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.

Spheroids present a biologically relevant three-dimensional model of avascular tumors and a unique tool for discovery of anticancer drugs. Despite being used in research laboratories for several decades, spheroids are not routinely used in the mainstream drug discovery pipeline primarily due to the difficulty of mass-producing uniformly sized spheroids and intense labor involved in handling, drug treatment, and analyzing spheroids. We overcome this barrier using a polymeric aqueous two-phase microtechnology to robotically microprint spheroids of well-defined size in standard 384-microwell plates. We use different cancer cells and show that resulting spheroids grow over time and display characteristic features of solid tumors. We demonstrate the feasibility of robotic, high-throughput screening of 25 standard chemotherapeutics and molecular inhibitors against tumor spheroids of three different cancer cell lines. This screening uses over 7000 spheroids to elicit high quality dose-dependent drug responses from spheroids. To quantitatively compare performance of different drugs, we employ a multiparametric scoring system using half-maximum inhibitory concentration (IC), maximum inhibition (E), and area under the dose-response curve (AUC) to take into account both potency and efficacy parameters. This approach allows us to identify several compounds that effectively inhibit growth of spheroids and compromise cellular viability, and distinguish them from moderately effective and ineffective drugs. Using protein expression analysis, we demonstrate that spheroids generated with the aqueous two-phase microtechnology reliably resolve molecular targets of drug compounds. Incorporating this low-cost and convenient-to-use tumor spheroid technology in preclinical drug discovery will make compound screening with realistic tumor models a routine laboratory technique prior to expensive and tedious animal tests to dramatically improve testing throughput and efficiency and reduce costs of drug discovery.

Solid tumors develop as three-dimensional tissue constructs. As tumors grow larger, spatial gradients of nutrients and oxygen and inadequate diffusive supply to cells distant from vasculature develops. Hypoxia initiates signaling and transcriptional alterations to promote survival of cancer cells and generation of cancer stem cells (CSCs) that have self-renewal and tumor-initiation capabilities. Both hypoxia and CSCs are associated with resistance to therapies and tumor relapse. Here, we conduct a systematic study to demonstrate that 3D cancer cell models, known as tumor spheroids, generated with a polymeric aqueous two-phase system (ATPS) technology capture these important biological processes. Similar to solid tumors, spheroids of triple negative breast cancer (TNBC) cells deposit major extracellular matrix proteins. Our molecular analysis establishes presence of hypoxic cells in the core region and expression of CSC gene and protein markers including CD24, CD133, and Nanog. Importantly, these spheroids resist treatment with chemotherapy drugs. We successfully target drug resistant spheroids through a combination treatment approach using a hypoxia-activated prodrug, TH-302, and a chemotherapy drug, doxorubicin. This study demonstrates that ATPS spheroids recapitulate important biological and functional properties of solid tumors and provide a unique model for studies in cancer research.

Cancer is one of the leading causes of death globally according to the World Health Organization. Although improved treatments and early diagnoses have reduced cancer related mortalities, metastatic disease remains a major clinical challenge. The local tumor microenvironment plays a significant role in cancer metastasis, where tumor cells respond and adapt to a plethora of biochemical and biophysical signals from stromal cells and extracellular matrix (ECM) proteins. Due to these complexities, there is a critical need to understand molecular mechanisms underlying cancer metastasis to facilitate the discovery of more effective therapies. In the past few years, the integration of advanced biomaterials and microengineering approaches has initiated the development of innovative platform technologies for cancer research. These technologies enable the creation of biomimetic in vitro models with physiologically relevant (i.e. in vivo-like) characteristics to conduct studies ranging from fundamental cancer biology to high-throughput drug screening. In this review article, we discuss the biological significance of each step of the metastatic cascade and provide a broad overview on recent progress to recapitulate these stages using advanced biomaterials and microengineered technologies. In each section, we will highlight the advantages and shortcomings of each approach and provide our perspectives on future directions.

Cell migration is a major process that drives metastatic progression of cancers, the major cause of cancer death. Existing chemotherapeutic drugs have limited efficacy to prevent and/or treat metastasis, emphasizing the need for new treatments. We focus on triple negative breast cancer (TNBC), the subtype of breast cancer with worst prognosis and no standard chemotherapy protocols. Here we demonstrate that a group of natural compounds, known as phytochemicals, effectively block migration of metastatic TNBC cells. Using a novel cell micropatterning technology, we generate consistent migration niches in standard 96-well plates where each well contains a cell-excluded gap within a uniform monolayer of cells. Over time, cells migrate into and occupy the gap. Treating TNBC cells with non-toxic concentrations of phytochemicals significantly blocks motility of cells. Using a molecular analysis approach, we show that anti-migratory property of phytochemicals is partly due to their inhibitory effects on phosphorylation of ERK1/2. This study provides a framework for future studies to understand molecular targets of phytochemicals and evaluate their effectiveness in inhibiting metastasis in animal models of cancer.