| Objective:Cancer-related anemia(CRA)is one of the common concomitant diseases of malignant tumors.With the emphasis on CRA in recent years,researches on the epidemiology and causative factors of CRA have gradually increased,but the pathogenesis of CRA is still unclear.Hematopoietic Stem Cells(HSCs)are a group of pluripotent stem cells that have the ability to self-renew and continuously produce all blood cells and immune cells in vivo.In this paper,we investigated the differentiation characteristics of erythroid cells from HSCs under tumor conditions to investigate the possible abnormal changes of this process and its potential molecular mechanisms by using a mouse lung cancer model.Methods:(1)Construction of mouse tumor model:6～8 weeks of age female C57BL/6J mice were used in this experiment,and Lewis lung carcinoma(LLC)was injected in mice subcutaneously as the experimental group,and PBS was injected in mice subcutaneously as the control group,which was used for subsequent experiments after 4 weeks.(2)Preparation of single cell suspension of bone marrow cells:After the mice were executed by cervical dislocation method,the mouse femur and tibia were separated by using medical scissors and forceps,and the bone marrow cells in the bone marrow cavity were repeatedly rinsed into DMEM complete medium by syringe.(3)Sorting of cells:Mouse bone marrow cells are incubated and labeled with specific combinations of fluorescein Euro-coupled antibodies,followed by flow cytometric sorting a sorting and purification of specific cell populations.(4)In vitro cell directed differentiation assay:cells of specific cell populations were sorted into semi-solid medium and cultured for 7～14 days,colonies were counted and the cells in the colonies were stained with Jimson’s stain and the cell types were determined by microscopic examination.(5)Detection of gene m RNA expression levels:RNA from cells was extracted using the TRIzol method and later reverse transcribed into c DNA using the c DNA First Strand Synthesis Kit for real-time fluorescence quantitative PCR(Real-Time PCR)detection.(6)Statistical method:Data were tallied by independent sample t-test using Prism software,and statistical results were expressed as mean±standard deviation.Result:(1)The changes of erythrocytes under tumor conditionsFirst,peripheral blood was obtained for Swiss Gipsa staining and the number of red blood cells(RBCs)was counted microscopically.Tumor-bearing mice and normal mice(control)origin cells were respectively denoted as T and N(same below).The number of T-RBCs and N-RBCs in the same field of view was 478.0±1.155 and 495.7±3.333,respectively,indicating a significant decrease in the number of RBCs in the tumor condition,but accompanied by a significant increase in nucleated RBCs.(2)Analysis of blood counts under tumor conditionsThe peripheral blood was analyzed in normal mice and tumor-bearing mice.The red blood cell(RBC),hemoglobin(Hb),red blood cell pressure(HCT)content and mean red blood cell volume(MCV)were counted in the peripheral blood of tumor-bearing and normal mice,respectively.The levels of T-RBC and N-RBC were 11.94±1.552×1012/L and 23.28±1.297×1012/L,respectively.the levels of T-Hb and N-Hb were 76.00±5.891(g/L)and 165.2±The size of T-MCV and N-MCV were 57.16±1.735(f L)and 45.50±0.4135(f L),respectively.It showed that the RBC,Hb,and HCT levels in peripheral blood of mice under tumor conditions were significantly reduced and MCV was significantly increased.(3)The changes of peripheral blood nucleated red blood cells in lung cancer patients under tumor conditionsThe abundance of nucleated red blood cells(NRBC)in peripheral blood of tumor patients was analyzed using flow analysis.The cells of tumor patient and normal human(control)origin were respectively denoted as T and N(same below).The results showed that T-NRBC and N-NRBC accounted for 0.2308±0.08761%and 0.0243±0.00408%of peripheral blood cells,respectively,indicating a significant increase in peripheral blood nucleated red blood cells in lung cancer patients.(4)The changes in the abundance of different progenitor cell populations upstream of the bone marrow erythroid differentiation pathway under tumor conditionsThe early stage of differentiation of hematopoietic stem cells to erythroid cells contains MPP2,common myeloid progenitor cells CMP,and erythroid/megakaryocyte progenitor cells MEP.Homozygous mice and normal mice(control)derived cells are respectively denoted as T and N(same below).Flow analysis showed that T-MPP2 and N-MPP2 accounted for 0.0083±0.00240%and 0.0023±0.00088%of total bone marrow cells,T-CMP and N-CMP accounted for 0.4165±0.08738%and 0.5068±0.02894%of total bone marrow cells,respectively.T-MEP and N-MEP accounted for 0.3158±0.09155%and 0.4160±0.06015%of the total bone marrow cells,respectively.The results indicated that there was no significant change in the abundance of different progenitor cells upstream of the bone marrow erythroid differentiation pathway under tumor conditions.(5)The changes in the abundance of different precursor cells downstream of the erythroid differentiation pathway in bone marrow and spleen under tumor conditionsThe late stage of differentiation of hematopoietic stem cells to erythrocytes contains proerythroblasts(Pro E),early erythroblasts(Basophilic erythroblasts,Baso E),intermediate erythroblasts(Polychromatic erythroblasts,Ply E),and late(Ortho E),Polychromatic erythroblasts(Ply E),and late erythroblasts(Ortho E).Cells of tumor-bearing and normal mouse(control)origin are respectively denoted as T and N(same below).Flow analysis showed that T-Pro E and N-Pro E accounted for 0.1593±0.02942%and 0.0713±0.01638%of the total bone marrow cells,respectively.T-Baso E and N-Baso E accounted for 20.87±1.485%and 27.50±0.9866%of the total bone marrow cells,respectively.T-Poly-E and N-Poly-E accounted for 1.041±0.2893%and 1.593±0.3284%of the total bone marrow cells,respectively.T-Ortho E and N-Ortho E accounted for 5.390±0.8075%and 16.90±1.858%of the total bone marrow cells,respectively.The results showed that the abundance of Baso E and Ortho E stage cells were significantly reduced in bone marrow under tumor conditions,indicating that the terminal differentiation stage of erythrocytes was impaired in bone marrow under tumor conditions.In mouse spleen,T-Pro E and N-Pro E accounted for 0.8193±0.2545%and 0.0400±0.02382%of the total splenic cells,respectively.T-Baso E and N-Baso E accounted for 35.75±3.082%and 1.023±0.2015%of the total splenic cells,respectively.T-Poly-E and T-Poly-E and N-Poly-E accounted for 3.150±0.5654%and 0.4790±0.1042%of the total spleen cells,respectively.T-Ortho E and N-Ortho E accounted for 3.833±0.6132%and 13.83±2.641%of the total spleen cells,respectively.The results showed that the abundance of Pro E,Baso E,and Ply E cells were significantly increased in the spleens of tumor-bearing mice,while the abundance of Ortho E cells was significantly decreased,indicating a large accumulation of Pro E,Baso E,and Ply E stage cells in the spleen under tumor conditions,but also an impairment in terminal erythrocyte differentiation.(6)Ability of different hematopoietic progenitor cells to differentiate and form erythroid cells in tumor-bearing miceAfter 7 days,the number of BFU-E and CFU-E colonies was determined.Tumor-bearing mice and normal mouse(control)derived cells were respectively denoted as T and N(same as below.)The number of CFU-E and BFU-E colonies was 75.67±2.186and 51.67±0.8819 for T-MPP2 cells in in vitro directed erythroid differentiation culture,respectively,and for N-MPP2 cells in in vitro directed erythroid differentiation culture colonies.The colony numbers of CFU-E and BFU-E were 75.67±1.202 and 48.33±0.8819,respectively.In vitro directed erythroid differentiation culture colony formation of T-CMP cells,the colony numbers of CFU-E and BFU-E were 20.67±1.764 and 56.00±0.5774.in vitro directed erythroid differentiation culture colony formation of N-CMP cells,the colony numbers of CFU-E and BFU-E were 20.67±1.764 and 56.00±0.5774,respectively.The colony numbers of CFU-E and BFU-E were 17.00±3.606 and 54.67±1.764,respectively,in the in vitro directed erythrocyte differentiation culture colony formation of T-MEP cells,19.33±2.333 and 35.67±1.667 for CFU-E and BFU-E,respectively.The number of CFU-E and BFU-E colonies was 15.67±1.453 and 41.67±2.963,respectively,in the in vitro directed erythroid differentiation colony formation of N-MEP cells.The results indicated that there was no significant change in the erythroid differentiation ability of progenitor cells including MPP2,CMP,and MEP cells at the stage of erythroid differentiation from hematopoietic stem cells to erythroid cells in tumor-bearing mice.(7)Ability of bone marrow erythroid differentiation pathway downstream precursor cells to differentiate and form erythroid cells under tumor conditionsAfter 2 days,the number of CFU-E colonies was measured.3.000±0.5774 CFU-E colonies were formed in T-erythroid precursor cells in vitro directed erythroid differentiation culture,and 48.33±2.404 CFU-E colonies were formed in N-erythroid precursor cells in vitro directed erythroid differentiation culture.The number of CFU-E in colony formation was 48.33±2.404,indicating a significant reduction in the ability of tumor-bearing mouse erythroid precursor cells to differentiate and form erythrocytes.(8)Swiss Jimsa staining of erythroid precursor cells for in vitro directed erythroid differentiationTo clarify the ability of erythroid precursor cells to differentiate and form erythroid cells under tumor conditions,we sorted Ter119~+CD71~+cells into erythroid-directed differentiation medium because the number of cells sorted at each stage of the downstream precursor cell differentiation pathway was too low.2 days later,Swiss Gipsa staining was performed to further determine the erythroid differentiation potential under tumor conditions.The results showed that the percentage of in vitro directed erythroid cell formation was 14.286%and 64.286%in tumor-derived Ter119~+CD71~+versus normal mouse-derived Ter119~+CD71~+,respectively.It indicates that the percentage of tumor-derived Ter119~+CD71~+differentiated to form erythrocytes was reduced.(9)Screening of regulatory factors for erythroid differentiation from erythroid precursor cells under tumor conditionsAnalysis using transcriptome cloud platform data showed that there were 6516differentially expressed genes in Ter119~+CD71~+cells of normal and tumor-bearing mice,among which 3091 genes were up-regulated and 3425 genes were down-regulated in Ter119~+CD71~+of tumor-bearing mice.These differential genes are mainly focused on biological functions such as cellular processes,single biological processes,bioregulation,stress response,metabolism;cellular composition such as cells,cellular components,organelles,cell membranes,organelles,macromolecular complexes;molecular functions such as catalytic activity,signal transduction,molecular transport,molecular sensor activity.In addition,these genes are mainly involved in cell cycle,apoptosis,NF-κB signaling pathway and other related.(10)The expression of SOX6 in erythroid precursor cells cells of normal mice and tumor-bearing miceThe expression levels of SOX6 m RNA in Ter119~+CD71~+cells of normal mice and tumor mice were detected by q RT-PCR,and the experimental results are shown in the figure,the expression of SOX6 in bone marrow Ter119~+CD71~+cells of tumor mice was0.1256±0.007 times higher than that of normal mice,and the SOX6 expression was significantly higher in normal mice than in tumor-bearing mice(P<0.05).Conclusion:CRA is a frequently observed disease accompanying malignant tumors.In this study,we investigated the red lineage cell differentiation characteristics of hematopoietic stem cells under tumor conditions utilizing a mouse lung cancer model to explore possible abnormal changes in this process and its underlying molecular mechanisms.We found that erythroid development is impaired in tumor conditions and that the impairment occurs at the stage of terminal erythroid differentiation.These findings provide new insights into the pathway of differentiation of hematopoietic stem cells to erythrocytes in the CRA disease state. |
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