The Role And Mechanism Of Metabolic Remodeling In Tumor-induced Impaired Differentiation Of Extramedullary Erythroid Precursor Cells

Background:Malignant tumor has a negative systemic impact on its hosts,and no cancer complication is more ubiquitous than anemia.More than 40%of all cancer patients will be anaemic regardless of treatment.Correction of anemia is not only linked to an improved quality of life,but also potentially improve the survival of cancer patients.Recent studies have shown that cancer-related anemia(CRA)has an adverse impact on immunotherapy,and a low hemoglobin level is an independent prognostic factor associated with tumor progression.Therefore,exploring the molecular mechanism of CRA will help to understand the impact of tumors on both hematopoietic and immune system,and provide new insights into improving anemia and enhancing the efficacy of immunotherapy.Although the postnatal erythropoiesis primarily occurs in BM,when faced with an accelerated demand of oxygen and nutrients,such as chronic infection and advanced tumor,hematopoietic stresses mobilize hematopoietic stem/progenitor cells(HSPCs)from the bone marrow to the liver,spleen,lymph nodes and initiate extramedullary hematopoiesis(EMH)of embryonic period.The spleen is both a hematopoietic and an immune organ,providing integral components of the tumor microenvironment and serving as a critical mediator of tumor-host interactions.Previous studies have shown that advanced cancer may initiate extramedullary hematopoiesis and lead to accumulation of a large number of erythroid precursor cells(EPCs),which are blocked in erythroid differentiation process in spleen.Among them,CD45~+subsets significantly inhibited the activation and response of CD8~+T cells;further research hightlighted the robust myeloid differentiation bias of CD45~+EPCs with strong immunosuppressive properties,thus impairing efficacy of immunotherapy.Thus,differentiation disorder of EPCs is one of the key mechanisms that inducing or exacerbating anemia in malignant circumstances,as well as increasing the risk of tumor progression and recurrence.However,the mechanism of extramedullary hematopoietic disorder is still inexplicit.Traditional view is that the lineage differentiation of HSCs is exquisitely regulated by a variety of cytokines,transcription factors and interactions between immune cells.However,beyond a role in energetic support,new evidence implicates nutrient response acts as mediators of crosstalk between HSCs cell fate and metabolic fluxes,intermediate metabolites,epigenetic modification and mitochondrial homeostasis.In our previous study,we found that immature erythroid precursor cells induced by EMH in the spleen secreted a large number of reactive oxygen species(ROS),and had numerous discrepancies in multiple mitochondrial metabolic pathways at transcriptional level.Therefore,this study intends to explore the mechanism of tumor-induced dysfunction of erythropoiesis from the perspective of mitochondrion-mediated metabolic remodeling,highlighting the urgent need to elucidate ineffective erythropoiesis in cancer patients and develop an optimal treatment strategy as well as a personalized approach to CRA management.Aims&Methods:(1)Explore the discrepancies in EMH between acute anemia and caner-related anemia.Animal models of acute anemia induced by phenylhydrazine(PHZ)and cancer-related anemia were generated.To identify the main sub-population and blockade period of erythroid differentiation,flow cytometry was used to detect the proportion and absolute number of immature erythroblasts in each periods(Pro E/Ery A/Ery B/Ery C).Furthermore,proliferation and apoptosis of blocked erythroid precursor cells were determined with Brdu and Annexin V/PI staining.(2)Mitochondrial dynamic and functional changes in tumor-induced splenic CD45~+EPCs and their role in extramedullary erythroid differentiation arrest.Splenic CD45~+EPCs of acute anemia and late-stage tumor mice were isolated by flow cytometry,and their mitochondrial morphological discrepancies were detected by using confocal microscope and transmission electron microscope.The mitochondrion content,mitochondrial membrane potential(MMP),ATP content and oxygen consumption rate(OCR)of CD45~+EPCs were then determined for mitochondrial functional detection.In vitro erythroid differentiation experiments were conducted to investigate the correlation between enhanced OXPHOS and erythropoiesis.Then,the effects of metabolic regulatory drugs on the splenic erythropoiesis and anemia were clarified by in vivo experiments.(3)Clarify the upstream mechanism of erythropoiesis arrest in tumor-induced extramedullary CD45~+EPCs.By screening RNA-SEQ data,mitophagy was determined and its key regulatory molecules were verified in vitro and in vivo.We further clarified the relationship between mitophagy and the mitochondrial dynamics and function of CD45~+EPCs by using mitophagy inducer Rapamycin.Then,we determined whether mitophagy inducer could improve erythroid maturation ex vivo by performing erythroid differentiation experiments.(4)The role of mitophagy enhancement in improving caner-related anemia.To evaluate the overall effect of mitophagy inducer on extramedullary erythropoiesis,we performed splenectomy on late-stage tumor mouse model.In order to explore new clinical prospects of CRA treatment,we tested the effects of mitophagy induer on remedying ineffective erthropoiesis in vivo.(5)Validation on clinical samples.The clinical samples of anemic patients with trauma or chronic renal failure as well as cancer in our hospital were collected to detect the erythropoietic periods of peripheral blood by flow cytometry.Mitochondrial characteristics and mitophagy status of CD45~+EPCs were as well detected.Results:(1)Tumor associated anemia initiated ineffective extramedullary hematopoiesis.With the increasing demand of nutrients and oxygen,the body usually initiates extramedullary hematopoiesis to compensate for the inadequate BM hematopoiesis.Under different erythropoietic stress,BM can rapidly triger mobilization of HSPCs to the extramedullary hematopoiesis site spleen,inducing splenomegaly and extensive proliferation of splenic erythroid precursor cells.We found that splenic erythropoiesis under tumor condition can not alleviate anemia,even the size of the spleen is adversely correlated with HGB level,indicating that ineffective splenic hematopoiesis in tumor-bearing mouse model.Further,we analyzed the proportion and quantity of Pro E/Ery A/Ery B/Ery C using Ter119/CD71 erythroid markers.Although the proportion and quantity of nucleated erythroid cells both increased significantly in mice of acute anemia and tumor with anemia,the proportion and quantity of Pro E and Ery A increased more robustly in mice with tumor,while the proportion and quantity of Ery B and Ery C were gradually decreased compared with acute counterparts.These data demonstrated that,unlike acute anemia,tumor-induced extramedullary hematopoiesis led to a blockade of erythropoiesis,and the retardation mainly occured in the Pro E/Ery A period.Although immature erythroid precursor cells are a heterogeneous population,we found that the CD45~+subgroup dominated in this retarded population.Moreover,the CD45 marker itself is also a well-established biomarker to distinguish immature erythroblasts,thus,we identified CD45~+EPCs as a key subset responsible for erythropoiesis blockade.Subsequently,in order to confirm this,we tested the proliferation and apoptosis of CD45~+EPCs,and found that there was no significant difference between acute anemia and tumor-associated anemia,suggesting that CD45~+EPCs play a vital role in defective extramedullary hematopoiesis under tumor condition.(2)Late-stage tumor-induced extramedullary CD45~+EPCs exhibited mitochondrial dynamic alterations and a decline of OXPHOS.Mito-tracker Green staining revealed an increase in the mitochondrion content of tumor-induced CD45~+EPCs.To examine the mitochondrial morphology,we employed cell confocal and transmission electron microscopy imaging,we found that mitochondrion in CD45~+EPCs of controls were mostly tubular in shape with regular arrangement of mitochondrial cristae.In contrast,mitochondrion of late-stage tumor-induced splenic CD45~+EPCs were significantly smaller and displayed a more circular shape,with irregular arrangement of mitochondrial cristae and enlarged cristae width.Mitochondrial dynamics often closely linked with its function,MMP and ATP production of tumor-derived CD45~+EPCs suggested that the mitochondrial OXPHOS was damaged compared with acute anemic mice.In addition,the expression level of key enzymes of the mitochondrial respiratory chain complexes in tumor-induced CD45~+EPCs was also decreased.The oxygen consumption rate(OCR)results further validated a weakened OXPHOS function of tumor-induced extramedullary CD45~+EPCs.These data suggested that tumor may jeopardize OXPHOS of CD45~+EPCs by remodeling the mitochondrial network of CD45~+EPCs.In order to clarify the effect of OXHPOS on erythroid differentiation,tumor-induced extramedullary CD45~+EPCs were initially isolated and then induced to differentiate into mature RBC ex vitro.With the property of promoting the tricarboxylic acid cycle,DCA was used to enhance OXPHOS,and we surprisingly found DCA could relieve erythroid differentiation arrest of tumor-induced CD45~+EPCs.Flow cytometry analysis also showed a higher proportion of Ter119~+CD71~-cells than that of control group.In subsequent studies,we found that DCA alleviated splenomegaly and CD45~+EPCs aggregation to a certain extent in vivo,however,can not effectively improve the anemia.Because DCA only promotes the tricarboxylic acid cycle and can not fundamentally improve integrity of mitochondrial structure and function,suggesting that the weakening of OXPHOS may only be a downstream regulatory factor,and the underlying mechanism affecting OXPHOS still need to be further explored.(3)Mitophagy disorder induced ineffective erythropoiesis of tumor-derived extramedullary CD45~+EPCs.The normal function of mitochondrial OXPHOS is inseparable from the dynamic regulation of mitochondrial network.By analyzing the difference of transcriptome between acute anemia and tumor-induced extramedullary CD45~+EPCs,we found that mitochondrial autophagy related genes,which are important mechanisms of mitochondrial"quality control"management,are generally declined under tumor-bearing conditions.Furthermore,by employing mitophagy dye,mitochondrion and lysosome colocalization,we confirmed that mitophagy in tumor-induced CD45~+EPCs was significantly impaired,and the expression of critical regulators of mitophay such as PINK1 and Parkin was also decreased.Given the above findings,we expected that mitophagy might potentially promote the function of mitochondrion in tumor-induced CD45~+EPCs.By inhibiting the m TOR pathway with administration of rapamycin,we noticed that compared to the saline control,the number of mitochondrial autophagosome and tubular mitochondrion was increased in tumor-induced CD45~+EPCs.Moreover,mitochondrial cristae became more regular with decreased cristae width.At the same time,the increased MMP and OCR suggested an overall improvement of OXPHOS in tumor-induced CD45~+EPCs,further validating that mitophagy was accounted for mitochondrial dynamics and metabolic remodeling.Most importantly,in vitro erythroid differentiation assay confirmed that interfering with m TOR and FOXO3 autophagic pathway can effectively promote the maturation of tumor-derived extramedullary CD45~+EPCs.(4)Accelerated mitophagy ameliorated tumor-associated anemia.Rapamycin in vivo treatment promoted the process of extramedullary erythroid differentiation,significantly ameliorated the accumulation of CD45~+EPCs in the spleen,and effectively improved splenomegaly.Through the critical observation of peripheral blood,it was also found that the peripheral blood cells and red blood cells increased after the drug intervention.The routine blood test showed that Rapamycin significantly delayed the occurrence of anemia,and effectively improved the anemic status of tumor-bearing mice,which manifested high HGB,RBC and HCT.On the other hand,by resecting the spleen in a LLC-bearing mouse model,we found that Rapamycin only played a role in improving anemia in the presence of spleen,indicating that mitochondrial autophagy dysfunction is the key to blocked extramedullary erythropoiesis under tumor condition.(5)Clinical validation.Having established the function,mechanism,and significance of tumor-induced extramedullary CD45~+EPCs in mouse models,we sought to assess the clinical relevance of erythropoiesis.In this section,we collected the peripheral blood samples of tumor patients with or with not anemia,and non-tumor traumatic and chronic renal failure anemic patients who were admitted in Xinqiao Hospital of the Army Military Medical University from October 2022 to February 2023.Flow cytometry was used to detect the erythroid differentiation of peripheral blood mononuclear cells(PBMC),as well as the mitochondrion content,MMP and mitophagy level of CD45~+EPCs.It was found that the erythroid differentiation of tumor patients was blocked,and the proportion of basophilic and polychromatic erythroblasts significantly increased,while mature erythrocytes decreased.In addition,mitochondrion content of CD45~+EPCs in tumor patients,especially those with anemia(HGB<120 g/L)was significantly increased,while MMP level decreased,which agreed with mouse models.Surprisingly,we found the level of mitophagy was increased in CD45~+EPCs of patients with mild anemia(HGB 100～120 g/L),however,when HGB<100g/L,that is,patients with moderate to severe anemia,the level of mitochondrial autophagy decreased significantly.Conclusion:In conclusion,although both acute anemia and cancer-related anemia induce EMH and the expansion of erythroid precursor cells in spleen,tumor-derived EMH can not effectively alleviate the anemic state,and extramedullary hematopoiesis-derived CD45~+EPCs exhibited differentiation disorder in response to tumor-promoting environmental stress.By exploring the underlying mechanism,we found fragmented mitochondrion accumulation and malfunction of OXPHOS in tumor-induced CD45~+EPCs.Mechanistically,restoring mitophagy facilitated mitochondrial dynamics and OXPHOS in tumor-derived CD45~+EPCs,thus improving erythropoiesis.Finally,we validated in clinical specimens that tumor patients exhibited blocked erythroid differentiation of peripheral blood,as well as increased mitochondrion content,decreased MMP and weakened mitophagy in CD45~+EPCs.Taken together,these findings highlighted the involvement of mitochondrial turnover in stress-induced expansion and differentiation of extramedullary erythroid cells.We also speculate on mitophagy pathways as potential biomarkers in selecting the most appropriate treatment strategy for CRA.