Organoid models of KRAS mutant cancer

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

BACKGROUND. Traditional cell culture method of growing epithelial cancer cells as a 2D monolayer on a stiff, adherent surface fails to capture the transformed phenotype of these cells. Indeed, a key hallmark that distinguishes cancer from normal epithelial cells is their ability to grow under anchorage-independent conditions. Alternatively, cancer cells that can be cultured under 3D anchorage-independent conditions, and under such conditions they often form a spheroid that mimic a microscope tumor. Such tumor spheroid culture has been difficult to scale up in the past, thus limiting their utility. A method to grow tumor spheroids on a larger scale with lower cost will enable the wider adoption of this model for in vitro studies. Recent development in organoid culture has made it possible to grow primary epithelial cells and maintain their lineage identify in vitro. In the lung, evidence suggests that alveolar type 2 (AT2) cells, which are the resident stem cells of the alveolar space, are also the cell of origin for lung adenocarcinoma. A scalable, murine lung organoid model of normal and KRAS mutant AT2 cells will provide better resemblance of lung cancer cell in vivo. These advanced cell models will be valuable for dissecting the function of the KRAS oncogene in the context of anchorage-independent cell growth. OBJECTIVES. 1) Develop a scalable tumor spheroid culture method for human cancer cells and elucidate the function of the KRAS oncogene in anchorage-independent cell growth and survival; 2) Develop a scalable, murine lung organoid model of KRAS mutant cells and investigate the function of the KRAS oncogene in the oncogenic transformation of lung epithelial cells; and 3) Use mouse xenograft and orthotopic transplant models to validate discoveries from organoid models. MAJOR ACTIVITIES, SIGNIFICANT RESULTS AND KEY OUTCOMES. 1) Elucidating KRAS oncogene function under anchorage-independent conditions. We have developed a tumor spheroid culture method that can be applied to many epithelial KRAS mutant cancer cell lines. Importantly, this culture method is scalable, and it is amenable to molecular and genetic dissection of cellular phenotypes. Using this system, we have identified features of KRAS oncogene signaling that is distinct from those in 2D cell culture. We are investigating the functional impact of these 3D-specific signaling from the KRAS oncogene and dissecting how they contribute to cellular transformation. 2) Using murine AT2 organoid to model lung adenocarcinoma in vitro. We have established organoid culture of KRAS mutant murine AT2 cells to study the biology of KRAS mutant lung adenocarcinoma. These organoids stably maintain their AT2 lineage markers, exhibit features of transformation, and are addicted to the KRAS oncogene for their growth. We are using this system to understand how mutant KRAS impact signaling, gene expression and stemness in AT2 cells. We are developing organoid culture of normal AT2 cells to understand how targeted therapies affect their viability.