Fig. 5.

The role of the Twist1-Prrx1-TNC PFL as a bistable switch was validated using an in vitro fibroblast line, cutaneous wound healing, and patient-derived CAFs. a Twist1 expression was induced in fibroblasts (MRC5) using a doxycycline-mediated induction system, and we examined the impact of the induced Twist1 on the Prrx1 and TNC expression profiles using multiplex immunohistochemistry (mIHC). Bimodal distribution of the triple gene expression, such as all positive vs. all negative, was evident after 7 days of induction. The insert images are enlarged picture of stained cells in each staining images. b We examined the Twist1, Prrx1, and TNC expressions during cutaneous wound healing using multiplex immunohistochemistry. The yellow box is a low power view of the mouse wound area. In this yellow box, dashed circle and arrow indicate area of interest (AOI) which is shown as a high power view. c Quantitative analysis of Twist1, Prrx1, and TNC expression in fibroblasts during wound healing. After we made circle-full-thickness cutaneous wounds (diameter of 5 mm) on the dorsal skin of mice, we determined the Twist1, Prrx1, and TNC expression in fibroblasts at multiple time points using mIHC. We noted highly dynamic changes in these three genes’ expression in fibroblasts. d The plot is generated based on data derived from Fig. 5b. Fibroblasts that were positive for all three genes were restricted to the granulation tissue formation stage of wound healing. e The GFP reporter, under the control of a Twist1 promoter, was stably integrated into the chromosome of two patient-derived CAFs. Then, we sorted CAFs into Twist1-positive and Twist1-negative CAFs, using flow cytometry. f Esophageal cancer-derived CAFs (ECAF#8) were sorted into Twist1-positive and Twist1-negative CAFs. Twist1-positive CAFs were specifically positive for Prrx1 and TNC and also exhibited increased colony-forming abilities. g The Twist1-high CAF (ECAF#8) showed resistance to doxorubicin-induced apoptosis