Developmental and activation pathways underlying mesenchymal stromal cell recruitment and differentiation in the context of neoplasias

Nogueira Gezer, Melanie (2023): Developmental and activation pathways underlying mesenchymal stromal cell recruitment and differentiation in the context of neoplasias. Dissertation, LMU München: Medizinische Fakultät

Abstract

Solid tumors are a leading cause of mortality in adults across the world. Treatment options are often limited trough toxicity in non-target tissues. Mesenchymal stem cells (MSCs) present a tropism toward the inflammatory microenvironments present within solid tumors. Once there, they condition the tumor environment and differentiate into components of the tumor stroma. Our group had previously demonstrated the effectiveness of genetically engineered stem cells containing a therapeutic transgene, using the CCL5 promoter as a delivery vehicle for the treatment of solid tumors. CCL5/Rantes is a proinflammatory cytokine produced by a variety of cells including MSCs within tumor microenvironments. The CCL5 gene promoter can be activated by many factors including proinflammatory stimuli but varies with the cell type studied. The tumor-associated stimuli that activate the CCL5 promoter in MSCs was poorly understood. The aim of this thesis was to better understand the mechanisms of activation of CCL5 in stem cells, specifically in response to various signals present in the tumor microenvironment. A construct containing the CCL5 promoter driving a Gaussia luciferase reporter gene was created using Gateway cloning and a vector platform designed in house for the efficient stable integration of complex constructs into target cells. The CCL5 reporter construct was used to verify that the CCL5 promoter is activated by TNFα, but not significantly by IFN-γ; however, the combination of TNFα and IFN-γ showed a more than additive activity than the individual components. Neither hypoxia nor TGF-β, whether alone or in combination with other stimuli, activated the CCL5 promoter in the in vitro setting. We then developed synthetic variants of the promoter. 3AB, a synthetic promoter containing three tandem NFkB sequences taken from the immediate upstream region of the human CCL5 promoter [(R)AB region] and previously shown to be important for the functional activity of the promoter was generated. The 3AB synthetic promoter responded more effectively to TNFα and IFN- γ, with higher fold induction. It also showed a response to hypoxia that was increased in conjunction with TNFα and IFN-γ. TGF-β was not effective in the activation of 3AB. A second synthetic promoter MegaRantes, in which the R(AB) element in the native promoter was exchanged for the triplicate 3AB element, showed the same approximate pattern of activation as native CCL5, suggesting upstream regulating elements counteract any advantage derived from the 3AB modification. The results presented provide more complete information as to CCL5 is activation in MSCs and suggest that the synthetic construct 3AB, through its more specific activation, may represent an attractive candidate promoter for the delivery of therapeutic transgenes in the context of MSC-based tumor therapy.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Stem cells, Neoplasia, Tumor
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 15. Juni 2023
• 1. Bericht­erstatter:in: Nelson, Peter