Fig 2: Heterogeneous CSC subtypes undergo STAT3/pY705-elicited EMT and STAT3/pS727-elicited MET to achieve metastatic colonization in a manner regulated by MSCs in vivo.A Diagram depicting the procedure to test whether MSC-elicited STAT3 signaling involved in lung cancer bone metastases. The details are summarized in Supplementary Materials and methods. B MSC recruitment assay. After lung orthotopic injection for 1 week, 0.5 × 106 MSC-RFP cells were injected intravenously. The primary lung tumors were harvested after 5 days for FACS analysis of MSC-RFP cells. Percent MSC-RFP cells in the primary tumor was shown. C–E Analysis of bone metastases. After MSC-RFP cells injection for 1 and 2 weeks, SCDCs were isolated from long bones after for FACS analysis of various cancer cells. The number of bone metastatic cells was shown in (C). Bone metastatic (CD133+/CD83+ and CD151+/CD38+) cells were incubated with isotype IgG (control) or antibody specifically recognizing p-ERK, pY705-STAT3, and pS727-STAT3 followed by labeling with Alexa Fluor 488-conjugated secondary antibody. Fluorescence intensity was determined with flow cytometry. The specific fluorescence index (SFI) was calculated as the ratio of the geometric mean fluorescence value obtained with the specific antibody and the isotype control antibody (shown in D). The performance status of the mice began to decrease, at which time the animals were sacrificed and autopsied. The orthotopic primary tumor and all major organs as well as the whole skeleton were explored. The incidence of bone metastases was shown in (E). ND not detectable. *P < 0.05; **P < 0.01.

Fig 3: CD133+/CD83+ early-response (HM20) cells undergo STAT3/pS727-elicited MET to achieve metastatic colonization in a manner regulated by BM-MSCs.A Transwell invasion assays were performed to assess invasion. Cells were seeded in the upper and lower chambers as indicated. B Cells were seeded in the upper and lower chambers as indicated. The mRNA expression levels in the lower chambers were determined by QPCR. C Cells were co-cultured as indicated for 24 h. After sorting by FACS, the expression levels of the mRNAs in the lower panel were determined by QPCR. D HM20 cells were pretreated (for 24 h) with or without inhibitors (IL6R blocking antibody or soluble LIFR (sLIFR) to block the LIF/LIFR interaction) prior to incubation with BM-MSC-CM for 4 h. The expression levels of STAT3 signaling-related proteins were determined by western blotting. The relative intensities of the bands are shown. E STAT3 mutants were overexpressed in HM20 and CL1-5 STAT3-KO [an sgRNA was designed to target human STAT3, and STAT3 knockout was performed by CRISPR/Cas9 technology] cells prior to incubation with BM-MSC-CM for 24 h. The expression levels of EMT signaling-related proteins were determined by western blotting. The relative intensities of the bands are shown. F HM20 and CL1-5 STAT3-KO cells overexpressing STAT3 mutants were pretreated with inhibitors (for 24 h) prior to incubation with BM-MSC-CM for 24 h. A luciferase reporter assay was then performed to assess CDH1 promoter activity. G, H STAT3 mutants were overexpressed in HM20 and CL1-5 STAT3-KO cells prior to incubation with BM-MSC-CM for 24 h. For ChIP, DNA was immunoprecipitated with anti-STAT3 (in G) and anti-GATA3 (in H). The extracted DNA was analysed by PCR using primers spanning the proximal promoter regions of GATA3 (in G), CCND1 and CDH1 (in H). I The mRNA expression levels of cells described in (G) were determined by QPCR. J The total number of viable cells described in (G) was determined. K Cells described in (G) were cultivated in ultra-low-attachment 96-well plates under sphere-forming conditions. The numbers of spheres were calculated using microscopic analysis after 7 days. The data in (A–C), (F), and (I–K) were derived from three independent experiments and are presented as the mean values ± s.ds. *P < 0.05; **P < 0.01; ***P < 0.005 (t-test).

Fig 4: A model for STAT3 phosphorylation at Ser727 and Tyr705 differentially regulating the EMT–MET switch and cancer metastasis.Model proposing a pathway in which heterogeneous CD151+/CD38+ and CD133+/CD83+ CSC subtypes undergo STAT3/pY705-elicited EMT and STAT3/pS727-elicited MET, respectively, to achieve metastatic colonization in a manner regulated by MSCs.