Fig. 4.

CD40 activated tumor-infiltrating macrophages mediate tumor regression. (A) KPC mice were treated with control IgG2a or FGK45 or FGK45 plus depletion of systemic macrophages by means of CELs. Shown is a waterfall plot displaying percent change in tumor volume from baseline to day +14 (in comparison with IgG2a, FGK45: P < 0.05; FGK45 + CELs: P = 1.00; Fisher’s exact test). (B) F4/80⁺ tumor-associated macrophages were isolated from KPC mice that had been treated with FGK45 (blue squares) or control IgG2a (green circles) and incubated with KPC-derived tumor cell lines. Tumor cell death in vitro was measured by 7-aminoactinomycin D (7-AAD) labeling and flow cytometric analysis at 24 hours. Shown is a representative assay from two independent experiments each performed in triplicate. Means ± SD are depicted; *P < 0.05, Student’s t test. (C) Cleaved caspase 3 expression on KPC tumors was determined by immunohistochemistry 18 hours after treatment with control IgG2a (top panel) or FGK45 (bottom panel). (D) to (L) KPC tumors were analyzed 18 hours after treatment with control IgG2a (D, F, and H), FGK45 (E, G, and I), or FGK45 + CEL (J, K, and L). Shown are hematoxylin-and-eosin histology (D, E, J); Masson’s trichrome stain to reveal extracellular matrix in blue [(F), (G), and (K)], and immunohistochemistry for collagen I [(H), (I), and (L)]. Scale bars, 50 μm.