| Background and purpose Epidemiological surveys have shown that brain metastases in lung cancer are the most common among various brain metastatic tumors,accounting for 30 to 50% of all cases.Especially small cell lung cancer has the tendency of brain metastasis at the early stage of the disease.About 50 to 80 percent of patients with small cell lung cancer who have been treated with clinical treatment have brain metastases after 2 years.The prognosis of the patients was poor and the average life period was less than six months.Therefore,it is of great significance to find relevant factors that affect the "preference to the brain" of lung cancer cells and elucidate its molecular mechanism to prevent and control the brain metastasis.Unlike other tumor metastases,the brain metastasis of tumor cells needs to pass through the blood-brain barrier(BBB)structure which is mainly composed of brain microvascular endothelial cells and tight junctions formed between them.The BBB strictly controls the transport of macromolecules and prevents foreign cells from entering the brain.The presence of the blood-brain barrier makes the brain tissue a relatively independent tissue area,serving as a dynamic regulatory barrier between the central nervous system and the peripheral blood system,transmitting signal communication between the outside of the brain and the brain,so as to maintain the brain homeostasis.However,lung cancer has its unique "preference to the brain" metastatic pathological features,and its specific molecular mechanism has always been our concern.The brain tissue lacks the lymphatic system,so blood metastasis becomes an important way for tumor cells to enter the brain tissue.Unlike the tumor metastasis mechanisms of other organs,the prerequisite for brain metastasis is that the tumor cells need to pass through a blood-brain barrier(BBB),a special barrier system located between the cerebral blood vessels and the brain parenchyma.BBB is mainly composed of brain microvascular endothelial cells,basement membranes,pericytes,and astrocyte foot processes.Its special intercellular connection and anatomical structure play an important role in maintaining the homeostasis of brain microenvironment,limiting exogenous macromolecules and cells entering the brain parenchyma.Although there is a special "gatekeeper"--the BBB,small cell lung cancer still shows a "preference to the brain" characteristic.According to Stephan Paget’s "soil and seed" theory,the formation of tumor brain metastasis is not random,but some tumor cells have the property of preferring the nervous system.The preference of tumor cells is the embodiment of the tumor heterogeneity,it can secrete factors that help regulate the microenvironment of target organs,create a suitable metastatic microenvironment for tumor cell growth.More and more evidences show that primary tumor cells can secrete some secretory factors to promote the change of microenvironment of secondary sites,making it easier to accept circulating tumor cells.This supporting microenvironment is called pre-metastatic microenvironment.However,this pre-metastatic microenvironment has both temporal and spatial characteristics relative to tumor cells.When the tumor cells are at the primary site and have not reached the secondary site,it is called pre-metastatic microenvironment.When the tumor comes to the secondary site and enters the pre-metastatic microenvironment,it is called metastatic microenvironment.At present,exosomes from tumor cells,diameter from 30 to 150 nm,are considered to be the most important "builders" of tumor microenvironment.Exosomes were initially defined as vesicular structures that transported intracellular garbage or debris in eukaryotic cells.However,in recent years,it has attracted much attention as a mediator between cells,celsl and microenvironment.It contains many kinds of bio-molecules from source cells.When tumor derived exosomes are internalized by target cells,exosomes release their substances into target cells,thereby changing various biological functions of target cells,which is helpful for the formation of pre-metastatic microenvironment.In other words,organ specific metastasis may depend not only on the ability of cancer cells to cross the biological barrier,but also on the formation of pre-metastatic microenvironment,such as the structure of the vascular system.However,there have been no reports on the mechanism of the regulation of the brain pre-metastatic microenvironment by exosomes derived from small cell lung cancer cells.Previous work results showed that Annexin A1 was highly expressed in primary cancer tissues of patients with small cell lung cancer.In vitro experiments showed that the expression of Annexin A1 in small cell lung cancer cells co-cultured with human brain microvascular endothelial cells was upregulated,and small cell lung cancer cells could secreted Annexin A1 into the extracellular;inhibiting the expression of Annexin A1 in small cell lung cancer cells can inhibit the formation of metastases in the brain of nude mice.Further experimental results show that Annexin A1 is present in the small cell lung cancer cell-derived exosomes,and it is speculated that Annexin A1 is secreted into the extracellular environment in the form of exosomes.It has been reported that Annexin A1 is contained in the vesicles of intestinal epithelial cells,and Annexin A1 is at a high level in the serum derived exudate of active enteritis patients,the vesicles containing Annexin A1 contribute to the repair of damaged intestinal epithelium.But whether Annexin A1 in exosomes is involved in the formation of the brain pre-metastatic microenvironment of small cell lung cancer and the mechanism are not clear.In this experiment,we extracted and purified the exosomes of small cell lung cancer cells.First,it is discussed whether the exocrine derived from small cell lung cancer enters the brain through the blood brain barrier and affects the microenvironment of the metastasis of the forebrain.Secondly,we discussed that whether Annexin A1 in exosomes regulated the function of cerebral vascular endothelial cells when small cell lung cancer cells interact with cerebrovascular microenvironment,so as to maintain the survival of small cell lung cancer cells migrating into the brain.Experimental methods1.The effect of small cell lung cancer cells derived exosomes on cerebral vascular microenvironment before metastasis.1.1 The exosomes in the culture supernatant of different cancer cells were purified by ultracentrifugation.Western blotting and flow cytometric analysis were used to identify the marker of exosomes.Transmission electron microscopy was used to observe its ultra micro-morphological features.The nano particle tracking analysis(NTA)analyzed the size of these vesicles.1.2 The human brain microvascular endothelial cells and the purified exosomes were labeled with live cell fluorescent dyes.Laser confocal microscopy was used to observe the endocytosis of the exosomes derived from small cell lung cancer by human brain microvascular endothelial cells.1.3 In vitro: The extraction of small cell lung cancer cell-derived exosomes was performed by ultracentrifugation to treat human brain microvascular endothelial cells.Metrigel angiogenesis was used to observe angiogenesis.The blood-brain barrier model was used to measure the permeability.1.4 Lewis lung cancer cell exosomes were extracted by ultracentrifugation and injected into the third ventricle using a mouse stereotaxic apparatus.After C57 mice were injected 8 times,paraformaldehyde was perfused and the brain tissue sections were prepared by a vibrating microtome.The expression of CD31 in brain tissue was detected by immunofluorescence combined with laser confocal fluorescence microscopy,and the distribution of cerebral blood vessels was observed.1.5 The extracellular exosomes of Lewis lung cancer cells were extracted by ultracentrifugation and injected into the left ventricle of C57 mice once every other day for 8 consecutive times.The brain tissue samples were collected and Western blot was used to detect the expression of tight junction-related proteins.1.6 The newborn mouse brain tissue was cut into a 250-300μm thick tissue section with a Vibrating slicer and established an in vitro culture system of living brain tissue(ex vivo).1.7 The Lewis lung cancer cell exosomes were extracted by ultracentrifugation and incubated with in vitro cultured brain tissue sections.The expression of the tight junction protein was detected by Western blot.2.How the small cell lung cancer-derived exosomes mediate Annexin A1 regulation of the function of human brain microvascular endothelial cells.2.1 After human brain microvascular endothelial cells were treated with recombinant human Annexin A1 recombinant protein,the proliferation of cells was detected by MTS proliferation kit.2.2 The permeability of blood brain barrier model in vitro was detected after the human Annexin A1 recombinant protein was used to treat the blood brain barrier model in vitro.2.3 In view of previous experimental results suggesting that Annexin A1 can promote the adhesion of small cell lung cancer cells,after treatment of human brain microvascular endothelial cells with Annexin A1 recombinant protein,Western blot and immunofluorescence were used to detect detect the expression of adhesion molecule Mac-1 on human brain microvascular endothelial cells.2.4 The expression and distribution of Annexin A1 receptor FPR1 in human brain microvascular endothelial cells were detected by cellular immunofluorescence and Western blot.2.5 The exosomes of small cell lung cancer cells were isolated and purified by ultracentrifugation,and the proteins in exosomes were analyzed by LC-MS/MS protein mass spectrometry.2.6 The exosomes derived from different tumor cells were extracted by ultracentrifugation,and the presence of Annexin A1 in exosomes was detected by Western blot.2.7 The Annexin A1-flag eukaryotic recombinant expression vector was constructed,and the NCI-H446 small cell lung cancer cell line with stable expression of Annexin A1-flag was established.The positive cell line was selected with G418.The expression of Annexin A1 in the positive cell line was detected by Western blot.2.8 Lentivirus-mediated RNAi interference technology was used to down-regulate the expression of Annexin A1 in small cell lung cancer cells.Puromycin was used to screen positive cell lines.Western blot was used to identify the down-regulation efficiency.2.9 NCI-H446 cell-derived exosomes were collected after the above Annexin A1 gene was intervened,and Western blot was used to identify the expression of Annexin A1.Human brain microvascular endothelial cells were treated with the aforementioned NCI-H446 cell exosomes to observe cerebrovascularization.2.10 The RNAi technology was used to down-regulate the expression of Annexin A1 in Lewis lung cancer cells,exosomes of the cells were collected,live brain tissue sections were processed in vitro,and the expression of the tight junction protein was detected by Western blot.3.The mechanism that Small cell lung cancer-derived exosomes containing Annexin A1 promote cerebral angiogenesis.3.1 Exosomes containing Annexin A1 derived from small cell lung cancer cells were treated with human brain microvascular endothelial cells,and the excretion of cytokines in human brain microvascular endothelial cells was analyzed by ELISAArray.3.2 After treatment of human brain microvascular endothelial cells with exosomes of small cell lung cancer cells that down-regulate Annexin A1 expression,the exocrine cytokines were analyzed by ELISAArray.3.3 We combined the contents of 1 and 2 to screen for cytokines that are significantly different and that are associated with brain microvessel production.3.4 The difference factor we obtained-CCL20.CCl20 was used to treat human brain microvascular endothelial cells and their angiogenesis was observed.Western blot was used to detect the tight protein expression.3.5 The human CCL20 recombinant protein was used to treat human small cell lung cancer cell NCI-H446 cells,and the vascular mimicry of tumor cells was observed.Experimental results1.Effect of cell-derived exosomes of small cell lung cancer on metastatic brain microenvironment.1.1 The exosomes in culture supernatants of different tumor cells(lung,breast,and ovarian cancer)were purified by ultracentrifugation.The expression of the exosome markers CD63 and CD9 was detected by Western blot.Transmission electron microscopy was observed the diameter of exosomes is about 30 nm,and the exosome vesicles had a double-layered membrane structure.The diameter of exosomes measured by the nanoparticle diameter analyzer was in the range of 10-100 nm,and the distribution peak appeared at 15-20 nm.The results of flow cytometry analysis demonstrated that the exosomal marker molecule CD63 was present on the surface of small cell lung cancer cells.In summary,we used ultracentrifugation to obtain exosomes derived from tumor cells.The exosome concentration ranged from 0.5 μg/μl to 1.6 μg/μl.1.2 We used a live cell dye(CFDA-SE)to pre-infect human brain microvascular endothelial cells,and labeled BODIPY? TR ceramide dyes to label small cell lung cancer cell-derived exosomes.After incubation,they were observed using a laser confocal microscope.Small cell lung cancer cell-derived exosomes can be endocytosed into cells by green fluorescence-labeled human brain microvascular endothelial cells.1.3 After the small cell lung cancer cell derived exosomes treated the human brain microvascular endothelial cells,the early endothelial cell permeability was enhanced and the later permeability was unchanged.After exosomes treatment,the brain microvascular endothelial cells gradually formed branched and tubular structures,indicating that exosomes promoted the angiogenesis of human brain microvascular endothelial cells in vitro.1.4 The Lewis lung cancer cell exosomes were purified by ultracentrifugation.After injection in the third ventricle of C57 mice,immunofluorescence was used to detect the expression of CD31,a marker of brain vascular endothelial cells.The results showed that the exosomes from the Lewis lung cancer cell that entered the brain could increase the local brain microvessel density suggests that it has a role in promoting cerebral microangiogenesis.1.5 The exosomes of Lewis lung cancer cells were extracted and labeled with red BODIPY? TR ceramide dye and injected into the left ventricle of C57 mice.Red exosomes were observed in brain tissue sections.It shows that exosomes of the Lewis lung cancer cell in peripheral blood circulation can enter the brain through the blood-brain barrier.Further Western blot results suggested that the exosomes entering the brain tissue up-regulated the expression of tight junction proteins in the brain vascular endothelial cells,suggesting that it has the role of promoting cerebral microangiogenesis.1.6 The neonatal mice were sacrificed,and the brain tissue was dissected after cervical dislocation.The brain tissues of neonatal mice were cut into 250-300 μm thick living tissue sections using a tissue vibrating microtome and placed in a Transwell culture chamber for continuous culture for 4 days.With an intact structure,no necrosis and autolysis occur in the central brain tissue,and an in vitro fresh brain tissue ex vivo system was established.1.7 After extracting Lewis lung cancer cell exosomes and co-cultured brain tissue sections in vitro,Western blot results suggested that small cell lung cancer-derived exosomes can increase the brain-associated tight junction protein,suggesting that it promoted the role of brain microvessel formation.2.How the small cell lung cancer-derived exosomes mediate Annexin A1 regulation of the function of human brain microvascular endothelial cells.2.1 Annexin A1 recombinant protein treated human brain microvascular endothelial cells,MTS proliferation experiments showed no significant effect on the proliferation of brain endothelial cells.Annexin A1 recombinant protein was used to treat the monolayer of brain microvascular endothelial cells cultured on Transwell.The results of permeation experiments showed that the Annexin A1 recombinant protein did not affect the permeability of the brain endothelial monolayer.In addition,after Annexin A1 recombinant protein treated human brain microvascular endothelial cells,Western blot results showed no significant changes in the expression of adhesion molecule Mac-1,immunofluorescence also prompted the results that Annexin A1 did not affect the surface of human brain microvascular endothelial cells Mac-1 expression,flow cytometry analysis yielded the same results as above.These results indicated that exocrine Annexin A1 may not regulate the function of brain microvascular endothelial cells through cytokine forms.2.2 TImmunofluorescence and Western blot were used to detect the presence of FPR1 in human brain microvascular endothelial cells.The results showed that there was no FPR1 on the surface of human brain microvascular endothelial cells.These results further suggested that the exocrine Annexin A1 may not regulate the function of brain microvascular endothelial cells through the mode of cytokine acting receptors.2.3 The analysis of protein in exosomes using LC-MS/MS protein mass spectrometry revealed that Annexin A1 was present in exosomes of small cell lung cancer.The ultracentrifugation method was used to separate and purify the exosomes of different tumor cells.Western blot results showed that there were Annexin A1 in different tumor exosomes.2.4 The Annexin A1-flag eukaryotic recombinant expression plasmid was constructed and transfected into NCI-H446 cells.The NCI-H446 cell line with stable and high expression of Annexin A1 was selected by G418.Lentivirus-mediated RNAi technology was used to down-regulate the expression of Annexin A1 in NCI-H446 cells.Puromycin was used to screen out the positive cells with low expression of Annexin A1.Western blotting results confirmed the expression of Annexin A1 in positive cell lines.2.5 The exosomes of the above Annexin A1 down-regulated cell line were purified by ultracentrifugation,and the expression of Annexin A1 was identified by Western blot.The results showed that the content of Annexin A1 in exosomes was decreased in NCI-H446 cells after down-regulation of Annexin A1.2.6 The exosomes of NCI-H446 cells after down-regulation and high expression of Annexin A1 were extracted by ultracentrifugation and treated with human brain microvascular endothelial cells to observe the angiogenesis.The high expression of Annexin A1 cell exosomes promoted angiogenesis compared with the blank control.Down-regulation of Annexin A1 had no promoting effect.2.7 Lewis lung cancer cells interfered with the expression of Annexin A1.Purified exosomes and incubated them with in vitro cultured brain tissue sections.The expression of claudin-5,a tight junction protein,was detected by Western blot.The results showed that the expression of tight junction protein claudin-5 was not promoted by the exosomes of lung cancer cells with low expression of Annexin A1.3.The mechanism that Small cell lung cancer-derived exosomes containing Annexin A1 promote cerebral angiogenesis.3.1 Using ELISAArray to analyze the exocrine cytokines in the supernatant of small cell lung cancer cell exosomes treatment of human brain microvascular endothelial cells,a differential exocrine factor CCL20 was found.3.2 Lentivirus-mediated RNAi technology down-regulated the expression of Annexin A1 in small cell lung cancer cells,collected exosomes,and processed human brain microvascular endothelial cells.The ELISAArray assay revealed a significant decrease in CCL20 content in the supernatant.3.3 Using human CCL20 recombinant protein to treat HBMEC,cerebral angiogenesis could be induced obviously.The expression of occludin was detected by Western blot.The results suggested that CCL20 can upregulate the expression of occludin in brain microvascular endothelial cells.These results suggested that CCL20 can promote the renewal of cerebral blood vessels.3.4 Using human CCL20 recombinant protein to treat NCI-H446 cells cultured in metrigel gel,the results suggested that CCL20 can also promote the formation of branches and tubular blood vessel morphology of NCI-H446 cells,which promoted the occurrence of tumor mimicry.Conclusions1.Small cell lung cancer cell-derived exosomes can be endocytosed by brain microvascular endothelial cells,which can promote local cerebral vascular microenvironment angiogenesis,providing a rich "soil" for the arrival of the tumor.2.The human brain microvessel microenvironment can induce small cell lung cancer cells to overexpress Annexin A1,and Annexin A1 can be secreted into the extracellular environment by the small cell lung cancer cells in the form of exosomes.3.Small cell lung cancer exosomes containing Annexin A1 can induce human brain microvascular endothelial cells to express and secrete high levels of CCL20.CCL20 can induce cerebral angiogenesis and at the same time promote the mimicry of small cell lung cancer.It also can help small cell lung cancer cells to form clonal lesions in the brain. |
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