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Development of a miniaturized, improved nucleic acid precursor incorporation assay for chemosensitivity testing of
Abstract:
Two technological problems limit the usefulness of chemosensitivity assays: low success rates (generally 30-60%); and the requirement for large numbers of tumor cells (5 X 10(5)/dish). To solve these problems, we developed a miniaturized, improved, nucleic acid precursor incorporation assay (MINI-assay). In this new assay, 0.3-1.5 X 10(5) tumor cells were plated in double-layer agarose in 16-mm wells of a Costar (No. 3524) 24-well cluster dish. After 72 h of incubation, 5 microCi [3H]thymidine were added to each well. After an additional 24 h of incubation, the trichloroacetic acid-precipitable material was collected and counted by liquid scintillation. We found that 280 of 351 (80%) human solid tumors gave evaluable chemosensitivity results. Labeling efficiency was optimum when the plating density was between 1.5 and 3 X 10(4) cells/well. Radioisotope uptake was less efficient in 35-mm Petri dishes and in the 7-mm wells. The MINI-assay was particularly suitable for small specimens (less than 1 g) and for tumor types that usually yield small numbers of viable tumor cells (19 of 30 breast cancers and 56 of 71 sarcomas were evaluable). The artifacts of colony counting (cell clumps, debris, clots) were also eliminated with this assay. With high evaluability rates, the requirement of fewer cells, a short duration (5 days), and ease of quantitation, the MINI-assay is widely applicable to chemosensitivity testing in human tumors.
Insights
A new miniaturized assay improves tumor chemosensitivity testing, increasing success rates to 80% and reducing cell requirements. This nucleic acid precursor incorporation assay is ideal for small or difficult tumor samples.
Area of Science:
- Oncology
- Biotechnology
- Molecular Biology
Background:
- Chemosensitivity assays are crucial for personalized cancer treatment.
- Current assays face limitations including low success rates (30-60%) and high tumor cell requirements (5 x 10^5 cells/dish).
Purpose of the Study:
- To develop an improved, miniaturized nucleic acid precursor incorporation assay (MINI-assay).
- To overcome the limitations of existing chemosensitivity testing methods for human solid tumors.
Main Methods:
- A miniaturized assay using 0.3-1.5 x 10^5 tumor cells in agarose within 16-mm wells.
- Incubation with [3H]thymidine followed by collection of trichloroacetic acid-precipitable material for scintillation counting.
- Optimization of plating density and well size for efficient radioisotope uptake.
Main Results:
- The MINI-assay achieved an 80% evaluability rate (280/351) for human solid tumors, significantly higher than conventional methods.
- Optimal labeling efficiency was observed at plating densities of 1.5-3 x 10^4 cells/well.
- The assay demonstrated suitability for small specimens (<1g) and tumor types yielding few cells, including breast cancers and sarcomas.
- Eliminated artifacts associated with colony counting, such as cell clumps and debris.
Conclusions:
- The MINI-assay offers high evaluability rates and reduced cell requirements for chemosensitivity testing.
- Its short duration (5 days) and ease of quantitation make it widely applicable to diverse human tumor types.
- This assay enhances the utility of chemosensitivity testing for guiding cancer therapy decisions.