3A). Five chemokines, CCL2, CCL7, CCL8, CXCL9 and CXCL10, were present at high levels in the supernatants of co-culture spheroids, but almost absent or significantly lower in the supernatants of both tumour spheroids and monocyte cultures, Sunitinib supplier indicating that co-culturing with tumour cells stimulated the production of these chemokines by the TAMs. When the supernatants of co-culture spheroids of other cancers (prostate, ovarian and breast) were assessed, CCL2, CCL7, CXCL9 and CXCL10 were present at significantly lower levels compared with that of colorectal

cancer (Supporting Information Fig. 5). This implies that TAMs in colorectal cancers secrete more chemokines to attract T cells than TAMs in other cancers Palbociclib supplier in which TAMs promote tumour growth. To ascertain that the chemokines present in the supernatants of the colorectal tumour

model were functionally capable of attracting T cells, we performed Transwell assays using two supernatants: supernatants from co-culture spheroids to mimic microenvironment of a tumour with macrophage infiltration, and supernatants from tumour spheroids to mimic microenvironment of a tumour without macrophage infiltration. Indeed, the supernatants of co-culture spheroids attracted significantly more of both CD4+ and CD8+ T cells than the supernatants of tumour spheroids (Fig. 3C), showing that the chemokines in the supernatants of co-culture spheroids were functionally able to attract T cells. As the TAM genes indicated that the

TAMs were involved in antigen presentation (Fig. 3A), and chemokines that attract T cells were present in the co-culture supernatants, we assessed colorectal TAMs for the expression of cell surface molecules involved in interaction with T cells. The TAMs expressed molecules for antigen presentation (HLA-DR, CD74), T-cell co-stimulation (CD40, CD80, CD86) and CD54 (or ICAM-1), an adhesion molecule that stabilises cell contact during T-cell co-stimulation (Fig. 4A, top panel) 15. To obtain an idea of the level of expression of these molecules on colorectal TAMs, we compared them with in vitro differentiated macrophages and freshly isolated monocytes. The median fluorescence intensity (MFI) of the expression of the molecules (Fig. 4A, middle panel) as well as the percentage MRIP of cells that expressed the molecules (Fig. 4A, bottom panel) were studied. Colorectal TAMs exhibited higher expression of all the molecules compared with in vitro MCSF-differentiated macrophages, and up-regulated the expression of all molecules except CD74 compared with freshly isolated monocytes. In addition, a significantly larger percentage of TAMs (than macrophages or monocytes) expressed CD74, CD40, CD80 and CD86. This observation indicated that co-culturing with colorectal tumour cells promoted the differentiation of monocytes to TAMs with enhanced expression of antigen presentation and T-cell co-stimulation molecules.