Lactate Promotes Metastasis Of Normoxic Colorectal Cancer Stem Cells Through PGC-1α-mediated Oxidative Phosphorylation

Part Ⅰ:The detection,enrichment,and identification of normoxic cancer stem cells in colorectal cancerObjectives:To detect the expression of normoxic CSCs in CRC.To enrich and identify normoxic CSCs in vitro.Methods:Two methods were used to detect normoxic CSCs:Method 1:Clinical specimens of CRC were collected for tissue immunofluorescence staining,based on the expression of CD31 and HIF-1ɑ,the normoxic areas(CD31~+HIF-1ɑ~-)and hypoxic areas(CD31~-HIF-1ɑ~+)inside CRC tissue were distinguished.The proportions of CD133~+cells were counted in the normoxic and hypoxic areas respectively,that is,the proportions of normoxic and hypoxic CSCs.Method 2:The clinical specimens of CRC were processed into single cells,and the proportion of normoxic CSCs(Ep CAM~+CD133~+HIF-1α~-)was detected by flow cytometry.In addition,by analyzing the expression of normoxic CSCs in different tumor stages,the association of normoxic CSCs with tumor progression and metastasis was explored.Two methods were used to enrich normoxic CSCs in vitro:Method 1:Sphere-forming assay was used to enrich normoxic CSCs.Method 2:After being infected by HRE-driven GFP(p-HRE-GFP)lentivirus,CD133~+GFP~-cells were selected by flow cytometry sorting.The expressions of CD133,Nanog,Sox2,and HIF-1ɑin the enriched cells were verified by Western-blot.Results:Tissue immunofluorescence and flow cytometry showed that there were normoxic and hypoxic CSCs in CRC.In addition,the proportion of normoxic CSCs increased with the progression of tumor stage.More importantly,the proportion of normoxic CSCs was higher than that of hypoxic CSCs in stage IV CRC.The results of Western-blot experiments showed that the enriched tumor cells highly expressed CD133,Sox2,and Nanog,and at the same time HIF-1αwas expressed at a low level.Conclusions:Both normoxic and hypoxic CSCs existed in CRC.The proportion of normoxic CSCs was positively related to tumor stage and metastasis.In vitro sphere-forming assay and flow sorting of CD133~+GFP~-cells could stably enrich normoxic CSCs.Part Ⅱ: Lactate promotes the invasion,migration,and metastasis of normoxic cancer stem cells via oxidative phosphorylation pathwayObjectives: To explore the effect of lactate on the metastasis of normoxic CSCs.To explore the differences in the metabolic characteristics of normoxic CSCs and other tumor cell subsets.To explore whether lactate affects the invasion,migration,and metastatic ability of normoxic CSCs through OXPHOS pathway.Methods: Transwell invasion assay and wound healing assay were used to detect the changes in the invasion and migration ability of normoxic CSCs after the treatment of lactate.The influence of lactate on epithelial-mesenchymal transitions(EMT)of normoxic CSCs was detected by immunofluorescence and Western-blot assay.The effect of lactate on the metastatic potential of normoxic CSCs was detected by pulmonary metastasis model.Hypoxia tumor cells were induced by Co Cl2 or culturing in the hypoxic condition.Then tumor cell subsets were enriched and Western-blot experiment was used to detect their metabolic characteristics.Lactate detection kit was used to detect lactate concentration in tumor cell subsets.Cell immunofluorescence,ATP detection,and Western-blot assay were used to detect the influence of conditioned medium on OXPHOS activity of normoxic CSCs.After knockdown of monocarboxylate transporter 1(MCT1)or lactate dehydrogenase B(LDHB),the effects of conditioned medium on the OXPHOS activity,invasion,and migration ability of normoxic CSCs were detected by oxygen consumption rate(OCR)assay,Transwell assay,and wound healing assay.Results: Transwell and wound healing assay showed that lactate promoted the invasion and migration ability of normoxic CSCs.The results of cellular immunofluorescence and Western-blot experiments showed that lactate promoted EMT of normoxic CSCs.Pulmonary metastasis model showed that lactate promoted the metastatic potential of normoxic CSCs.Western-blot experiments showed that HIF-1α was highly expressed in cells after hypoxic treatment,and tumor cell subsets could be enriched in vitro though sphere-forming assay or flow cytometry sorting.Lactate concentration in the culture medium of normoxic CSCs was lower than that of other tumor cell subsets,while the expression of TOM20 was higher.Cell immunofluorescence,ATP detection,and Westernblot showed that conditioned medium enhanced the OXPHOS activity of normoxic CSCs.The results of OCR assay showed that after knocking down MCT1 or LDHB,the effect of conditioned medium on enhancing the OXPHOS activity of normoxic CSCs was impaired.In addition,Transwell invasion assay and wound healing assay showed that after knocking down MCT1 or LDHB,the effect of conditioned medium on promoting migration and invasion of normoxic CSCs was also impaired.Conclusions: Lactate promoted invasion,migration,EMT,and metastasis ability of normoxic CSCs.Hypoxic treatment,combined with sphere-forming assay or flow cytometry sorting could enrich tumor cell subsets.Normoxic CSCs obtained high OXPHOS activity,whereas other tumor cell subsets were prone to glycolysis.Conditioned medium enhanced OXPHOS activity of normoxic CSCs.After inhibiting lactate uptake,the effect of conditioned medium on promoting the invasion and migration of normoxic CSCs was impaired.Part Ⅲ: Lactate promotes normoxic cancer stem cell metastasis through PGC-1α-regulated oxidative phosphorylation pathwayObjectives: To explore the regulation of PGC-1α on mitochondrial biosynthesis and OXPHOS activity in normoxic CSCs.To explore whether lactate affects the OXPHOS activity and metastatic ability of normoxic CSCs via PGC-1α.To explore the influence of HIF-1α on OXPHOS activity,invasion,and migration ability of normoxic CSCs.Methods: Western-blot assay was used to detect the expression of PGC-1α in normoxic CSCs.After knocking down PGC-1α,RT-PCR was used to detect the changes in m RNA expression of mitochondrial biosynthesis-related genes in normoxic CSCs.Electron microscopy detected the changes in mitochondrial numbers in normoxic CSCs.After knockdown of PGC-1α,the effects of conditioned medium on the OXPHOS activity,invasion,and migration of normoxic CSCs were detected by OCR assay,transwell invasion assay,and wound healing assay.After knocking down PGC-1α,the effect of conditioned medium on the metastatic potential of normoxic CSCs was detected by pulmonary metastasis model.Treatment of normoxic CSCs with prolyl hydroxylase domain 2(PHD2)inhibitors to inhibit the degradation of HIF-1α,OCR assay was used to detect changes in the OXPHOS activity of normoxic CSCs and Transwell invasion assay and wound healing assay were used to detect the changes of invasion and migration ability of normoxic CSCs.Results: Western-blot showed that the expression of PGC-1α in normoxic CSCs was higher than that of other tumor cell subsets.After knocking down PGC-1α,the results of electron microscopy and RT-PCR showed that the number of mitochondria and the expression of mitochondrial biosynthesis genes in normoxic CSCs were significantly reduced.OCR assay and ATP detection showed that after knockdown of PGC-1α,the ability of conditioned medium to enhance the OXPHOS activity of normoxic CSCs was impaired.Transwell invasion assay,wound healing assay,and pulmonary metastasis model showed that after knockdown of PGC-1α,the effect of conditioned medium on enhancing invasion,migration,and metastatic potential of normoxic CSCs was also impaired.Western-blot showed that after inhibiting the degradation of HIF-1α,the expression of HIF-1α in normoxic CSCs increased,while the expression of PGC-1α decreased.OCR assay,Transwell invasion assay,and wound healing assay showed that after high expression of HIF-1α,the effect of conditioned medium on enhancing OXPHOS activity,invasion ability,and migration ability of normoxic CSCs was impaired.Conclusions: PGC-1α regulated mitochondrial biosynthesis and OXPHOS activity in normoxic CSCs.Lactate regulated the OXPHOS activity of normoxic CSCs via PGC-1α,thereby enhancing metastatic potential.HIF-1α negatively regulated the expression of PGC-1α.After high expression of HIF-1α,the effects of conditioned medium on enhancing OXPHOS activity,invasion,and migration of normoxic CSCs were impaired.