miR-200-mediated inactivation of cancer-associated fibroblasts via targeting of NRP2-VEGFR signaling attenuates lung cancer invasion and metastasis

Cancer-associated fibroblasts (CAFs) play a substantial role in promoting cancer cell motility, drug resistance, angiogenesis, and metastasis; therefore, extensive research has been conducted to determine their mode of activation. We aimed to identify whether miRNA-200 (miR-200), a widely recognized suppressor of epithelial-mesenchymal transition, prevents CAFs from promoting cancer progression. Overexpression of miR-200 prevented CAFs from promoting lung cancer cell migration, invasion, tumorigenicity, and metastasis. Additionally, miR-200 suppressed the ability of CAFs to recruit and polarize macrophages toward the M2 phenotype, as well as the migration and tube formation of vascular endothelial cells. NRP2, a co-receptor of vascular endothelial growth factor receptor (VEGFR), was confirmed to be a target of miR-200, which mediates the functional activity of miR-200 in CAFs. NRP2-VEGFR signaling facilitates the secretion of VEGF-D and pleiotrophin from CAFs, leading to the activation of cancer cell migration and invasion. These findings suggest that miR-200 remodels CAFs to impede cancer progression and metastasis and that miR-200 and NRP2 are potential therapeutic targets in the treatment of lung cancer.

D. Transwell migration assay of 344SQ cells treated with CM from CAFs transfected with NTC or siVegfd.344SQ cells were seeded in the upper inserts, and CAF-CM were added to the bottom wells.
After 24 h, the migrated 344SQ cells were photographed and counted.Mean + SD (n = 3).Table S3.The raw data for all graphs in the manuscript.An Excel file containing the raw data has been uploaded separately.
Figure S1.miR-200 downregulated in CAFs suppresses the expression of CAF markers. A. RT-qPCR analysis of miRNAs differentially expressed between LFs and CAFs.miRNA levels were normalized to RNU6B snoRNA levels, and values relative to those of LFs (set at 1.0) are presented.The results of miR-200 family members are shown on the right.Mean + SD (n = 3).* P < 0.05, ** P < 0.01; two-tailed Student's t-test.B. A heatmap showing the expression of inflammatory CAF makers in RNA sequencing data from CAFvec and CAF-200.C. RT-qPCR analysis of myofibroblastic (myCAF) and inflammatory CAF (iCAF)-specific markers in LFs transfected with miR-200b inhibitor.The expression was normalized to the Rpl32 mRNA level, and log2 fold-change values (miR-200 inhibitor vs. control) are presented in the graphs.Mean + SD (n = 3).*P < 0.05, **P < 0.01; two-tailed Student's t-test.D. Rank-rank hypergeometric overlap analysis of two profiling data sets: CAF-200 vs. CAF-vec and LFs vs. CAFs.Negative values of log10-transformed hypergeometric p-values are indicated in the color scale.

Figure S6 .
Figure S6.Cytokines and growth factors are downregulated by miR-200 in CAFs. A. List of downregulated cytokines and growth factors in CAF-200 compared with those in CAF-vec based on the RNA sequencing data (log2 fold-change > 1, FDR < 0.01).B. Functional protein association network using STRING database (https://string-db.org)between cytokines and growth factors downregulated by miR-200 in CAFs.C. RT-qPCR analysis of cytokines ang growth factors in CAF-vec and CAF-200.The expression levels were normalized to the Rpl32 mRNA level, and the expression ratios of CAF-200 to CAF-vec are presented.Mean + SD (n = 3).* P < 0.05, ** P < 0.01; two-tailed Student's t-test.

Table S1 .
. Spheroid invasion assay of 344SQ cells with CM of CAFs transfected with NTC or siVegfd.344SQ spheroids were incubated with CAF-CM for 24 h.Mean ± SD (+NTC, n = 26; +siVegfd #2, n = 22; +siVegfd #3, n = 22).P, two-tailed Student's t-test.Figure S7.miR-200 attenuates CAF activity to recruit macrophages and vascular endothelial cells.A. RT-qPCR analysis of M1 and M2 macrophage markers in Raw264.7 cells treated with CM of CAFvec and CAF-200.The expression was normalized to the Rpl32 mRNA level, and values relative to List of siRNAs used in this study P, twotailed Student's t-test.E

Table S2 .
List of RT-qPCR primers used in this study