Liquid-based three-dimensional tumor models for cancer research and drug discovery

Abstract

Tumors are three-dimensional tissues where close contacts between cancer cells, intercellular interactions between cancer and stromal cells, adhesion of cancer cells to the extracellular matrix, and signaling of soluble factors modulate functions of cancer cells and their response to therapeutics. Three-dimensional cultures of cancer cells overcome limitations of traditionally used monolayer cultures and recreate essential characteristics of tumors such as spatial gradients of oxygen, growth factors, and metabolites and presence of necrotic, hypoxic, quiescent, and proliferative cells. As such, three-dimensional tumor models provide a valuable tool for cancer research and oncology drug discovery. Here, we describe different tumor models and primarily focus on a model known as tumor spheroid. We summarize different technologies of spheroid formation, and discuss the use of spheroids to address the influence of stromal fibroblasts and immune cells on cancer cells in tumor microenvironment, study cancer stem cells, and facilitate compound screening in the drug discovery process. We review major techniques for quantification of cellular responses to drugs and discuss challenges ahead to enable broad utility of tumor spheroids in research laboratories, integrate spheroid models into drug development and discovery pipeline, and use primary tumor cells for drug screening studies to realize personalized cancer treatment.

Keywords: 3D tumor models; anti-cancer drug discovery; cancer cell spheroids; cancer stem cells; co-culture spheroids; tumor microenvironment.

Figure 1

Tumor is a complex heterocellular microenvironment where cancer cells are in constant communications with the stromal cells, the extracellular matrix, and biochemical signaling molecules. Tumor stroma plays a major role in regulating functions of cancer cells and their responses to therapeutic compounds. (Adapted from Joyce JA and Pollard JW, Nat Rev Cancer 2009). (A color version of this figure is available in the online journal.)

Figure 2

Three major liquid-based methods of culture of cancer cell spheroids are shown (a) Rotary wall vessel and spinner flask systems use rotary devices to constantly keep cells in suspension to aggregate into spheroids of random size. (b) Hanging drop array method uses gravitational-mediated aggregation of cells at the apex region of drops hanging from a plate containing through-holes and micro-rings to result in one spheroid per drop. (c) Aqueous two-phase system (ATPS) method uses two immiscible polymeric aqueous phases, where cancer cells are confined in the drop phase surrounded by the immiscible immersion phase. Cells spontaneously aggregate to form a compact spheroid of well-defined size. Panel a is reproduced from Molecular & Cancer Therapeutics 2007;6:2505–14. (A color version of this figure is available in the online journal.)

Figure 3

(a) Concentration gradients of oxygen, nutrients, and metabolites generate distinct concentric zones in spheroids: An outer zone containing proliferative cells, a middle zone with quiescent cells, and an inner zone containing necrotic cells. Abundance of oxygen and glucose at the outer zone and efficient removal of waste products facilitate cell proliferation, whereas low oxygen levels and a buildup of toxic metabolites such as carbon dioxide and lactate generate a necrotic core. (b,c) Development of hypoxia and anoxia is shown in spheroids. Sections taken from spheroids grown for (b) four days and (c) six days stained for the proliferation marker Ki-67 (green) and hypoxia (purple). On day 4 of growth, central hypoxia is observed and on day 6 of culture, an anoxic core develops. Panels b and c are reproduced from Journal of The Royal Society Interface, 20146;11: 20131124. (A color version of this figure is available in the online journal.)

Figure 4

(a) Tumors contain a subpopulation of cells, cancer stem cells (CSCs), with the ability to initiate and regenerate tumors. Conventional chemotherapy targets rapidly dividing cells and considerably diminishes the tumor. However, unaffected CSCs repopulate the tumor growth and result in recurrence of cancer, which is often resistant to therapies. Novel therapies that target CSCs are critical to eliminate tumorigenic cells and potentially eradicate the cancer. (b,c) Expression of CSCs markers CD44 and ALDH1 in HT29 colorectal tumor spheroids detected using immunostaining of sections of spheroids. Panels b and c are reproduced with permission from BMC Cancer, 2010;10:106. (A color version of this figure is available in the online journal.)

Figure 5

Workflow of anti-cancer drug discovery. (A color version of this figure is available in the online journal.)

Figure 6

High throughput dose-dependent testing of paclitaxel against monolayer and spheroids of (a) A431.H9 skin cancer cells and (b) MDA-MB-157 triple negative breast cancer cells generated using the aqueous two-phase technology. (a) 2D cultures of A431.H9 cells (triangles) show a sigmoidal response with an LD₅₀ value of 22.1 nM, whereas cells in spheroid culture (circles) show greater resistance and result in an LD₅₀ value of 178.5 nM. (b) Monolayer of MDA-MB-157 cells (triangles) also shows a sigmoidal response with an LD₅₀ value of 8.0 nM. In contrast, these cells in spheroid culture (circles) show complete resistance to paclitaxel. Error bars indicate standard error of the mean. Dashed lines represent sigmoidal fit generated using GraphPad Prism