Figure 8. IFNγ and TLRa promote tumor-inhibitory properties in human monocytes.

A. Human breast cancer cell line MDA-MB-231 tGFP (2×10⁴) was cultured in the presence or absence of enriched human monocytes from healthy donors (1×10⁵), and IFNγ, resiquimod (R848) and/or LPS were variably added to the assays at time 0; all wells received GM-CSF to promote sustained viability. Upper panel displays treatment with the above factors but without monocytes (M-); lower panel displays the same factors but also with human monocytes (M+). The 96h time point is shown, representative of four healthy monocyte donors. For each factor or factor combination, % tumor inhibition was enumerated for six non-overlapping HPF with or without monocytes (black boxes). Monocyte-attributable inhibition of MDA-MB-231 cells (shown in red boxes) was calculated for each factor set by subtracting background inhibition in the absence of monocytes. The red boxes also indicate the degree of significance between M- and M+ groups for each factor analyzed by unpaired Student's t-Test. Confluence of MDA-MB-231 cells was variably delayed by the tested factors even in the absence of monocytes but to a significantly greater degree when monocytes were also present (* p < 0.05, **p < 0.01, ***p < 0.001), the exception being wells which received only GMCSF+R848 (ns, non-significant). B. Bright field examination of MDA-MB-231 cells exposed to GMCSF+IFNγ+LPS+R848 either without (upper panel) or with (lower panel) co-exposure to human monocytes (same treatments as far right group in A). Upper panel shows nearly confluent tumor outgrowth whereas lower panel shows largely eradicated MBA-MB-231 cells with prominent acellular spaces and monocytes clustering on residual tumor cells. C. Same as B except that the target is 3T3 non-transformed mouse fibroblasts. 3T3 cells achieve near confluence regardless of GMCSF+IFNγ+LPS+R848 exposure without monocytes (upper panel) or with monocytes (lower panel), the monocytes failing to form clusters with the 3T3 cells.