Surgical Induction Of Bone Marrow Immature Cell-derived Exosomes To Promote Tumor Growth And Metastasis

Part I Study on the relationship between surgically induced bone marrow immature cell-derived exosomes in tumor growth and metastasisObjective:Surgery can cause significant changes in metabolism,inflammation and immune stress.Immature myleiod cells(IMCs)are a population of heterogeneous cells of bone marrow origin that usually proliferate in inflammation or cancer.Exosome play an important role in the occurrence and development of tumors.However,the effect of surgery-induced bone marrow-derived Ly6G IMC and its secreted exosomes on tumor progression remains unclear.We studied the relationship between bone marrow-derived Ly6G IMC and tumor growth and metastasis by in vivo and in vitro experiments,and further isolated Ly6G+IMC-derived exosomes.In vivo and in vitro experiments were conducted to investigate the effects of Ly6G IMC-derived exosomes on tumor growth and metastasis.Methods:1.Construction of postoperative metastasis model of tumor-bearing mice:Lewis lung Carcinoma(LLC)(1×106)were inoculated into the dorsal middle of C57BL/6 female mice.and the tumor was resected after the tumor volume reached 1200 mm3.and the incision was sutured.Continue to raise,and the mice were sacrificed on the 23rd day after surgery to observe the metastasis in vivo.2.Effects of surgical induction of bone marrow-derived immature cells on tumor growth and metastasis:LLC(1 ×106)were inoculated into the dorsal middle of C57BL/6 female mice and the tumor was resected after the tumor volume reached 1200 mm3.Flow cytometry was used to detect the changes of bone marrow Ly6G+IMC in tumor-bearing mice at 0 H,24 H,48 H,72 H,Day,and Day7.According to the flow results,a postoperative time point was selected.Magnetic activated cell sorting(MACS)was used to sort the surgical and non-surgical tumor-bearing mice bone marrow-derived Ly6G+IMC.LLC(5×104)were inoculated into the 24-well plate and were divided into three groups:add the bone marrow-derived Ly6G+IMC of the surgical tumor-bearing mice(LLC+TF6G),add the bone marrow-derived Ly6G+IMC of the non-surgical tumor-bearing mice(LLC+TB6G);put into the Transwell insert,and add the bone marrow-derived Ly6G+IMC of the surgical tumor-bearing mice(LLC+Transwell+TF6G);put into the Transwell insert,and add the bone marrow-derived Ly6G+IMC of the surgical tumor-bearing mice and GW4869(10 μmol/L)(LLC+Transwell+TF6G+GW4869);add PBS(LLC).The absorbance of LLC cells was determined by cck-8 cell proliferation assay.LLC(1×106)were inoculated into the dorsal middle of C57BL/6 female mice,and the caudal vein injection was started on the third day after inoculation,and the mice were divided into three groups:caudal vein injection of bone marrow-derived Ly6G+IMC of the surgical tumor-bearing mice(TF6G),caudal vein injection of bone marrow-derived Ly6G+IMC of the surgical tumor-bearing mice and intraperitoneal injection of GW4869(TF6G+GW4869),caudal vein injection of PBS(PBS).The volume of tumor was measured on days 7,10 and 14 of the caudal vein injection,respectively.3.Effects of surgery-induced bone marrow immature cell-derived exosomes on tumor growth and metastasis:LLC(1×106)were inoculated into the dorsal middle of C57BL/6 female mice and the tumor volume reached 1200 mm3 divided into two groups:the surgical group removed the tumor,the non-the surgical group does not process.Two groups of mice were sacrificed at 48H after surgery,the bone marrow was flushed out with DMEM medium under sterile conditions for MACS to obtain four groups of cells:the bone marrow-derived Ly6G+IMC of the surgical tumor-bearing mice,the bone marrow-derived Ly6C+IMC of the surgical tumor-bearing mice,the bone marrow-derived Ly6G+IMC of the non-the surgical tumor-bearing mice and the bone marrow-derived Ly6C+IMC of the non-the surgical tumor-bearing mice.Four groups of cells were cultured in vitro for 72 H,and the supernatant was collected.ExoEasy Maxi Kit(Qiagen)was used to extract four groups of cell-derived exosomes.LLC are inoculated into the cell culture plate and are divided into five groups after the cells are attached to the cells:add the bone marrow-derived Ly6G+IMC of the surgical tumor-bearing mice(TF6G),add the bone marrow-derived Ly6G+IMC of the non-surgical tumor-bearing mice(TB6G);add the bone marrow-derived Ly6C+IMC of the surgical tumor-bearing mice(TF6C);add the bone marrow-derived Ly6G+IMC of the non-surgical tumor-bearing mice(TB6C);add PBS(Negative).PI single-stain method,CCK-8 cell proliferation assay and Transwell cell invasion assay were used to detect LLC cycle progression,absorbance and number of cell invasion,respectively.LLC(1×106)were inoculated into the dorsal middle of C57BL/6 female mice,and the caudal vein injection was started on the third day after inoculation,and the mice were divided into five groups:caudal vein injection of the bone marrow Ly6G+IMC derived exosome of the the surgical tumor-bearing mice(TF6G),caudal vein injection of the bone marrow Ly6G+IMC derived exosome of the non-surgical tumor-bearing mice(TB6G),caudal vein injection of the bone marrow Ly6C+IMC derived exosome of the surgical tumor-bearing mice(TF6C),caudal vein injection of the bone marrow Ly6C+IMC derived exosome of the non-surgical tumor-bearing mice(TB6C)and caudal vein injection of PBS(PBS).The volume of tumor was measured on days 6,12 and 18 of the caudal vein injection,respectively.Results:1.Construction of postoperative metastasis model of tumor-bearing mice:C57BL/6 female mice were inoculated with LLC for about two weeks,and a 1200 mm3 mass was formed on the back.The back mass was removed,and on the 23th day after surgery,the liver,lungs,and kidneys formed a macroscopic metastasis.2.Effects of surgical induction of bone marrow-derived Ly6G+IMC on tumor growth:Flow cytometry results showed that the bone marrow-derived Ly6G+IMC increased gradually after surgery,and reached a peak at 48 H after surgery(67.83±7.46)%,and presented a downward trend after 48 H.According to the results of flow cytometry,the selection of the bone marrow-derived Ly6G+IMC of the surgical tumor-bearing mice was performed at 48 H after surgery.The results of CCK-8 cell proliferation assay showed that the absorbance of LLC in LLC+TF6G group was significantly higher than that in LLC+TB6G group and LLC group(24 H:F=6.122,P=0.036;48 H:F=232.418,P=0.000;72 H:F=267.148,P=0.000),The absorbance of LLC in the LLC+Transwell+TF6G+GW4869 group was significantly lower than that in the LLC+Transwell+TF6G group(24 H:t=8.215,P=0.001;48 H:t=33.549,P=0.000;72 H:t=40.379,P=0.000).Caudal vein injection of Ly6G+IMC showed that on the 7th day,the volume of the subcutaneous mass of the tumor-bearing mice in the TF6G group was significantly larger than that of the TF6G+GW4869 group(F=7.569,P=0.012),and on 10th and 14th day,the subcutaneous mass of the tumor-bearing mice in the TF6G group was significantly larger than that of the TF6G+GW4869 group and the PBS group(F=12.89,P=0.002;F=6.743,P=0.016).3.Effects of surgery-induced bone marrow Ly6G+IMC derived exosomes on tumor growth and metastasis:the results of LLC cycle detection by PI single-stain method showed that the percentage of LLC in G2/M phase at 48 H in TF6G group was significantly higher than that in TB6G group,TF6C group,TB6C group and Negative group(F=40.398,P=0.000).The results of CCK-8 cell proliferation assay showed that at 24 H,the absorbance of LLC in the TF6G group was significantly higher than that in the TB6G group(F=29.65,P=0.000),48 H and 72 H,and the TF6G group had significantly higher LLC absorbance values than Negative.Group,TB6G group,TF6C group and TB6C group(F=43.919,P=0.000;F=705.777,P=0.000).The results of Transwell cell invasion assay showed that the number of LLC invasion in TF6G group was significantly higher than that in Negative group,TB6G group,TF6C group and TB6C group(F=8.045,P=0.004).The results of caudal vein injection of exosomes showed that on the 12th day,the volume of subcutaneous mass in the tumor-bearing mice of TF6G group was larger than that of TB6G group,TF6C group and TB6C group(F=6.455,P=0.002),on the 18th day,the volume of the subcutaneous mass of the tumor-bearing mice in the TF6G group was greater than that in the PBS group,the TB6G group,the TF6C group,and the TB6C group(F=16.388,P=0.000).Conclusions:1.Surgery may promote tumor metastasis.2.Surgery can cause stress increase of the bone marrow-derived Ly6G+IMC of tumor-bearing mice.3.Surgery-induced bone marrow-derived Ly6G+IMC can promote tumor proliferation.4.Surgery-induced bone marrow-derived Ly6G+IMC do not rely on cell-cell direct contact to promote tumor cell proliferation.5.Surgery-induced bone marrow-derived Ly6G+IMC exosomes promote tumor cell cycle progression.6.Surgery-induced bone marrow-derived Ly6G+IMC exosomes promote tumor cell proliferation.7.Surgery-induced bone marrow-derived Ly6G+IMC exosomes promote tumor cell invasion.8.Surgery-induced bone marrow-derived Ly6G+IMC exosomes promote tumor growth in vivo.Part II Study on the relationship between surgical induction of exosomal non-coding RNA from immature bone marrow cells and tumor proliferation and metastasisObjective:Non-coding RNA(ncRNA)is a class of RNA molecules that cannot be translated into proteins.ncRNA can cause a variety of diseases including cancer.Exosome derived ncRNA plays an important role in tumor progression.The mechanism of surgically induced bone marrow Ly6G+IMC exosome derived ncRNA in tumor growth and metastasis remains unclear.the bone marrow-derived Ly6G+IMC of the surgical tumor-bearing mice In this study,sequencing analysis of expression of the ncRNAs from the bone marrow-derived IMC exosomes of the surgical and non-surgical tumor-bearing mice,and the data were analyzed by biological information,and the ncRNA differentially expressed in the bone marrow-derived IMC exosomes of the surgical and non-surgical tumor-bearing mice were screened and verified,so as to further explore the mechanism of the differential ncRNA between the bone marrow-derived IMC exosomes of the surgical and non-surgical tumor-bearing mice in tumor growth and metastasis.Methods:LLC(1×106)were inoculated into the dorsal middle of C57BL/6 female mice and the tumor volume reached 1200 mm3 divided into two groups:the the surgical group removed the tumor,the non-the surgical group does not process.Two groups of mice were sacrificed at 48 H after operation,the bone marrow was flushed out with DMEM medium under sterile conditions for MACS to obtain four groups of cells:the bone marrow-derived Ly6G+IMC of the surgical tumor-bearing mice,the bone marrow-derived Ly6C+IMC of the surgical tumor-bearing mice,the bone marrow-derived Ly6G+IMC of the non-the surgical tumor-bearing mice and the bone marrow-derived Ly6C+IMC of the non-the surgical tumor-bearing mice.Four groups of cells were cultured in vitro for 72 H,and the supernatant was collected.ExoEasy Maxi Kit(Qiagen)was used to extract four groups of cell-derived exosomes.ExoRNeasy Serum/Plasma Starter Kit(Qiagen)was used to extract four groups of exosomes RNA.Four groups of the bone marrow-derived IMC exosomal non-coding RNAs were sequenced and analyzed,and bioinformatics analysis of circular RNA(circRNA)of the bone marrow-derived Ly6G+IMC exosomes of the surgical and non-surgical tumor-bearing mice and the bone marrow-derived Ly6G+IMC exosomes of the surgical and non-surgical tumor-bearing mice.Combined with the first part of the experimental results,the differentially expressed circRNAs in the bone marrow-derived Ly6G+IMC exosomes of the surgical and non-surgical tumor-bearing mice were screened.The differential circRNA primers were designed according to the screening results,and the difference was verified by qRT-PCR.Results:the the bone marrow-derived Ly6G+IMC exosomes of the surgical and non-surgical tumor-bearing mice differentially expressed 319 circRNA.the the bone marrow-derived Ly6C+IMC exosomes of the surgical and non-surgical tumor-bearing mice differentially expressed 82 circRNAs.Eighteen representative circRNAs were screened from the exosomes of the bone marrow-derived Ly6G+IMC in the surgical and non-surgical tumor-bearing mice.The designed primers were differentially verified by qRT-PCR.Conclusion:The number of circRNAs differentially expressed in the surgical and non-surgical tumor-bearing mice bone marrow-derived Ly6G+IMC exosomes was the highest,and the results of previous work demonstrated that surgery-induced bone marrow-derived Ly6G+IMC exosomes could promote tumor proliferation and invasion,suggesting that circRNA with differential expression of the surgical and non-surgical tumor-bearing mice bone marrow-derived Ly6G+IMC exosomes were associated with tumor proliferation or metastasis.