Fig. 7. The HSP70-TLR4 signaling axis in lung epithelial tissues promotes primary tumor progression and anti–PD-1 immunotherapy resistance.

(A) Control BRAF^V600EPTEN^−/− TGRs in TLR4^+/+ and SPC-TLR4^−/− mice during IgG Ctrl or anti–PD-1 treatment. Growth is expressed as a ratio during anti–PD-1 therapy relative to IgG Ctrl (n = 6). (B) Flow cytometry analysis of PMN-MDSCs in the blood of BRAF^V600EPTEN^−/− tumor-bearing TLR4^+/+ and SPC-TLR4^−/− mice (n = 5). (C) Representative flow cytometry dot plot of PD-1 expression by circulating PMN-MDSCs in TLR4^+/+ and SPC-TLR4^−/− transgenic mice (n = 5 mice). (D) Csf3 qrt-PCR analysis of CD45⁺EpCAM⁻ and CD45⁻EpCAM⁺ cells FACS-sorted from the lungs of non–tumor-bearing and BRAF^V600EPTEN^−/− tumor–bearing mice (n = 3). Csf3 encodes G-CSF. Statistical analysis performed by two-way ANOVA followed by Tukey’s multiple comparisons test. (E) Csf3 qrt-PCR analysis of lung tissues harvested from TLR4^+/+ and SPC-TLR4^−/− mice (n = 3). (F) Representative G-CSF IHC of lung tissues harvested from TLR4^+/+and SPC-TLR4^−/− mice (n = 3). 20×; scale bars, 50 μm. (G) G-CSF ELISA analysis of the plasma of TLR4^+/+ and SPC-TLR4^−/− tumor–bearing transgenic mice (n = 3). (H) Left: G-CSF ELISA analysis of the plasma of transgenic BRAF^V600EPTEN^−/− mice after anti–PD-1 ± HSP70i treatment (n = 4). Right: Csf3 qrt-PCR analysis of the lung tissues of tumor-bearing transgenic BRAF^V600EPTEN^−/− mice after anti–PD-1 ± HSP70i treatment (n = 4). (I) Representative G-CSF Western blot analysis of MLE12 lung epithelial cells after treatment with recombinant Wnt5a. All two-group comparisons were analyzed using unpaired t tests. All data are representative of two to three independent experiments and expressed as mean values ± SEM. *P < 0.05, **P < 0.005, and ***P < 0.0005.