Sensitivity and resistance of human tumor cells to interferon and rIn. a quiescent state as indicated by the downregulation of cleaved caspase 3 (Supplementary Figure 3C). Probably, NCI-H1299 expresses high but non-functional TLR3 protein that does not engage polyI:C. More importantly, our findings suggest that low-to-medium level of functional TLR3 protein expressed in A549, NCI-H292 and NCI-H358 appeared to support the susceptibility of these cells to polyI:C treatment. For example, A549 and NCI-H292 expressed low but adequate TLR3 protein (Figure ?(Figure1B)1B) for binding with polyI:C, resulting in suppressions of survival (Figure ?(Figure1E),1E), oncogenicity (Figure 2A, 2B) and metastasis (Figure 2CC2E). PolyI:C induces apoptosis of A549, NCI-H292, and NCI-H358 via direct activation of TLR3-caspase 3/8-dependent apoptosis pathway. Furthermore, TLR3 antibody-neutralization (Figure ?(Figure3)3) and TLR3 siRNA knockdown (Figure ?(Figure4)4) reversed the polyI:C-suppression of survival and metastasis of A549 and NCI-H292, suggesting that polyI:C specifically acts on TLR3 protein to exert anti-cancer functions. Consistent with the anti-cancer activity of polyI:C [45], our findings reveal how polyI:C alone exerts pro-apoptotic, anti-proliferative and anti-metastatic activities in susceptible lung cancer cells, to suppress survival and oncogenicity of A549, NCI-H292, and NCI-H358. PolyI:C stimulation has been reported to activate inflammatory response through production of pro-inflammatory cytokines (IL-1, IL-6, and IL-8) [47, 48]. Here, we showed that stimulation of different lung cancer cell lines with polyI:C induced differential secretion of inflammatory cytokines in a cell type-specific manner. Notably, NCI-H358, which expresses medium level of TLR3 MC 1046 protein and produces abundant endogenous IL6 and IL8, was not further induced by polyI:C to produce more of these cytokines (Figure ?(Figure5).5). NCI-H358, which expresses high endogenous level of IL-6 protein, underwent IL6-independent suppression of metastasis when treated with polyI:C, and this was mediated indirectly through inactivation of IL6/JAK2/STAT3 signalling (Supplementary Number 3C). Hence, NCI-H358 was unaffected from the inhibition of cytokine-dependent metastasis. On the other hand, NCI-H1299, which also expresses high endogenous level of TLR3, was insensitive/unresponsive to polyI:C activation, and did not secrete any pro-inflammatory cytokines (Number ?(Number5).5). The apparent resistance/unresponsiveness of NCI-H1299 to polyI:C may be due to both the quiescence of TLR3 signalling pathway and the inactivation of IL6/JAK2/STAT3 signalling (Supplementary Number 3C). Concordantly, A549 and NCI-H292 cells which communicate low but adequate levels of TLR3, were sensitive to polyI:C activation, producing high levels of pro-inflammatory cytokines (IL6, IL8 and GRO) associated with survival and metastasis (Number ?(Number5C).5C). IL6 was reported to stimulate STAT3 IFN-alphaJ activity which promotes tumor growth and survival of NSCLC via JAK/STAT3 signalling [49]. Consistently, we found that inhibition of STAT3 by Stattic suppressed polyI:C-induced IL6 secretion in A549, indicating that polyI:C activates JAK2/STAT3 signalling to enhance the production of IL6 (Number ?(Figure6E).6E). Therefore, our findings suggest that polyI:C kills A549 via both activation of IL6/JAK2/STAT3 and TLR3-caspase-3/8 apoptosis pathways. PolyI:C can be used as an anti-cancer therapy or a vaccine adjuvant. Combinatorial therapy with Hiltonol MC 1046 and siltuximab is known to control tumor growth and enhance local immune response, providing evidence that they not only attenuate survival and proliferation of malignancy cells but also activate infiltration of immune cells [50]. Herein, we shown that combinatorial treatment with polyI:C and anti-IL6 antibody enhanced polyI:C-mediated suppressions of survival, oncogenicity, and metastatic potential of A549 (Number MC 1046 ?(Number7,7, Number ?Number8).8). Furthermore, blockade of the JAK2 and STAT3 activities enhanced the polyI:C-suppressions of survival, oncogenicity, and metastasis of A549 (Number ?(Number7,7, Number ?Number8)8) and NCI-H292 (Supplementary Number 4, Supplementary Number 5). Our data suggest that enhancement of polyI:C-killing of A549 resulted from your blockade of IL6-dependent JAK2/STAT3 signalling, but polyI:C-killing of NCI-H292 resulted from your blockade of IL6-self-employed JAK2/STAT3 signalling. We postulate a model to illustrate this mechanism (Number ?(Number9).9). It is conceivable that as long as a malignancy cell (e.g. A549, NCI-H292, and NCI-H358) expresses a low-to-medium level of practical TLR3 protein, it will participate polyI:C and becomes responsive to polyI:C treatment, which activates the TLR3 signalling to consequently destroy the lung carcinoma. Thus, we propose that the manifestation of TLR3 and secretion of pro-/anti-inflammatory cytokines would correlate with the effectiveness of polyI:C (and possibly, Hiltonol) treatment of lung malignancy cells. Combination of polyI:C and anti-IL6 antibody enhanced polyI:C-suppressions of survival, oncogenicity, and metastasis of A549.