Therapeutic mechanism and post treatment evaluation of acute leukemia by single cell inductively coupled plasma mass spectrometry (ICP-MS)

Abstract

Cell is a complex dynamic system whose components change with space and time, and heterogeneity is a widespread event existing in most cells. Compared with bulk analysis, which uses the stochastic average results to demonstrate the biological phenomena, single-cell analysis more precisely represents cell-to-cell variations which could help us better understand the fundamental biological principle. Arsenic trioxide (ATO) has long been used in clinic for the treatment of Acute Promyelocytic Leukemia (APL). Exploration of intrinsic mechanisms of ATO in the treatment of APL at a single cell level will facilitate the development of new efficient arsenic drugs. In this thesis, a new approach was developed to investigate the uptake of ATO by two different leukemia cell lines and examine their corresponding cytotoxicity at a single cell level via time-resolved ICP-MS (TR ICP-MS) combined with cisplatin as viability dye and lanthanide-tags for apoptosis and differentiation monitoring. Different uptake capability of ATO shows in different cell lines which may account for the corresponding sensitivity towards ATO. The results show that the ATO kill leukemia cells mainly through inducing apoptosis instead of stimulating differentiation. The positive correlation among intracellular arsenic contents and apoptosis, death cell ratios may provide a basis for the design of more potent arsenic drugs. Cell cycle has been commonly known to play a critical role for cancer treatment and diagnosis. The uptake of two arsenic-based drugs (ATO and ZIO-101) across the cell cycle in single leukemia cells were further evaluated using TR ICP-MS. The maximal amounts of both ATO and ZIO-101 shows in G2/M phase and minimum in S phase. The variation of the expression level of related transporters and cytotoxicity across the cell cycle provide the first evidence on cell cycle dependent uptake and cytotoxicity of arsenic-based drugs at single cell levels, may have general implications for precise evaluation of other anticancer drugs by considering cell cycle phase. The detection of minimal residual disease (MRD) provides valuable information of prognosis which could guide the risk stratification and treatment decisions. To overcome the problem of fluorophore overlapping existing in flow cytometry, mass cytometry was introduced for MRD detection and multidrug resistance (MDR) characterization of B-cell acute lymphoblastic leukemia (B-ALL). Compared with normal bone marrow samples, evident overexpression of BCRP, Bcl-2, MRP1 and P-gp are observed in B-ALL sample with slightly under expression of p53. A positive relationship is observed between BCRP, Bcl-2, MRP1 and P-gp. The cell subpopulation with overexpressed MDR shows the immune phenotype of CD10dimCD34+CD38int and CD10+CD34+CD38dim which may play a role as potential leukemia stem cell population of B-ALL. This study could provides more precise and systematic information to recognize the immune phenotype of MRD in clinical samples and provide an alternative strategy to develop targeted therapy to the potential stem cell subpopulation on the basis of the molecule difference shown in CD10dimCD34+CD38int and CD10+CD34+CD38dim cell subpopulation, facilitating the diagnosis and prognosis of B-ALL and a better understanding of the role of MRD in clinical outcomes.