Decades of Discovery: How the NCI-60 Revolutionized Cancer Drug Screening

The National Cancer Institute’s NCI-60 drug screening panel, comprised of 60 diverse human cancer cell lines, has been a cornerstone in advancing cancer research and drug discovery since its inception in the late 1980s. Developed in response to the need for more predictive and comprehensive preclinical models, the NCI-60 facilitates the screening of thousands of compounds annually, aiming to identify potential anti-cancer drugs across a broad spectrum of human cancers. This article traces the origins, development, and evolution of the NCI-60 panel, highlighting its significant role in advancing our understanding of cancer and therapeutic agents.  


The NCI-60 is a panel of 60 human cancer cell lines used by the National Cancer Institute (NCI) for in vitro screening of up to 7,000 small molecules (synthetic or purified natural products) annually to identify potential anti-cancer drugs. This drug screening panel represents a diverse cross-section of human cancers, including leukemia, melanoma, and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney. The development and use of the NCI-60 panel has played a significant role in cancer research and drug discovery, providing valuable insights into the molecular underpinnings of cancer and the mechanisms of action of therapeutic agents.

Origins and Development

Although the initial discovery of many key chemotherapy agents took place in the labs of major pharmaceutical corporations, the development of almost all significant drugs can be attributed to clinical trials sponsored by the NCI (Chabner and Roberts, 2005). The NCI-60 panel was developed in the late 1980s as part of an effort to improve the drug discovery process for cancer treatments (Alley et al., 1988). Between 1950 and 1990, the advancement in cancer treatment through pharmacological means was slow, with only one or two new chemical compounds receiving approval annually. There was also mounting frustration in the field about the prolonged journey from drug discovery to market and the failure of existing preclinical models to forecast clinical efficacy in cancer broadly. These concerns and increasing awareness that cancer is a collection of related but genetically and phenotypically diverse diseases inspired the creation of the NCI-60 panel. By creating a panel that included a wide range of cancer types, the NCI aimed to identify compounds with specific activity against certain cancers and to uncover broader patterns of sensitivity and resistance.

Evolution of NCI’s Drug Screening Operations

Since its establishment in 1955, the NCI screening program heavily relied on mouse leukemia models to screen compounds for anticancer activity. In fact, from 1975 until 1985, the in vivo P388 mouse leukemia model served as the predominant primary screen where agents showing minimal or no anticancer activity in this system would not be passed for further evaluation by the NCI (Boyd, 1997). While treatment outcomes for human leukemias and lymphomas saw significant improvements through the early 1970s, the efficacy of clinical treatments for most human solid tumors remained relatively disappointing. NCI’s Division of Cancer Treatment responded to this outcome in 1976 by launching a new tumor panel, which later served as a secondary screen, that included transplantable solid tumor models mimicking the primary histological cancer types common in the United States during that period (Suggitt and Bibby, 2005). Though additional agents were discovered using this adjusted approach, promising results obtained from preclinical screening oftentimes failed to translate into the clinic. These methods were not predictive of activity in human tumors, leading to the search for a more diverse and representative set of models.

By 1985, growing awareness of the molecular diversity and histological differences of human solid tumors in addition to resistance from outside parties to continue current screening methods spurred the development of the NCI-60. At this time, NCI began to downsize its P388 in vivo screen as well as their human/murine tumor panels. NCI-60, the new in vitro human tumor cell line screen, was formally implemented in 1990, shifting the screen from “compound oriented” to “disease oriented”. The first step to its adoption was optimizing the screening methods. The initial landmark paper published in Cancer Research in 1988 by Alley and colleagues showcased the practicality of conducting high-throughput screening using a 96-well plate. This approach necessitated the large-scale cultivation of verified cell lines, the automation of liquid handling, and the implementation of a miniaturized microculture assay. It also involved initially employing a colorimetric tetrazolium-based metabolic dye reduction method also known as the MTT assay as an endpoint to measure cell viability after incubating with a drug for several days. Additionally, it demonstrated the ability to achieve stable and reproducible sensitivity patterns over time and introduced techniques for analyzing data on a large scale.  

At the time of NCI-60’s conception, practical endpoints for microplate cytotoxicity assays did not exist. While the MTT assay was the most cost-effective option and appeared to have better adaptability for their application, the tetrazolium assay posed practical challenges for large-scale screening. For instance, the MTT assay’s end product, MTT formazan, was significantly influenced by a number of assay conditions including specific cell line used and culture age, impairing the assay’s reliability (Vistica et al., 1991). Consequently, alternative measures of cell viability were explored including a method that used sulforhodamine B (SRB) as a protein-binding dye, which was selected for use in the screen. Both the SRB and MTT assays yielded comparable results when used as an endpoint assay in drug sensitivity testing. However, unlike the MTT assay, the SRB assay had superior sensitivity and better correlation between staining and cell number (Keepers et al., 1991). The SRB assay could also be interrupted throughout the protocol and still provide results that were stable and able to be stored for up to several months.

Since the early 1990s, NCI has been relying on the SRB assay for its endpoint assay to measure drug-induced cytotoxicity during its screening procedures until recently. With scientific advancement, the methods for quantifying cytotoxicity and cell viability have been further optimized and recently led the NCI to adopt Promega’s CellTiter-Glo® assay for their detection endpoint assay.

Advantages of the CellTiter-Glo® Assay

Both the SRB and CellTiter-Glo® assays are popular methods for assessing cell viability and cytotoxicity. The SRB assay is based on the ability of the SRB dye to bind to protein components of cells that have been fixed onto a culture plate. The amount of dye absorbed is directly proportional to the cell density, providing an absorbance-based measure of cell proliferation (Skehan et al., 1990). Quantitative measures of absorbance are relatively straightforward but can be impacted by factors such as uneven cell distribution or incomplete washing. Furthermore, the SRB dye cannot distinguish between live and dead cells, as it measures total cell mass. On the contrary, the CellTiter-Glo® assay measures the amount of ATP present, which is an indicator of metabolically active cells. The luminescent signal produced is detectable within a broad dynamic range and is directly proportional to the amount of intracellular ATP, thus reflecting the number of viable cells.

While the SRB assay provided a simpler, faster, and more sensitive option than the MTT assay the NCI initially started screening with, the SRB assay’s limitations have been overcome by the newly implemented CellTiter-Glo® workflow. For example, the measure of intracellular ATP in the CellTiter-Glo® assay provides a direct measure of viable cells and its luminescent output yields a stable result with greater sensitivity in both high and low-density cell cultures. The CellTiter-Glo® assay further expands sample options for its users by enabling them to measure cell viability and cytotoxicity in non-adherent cells, which cannot be achieved with the SRB assay.

Certain features of the CellTiter-Glo® assay also make it more amenable to high-throughput screening (HTS) workflows. Unlike the SRB assay that requires multiple steps including fixation and washing, the CellTiter-Glo® assay only involves adding a reagent directly to cells with a short incubation period prior to measuring the luminescent results. The high sensitivity and signal to background ratio of the CellTiter-Glo® assay also enables fewer cells and lower sample volumes to be used, allowing the NCI to adopt higher well number plate formats for their HTS screening protocols. Furthermore, the output of the CellTiter-Glo® assay yields a stable luminescent signal with a half-life of greater than five hours. This extended half-life further improves the HTS process by eliminating the need for reagent injectors and providing flexibility for batch processing of multiple plates, reducing pipetting errors that may be introduced during multi-step methods such as the SRB assay.

Impact on Drug Discovery and Research

Since its inception, the NCI-60 panel has been used to screen over 100,000 compounds, including chemotherapeutic drugs, natural products, and other biologically active agents. The data generated from these screenings have been made publicly available, serving as a rich resource for researchers worldwide.

The NCI-60 continues to be a valuable tool for cancer research and drug discovery, and its ongoing efforts to update and expand the molecular characterization of the panel will further enhance its utility. Likewise, NCI’s recent implementation of the CellTiter-Glo® assay highlights the organization’s efforts aimed at enhancing the efficiency, reliability, and sensitivity of drug screening processes to continue providing high-quality screening data. As cancer research moves towards more personalized and targeted therapies, the NCI-60 panel remains a critical resource, embodying the progress and collaborative effort in the ongoing battle against cancer. Overall, the NCI-60 has had a profound impact on the field of cancer research, providing a foundational resource for the identification of new cancer therapies and the exploration of the molecular landscape of human cancers.

Learn more about the CellTiter-Glo® Cell Viability Assay or view our entire portfolio of Cell Viability and Cytotoxicity Assays.


Alley, M.C. et al. (1988) Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Research, 48(3), 589-601.

Boyd, M.R. (1997) The NCI In Vitro Anticancer Drug Discovery Screen. In Teicher, B.A. (Eds.) Anticancer Drug Development Guide. Cancer Drug Discovery and Development. (pp. 23-42). Humana Press, Totowa, NJ.

Chabner, B.A., and Roberts, T.G., Jr. (2005) Chemotherapy and the war on cancer. Nature Reviews Cancer, 5(1), 65-72.

Keepers, Y.P. et al. (1991) Comparison of the sulforhodamine B protein and tetrazolium (MTT) assays for in vitro chemosensitivity testing. European Journal of Cancer, 27(7), 897-900.

Skehan, P. et al. (1990) New Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening. Journal of the National Cancer Institute, 82(13), 1107-1112.

Suggitt, M., and Bibby, M.C. (2005) 50 years of preclinical anticancer drug screening: empirical to target-driven approaches. Clinical Cancer Research, 11(3), 971-981.

Vistica, D.T. et al. (1991) Tetrazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan production. Cancer Research, 51(10), 2515-2520.

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