Fig. 5: Hpa2-KO macrophages fail to populate tumor spheroids.
A Macrophages were isolated from thioglycolate-treated WT and Hpa2-KO mice, labeled with Crystal Trace Violet (CTV) and were added (1 × 105) to pre-established spheroids of LLC cells. Spheroids were collected 24 h thereafter, and the presence of macrophages within tumor spheroids was examined by microscopy (A, left panels) and quantified (A, right panel). Shown are images captured by light (×10; left) and confocal microscopy (×10; z-stack; right). Note that Hpa2-KO macrophages fail to penetrate the tumor spheroids. B LDH activity. Conditioned medium was collected after 4 and 24 from corresponding cultures and LDH activity, indicative of cytotoxic activity, was quantified. Note that Hpa2-KO macrophages failed to populate tumor spheroids and exhibited reduced cytotoxic activity typical of M1 macrophages. C qPCR. Total RNA was extracted from WT and Hpa2-KO macrophages and was subjected to qPCR analyses applying primers sets specific for IL-10 and IL-6. Cytokine expression by Hpa2-KO macrophages is presented in relation to WT macrophages, set arbitrarily to a value of 1, and normalized to the expression of actin. Note increased expression of pro-tumorigenic IL-10 and IL-6 by Hpa2-KO macrophages. D Cell migration. Medium conditioned by WT and Hpa2-KO macrophages was used as a chemoattractant in the Boyden chamber migration assay of RAW264.7 mouse macrophage cell line. Representative images of the migration assay are shown in the left panels; Quantification of cell migration is shown graphically in the right panel. E MIP2 expression. Total RNA was extracted from WT and Hpa2-KO macrophages and was subjected to qPCR analysis applying primers specific for MIP2. MIP2 expression in Hpa2-KO macrophages is presented relative to WT macrophages, set arbitrarily to a value of 1, and after normalization to the expression of actin.
