Musashi-2 in cancer-associated fibroblasts promotes non-small cell lung cancer metastasis through paracrine IL-6-driven epithelial-mesenchymal transition

Background Lung cancer, the most common cause of cancer-related mortality worldwide, is predominantly associated with advanced/metastatic disease. The interaction between tumor cells and cancer-associated fibroblasts (CAFs) in tumor microenvironment is known to be essential for regulating tumor progression and metastasis, but the underlying mechanisms, particularly the role of RNA-binding protein Musashi-2 (MSI2) in CAFs in promoting non-small cell lung cancer (NSCLC) invasiveness and metastatic spread, remain obscure. Methods Genomic and proteomic database analyses were performed to evaluate the potential clinical significance of MSI2 in NSCLC tumor and stromal clinical specimens. Molecular approaches were used to modify MSI2 in CAFs and determine its functional role in NSCLC cell motility in vitro using 2D and 3D models, and in metastasis in a xenograft mouse model using live-cell imaging. Results MSI2, both gene and protein, is upregulated in NSCLC tissues and is associated with poor prognosis and high metastatic risk in patients. Interestingly, MSI2 is also upregulated in NSCLC stroma and activated fibroblasts, including CAFs. Depletion of MSI2 in CAFs by CRISPR-Cas9 strongly inhibits NSCLC cell migration and invasion in vitro, and attenuates local and distant metastatic spread of NSCLC cells in vivo. The crosstalk between CAFs and NSCLC cells occurs via paracrine signaling, which is regulated by MSI2 in CAFs via IL-6. The secreted IL-6 promotes epithelial-mesenchymal transition in NSCLC cells, which drives metastasis. Conclusion Our findings reveal for the first time that MSI2 in CAFs is important in CAF-mediated NSCLC cell invasiveness and metastasis via IL-6 paracrine signaling. Therefore, targeting the MSI2/IL-6 axis in CAFs could be effective in combating NSCLC metastasis. Supplementary Information The online version contains supplementary material available at 10.1186/s13578-023-01158-5.

at 0 and 48 h under an inverted phase-contrast microscope.Wound spaces were measured using ImageJ software.

Cytokine antibody array
Cytokines and chemokines in CAF-CM were analyzed using the RayBiotech human cytokine antibody array C1000, according to the manufacturer's instructions.Briefly, membranes were blocked with the supplied blocking buffer, then incubated with CAF-CM overnight at 4 °C.
Next, biotin-conjugated antibody and HRP-streptavidin were added and the membranes were detected using chemiluminescence.

ELISA
An ELISA kit (R&D systems) was used to quantify CAF-secreted IL-6 in CM according to the manufacturer's instructions.Briefly, ELISA plates coated with IL-6 antibody were incubated with CAF-CM containing antibody cocktail for 1 h.The plates were washed three times with washing buffer, and detection substrate and stop solution were added.Then, the signals were read at 450 nm.

Histopathology
For immunostaining of mouse xenograft tumors, mouse tissue specimens were fixed with 10% neutral buffered formalin, embedded in paraffin, and sliced sections were subjected to routine staining with hematoxylin and eosin (H&E), or immunohistochemical (IHC) staining with the indicated antibodies.All histopathological processing was performed at the West Virginia University Pathology Research Laboratories.All images were obtained by an inverted phasecontrast microscope and quantified using ImageJ software.

Proliferation assays
A total of 4 × 10 4 cells/well was seeded into 6-well plates and cultured in culture medium.On the next day, the medium was removed, and the cells were washed with RPMI 1640 medium.
Then, CAF-CM and RPMI 1640 containing 5% FBS were mixed in a 1:1 ratio and added to the wells.Cells were counted at day 1, 3, and 5 after specific treatments using an automated cell counter.

Colony formation assay
A total of 500 cells/well was seeded into 6-well plates and cultured in culture medium.On the next day, the medium was removed, and the cells were washed with RPMI 1640 medium.Then, CAF-CM and RPMI 1640 containing 5% FBS were mixed in a 1:1 ratio and added to the wells.
One week after plating, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet.Whole well images were taken using a digital imaging system and counted by ImageJ software.

Sphere formation assay
Following specific treatments, a total of 500 cells/well was seeded into 24-well ultra-low attachment plates in MethoCult TM H4100 containing 20 ng/mL EGF, 20 ng/mL FGF, and 4 mg/mL insulin.One week after plating, micrographs were taken under an inverted phasecontrast microscope, and sphere number and size were quantified using ImageJ software.

CCK-8 assay
Following specific treatments, a total of 3 × 10 3 cells/well was seeded into 96-well plates.At 0, 24, and 48 h, a total of 10 μL/well of a CCK-8 solution was added and incubated for 4 h at 37 °C.Optical absorbance was then measured at the wavelength of 450 nm using a microplate reader and corrected for background reading.

Overexpression plasmid and transfection
CAFs were transfected with either MSI2 (Genscript) or control pcDNA3.1 plasmid using Lipofectamine 3000 reagent according to the manufacturer's instructions.At 48 h posttransfection, cells were subjected to Western analysis for MSI2 and IL-6 levels to investigate their potential correlation.

Quantitative real-time PCR (RT-qPCR)
Total RNA was extracted from Ctrl and gMSI2 CAFs by using Tri reagent, followed by quantification using a NanoDrop 2000 spectrophotometer.The generation of complementary DNA (cDNA) was performed using RevertAid First Strand cDNA Synthesis Kit according to the manufacturer's instructions.RT-qPCR was performed using the CFX384 Touch™ Real-Time PCR Detection System with SYBR™ Select Master Mix.Additional file 1: Table S3 shows the primer sequences that were used in this study.Relative expression of each gene was normalized to GAPDH, which served as a housekeeping gene.