DLDH-containing extracellular vesicles from CAFs reduced DOX sensitivity in triple-negative breast cancer

DLDH-containing extracellular vesicles from CAFs reduced DOX sensitivity in triple-negative breast cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article DLDH-containing extracellular vesicles from CAFs reduced DOX sensitivity in triple-negative breast cancer Zhi Xu, Junchen Hou, Hehua Ma, Yu Zhao, Fei Fei, Runbin Sun, Juan Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2262675/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Chemo-resistance is a major obstacle in the control of advanced triple-negative breast cancer (TNBC). Cancer-associated fibroblasts (CAFs)-derived extracellular vesicles (EVs) were critical for tumor progression. Herein, we demonstrated that CAFs/TNBC-derived EVs could suppress doxorubicin (DOX) sensitivity in breast cancer both in vitro and in vivo. The protein array revealed that dihydrolipoamide dehydrogenase (DLDH) was enriched in CAFs/TNBC-derived EVs, which was the E3 component of the 2-oxoglutarate dehydrogenase complex (α-KGDC). EVs-DLDH was transported into mitochondria and enhanced mitochondrial respiration through increasing α-KGDC activity and NADH content. Inhibiting DLDH reduced oxidative phosphorylation (OXPHOS) and CAFs-derived EVs-induced drug resistance in the recipient cells. It was also shown that the EVs-reduced sensitivity of DOX was due to increased drug efflux driven by OXPHOS. Additionally, suppression of ATP-binding cassette transporters or mitochondrial respiration conferred the recipient cells with increased susceptibility to DOX. These results elaborated that CAFs-derived EVs inhibit the DOX sensitivity of TNBC through increasing drug efflux driven by DLDH-induced OXPHOS. Inhibiting EVs-DLDH provides a potential therapeutic application to enhance the responsiveness to chemotherapy in TNBC. Biological sciences/Cancer/Breast cancer Biological sciences/Cancer/Cancer therapy/Cancer therapeutic resistance Biological sciences/Cancer/Cancer microenvironment Biological sciences/Cancer/Cancer metabolism Triple-negative breast cancer extracellular vesicles doxorubicin resistance cancer-associated fibroblasts dihydrolipoamide dehydrogenase Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Highlights CAFs-derived EVs suppressed doxorubicin sensitivity in breast cancer. EVs transported DLDH into the mitochondria of recipient cells leading to increased OXPHOS. EVs-DLDH reduced doxorubicin sensitivity through increasing drug efflux driven by OXPHOS. Introduction Due to the absence of effective targeted therapy, the combination of surgery and chemotherapy or radiotherapy is the mainstay of triple-negative breast cancer (TNBC) treatment [ 1 , 2 ]. Despite the efficacy of cytotoxic chemotherapy, TNBCs are usually more aggressive, showing strong resistance to therapy [ 3 , 4 ]; the median survival of advanced TNBC patients is approximately 13 to 20 months [ 5 ]. Therefore, it is extremely urgent to improve the efficacy of TNBC therapies and prolong the patient survival time. Doxorubicin (DOX) is widely used for the treatment of cancers[ 6 ]. Multiple mechanisms have been proposed for DOX resistance, including p53 or topoisomerase II mutation, altered cellular accumulation, increased drug inactivation, and signaling pathway activation [ 7 , 8 ]. Additionally, growing evidence has indicated that the tumor microenvironment (TME) influences therapeutic response and drug resistance in tumor cells[ 9 , 10 ]. Cancer-associated fibroblasts (CAFs) as an important component of TME promote tumor progression, including proliferation, migration, invasion, epithelial-to-mesenchymal transition (EMT), and metabolic reprogramming [ 11 , 12 ]. It was reported that the depletion of CAFs could provide survival benefits in TNBC murine models by reducing intratumoral collagen [ 13 ]. Another study has indicated that targeting CAFs could protect MCF-7 cells against apoptosis induced by DOX [ 14 ]. However, the mechanism that CAFs direct deploy in DOX resistance in breast cancer (BC) has not been studied in greater detail. Extracellular vesicles (EVs) are microvesicles composed of lipid bilayers containing bioactive molecules, such as proteins, bioactive lipids, and small RNAs[ 15 ]. Currently, multiple studies have demonstrated that CAFs-mediate EVs could facilitate BC progression. It was found that CAFs-secreted EVs mediated stromal mobilization of autocrine Wnt-PCP signaling in BC cell migration [ 12 ]. A study of the metabolomic analysis provided that CAFs-derived EVs containing intact metabolites (amino acids, lipids, and tricarboxylic acid (TCA) cycle intermediates) and modulating cancer cell metabolism [ 16 ]. In addition, it was demonstrated that miRNAs in CAFs could be delivered to BC cells via EVs, thereby inhibiting ER expression and reducing the sensitivity to hormone therapy [ 17 ]. The present study revealed that CAFs-derived EVs enhance BC cells’ resistant capacity of DOX by delivering dihydrolipoamide dehydrogenase (DLDH). DLDH is a core component of the α-ketoglutarate dehydrogenase complex (α-KGDC, a key rate-limiting enzyme of the TCA cycle) [ 18 ]. The results showed that EVs-DLDH could translocate into mitochondria of BC cells and promote DOX resistance via promoting adenosine triphosphate (ATP) synthesis and enhancing the efflux function of ATP-binding cassette (ABC) transporters. This finding suggests that DLDH in CAF-derived EVs contributes to protecting cancer cells from chemotherapy and that DLDH could be a relevant therapeutic target. Results CAFs reduced the sensitivity of BC cells to DOX. It has recently been suggested that the TME may confer resistance to therapy[ 19 ]. In this study, we examined the role of CAFs in modulating the response of BC cells (MCF-7, BT-549, and MDA-MB-231) to DOX. The fibroblastic nature of tissue-derived cell cultures has been verified through their characteristic fibroblastic morphology and expression of fibroblastic markers such as fibronectin (FN), fibroblast activation protein (FAP), vimentin, and α-smooth muscle actin (α-SMA) (Fig.S1). The primary fibroblasts were subsequently co-cultured with BC cells. The cytotoxicity tests indicated that DOX-induced cell death in BC cell monocultures and this cytotoxic effect inhibited in co-culture with CAFs but not with NFs (Fig. 1 A-E and Fig.S2). Notably, the effect was more pronounced for CAFs/TNBC than for CAFs/LuA in the above experiments. To investigate the cause of the DOX resistance observed in the CAFs co-culture system, the BC cells were cultured in a fibroblasts-conditioned medium with EVs (CM-CAFs/LuA, CM-CAFs/TNBC) or without EVs (non-EVs CM-CAFs/LuA, non-EVs CM-CAFs/TNBC), and followed by multiple doses of DOX treatment (Fig. 1 F). MTT assay showed that the cytotoxic effect and DOX IC50 value were only increased in the group of incubation with CM-CAFs, especially CM-CAFs/TNBC (Fig. 1 G, H and Fig.S3A-F). Colony survival assay further confirmed that there is no improved survival by incubation with non-EVs CM (Fig. 1 I, J and Fig.S3G-I). These results supported the hypothesis that CAFs suppressed the DOX sensitivity of BC cells and that CAFs-derived EVs might play a potentially more important role than soluble factors. CAFs-derived EVs induced DOX resistance of BC cells. To determine whether CAFs-derived EVs attenuate DOX sensitivity in BC cells, equal amounts of EVs extracted from fibroblast cell cultural supernatants were pretreated in BC cells for 48h (Fig.S4A). Before being co-cultured with BC cells, EVs were verified using TEM, NTA, and immunoblots (CD63, Alix, and TSG101), which suggested that these EVs isolated using ultracentrifugation were exosome-enriched EVs (Fig. 2 A-C). The MTT assay showed that the cell’s uptake of EVs CAFs/TNBC remarkably improved survival compared to no survival improvement by uptake of EVs NFs and EVs CAFs/LuA (Fig. 2 F and Fig.S4E). Consistent with cell viability, the DOX IC50 value was higher in the uptake of EVs CAFs/TNBC than in the uptake of EVs NFs and EVs CAFs/LuA (Fig.S4B-D). In addition, the colony survival assay further confirmed that the uptake apparently enhanced the DOX resistance of the recipient cells compared to the uptake of EVs NFs (Fig. 2 F, G and Fig.S4F). To determine whether EVs entered the receiving cells, red fluorescent PKH26 dye-labeled EVs were co-cultured with GFP-labeled BC cells (Fig. 2 H and Fig.S4G). After incubation of cells with EVs CAFs/TNBC , as expected, the labeled EVs can be detected in the recipient cells, which are mainly localized in the cytoplasm. Next, PKH26-labeled EVs CAFs/TNBC were pretreated with trypsin (Trypsinized-EVs) to cleave membrane proteins before being incubated with BC cells (Fig. 2 I). Trypsinized-EVs-treated cells showed decreased fluorescent signals in the recipient cells, compared with Control-EVs-treated cells (Fig. 2 L). Meanwhile, the cytotoxicity assay showed that compared with EVs-treated cells, there was almost no difference between Trypsinized-EVs-treated cells and untreated cells (Fig. 2 J, K and Fig.S4H), suggesting EVs play a significant role in CAFs-induced DOX resistance. To further evaluate the role of EVs CAFs/TNBC in promoting cell DOX resistance in vivo, a zebrafish tumor experimental model was established. BC cells incubated with EVs were injected into zebrafish embryos, and DOX was added to nutrient solutions when the fishes were born. Tumors formed as the fish grown up and were daily tracked by fluorescent imaging. Consistent with the results from in vitro experiments, DOX treatment efficiently inhibited tumor growth in the group injected with BC cells, but the anticancer efficacy was significantly reduced in the group injected with EVs CAFs/TNBC− treated BC cells (Fig. 4 K, L). DLDH is enriched in CAFs/TNBC-derived EVs and transmitted to BC cells EVs perform various functions that involve interactions within the cellular microenvironment due to their ability to carry lipids, proteins, nucleic acids, and metabolites[ 15 ]. Since CAFs-derived EVs are recognized as potent factors for the acquisition of drug resistance in BC, the protein profiling in EVs CAFs/LuA vs. in EVs CAFs/TNBC was quantified using protein arrays. A majority of proteins were increased in EVs CAFs/TNBC compared to EVs CAFs/LuA , and several genes relating to cell binding signals and metabolic pathways were upregulated in EVs CAFs/TNBC (Fig.S5A). Among them, DLDH, one of the critical proteins regulating cellular ATP synthesis[ 18 ], was highly enriched in EVs CAFs/TNBC (Fig. 3 A). DLDH is a core component of the α-KGDC that is a key rate-limiting enzyme in the cellular TCA cycle[ 20 ]. Notably, it was identified that a significant enrichment for DLDH in CAFs/TNBC-derived EVs, whose amount was consistent with the constitutive levels of DLDH in CAFs/TNBC cells vs. CAFs/LuA cells (Fig.S5B-D). In addition, the results from the immunoblots, immunofluorescence, and ELISA assay confirmed that DLDH contents were enhanced in EVs CAFs/TNBC (Fig. 3 B, C). To determine that EVs can carry and enrich DLDH, the purified EVs from the CAFs/LuA and CAFs/TNBC cells were incubated with cells, and the internalized EVs-transported DLDH (EVs-DLDH) was determined in the recipient cells. As expected, DLDH was co-localized with PKH26-labeled EVs CAFs/TNBC in the cytoplasm (Fig. 3 D). Intriguingly, the EVs were co-localized with TOM20 (the mitochondrial outer membrane protein, labeled with green secondary antibody) (Fig. 3 E), indicating that EVs could deliver DLDH into the mitochondria of recipient cells. Furthermore, after incubation with EVs, the expression of DLDH was increased markedly in the EVs CAFs/TNBC –treated cells (Fig. 3 F-H). Results showed that EVs secreted from CAFs cells could alter the content of DLDH in BC cells via the delivery of EVs. EVs-mediated DLDH delivery contributes to DOX resistance of BC cells. To verify that EVs-DLDH could confer the DOX resistance to BC cells, DLDH was ectopically expressed into fibroblasts with a low level of constitutive DLDH (Fig.S6A, B). And the level of DLDH in CAFs-derived EVs was measured by western blots and ELISA assay (Fig. 4 A, B). After incubation with EVs, the DLDH level was increased in EVs CAFs/LuA−DLDH -treated BC cells versus EVs CAFs/LuA−NC -treated cells (Fig. 4 C-F). Subsequently, the MTT assay showed that DLDH-overexpressed EVs-treated cells remarkably improved survival compared to the lack of improvement in survival by control groups (Fig. 4 G, H and Fig.S6C-F). Consistent with cell viability, the results from the colony survival assay further confirmed that uptake of DLDH-overexpressed EVs enhanced DOX resistance of the recipient cells (Fig. 4 I, J). In parallel, DLDH was silenced in CAFs/TNBC cells using a DLDH siRNA (Fig.S6A, B). The level of DLDH in CAFs/TNBC cells and EVs CAFs/TNBC−siDLDH -treated BC cells were reduced significantly. Accordingly, the drug resistance was alleviated in the DLDH-silenced EVs treated cells compared to the scrambled control (Fig. 4 and Fig.S6). Furthermore, to validate that inhibiting EVs-DLDH could increase the susceptibility for DOX of BC cells, TNBC cells were incubated with EVs NFs , EVs CAFs/TNBC and DLDH-silenced EVs CAFs/TNBC prior to injection into the zebrafish embryos (Fig. 4 K, L and Fig.S6G, H). As expected, compared to the EVs NFs -treated group, the drug sensitivity was decreased in the EVs CAFs/TNBC group, but eliminated with the use of EVs isolated from the DLDH-silenced CAFs/TNBC cells. EVs transported DLDH enhanced mitochondrial respiration in the recipient cells DLDH is a component of α-KGDC, which is a mitochondrial enzyme complex catalyzing the α-ketoglutarate to succinyl-CoA[ 21 ]. The immunofluorescence experiment also demonstrated that EVs could transport DLDH into the mitochondrial of recipient cells (Fig. 3 D, E). Recent evidence suggests that the metabolic pattern is crucial for chemo-resistance in cancers[ 22 ]. As a rate-limiting enzyme, α-KGDC activity may induce alterations in the flux of metabolites through TCA. Thus, the α-KGDC activity assay was performed to confirm that the CAFs/TNBC-secreted EVs were able to enhance α-KGDC activation (Fig. 5 A, B). Additionally, suppression of EVs-DLDH could neutralize EVs CAFs -mediated activation of α-KGDC. Because α-KGDC plays an important role in NAD + conversion into NADH, EVs CAFs -upregulated oxidative flux of the TCA cycle led to increased NADH levels (Fig. 5 C, D). Furthermore, we examined the mitochondrial respiratory capacity of BC cells after incubating with EVs CAFs . In EVs CAFs/TNBC -treated cells, the basal and maximal respiratory capacities were higher than in EVs CAFs/LuA -treated cells, suggesting activated oxidative phosphorylation (OXPHOS) (Fig. 5 E, F and Fig.S7A, B). Intriguingly, glycolysis was not significantly different between the two groups (Fig.S7C, D). Next, we measured cellular levels of reactive oxygen species (ROS), which could elevate through the activation of the electron transport chain (ETC) in the mitochondria. The level of hydroxyl radical and H2O2 was elevated in EVs CAFs/TNBC -treated cells compared to EVs CAFs/LuA -treated cells, in line with a hyperactive OXPHOS (Fig. 5 G, H). EVs CAFs/TNBC -treated cells also exhibited elevated superoxide anion level that was comparable to EVs CAFs/LuA -treated cells (Fig. 5 I). Correspondently, suppression of EVs-DLDH could neutralize EVs CAFs -mediated increased ROS level. These results suggested that EVs CAFs/TNBC exhibit hyperactive OXPHOS which was associated with the level of DLDH delivered by EVs CAFs . In addition, a lipoate analog CPI-613 was used to inhibit α-KGDC function through reduced DLDH activity [ 23 ]. CPI-613 significantly inhibited the α-KGDC activity and NADH level in EVs-treated cells (Fig.S7E-H). The experimental results are demonstrated in Fig. 6 , which reveal DLDH inhibitors have been shown to inhibit mitochondrial OXPHOS and cell viability. EVs-DLDH-mediated reduction of DOX sensitivity was driven by OXPHOS. It has been widely recognized that the induction of drug efflux by ABC transporters is the leading cause of decreased intracellular drug accumulation thereby lowering the efficacy of DOX[ 24 ]. To examine whether the lower response to DOX in EVs-treated BC cells was associated with drug accumulation changes by enhanced OXPHOS, the intracellular DOX content was quantified by flow cytometry after cell-EVs incubation and DOX treatment (Fig. 6 A, B and Fig.S8A, B). As shown in Fig. 7 , the DOX accumulation was significantly reduced in EVs CAFs/TNBC -incubated cells compared to the EVs CAFs/LuA -incubated cells. EVs-DLDH also increased ATP synthesis in BC cells (Fig. 6 C, D). After being treated with Rotenone or Antimycin A, which was used to inhibit OXPHOS in the recipient cells, the EVs CAFs/TNBC -induced drug efflux and DOX resistance were significantly reduced (Fig. 6 E-G and Fig.S8C, D). To further validate the EVs-mediated reduction of DOX sensitivity was driven by OXPHOS, ABCG2-siRNA and ABCB1-siRNA were employed to inhibit the ABC transporters of the recipient cells (Fig.S8E, F). Cells were co-incubated with EVs following the knockdown of ABC transporter protein and subjected to detection of DOX cytotoxic effects on cells. The data demonstrated that ABC transporters inhibition could attenuate EVs CAFs -reduced DOX sensitivity of BC cells (Fig. 6 J-L). Consistently, inhibition of the ABC transporters improves DOX accumulation in EVs-treated cells (Fig. 6 H, I and Fig.S8G, H), suggesting that these transporters participate in the efflux of the drug. These results suggest that EVs-DLDH-mediated DOX sensitivity inhibition resulting from increased drug efflux driven by OXPHOS. Discussion BC is one of the most common malignancies in the world, TNBC comprises 15–20% of all BC patients, yet accounts for 30% of BC deaths[ 25 ]. Unlike ER or HER2-positive BC, which benefit from targeted therapies, TNBC patients mostly rely on surgery and systemic chemotherapy[ 26 ]. However, distant-organ metastasis and drug resistance-related malignant recurrence significantly decreased the long-term survival in TNBC[ 27 , 28 ]. Recently, CAFs in TME are thought to play a pivotal role in the progression of chemo-resistance [ 29 ] [ 30 ]. The present study constructed a co-culture system to evaluate the in vitro effect of CAFs on the drug sensitivity of BC cells. The results showed that CAFs reduced the sensitivity of BC cells to DOX (Fig. 1 ), proving the adverse effect of CAFs on TNBC chemotherapy. CAFs could contribute to tumor cell proliferation, EMT, angiogenesis, and ECM remodeling through the secretion of EVs or direct transmission of paracrine signals [ 11 , 31 , 32 ]. Indeed, CAFs have been shown to promote therapy resistance to BC cells by generating soluble factors through paracrine mechanisms [ 33 ]. CAFs/TNBC induced stemness features of tumor cells and decreased sensitivity to docetaxel via the secretion of FGF5 and production of fibrillar collagen[ 34 ]. And CAFs from HER2 + BC patients directly contributed to trastuzumab resistance through secreted high levels of NRG1 and activated HER3/AKT pathway[ 35 ]. In addition, CAFs could secrete EVs to transfer intracellular components to cancer cells and modify their treatment resistance. CAFs expanded BC subpopulations adept at resisting therapy by utilizing EVs to instigate the juxtacrine NOTCH3 pathway[ 36 ]. In BC endocrine treatment, EVs derived by CAFs administered substantial amounts of onco-miR-221 for fostering hormonal therapy resistance[ 37 ]. Our experimental results showed that CAFs-reduced DOX sensitivity of BC cells was attenuated as a result of the destruction or deletion of EVs in the culture medium (Fig. 2 ), indicating that EVs CAFs -mediated delivery of bioactive small molecules played a major role in the CAFs-induced of DOX resistance. Recent studies have demonstrated that distinct subtypes of CAFs could play varying roles in the tumor microenvironment. A study revealed that four subsets (S1–S4) of the CAFs were present in BC and metastatic lymph nodes of BC[ 38 ]. CAF-S1 favored an immunosuppressive microenvironment by secreting CXCL12 and facilitating regulatory T-cell differentiation. Whereas cell cancer migration and invasion were stimulated by CAF-S1-initiated EMT and CAF-S4-activated Notch signaling, respectively. This suggests distinct CAF subpopulations appear to perform different functions in tumor progression. Our data indicated that EVs derived by CAFs/TNBC reduced the sensitivity of BC cells to DOX more significantly (Fig. 2 , 3 ), suggesting that the heterogeneity of CAFs might be a key factor promoting drug-resistance phenotypes of BC. Thus, a protein array was used to screen EVs isolated from different CAFs and revealed that DLDH was significantly enriched in EVs secreted by CAFs/TNBC. While decreasing the level of EVs-DLDH, the drug sensitivity reduced by EVs CAFs was significantly prevented (Fig. 4 , 5 ). These findings suggested that DLDH in CAF-derived EVs contributed to protecting cancer cells from chemotherapy and that DLDH could be a relevant therapeutic target. α-KGDC represents a major modulator of ETC activity and TCA cycle flux, and is a pivotal enzyme in the metabolic reprogramming[ 20 ], and its activation suppressed via the hypoxia-inducible factor 1 (HIF1) could impede tumor growth in vivo [ 21 ]. DLDH, as its E3 subunit, is involved in the decarboxylation of pyruvate to form acetyl-CoA and reduced NAD to NADH during the cascade of glucose metabolism and mitochondrial ATP production [ 18 , 22 ]. Our study revealed that EVs-DLDH was delivered and enriched in the mitochondria of the recipient cells, promoting the activity of α-KGDC and NADH production (Fig. 6 ). In parallel, the elevated DLDH levels were sufficient to promote OXPHOS without affecting glycolysis in BC cells. DLDH is also a key enzyme of branched-chain α-keto dehydrogenase, α-ketoadipate dehydrogenase, and glycine decarboxylase complexes, hence the dysfunction of DLDH, which simultaneously incapacitates several central metabolic pathways[ 39 ]. Cancer cells tend toward glycolysis metabolism even in the presence of oxygen, or the “Warburg effect”[ 40 ]. However, in some tumors, the stromal cells drive cancer cell mitochondria to conduct OXPHOS when interacting with cancer cells, known as the “reverse Warburg effect”[ 41 ]. There is growing evidence to support the role of metabolic alteration in the induction of tumor drug resistance[ 42 ]. Recent studies showed that cancer cells with hybrid glycolysis/OXPHOS phenotype might facilitate metabolic plasticity and be specifically associated with metastasis and therapy resistance[ 43 , 44 ]. The present study provides experimental evidence supporting this notion. Moreover, we observed that high DLDH levels increased ATP synthesis and decreased intracellular DOX content (Fig. 7 ), which might be associated with the enhanced efflux function of the ATP-binding cassette transporter. As expected, the sensitivity of cells to DOX was significantly attenuated after inhibition of intracellular OXPHOS, demonstrating that CAFs-secreted EVs-DLDH induced the DOX resistance of BC cells due to a shift to OXPHOS metabolism. Even though chemotherapy is an effective option to treat TNBC, its therapeutic efficacy eventually decreases when BC develops chemo-resistance. Since the local microenvironment might be a crucial cause for increasing chemo-resistance of TNBC, it is imperative that the combination of CAFs intervention measures with traditional chemotherapy may be beneficial to improve TNBC treatment. The present study demonstrated that DLDH was highly expressed in CAFs/TNBC-secreted EVs and its enrichment was negatively correlated with DOX sensitivity in TNBC cells. EVs-delivered DLDH enhanced α-KGDC activity and promoted mitochondrial OXPHOS in TNBC cells, thereby increasing DOX efflux and reducing its accumulation to induce chemo-resistance (Fig. 7 ). This finding suggests that DLDH in CAFs-secreted EVs contributes to preserving cancer cells from chemotherapy and could be a relevant therapeutic target to overcome DOX resistance in TNBC patients. Conclusion The present study illustrated that CAFs derived EVs in regulating the chemo-sensitive by transporting DLDH, which promoted a shift of TNBC cells from a low- to a high- OXPHOS status. This data also cast a good light on EVs CAFs intervention as a mechanism that has significant potential for TNBC therapeutic development. Materials And Methods Cell culture and gene manipulation Primal cultures of human mammary fibroblasts have been established as described [ 45 ]. In brief, fresh breast tissue was collected from grossly malignant and benign areas of the cut surfaces of the modified radical mastectomy samples. Once resected, the tissue samples were cut into 2 mm 3 pieces, rinsed with the PBS solution, and then digested with 1 mg/ml of Type II collagenase for 2h at 37°C. Following filtration and centrifugation, cell precipitation was collected and seeded in 6-well culture plates. 30 minutes later, the medium was replaced with a fresh medium to remove non-adherent cells to obtain pure fibroblasts. Fibroblasts from malignant areas were termed CAFs, and those of benign regions were termed normal fibroblasts (NFs). The human breast cell lines were purchased from the American Type Culture Collection (ATCC, VA, USA) including ER-positive BC cell lines (MCF-7), and TNBC cell lines (MDA-MB-231 and BT549). All the cells were cultured in DMEM (Gibco, CA, USA) supplemented with 10% FBS (Gibco) and penicillin/streptomycin (Beyotime, China), and tested regularly and negative for mycoplasma. DLDH was ectopically expressed in fibroblasts with low constitutive DLDH. Conversely, CAFs/TNBC cells were transfected with DLDH shRNA and its scramble control (GenePharma, China) to silence DLDH using a transfectamine reagent (ThermoFisher, MA, USA) according to the manufacturer’s instructions. BC cells were transfected with ABCB1 or ABCG2 small interfering RNA (siRNA, Santa Cruz Biotechnology, CA, USA) using the reagent following the manufacturer's instructions. EVs isolation and characterization Fibroblast-secreted EVs isolation was performed as previously described [ 46 ]. Cells were grown at sub-confluence in growth media containing EVs-depleted FBS (prepared overnight ultracentrifugation at 100,000 g at 4ºC) for 18 h. The conditioned medium was then collected and centrifuged at 500 g for 15 min, 2,000 g for 15 min, and 5,000 g for 15 min to remove cells and cell debris, followed by centrifugation at 12,000 g for 30 min for removal of large vesicles. EVs were precipitated by ultracentrifugation at 100,000 g for 90 min at 4°C. The pelleted EVs were collected and resuspended in PBS. To remove contaminated proteins such as cytokines and signaling molecules, the precipitated EVs were finally purified by passing an Amicon Ultra-0.5 tube (Millipore, MA, USA). All steps were performed at 4 ℃. For the characterization of EVs, particle size analysis was performed using a nanoparticle tracking analysis (NTA). The morphology of EVs was observed via transmission electron microscopy (TEM), and the protein content of EVs was determined by western blots. The protein concentration of EVs was measured using a BCA assay (Beyotime). EVs labeling and trafficking To monitor EV trafficking, EVs were labeled with a red fluorescent dye PKH26 (Sigma-Aldrich, MO, USA). After staining for 5 min, the EVs were washed with PBS and collected by ultracentrifugation (100,000 g for 20 min) at 4 ℃. The GFP-labeled cells were grown on a confocal dish with an EVs-free medium and incubated subsequently with PKH26-labeled EVs for 24 h. Cells were fixed with ice-cold 4% paraformaldehyde for 15 min followed by permeabilization with 0.5% Triton X-100 for 10 min at room temperature. The samples were subjected to probing with the appropriate primary and secondary antibodies. The nuclei were counterstained with DAPI (Cell Signaling Technology, MA, USA). To cleave the membrane proteins of EVs, EVs were pretreatment with trypsin (0.025% EDTA) for 10 min before being labeled with PKH26 fluorescent dye. The fluorescent images were analyzed using a Leica DMi8 microscope using the Thunder Imaging System (Leica Microsystems). Cell survival analysis MTT and colony survival assays were performed to quantify cytotoxicity after BC cells were treated with EVs, DOX, or combination. For transwell co-culture assays, BC cells were plated into 6-well plates and allowed to adhere overnight in a DMEM medium. Fibroblasts were plated in cell culture inserts using the same medium and placed on top into the same 6-well plates after overnight adhering. The cells were incubated together for 48h before drug treatments. For the MTT assay, the cells were pretreated with EVs or not for 48h and then plated into 96-well plates at 4×10 3 cells per well. After 12 h, the medium was replaced by media containing a serial of dilutions of DOX. To inhibit the oxidative phosphorylation pathway, the cells were pretreated with Rotenone (5 µM) or Antimycin A (10 µM) for 3 h prior to DOX treatments. The cells were further cultured for 48 h, and an MTT assay was conducted using a microplate reader (Thermo Multiskan) at 562 nm. The percentage of cell viability at 0.5 µM DOX was calculated using the ratio between treated and untreated cells. For the colony survival assay, 200 cells were plated into 6-well plates and maintained for 24h before EVs and DOX treatment at relating doses corresponding to the MTT assay. The treated cells were cultured for 14–21 days allowing colony formation. The colonies were stained with 1% crystal violet and then counted. Tumor formation in zebrafish A zebrafish tumor experimental model was established for In vivo study. Before injection, BC cells were incubated with EVs in serum-free media for 3 days. Then, cells were resuspended and stained with lipophilic dyes CM-DiL (ThermoFisher) for 20 min at 37°C. After washing with PBS, PKH 26-labeled cells were collected, washed, and counted. For zebrafish xenotransplantation, 48h post-fertilization (hpf) zebrafish embryos were anesthetized with tricaine (Sigma-Aldrich) and 10 nL of cell suspension (approximately 200 labeled cells) were injected into the perivitelline cavity of each embryo. The injected embryos were incubated at 28°C for 1 h and then turned to 34°C to continue the cultivation, and allowed to recover in the presence of N-phenylthiourea (Sigma-Aldrich) to inhibit melanocyte formation. 24 h after cell injection, 5 µM DOX was added into zebrafish nutrient solution to inhibit tumor growth. 2 days after treatment, the tumors formed in adult fishes were anesthetized with tricaine, imaged using a fluorescent microscope (Nikon, Japan), and quantified using Image J software. Protein array and immunoblot analysis Concentrations of secreted proteins in the EVs were measured using the Human L2000 protein array (RayBiotech, GA, USA) according to the manufacturer's protocol. Whole-cell and EVs lysates were prepared and subjected to Western blot analysis for FN, FAP, α-SMA, and DLDH, respectively, as previously described. Primary antibodies used in this study were FN, FAP, α-SMA, DLDH (Santa Cruz Biotechnology), TOM20, and Vimentin (Cell Signaling Technology). All blots were detected using the enhanced chemiluminescence (ECL, Epizyme, China) with ChemiDoc™ XRS + imaging system (Bio-Rad, USA). Immunofluorescence The pretreated cells were seeded into confocal dishes at 10 4 cells. 12 h later, cells were fixed with ice-cold 4% paraformaldehyde for 15 min, permeabilization with 0.5% Triton X-100 for 10 min, blocked in 5% BSA for 1 h, and probed with primary and secondary antibodies (Cell Signaling Technology) sequentially. The nuclei were counterstained with DAPI according to the manufacturer’s instructions. The fluorescence was visualized and captured with a Leica DMi8 microscope. Enzyme-linked immunosorbent assay (ELISA) The levels of DLDH were measured using the Human DLDH ELISA Kit (Abbkine, China) according to the manufacturer’s instructions. The cell or EVs lysates were added into a 96-well plate coated with a monoclonal antibody against DLDH. After incubation, a substrate solution for the immunoperoxidase reaction was added to develop a color based on the amount of DLDH antigen present in the samples. The reaction was terminated by adding a stop solution. DLDH concentration was quantitated by measuring the absorbance of each well at 450 nm based on the standard curve. Biochemical analysis The α-KGDC activity was determined as previously described [ 47 ]. Briefly, sample protein was extracted by grinding harvested cells in liquid nitrogen. The lysate was cleared by ultracentrifugation at 12,000 g for 30min. α-KGDC activity was detected in 1 mL of reaction mixture containing 2.5 µM rotenone and 0.05% Triton X-100 upon addition of 5.0 mM MgCl2, 2.5 mM α-ketoglutarate, 0.1 mM CoASH, 0.2 mM thiamine pyrophosphate (TPP), 1.0 mM NAD+, and 5 µg of protein extract. The increase in absorbance due to NADH synthesis was detected at 340 nm for 5 min. α-KGDC activity was calculated using the extinction coefficient of NADH (ε = 6.22 mm-1 cm-1). Sample protein concentrations were determined by the BCA method. NADH levels were measured using an NADH assay kit from Abcam. Briefly, 10 5 cells were rapidly homogenized with 200 µl ice-cold NADH extraction buffer for 10 min on ice, and the supernatant was collected by centrifuge at 12,000 g for 5 min. Samples were incubated at 60℃ for 30 min to completely decompose the NAD and cooled on ice. Then, 50 µl of each sample was mixed with 100 µl of the reaction mix, incubated at room temperature for 30 min, and then fluorescence was measured in a microplate reader (Ex/Em = 535/587 nm). The intracellular ATP level of cells was detected with the Luminescent ATP detection assay kit (Abcam) following the instructions. In brief, 10 5 cells were lysed by the detergent provided in the kit, followed by the addition of reconstituted substrate solution and measurement of luminescence with a plate reader. OCR and ECAR quantifications Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured using Seahorse XF Analyzer (Agilent, USA). The EVs-treated cells were transferred into a 96-well XF96 plate at a cell density of 5×10 3 cells/well and incubated overnight. Cartridge plates for metabolic stress injections were hydrated for 24 h at 37°C without CO 2 in calibrant solution. 1 h before the assay, the culture medium in the XF96 plate was replaced by a Seahorse Assay medium. OCR was measured under four following conditions: basal, oligomycin, FCCP, and rotenone + antimycin. For quantifying ECAR, 2-DG, an inhibitor of glycolysis is injected to stop glycolytic acidification. OCR and ECAR were normalized by the total protein. Flow cytometry analysis For the determination of protein levels, the fixed cells were first incubated with a DLDH antibody (Santa Cruz Biotechnology). Then washed cells were incubated with Alexa 488 anti-mouse IgG (H + L) conjugate (Cell Signaling Technology). Flow cytometry was performed using a BD FACSMelody instrument (BD, USA). Green fluorescence was excited at 488 nm and detected at 500–550 nm. For intracellular DOX concentration determination, cells were seeded into 6-well plates and pretreated. Then, DOX was added and incubated with cells for 0.5 h or 3 h at 37°C. After cells were digested with trypsin, rinsed with cold PBS, and suspended in 400 µl of PBS, the DOX fluorescence was determined using a flow cytometer. The number of cells collected was 10,000. A gate is set for each sample with a majority of the cells, only excluding small fragments of cells. Measurement of total and mitochondrial ROS The generation of cellular hydroxyl radical was measured using DCFHDA as described previously [ 48 ]. Briefly, 48h after EVs-cells incubation, cells were labeled by both DCFHDA (2,7-Dichlorodi-hydrofluorescein diacetate, Sigma-Aldrich) at a final concentration of 5 µM in a culture medium at 37℃ for 30 min. Fluorescent images of DCF in the cells were observed using a fluorescence microscope (Olympus, Japan) and then quantified by λex-495nm and λem -520nm using a TECAN Spark fluorescence spectrometer (Tecan, Switzerland). Hydrogen peroxide (H2O2) was fluorometrically detected using an H2O2 Assay (Abcam) according to the manufacturer’s protocol. Briefly, 10 6 cells were incubated with EVs for 48 h. The protein lysates from each group were used to measure the cellular content of hydrogen peroxide. The red-fluorescent dye was measured using a microplate reader (Ex/Em = 485/535 nm) and absolute amounts of H2O2 were calculated. The generation of superoxide anion 48h after each treatment was measured using DHE (Beyotime). In brief, after loading of DHE, the cellular fluorescence in 10 5 cells was analyzed using a spectrofluorometer (λex-385nm, λem -565nm). The amplitudes of fluorescence intensity are presented as the relative fold changes in each group to that of the control. Statistical analysis All experiments were representative of multiple independent repeat experiments. Unpaired two-tailed Student's t-test was used to analyze the significance between the two groups. One-way analysis of variance (ANOVA) followed by Dunnett’s or Bonferroni’s test was used to analyze the variance of multiple groups with normal distribution and equal variances. Data were analyzed using GraphPad Prism (USA), and statistical significance was defined as P < 0.05. Abbreviations BC, breast cancer; TNBC, triple-negative breast cancer; EVs, extracellular vesicles; DOX, doxorubicin; TME, tumor microenvironment; CAFs, cancer-associated fibroblasts; EMT, epithelial-to-mesenchymal transition; NTA, nanoparticle tracking analysis; TEM, transmission electron microscopy; NFs, normal fibroblasts; FAP, fibroblast activation protein; FN, fibronectin; α-SMA, α-smooth muscle actin; TCA cycle, tricarboxylic acid cycle; α-KGDC, α-ketoglutarate dehydrogenase complex; DLDH, dihydrolipoamide dehydrogenase; hpf, hour post-fertilization; ECL, enhanced chemiluminescence; ROS, reactive oxygen species; H2O2, hydrogen peroxide; OXPHOS, oxidative phosphorylation; OCR, oxygen consumption rates; ECAR, extracellular acidification rates; ATP, adenosine triphosphate; ABC transporter, ATP-binding cassette transporter; ETC, electron transport chain Declarations Ethics approval and consent to participate The Research Committee and the Institutional Animal Care of and Use of Nanjing University Medical School have approved animal studies conducted in this study. Consent for publication Not applicable. Availability of supporting data All materials made and datasets analyzed in this study are available for reasonable requests to the corresponding author, Dr. Juan Li. Competing interests No potential competing interest were disclosed. Authors' contributions Conception and design: ZX and JL; Development of methodology: ZX, YZ, FF and RS; Acquisition of data: ZX; Analysis and interpretation of data: ZX and HM; Administrative, technical, or material support: ZX, JH and JL; Writing, review, and/or revision of the manuscript: ZX, RS and JL; Study supervision: RS and JL. All authors have read and approved the final manuscript. Acknowledgements This study was supported by the National Natural Science Foundation of China (No. 31371399) to J Li. Authors' information Phase I Clinical Trials Unit, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, 210008, China. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2262675","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":155550084,"identity":"6a0c29bd-79b6-4e06-8c33-bda1ad0c97ed","order_by":0,"name":"Zhi Xu","email":"","orcid":"https://orcid.org/0000-0003-2722-1471","institution":"The Affiliated Drum Tower Hospital of Nanjing University Medical School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhi","middleName":"","lastName":"Xu","suffix":""},{"id":155550085,"identity":"6a99e23f-c7e6-4a41-89a4-f673daab4210","order_by":1,"name":"Junchen Hou","email":"","orcid":"","institution":"The First Affiliated Hospital with Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Junchen","middleName":"","lastName":"Hou","suffix":""},{"id":155550086,"identity":"f1cb7c6d-420a-4002-966f-04c3fb910b3e","order_by":2,"name":"Hehua Ma","email":"","orcid":"","institution":"The Affiliated Hospital of Nanjing University Medical School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hehua","middleName":"","lastName":"Ma","suffix":""},{"id":155550087,"identity":"4af5f8b4-5d7e-43ba-b004-5231d7d774a0","order_by":3,"name":"Yu Zhao","email":"","orcid":"","institution":"The Affiliated Hospital of Nanjing University Medical School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Zhao","suffix":""},{"id":155550088,"identity":"3874ccf7-4128-4995-82d4-e0ee01967595","order_by":4,"name":"Fei Fei","email":"","orcid":"","institution":"The Affiliated Hospital of Nanjing University Medical School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Fei","suffix":""},{"id":155550089,"identity":"ce23be50-313f-41ee-8d4c-1a1ab56ad631","order_by":5,"name":"Runbin Sun","email":"","orcid":"","institution":"The Affiliated Hospital of Nanjing University Medical School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Runbin","middleName":"","lastName":"Sun","suffix":""},{"id":155550090,"identity":"a95bad81-480f-4cde-ad7a-43570fb55708","order_by":6,"name":"Juan Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYBACxgYg8cDAhoeNv/nAgQ8/iNWSYJAmxy9xLPHgzB5irUpgOGws2ZBjfJiDjQjVzDOSnz1IKGBO3HDgzIfDDDwM8vxiBwg4bEaauUGCAVvihsO9Gw4XWDAYzpydQEhLgplEggEP0JazGw7P4AH66zZBLenfgFokgFpyHhzmYSNKSw7IFgOQ9xmI1NLzpgyoJQEUyAbAQJYg7BfD9vRtEh/+/AdF5eMPH37YyPNLE9LSgMqXwK8cBOQJKxkFo2AUjIIRDwDCJUjwh3wnFwAAAABJRU5ErkJggg==","orcid":"","institution":"The Affiliated Drum Tower Hospital of Nanjing University Medical School","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2022-11-11 10:26:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2262675/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2262675/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":29738684,"identity":"39d8693b-f204-4deb-b4de-9c345524d546","added_by":"auto","created_at":"2022-11-30 19:41:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1909962,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCAFs inhibited the DOX sensitivity in BC cells.\u003c/strong\u003e (\u003cstrong\u003eA)\u003c/strong\u003eExperimental scheme. (\u003cstrong\u003eB) \u003c/strong\u003eMCF-7 cells were incubated with NFs or CAFs and then treated with different concentrations of DOX as indicated. The cell viability after normalized with cell plating efficiency was analyzed by MTT assay.\u003cstrong\u003e(C)\u003c/strong\u003e The cell viability of DOX in MDA-MB-231 cells. (\u003cstrong\u003eD, E)\u003c/strong\u003e After cell-fibroblasts incubation and drug treatment, DOX resistance was analyzed by colony survival assay. (\u003cstrong\u003eF)\u003c/strong\u003eExperimental scheme. (\u003cstrong\u003eG, H)\u003c/strong\u003e Cell viability of BC cells post 0.5μM DOX treatment for 48 h. (\u003cstrong\u003eI, J) \u003c/strong\u003eThe cell survival rate was determined using a colony survival assay. Data shown represent the results of at least three biologically independent experiments. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-2262675/v1/3037e09c9886d78f3c3adb3e.png"},{"id":29738686,"identity":"6068bf22-f82e-480a-b11b-8c1603b64f9b","added_by":"auto","created_at":"2022-11-30 19:41:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3641462,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCAFs-derived EVs enhanced DOX resistance of BC cells.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e The isolated EVs were examined by the determination of EVs-specific proteins CD63, TSG-101, and Alix by western blots. Calnexin serves as a cytosolic protein control. \u003cstrong\u003e(B) \u003c/strong\u003eEVs were isolated from the culture medium of NFs or CAFs cells and characterized by TEM. \u003cstrong\u003e(C)\u003c/strong\u003e Size distribution profile of EVs were obtained by NTA. \u003cstrong\u003e(D)\u003c/strong\u003e Experimental scheme. \u003cstrong\u003e(E)\u003c/strong\u003e Cell viability of EVs-treated BC cells post 0.5μM DOX treatment. \u003cstrong\u003e(F, G)\u003c/strong\u003e The cellular survival was examined by colony formation assay. \u003cstrong\u003e(H)\u003c/strong\u003e BC cells were labeled with GFP, and EVs\u003csup\u003eCAFs\u003c/sup\u003e were labeled with PKH26 (red). After 48 h incubation with EVs, the recipient cells were observed using a confocal microscope. \u003cstrong\u003e(I)\u003c/strong\u003e Experimental scheme.\u003cstrong\u003e (J)\u003c/strong\u003e PKH26-labeled EVs were pretreated with trypsin to cleave membrane proteins before being incubated with BC cells. Cell viability was analyzed by MTT assay. \u003cstrong\u003e(K)\u003c/strong\u003e The cell survival rate was determined using a colony survival assay. \u003cstrong\u003e(L) \u003c/strong\u003eThe representative confocal images of EVs PKH26 and recipient cells. \u003cstrong\u003eA, E-G, J, K\u003c/strong\u003e Data shown represent the results of at least three biologically independent experiments. Image representative of at least two independent experiments. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-2262675/v1/04627b917e01fa6ffe5bb0ba.png"},{"id":29739407,"identity":"f57007ed-9803-4ef5-b630-cf6bc36a97ec","added_by":"auto","created_at":"2022-11-30 19:49:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1606557,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe DLDH-enriched EVs efficiently deliver into the mitochondrial of BC cells. (A) \u003c/strong\u003eEVs isolated from CAFs/LuA and CAFs/TNBC were subjected to a human protein array. A red box indicated DLDH profiling. \u003cstrong\u003e(B, C)\u003c/strong\u003e The amounts of DLDH in EVs\u003csup\u003eCAFs/LuA\u003c/sup\u003e and EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e were quantified using an ELISA kit and western blots. \u003cstrong\u003e(D)\u003c/strong\u003e Co-localization of DLDH with EVs\u003csup\u003eCAFs\u003c/sup\u003e in recipient cells was analyzed by confocal imaging.\u003cstrong\u003e (E)\u003c/strong\u003e Co-localization of TOM20 with EVs\u003csup\u003eCAFs\u003c/sup\u003e in the mitochondria of the recipient cells was examine. \u003cstrong\u003e(F-H)\u003c/strong\u003e The level of DLDH in EVs-treated BC cells was confirmed by immunofluorescence, ELISA and immunoblots, respectively. \u003cstrong\u003eB-H\u003c/strong\u003e Data shown represent the results of at least three biologically independent experiments. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-2262675/v1/fd46fd55605d7342af7393d5.png"},{"id":29739406,"identity":"531bdb7a-999c-47ef-800b-f870e150e7d5","added_by":"auto","created_at":"2022-11-30 19:49:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2286964,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of EVs-DLDH on intercellular DOX-resistance enhancement. (A, B)\u003c/strong\u003e EVs were isolated from the DLDH-silenced CAFs/TNBC cells and the DLDH-overexpressed CAFs/LuA cells, the levels of EVs-DLDH were confirmed by western blots and ELISA. \u003cstrong\u003e(C, E)\u003c/strong\u003e The amounts of DLDH in EVs-treated cells were quantified using western blots. \u003cstrong\u003e(D, F)\u003c/strong\u003e After incubation with EVs derived from DLDH-manipulated CAFs, DLDH levels in the recipient cells were measured by ELISA. \u003cstrong\u003e(G, I)\u003c/strong\u003eCell viability of the recipient cells post 0.5μM DOX treatment. (\u003cstrong\u003eH, J) \u003c/strong\u003eThe cell survival rate was determined using a colony survival assay. \u003cstrong\u003e(K) \u003c/strong\u003eAfter incubation with EVs, the CM-DiL-labeled cells were injected into the perivitelline cavity of zebrafish embryos. Fishes were grown with 5μM DOX or left the drug as a negative control. The formed tumors in the fish bodies were screened as indicated. \u003cstrong\u003e(L) \u003c/strong\u003eThe tumor volumes were calculated and plotted, n≧6. \u003cstrong\u003eA-J\u003c/strong\u003e Data shown represent the results of at least three biologically independent experiments. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-2262675/v1/412b0da27f5d963173a44aa9.png"},{"id":29740764,"identity":"ee1e43c7-82ec-49fb-97d0-151dc10ab059","added_by":"auto","created_at":"2022-11-30 20:05:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2859164,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEVs-DLDH contributed to CAFs-increased OXPHOS in BC cells. (A, B) \u003c/strong\u003eThe α-KGDC activity was quantified in EVs-treated cells. \u003cstrong\u003e(C, D)\u003c/strong\u003e NADH level of the recipient cells was measured. \u003cstrong\u003e(E, F)\u003c/strong\u003e As indicated, the effects of EVs on the OCR of recipient cells were quantified by Seahorse XF Analyzer and representative of two independent experiments. Mitochondrial reparation at basal, ATP-linked, or maximal condition was tracked. Reverse capacity was calculated by subtracting the basal value from the maximal value. \u003cstrong\u003e(G-I)\u003c/strong\u003e After EVs-cells incubation, the effect of DLDH on EVs\u003csup\u003eCAFs\u003c/sup\u003e-induced ROS generation in BC cells was determined by DCFHDA probe\u003cstrong\u003e (G)\u003c/strong\u003e, H2O2 \u003cstrong\u003e(H)\u003c/strong\u003e, and DHE staining\u003cstrong\u003e (I)\u003c/strong\u003e. DCFHDA was rapidly de-esterified intracellularly and then oxidized in the presence of ROS into fluorescent DCF (Green), and the nuclei were counterstained with DAPI (Blue). DHE formed a red fluorescent product (ethidium) upon reaction with superoxide anions and intercalated with DNA. \u003cstrong\u003e(J)\u003c/strong\u003e After 3 h of CPI-613 treatment, which inhibited α-KGDC function, the cellular ROS levels were quantified using a DCFHDA probe. \u003cstrong\u003e(K)\u003c/strong\u003e EVs-incubated cells were treated with CPI-613 for 30 min followed by DOX treatment. Cell viability was analyzed by the MTT assay. DCW, dry cell weight. \u003cstrong\u003eA-D, G-K\u003c/strong\u003e Data shown represent the results of at least three biologically independent experiments. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-2262675/v1/1be4278ab5641a4763542573.png"},{"id":29739959,"identity":"62dabc19-c93d-40fd-ac3d-43b41f4b689c","added_by":"auto","created_at":"2022-11-30 19:57:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1892562,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCAFs-derived EVs increased DOX efflux was driven by OXPHOS. (A, B)\u003c/strong\u003e After 3 h treatment with 0.5 μM DOX, the intracellular DOX content was quantified by flow cytometry and the DOX efflux ratio was calculated. \u003cstrong\u003e(C, D)\u003c/strong\u003e After EVs-cells incubation, the cellular level of ATP was measured. \u003cstrong\u003e(E, F)\u003c/strong\u003e The EVs-incubated cells were pretreated with Rotenone or Antimycin Afor 30 min to inhibit intracellular OXPHOS, and an MTT assay was used to determine the inhibition rate of DOX on cell viability. \u003cstrong\u003e(G) \u003c/strong\u003eCell survival rate was determined using a colony survival assay. \u003cstrong\u003e(H, I)\u003c/strong\u003e ABCG2-siRNA and ABCB1-siRNA were employed to inhibit the ABC transporters of the recipient cells. After transfection, the cells were incubated with CAFs/TNBC-derived EVs followed by DOX treatment. The intracellular DOX content was quantified and the DOX efflux ratio was calculated. \u003cstrong\u003e(J)\u003c/strong\u003e Cell viability was analyzed by the MTT assay. \u003cstrong\u003e(K, L) \u003c/strong\u003eCell survival rate was determined using a colony survival assay. Data shown represent the results of at least three biologically independent experiments. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-2262675/v1/399edf26eec6900d5fe0d29f.png"},{"id":29738690,"identity":"8ca0e8ad-87f8-4468-bf17-965a3cd7dfa0","added_by":"auto","created_at":"2022-11-30 19:41:21","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1380591,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDepiction of the suggested mechanism involved in EVs\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eCAFs\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e-deliveried DLDH reduced DOX sensitivity of BC cells.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-2262675/v1/7856c91d0233d91f434e4270.png"},{"id":40945705,"identity":"fa4fdb9a-d347-4532-963f-7e0c55214df6","added_by":"auto","created_at":"2023-08-02 12:01:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4809438,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2262675/v1/ed1fd5e3-a77c-4fe1-9580-b4a6ecfb1296.pdf"},{"id":29738691,"identity":"4e320006-8b19-40ec-a509-6ac51ba6e7e3","added_by":"auto","created_at":"2022-11-30 19:41:21","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2923303,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"supplementaldata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2262675/v1/989e20fb33bb66e9bc47c178.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"DLDH-containing extracellular vesicles from CAFs reduced DOX sensitivity in triple-negative breast cancer","fulltext":[{"header":"Highlights","content":"\u003cul start=\"12\"\u003e\n \u003cli\u003eCAFs-derived EVs suppressed doxorubicin sensitivity in breast cancer.\u003c/li\u003e\n \u003cli\u003eEVs transported DLDH into the mitochondria of recipient cells leading to increased OXPHOS.\u003c/li\u003e\n \u003cli\u003eEVs-DLDH reduced doxorubicin sensitivity through increasing drug efflux driven by OXPHOS. \u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eDue to the absence of effective targeted therapy, the combination of surgery and chemotherapy or radiotherapy is the mainstay of triple-negative breast cancer (TNBC) treatment [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite the efficacy of cytotoxic chemotherapy, TNBCs are usually more aggressive, showing strong resistance to therapy [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]; the median survival of advanced TNBC patients is approximately 13 to 20 months [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, it is extremely urgent to improve the efficacy of TNBC therapies and prolong the patient survival time.\u003c/p\u003e \u003cp\u003eDoxorubicin (DOX) is widely used for the treatment of cancers[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Multiple mechanisms have been proposed for DOX resistance, including p53 or topoisomerase II mutation, altered cellular accumulation, increased drug inactivation, and signaling pathway activation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Additionally, growing evidence has indicated that the tumor microenvironment (TME) influences therapeutic response and drug resistance in tumor cells[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Cancer-associated fibroblasts (CAFs) as an important component of TME promote tumor progression, including proliferation, migration, invasion, epithelial-to-mesenchymal transition (EMT), and metabolic reprogramming [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It was reported that the depletion of CAFs could provide survival benefits in TNBC murine models by reducing intratumoral collagen [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Another study has indicated that targeting CAFs could protect MCF-7 cells against apoptosis induced by DOX [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, the mechanism that CAFs direct deploy in DOX resistance in breast cancer (BC) has not been studied in greater detail.\u003c/p\u003e \u003cp\u003eExtracellular vesicles (EVs) are microvesicles composed of lipid bilayers containing bioactive molecules, such as proteins, bioactive lipids, and small RNAs[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Currently, multiple studies have demonstrated that CAFs-mediate EVs could facilitate BC progression. It was found that CAFs-secreted EVs mediated stromal mobilization of autocrine Wnt-PCP signaling in BC cell migration [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. A study of the metabolomic analysis provided that CAFs-derived EVs containing intact metabolites (amino acids, lipids, and tricarboxylic acid (TCA) cycle intermediates) and modulating cancer cell metabolism [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In addition, it was demonstrated that miRNAs in CAFs could be delivered to BC cells via EVs, thereby inhibiting ER expression and reducing the sensitivity to hormone therapy [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe present study revealed that CAFs-derived EVs enhance BC cells\u0026rsquo; resistant capacity of DOX by delivering dihydrolipoamide dehydrogenase (DLDH). DLDH is a core component of the α-ketoglutarate dehydrogenase complex (α-KGDC, a key rate-limiting enzyme of the TCA cycle) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The results showed that EVs-DLDH could translocate into mitochondria of BC cells and promote DOX resistance via promoting adenosine triphosphate (ATP) synthesis and enhancing the efflux function of ATP-binding cassette (ABC) transporters. This finding suggests that DLDH in CAF-derived EVs contributes to protecting cancer cells from chemotherapy and that DLDH could be a relevant therapeutic target.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCAFs reduced the sensitivity of BC cells to DOX.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt has recently been suggested that the TME may confer resistance to therapy[\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. In this study, we examined the role of CAFs in modulating the response of BC cells (MCF-7, BT-549, and MDA-MB-231) to DOX. The fibroblastic nature of tissue-derived cell cultures has been verified through their characteristic fibroblastic morphology and expression of fibroblastic markers such as fibronectin (FN), fibroblast activation protein (FAP), vimentin, and \u0026alpha;-smooth muscle actin (\u0026alpha;-SMA) (Fig.S1). The primary fibroblasts were subsequently co-cultured with BC cells. The cytotoxicity tests indicated that DOX-induced cell death in BC cell monocultures and this cytotoxic effect inhibited in co-culture with CAFs but not with NFs (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA-E and Fig.S2). Notably, the effect was more pronounced for CAFs/TNBC than for CAFs/LuA in the above experiments. To investigate the cause of the DOX resistance observed in the CAFs co-culture system, the BC cells were cultured in a fibroblasts-conditioned medium with EVs (CM-CAFs/LuA, CM-CAFs/TNBC) or without EVs (non-EVs CM-CAFs/LuA, non-EVs CM-CAFs/TNBC), and followed by multiple doses of DOX treatment (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF). MTT assay showed that the cytotoxic effect and DOX IC50 value were only increased in the group of incubation with CM-CAFs, especially CM-CAFs/TNBC (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eG, H and Fig.S3A-F). Colony survival assay further confirmed that there is no improved survival by incubation with non-EVs CM (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI, J and Fig.S3G-I). These results supported the hypothesis that CAFs suppressed the DOX sensitivity of BC cells and that CAFs-derived EVs might play a potentially more important role than soluble factors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCAFs-derived EVs induced DOX resistance of BC cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether CAFs-derived EVs attenuate DOX sensitivity in BC cells, equal amounts of EVs extracted from fibroblast cell cultural supernatants were pretreated in BC cells for 48h (Fig.S4A). Before being co-cultured with BC cells, EVs were verified using TEM, NTA, and immunoblots (CD63, Alix, and TSG101), which suggested that these EVs isolated using ultracentrifugation were exosome-enriched EVs (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). The MTT assay showed that the cell\u0026rsquo;s uptake of EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e remarkably improved survival compared to no survival improvement by uptake of EVs\u003csup\u003eNFs\u003c/sup\u003e and EVs\u003csup\u003eCAFs/LuA\u003c/sup\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF and Fig.S4E). Consistent with cell viability, the DOX IC50 value was higher in the uptake of EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e than in the uptake of EVs\u003csup\u003eNFs\u003c/sup\u003e and EVs\u003csup\u003eCAFs/LuA\u003c/sup\u003e (Fig.S4B-D). In addition, the colony survival assay further confirmed that the uptake apparently enhanced the DOX resistance of the recipient cells compared to the uptake of EVs\u003csup\u003eNFs\u003c/sup\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF, G and Fig.S4F).\u003c/p\u003e\n\u003cp\u003eTo determine whether EVs entered the receiving cells, red fluorescent PKH26 dye-labeled EVs were co-cultured with GFP-labeled BC cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eH and Fig.S4G). After incubation of cells with EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e, as expected, the labeled EVs can be detected in the recipient cells, which are mainly localized in the cytoplasm. Next, PKH26-labeled EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e were pretreated with trypsin (Trypsinized-EVs) to cleave membrane proteins before being incubated with BC cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eI). Trypsinized-EVs-treated cells showed decreased fluorescent signals in the recipient cells, compared with Control-EVs-treated cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eL). Meanwhile, the cytotoxicity assay showed that compared with EVs-treated cells, there was almost no difference between Trypsinized-EVs-treated cells and untreated cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eJ, K and Fig.S4H), suggesting EVs play a significant role in CAFs-induced DOX resistance.\u003c/p\u003e\n\u003cp\u003eTo further evaluate the role of EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e in promoting cell DOX resistance in vivo, a zebrafish tumor experimental model was established. BC cells incubated with EVs were injected into zebrafish embryos, and DOX was added to nutrient solutions when the fishes were born. Tumors formed as the fish grown up and were daily tracked by fluorescent imaging. Consistent with the results from in vitro experiments, DOX treatment efficiently inhibited tumor growth in the group injected with BC cells, but the anticancer efficacy was significantly reduced in the group injected with EVs\u003csup\u003eCAFs/TNBC\u0026minus;\u003c/sup\u003etreated BC cells (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eK, L).\u003c/p\u003e\n\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003cp\u003e\u003cstrong\u003eDLDH is enriched in CAFs/TNBC-derived EVs and transmitted to BC cells\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eEVs perform various functions that involve interactions within the cellular microenvironment due to their ability to carry lipids, proteins, nucleic acids, and metabolites[\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]. Since CAFs-derived EVs are recognized as potent factors for the acquisition of drug resistance in BC, the protein profiling in EVs\u003csup\u003eCAFs/LuA\u003c/sup\u003e vs. in EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e was quantified using protein arrays. A majority of proteins were increased in EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e compared to EVs\u003csup\u003eCAFs/LuA\u003c/sup\u003e, and several genes relating to cell binding signals and metabolic pathways were upregulated in EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e (Fig.S5A). Among them, DLDH, one of the critical proteins regulating cellular ATP synthesis[\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e], was highly enriched in EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). DLDH is a core component of the \u0026alpha;-KGDC that is a key rate-limiting enzyme in the cellular TCA cycle[\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. Notably, it was identified that a significant enrichment for DLDH in CAFs/TNBC-derived EVs, whose amount was consistent with the constitutive levels of DLDH in CAFs/TNBC cells vs. CAFs/LuA cells (Fig.S5B-D). In addition, the results from the immunoblots, immunofluorescence, and ELISA assay confirmed that DLDH contents were enhanced in EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, C).\u003c/p\u003e\n \u003cp\u003eTo determine that EVs can carry and enrich DLDH, the purified EVs from the CAFs/LuA and CAFs/TNBC cells were incubated with cells, and the internalized EVs-transported DLDH (EVs-DLDH) was determined in the recipient cells. As expected, DLDH was co-localized with PKH26-labeled EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e in the cytoplasm (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). Intriguingly, the EVs were co-localized with TOM20 (the mitochondrial outer membrane protein, labeled with green secondary antibody) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE), indicating that EVs could deliver DLDH into the mitochondria of recipient cells. Furthermore, after incubation with EVs, the expression of DLDH was increased markedly in the EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e\u0026ndash;treated cells (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF-H). Results showed that EVs secreted from CAFs cells could alter the content of DLDH in BC cells via the delivery of EVs.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eEVs-mediated DLDH delivery contributes to DOX resistance of BC cells.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTo verify that EVs-DLDH could confer the DOX resistance to BC cells, DLDH was ectopically expressed into fibroblasts with a low level of constitutive DLDH (Fig.S6A, B). And the level of DLDH in CAFs-derived EVs was measured by western blots and ELISA assay (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). After incubation with EVs, the DLDH level was increased in EVs\u003csup\u003eCAFs/LuA\u0026minus;DLDH\u003c/sup\u003e-treated BC cells versus EVs\u003csup\u003eCAFs/LuA\u0026minus;NC\u003c/sup\u003e-treated cells (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC-F). Subsequently, the MTT assay showed that DLDH-overexpressed EVs-treated cells remarkably improved survival compared to the lack of improvement in survival by control groups (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG, H and Fig.S6C-F). Consistent with cell viability, the results from the colony survival assay further confirmed that uptake of DLDH-overexpressed EVs enhanced DOX resistance of the recipient cells (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eI, J). In parallel, DLDH was silenced in CAFs/TNBC cells using a DLDH siRNA (Fig.S6A, B). The level of DLDH in CAFs/TNBC cells and EVs\u003csup\u003eCAFs/TNBC\u0026minus;siDLDH\u003c/sup\u003e-treated BC cells were reduced significantly. Accordingly, the drug resistance was alleviated in the DLDH-silenced EVs treated cells compared to the scrambled control (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig.S6).\u003c/p\u003e\n \u003cp\u003eFurthermore, to validate that inhibiting EVs-DLDH could increase the susceptibility for DOX of BC cells, TNBC cells were incubated with EVs\u003csup\u003eNFs\u003c/sup\u003e, EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e and DLDH-silenced EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e prior to injection into the zebrafish embryos (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eK, L and Fig.S6G, H). As expected, compared to the EVs\u003csup\u003eNFs\u003c/sup\u003e-treated group, the drug sensitivity was decreased in the EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e group, but eliminated with the use of EVs isolated from the DLDH-silenced CAFs/TNBC cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003cp\u003e\u003cstrong\u003eEVs transported DLDH enhanced mitochondrial respiration in the recipient cells\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eDLDH is a component of \u0026alpha;-KGDC, which is a mitochondrial enzyme complex catalyzing the \u0026alpha;-ketoglutarate to succinyl-CoA[\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. The immunofluorescence experiment also demonstrated that EVs could transport DLDH into the mitochondrial of recipient cells (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD, E). Recent evidence suggests that the metabolic pattern is crucial for chemo-resistance in cancers[\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. As a rate-limiting enzyme, \u0026alpha;-KGDC activity may induce alterations in the flux of metabolites through TCA. Thus, the \u0026alpha;-KGDC activity assay was performed to confirm that the CAFs/TNBC-secreted EVs were able to enhance \u0026alpha;-KGDC activation (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Additionally, suppression of EVs-DLDH could neutralize EVs\u003csup\u003eCAFs\u003c/sup\u003e-mediated activation of \u0026alpha;-KGDC. Because \u0026alpha;-KGDC plays an important role in NAD\u003csup\u003e+\u003c/sup\u003e conversion into NADH, EVs\u003csup\u003eCAFs\u003c/sup\u003e-upregulated oxidative flux of the TCA cycle led to increased NADH levels (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). Furthermore, we examined the mitochondrial respiratory capacity of BC cells after incubating with EVs\u003csup\u003eCAFs\u003c/sup\u003e. In EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e-treated cells, the basal and maximal respiratory capacities were higher than in EVs\u003csup\u003eCAFs/LuA\u003c/sup\u003e-treated cells, suggesting activated oxidative phosphorylation (OXPHOS) (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE, F and Fig.S7A, B). Intriguingly, glycolysis was not significantly different between the two groups (Fig.S7C, D).\u003c/p\u003e\n \u003cp\u003eNext, we measured cellular levels of reactive oxygen species (ROS), which could elevate through the activation of the electron transport chain (ETC) in the mitochondria. The level of hydroxyl radical and H2O2 was elevated in EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e-treated cells compared to EVs\u003csup\u003eCAFs/LuA\u003c/sup\u003e-treated cells, in line with a hyperactive OXPHOS (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG, H). EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e-treated cells also exhibited elevated superoxide anion level that was comparable to EVs\u003csup\u003eCAFs/LuA\u003c/sup\u003e-treated cells (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eI). Correspondently, suppression of EVs-DLDH could neutralize EVs\u003csup\u003eCAFs\u003c/sup\u003e-mediated increased ROS level. These results suggested that EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e exhibit hyperactive OXPHOS which was associated with the level of DLDH delivered by EVs\u003csup\u003eCAFs\u003c/sup\u003e. In addition, a lipoate analog CPI-613 was used to inhibit \u0026alpha;-KGDC function through reduced DLDH activity [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. CPI-613 significantly inhibited the \u0026alpha;-KGDC activity and NADH level in EVs-treated cells (Fig.S7E-H). The experimental results are demonstrated in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, which reveal DLDH inhibitors have been shown to inhibit mitochondrial OXPHOS and cell viability.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eEVs-DLDH-mediated reduction of DOX sensitivity was driven by OXPHOS.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eIt has been widely recognized that the induction of drug efflux by ABC transporters is the leading cause of decreased intracellular drug accumulation thereby lowering the efficacy of DOX[\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. To examine whether the lower response to DOX in EVs-treated BC cells was associated with drug accumulation changes by enhanced OXPHOS, the intracellular DOX content was quantified by flow cytometry after cell-EVs incubation and DOX treatment (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA, B and Fig.S8A, B). As shown in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e, the DOX accumulation was significantly reduced in EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e-incubated cells compared to the EVs\u003csup\u003eCAFs/LuA\u003c/sup\u003e-incubated cells. EVs-DLDH also increased ATP synthesis in BC cells (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC, D). After being treated with Rotenone or Antimycin A, which was used to inhibit OXPHOS in the recipient cells, the EVs\u003csup\u003eCAFs/TNBC\u003c/sup\u003e-induced drug efflux and DOX resistance were significantly reduced (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE-G and Fig.S8C, D). To further validate the EVs-mediated reduction of DOX sensitivity was driven by OXPHOS, ABCG2-siRNA and ABCB1-siRNA were employed to inhibit the ABC transporters of the recipient cells (Fig.S8E, F). Cells were co-incubated with EVs following the knockdown of ABC transporter protein and subjected to detection of DOX cytotoxic effects on cells. The data demonstrated that ABC transporters inhibition could attenuate EVs\u003csup\u003eCAFs\u003c/sup\u003e-reduced DOX sensitivity of BC cells (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eJ-L). Consistently, inhibition of the ABC transporters improves DOX accumulation in EVs-treated cells (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eH, I and Fig.S8G, H), suggesting that these transporters participate in the efflux of the drug. These results suggest that EVs-DLDH-mediated DOX sensitivity inhibition resulting from increased drug efflux driven by OXPHOS.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBC is one of the most common malignancies in the world, TNBC comprises 15\u0026ndash;20% of all BC patients, yet accounts for 30% of BC deaths[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Unlike ER or HER2-positive BC, which benefit from targeted therapies, TNBC patients mostly rely on surgery and systemic chemotherapy[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, distant-organ metastasis and drug resistance-related malignant recurrence significantly decreased the long-term survival in TNBC[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Recently, CAFs in TME are thought to play a pivotal role in the progression of chemo-resistance [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The present study constructed a co-culture system to evaluate the in vitro effect of CAFs on the drug sensitivity of BC cells. The results showed that CAFs reduced the sensitivity of BC cells to DOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), proving the adverse effect of CAFs on TNBC chemotherapy.\u003c/p\u003e \u003cp\u003eCAFs could contribute to tumor cell proliferation, EMT, angiogenesis, and ECM remodeling through the secretion of EVs or direct transmission of paracrine signals [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Indeed, CAFs have been shown to promote therapy resistance to BC cells by generating soluble factors through paracrine mechanisms [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. CAFs/TNBC induced stemness features of tumor cells and decreased sensitivity to docetaxel via the secretion of FGF5 and production of fibrillar collagen[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. And CAFs from HER2\u0026thinsp;+\u0026thinsp;BC patients directly contributed to trastuzumab resistance through secreted high levels of NRG1 and activated HER3/AKT pathway[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In addition, CAFs could secrete EVs to transfer intracellular components to cancer cells and modify their treatment resistance. CAFs expanded BC subpopulations adept at resisting therapy by utilizing EVs to instigate the juxtacrine NOTCH3 pathway[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In BC endocrine treatment, EVs derived by CAFs administered substantial amounts of onco-miR-221 for fostering hormonal therapy resistance[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Our experimental results showed that CAFs-reduced DOX sensitivity of BC cells was attenuated as a result of the destruction or deletion of EVs in the culture medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), indicating that EVs\u003csup\u003eCAFs\u003c/sup\u003e-mediated delivery of bioactive small molecules played a major role in the CAFs-induced of DOX resistance.\u003c/p\u003e \u003cp\u003eRecent studies have demonstrated that distinct subtypes of CAFs could play varying roles in the tumor microenvironment. A study revealed that four subsets (S1\u0026ndash;S4) of the CAFs were present in BC and metastatic lymph nodes of BC[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. CAF-S1 favored an immunosuppressive microenvironment by secreting CXCL12 and facilitating regulatory T-cell differentiation. Whereas cell cancer migration and invasion were stimulated by CAF-S1-initiated EMT and CAF-S4-activated Notch signaling, respectively. This suggests distinct CAF subpopulations appear to perform different functions in tumor progression. Our data indicated that EVs derived by CAFs/TNBC reduced the sensitivity of BC cells to DOX more significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e), suggesting that the heterogeneity of CAFs might be a key factor promoting drug-resistance phenotypes of BC. Thus, a protein array was used to screen EVs isolated from different CAFs and revealed that DLDH was significantly enriched in EVs secreted by CAFs/TNBC. While decreasing the level of EVs-DLDH, the drug sensitivity reduced by EVs\u003csup\u003eCAFs\u003c/sup\u003e was significantly prevented (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These findings suggested that DLDH in CAF-derived EVs contributed to protecting cancer cells from chemotherapy and that DLDH could be a relevant therapeutic target.\u003c/p\u003e \u003cp\u003eα-KGDC represents a major modulator of ETC activity and TCA cycle flux, and is a pivotal enzyme in the metabolic reprogramming[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and its activation suppressed via the hypoxia-inducible factor 1 (HIF1) could impede tumor growth in vivo [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. DLDH, as its E3 subunit, is involved in the decarboxylation of pyruvate to form acetyl-CoA and reduced NAD to NADH during the cascade of glucose metabolism and mitochondrial ATP production [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Our study revealed that EVs-DLDH was delivered and enriched in the mitochondria of the recipient cells, promoting the activity of α-KGDC and NADH production (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In parallel, the elevated DLDH levels were sufficient to promote OXPHOS without affecting glycolysis in BC cells. DLDH is also a key enzyme of branched-chain α-keto dehydrogenase, α-ketoadipate dehydrogenase, and glycine decarboxylase complexes, hence the dysfunction of DLDH, which simultaneously incapacitates several central metabolic pathways[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Cancer cells tend toward glycolysis metabolism even in the presence of oxygen, or the \u0026ldquo;Warburg effect\u0026rdquo;[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, in some tumors, the stromal cells drive cancer cell mitochondria to conduct OXPHOS when interacting with cancer cells, known as the \u0026ldquo;reverse Warburg effect\u0026rdquo;[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. There is growing evidence to support the role of metabolic alteration in the induction of tumor drug resistance[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Recent studies showed that cancer cells with hybrid glycolysis/OXPHOS phenotype might facilitate metabolic plasticity and be specifically associated with metastasis and therapy resistance[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The present study provides experimental evidence supporting this notion. Moreover, we observed that high DLDH levels increased ATP synthesis and decreased intracellular DOX content (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), which might be associated with the enhanced efflux function of the ATP-binding cassette transporter. As expected, the sensitivity of cells to DOX was significantly attenuated after inhibition of intracellular OXPHOS, demonstrating that CAFs-secreted EVs-DLDH induced the DOX resistance of BC cells due to a shift to OXPHOS metabolism.\u003c/p\u003e \u003cp\u003eEven though chemotherapy is an effective option to treat TNBC, its therapeutic efficacy eventually decreases when BC develops chemo-resistance. Since the local microenvironment might be a crucial cause for increasing chemo-resistance of TNBC, it is imperative that the combination of CAFs intervention measures with traditional chemotherapy may be beneficial to improve TNBC treatment. The present study demonstrated that DLDH was highly expressed in CAFs/TNBC-secreted EVs and its enrichment was negatively correlated with DOX sensitivity in TNBC cells. EVs-delivered DLDH enhanced α-KGDC activity and promoted mitochondrial OXPHOS in TNBC cells, thereby increasing DOX efflux and reducing its accumulation to induce chemo-resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This finding suggests that DLDH in CAFs-secreted EVs contributes to preserving cancer cells from chemotherapy and could be a relevant therapeutic target to overcome DOX resistance in TNBC patients.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study illustrated that CAFs derived EVs in regulating the chemo-sensitive by transporting DLDH, which promoted a shift of TNBC cells from a low- to a high- OXPHOS status. This data also cast a good light on EVs\u003csup\u003eCAFs\u003c/sup\u003e intervention as a mechanism that has significant potential for TNBC therapeutic development.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and gene manipulation\u003c/h2\u003e \u003cp\u003ePrimal cultures of human mammary fibroblasts have been established as described [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In brief, fresh breast tissue was collected from grossly malignant and benign areas of the cut surfaces of the modified radical mastectomy samples. Once resected, the tissue samples were cut into 2 mm\u003csup\u003e3\u003c/sup\u003e pieces, rinsed with the PBS solution, and then digested with 1 mg/ml of Type II collagenase for 2h at 37\u0026deg;C. Following filtration and centrifugation, cell precipitation was collected and seeded in 6-well culture plates. 30 minutes later, the medium was replaced with a fresh medium to remove non-adherent cells to obtain pure fibroblasts. Fibroblasts from malignant areas were termed CAFs, and those of benign regions were termed normal fibroblasts (NFs). The human breast cell lines were purchased from the American Type Culture Collection (ATCC, VA, USA) including ER-positive BC cell lines (MCF-7), and TNBC cell lines (MDA-MB-231 and BT549). All the cells were cultured in DMEM (Gibco, CA, USA) supplemented with 10% FBS (Gibco) and penicillin/streptomycin (Beyotime, China), and tested regularly and negative for mycoplasma. DLDH was ectopically expressed in fibroblasts with low constitutive DLDH. Conversely, CAFs/TNBC cells were transfected with DLDH shRNA and its scramble control (GenePharma, China) to silence DLDH using a transfectamine reagent (ThermoFisher, MA, USA) according to the manufacturer\u0026rsquo;s instructions. BC cells were transfected with ABCB1 or ABCG2 small interfering RNA (siRNA, Santa Cruz Biotechnology, CA, USA) using the reagent following the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEVs isolation and characterization\u003c/h2\u003e \u003cp\u003eFibroblast-secreted EVs isolation was performed as previously described [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Cells were grown at sub-confluence in growth media containing EVs-depleted FBS (prepared overnight ultracentrifugation at 100,000 g at 4\u0026ordm;C) for 18 h. The conditioned medium was then collected and centrifuged at 500 g for 15 min, 2,000 g for 15 min, and 5,000 g for 15 min to remove cells and cell debris, followed by centrifugation at 12,000 g for 30 min for removal of large vesicles. EVs were precipitated by ultracentrifugation at 100,000 g for 90 min at 4\u0026deg;C. The pelleted EVs were collected and resuspended in PBS. To remove contaminated proteins such as cytokines and signaling molecules, the precipitated EVs were finally purified by passing an Amicon Ultra-0.5 tube (Millipore, MA, USA). All steps were performed at 4 ℃. For the characterization of EVs, particle size analysis was performed using a nanoparticle tracking analysis (NTA). The morphology of EVs was observed via transmission electron microscopy (TEM), and the protein content of EVs was determined by western blots. The protein concentration of EVs was measured using a BCA assay (Beyotime).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEVs labeling and trafficking\u003c/h2\u003e \u003cp\u003eTo monitor EV trafficking, EVs were labeled with a red fluorescent dye PKH26 (Sigma-Aldrich, MO, USA). After staining for 5 min, the EVs were washed with PBS and collected by ultracentrifugation (100,000 g for 20 min) at 4 ℃. The GFP-labeled cells were grown on a confocal dish with an EVs-free medium and incubated subsequently with PKH26-labeled EVs for 24 h. Cells were fixed with ice-cold 4% paraformaldehyde for 15 min followed by permeabilization with 0.5% Triton X-100 for 10 min at room temperature. The samples were subjected to probing with the appropriate primary and secondary antibodies. The nuclei were counterstained with DAPI (Cell Signaling Technology, MA, USA). To cleave the membrane proteins of EVs, EVs were pretreatment with trypsin (0.025% EDTA) for 10 min before being labeled with PKH26 fluorescent dye. The fluorescent images were analyzed using a Leica DMi8 microscope using the Thunder Imaging System (Leica Microsystems).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell survival analysis\u003c/h2\u003e \u003cp\u003eMTT and colony survival assays were performed to quantify cytotoxicity after BC cells were treated with EVs, DOX, or combination. For transwell co-culture assays, BC cells were plated into 6-well plates and allowed to adhere overnight in a DMEM medium. Fibroblasts were plated in cell culture inserts using the same medium and placed on top into the same 6-well plates after overnight adhering. The cells were incubated together for 48h before drug treatments. For the MTT assay, the cells were pretreated with EVs or not for 48h and then plated into 96-well plates at 4\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells per well. After 12 h, the medium was replaced by media containing a serial of dilutions of DOX. To inhibit the oxidative phosphorylation pathway, the cells were pretreated with Rotenone (5 \u0026micro;M) or Antimycin A (10 \u0026micro;M) for 3 h prior to DOX treatments. The cells were further cultured for 48 h, and an MTT assay was conducted using a microplate reader (Thermo Multiskan) at 562 nm. The percentage of cell viability at 0.5 \u0026micro;M DOX was calculated using the ratio between treated and untreated cells. For the colony survival assay, 200 cells were plated into 6-well plates and maintained for 24h before EVs and DOX treatment at relating doses corresponding to the MTT assay. The treated cells were cultured for 14\u0026ndash;21 days allowing colony formation. The colonies were stained with 1% crystal violet and then counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTumor formation in zebrafish\u003c/h2\u003e \u003cp\u003eA zebrafish tumor experimental model was established for In vivo study. Before injection, BC cells were incubated with EVs in serum-free media for 3 days. Then, cells were resuspended and stained with lipophilic dyes CM-DiL (ThermoFisher) for 20 min at 37\u0026deg;C. After washing with PBS, PKH 26-labeled cells were collected, washed, and counted. For zebrafish xenotransplantation, 48h post-fertilization (hpf) zebrafish embryos were anesthetized with tricaine (Sigma-Aldrich) and 10 nL of cell suspension (approximately 200 labeled cells) were injected into the perivitelline cavity of each embryo. The injected embryos were incubated at 28\u0026deg;C for 1 h and then turned to 34\u0026deg;C to continue the cultivation, and allowed to recover in the presence of N-phenylthiourea (Sigma-Aldrich) to inhibit melanocyte formation. 24 h after cell injection, 5 \u0026micro;M DOX was added into zebrafish nutrient solution to inhibit tumor growth. 2 days after treatment, the tumors formed in adult fishes were anesthetized with tricaine, imaged using a fluorescent microscope (Nikon, Japan), and quantified using Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eProtein array and immunoblot analysis\u003c/h2\u003e \u003cp\u003eConcentrations of secreted proteins in the EVs were measured using the Human L2000 protein array (RayBiotech, GA, USA) according to the manufacturer's protocol. Whole-cell and EVs lysates were prepared and subjected to Western blot analysis for FN, FAP, α-SMA, and DLDH, respectively, as previously described. Primary antibodies used in this study were FN, FAP, α-SMA, DLDH (Santa Cruz Biotechnology), TOM20, and Vimentin (Cell Signaling Technology). All blots were detected using the enhanced chemiluminescence (ECL, Epizyme, China) with ChemiDoc\u0026trade; XRS\u0026thinsp;+\u0026thinsp;imaging system (Bio-Rad, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eThe pretreated cells were seeded into confocal dishes at 10\u003csup\u003e4\u003c/sup\u003e cells. 12 h later, cells were fixed with ice-cold 4% paraformaldehyde for 15 min, permeabilization with 0.5% Triton X-100 for 10 min, blocked in 5% BSA for 1 h, and probed with primary and secondary antibodies (Cell Signaling Technology) sequentially. The nuclei were counterstained with DAPI according to the manufacturer\u0026rsquo;s instructions. The fluorescence was visualized and captured with a Leica DMi8 microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eThe levels of DLDH were measured using the Human DLDH ELISA Kit (Abbkine, China) according to the manufacturer\u0026rsquo;s instructions. The cell or EVs lysates were added into a 96-well plate coated with a monoclonal antibody against DLDH. After incubation, a substrate solution for the immunoperoxidase reaction was added to develop a color based on the amount of DLDH antigen present in the samples. The reaction was terminated by adding a stop solution. DLDH concentration was quantitated by measuring the absorbance of each well at 450 nm based on the standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical analysis\u003c/h2\u003e \u003cp\u003eThe α-KGDC activity was determined as previously described [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Briefly, sample protein was extracted by grinding harvested cells in liquid nitrogen. The lysate was cleared by ultracentrifugation at 12,000 g for 30min. α-KGDC activity was detected in 1 mL of reaction mixture containing 2.5 \u0026micro;M rotenone and 0.05% Triton X-100 upon addition of 5.0 mM MgCl2, 2.5 mM α-ketoglutarate, 0.1 mM CoASH, 0.2 mM thiamine pyrophosphate (TPP), 1.0 mM NAD+, and 5 \u0026micro;g of protein extract. The increase in absorbance due to NADH synthesis was detected at 340 nm for 5 min. α-KGDC activity was calculated using the extinction coefficient of NADH (ε\u0026thinsp;=\u0026thinsp;6.22 mm-1 cm-1). Sample protein concentrations were determined by the BCA method.\u003c/p\u003e \u003cp\u003eNADH levels were measured using an NADH assay kit from Abcam. Briefly, 10\u003csup\u003e5\u003c/sup\u003e cells were rapidly homogenized with 200 \u0026micro;l ice-cold NADH extraction buffer for 10 min on ice, and the supernatant was collected by centrifuge at 12,000 g for 5 min. Samples were incubated at 60℃ for 30 min to completely decompose the NAD and cooled on ice. Then, 50 \u0026micro;l of each sample was mixed with 100 \u0026micro;l of the reaction mix, incubated at room temperature for 30 min, and then fluorescence was measured in a microplate reader (Ex/Em\u0026thinsp;=\u0026thinsp;535/587 nm).\u003c/p\u003e \u003cp\u003eThe intracellular ATP level of cells was detected with the Luminescent ATP detection assay kit (Abcam) following the instructions. In brief, 10\u003csup\u003e5\u003c/sup\u003e cells were lysed by the detergent provided in the kit, followed by the addition of reconstituted substrate solution and measurement of luminescence with a plate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eOCR and ECAR quantifications\u003c/h2\u003e \u003cp\u003eOxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured using Seahorse XF Analyzer (Agilent, USA). The EVs-treated cells were transferred into a 96-well XF96 plate at a cell density of 5\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well and incubated overnight. Cartridge plates for metabolic stress injections were hydrated for 24 h at 37\u0026deg;C without CO\u003csub\u003e2\u003c/sub\u003e in calibrant solution. 1 h before the assay, the culture medium in the XF96 plate was replaced by a Seahorse Assay medium. OCR was measured under four following conditions: basal, oligomycin, FCCP, and rotenone\u0026thinsp;+\u0026thinsp;antimycin. For quantifying ECAR, 2-DG, an inhibitor of glycolysis is injected to stop glycolytic acidification. OCR and ECAR were normalized by the total protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry analysis\u003c/h2\u003e \u003cp\u003eFor the determination of protein levels, the fixed cells were first incubated with a DLDH antibody (Santa Cruz Biotechnology). Then washed cells were incubated with Alexa 488 anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) conjugate (Cell Signaling Technology). Flow cytometry was performed using a BD FACSMelody instrument (BD, USA). Green fluorescence was excited at 488 nm and detected at 500\u0026ndash;550 nm.\u003c/p\u003e \u003cp\u003eFor intracellular DOX concentration determination, cells were seeded into 6-well plates and pretreated. Then, DOX was added and incubated with cells for 0.5 h or 3 h at 37\u0026deg;C. After cells were digested with trypsin, rinsed with cold PBS, and suspended in 400 \u0026micro;l of PBS, the DOX fluorescence was determined using a flow cytometer. The number of cells collected was 10,000. A gate is set for each sample with a majority of the cells, only excluding small fragments of cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of total and mitochondrial ROS\u003c/h2\u003e \u003cp\u003eThe generation of cellular hydroxyl radical was measured using DCFHDA as described previously [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Briefly, 48h after EVs-cells incubation, cells were labeled by both DCFHDA (2,7-Dichlorodi-hydrofluorescein diacetate, Sigma-Aldrich) at a final concentration of 5 \u0026micro;M in a culture medium at 37℃ for 30 min. Fluorescent images of DCF in the cells were observed using a fluorescence microscope (Olympus, Japan) and then quantified by λex-495nm and λem -520nm using a TECAN Spark fluorescence spectrometer (Tecan, Switzerland).\u003c/p\u003e \u003cp\u003eHydrogen peroxide (H2O2) was fluorometrically detected using an H2O2 Assay (Abcam) according to the manufacturer\u0026rsquo;s protocol. Briefly, 10\u003csup\u003e6\u003c/sup\u003e cells were incubated with EVs for 48 h. The protein lysates from each group were used to measure the cellular content of hydrogen peroxide. The red-fluorescent dye was measured using a microplate reader (Ex/Em\u0026thinsp;=\u0026thinsp;485/535 nm) and absolute amounts of H2O2 were calculated.\u003c/p\u003e \u003cp\u003eThe generation of superoxide anion 48h after each treatment was measured using DHE (Beyotime). In brief, after loading of DHE, the cellular fluorescence in 10\u003csup\u003e5\u003c/sup\u003e cells was analyzed using a spectrofluorometer (λex-385nm, λem -565nm). The amplitudes of fluorescence intensity are presented as the relative fold changes in each group to that of the control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were representative of multiple independent repeat experiments. Unpaired two-tailed Student's t-test was used to analyze the significance between the two groups. One-way analysis of variance (ANOVA) followed by Dunnett\u0026rsquo;s or Bonferroni\u0026rsquo;s test was used to analyze the variance of multiple groups with normal distribution and equal variances. Data were analyzed using GraphPad Prism (USA), and statistical significance was defined as P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBC, breast cancer; TNBC, triple-negative breast cancer; EVs, extracellular vesicles; DOX, doxorubicin; TME, tumor microenvironment; CAFs, cancer-associated fibroblasts; EMT, epithelial-to-mesenchymal transition; NTA, nanoparticle tracking analysis; TEM, transmission electron microscopy; NFs, normal fibroblasts; FAP, fibroblast activation protein; FN, fibronectin; \u0026alpha;-SMA, \u0026alpha;-smooth muscle actin; TCA cycle, tricarboxylic acid cycle; \u0026alpha;-KGDC, \u0026alpha;-ketoglutarate dehydrogenase complex; DLDH, dihydrolipoamide dehydrogenase; hpf, hour post-fertilization; ECL, enhanced chemiluminescence; ROS, reactive oxygen species; H2O2, hydrogen peroxide; OXPHOS, oxidative phosphorylation; OCR, oxygen consumption rates; ECAR, extracellular acidification rates; ATP, adenosine triphosphate; ABC transporter, ATP-binding cassette transporter; ETC, electron transport chain\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eThe Research Committee and the Institutional Animal Care of and Use of Nanjing University Medical School have approved animal studies conducted in this study.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eAvailability of supporting data\u003c/h2\u003e\n\u003cp\u003eAll materials made and datasets analyzed in this study are available for reasonable requests to the corresponding author, Dr. Juan Li.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eNo potential competing interest were disclosed.\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026apos; contributions\u003c/h2\u003e\n\u003cp\u003eConception and design: ZX and JL; Development of methodology: ZX, YZ, FF and RS; Acquisition of data: ZX; Analysis and interpretation of data: ZX and HM; Administrative, technical, or material support: ZX, JH and JL; Writing, review, and/or revision of the manuscript: ZX, RS and JL; Study supervision: RS and JL. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (No. 31371399) to J Li.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026apos; information\u003c/h2\u003e\n\u003cp\u003ePhase I Clinical Trials Unit, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, 210008, China. Zhi Xu, Hehua Ma, Yu Zhao, Fei Fei, Runbin Sun, Juan Li\u003c/p\u003e\n\u003cp\u003eDepartment of General Surgery, the First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, P. R. China. Junchen Hou\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBianchini G, De Angelis C, Licata L, Gianni L. 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Cancer-associated fibroblasts (CAFs)-derived extracellular vesicles (EVs) were critical for tumor progression. Herein, we demonstrated that CAFs/TNBC-derived EVs could suppress doxorubicin (DOX) sensitivity in breast cancer both in vitro and in vivo. The protein array revealed that dihydrolipoamide dehydrogenase (DLDH) was enriched in CAFs/TNBC-derived EVs, which was the E3 component of the 2-oxoglutarate dehydrogenase complex (α-KGDC). EVs-DLDH was transported into mitochondria and enhanced mitochondrial respiration through increasing α-KGDC activity and NADH content. Inhibiting DLDH reduced oxidative phosphorylation (OXPHOS) and CAFs-derived EVs-induced drug resistance in the recipient cells. It was also shown that the EVs-reduced sensitivity of DOX was due to increased drug efflux driven by OXPHOS. Additionally, suppression of ATP-binding cassette transporters or mitochondrial respiration conferred the recipient cells with increased susceptibility to DOX. These results elaborated that CAFs-derived EVs inhibit the DOX sensitivity of TNBC through increasing drug efflux driven by DLDH-induced OXPHOS. Inhibiting EVs-DLDH provides a potential therapeutic application to enhance the responsiveness to chemotherapy in TNBC.\u003c/p\u003e","manuscriptTitle":"DLDH-containing extracellular vesicles from CAFs reduced DOX sensitivity in triple-negative breast cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-30 19:41:15","doi":"10.21203/rs.3.rs-2262675/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9b648179-0803-4317-8be7-4e328b90bd76","owner":[],"postedDate":"November 30th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":17282934,"name":"Biological sciences/Cancer/Breast cancer"},{"id":17282935,"name":"Biological sciences/Cancer/Cancer therapy/Cancer therapeutic resistance"},{"id":17282936,"name":"Biological sciences/Cancer/Cancer microenvironment"},{"id":17282937,"name":"Biological sciences/Cancer/Cancer metabolism"}],"tags":[],"updatedAt":"2023-08-02T11:53:23+00:00","versionOfRecord":[],"versionCreatedAt":"2022-11-30 19:41:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2262675","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2262675","identity":"rs-2262675","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}