Understanding the complex molecular pathways leading to disease is critical for the development of effective treatment regimes and novel drug targets. Due to research and resource limitations associated with the use of primary patient material, pre-clinical models are essential to expand our knowledge of cancer biology and for the evaluation of new drugs. For pre-clinical testing, cell lines cultured in vitro have been extensively used but their ability to recapitulate primary disease is limited. Therefore, more relevant disease models are of critical importance.
An ideal model would mimic the proliferation and dissemination of cancer cells that occur in vivo and behave in a similar manner in response to chemotherapeutic drug treatment. The non-obese diabetic/severe combined immunodeficient (NOD/SCID) xenograft mouse model is currently one of the most successful models with which to study haematological malignancies such as acute lymphoblastic leukaemia (ALL) , whereby patient bone marrow leukaemia cells are directly transplanted into recipient NOD/SCID mice . The kinetics of engraftment reflects the human disease, leading to bone marrow (BM) infiltration, followed by migration to the spleen, peripheral blood and other haematopoietic organs [2–4].
For ALL, although cure rates are exceeding 75%, the development of drug resistance is poorly understood and remains a major cause of morbidity and mortality in children . Importantly, much of our knowledge of the mechanisms underlying drug resistance has been generated in vitro using immortalised cancer cell lines. The extent to which cell lines retain features of the original disease in vivo is a matter of debate . Thus relevant in vivo, pre-clinical models that recapitulate human disease are critical to delineate resistance mechanisms and improve survival.
Primary leukaemia cells engrafted into NOD/SCID mice appear to retain many of the phenotypic and genotypic features of the original specimen [2, 7–10]. Moreover, their drug resistance profile to conventional chemotherapeutics mirrors that of the patient clinical response [2, 10]. Importantly, comparisons have shown that such xenografts more closely resemble their tumour type of origin than in vitro cell lines and have been accurate in predicting efficacious drug combinations and clinically active therapeutics [11–14].
Continuous xenografts can be established by transplanting cells harvested from the spleen of engrafted animals into secondary and tertiary recipient mice . Utilising continuous ALL NOD/SCID xenografts the effects of chemotherapy drugs can be assessed at the molecular level. Thus, the aim of the current study was to characterise gene expression profiling in the continuous ALL xenograft so that it can be used as a model for the development of therapy resistance in vivo. We have previously demonstrated the clinical relevance of gene-expression profiling through the successful identification of markers predictive of ALL disease outcome, drug-resistance and relapse in a number of primary ALL patient cohorts [15–21]. However, to validate the xenograft model system for transcriptional analysis three critical issues needed to be addressed. Firstly, we needed to determine the most appropriate engrafted xenograft tissue for analysis. BM is more commonly isolated from patients, however, the spleen in xenograft mice contains at least seven-fold more leukaemia cells, which makes isolation of these cells more practical for analysis. Thus, we were interested in establishing whether the same gene expression profiles can be obtained from engrafted spleen and BM. Secondly, we wanted to address the reproducibility of the engraftment in both the BM and spleen of independent mice. Phenotypic evidence suggests engraftment in the continuous mouse model is reproducible , however, this has not been examined at the transcriptional level. Finally, when testing the expression of human xenografts we wanted to measure the effect of the host murine tissue. Studies from other xenograft models have demonstrated cross-species hybridisation of mouse RNA to human specific microarrays. Although not extensively characterised, such studies suggest the potential skewing of human gene expression profiles [22–26]. The previous studies were performed using Affymetrix expression arrays designed to target the 3' end of the gene. This region shows the most divergence between mouse and human. To date, the extent of cross hybridisation using the Affymetrix whole transcript Human Gene 1.0 ST array has not been assessed. The goal of the present study was therefore to validate the use of gene expression profiling in the ALL NOD/SCID xenograft model so that it can be used as a pre-clinical model of relapse.