Characterization of in vivo tumorigenicity tests using severe immunodeficient NOD/Shi-scid IL2Rγnull mice for detection of tumorigenic cellular impurities in human cell-processed therapeutic products

Abstract

The contamination of human cell-processed therapeutic products (hCTPs) with tumorigenic cells is one of the major concerns in the manufacturing and quality control of hCTPs. However, no quantitative method for detecting the tumorigenic cellular impurities is currently standardized. NOD/Shi-scid IL2Rγ^null (NOG) mice have shown high xeno-engraftment potential compared with other well-known immunodeficient strains, e.g. nude mice. Hypothesizing that tumorigenicity test using NOG mice could be a sensitive and quantitative method to detect a small amount of tumorigenic cells in hCTPs, we examined tumor formation after subcutaneous transplantation of HeLa cells, as a model of tumorigenic cells, in NOG mice and nude mice. Sixteen weeks after inoculation, the 50% tumor-producing dose (TPD₅₀) values of HeLa cells were stable at 1.3 × 10⁴ and 4.0 × 10⁵ cells in NOG and nude mice, respectively, indicating a 30-fold higher sensitivity of NOG mice compared to that of nude mice. Transplanting HeLa cells embedded with Matrigel in NOG mice further decreased the TPD₅₀ value to 7.9 × 10 cells, leading to a 5000-fold higher sensitivity, compared with that of nude mice. Additionally, when HeLa cells were mixed with 10⁶ or 10⁷ human mesenchymal stem cells as well as Matrigel, the TPD₅₀ values in NOG mice were comparable to those of HeLa cells alone with Matrigel. These results suggest that the in vivo tumorigenicity test using NOG mice with Matrigel is a highly sensitive and quantitative method to detect a trace amount of tumorigenic cellular impurities in human somatic cells, which can be useful in the quality assessment of hCTPs.

Keywords: Cellular therapy; NOG mice; Quality control; Regenerative medicine; Tumorigenicity test.

Fig. 1

Tumor incidence of HeLa cells in nude and NOG mice. The tumor formation of HeLa cells transplanted into the subcutaneous spaces of mice was examined for 16 weeks. The relationships between the dose and the tumorigenic incidence of HeLa cells in nude mice (a) and NOG without (b) or with (c) Matrigel are presented (n = 10 in each group).

Fig. 2

Characterization of subcutaneous tumors formed by transplantation with HeLa cells in nude and NOG mice. Growth curves of subcutaneous tumors formed by inoculation with various dosages of HeLa cells were presented in respective mice (a: nude; b: NOG w/o Matrigel; c: NOG w/Matrigel). The tumor volume (TV) was calculated using the formula volume = 1/2 × length (mm) × (width [mm])². The successive engraftment was determined according to progressive nodule growth at the injection site. Mice were euthanized and necropsied when tumors reached approximately 20 mm in any dimension or when a sign of deconditioning was noted. Representative images from histology and immunohistochemistry analyses of subcutaneous tumors in NOG mice formed by transplantation with 1.0 × 10² HeLa cells suspended in Matrigel (d and e). Serial sections were stained with H&E (d) and HLA antibody (e) (magnification, 40×; scale bars, 1 mm).

Fig. 3

Detection of tumors in NOG mice transplanted with HeLa cells spiked into hMSCs. The cohorts inoculated HeLa cells spiked in 10⁶ and 10⁷ hMSCs cells were observed for 16 weeks, respectively. The relationships between the cell dose and the tumorigenic incidence of HeLa spiked in 1.0 × 10⁶ (a) and 1.0 × 10⁷ (b) hMSCs cells in NOG mice are presented (n = 6 in each group). Representative images from histology and immunohistochemistry analyses of subcutaneous tumors in NOG mice formed by transplantation with 1.0 × 10⁴ HeLa cells mixed in 1.0 × 10⁶ hMSCs suspended in Matrigel (c, d, and e). Serial sections were stained with H&E (c), HLA antibody (d), and Vimentin antibody (e) (magnification, 100×; scale bars, 500 μm).

Fig. 4

Comparative development of HeLa cell tumors in nude and NOG mice under various conditions. HeLa cell tumor development in nude and NOG mice under various conditions are expressed as transition of TPD₅₀ at weekly intervals. MG, Matrigel.

Fig. 5

Soft agar colony formation assay of HeLa cells. HeLa cells enclosed in soft agar showed progressive formation of colonies (a), whereas hMSCs did not form any colonies in a soft agar media with 1.0 × 10⁴ cells/well by day 20 (b). Arrows indicate the colonies of HeLa cells (magnification, 100×; scale bars, 250 μm). HeLa cells (0%, 0 cells; 0.1%, 10 cells; 0.2%, 20 cells; 0.3%, 30 cells; 0.5%, 50 cells; 1%, 100 cells; 3%, 300 cells; 10%, 1000 cells) were spiked into 1.0 × 10⁴ hMSCs and grown in soft agar for 10 and 20 days (c). HeLa cells spiked into hMSCs and three lots of hMSCs were grown in soft agar for 20 days (d). The lower limit of detection (LLOD) was calculated as the mean plus 3.3 fold the standard deviation of the measurement of the three lots of hMSCs. Quantification of the results is described in (d). Cell growth was quantified using a CytoSelect™ kit. Results were expressed as a relative fold change of the value of negative control (hMSC lot A). Error bars represent the standard deviation of the measurements (n = 3).