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. 2021 Feb 6;13(4):654. doi: 10.3390/cancers13040654

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© 2021 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Effect of CAF-P and CAF-D on Matrigel invasion of FaDu spheroids. (A) To evaluate the effect of CAF-derived CM on 3D FaDu spheroid invasion, spheroid formation was performed in 96-well U-bottom Ultra-Low Attachment plate for two days (400 µm in diameter), followed by medium change with CM and Matrigel addition. (B) The spheroid invasion under CM from CAF-P was monitored for 14 days using phase-contrast microscopy (5× magnification). (C) Cell invasion was quantified by measuring the mean number of tube-like structures extending from the surface of each spheroid. The spheroid size was quantified using Cell³iMager. (D) MMPs mRNA expression in FaDu spheroids was analyzed using qPCR on day 14. mRNA expression of representative EMT markers was also compared at the same condition. (E–G) Same experiments were performed with CM derived from CAF-D. Results represent the mean ± standard deviation of 2–3 experiments (* p < 0.05, ** p < 0.01, *** p < 0.005).

Effect of CAF-P and CAF-D on Matrigel invasion of FaDu spheroids. (A) To evaluate the effect of CAF-derived CM on 3D FaDu spheroid invasion, spheroid formation was performed in 96-well U-bottom Ultra-Low Attachment plate for two days (400 µm in diameter), followed by medium change with CM and Matrigel addition. (B) The spheroid invasion under CM from CAF-P was monitored for 14 days using phase-contrast microscopy (5× magnification). (C) Cell invasion was quantified by measuring the mean number of tube-like structures extending from the surface of each spheroid. The spheroid size was quantified using Cell³iMager. (D) MMPs mRNA expression in FaDu spheroids was analyzed using qPCR on day 14. mRNA expression of representative EMT markers was also compared at the same condition. (E–G) Same experiments were performed with CM derived from CAF-D. Results represent the mean ± standard deviation of 2–3 experiments (* p < 0.05, ** p < 0.01, *** p < 0.005).