Sensitive PCR method for the detection and real-time quantification of human cells in xenotransplantation systems

Abstract

The sensitive detection of human cells in immunodeficient rodents is a prerequisite for the monitoring of micrometastasis of solid tumours, dissemination of leukaemic cells, or engraftment of haematological cells. We developed a universally applicable polymerase chain reaction method for the detection of a human-specific 850-bp fragment of the alpha-satellite DNA on human chromosome 17. The method allows the detection of one human cell in 10(6) murine cells and could be established as both, a conventional DNA polymerase chain reaction-assay for routine screening, and a quantitative real-time polymerase chain reaction-assay using TaqMan-methodology. It was applied to the following xenotransplantation systems in SCID and NOD/SCID mice: (1) In a limiting dilution assay, cells of the MDA-MB 435 breast carcinoma were injected into the mammary fat pad of NOD/SCID mice. It could be shown that 10 cells mouse(-1) were sufficient to induce a positive polymerase chain reaction signal in liver and lung tissue 30 days after transplantation as an indicator for micrometastasis. At this time a palpable tumour was not yet detectable in the mammary fat pad region. (2) Cells of a newly established human acute lymphatic leukaemia were administered intraperitoneally to SCID mice. These cells apparently disseminated and were detectable as early as day 50 in the peripheral blood of living mice, while the leukaemia manifestation was delayed by day 140. (3) In a transplantation experiment using mature human lymphocytes we wanted to standardise conditions for a successful survival of these cells in NOD/SCID mice. It was established that at least 5 x 10(7) cells given intravenously were necessary and that the mice had to be conditioned by 2 Gy body irradiation to get positive polymerase chain reaction bands in several organs. (4) Engraftment studies with blood stem cells originating from cytapheresis samples of tumour patients or from cord blood were undertaken in NOD/SCID mice in order to define conditions of successful engraftment and to use this model for further optimisation strategies. The polymerase chain reaction method presented allowed a reliable prediction of positive engraftment and agreed well with the results of immunohistochemical or FACS analysis. All together, the polymerase chain reaction method developed allows a sensitive and reliable detection of low numbers of human cells in immunodeficient hosts. In combination with real-time (TaqMan) technique it allows an exact quantification of human cells. As this method can be performed with accessible material of living animals, follow up studies for the monitoring of therapeutic interventions are possible in which the survival time of mice as evaluation criteria can be omitted.

Figure 1

Human specific DNA-PCR of an α-satellite sequence of chromosome 17 used as a screening assay for human cells in the mouse. Sensitivity of the PCR method proved by logarithmic dilution steps of human MCF-7 cells in mouse P388 ascitis cells.

Figure 2

TaqMan created calibration curve using a dilution series of human MCF-7 cells in mouse P388 ascitis cells as processed with each TaqMan PCR. C_T describes the threshold cycle number at which fluorescence exceeds 10-fold the standard deviation of the fluorescence detected during PCR cycles 3–15. This calibration curve serves to calculate the amount of human DNA reflecting the number of human cells.

Figure 3

Example for the detection of micrometastases in mouse liver and lung in the MDA-MB 435 xenotransplantation experiment performed by both the conventional DNA PCR and the quantitative real-time PCR. The inoculation of 100 (A), 10 (B) and 1 (C) human tumour cells per mouse correlated with a decrease of the PCR signal in the organs (same as experiment documented in Table 2).

Figure 4

FACS analysis of bone marrow of mice 12 weeks after transplantation of cord blood MNC (A) or sorted CD 34 cells (B) from experiment II, Table 4. FL-1=CD 19⁻ FITC, FL-2=CD 45⁻ PE.

Figure 5

Immunohistochemistry of bone marrow of mice 12 weeks after transplantation of cord blood MNC (A, B) or CD 34 cells (C, D) of experiment II (Table 4) (A, C) HLA-I positive cells; (B, D) CD45 positive cells.