| BackgroundThere is no doubt that the morbidity and mortality of cancer is still the highest in the world. It is vital that new technologies emerge for accurate detection and treatment of malignant tumor in medical research field. Breast cancer, one of the most common malignant tumors of female, negatively influences normal mental and physical functions and even endangers patients’ life. Patients with cancer frequently suffer from a multifactorial anemia, which negatively influences normal mental and physical functions with debilitating symptoms, such as fatigue, headache, dizziness, nausea and depression. These symptoms directly affect the patient’s mental health and quality of life. The etiology and treatment for cancer-related anemia remain controversial, and the complexity of the interactions between tumors and erythropoiesis system has been examined and discussed. Cancer-related anemia and its mechanism provide important theoretical basis for clinical treatment of cancer.Erythropoiesis is a process through which the hematopoietic stem cells (HSCs) develop into red blood cells (RBCs). At steady state, medullar erythropoiesis is primarily homeostatic, whereas stress erythropoiesis predominates in the spleen or liver after several disorders, such as acute anemia, Anemia of inflammation (AI). The increase of Epo can stimulate pathological erythropoiesis in spleen or liver to ease anemia status by elevating the red blood cells in circulation system. More recently, studies have shown that macrophages support pathological erythropoiesis in polycythemia vera and β-thalassemia. The spleen contains a unique microenvironment that can support stress-erythropoiesis. Macrophages play a crucial role in stress erythropoiesis that develops in the spleen. To get a better insight into the link between cancer, anemia and erythropoiesis, we examined the role of spleen in murine breast tumor models.Objective:1、Under physiologic conditions, erythropoiesis is regulated by a complex feedback mechanism to maintain the mass of circulating RBCs at an optimum level. Anemia of inflammation (AI), one of the most common forms of anemia seen clinically, develops during chronic inflammatory conditions, such as chronic infections, malignancies, and chronic kidney disease. Nutritional deficiencies of iron or severe protein calorie malnutrition, decreased red cell survival, reduced EPO levels and impaired bone marrow hematopoietic function may be the pathogenesis of anemia in patients with cancer. So, cancer-related anemia and its mechanism is one of the purposes of this study.2、At steady state, hamatopoietic stem cells (HSCs) reside in bone marrow (BM) and produce all cells required to replenish the blood. Stress erythropoiesis predominates in the spleen after several disorders. So in this study, we aim to investigate the effect of tumor on erythropoiesis in bone marrow and the pathological erythropoiesis in spleen of murine breast tumor models.3、Bone morphogenetic protein 4 (BMP4) can stimulate the expansion of stress-erythroid progenitors in the spleen and promote their differentiation into burst-forming units-erythroid (BFU-E) and differentiate rapidly into erythroblasts. So another objective of the work is to explore the molecular processes responsible for splenic erythropoiesis in tumor-bearing mice.Methods:Part 1:The consequences of tumor development on erythropoiesis and the evaluation of the causes of anemia1. Induction of murine breast tumor and the hematologic analysis1.1 Different groups were allocated in a randomized manner, the breast tumor models were induced by implanting tumor cells into the right fourth mammary gland.1.2 Mice were anesthetized with sodium pentobarbital at day 28 after tumor cells transplantation. Blood was collected on ethylenediaminetetraacetic acid (EDTA) by cardiac puncture. Red blood cell (RBC) counts, reticulocytes, hemoglobin (Hb) concentrations and hematocrit were measured on the SYSMEX XT-2000iV automated blood cell analyzer.2. Measurement of serum proinflammatory cytokinesDetermination of serum concentrations of IL-6, IFN-γ and TNF-α were carried out after tumor cells transplantation using commercially available enzyme-linked immunosorbent assay kits.3. Serum iron parameters in tumor-bearing miceSerum iron and unsaturated iron binding capacity were measured using an Iron/TIBC reagent set, and transferrin saturation was calculated according to the manufacture’s instructions.4. Measurement of RBC lifespan in tumor-bearing miceRBC lifespan was determined by labeling RBCs with sulfo-NHS-biotin reagent using flowcytometry.5. Serum G-CSF was examined in tumor-bearing miceG-CSF concentrations were measured on days 1,14,21 and 28 of tumor cells implantation by flow cytometry using the mouse G-CSF Flex-Set bead array. Meanwhile, G-CSF levels in 4T1 supernatant were also examined.Part 2:The effect of tumor development on bone marrow erythropoiesis.1. The appearance of femur dissected from tumor-bearing mice1.1 Mice were sacrificed by cervical dislocation. The femurs were stripped and photographed.1.2 Bone marrow cells were flushed out of femurs, and cells were resuspended in Iscove’s medium. Total cells of bone marrow (BM) in one femur were counted under a microscope using a Counting Chamber Set.2. The functional erythroid progenitors in bone marrow of tumor-bearing miceThe numbers of BFU-E and CFU-E were measured by plating nucleated cells from bone marrow in methylcellulose media. BFU-E and CFU-E colonies were counted under a microscope.3. The erythroblast terminal differentiation in bone marrow of tumor-bearing miceCD71 and Ter119 reflect the erythroblast terminal differentiation. The percentage of Ter119-positive and CD71-postitive cells in bone marrow of tumor-bearing mice were measured using flow cytometry.4. The effect of G-CSF on bone marrow erythropoiesis4.1 Wild-type mice were treated with G-CSF and the femurs were dissected and photographed. Total cells of bone marrow in one femur from G-CSF treated mice were counted.4.2 Bone marrow cells were isolated from G-CSF treated mice and then plated in methylcellulose media. BFU-E and CFU-E colonies were counted under a microscope to explore the influence of G-CSF on functional erythroid progenitors.4.3 To investigate the role of G-CSF on cells terminal differentiation in bone marrow using flow cytometry with CD71 and Ter119 markers.Part 3:The role of spleen in stress erythropoiesis of tumor-bearing mice1. The appearance of spleen from tumor-bearing mice1.1 Mice were sacrificed by cervical dislocation. The spleen was harvested, photographed and weighted.1.2 Splenocytes were isolated by mechanical dissociation of the spleen. Cells were resuspended in Iscove’s medium and counted under a microscope.2. The erythroid progenitors in the spleen response to tumor burdenTo measure the numbers of BFU-E and CFU-E in spleen of the tumor-bearing mice, splenocytes were plated in methylcellulose. BFU-E and CFU-E colonies were counted under a microscope.3. The differentiation profiling of terminal erythroid precursors from spleen in tumor-bearing miceTo examine the erythroblast terminal differentiation, the percentage of Ter119-positive and CD71-postitive cells in spleen of tumor-bearing mice were measured using flow cytometry.4. In wild-type mice, splenic erythropoiesis during G^CSF administration4.1 The changes of spleen weight and splenocyte numbers after treatment of wild-type mice with G-CSF.4.2 Splenocyte from G-CSF treated mice were harvested and plated in methylcellulose media. BFU-E and CFU-E colonies were counted under a microscope.4.3 Splenic erythroid terminal differentiation using flow cytometry with CD71 and Ter119 markers after treatment of wild-type mice with G-CSF.Part 4:The effect of splenectomy on stress erythropoiesis of tumor-bearing mice1. The analysis of peripheral blood from splenectomized and sham-operated tumor-bearing mice1.1 Splenectomy was performed and the mice were allowed to recover for at least 2 weeks before injeceted tumor cells.1.2 Blood was collected on days 7,14,21 and 28 after tumor cells implantation. Red blood cell (RBC) and white blood cell (WBC) counts, reticulocytes, hemoglobin (Hb) concentrations, granulocyte, lymphocyte, monocyte, hematocrit were measured on the automated blood cell analyzer.2. Serum levels of proinflammatory cytokines in splenectomized 4T1 tumor-bearing mice.Serum concentrations of IFN-y and IL-6 were analyzed by ELISA in splenectomized 4T1 tumor-bearing mice.3. Serum iron in splenectomized 4T1 tumor-bearing miceSerum iron and transferring saturation in splenectomized 4T1 tumor-bearing mice were measured using an Iron/TIBC reagent set.4. Serum levels of G-CSF in splenectomized 4T1 tumor-bearing miceSerum levels of G-CSF in splenectomized 4T1 tumor-bearing mice were analyzed by flow cytometry using the mouse G-CSF Flex-Set bead array at indicated times after tumor cells implantation.5. Hematological parameters in G-CSF-treated mice without spleenBlood was collected from G-CSF-treated mice with or without spleen, and RBC counts, reticulocytes, hemoglobin (Hb) concentrations and hematocrit were measured on the automated blood cell analyzer.Part 5:The role of macrophages during tumor-stress erythropoiesis1. The analysis of peripheral blood in tumor-bearing mice depleted macrophages in vivo.1.1 The macrophages in vivo were depleted by administering liposomal clodronate on days 7 and 18 of 4T1 tumor cells implantation.1.2 By day 28 tumor cells transplantation, RBC numbers, hemoglobin levels and hematocrit were measured on the automated blood cell analyzer after clodronate treatment.2. The effect of clodronate on splenic erythropoiesis in tumor-bearing mice2.1 Splenocytes in tumor-bearing mice treated with clodronate were isolated and the BFU-E and CFU-E were analyzied.2.2 In the absence of macrophages, splenic erythroid terminal differentiation was determined using flow cytometry with CD71 and Ter119 markers at day 20 of tumor cells transplantation.3. The mechanisms underlying macrophages mediated-erythropoiesis in the spleen of tumor-bearing mice3.1 The circulating Epo levels in tumor-bearing mice were measured by Elisa.3.2 BMP4 expression in the spleen of breast tumor models was assessed by RT-PCR and immunohistochemistry;3.3 Colocalization of BMP4 and F4/80 in the spleen of tumor-bearing mice was analyzed by confocal immunofluorescence.3.4 The expression of BMP4 in spleen of tumor bearing mice after macrophage depletion with cloldronate was analyzed by RT-PCR, immunohistochemistry and immunofluorescence.4. The effect of splenic erythropoiesis on tumor growthTumor volume was recorded for splenectomy and clodronate treated mice every two days at sixth day after tumor cells implantation. On day 28 of tumor cells injection, mice were sacrificed, the tumors were excised from the mice and weighed.Results:Part 1:Tumor-bearing mice display anemia and high serum levels ofG-CSF.1. Induction of murine breast tumor and the hematologic analysis4T1 tumor-bearing mice had anemic phenotype, with lower RBC count, hematocrit and Hb levels, as well as increased reticulocytosis compared to control mice.2. Measurement of serum proinflammatory cytokinesThe serum levels of IL-6 were only slightly affected by tumor at day 28, while the serum levels of IFN-y were not increased, and serum TNF-a was undetectable in tumor-bearing mice up to day 28 after tumor cells implantation.3. Serum iron parameters in tumor-bearing miceThe serum iron and transferrin saturation did not change significantly in tumor-bearing mice compared to control mice.4. Measurement of RBC lifespan in tumor-bearing miceThe percent of biotinylated RBCs in tumor-bearing mice is similar to control mice at different time, indicating RBC lifespan is not reduced.5. Serum G-CSF was examined in tumor-bearing miceSerum G-CSF values were significantly increased in tumor-bearing mice in comparison with control mice. G-CSF levels in 4T1 supernatant were higher than that in normal medium.Part 2:G-CSF is associated with medullar erythropoiesis repression induced by 4T1 tumor1. The appearance of femur dissected from tumor-bearing miceThe long bones of the tumor-bearing mice were paler than those of control mice. The total number of cells in femurs was also reduced along with tumor growth.2. The functional erythroid progenitors in bone marrow of tumor-bearing miceBoth BFU-E and CFU-E colony numbers of bone marrow decreased along with tumor growth, indicating that there are fewer functional erythroid progenitors in bone marrow of tumor-bearing mice.3. The erythroblast terminal differentiation in bone marrow of tumor-bearing miceThe proportions of Ter119+ cells and CD71+ cells in bone marrow of tumor-bearing mice were both significantly lower than in control mice, which indicate tumor-stress impaired the erythroblast terminal differentiation in bone marrow. Meanwhile, the total number of cell subsets expressing CD71 or Ter119 staining in bone marrow was also decreased in tumor-bearing mice.4. The effect of G-CSF on bone marrow erythropoiesisG-CSF can mobilize HSCs from bone marrow and suppress medullar erythropoiesis. As positive control, treatment of wild-type mice with G-CSF also led to bone marrow erythropoiesis repression, the femurs of G-CSF-treated mice were paler, the total number of cells in femurs was reduced, BFU-E and CFU-E colony numbers decreased, and the percentage of Ter119-positive and CD71-postitive cells were lower than those of control mice.Part 3:G-CSF promotes stress erythropoiesis in spleen of tumor-bearing mice1. The appearance of spleen from tumor-bearing miceSpleen was harvested at different time after tumor cells injection. One week after tumor cells implantation, the average spleen weight and splenocyte numbers increased markedly and continued to increase over subsequent weeks.2. The erythroid progenitors in the spleen response to tumor burdenThe numbers of BFU-E and CFU-E of spleen were increased in the tumor-bearing mice, indicating that expansion of erythroid progenitors in the spleen responses to tumor burden.3. The differentiation profiling of terminal erythroid precursors from spleen in tumor-bearing miceTumor development triggered increases of CD71+and Ter119+ cells. The percentage and the number of Ter119+ cells and CD71+ cells in spleen of tumor-bearing mice were both significantly increased than in control mice.4. In wild-type mice, splenic erythropoiesis during G-CSF administrationAs positive control, splenomegaly and splenic erythropoiesis developed during G-CSF administration. The total number of splenocyte increased, BFU-E and CFU-E colony numbers elevated, and the percentage of Ter119-positive and CD71-postitive cells were higher than those of control mice.Part 4:Splenectomy aggravates tumor-induced anemia1. The analysis of peripheral blood in either splenectomized or sham-operated tumor-bearing micePeripheral blood analysis revealed that splenectomy significantly reduced the number of RBCs, reticulocytes, hematocrit and down regulated the Hb levels in tumor-bearing mice, indicating that the anemia was of greater severity in the tumor-bearing mice undergoing splenectomy than in the sham-operated tumor-bearing mice. WBCs, granulocyte, lymphocyte and monocyte were higher in splenectomy tumor-bearing mice than in the sham-operated tumor-bearing mice. As control, splenectomized normal mice did not develop an overt peripheral blood anemia, indicating that splenic function is not essential for steady erythropoiesis in normal mice.2. Serum levels of proinflammatory cytokines in splenectomized 4T1 tumor-bearing miceIFN-y and IL-6 in serum of splenectomized tumor-bearing mice were similar to sham-operated tumor-bearing mice.3. Serum iron in splenectomized tumor-bearing miceSerum iron and transferring saturation in splenectomized tumor-bearing mice were also similar to sham-operated tumor-bearing mice. 4. Serum levels of G-CSF in splenectomized tumor-bearing miceThe serum concentration of G-CSF in splenectomized 4T1 tumor-bearing mice was increased, which is similar to sham-operated tumor-bearing mice.5. Hematological parameters in G-CSF-treated mice without spleenThe hematologic parameters of wild-type mice treated with G-CSF revealed that splenectomy significantly reduced the number of RBCs, reticulocytes, hematocrit and down regulated the Hb levels compared to G-CSF-treated mice with spleen.Part 5:Macrophage depletion impairs tumor-driven erythropoiesis in spleen1. The analysis of peripheral blood in tumor-bearing mice depleted macrophages in vivo.Liposomal clodronate was used to reduce the number of macrophages in vivo. Peripheral blood analysis showed that clodronate treatment decreased RBC numbers, hemoglobin levels and hematocrit.2. The effect of clodronate on splenic erythropoiesis in tumor-bearing miceThe spleen weight decreased significantly when macrophages were depleted in tumor-bearing mice. Macrophage depletion reduced the numbers of splenic-stress BFU-E and CFU-E, indicating that there are fewer functional erythroid progenitors in the spleen under the conditions of macrophage deficiency. We also found that absence of macrophages impacted splenic erythroid terminal differentiation using flow cytometry with CD71 and Ter119 markers at day 20 of tumor cells transplantation.3. The mechanisms underlying macrophages mediated-erythropoiesis in the spleen of tumor-bearing mice3.1 Epo concentrations elevated in the serum of mice with tumor burden compared to normal controls.3.2 BMP4 in the spleen was increased at the mRNA and protein level under tumor-stress conditions.3.3 Double staining for F4/80 and BMP4 showed that the majority of spleen macrophages expressing F4/80 were associated with BMP4 expression in tumor-bearing mice.3.4 Macrophage depletion with cloldronate reduced the induction of splenic BMP4, BMP4 expression was decreased at the mRNA and protein level. The colocalization of BMP4 and F4/80 by confocal immunofluorescence also showed that macrophages expressing F4/80 and BMP4 were reduced in spleen of cloldronate treated tumor-bearing mice.4. The effect of splenic erythropoiesis on tumor growthOur results showed that splenectomy and clodronate treatment abolish splenic erythropoiesis and attenuates tumor growth.Conclusions1、Tumor-bearing mice had an anemic phenotype. G-CSF, mainly synthesized by tumor cells, mobilizes HSC to the blood and disrupts bone marrow erythropoiesis.2、Splenic erythropoiesis was stimulated under tumor-stress conditions. G-CSF is involved in tumor-induced anemia and mediated stress erythropoiesis. The spleen is indispensable for tumor-induced stress erythropoiesis.3、Tumor-stress stimulates Epo expression in vivo. Under conditions of stress erythropoiesis, splenic macrophages synthesize BMP4 in response to Epo, which induces proliferation of stress BFU-E that matures into an erythroid colony-forming unit (CFU-E) before extending the proerythroblast stage.4、Splenectomy and clodronate treatment aggravate tumor-induced anemia, attenuates tumor growth, which suggest that pathological splenic erythropoiesis promotes the growth of breast tumors. |
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