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The tumor microenvironment (TME) comprises a complex milieu of different cell types, including cancer associated fibroblasts (CAFs) and immune cells, blood vessels, and the extracellular matrix. Through its interaction with cancer cells, it plays an essential role in cancer invasion and metastasis. The inherent complexity of the TME presents a challenge to study it within experimental model systems. It underscores the importance of complementing such research with observation from human tumor tissues, wherein this intricate complexity is preserved. In Paper IV, we introduce a new software designed to explore the Human Protein Atlas, an online database that includes image data on the protein expression across normal and cancerous tissues from immunohistochemically (IHC) stained tissues. In Paper I, we use this software to identify 12 novel proteins expressed in cancerassociated fibroblasts, four revealing connections to Rho-kinase signaling. We contrast their expression across various tumors and against normal tissue fibroblasts, uncovering expression variability among cancer types and confirm their similarities with the myofibroblastic phenotype. In Paper II, we explore the expression of the proteoglycan Decorin, abundantly present in normal connective tissue and having tumor inhibitory properties, showing its downregulation in the connective tissue surrounding tumors. In Paper III, based on our observations in Paper I of the connection of Rhosignaling in CAFs, we study the effects of knocking out the related RhoA in fibroblasts both in vitro and in vivo models. We demonstrate that the knockout fibroblasts compromise their tumor inhibitory capacity, enhancing cancer cell growth, migration, and metastasis. In Paper VI, we develop a new method for analyzing the extensive data within the Human Protein Atlas by developing a deep-learning-based image classifier. Utilizing a limited training image set, we classify all images available for the prostate, identifying 44 new

Fibroblasts are a main player in the tumor-inhibitory microenvironment. Upon tumor initiation and progression, fibroblasts can lose their tumor-inhibitory capacity and promote tumor growth. The molecular mechanisms that underlie this switch have not been defined completely. Previously, we identified four proteins over-expressed in cancer-associated fibroblasts and linked to Rho GTPase signaling. Here, we show that knocking out the Ras homolog family member A (RhoA) gene in normal fibroblasts decreased their tumor-inhibitory capacity, as judged by neighbor suppression in vitro and accompanied by promotion of tumor growth in vivo. This also induced PC3 cancer cell motility and increased colony size in 2D cultures. RhoA knockout in fibroblasts induced vimentin intermediate filament reorganization, accompanied by reduced contractile force and increased stiffness of cells. There was also loss of wide F-actin stress fibers and large focal adhesions. In addition, we observed a significant loss of a-smooth muscle actin, which indicates a difference between RhoA knockout fibroblasts and classic cancer-associated fibroblasts. In 3D collagen matrix, RhoA knockout reduced fibroblast branching and meshwork formation and resulted in more compactly clustered tumor-cell colonies in coculture with PC3 cells, which might boost tumor stem-like properties. Coculturing RhoA knockout fibroblasts and PC3 cells induced expression of proinflammatory genes in both. Inflammatory mediators may induce tumor cell stemness. Network enrichment analysis of transcriptomic changes, however, revealed that the Rho signaling pathway per se was significantly triggered only after coculturing with tumor cells. Taken together, our findings in vivo and in vitro indicate that Rho signaling governs the inhibitory effects by fibroblasts on tumor-cell growth.

Decorin is a small leucine-rich proteoglycan, synthesized and deposited by fibroblasts in the stroma where it binds to collagen I. It sequesters several growth factors and antagonizes numerous members of the receptor tyrosine kinase family. In experimental murine systems, it acted as a potent tumor suppressor. Examining the Human Protein Atlas online database of immunostained tissue samples we have surveyed decorin expression in silico in several different tumor types, comparing them with corresponding normal tissues. We found that decorin is abundantly secreted and deposited in normal connective tissue but its expression is consistently decreased in the tumor microenvironment. We developed a software to quantitate the difference in expression. The presence of two closely related proteoglycans in the newly formed tumor stroma indicated that the decreased decorin expression was not caused by the delay in proteoglycan deposition in the newly formed connective tissue surrounding the tumor.