Tumor Cell-released LC3~+ EVs Promote Lung Premetastatic Niche Formation Of Breast Cancer By Activating Lung Fibroblasts And Its Underlying Mechanisms

Breast cancer（BC）is the most common diagnosed malignancy in women worldwide.Most BC-related deaths are caused by metastasis in vital organs including the lungs.Development of supportive metastatic microenvironments,referred to as premetastatic niches（PMN）,in certain distant organs before metastasis,is a key initial step of metastasis.However,the mechanisms of PMN formation are not fully clear.Recently,we have found extracellular secretory autophagosomes from the supernatant of tumor cells,which is a new extracellular vesicles,and have termed such tumor-released autophagosomes.In tumor microenvironment,we confirmed that LC3⁺EVs which carried damage associated molecular pattern（DAMP）could modulate the immunosuppressive activities of B cells,neutrophils,tumor-associated macrophages and CD4⁺T cells and enhance primary tumor growth.However,it is unclear whether these tumor cell-released circulating LC3⁺EVs（cLC3⁺EVs）could modulate PMN formation in distant organ and affect metastasis.ObjectivesTo explore the mechanism by which cLC3⁺EVs activate lung fibroblasts to induce PMN formation,aiming to clarify the role of cLC3⁺EVs in PMN formation and provide promising therapeutic targets or treatment thoughts for tumor immunotherapy.Methods1.Investigation of primary tumor cell-released cLC3⁺EVs promote lung PMN formationBecn1 knockdown mouse breast cancer 4T1 cells that stably express sh RNA targeting the central autophagy regulator Beclin-1（Becn1 KD-4T1）and luciferase-labeled 4T1 cells（luc⁺4T1cells）,and the subcutaneous tumor bearing model were established.The LC3II levels of cell lysates and supernatant sediments of Becn1 KD-4T1 cells were determined by Western blot.For defining the premetastatic lungs,the lung tissues were harvested from luc⁺4T1-bearing mice and tumor-free mice at the indicated day for detecting of luciferase by qRT-PCR.The peripheral blood samples from Becn1 NC-and Becn1 KD-4T1 bearing mice and tumor-free mice were collected at the indicated day for counting the number of cLC3⁺EVs by flow cytometry（FCM）.BALB/c mice were s.c.injected with Becn1 NC-or Becn1 KD-4T1 cells.On day 10,the proportion of CD11b⁺ly6c^himonocytes（Mo）and F4/80⁺CD11b^hiCD11c^-interstitial macrophages（IMφ）in the lung tissue,PD-L1 expression on Mo in blood and lung tissues,and IMφin lung tissues were detected by FCM.The lung infiltrating lymphocytes were extracted and stimulated with anti-CD3/CD28 m Ab for 3days,intracellular IFN-γexpression of CD4⁺T cells and CD8⁺T cells were analyzed by FCM.IFN-γsecretion in supernatants were detected by ELISA.Becn1 NC-and Becn1 KD-4T1 bearing mice and tumor-free mice were were received an intravenous injection of Dextran-FITC for detecting lung vascular leakage.On day 35,lung metastasis was analyzed by counting the number of scattered macroscopic tumors.LC3⁺EVs-educated mice model was established.LC3⁺EVs distribution in the lungs detected by ex vivo bioluminescence imaging 24 h after Di R-labeled LC3⁺EVs i.v.injection.LC3⁺EVs distribution in the lungs detected by fluorescence microscope 24 h after PKH67-labeled LC3⁺EVs i.v.injection.BALB/c mice were i.v.injected with LC3⁺EVs every other day for 5 times.The proportion of lung Mo and IMφin the lung,PD-L1 expression on Mo in blood and lung tissues,and IMφin lung tissues were detected by FCM.The lung infiltrating lymphocytes were isolated and stimulated with anti-CD3/CD28 m Ab,intracellular IFN-γexpression of CD4⁺T cells and CD8⁺T cells were analyzed by FCM.IFN-γsecretion in supernatants were detected by ELISA.BALB/c mice were received an i.v.injection of Dextran-FITC for detecting lung vascular leakage by fluorescence microscopy.BALB/c mice were i.v.injected with LC3⁺EVs every other day for 5times,and then 4T1 tumor cells were i.v.injected into mice.21 days later,lungs were harvested for counting the number of macroscopic metastases.2.Study on the mechanism of primary tumor cell-released cLC3⁺EVs promoting lung PMN formation2.1 Investigation of LC3⁺EVs induced CCL2 production in lung fibroblastsBALB/c mice were i.v.injected with PKH67-labeled LC3⁺EVs.PKH67-positive cells in the lung single cell suspensions and the proportion of fibroblasts,macrophages,endothelial cells,neutrophils in PKH67 positive cells were analyzed by FCM.Confocal imaging and FCM analysis were used to detect cytoplasmic localization of PKH67-labeled LC3⁺EVs in lung fibroblasts.The lung fibroblasts treated with or without LC3⁺EVs underwent RNA sequencing assay,which was followed by functional enrichment analysis of the differentially expressed genes.CCL2 level was detected by ELISA.Western blot analyses of the phosphorylation of IKKα/β,IκB,p65 in primary lung fibroblasts treated with LC3⁺EVs for the indicated time.The lung fibroblasts were pretreated with NF-κB inhibitor at the indicated concentration for 1 h and then co-cultured with LC3⁺EVs for48 h.CCL2 levels in supernatants were determined by ELISA.The lung fibroblasts purified from WT,Tlr4^-/-,Tlr2^-/-,Myd88^-/-mice were incubated with LC3⁺EVs for 48 h.The lung fibroblasts were pretreated with the TLR2、TLR4 or MyD88 inhibitors for 1 h and then incubated with LC3⁺EVs for 48 h.CCL2 levels in supernatants were determined by ELISA.The lung fibroblasts were isolated from lung tissues of Becn1 NC-or Becn1 KD-4T1 tumor bearing mice or after LC3⁺EVs i.v.injection and cultured for 48 h.On day 10,lung fibroblasts were isolated from lung tissue,and CCL2 levels in supernatants of lung fibroblasts were detected by ELISA.2.2 CCL2 recruits monocytes and macrophages to promote lung PMN formationCell counting was conducted to identify migration of Raw 264.7 towards supernatants from control,LC3⁺EVs or LC3⁺EVs treated with anti-CCL2 antibody.BALB/c mice were were administrated with subcutaneous injection of 4T1 cells and then with intraperitoneal injection of anti-mouse CCL2 antibody（10mg/kg/mouse）every other day for 4 times from the second day of tumor inoculation.On day 10,the proportion of Mo,IMφand T cells from lung tissue were analyzed by FCM.The infiltrating lymphocytes were extracted from lung tissue and stimulated with anti-CD3/CD28 m Ab for 3 days,intracellular IFN-γexpression of CD4⁺T cells and CD8⁺T cells were analysed by FCM and IFN-γsecretion in supernatants were detected by ELISA.On day 35after 4T1 cell inoculation,lungs were harvested for counting the number of macroscopic metastases.2.3 Study on the mechanism of membrane-bound HSP60 on tumor cell-released cLC3⁺EVs inducing lung PMN formationThe lung fibroblasts were treated with LC3⁺EVs,sonicated LC3⁺EVs,proteinase K-digested LC3⁺EVs,DNase I-digested LC3⁺EVs or RNase-digested LC3⁺EVs for 48 h.The lung fibroblasts treated with LC3⁺EVs or blocking antibody-pretreated LC3⁺EVs（anti-HSP60,anti-HSP90α,anti-HSP70,anti-HSP27,anti-HMGB1 antibodies）for 48 h.CCL2 secretion in the above supernatants were tested by ELISA.The phosphorylation of NF-κB p65 in lung fibroblasts treated with Hsp60 NC-or Hsp60 KD-4T1 cell-derived LC3⁺EVs were assessed by Western blot,and the levels of CCL2 were detected by ELISA.BALB/c mice were s.c.injected with Hsp60 NC-or Hsp60 KD-4T1 cells.On day 10,the peripheral blood samples were collected for detection of cLC3⁺EVs number and its HSP60 on cLC3⁺EVs by FCM.The lung fibroblasts were purified from lung tissues and then cultured for 48 h.CCL2 levels in supernatants were determined by ELISA.The proportion of Mo and IMφin the lung were analyzed by FCM.On day 35,mice lungs were harvested for counting the number of scattered macroscopic tumors.3.Clinical study on the correlation between cLC3⁺EV and HSP60 on cLC3⁺EV and lung metastasis in patients with breast cancerHuman fetal lung fibroblasts（HFL1）were cultured with different concentrations of h LC3⁺EVs derived from human breast cancer cells（MDA-MB-231）or h LC3⁺EVs（10μg/m L）for different time.HFL1 treated with LC3⁺EVs with or without anti-HSP60 m Ab pretreatment for 48h.CCL2 levels in supernatants were determined by ELISA.HFL1 were co-incubated LC3⁺EVs,and the expression of activation markerαSMA in HFL1 was detected by Western blot.The peripheral blood sample from breast cancer（BC）patients at diagnosis and in parallel from age-and sex-matched healthy donors（HDs）were collected.The number of cLC3⁺EV,the expression of epithelial adhesion molecule（Ep CAM）and HSP60 on cLC3⁺EVs were determined by FCM.Binary logistic regression analysis and receiver operating characteristic curve（ROC）were used to analyze the diagnostic value of cLC3⁺EV number and s HSP60 on cLC3⁺EVs of lung metastasis.Results1.Investigation of primary tumor cell-released cLC3⁺EVs promote lung PMN formationIn vivo experiment showed that cLC3⁺EVs of Becn1 KD-4T1-bearing mice were significantly reduced compared with that of Becn1 NC-4T1-bearing mice.More importantly,both in vivo luciferase-based bioluminescence imaging and macroscopic observation revealed significant reduction of spontaneous lung metastasis in Becn1 KD-4T1-bearing mice compared with that in Becn1 NC-4T1-bearing mice at day 35,suggesting a promoting role of cLC3⁺EVs in the process of spontaneous lung metastasis in the breast cancer mouse model.Lung tissues were collected for luciferase m RNA detection by qRT-PCR every three days after the subcutaneous implantation of luc⁺4T1 cells.No luciferase m RNA could been detected in lung tissue within 14 days after luc⁺4T1 cell-inoculation,indicating this period is the premetastatic phase.On the day 10 after 4T1 cells implantation,the frequency of Mo and IMφand their surface PD-L1 expression in lung tissue of Becn1 NC-4T1 tumor-bearing mice were significantly increased compared with tumor-free mice.Interestingly,compared with Becn1 NC-4T1-bearing mice,the frequency of Mo and IMφand their PD-L1 expression of Becn1 KD-4T1-bearing mice were dramatically decreased,while PD-L1 expression on monocytes in peripheral blood remained the same in tumor-free mice,Becn1 NC-4T1-bearing mice and Becn1 KD-4T1-bearing mice.Meanwhile,when the extracted lung infiltrating lymphocytes were stimulated with anti-CD3/CD28m Ab,IFN-γsecreting CD4⁺T cells,IFN-γsecreting CD8⁺T cells and total IFN-γsecretion from tumor-bearing mice were significantly decreased compared to tumor-free mice,while the proportion of CD4⁺T cells and CD8⁺T cells in lung tissues did not change significantly.Compare with Becn1NC-4T1-bearing mice,the frequencies of IFN-?secreting CD4⁺T cells,IFN-γsecreting CD8⁺T cells and total IFN-γsecretion were partially recovered in Becn1 KD-4T1-bearing mice.Moreover,the enhanced vascular permeability in the lung of Becn1 NC-4T1-bearing mice was observed by the extravasation of intravenous injecting Dextran-FITC compared with tumor-free mice,while the vascular permeability in the lung of Becn1 KD-4T1-bearing mice was decreased.Collectively,these results show that on the day 10 after 4T1 cell subcutaneous injection,lung PMN infiltrated with Mo and IMφ,enhanced lung permeability,and suppressed T cell function has been formed.More importantly,the PMN in the lung is diminished in Becn1 KD-4T1-bearing mice,suggesting an enhancing role of cLC3⁺EVs in the lung PMN formation.The bioluminescence imaging and fluorescence microscope observation revealed that LC3⁺EVs could migrate into the lung tissue.Pre-injection of LC3⁺EVs promoted lung vascular leakiness of mice.Compared with normal saline group,the proportion of lung-infiltrating Mo and IMφand their PD-L1 levels were dramatically elevated in LC3⁺EVs-pretreated mice.A lower frequency of IFN-γsecreting T cells and decreased IFN-γsecretion were observed in LC3⁺EVs-pretreated mice.Pretreatment of mice with LC3⁺EVs increased lung metastasis following intravenous injection of4T1 tumor cells.Taken together,these results further demonstrated that cLC3⁺EVs could induce a proinflammatory and immunosuppressive PMN formation and pave the way for lung metastasis.2.Study on the mechanism of primary tumor cell-released cLC3⁺EVs promoting lung PMN formation2.1 Investigation of LC3⁺EVs induced CCL2 production in lung fibroblastsFCM analysis showed that 3.3%of the lung cells were PKH67 positive,among which 46.8%and 33.0%were macrophages and fibroblasts,indicating that LC3⁺EVs entering the lungs were mainly taken up by macrophages and fibroblasts.Confocal imaging and FCM analysis showed a cytoplasmic localization of PKH67-labeled LC3⁺EVs in lung fibroblasts.RNA sequencing result showed 666 up-regulated genes highly enriched in LC3⁺EVs stimulated fibroblasts as compared to normal fibroblasts.Gene Ontology（GO）analysis revealed that differentially genes were significantly enriched in the biological processes of inflammatory response.CCL2 was the most upregulated abundant gene involved in the biological processes of inflammatory response.Ex vivo results showed that CCL2 produced by lung fibroblasts from Becn1 KD 4T1-bearing mice was significantly decreased compared to that from Becn1 NC tumor-bearing mice.CCL2 produced by lung fibroblasts from LC3⁺EVs-preinjected mice was significantly increased compared to that from mice with NS injection,indicating that LC3⁺EVs could induce lung fibroblasts to produce CCL2.Pretreatment of lung fibroblasts with an inhibitor of TLR2 or MyD88 attenuated LC3⁺EVs-induced secretion of CCL2,whereas LC3⁺EVs-induced secretion of CCL2 was not affected by inhibition of TLR4.Consistently,Tlr2^-/-and Myd88^-/-mice-derived lung fibroblasts were completely defective in producing CCL2 in response to LC3⁺EVs.LC3⁺EVs treatment of lung fibroblasts resulted in the phosphorylation of NF-κB pathway,and inhibition of NF-κB pathway repressed the induction of CCL2.These data collectively indicate that LC3⁺EVs could induce CCL2 secretion from lung fibroblasts through TLR2-MyD88-NF-κB pathway.2.2 CCL2 recruits monocytes and macrophages to promote lung PMN formationIn comparison with cells migrating toward control media,conditioned media from LC3⁺EVs-treated fibroblast significantly increased Raw 264.7 migration.Neutralizing CCL2significantly inhibited LC3⁺EVs-stimulated lung fibroblasts induced chemotaxis of Raw 264.7.CCL2 n Ab treatment in tumor-bearing mice suppressed the recruitment of lung-infiltrating Mo and IMφ.The proportion of lung-infiltrating CD4⁺T cells and CD8⁺T cells did not change significantly,however,IFN-γsecreting CD4⁺T cells,IFN-γsecreting CD8⁺T cells and their total IFN-γsecretion were significantly increased post intraperitoneal injection of CCL2 n Ab.CCL2blockade inhibited spontaneous lung metastasis of 4T1 tumor-bearing mice.Above results hint that the CCL2 is the major driving force for LC3⁺EVs-induced inflammatory and immunosuppressive PMN formation in vitro and in vivo.2.3 Study on the mechanism of membrane-bound HSP60 on cLC3⁺EVs inducing lung PMN formationPretreatment with proteinase K digestion and sonication,but not DNase or RNase digestion,impaired the ability of LC3⁺EVs to stimulate the CCL2 production from lung fibroblasts,indicating that proteins on the surface,but not DNA or RNA,of LC3⁺EVs are involved in the CCL2 induction of fibroblasts.Blocking HSP60 partially reduced LC3⁺EVs-induced CCL2secretion.Moreover,levels of P65 NF-κB phosphorylation,and levels of CCL2 secretion in lung fibroblasts post stimulation by LC3⁺EVs from Hsp60 KD-4T1 cells were significantly lower as compared to those stimulated from LC3⁺EVs derived from Hsp60 NC-4T1 cells.Taken together,these results show that membrane-bound HSP60 on intact LC3⁺EVs plays a crucial role in CCL2induction of lung fibroblasts.Hsp60 KD-or Hsp60 NC-4T1 cells were s.c.injected into mice.On day 10,there was no significant difference in cLC3⁺EVs number in plasma from Hsp60 KD 4T1-bearing mice,but the HSP60 level on cLC3⁺EVs in plasma from Hsp60 KD 4T1-bearing mice was markedly decreased,compared to Hsp60 NC 4T1-bearing mice.Furthermore,a pronounced reduction of CCL2 secretion by lung fibroblasts and a substantial decrease in frequency of lung-infiltrating Mo and IMφwere evidenced in Hsp60 KD 4T1-bearing mice.Moreover,a marked reduction in spontaneous lung metastasis was found in Hsp60 KD 4T1-bearing mice,compared to Hsp60 NC 4T1-bearing mice.These results suggest that membrane-bound HSP60 on cLC3⁺EVs is the major player responsible for cLC3⁺EVs-induced lung PMN formation.3.Clinical study on the correlation between cLC3⁺EV and HSP60 on cLC3⁺EV and lung metastasis in patients with breast cancerELISA results showed that h LC3⁺EVs induced HFL1 to upregulateαSMA expression.h LC3⁺EVs-induced CCL2 secretion by HFL1 was partly abolished by pretreatment of h LC3⁺EVs with an anti-HSP60 blocking antibody.Altogether,these results indicate that HSP60 on the surface of h LC3⁺EVs is a key molecule that induces the expression of CCL2 in HFL1.FCM analysis revealed the cLC3⁺EVs from BC patients contained higher levels of Ep CAM compared with that from HDs,suggesting a BC cell origin of most of the cLC3⁺EVs from BC patients.The number of cLC3⁺EVs and HSP60 on cLC3⁺EVs in peripheral blood of BC patients were positively correlated with disease progression and lung metastasis.Binary logistic regression and ROC curve analysis showed that cLC3⁺EVs number and HSP60 on cLC3⁺EVs were risk factors for lung metastasis of BC.These results suggest that cLC3⁺EVs number and HSP60 on cLC3⁺EVs in the plasma could be used as biomarkers for risk stratification of lung metastasis in patients with BC.Conclusions1.Primary tumor cell-released cLC3⁺EVs is crucial for inducing CCL2 production in lung fibroblast via a HSP60-TLR2-MyD88-NF-κB signal cascade and induce a proinflammatory and immunosuppressive lung PMN formation promoting lung metastasis.2.cLC3⁺EVs number and HSP60 on cLC3⁺EVs in plasma of patients with breast cancer are risk factors for lung metastasis.The combination detection of cLC3⁺EVs number and HSP60 on cLC3⁺EVs,have potential diagnostic value for lung metastasis in patients with breast cancer.