescent secondary antibodies diluted in 0.5x blocking buffer.

Techniques: Immunohistochemistry, Quantitation Assay, Enzyme-linked Immunosorbent Assay

Journal: Cancer cell

Article Title: Complement C5a Fosters Squamous Carcinogenesis and Limits T Cell Response to Chemotherapy

doi: 10.1016/j.ccell.2018.09.003

Figure Lengend Snippet: (A) Casein/plasminogen zymogram of nontransgenic (n), hyperplastic (h), dysplastic (d), and SCC (t) tissue extracts at indicated ages. Protein identity based on molecular weight. lmw, low molecular weight; hmw, high molecular weight. (B) SCC growth kinetics from PDSC5 cells, implanted in uPA+/− versus uPA−/− mice from two independent experiments (n≥17 mice/group). Significance determined by two-way ANOVA with Bonferroni post-test for multiple comparisons. (C) Co-IF analysis of uPA (green) with indicated lineage markers in dysplastic ear skin from HPV16 mice. Representative images shown. Boxed areas on top are shown at higher magnification on right. (D) Schematic of sample preparation for LC-MS/MS analysis. (E) Quantitation of fold change in protein levels of factors associated with plasminogen processing in M2/M(IL-4) and M1/M(LPS), as compared to M0/M(CSF-1) macrophages. (F) In vitro complement deposition assay of BMMΦs from uPA+/− or uPA−/− mice co-cultured with PDSC5 cells, and IF-stained for C5a (red) and F4/80 (green) (top) and quantified (bottom). Data points shown reflect two ROIs/well with each experimental condition replicated in 3–4 wells, and the experiment repeated twice. (G) IF staining of C5a (green) in tissue sections reflecting high grade dysplasia from HPV16/uPA+/− and HPV16/uPA−/− mice. Representative images are shown. (H) Model of complement activation. ‘M2’-type macrophages upregulate plasminogen processing to generate C5a, while ‘M1’-type macrophages produce negative regulators of plasminogen processing. Data are represented as means ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. See also Figure S3.

Article Snippet: Primary antibodies including rat anti-mouse PECAM1/CD31 (MEC 13.3; BioLegend; 1:50), CD45 (30-F11; BD Pharmingen; 1:100), F4/80, C5aR1 (M19; Santa Cruz; 1:100), C5a (L-14; Santa Cruz; 1:50), CD3 (17A2; BD Pharmingen; 1:100), PDGFRa (APA5; eBioscience; 1:100), CD117 (ACK45; BD Pharmingen; 1:50), CD11c (NB110–97871; BD Pharmingen; 1:100) were diluted in 0.5x blocking buffer and incubated overnight at 4 o C. Slides were washed and incubated with appropriate fluorescent secondary antibodies diluted in 0.5x blocking buffer.

Techniques: Molecular Weight, Sample Prep, Liquid Chromatography with Mass Spectroscopy, Quantitation Assay, In Vitro, Cell Culture, Staining, Activation Assay