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 a-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 71 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 5610 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.

Transfection of Keratin 18 Gene in Human Breast Cancer Cells Causes Induction of Adhesion Proteins and Dramatic Regression of Malignancy In vitro and In vivo

“…We previously showed in a retrospective study that high K18 expression in breast tumors was associated with a markedly improved prognosis for the patient, with the majority of patients with high K18 remaining recurrence-free throughout an observation period of >8 years (8). A similar correlation between K8/K18 expression and reduced tumorigenicity was also reported by Pankov et al (18) for murine pancreatic carcinoma cells in a murine syngeneic transplant model.…”

“…Lung, brain and bone marrow tissue from all five animals showed a positive PCR signal for human DNA with the strongest signals found in lung and liver. The method is described elsewhere in detail (8). Mice that had received MDA231-K18 or MDA231-E cells had no histologically detectable metastases.…”

“…Expression of keratins 7, 17, and 19 is variable but generally low (5,7). We have previously shown that 80% of all breast carcinomas exhibited a loss of the differentiation-associated keratins 8 and 18, and that loss of these keratins was associated with a significantly worse prognosis (8,9). In breast cancer cell lines, invasiveness in vitro and metastatic potential in athymic mice also correlated inversely with the degree of K18 expression (8).…”

“…We have previously shown that 80% of all breast carcinomas exhibited a loss of the differentiation-associated keratins 8 and 18, and that loss of these keratins was associated with a significantly worse prognosis (8,9). In breast cancer cell lines, invasiveness in vitro and metastatic potential in athymic mice also correlated inversely with the degree of K18 expression (8). Similar results were obtained by Sommer and Thompson, who showed that breast cancer cell lines became more aggressive as keratin filaments were replaced by vimentin, the intermediate filament-protein of mesenchymal cells (2)(3)(4).…”

Cell entry targeting restricts biodistribution of replication-competent retroviruses to tumour tissue

“…To test the DNA samples for the presence of DNA from HT1080 cells, a PCR detecting specifically human DNA was performed. 22 Indeed, all tissues were positive for invaded HT1080 cells (Figure 3). It is thus likely that especially the weak PCR signals obtained in some organs with the MMP-activatable viruses were in fact derived from the invasion of infected HT1080 cells, although the PCR signals obtained with the much less- A second non-infected target tumour was implanted into the opposite flank.…”