Summary Expression of the initiator methionine tRNA (tRNA i Met ) is deregulated in cancer. Despite this fact, it is not currently known how tRNA i Met expression levels influence tumor progression. We have found that tRNA i Met expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNA i Met in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNA i Met contributes to tumor progression, we generated a mouse expressing additional copies of the tRNA i Met gene (2+tRNA i Met mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNA i Met mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNA i Met mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNA i Met significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNA i Met -overexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNA i Met -driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNA i Met mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNA i Met levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis. [ABSTRACT FROM AUTHOR]