Acute lymphoblastic leukemia (ALL) is the most common malignancy diagnosed in children, accounting for 30% of cancer diagnoses in patients under the age of 15 years. Of these cases, roughly 15% derive from the T-cell lineage (T-ALL). Steady improvements in treatment protocols have recently led to survival rates near 80%; however, the remaining 20% have uniformly poor outcomes. Thus far, the therapeutic strategies implemented have focused on intensifying multiagent chemotherapy. As undesirable toxicity arises with additional intensification of standard chemotherapy agents, more targeted molecular agents for T-ALL are urgently needed to further improve outcomes of this disease.
Preclinical studies have largely relied on a relatively small number of established T-ALL cell lines that have been maintained in culture for long periods of time, as well as primary T-ALLs xenografted in immunodeficient mice. Recently, research efforts have focused on the development of robust in vitro culture models for efficient testing and validation of novel therapeutics on primary human T-ALL blasts. A relatively simple and efficient in vitro culturing system would in part eliminate the concerns of using cell lines that might not reflect the biology of a typical disease in patients, and mitigate the costly and time-consuming endeavor of xenografting T-ALL blasts. Moreover, personalized medicine initiatives would be dramatically improved by the ability to assess drug sensitivity profiles on patients' own tumor cells in direct in vitro culture assays.
Several groups have devised methods for in vitro culture of patient T-ALL cells, the most successful of which have utilized stromal feeders. These methods have relied also upon serum-containing medium and thus have yielded variable results due in part to lot-to-lot variation in serum quality. Using a defined, serum-free media based on the WIT formulation initially described for in vitro propagation of normal and transformed human mammary epithelial cells, we have now optimized culture conditions required for primary human T-ALL samples. We demonstrate this serum-free culture system to consistently outperform serum-containing medium, supporting expansion of primary T-ALL samples 2- to 200-fold within 3 weeks of initial explantation from either xenografted mice or patient biopsy material and with only limited cell death. When carried for longer periods of time (up to 6 weeks), cultures maintained at higher cell densities attained maximal expansions of 40- to 100-fold while those maintained at lower cell densities expanded up to 10,000-fold. Importantly, T-cell receptor γ heteroduplex analysis confirmed expansion of the original clone throughout the culture period and flow cytometric analysis confirmed cultures to be composed of immature human T-lineage cells. Of note, cultures tended to regress after 30–40 days in vitro, suggesting that long-term renewing cells are not supported under these conditions.
The use of defined, serum-free medium in this culture system has facilitated systematic characterization of growth factor requirements. We show that supplemental IL-7 and insulin/IGF1 are critical for short-term culture expansion, whereas SCF and Flt3L are dispensable. This culture system will also enable us to assess other “stem” supporting factors and genes (by lentiviral transduction) for their contribution to long-term culture maintenance. Finally, we demonstrate the facility of this culture system for testing of novel therapeutic compounds. By allowing direct study of primary samples in vitro, this culture system will advance basic mechanistic studies in T-ALL biology as well as preclinical studies aimed at developing individually tailored therapy protocols.
Ince:Stemgent (2008–10): Consultancy, TAI receives royalties for WIT-P, -I, and -T media Other.
Asterisk with author names denotes non-ASH members.