Influences Of Chemotherapy On Primitive Hematopoietic Cells And Their Leukemic Counterparts:Studies In A T-ALL Therapy Mouse Model

Primitive hematopoietic cells are ancestors of all blood cells, and they have the abilities of self-renewal, multi-lineages differentiation and long-term hematopoietic reconstitution. Normally these cells mainly reside in the bone marrow (BM) micro-environment, usually called the "niche", and their proliferation and differentiation are tightly regulated to keep hematopoietic homeostasis However, when influenced by intrinsic or external factors, such as leukemia or infection, complex changes seemed unavoidable. In clinic, leukemia patients usually die of hematopoietic failure or side-effects brought by therapy, which are mainly composed of chemotherapy and stem cell transplantation. And for those relapsing patients, they seemed much more fragile for the next course of chemotherapy. They usually showed prolonged hematopoietic depression or incomplete hematopoietic recovery post therapy. Seemingly, normal hematopoieis had been impaired in the relapsing patients. Studies of hematopoietic ancestors in this setting may provide us better understandings of this disease, and even new concepts for treatment.While, for changes in context of leukemia, they had already been reported. For example, in 2009 Hu had tested kinetics of primitive hematopoietic cells in an irradiated T-ALL mice model, in which they demonstrated that these cells quantitatively decreased while functionally reserved their repopulating capability. Reports about chemotherapy on leukemia had also been presented. However, changes of primitive hematopoietic cells especially comparison with leukemia cells during this course have not been clarified yet. Here in this article, we used a non-irradiated T-ALL mice model, combined with chemotherapy to set up a leukemia-therapy model and studied changes of primitive hematopoietic cells plus their leukemic companions.MethodsMice and cellsC57/BL6J mice (CD45.2+,8-week-old) were used as leukemia hosts, while B6.SJL mice (CD45.1+,8-week-old) were used as recipients and source of competitive cells in the competitive bone marrow transplantation (c-BMT) assays. The T-ALL cells were derived from bone marrow CD45.1+Lin- hematopoietic cells, induced by Notch-1 ICN-GFP over-expression, and kindly offered by professor Tao Chen group of the State Key Laboratory of Experimental Hematology, Tianjin, China.Chemotherapeutic agentsChemotherapeutic agents were Cyclophosphamide (CTX, Jiangsu Hengrui Medicine, CO., LTD) and Cytosine arabinoside (Ara-C, Pfizer Italia s.r.l). Agents were dissolved in PBS and stored at -20℃. Storage concentrations were 50 mg/ml for both drugs.Determination of maximum tolerant dose (MTD) in C57/BL6J miceMTD of therapeutic agent was defined as the maximum dose that caused no death and no more than 10% weight loss. This was determined by treating wide-type,8-week-old female C57/BL6J mice (n=5) for four consecutive days. Finally, MTD gained was 100 mg/kg for CTX and 150 mg/kg for Ara-C when mice were treated for four consecutive days. These doses were then used for primary chemotherapeutic testing in leukemic mice.Flow cytometric (FCM) analysis and cell sortingMurine BM cells were obtained by flushing iliums, femurs and tibias with PBS or PBE. Immuno-phenotypes were used as follows:for murine HSC was Lin-c-Kit+Sca-1+ (LKS+), including long-term repopulating HSC (LT-HSC; CD34Flk2-LKS+), short-term repopulating HSC (ST-HSC; CD34+Flk2-LKS+) and multi-potential progenitor (MPP; CD34+Flk2+LKS+); for murine HPC was Lin-c-Kit+Sca-1-(LKS-), sub-divided as granulocyte¯ophage progenitor (GMP; CD34+CD16/32+LKS-), common myeloid progenitor (CMP; CD34+CD16/32-LKS-) and megakaryocytic&erythroid progenitor (MEP; CD34-CD16/32-LKS-). Normal hematopoietic cells and leukemic cells were discriminated by different expression patterns of CD45.2 and CD45.1. All analysis was performed on an LSR Aria Ⅱ flow cytometor.For normal HSC&HPC isolation, bone marrow cells were firstly enriched for c-Kit expression by immuno-selection with CD117 conjugated micro-magnetic beads according to the manufacturer’s instructions. Enriched cells were then stained with PE-cy7 conjugated with a mixture of lineage antibodies, PE conjugated Sca-1, APC conjugated c-Kit and Percp-cy5.5 conjugated CD45.2. Then CD45.2+LKS+ cells were directly sorted into tubes and lysed for gene expression analysis. Leukemic cells were sorted by expression of GFP. During the sorting procedure, DAPI was used to exclude dead cells.Analysis of apoptosis by flow cytometryFor apoptosis assays, staining was performed in the accessory staining buffer with 7-AAD and FITC conjugated Annexin-V (5ul/ml for both dyes; BD PharmigenTM FITC Annexin V Apoptosis Detection Kit) at 37℃ for 15 minutes according to the user’s manual and analyzed by flow cytometry in less than one hour after staining completed.Cell cycle analysis by flow cytometryFor cell-cycle analysis, cells were stained with 5ul/ml PE conjugated Ki-67 (BD Bioscience) at 37℃ for 30 minutes, then 5ug/ml Hoechst 33342 (Invitrogen) was added right before flow cytometric analysis. The staining pattern discriminated cells in GO (Hochestlow Ki-67low), G1 (Hochestlow Ki-67high) and G2-S-M (incorporation of both Hochest and Ki-67) phase.In vitro colony-forming assayCD45.2+ GFP- BM cells from therapy and leukemia groups were sorted by flow cytometry on day 1,2,5,12 after chemotherapy for in vitro colony-forming assay, respectively. Sorted cells (with a concentration of 1.2×106/ml and a volume of 50 ul) were seeded in methylcellulose medium M3434 (3 ml) and plated in 24-well plates with a 0.5 ml volume of the mixture at a cell density of 20000/ml. Five replicated wells each. Cells were cultured at 37℃, with 5% CO2 and ≥95% humanity. After ten days of culture, colonies were counted under an inverted microscope and recorded in specific lineages.Competitive bone marrow transplantationCD45.2+GFP- BM cells were sorted from the one-day treated leukemic mice on the 1st, 2nd,5th and 12th day post therapy for c-BMT assay, respectively. A total number of 5×105 sorted CD45.2+ cells together with an equal number of viable CD45.1+ competitive cells (from wide-type untreated 8-week-old B6.SJL female mice) were co-transplanted into lethally irradiated (9.5Gy) female B6/SJL mice (n=9/group,8-week-old) through tail-vein injection six hours after irradiation. After transplantation, tail-vein blood was tested for donor contribution and lineage differentiation one month later and monthly for four consecutive months since. Relative contributions of tested (CD45.2+) and competitive cells (CD45.1+) were analyzed by FCM using FITC conjugated CD45.2 and Percp-cy5.5 conjugated CD45.1. Differentiation status was analyzed using following lineage markers: APC conjugated Mac-1 for myeloid lineage, PE conjugated CD3 for T lineage and PE-cy7 conjugated B220 for B lineage. Analysis was done by flow cytometry.Senescence analysis by flow cytometrySenescent status of cells was examined according to the manufacture’s instruction of the ImaGene GreenTM C12-FDG lacZ Gene Expression Kit (Molecular Probes, Inc.), and further guided by a Nature Protocol suggested method.Quantitative reverse transcriptase PCR (qRT-PCR)A total number of 2×104 CD45.2+LKS+ cells or GFP+ cells were sorted directly into the lysis buffer. Total RNA were extracted with the RNA nano-prep kit according to the manufacturer’s instructions. Reverse transcription was achieved using oligo-dT and M-MLV reverse transcriptase. Real-time polymerase chain reaction (PCR) was done with SYBR green Master Mix, using a Real-time Quantitative PCR 7500 (ABI) machine.Statistical analysisData were all presented as mean±SEM if not indicated elsewise. Survival status was analyzed using Kaplan-Meier log-rank analysis. Differences between two groups were analyzed using two-tail unpaired Student t test. For comparison of multiple groups, one-way ANOVA was used and followed by Dunnett analysis between each two groups. Differences with a P-value< 0.05 were considered as statistically significant.ResultsEstablishment of a T-ALL like disease in non-irradiated mice. C57/BL6J mice (8-week-old, female, non-irradiated) received T-ALL cells injection 100% developed T-ALL, characterized by shortened survival (median survival days post injection:29), changes of peripheral blood, leukemic infiltration, gradually increased leukemic burden in bone marrow (nearly 10% in BM on the 12th day post injection) and a T-ALL phenotype (CD45.1+GFP+CD3+CD4+CD8+) confirmed by flow cytometry.Therapeutic dose&course dependent leukemia-therapy model. The T-ALL mice received different doses and courses of chemotherapy composed of Ara-C and CTX since the 12th day after leukemic cells injection. All groups of T-ALL mice which received chemotherapy showed longer survival compared to the leukemia-only group, however, they responded differently due to different doses and courses:heavier dose or longer duration of therapy led to longer disease-free-survival (DFS), together with heavier suppression of normal hematopoiesis. One-day therapy combined of Ara-C (150mg/kg) plus CTX (100mg/kg) led to a MRD-negative CR to relapse leukemia-therapy model, while a lower dose of one-day therapy combined of Ara-C (75mg/kg) plus CTX (50mg/kg) led to a MRD-positive CR to relapse model.HPSCs showed an earlier regeneration than leukemic cells in bone marrow after therapy in the one-day heavy-dose therapy model. HSC and HPC both decreased on the 1st day post therapy, regenerated since the 2nd day, and decreased again after the 5th day when the leukemic cell started to show their recurrence. Post therapy, HPC showed a GMP-biased differentiation pattern since the hematopoietic recovery phase, while HSC showed an additional consumption in the end stage of the leukemia relapse. Leukemia cells decreased right after therapy, and regenerated since the 5th day post therapy.Cell cycle changes of HSCs instead of apoptosis seemed responsible. Analysis of cell cycle on the 1st,2nd,5th and 12th day after therapy showed HSCs experienced complex cell-cycle changes after therapy. Frequency of HSCs cells in GO phase dropped from the 1st day after therapy, sustained at a low level and began to increase from the 5th day when leukemia relapsed. While the frequency of HSC in G2-S-M phase dropped on the 1st day, started growing from the 2nd day, decreased on the 5th day, and elevating again on the 12th day. For the leukemic cells, in their regenerating phase, cells in G2-S-M phase kept in a low frequency until the 7th day. However, on the 1st day post therapy, a large proportion of leukemic cells was in G2-S-M phase with a sharply decreased G1 percentage. Gene expression patterns analysis of cell cycle related genes in these cells had found coincident changes. When focused on apoptotic status of HSPCs on day 0.4,1,2,5 and 12, though with no significance, there was obvious increase of apoptosis on day 0.4 and 1 when compared to normal mice, but showed no changes since the hematopoietic recovery phase (on the 2nd day).Gradually lost repopulating capability of HSPCs involved in the leukemia-therapy model. CD45.2+ hematopoietic cells were sorted from the therapeutic leukemia mice on the 1st,2nd,5th and 12th day after therapy, and then co-transplanted with an equal number of CD45.1+ competitive cells from normal control into lethally irradiated B6.SJL mice (CD45.1+). CD45.1/CD45.2 and lineage differentiation were inspected monthly for 4 consecutive months. On the 4th month, contributions of the testing cells were 70.32%,79.32%,50.13% and 1.57% for one sorted on the 1st,2nd,5th and 12th day, respectively. Differences were more dramatic when comparing donor contribution of the primitive hematopoietic cells, indicating a gradually lost repopulating capability of the primitive hematopoietic cells. Lineage differentiation analysis showed a gradual inhibition of myeloid cells.Senescence confirmed in LKS+ hematopoietic cells post therapy. Senescent status of CD45.2+LKS+ hematopoietic cell in our model on the 3rd and 5th day post therapy were tested, and compared to normal control. CD45.2+LKS+ hematopoietic cells of leukemia-therapy mice on the 3rd and 5th day post therapy showed higher mean fluorescence intensity (MFI) of C12-FDG, indicating higher levels of senescence. We then analyzed gene expressions of P16, P21 and P53, three key genes involved in the cellular senescence. Data showed higher expression levels of P16 and P21 in CD45.2+LKS+ hematopoietic cells on both the 3rd and 5th day post therapy compared to normal control. We also observed down-regulated expressions of EGR1 and FOS on these two days.ConclusionsUsing a non-irradiated T-ALL mouse model combined with traditional chemotherapeutic drugs, we successfully established a therapeutic dose&course dependent leukemia-therapy model, including the MRD-negative and MRD-positive CR to relapse subgroups.In this leukemia-therapy model, we proved that HSPCs regenerated earlier than leukemic cells post therapy, with a GMP-biased differentiation pattern and a decreased frequency of LT-HSC in the hematopoietic compartment.Cell cycle was responsible for changes of hematopoietic stem and progenitors post therapy, while influences of apoptosis were negligible.HSPCs post therapy in the leukemia-therapy model gradually lost their reconstitution capacity, possibly due to over-proliferation related cellular senescence.