Tracking Tumor Colonization in Xenograft Mouse Models Using Accelerator Mass Spectrometry

Abstract

Here we introduce an Accelerator Mass Spectrometry (AMS)-based high precision method for quantifying the number of cancer cells that initiate metastatic tumors, in xenograft mice. Quantification of ¹⁴C per cell prior to injection into animals, and quantification of ¹⁴C in whole organs allows us to extrapolate the number of cancer cells available to initiate metastatic tumors. The ¹⁴C labeling was optimized such that 1 cancer cell was detected among 1 million normal cells. We show that ~1-5% of human cancer cells injected into immunodeficient mice form subcutaneous tumors, and even fewer cells initiate metastatic tumors. Comparisons of metastatic site colonization between a highly metastatic (PC3) and a non-metastatic (LnCap) cell line showed that PC3 cells colonize target tissues in greater quantities at 2 weeks post-delivery, and by 12 weeks post-delivery no ¹⁴C was detected in LnCap xenografts, suggesting that all metastatic cells were cleared. The ¹⁴C-signal correlated with the presence and the severity of metastatic tumors. AMS measurements of ¹⁴C-labeled cells provides a highly-sensitive, quantitative assay to experimentally evaluate metastasis and colonization of target tissues in xenograft mouse models. This approach can potentially be used to evaluate tumor aggressiveness and assist in making informed decisions regarding treatment.

Figure 1

Workflow and validation of ¹⁴C-labeling cancer colonization assay. (A) Schematic of colonization assay workflow. Cells were first cultured with ¹⁴C-thymidine media to achieve single cell resolution and injected into NSG mice via tail vein (TV), heart (IC) or subcutaneous (SQ) routes of delivery. Injected cells were allowed to metastasize for up to 12 weeks. Tissues were harvested at early (2 weeks post injection) and late (12 weeks post injection) time points and DNA was isolated and quantified using AMS. In parallel, the activity of ¹⁴C-thymidine label in cultured cells was quantified using liquid scintillation counting (LSC). AMS measurements and LSC readings were combined to calculate the number of colonized cells per each organ examined. (B) Optimization of ¹⁴C-thymidine labeling in vitro using various concentrations of radioactivity in cell culture media (n = 3). (C) Correlation of experimental versus theoretical values of ¹⁴C-thymidine in labeled cells. (D) Pharmacokinetics of free ¹⁴C-thymidine (n = 6): mean concentration-time profile of thymidine in blood following a single intravenous administration of 700 pCi/animal in C57Bl/6 male mice. Data is expressed as the average per time point ± standard error. (E) ¹⁴C-thymidine dosing solution administered to C57Bl/6 male mice, thymidine retention time = 16 min. (F,G) HPLC chromatographs of mouse urine collected at 24 h post dose from 2 mice exposed to ¹⁴C-thymidine. Open bars indicate thymidine retention time. (H) Stability of ¹⁴C-thymidine in labeled cells transferred to non-¹⁴C-thymidine media.

Figure 2

Quantification of subcutaneous tumor forming cells. Comparison of subcutaneously injected cells in relation to the number of colonized cells quantified using AMS (A) from the harvested tumor. Percentage of initially injected ¹⁴C-labeled cells retained in final tumor (B). At least 4 replicates were analyzed for each quantity of cells injected with the exception of 1.00E + 03 cells due to ¹⁴C signal below the level of detection (++).

Figure 3

Colonization of tissue from PC3 and LnCap cells. Quantity of colonized founder cells calculated from ¹⁴C signal in DNA from target tissues isolated (A) 2 weeks post injection (n ≥ 5) and (B) 12 weeks post injection (n ≥ 4). Error bars represent SEM. *p < 0.05. (C) Representative pictures of kidneys and livers from PC3 or LnCap injected animals.

Figure 4

Tail vein and intracardiac injected cancer cell colonization. Profile of colonized cells in target tissues calculated from ¹⁴C signal in DNA from target tissues isolated at 2-week post injection, 7-week for intracardiac (IC), or 12-week for tail vein (TV) (n = 5).

Figure 5

Tumorigenic and ¹⁴C activity in lung and bone metastasis. (A) Lung metastases of differing tumor burden 7-week following PC3 intracardiac injection from 4 mice and (B) quantification of colonized cells from AMS analysis of ¹⁴C signal in tissue DNA. (C) X-ray images of femurs from 4 animals 7-week post injection following PC3 intracardiac injection. Red arrows denote sites of osteolytic activity induced by cancer cells and (D) associated quantities of colonized cells in each femur.