Fig 2: 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

Fig 3: Sustained switching ON of the Twist1-Prrx1-TNC PFL triggers persistent fibroblast activation, forming fibrotic nodules in vivo during wound healing. a Schematic diagram of the experimental design. We injected TNC protein or PBS (control) into the wound area of WT and fibroblast-specific Twist1 knockout mice. The experiments were conducted three times (N = 5 mice in each experimental group). Experimental timeline for the detailed procedures is indicated. b The maintenance of the PFL activation by TNC injection (FSP1-cre; Twist1 WT mice, left) and disruption of PFL by fibroblast-specific Twist1 deficiency despite the TNC injection (FSP1- Cre;Twist1fl/fl mice, right). c H&E staining images showing the wound areas of each group (top). In Twist1 WT mice (depicted as iii, FSP1-cre; Twist1 WT), we observed fibroblastic nodules (red arrow) comprising hyper-proliferative immature fibroblasts and epithelial cell hyperplasia (blue dotted line). TNC is strongly expressed in most fibroblastic nodules as mentioned above in which Twist1 and Prrx1 are also strongly coexpressed. These results were obtained through multiplex IHC staining and visualized using ImageJ Fiji (Twist1-green, Prrx1-red, TNC-yellow, and hematoxylin-blue). We performed quantitative analysis of these results using FCS Express 6 image cytometry analysis software, and we presented the outcomes as an FACS plot (x axis-Twist1 green, y axis-Prrx1 red and contour indicated with TNC) (bottom). The number of fibroblasts displaying Twist1/Prrx1 double positivity in the TNC- or PBS-injected wound areas (150 µm × 150 µm square) is indicated by box plot (bottom right of Fig. 6c). Data are presented as the mean ± SEM; N = 6 independent measurements (two-tailed t test: **p<0.005, ***p<0.001)

Fig 4: Summary of our study. The Twist1-Prrx1-TNC positive feedback regulation provides clues for understanding the activation of fibroblasts during wound healing under normal conditions, as well as abnormally activated fibroblasts in pathological conditions such as cancerous and fibrotic diseases. Under normal conditions, the PFL acts as a reversible bistable switch by which the activation of fibroblasts is “ON" above a sufficient level of stimulation and “OFF" for the withdrawal of the stimulus. However, this switch can be permanently turned on under pathologic conditions by continued activation of the PFL, resulting in sustained proliferation of fibroblasts

Fig 5: High TNC is essential for activating the irreversible PFL. a In the fibrotic nodule generated by exogenous TNC, we observed a TNC concentration gradient (top). In-depth analysis of this nodule using multiplex IHC showed that Twist1 and Prrx1 were strongly positive and coexpressed in most fibroblasts within strongly TNC-positive zone1, and this pattern disappeared as the TNC concentration decreased (bottom left) (Twist1-green, Prrx1-red, TNC-yellow and hematoxylin-blue). We measured each integrated density per zone representing the expression level of each gene using ImageJ Fiji (bottom right). Data are presented as the mean ± SEM; N = 3 independent measurements (two-tailed t test: *p<0.05, **p<0.001, ***p<0.0001). b We treated mouse dermal fibroblasts isolated from fresh Balb/c mouse tissue with TNC at different concentrations (0, 0.1, 1, 2, and 5 µg) and then evaluated Twist1 and TNC expression by western blotting (top). We estimated the kinetic parameter values of the mathematical model based on this western blotting data on the Twist1 and TNC fold changes along with the increase of exogenous TNC (bottom). Discrete data marked with squares indicate experimental measurements of the corresponding genes (N = 3, error bars indicate s.d.). c Phase diagram illustrating the irreversibility of TNC (represented by color intensity) with respect to the feedback strength (V) and the TNC level for feedback activation (Km1, the Michaelis-Menten constant) (top left). The hysteretic responses of TNC to the TNC injection in the irreversible ((Km1, V) = (5, 30)) and reversible ((Km1, V) = (10, 15)) states are shown in the top right and bottom left figures, respectively. Given that the stimulus increases from zero to TNCmax and then decreases back to zero, we compared the irreversibility of TNC, ?TNC, with that of the reversible state (bottom right). The inlet shows examples of obtaining ?TNC for the TNCmax