The utility of 3D models to study cholesterol in cancer: Insights and future perspectives

Abstract

Cholesterol remains a vital molecule required for life; however, increasing evidence exists implicating cholesterol in cancer development and progression. Numerous studies investigating the relationship between cholesterol and cancer in 2-dimensional (2D) culture settings exist, however these models display inherent limitations highlighting the incipient need to develop better models to study disease pathogenesis. Due to the multifaceted role cholesterol plays in the cell, researchers have begun utilizing 3-dimensional (3D) culture systems, namely, spheroids and organoids to recapitulate cellular architecture and function. This review aims to describe current studies exploring the relationship between cancer and cholesterol in a variety of cancer types using 3D culture systems. We briefly discuss cholesterol dyshomeostasis in cancer and introduce 3D in-vitro culture systems. Following this, we discuss studies performed in cancerous spheroid and organoid models that focused on cholesterol, highlighting the dynamic role cholesterol plays in various cancer types. Finally, we attempt to provide potential gaps in research that should be explored in this rapidly evolving field of study.

Keywords: 3D culture; cancer; cholesterol; organoids; spheroids.

Figure 1

Cholesterol as a Contributing Factor to Drug Resistance. Molecular mechanisms of drug resistance in cancer include tumors having enhanced drug efflux capabilities, drug target mutations, hyperactivation of survival pathways, increased metabolism of xenobiotics leading to drug detoxification and concomitant inactivation can cause drug resistance in cancer. Additionally inherited genetic instability, epithelial-mesenchymal transition (EMT), inactivation of apoptotic pathways and mutagenicity of tumor cells. Cholesterol is also a contributing factor due to altered cholesterol metabolism that can cause increased lipid mediators that retain proliferative signaling, increased tumor growth, enhanced risk of metastasis and increased cancer aggressiveness.

Figure 2

Deregulated Cholesterol Homeostasis in Cancer. (A) In healthy cells, normal cholesterol synthesis is required for various metabolic requirements. Cholesterol synthesis is initiated as citrate is converted to acetyl-coenzyme A and this is converted to lanosterol in a series of reactions that occur in the endoplasmic reticulum. Sterol regulatory element binding proteins (SREBPs) are a group of transcription factors that are responsible for regulating lipogenesis and lipid uptake. When cholesterol levels within the cells are low, cholesterol biosynthesis will be induced by SREBP2 and the lipid isoform (nSREBP2) will bind to the sterol response elements to trigger the expression of lipogenesis target genes, which include HMGCR, LDLR and PCSK-9. HMGCR catalyzes the rate-limiting step in cholesterol biosynthesis and LDLR imports cholesterol from the cellular environments, of which both will result in increased sterol levels within the cell. SREBP cleavage-activating protein (SCAP) is responsible for controlling intracellular biosynthesis of cholesterol, fatty acids and triglycerides whereas PCSK-9 is responsible for regulating LDLR by lysosomal degradation or inhibition of endocytic recycling of LDLR. To prevent excess accumulation of cholesterol within cells, acyl-CoA:cholesterol acyltransferase (ACAT) will convert excess free cholesterol into cholesterol esters (CEs). CEs will be shuttled by cholesteryl ester transfer protein (CETP) to lipid droplets that may be stored. Additionally, ATP-binding cassette subfamily C member 1 will transport cholesterol to HDL where cholesterol may be eliminated instead of being stored and scavenger receptor, class B type 1 (SRB1) will mediate the selective uptake of HDL-derived CEs into cells as required. (B) In cancerous cells, abnormal cholesterol synthesis is necessary to maintain their metabolic requirements. Cancerous cells can upregulate cholesterol synthesis to allow for rapid cell division and growth. HMGCR and LDLR are upregulated to ensure sufficient cholesterol biosynthesis and uptake, respectively, while ACAT and CETP will also be upregulated to ensure the excess cholesterol is safely stored. Lipid rafts have also been shown to be upregulated in cancerous cells to potentially allow for drug resistance, metastasis, and development due to the role that lipid rafts play in signaling.

Figure 3

Spheroid Culture vs. Organoid Culture. (A) Spheroid culture. Spheroids are cultured by obtaining single cells from primary cells or from stable cell lines. The cells are naturally able to form aggregates and can do so in suspension culture. The aggregates will become more defined spheroids as the culture progresses. (B) Organoid culture. Tissue that is biopsied from patients will be digested into cells and the cells of interest can be harvested. These cells are then seeded in a ECM-mimicking substrate (BME), which will result in the formation of domes. The stem cells will be present in the domes and as the culture progresses it will give rise to organoids.

Figure 4

Summary of Cholesterol Related Research Utilizing 3D in-vitro Models. Based on literature, studies investigating the relationship between breast cancer and cholesterol, it was evident that increased cholesterol synthesis occurs via SREBP2 in organoids and that there is increased cholesterol biosynthesis gene expression in spheroids. The relationship between cholesterol synthesis and ovarian cancer has yet to be explored in organoids, but in a small study utilizing spheroids, it was shown that the ovarian cancer spheroids are dependent on cholesterol. In prostate cancer, the utilization of organoids has illustrated that cholesterol sulphate decreases SULT2B1b, which correlates to increased cancer progression and in spheroids it has illustrated increased expression of cholesterol biosynthesis genes and sterol biosynthesis genes. The use of intestinal organoids has shown that SQLE increases CRC progression, increased cellular cholesterol promoting ISC proliferation and increased ABCG1 promotes aggregation and growth of intestinal cancer cells, whereas spheroids have illustrated increased expression of cholesterol biosynthesis genes. In glioblastoma, the use of spheroids have illustrated increased expression of cholesterol biosynthesis genes and in organoids it was shown that there was increased expression of cholesterol homeostasis genes at the organoid core. In pancreatic cancer, organoids have been used to illustrate increased LDLR, SOAT1 sustains the mevalonate pathway and there is increased cholesterol esters present. In lung cancer, there are limited studies that explore the link between cholesterol and cancer, but a small study has illustrated that utilizing a cyclodextrin as a drug vehicle for delivery in cholesterol-rich erlotinib resistant small cell lung cancer cells can be a potentially useful therapeutic mechanism.