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Apr 15, 2014 (Vol. 34, No. 8)

Cellular Models for Finer Screening

  • The cost of bringing a new drug to the market has recently been estimated by Forbes Magazine at $5 billion. This figure derives in large part from high attrition rates in the drug development pipeline that arise from failures in drug efficacy or adverse toxic events, such that in excess of 90% of all small molecules that enter Phase I testing are ultimately not approved.

    Accordingly, significant effort is being invested in developing new in vitro models that more accurately recapitulate the effect of small molecule drug candidates on cellular pathways and processes in vivo.
    At “Physiologically Relevant Cellular Models for Drug Discovery,” a CHI conference held recently in La Jolla, CA, several speakers described advances in the characterization of cell-based platforms for drug screening, and in the use of pathway analysis to model the effect of drug candidates on critical cellular processes.
    While convenient, traditional two-dimensional (2D) monolayer cell culture techniques fail to fully recapitulate the mechanical and chemical gradients to which cells are exposed in the body, and which regulate pathways controlling cell proliferation, polarity, and differentiation. As a result, effects that a drug candidate might have in the context of such gradients might not be apparent until the drug is tested in vivo. One solution to this issue is the use of 3D hanging drop spheroids, in which cells suspended in a droplet settle at the apex of the droplet and establish appropriate cell-cell interactions.
  • Collagen Contraction

    “The major advantage of using 3D spheroids over 2D monolayer cultures as a drug discovery model is that its geometry and avascular nature mimics the transport barrier of native tissue, where drugs and other molecules experience diffusion limitation, as they get farther away from blood vessels,” said Shiuchi Takayama, Ph.D., professor of biomedical engineering at the University of Michigan.

    “The gradients that are established in the cellular microenvironments in the 3D spheroids produce drug response profiles that more closely resemble in vivo responses compared to flat, 2D monolayer cultures,” Dr. Takayama added. Collagen contraction is one of the processes by which cells remodel the surrounding extracellular matrix, which is known to be an important feature of disease progression.

    Dr. Takayama’s group has developed a 3D spheroid-based collagen contraction assay that, by miniaturizing the conventional 2D assay, more closely models in vivo diffusion of nutrients and soluble cues. In addition, the assay is more amenable to the high throughput required for efficient drug screening.

    Cell-mediated contraction of the collagen matrix is known to be regulated by transforming growth factor-β (TGF-β), which signals via the Smad pathway to modulate the expression of downstream effectors such as connective-tissue growth factor. “We found that brief bursts of TGF-β in the 3D spheroid assay prompted contraction of the matrix, a phenomenon we did not observe in the conventional 2D assay,” remarked Dr. Takayama. Though such modeling of cell-mediated contraction, the 3D assay can  more accurately contextualize in vivo biological processes.

  • Tumor Spheroids

    Click Image To Enlarge +
    In assays run by Janssen Research and Development, cMET but not EGFR inhibitors reduced cell migration in hepatocyte growth factor (HGF)-stimulated lung tumor spheroids. As shown in these representative images, crizotinib inhibits cell migration pathways in HGF-stimulated lung tumor spheroids. Day four 3D lung spheroids were treated with crizotinib or compound in the presence of 20 ng/mL of HGF for 48 hours to stimulate cell migration pathways. Total area of migrating and spheroid were determined by using bright-field images in a high-throughput Operetta high-content imaging system (PerkinElmer). Magnification: 2× objective.

    Fredika A. Robertson, Ph.D., executive director for clinical research services at Virginia Commonwealth University’s Center for Clinical and Translational Research, described the development of a tumor spheroid system for small compound screening. This system was developed using tumor cells isolated from the pleural fluid of breast cancer patients.

    “These freshly isolated metastatic tumor cells spontaneously form 3D multilayered tumor spheroids, and have several advantages over 2D monolayer cultures for screening,” Dr. Robertson pointed out. These advantages include the enrichment in the 3D spheroids of cancer stem cells or tumor-initiating cells expressing markers such as CD44+, ALDH-1+, and CD133, as well as the recapitulation by the spheroids of the necrotic center that exists in tumors beyond 1–2 mm in size.

    “We found that our 3D tumor spheroid system mimics activation of multiple cellular signaling pathways, allowing us to identify the activation of specific signaling pathways in breast metastasis,” said Dr. Robertson. With her group, Dr. Robertson showed that the receptor tyrosine kinase anaplastic lymphoma kinase (ALK) is activated in preclinical models of inflammatory breast cancer, where it signals through a variety of downstream pathways, including JAK1/STAT3, AKT, and mTOR.

    “We found that the ALK inhibitor, Crizotinib [Xalkori®, Pfizer] effectively eradicates the 3D tumor spheroids from patients with ALK+ tumors,” Dr. Robertson pointed out. “This is an example of how the identification of the biological signaling pathways that are activated in our patient-derived 3D tumor spheroid systems directly led to clinical trials using this targeted therapeutic.”

    Tumor spheroids were also the subject of a presentation by Jason Ekert, Ph.D., senior research scientist at Janssen Research and Development. Dr. Ekert described efforts to develop a tumor model that more faithfully recapitulated the role of cancer signaling pathways in patient tumors.

    Pathways involving the transmembrane receptor kinases epidermal growth factor receptor (EGFR) and cMET are involved in the transduction of growth factor signals to the cell, and are thought be active in a large number of lung tumors.

    “We were interested in better understanding the differences between 2D monolayer and 3D spheroid lung tumor cultures in the context of the EGFR-cMET cancer biology pathway,” explained Dr. Ekert. “Our flow cytometry data told us that EGFR and cMET expression was reduced in spheroid cultures compared to monolayer cultures, and that basal phosphorylation of EGFR and cMET was higher in spheroid cultures.”

    Dr. Ekert suggests that such subtle microenvironment-induced changes in lung tumor cell physiology add a level of complexity to cell-based assays that may be more representative of the in vivo tumor microenvironment, thereby improving the predictive capability of screening assays. He went on to highlight the ability of the 3D system to discriminate between different EGFR and cMET mutants that inhibited cell migration and proliferation.

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