Differential effect of cancer-associated fibroblast-derived extracellular vesicles on cisplatin resistance in oral squamous cell carcinoma via miR-876-3p

Rationale: Platinum-based chemotherapy is commonly used for treating solid tumors, but drug resistance often limits its effectiveness. Cancer-associated fibroblast (CAF)-derived extracellular vesicle (EV), which carry various miRNAs, have been implicated in chemotherapy resistance. However, the molecular mechanism through which CAFs modulate cisplatin resistance in oral squamous cell carcinoma (OSCC) is not well understood. We employed two distinct primary CAF types with differential impacts on cancer progression: CAF-P, representing a more aggressive cancer-promoting category, and CAF-D, characterized by properties that moderately delay cancer progression. Consequently, we sought to investigate whether the two CAF types differentially affect cisplatin sensitivity and the underlying molecular mechanism. Methods: The secretion profile was examined by utilizing an antibody microarray with conditioned medium obtained from the co-culture of OSCC cells and two types of primary CAFs. The effect of CAF-dependent factors on cisplatin resistance was investigated by utilizing conditioned media (CM) and extracellular vesicle (EVs) derived from CAFs. The impacts of candidate genes were confirmed using gain- and loss-of-function analyses in spheroids and organoids, and a mouse xenograft. Lastly, we compared the expression pattern of the candidate genes in tissues from OSCC patients exhibiting different responses to cisplatin. Results: When OSCC cells were cultured with conditioned media (CM) from the two different CAF groups, cisplatin resistance increased only under CAF-P CM. OSCC cells specifically expressed insulin-like growth factor binding protein 3 (IGFBP3) after co-culture with CAF-D. Meanwhile, IGFBP3-knockdown OSCC cells acquired cisplatin resistance in CAF-D CM. IGFBP3 expression was promoted by GATA-binding protein 1 (GATA1), a transcription factor targeted by miR-876-3p, which was enriched only in CAF-P-derived EV. Treatment with CAF-P EV carrying miR-876-3p antagomir decreased cisplatin resistance compared to control miRNA-carrying CAF-P EV. On comparing the staining intensity between cisplatin-sensitive and -insensitive tissues from OSCC patients, there was a positive correlation between IGFBP3 and GATA1 expression and cisplatin sensitivity in OSCC tissues from patients. Conclusion: These results provide insights for overcoming cisplatin resistance, especially concerning EVs within the tumor microenvironment. Furthermore, it is anticipated that the expression levels of GATA1 and miR-876-3p, along with IGFBP3, could aid in the prediction of cisplatin resistance.


Figure S2 .
Figure S2.Effect of IGFBP3 overexpression on cisplatin sensitivity in CAF-P CM. (A)In control CM from FaDu or UMSCC1, OSCC cells were transfected with an IGFBP3 overexpression vector for 24 h and treated with cisplatin for another 24 h.The pCMV3 vector was used as a control vector.mRNA and protein expression of IGFBP3 was analyzed.(B) Cell viability was evaluated using MTT assay under the same condition.(C) FaDu and UMSCC1 cells were transfected with an IGFBP3 overexpression vector for 24 h, followed by medium change with each CAF-P CM.After 16 h, cisplatin was added for another 24 h, and IGFBP3 mRNA expression was analyzed.(D) Cell viability was evaluated under the same condition using an MTT assay.(E) FaDu and UMSCC1 spheroids were formed in 96-well Ubottom ultra-low attachment plates for two days.After transfection with IGFBP3 overexpression vector for 24 h, the medium was replaced with CAF-P CM.After 16 h, cisplatin was added for another 14 days, and IGFBP3 expression was analyzed on the mRNA and

Figure S3 .
Figure S3.IHC and IF analysis of mouse tumor tissues.(A) Tissue sections were stained with anti-Ku80 antibody to determine whether these are derived from human cells.Representative Ku80-positive fibroblast cells were indicated with red arrows.Tissues were counterstained with H&E.(B) IF staining of mouse tumor tissues was performed with the KRT13 antibody to confirm squamous epithelial cell origin.(C) The mRNA and protein expression of IGFBP3 in the xenografts were evaluated via qPCR analysis and IF staining, respectively.Results were presented as the mean ± standard deviation of three experiments.*p < 0.05; **p < 0.01.

Figure S4 .
Figure S4.Effect of CAF-derived EV on cisplatin resistance in OSCC cells.(A)Two different CAF-P cell lines were cultured with GW4869, an inhibitor of EV production, for 2 days, followed by CM collection.Cell viability was compared between DMSO-treated CM and GW4869-treated CM after cisplatin treatment for 24 h.CM from OSCC cells were compared as controls.(B) EV-loaded IGFBP3 protein level was analyzed via western blot analysis.The level of CD63, a representative EV marker, was analyzed under the same condition.(C) Spheroids were formed in 96-well U-bottom ultra-low attachment plates for two days, followed by EV treatment for 16 h (1 ͯ 10 7 particles/mL, MOI = 100).Cisplatin was added for another 14 days.Spheroids were imaged using phase-contrast microscopy, and their size (surface

Figure S5 .
Figure S5.IGFBP3 siRNA-or overexpression vector-dependent cisplatin sensitivity and mRNA expression in organoids.(A) The effect of IGFBP3 knockdown on cisplatin sensitivity in FaDu and UMSCC1 organoids without any CM treatment was compared as a control.(B) The effect of IGFBP3 overexpression on cisplatin sensitivity in FaDu and UMSCC1 organoids without any CM treatment was compared as a control.(C) Organoids derived from FaDu or UMSCC1 xenografts were transfected with siIGFBP3 for 24 h, followed by CAF-D EV treatment (MOI = 100) for another 16 h.After seven days of cisplatin treatment, IGFBP3 mRNA expression was compared via qPCR.(D) IGFBP3 overexpression vectorpretreated organoids treated with cisplatin for seven days were analyzed for IGFBP3 mRNA expression via qPCR.Results are presented as the mean ± standard deviation of three experiments.*p < 0.05; **p < 0.01.Scale bars: A-B 100 μm.

Figure S6 .
Figure S6.Effect of siGATA1 on cisplatin sensitivity in cells or spheroids under CAF-D EV treatment.(A)OSCC cells were transfected with siGATA1 for 24 h, followed by cisplatin treatment for another 24 h.The mRNA and protein expression of GATA1 was analyzed.(B) Cell viability was evaluated using MTT assay under the same condition.(C) OSCC cells were transfected with siGATA1 for 24 h, followed by CAF-D EV treatment (MOI = 100) for 16 h.After cisplatin treatment for another 24 h, cell viability was evaluated using MTT assay.(D) FaDu and UMSCC1 spheroids were formed via culture in 96-well U-bottom ultra-low attachment plates for two days.After transfection with siGATA1 for 24 h, CAF-D EV treatment was carried out for 16 h.After cisplatin treatment for another 14 days, mRNA and protein expression was analyzed.(E) Spheroids were imaged using phase-contrast microscopy, and their sizes were measured using Cell 3 iMager.Each experimental group consisted of eight spheroids, and a representative image is presented.Results were presented as the mean ± standard deviation of three experiments.*p < 0.05; **p < 0.01.Scale bars: D-E 500 μm.

Figure S7 .
Figure S7.siRNA-dependent reduction of GATA1 mRNA and protein expression in organoids.Organoids derived from OSCC xenografts were transfected with siGATA1 for 24 h, followed by CAF-D EV treatment (MOI = 100) for another 16 h.(A) After cisplatin treatment for seven days, GATA1 protein expression was compared via IF staining.(B) GATA1 mRNA expression was analyzed using qPCR under the same condition.Results are presented as the mean ± standard deviation of three experiments.Scale bars: A 100 μm.

Figure S8 .
Figure S8.Xenograft characterization and IF staining of GATA1 and IGFBP3.(A) mRNA and protein expression of IGFBP3 and GATA1 in xenografts were analyzed using qPCR and IF staining, respectively.(B) IHC analysis of mouse tumor tissues was performed with an antibody against Ku80 to evaluate whether these are derived from human cells.Tissues were counterstained with H&E.(C) IF staining with the anti-KRT13 antibody revealed squamous epithelial xenograft characteristics.

Table S4 . Clinicopathological parameters of OSCC tissues from patients used for IGFBP3 and GATA1 immunostaining
low attachment plates for two days.After transfection with siIGFBP3 for 24 h, the medium was replaced with CAF-D CM.After 16 h, cisplatin was added for another 14 days, and IGFBP3 expression was analyzed at the mRNA and protein level via qPCR and IF staining, respectively.*p < 0.05; **p < 0.01.Scale bars: E 500 μm.