Fig 1: HSP27/IL-6 establishes a positive feedback loop between OSCC cells and M2 TAMs. A-B iNOS, IL-12, TNF-α, Arg-1, IL-10, and IL-6 mRNA levels in RAW264.7cells under stimulation with the TCM of the CAL27 cells pretreated without or with HSP27–siRNA, detected by RT-qPCR. C-D iNOS, IL-12, Arg-1, CD163, and IL-6 mRNA levels in RAW264.7cells pretreated without or with rhHSP27 (2 μg/mL) in the presence of CAL27-CM for 12 h, detected by RT-qPCR. E–F IL-12, Arg-1, and IL-6 mRNA and IL-6 protein levels in RAW264.7cells pretreated without or with TAK-242 (1 µM) under the stimulation of rhHSP27 (2 μg/mL) in the presence of CAL27-CM for 12 h, detected by RT-qPCR (E) and ELISA (F). G-H IL-6 mRNA, protein levels in THP-1 cells with NC-siRNA or HSP27-siRNA induced TCM, detected by RT-qPCR (G) and ELISA (H). I-J IL-6 mRNA, protein levels in THP-1 cells without or with rhHSP27 (2 μg/mL) stimulated, detected by RT-qPCR (I) and ELISA (J). K-L IL-6 mRNA, protein levels in RAW264.7 cells with NC-siRNA or HSP27-siRNA induced TCM, detected by RT-qPCR (K) and ELISA (L). M–N), IL-6 mRNA, protein levels in Raw264.7 cells without or with rhHSP27 (2 μg/mL) stimulated, detected by RT-qPCR (M) and ELISA (N). O-P HSP27 mRNA levels in CAL27 cells without or with IL-6 (100 ng/mL) treated (O), with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated (P), detected by RT-qPCR. (Q-S), HSP27protein levels in CAL27 cells without or with IL-6 (100 ng/mL) treated, with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated, detected by Western blotting in the whole-cell lysates (Q) and ELISA (R-S). T-U HSP27 mRNA levels in SCC9 cells without or with IL-6 (100 ng/mL) treated (T), with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated (U), detected by RT-qPCR. V-X HSP27 protein levels in SCC9 cells without or with IL-6 (100 ng/mL) treated, with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated, detected by Western blotting in the whole-cell lysates (V) and ELISA (W-X). T-tests and one-way ANOVA. Means ± SD of at least three separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001
Fig 2: Paracrine HSP27 promotes OSCC chemoresistance through TAMs. A Schematic presentation of the analyzed tumor models, Vehicle or HSP27-shRNA OSCC cells were orthotopically injected into BALB/c nude mice. B-C Tumor pictures (B) and growth analysis (C) over 21 days show the mean tumor volume at the indicated time points following implantation, statistical significance was determined by 2-way ANOVA. Volume of individual tumors at day 21 is shown in the box plot; statistical significance was determined by Welch t-test. D-E HSP27 mRNA (D), protein (E) levels in CAL27 cells with NC-siRNA or HSP27-siRNA induced TAMs-CM, detected by RT-qPCR and Western blotting in the whole-cell lysates. F Apoptosis relative gene BAX, BIM, BAD and Caspase3 mRNA levels in CAL27 cells with NC-siRNA or HSP27-siRNA induced TAMs-CM, detected by RT-qPCR. G Apoptosis relative gene BAX, BIM, BAD and Caspase3 mRNA levels in CAL27 cells without or with rhHSP27 (2 μg/mL) induced TAMs-CM, detected by RT-qPCR. H Relative cell apoptosis level in CAL27 cells with NC-siRNA or HSP27-siRNA induced TAMs-CM, detected by Flow Cytometry. I Relative cell apoptosis level in CAL27 cells without or with rhHSP27 (2μg/mL) induced TAMs-CM, detected by Flow Cytometry. J Analysis of (H), determined with relative ratio of apoptosis in total per 104cells. K Apoptosis relative BAX, BIM, BAD and Cleaved-caspase3 protein levels in CAL27 cells with NC-siRNA or HSP27-siRNA induced TAMs-CM, detected by Western blotting in the whole-cell lysates. L Analysis of (I), determined with relative ratio of apoptosis in total per 10.4cells. M Apoptosis relative BAX, BIM, BAD and Cleaved-caspase3 protein levels in CAL27 cells without or with rhHSP27 (2 μg/mL) induced TAMs-CM, detected by Western blotting in the whole-cell lysates. N–O Analysis of (K) and (M). T-tests and two-way ANOVA. Means ± SD of at least three separate experiments. ns, no significance. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001
Fig 3: TAMs-derived IL-6 orchestrates stemness and chemoresistance through upregulating β-catenin and CD44. A–B CAL27 cells are stimulated with IL-6 (100 ng/mL) for 12 h. The relationship between HSP27 expression, metatasis, and the β-catenin signaling enrichment score is assayed by RNA sequencing, each circle represents a sample of RNA sequencing. C Kaplan–Meier overall survival curves for HNSCC patients according to the IL-6 expression in the TCGA cohort. D, F β-catenin, c-myc and cyclin D1mRNA levels in CAL27 cells without or with IL-6 (100 ng/mL) treated (D), with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated (F), detected by RT-qPCR. E, G β-catenin, c-myc and cyclind1 mRNA levels in SCC9 cells without or with IL-6 (100 ng/mL) treated (E), with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated (G), detected by RT-qPCR. H β-catenin protein levels in CAL27 cells without or with IL-6 (100 ng/mL) treated, with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated, Western blotting in the whole-cell lysates. I β-catenin protein levels in SCC9 cells without or with IL-6 (100 ng/mL) treated, with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated, Western blotting in the whole-cell lysates. J-K CD44 mRNA levels in CAL27 and SCC9 cells without or with IL-6 (100 ng/mL) treated (J), with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated (K) detected by RT-qPCR. L-M CD44 protein levels in CAL27 (L) and SCC9 (M) cells without or with IL-6 (100 ng/mL) treated, with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated, detected by Western blotting in the whole-cell lysates. (N–O), Analysis of (L) and (M). P-Q OCT4 mRNA levels in CAL27 and SCC9 cells without or with IL-6 (100 ng/mL) treated (P), with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated (Q), detected by RT-qPCR. T-tests and one-way ANOVA. Means ± SD of at least three separate experiments. ns, no significance. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001
Fig 4: Higher expression of HSP27 in OSCC cells and its correlation. A-B UMAP plot showing the cell clusters in the TME. C The expression patterns of HSP27 in different cell types were analyzed using single-cell RNA sequencing data. D-E HSP27 mRNA levels in Hacat and CAL27 cells without (D) or with (E) TAMs-CM stimulation detected by RT-qPCR. F HSP27 protein levels in Hacat and CAL27 cells, detected by Western blotting in the whole-cell lysates. G HSP27 protein levels in cell TCM, detected by ELISA. H HSP27 mRNA levels in the RAW264.7 cells, TAMs and CAL27 cells, detected by RT-qPCR. I-J HSP27 mRNA levels in CAL27 cells (I) and SCC9 (J) cells without or with TAMs-CM stimulation, detected by RT-qPCR. K HSP27 protein levels in CAL27 cells and SCC9 cells without or with TAMs-CM stimulation, detected by Western blotting in the whole-cell lysates. L-M HSP27 protein levels in CAL27 cells (L) and SCC9 cells (M) without or with TAMs-CM stimulation, detected by ELISA. N–O MDR1 mRNA levels in CAL27 cells (N) and SCC9 cells (O) without or with TAMs-CM stimulation, detected by RT-qPCR. P-R Representative images of immunohistochemistry (IHC) staining showing HSP27, CD163, and P-gp expressions in tumor tissues form non-metastatic and metastatic OSCC patients. S Semi-quantitative analysis of IHC staining. T-tests and one-way ANOVA. Means ± SD of at least three separate experiments. ns, no significance. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001
Fig 5: HSP27/IL-6 axis promotes OSCC chemoresistance, invasion, and migration by orchestrating macrophages via a positive feedback loop
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