• immunohistochemistry
• myofibroblast
• proteomics
• thymosin

During tumour development cancer cells secrete growth factors (such as TGF-β1) into the extra cellular matrix. Herein, fibroblasts are activated by TGF-β1 and transition to cancer associate fibroblasts (CAFs) or myofibroblasts, characterized by the expression of α-smooth muscle actin (α-SMA). These cells promote tumour progression through specific communication with cancer cells. It has previously been shown that treatment with thymosin β4 (Tβ4) can prevent the fibroblast to myofibroblast transition, because its of ability to affect the degree of actin polymerization. Moreover, fibroblasts, myofibroblasts and myofibroblasts treated with Tβ4 are characterized by different thymosin proteoforms. Mass spectrometry-based on top-down proteomics identified Tβ4, Tβ10 and shorter-proteoforms in all cellular states. Relative quantification indicated that myofibroblast differentiated by TGF-β1 was associated with a larger Tβ10*/Tβ10 ratio (where Tβ10* denotes shortened proteoforms) than fibroblasts but lower than myofibroblasts treated with Tβ4. In the latter case shorter-Tβ4 proteoforms were also identified.