In addition, T cells of the type-1 inflammatory phenotype were present. Clinical data of the patients strongly support the findings that TAMs, together with tumour-infiltrating T cells, exert tumour-suppressive effects. For the first time, we demonstrated the tumour-suppressive properties of TAMs and have begun to dissect the

underlying processes. These findings will help us understand the potential beneficial actions of TAMs, so that future cancer immunotherapy can be developed based on enhancing these tumour-suppressive effects of TAMs to boost anti-tumour immune responses. We co-cultured HSP inhibitor cancer human primary monocytes with a human colorectal cell line, HT29, as MCTSs for 8 days (this set-up will be referred to as ‘co-culture spheroids’

check details hereafter). To mimic tumours with no macrophage infiltration, we cultured tumour cells alone as spheroids (hereafter referred to as ‘tumour spheroids’). To determine if monocytes co-cultured with tumour cells differentiated into macrophages, we checked the expression of CD68 and CD14, markers up-regulated and maintained, respectively, during monocyte-to-macrophage differentiation. In contrast, CD68 and CD14 expression are down-regulated in monocyte-to-dendritic cell (DC) differentiation (Supporting Information Fig. 1A–C). All the monocytes (CD45+) co-cultured with tumour cells for 8 days up-regulated the expression of CD68 (Fig. 1A) and maintained the expression of CD14 (Fig. 1B), compared with freshly isolated monocytes (Supporting Information Fig. 1A), indicating that the monocytes have differentiated into macrophages. Monocyte cultured alone for 8 days under the same conditions, in the absence of tumour cells, do not spontaneously differentiate (Supporting Information Fig. 1D). In addition, from day 4 to 8, CD68+ cells in the co-culture spheroids displayed increase in size, number of cytoplasmic granules and heterogeneity of cell shape characteristic of monocyte-to-macrophage differentiation (Fig. 1C). Together, these observations indicated that the monocytes have differentiated into macrophages after 8 days

of co-culture with tumour cells. To study the interaction between tumour cells and macrophages, we carried out global gene expression profiling on three groups of cells: (I) tumour cells Quinapyramine from tumour spheroids; (II) tumour cells sorted out from co-culture spheroids and (III) tumour cells and TAMs from co-culture spheroids (Fig. 2A). To assess the changes induced in the tumour cells upon co-culture with macrophages, we compared the gene expression profiles of (I) and (II), which gave 286 differentially expressed genes (DEGs; Supporting Information Table 1). Sorted tumour cells in (II) had a purity of 92.6±4.2%, with only 0.5±0.2% TAMs remaining (Supporting Information Fig. 2), making the comparison valid. Twenty-eight of the 286 DEGs (10%) were associated with proliferation and apoptosis (Fig. 2B).