Hematopoietic Stem Cell Development In Cancer

Purpose:Adult hematopoietic stem cells are present in bone marrow and persist throughout life.They give rise to all the cell types of immune system.In cancer and other disease conditions,hematopoietic stem cells undergo characteristic changes,leading to decreased immunity and immune escape of tumors.Currently,the cellular and molecular basis underlying tumor-induced changes of hematopoietic stem cell development remains unclear.The present study focuse comparing hematopoietic stem/progenitor cells of tumor-bearing mice and normal mice for cell numbers,cell cycles,proliferation and expression of related genes,in order to better define the patterns of developmental changes and regulatory mechanisms of hematopoietic stem/progenitor cells during tumor progression.The results from this study will also enhance our understanding of the mechanisms involved in the evasion of tumor cells from immune surveillance by interfering the process of hematopoietic stem/progenitor cell differentiation into effector immune cells,and provide a knowledge basis of development of immunotherapies for tumor diseases.Methods:(1)Tumor model: The present study utilized the murine Lewis lung carcinoma(Lewis lung carcinoma line,LLC)model that has been widely validated previously.Lewis lung carcinoma cells in logarithmic phase of growth were harvested and 8 to 10 weeks’ old mice were inoculated subcutaneously with Lewis lung carcinoma line cells in 200 ul PBS in the left axillary.4 weeks later,mice with proper sizes of tumor(around 1.5 cm)were chosen for experiments.(2)Isolation and preparation of bone marrow cells.Mice were sacrificed by cervical dislocation,tibia and femur were collected and flushed with medium for obtaining bone marrow.After depleting red cells via lysis method,bone marrow cells were finally prepared into single cell suspension.(3)Cell phenotyping and sorting.Cells were labeled with anti-surface marker antibodies conjugated with the fluorescent dye,cell parameters were assessed by flow cytometry analyzer,specific cell populations were sorted by flow cytometry sorter.(4)In vivo proliferation of cells: EdU was dissolved in PBS at a concentration of 2.5mg/ml,and mice were injected intraperitoneally at a dose of 20mg/Kg body weight.10 hours later,mice were sacrificed and bone marrow cells were isolated.Cells were labeled with anti-surface maker antibodies conjugated with the fluorescent dye.After fixing and permeabilizing cells,EdU was stained by Click-iT kit according to manufacture’s manual.(5)Cell cycle analysis: Cells were labeled with anti-surface maker antibodies conjugated with the fluorescent dye,then after fixing and permeabilizing,DNA was stained by 7-aminoactinomycin D(7-AAD).The proportions of cells in different cell cycle phases were determined by flow cytometry analyzer.Data were analyzed by using Flowjo 7.6.1.(6)The detection of mRNA levels of hematopoietic stem/progenitor cell development-related genes: Total mRNA was extracted,then cDNA was generated,PCR products were examined by agarose gel electrophoresis.The RT-PCR data for parts of examined genes was confirmed by Real-time PCR.(7)Analysis of irf8 protein expression in GMPs: Bone marrow cells were isolated for tumorbearing mice and normal mice,then GMPs were purified by flow cytometry sorter.Proteins were extracted,and β-actin were used as an internal reference,IRF8 proteins were determined by Western blot.(8)Statistical Methods: T test was applied for all data analysis using SPSS software,and data was showed as Mean ± standard deviation.Results:(1)Hematopoietic stem/progenitor cells displayed characteristic changes during tumor development.To understand the changes of hematopoietic stem/progenitor cells in cancer,we measured simultaneously via flow cytometry method the contents of various hematopoietic stem/progenitor cell populations isolated from tumor-bearing mice and normal mice.In tumor-bearing mice,the frequency of GMPs was 1.307±0.136%,which was significantly higher than that of normal mice(0.505±0.174%,P<0.01),the absolute numbers of GMPs were 4.088±0.425 × 105 and 1.320±0.456×105(P<0.01),respectively,for tumor-bearing mice and normal mice.Both percentage and absolute number of CMPs were reduced in tumor-bearing mice compared to normal mice(cell percentages: 0.154±0.032% VS 0.237±0.029%,P<0.05;cell numbers: 0.482±0.101 ×105 VS normal CMPs: 0.621 ±0.076 ×105,P>0.05).For MPPs in normal and tumor-bearing mice,the percentages were 0.033±0.003% and 0.199±0.040%(P<0.01),and the absolute number were0.087±0.008 ×105 and 0.622±0.124 ×105(P<0.01),respectively.The percentages of HSCs were0.176±0.014% and 0.033±0.007%(P<0.001),and the absolute numbers of cells were 0.550±0.042×105 and 0.087±0.018 × 105(P<0.001)in tumor-bearing mice and normal mice,respectively.These results indicated that during tumor progression,all the hematopoietic stem/progenitor cells including HSCs,MPPs and GMPs,but no CMPs,underwent extensive amplification.(2)Proliferative changes of hematopoietic stem/progenitor cells during tumor development.Flucuations in hematopoietic stem/progenitor cell populations abundances observed between tumor-bearing mice and normal mice could be caused by changes in proliferation abilities of cells.Hence,we further examined the proliferation of hematopoietic stem/progenitor cells from tumorbearing and physiologically normal mice.To this end,EdU was applied to directly label nuclear DNA of cells in vivo by intraperitoneal injection.Overal,HSCs,MPPs and GMPs exhibited a significantly increased proliferation during tumor process.Under the tumor and physiological statuses,the proportions of EdU positive cells were 31.806±3.031% and 13.530±12.413%(P<0.01)for HSCs,50.375±1.289% and 46.009±0.671%(P<0.05)for MPPs,65.301±0.124% and52.4351±0.967%(P<0.01)for GMPs,respectively.In contrast,CMPs proliferation appeared to be reduced during cancer.The proportions of EdU positive cells in CMPs were 50.272±1.217% and62.308±8.011 %(P<0.05),respectively in normal and tumor-bearing mice.These data suggested that the changes in abundance of hematopoietic stem/progenitor cell populations in cancer could be a result of changes in their proliferation capacities.(3)Cell cycle changes of bone marrow hematopoietic stem/progenitor cells during tumor growth.In view of changes in hematopoietic stem/progenitor cell proliferation during tumor growth,we moved on to analyze the cell cycle distribution of hematopoietic stem/progenitor cells.Cell cycle analysis was performed using cell surface antigens combined with 7-AAD nuclear staining.The distribution G0/Gi phase,S phase and G2/M phase cells within HSCs accounted for81.795±1.378%,11.308±2.603% and 7.208±1.831%,respectively,in tumor-bearing mice,and the percentages of corresponding phase cells changed to 87.250±0.779%,6.442±0.398%,6.624±0.709% in normal mice.MPPs and GMPs also showed similar changes.The percentages of G0/G1 phase,S phase and G2/M phase cells were 63.849±5.062%,20.095±3.967% and14.299±3.055% for MPPs,and 54.474±0.983%,32.678±2.044% and 15.112±1.004% for GMPs,respectively;in tumor-bearing mice,where as these percentages 70.203± 1.691 %,17.141 ±3.438%,12.632±1.526% for MPPs,and 56.655±2.491%,31.617±2.331%,12.081±0.477% for GMP,respectively,in normal mice.Consistent with the cellular content changes,the proportions of G0/G1 phase,S phase and G2/M phase cells within CMPs were 56.497±6.581%,27.787±3.944%,14.730±9.071%,repectively,in tumor-bearing mice,and 63.995±2.703%,26.195±3.959%,9.971±2.082%,repectively,in normal mice.These data not only supported well the results of frequency and proliferation analysis of hematopoietic stem/progenitor cell populations,but also suggested that tumor cells might promote division of HSCs,MPPs and GMPs by stimulating them entering into cycling;on the one hand,and reduced CMP proliferation by keeping cells to stay out of or exit from cell cycle,on the other hand.(4)Changes in expression of hematopoietic stem/progenitor cell development-related genes.To dissect the molecular basis underlying hematopoietic stem/progenitor cells development changes in tumor progression,we examined the several genes known to have a role in HSCs,MPPs,CMPs and GMPs.Semi-quantitative PCR and quantitative PCR analysis showed that the expressions of RUNX1 was significantly down-regulated in HSCs and MPPs under tumor condition.The mRNA levels of Tumor HSCs and MPPs were only 0.194±0.065(P<0.01)and0.2531±0.066 folds(P<0.01),respectively,of normal cells.The expression of PU.1 in MPPs and CMPs also decreased in tumor-bearing mice,and the mRNA levels of Tumor HSCs and MPPs were 0.448±0.191 times(P<0.01)and 0.272±0.106 times(P<0.05),respectively,of normal mice.The expression of IRF8 was down-regulated significantly in HSCs,MPPs,CMPs,GMPs,the Mrna levels of were 0.283±0.048 fold(P<0.01),0.403±0.094 fold(P<0.01),0.081±0.070 fold(P<0.01),0.205±0.092 fold(P<0.01)respectively,of normal cells.These results suggested that distince transcription factors might take part in the developmental regulation of hematopoietic stem/progenitor cells at different stages.Conclusion:(1)The capacity of mouse hematopoietic stem/progenitor cells to form immune cells is substantially enhanced during tumor procession.This increase is not systemic but limited specifically to the myeloid lineages.Results from our systematic analysis of hematopoietic stem/progenitor cells at different developmental stages indicated that except CMPs,HSCs,MPPs,GMPs under tumor condition proliferate much faster than normal cells,suggesting that HSCs might be activated and,after passing the CMP stage briefly,differentiate into GMPs.It is well established that myeloid cells at different stages of the myeloid developmental pathway,including down stream populations such as monocytes and granulocytes and early myeloid progenitors,are endowed with potent immune-suppressive activities.Our findings may provide a plausible explaination for the remarkable expansion accompying tumor progression.(2)The development of mouse hematopoietic stem/progenitor cells is subject to regulation of specific transcription factors.An examination a panel of hematopoiesis-associated genes showed that RUNX1,PU.1 and IRF8 were all downregulated during tumor development.Given the specific cell types in which these genes pxpress,it is likely that RUNX1 may function mainly in HSCs,PU.1 regulate CMPs development from its precursor,and IRF8 may play a role in different stages particularly in the development of cell populations from HSCs to GMPs.These findings not only enhance our understanding of molecular mechanisms responsible for bone marrow hematopoietic stem/progenitor cell development in cancer,but also may prove useful for creating specific immune intervention approaches or gene therapies for tumor clinics.