Fig. 5

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 TGFβ mRNA expression. Total RNA was extracted from MDA-MB-231 tumor cell xenografts at the end of treatment and TGFβ 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 IFNγ 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)