Fig. 1.

E0771 breast cancer cells do not produce nor respond to TNF-$\alpha$. (a): In vitro TNF-$\alpha$ production: Supernatants from E0771 tumor cells showed no detectable TNF-$\alpha$ in vitro (E0771 bar). Supernatants from $\mathrm{CD}11\mathrm{b}+$ TAMs isolated from E0771 tumors, in contrast, did produce detectable TNF-$\alpha$ ($\mathrm{E}0771/\mathrm{CD}11\mathrm{b}+$ bar). To confirm sensitivity of the assay, RAW264.7-transformed murine macrophages produced detectable TNF-$\alpha$ both when unstimulated (RAW264.7 bar) and upon activation with $100\mathrm{ng}/\mathrm{mL}$ LPS for 24 h (RAW264.7+LPS bar). TNF-$\alpha$ ELISA sensitivity was $5.1\mathrm{pg}/\mathrm{mL}$, $n=8$ samples for all but $\mathrm{E}0771/\mathrm{CD}11{\mathrm{b}}^{+}$, where $n=5$. Both the media alone control (media bar) and E0771 supernatants (E0771 bar) registered below sensitivity (not detectable). (b): In vivo TNF-$\alpha$ production: also by this same ELISA assay, TNF-$\alpha$ was detectable in E0771 tumors grown in C57Bl/6 mice (wt bar) but not in mice lacking TNF-$\alpha$ [TNF-${\alpha }^{(-/-)}$ bar]. (c): Proliferation responses to TNF-$\alpha$: three breast tumor cell lines (T47D, MCF-7, and E0771) were treated with media control or $20\mathrm{ng}/\mathrm{mL}$ TNF-$\alpha$. Pairwise comparisons indicate that T47D proliferation was significantly elevated by $20\mathrm{ng}/\mathrm{mL}$ TNF-$\alpha$ at 48 h ($p<0.01$), MCF-7 proliferation was significantly decreased ($p<0.05$), whereas only E0771 proliferation was unchanged in response to TNF-$\alpha$. Proliferation was assessed by fluorescent intensity of CyQuant DNA-binding dye standardized to that of cells in untreated media, $n=10$ per group.