Effect of siRNA-mediated LGR5 Inhibition on the Expression of Stemness-related Genes in Triple-negative Breast Cancer Spheroid Model.

INTRODUCTION
Breast cancer (BC) is a major health concern worldwide, accounting for approximately 24.5% of all cancer cases and 15.5% of cancer deaths among women.[1] Within this category, triple-negative breast cancer (TNBC) constitutes 10%–20% of BCs and is characterized by its high recurrence rate, propensity for distant metastases, and lower overall survival rates compared to other BC subtypes.[2] TNBC’s aggressive nature and lack of targeted therapeutic options leave chemotherapy as the primary treatment strategy.[3] Despite a relatively high initial response rate to chemotherapy, TNBC patients often face poor long-term outcomes, with a significant risk of relapse and development of chemoresistance, particularly in metastatic cases.[4] Therefore, understanding the molecular mechanisms underlying TNBC’s resistance to treatment and relapse is crucial for developing new treatment approaches.
Cancer stem cells (CSCs), or tumor-initiating cells, possess stem-like qualities that are central to cancer’s ability to grow and spread. Evidence suggests that CSCs are key contributors to chemoresistance and cancer recurrence due to their capacity for self-renewal and differentiation into various cancer cell lineages upon chemotherapy exposure.[5] Furthermore, chemotherapy itself has been shown to promote CSC proliferation.[6] The Wnt/b-catenin signaling pathway, known for its role in maintaining CSCs and its aberrant activation in cancer development, is significantly implicated in TNBC.[7] It is associated with tumor growth, spread, and chemotherapy resistance due to its influence on cell stemness and survival.[8] Thus, targeting the Wnt/b-catenin pathway and its regulatory genes presents a promising approach for creating novel therapeutic strategies against TNBC, aiming to overcome the limitations of current treatments.
The leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), also recognized as GPR49, belongs to the expansive family of G protein-coupled receptors and is identified as a gene activated by the Wnt pathway.[9] Significantly, studies have highlighted LGR5’s involvement in advancing tumor development, likely via mutations that activate the Wnt signaling pathway.[10] LGR5 amplifies the beta-catenin/Wnt signaling, which in turn promotes the proliferation and self-renewal capabilities of CSCs.[11] It is noteworthy that LGR5 is overexpressed in a variety of cancers, including TNBC, hepatocellular carcinoma (HCC), colorectal cancer, BC, and ovarian cancer, serving as a specific indicator of CSCs presence.[12] Furthermore, a link between elevated LGR5 levels and reduced survival rates in TNBC patients has been observed.[13] Recent studies also underline the association between LGR5 expression and chemotherapy resistance. For instance, Ma et al. illustrated that LGR5 can initiate tumor growth, enhance cell mobility, and trigger epithelial-mesenchymal transition (EMT) in HCC, thus fostering resistance to doxorubicin.[14] Similarly, Clark-Corrigall et al. found a connection between LGR5 expression and increased chemotherapy resistance in neuroblastoma.[15] These findings collectively suggest a pivotal role for LGR5 in both cancer genesis and resistance to chemotherapy, although the specific mechanisms of LGR5’s action in TNBC remain to be fully understood.
In light of the pivotal role of LGR5 in cancer, the exploration of innovative therapeutic strategies becomes imperative. Nowadays, siRNA (small interfering RNA)-based drugs have become a hot topic in anticancer drug therapy. Despite the potential of siRNA to silence specific genes involved in cancer pathology, its clinical application faces significant obstacles, including a short circulating half-life, limited ability to penetrate cell membranes, and inefficient release from endosomes.[16] In this context, exosomes, naturally occurring nanoparticles released by various cell types that facilitate intercellular communication, have garnered attention as potential vehicles for siRNA delivery. Their biocompatibility, ability to evade immune detection, and resistance to lysosomal and enzymatic breakdown make exosomes ideal candidates for ferrying siRNA directly to cancer cells. This innovative approach leverages exosomes’ natural roles to overcome the delivery challenges associated with siRNA-based drugs, opening new avenues for targeted cancer therapy.[17]
Cancer is a highly heterogeneous disease with a complex and dynamic tumor microenvironment. The tumor microenvironment plays a vital role in the initiation and progression of cancer.[18] In BC, studies focusing on the interaction between cancer cells and the tumor microenvironment have highlighted the important role of stromal components such as cancer-associated fibroblasts.[19] Consequently, clinical models that mimic tumor biology in vivo are critical for accurate assessment of therapeutic toxicity and efficacy. Therefore, multicellular tumor spheroid (MCTS) models have been designed with different types of cells, including fibroblasts, because they better mimic tumor characteristics in vivo. MCTS are a collection of tumor cells that grow simultaneously with other types of cells in a suspension or scaffold in a three-dimensional environment that closely mimics the natural cell microenvironment.[20] As a result, the expression profiles generated by MCTS are similar to physiological conditions in humans, which are suitable for experimental evaluation.
Considering the importance of the LGR5 gene in chemoresistance and its role in promoting the activation of Wnt/b-catenin signaling, the present study was conducted to investigate the suppressive effect of the LGR5 on the three-dimensional (3D) multicellular spheroid models of TNBC cell line compared to the effect of the chemotherapy drug by examining the expression of stemness markers. We also investigated whether exosomes could serve as a carrier for siRNA delivery. Furthermore, our results showed that the use of the chemotherapy drug alone leads to an increase in the expression of SOX9, OCT4, and NANOG. However, inhibiting the LGR5 leads to a significant decrease in SOX9 and OCT4 genes. In addition, our results showed that exosome is a suitable carrier for siRNA delivery. This suggests that LGR5 plays a critical role in regulating specific stemness-associated genes in TNBC, highlighting the potential therapeutic benefits of targeting LGR5 in this aggressive cancer subtype.

MATERIALS AND METHODS

Cell culture
MDA-MB-231 TNBC cell line and human foreskin fibroblast cell line (HFFF2) were purchased from the Iran Biological Resources Center. HFFF2 and MDA-MB231 cell lines were grown in high-glucose Dulbecco’s modified eagle medium (DMEM) (Gibco, UK). This medium was supplemented with 10% fetal bovine serum (FBS) (Bioidea, Iran) and 1% penicillin-streptomycin (Bioidea, Iran). The cells were then incubated at 37°C in a CO2 incubator with a 5% CO2 environment.

Spheroid formation
The liquid overlay technique was used to generate 3D multicellular spheroids from MDA-MB-231 and HFFF2 fibroblast cell lines.[21] In this method, agarose coating prevents cell attachment to culture flasks and promotes cell-to-cell attachment. Briefly, a 1% agarose solution was prepared in a DMEM culture medium. Next, the prepared solution was sterilized in an autoclave. Next, the wells of 96 microplates were covered with agarose solution. Time was given for solidification. The MDA-MB-231 and HFFF2 cell lines were separated from the flask with 2 mL trypsin-0.5% EDTA (Bioidea, Iran) for co-culture to prepare multicellular spheroids. The MDA-MB-231 and HFFF2 cell lines were seeded at a 1:1 ratio in a low-attachment 96-well plate, precoated with a 1% agarose solution, to promote spheroid formation and incubated at 37°C in a humidified incubator with 5% CO2 atmosphere. At the end of the third day, the cell culture medium was refreshed. Spheroids were allowed to form over 7 days until reaching a minimum diameter of 500 mm.

Exosomes isolation
Adipose tissues were collected from the voluntary donors who were admitted to the surgery ward for liposuction surgery. Strict informed written consent was obtained from the donor patients. Separation of human adipose mesenchymal stem cells (ADMSCs) was performed as described previously.[18] The Ethics Committee of Isfahan University of Medical Sciences approved all methods. Briefly, to isolate mesenchymal stem cells from adipose tissue, subcutaneous fat samples were first prepared from several people with the same conditions who were hospitalized for abdominal fat liposuction. The samples were transferred to the laboratory. Subsequently, collagenase (Bioidea) enzyme was added to the sample and placed in the incubator for 30 minutes. Next, the enzyme was neutralized by DMEM culture medium (Bioidea) and then centrifuged at 1500 rpm. Next, the cell mass was cultured in a 25-mL flask. To extract exosomes from the cell culture medium of ADMSCs, stem cells were starved in serum-free media for 48 hours. After that, the culture media overlay was collected and centrifuged for 5 minutes at 300 g. The supernatant was passed through a 0.2-mm syringe filter and then used for exosome isolation. The manufacturer’s instruction manual for the Exo-spin™ kit (Cell Guidance Systems, UK) was followed to separate the exosomes. Briefly, the precipitation buffer provided in the kit was mixed with pre-centrifuged (30 min at 14,000 g) cell culture medium in a 2:1 ratio and incubated overnight at 4°C in a refrigerator. The samples were spun at 20,000 g for 40 minutes the next day, and the pellet was suspended in 100 µL of PBS after the supernatant was discarded. Additional purification of the sample was performed using the provided columns. For the final elution of exosomes, 200 µL of cold sterile PBS was used.

EXOSOMES CHARACTERIZATION

Electron microscopy
A transmission electron microscope (TEM) was used to study the exosomes’ morphology. Briefly, the refined exosomes were first fixed in 2% paraformaldehyde (PFA) for 30 minutes. A tiny droplet of exosome suspension was placed on the coated copper grid and left for 2 minutes at room temperature. Uranyl acetate was used to stain and then air-dried for a few minutes. The grid was then examined with TEM (Philips CM30, Eindhoven, Netherlands) and photographed.

Dynamic light scattering
The size and zeta potential measurements of exosomes were calculated by dynamic light scattering (DLS) using a Nano Zeta sizer (Malvern Instruments Ltd, UK). To calculate DLS, 50 mL of exosomes were first diluted in PBS. Next, it was transferred to the Nano Zeta sizer, and the results were analyzed.

Bradford assay
The Bradford method was used to calculate the total proteins in the exosome. First, exosomes were digested using radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, USA). Then, the prepared solution was incubated for 15 minutes at 70°C. Next, Bradford’s reagent was mixed with the solution. After that, it was incubated for 15 minutes at room temperature in the dark. After 15 minutes, an ultraviolet-visible spectrophotometer was used to measure the solution’s absorbance at 595 nm.

siRNA transfection
Short-interfering RNA (siRNA) oligonucleotides targeting LGR5 and scrambled siRNA were acquired from Applied Biological Materials (ABM) (Richmond, Canada). Using Lipofectamine RNAiMAX (Life Technologies), transfection followed the provided guidelines. Six spheroids were used for each experiment. In DMEM with 10% FBS, cells were cultured to form spheroids. A mixture of 6 mL siRNA and 2 mL Lipofectamine 2000 in 600 mL DMEM was incubated for 20 minutes. Aliquots of this solution were then added to each well. Spheroids were transfected with scrambled siRNA and used as the negative control. Analysis occurred 48 hours post transfection.

siRNA loading into exosomes
LGR5 siRNA was encapsulated in the exosomes by using Lipofectamine RNAiMAX transfection reagent (Life Technologies) following the manufacturer’s indications. The formulation including 18 mL exosomes and 6 mL siRNA was mixed with Lipofectamine and incubated for 4 hours at 37°C. Aliquots of this solution were then added to each well. Free siRNAs were omitted using size exclusion chromatography (Exo-spin™ mini-column).[22]

Determination of the amount of siRNA incorporated in exosomes
To estimate the amount of siRNA loaded in exosomes derived from MSC, first, the initial siRNA concentration for binding was determined by measuring absorbance at 260 nm (Abs260) by nanodrop. After loading the exosomes, the free siRNA was removed with the help of a chromatography column. Then, the exosomes were destroyed with the help of RIPA buffer, and the siRNA inside was released into the environment. Next, again, the absorbance was measured at 260 nm, and by calculating the observed difference in concentration, the percentage of siRNA loading was calculated.[23]
Encapsulated percentage (%) =100 − Abs 260 after size exclusion chromatography/Abs260 before size exclusion chromatography × 100

Drug treatment
The spheroids were treated with cisplatin. After treatment for 48 hours, the expression of SOX9, NANOG, and OCT4 was checked by the real-time method. The half-maximal inhibitory concentration (IC50) for cisplatin in 3D spheroid models was investigated by the MTT method and determined by drawing a linear regression curve and was used to load solutions in the treatment of spheroids. The IC50 value for the cisplatin drug was calculated to be 25.55 mM.[24]

RNA isolation and RT-qPCR
The treated multicellular spheroids were lysed 48 hours post treatment for the quantitative RT-PCR assay of each group. Total RNA was extracted using TRIzol reagent (Invitrogen, USA) in accordance with the manufacturer’s procedure. The OD 260/280 spectrophotometry (Synergy H1M, BioTek instruments) (BioTek, USA) was utilized to quantify the quantity and quality of extracted RNA. Using a reverse transcriptase kit (TaKaRa, Osaka, Japan) and random hexamer primers, 2 mg of extracted RNA was utilized to synthesize cDNA. Using a 25-mL reaction volume and a SYBR Green Master Mix (Roche), real-time q-PCR was evaluated in accordance with the manufacturer’s instructions. First, denaturation took place at 95°C for 5 minutes. This was followed by 40 cycles of denaturation, which included 20 seconds at 94°C, 20 seconds at 60°C, and 20 seconds at 72°C. Table 1 lists the primer sequences that were utilized to amplify the target genes. As an internal control for the normalization of expression data, GAPDH was employed. Using the 2 − DDCt method, the relative expression between groups was computed.

Statistical analysis
The tests were run in triplicate and independently three more times. For all statistical analyses, SPSS 21.0 (IBM Corp. Armonk, NY, USA) was utilized. All group comparisons were done with a one-way analysis of variance (ANOVA). Values of P < 0.05 were considered to represent statistically significant outcomes in all cases.

RESULTS

Spheroid formation
MDA-MB-231 cell lines with fibroblasts (HFFF2 cell line) were capable of forming spheroids. The spheroids formed on day 5, with increased diameter on day 7 [Figure 1]. After 7 days, the spheroids were used for the experiment.

Exosome isolation and characterization
Exosomes were extracted from ADMSCs.[24] According to the results of TEM imaging, the vesicular morphology of exosomes with a diameter of less than 200 nm was confirmed [Figure 2]. In addition, according to the results of DLS and zeta potential, the exosomes were approximately 155 nm in diameter [Figure 3a], and the zeta potential was approximately −4.5 mV [Figure 3b]. The protein content of exosomes was 3800 mg/mL based on Bradford test results.

siRNA loading
LGR5 siRNA was encapsulated in the exosomes by using the transfection method. After removal of the unloaded siRNA by the Exo-spin™ column, measuring absorbance at 260 nm (Abs260) by nanodrop revealed that approximately 45 ± 0.5% of siRNA was loaded into exosomes.

LGR5 mRNA level was reduced via siRNA-LGR5
To investigate the effective suppression of LGR5, we treated the 3D multicellular spheroids MDA-MB-231 cell line, and after 48 hours, the mRNA level of the LGR5 gene in the studied groups using the real-time PCR method. The results of LGR5 expression in the studied groups are shown in Figure 4a. According to our results, LGR5 siRNA can effectively suppress LGR5 expression. LGR5 gene expression in the si-LGR5 group and EXO-si-LGR5 groups significantly decreased compared to the control groups. The exosome demonstrated efficient delivery of LGR5, as demonstrated by a statistically significantly greater decrease in LGR5 mRNA levels in the EXO-si-LGR5 group than in the other groups. Considering that the amount of siRNA loaded in the exosome was only 50%, its performance was more effective.

Silencing of LGR5 reduces the expression of CSC-related genes
It is well known that genes associated with CSCs play an important role in the stemness of CSCs. Using the real-time PCR method, the expression level of SOX9, OCT4, and NANOG genes in the studied groups was checked 48 hours after treated multicellular spheroids MDA-MB-231 cell line, and the results are shown in Figure 4b–d. In general, the results showed that chemotherapy of multicellular spheroids with cisplatin causes a significant increase in the expression of all three genes. This finding shows that chemotherapy can induce CSCs state. On the contrary, our results showed that in the si-LGR5 group and the EXO-si-LGR5 group compared to other groups, only SOX9 expression and OCT4 expression decreased in the EXO-si-LGR5 group, and no significant difference was observed in NANOG, thus suggesting that LGR5 might play a prominent role in the stemness.

DISCUSSION
Chemoresistance poses a significant challenge in treating TNBC and often leads to a more aggressive or metastatic form. Although chemotherapy is a primary strategy for eliminating tumor cells, the persistence of chemo-resistant cells, known as minimal residual disease (MRD), along with processes such as EMT that promote CSC induction, contribute to treatment failure and an increased risk of cancer recurrence. Research has consistently shown that the survival of CSCs underlies cancer recurrence and resistance to chemotherapy.[25] Studies by Oliveira et al.[26] have demonstrated that inhibiting the Wnt signaling pathway in TNBC cell lines reduces CSC markers and enhances chemotherapy responsiveness. Moreover, the CSC marker LGR5 has been implicated in increasing cancer cell mobility, tumor growth, and metastasis in TNBC by activating the beta-catenin/Wnt signaling pathway.[27] This highlights the critical need for comprehensive research aimed at developing and refining new treatment modalities that can effectively target drug-resistant cells, thereby enhancing the overall efficacy of cancer therapies.
Our findings demonstrate that siRNA, both in its free form and encapsulated within the exosomes, effectively targets and suppresses the LGR5 gene. This gene has been identified as playing a significant role in both treatment resistance and the initiation of tumors.[28] Research by Lin et al.[29] supports this conclusion, revealing that LGR5 inhibition via siRNA not only decreases b-catenin levels in colon cancer cells but also inhibits their proliferation. This suggests that LGR5’s activity is essential for the growth of colorectal cancer cells, aligning it as a potential novel target for therapeutic intervention. Furthermore, LGR5 has been implicated in modulating the chemotherapy sensitivity of nasopharyngeal carcinoma (NPC) cells. Studies indicate that NPC cells with elevated LGR5 expression exhibit enhanced tumorigenic capabilities and increased expression of stem cell markers. Importantly, LGR5 silencing markedly reduces cisplatin resistance in CNE1 cells, concurrently leading to a diminished expression of genes associated with stemness. These results underscore the therapeutic potential of targeting LGR5 to overcome drug resistance and impair the propagation of cancer cells.[30]
In addition, it was shown that delivering siRNA via exosomal formulation significantly enhances its effectiveness compared to traditional transfection methods for free siRNA transfer. The field of siRNA-based drugs has gained considerable attention for its potential in anticancer therapy.[31] Nonetheless, the efficient cellular uptake of siRNAs presents challenges, including their rapid degradation in the bloodstream, limited ability to cross cell membranes, and ineffective release from endosomes, highlighting the need for innovative delivery mechanisms to achieve successful gene silencing.[32] Exosome-mediated gene delivery has emerged as a promising strategy for transporting siRNA into cancer cells. Exosomes, equipped with specific membrane proteins, are naturally adept at facilitating cellular entry, thereby addressing some of the main obstacles in siRNA delivery.[33] Studies such as Zhao et al.’s[34] have highlighted the advantages of exosome-based delivery systems, demonstrating superior stability and efficacy of siRNA targeting crucial genes involved in metastasis within TNBC models. Furthermore, research by Liu et al.[35] revealed that exosomes loaded with DARS-AS1 siRNA not only significantly impede TNBC cell proliferation but also reduce liver metastasis, suggesting their potential utility as siRNA carriers in clinical settings. These findings point toward the viability of exosomes as a method for enhancing the delivery and therapeutic impact of siRNA in cancer treatment, offering new avenues for combating drug resistance and tumor progression.
Our investigation into the expression profiles within 3D cell culture models has highlighted their enhanced capacity for mirroring the tissue-specific markers, functionalities, and gene expressions distinct from those observed in traditional 2D cultures. In this study, we cultured cancer cells alongside fibroblast cells in a 3D environment to better replicate the tumor microenvironment, including cell-cell interactions and the ECM, thereby providing insights more aligned with in vivo conditions.[36]
In addition, the findings revealed that treatment with cisplatin led to a significant increase in the expression of the SOX9, OCT4, and NANOG genes compared to both the control group and those treated with siRNA targeting LGR5. These genes are recognized as pivotal markers of CSCs, playing crucial roles in regulating cell proliferation and differentiation. Their expression is considered indicative of a potential for poor prognosis across various cancers, including BC, underlining their value as biomarkers for disease progression and response to treatment.[37] To support our findings, research conducted by Voronkova et al.[38] demonstrated a notable increase in SOX9 levels in non-small cell lung cancer (NSCLC) cells following cisplatin administration, establishing a link between elevated SOX9 expression and decreased overall survival rates among lung cancer patients. It was also discovered that diminishing SOX9 levels could heighten the sensitivity of cells to cisplatin, while its overexpression tends to strengthen resistance to the treatment. In a similar investigation, Huang et al.[39] observed a post-cisplatin treatment surge in OCT4 expression within bladder cancer cells, with a specific increase among the CD44+ cell population expressing OCT4. This observation indicates that cisplatin therapy may unintentionally favor the proliferation of cells with stem cell-like properties, thus promoting an environment conducive to chemotherapy resistance. Furthermore, the work of Wiechert et al.[40] in the context of ovarian cancer reveals that CSCs resistant to cisplatin and expressing NANOG are more adept at combating the drug. Interestingly, cisplatin therapy was found to provoke a CSC-like phenotype in cells previously lacking NANOG expression, suggesting that cisplatin itself may play a role in encouraging chemoresistance by promoting CSC-like qualities. Collectively, these findings underscore the intricate challenges of cancer therapy and highlight the urgent need for targeted treatments that can effectively target and neutralize the CSC population, thereby enhancing the success of therapeutic interventions.
Notably, our findings indicate that silencing the LGR5 gene via siRNA effectively reduces the expression of SOX9 in both the si-LGR5 and EXO-si-LGR5 groups, and similarly decreases OCT4 expression in the EXO-si-LGR5 group, without significantly impacting NANOG levels. This supports the concept that exosome-mediated siRNA delivery is superior. The limited changes observed may be attributed to the transient nature of gene inhibition, suggesting that extended silencing might unveil more significant alterations. Another consideration is the specific reduction in SOX9 expression, which plays a pivotal role in defining mammary stem cell identity in both healthy and cancerous breast tissue.[41] The accumulation of SOX9 in the cytoplasm is known to markedly enhance the proliferation and metastatic potential of ductal carcinoma in human BC.[42] The importance of SOX9 as a key regulator of TNBC growth, survival, and metastasis has been underscored in multiple studies. Suppressing SOX9 expression has been shown to lead to TNBC cell death, alongside a decrease in invasiveness in vitro, and a reduction in tumor growth and metastasis in vivo.[43] Ma et al.’s[44] research further demonstrated SOX9’s role in promoting the growth and invasion of TNBC cells, with the knockdown of SOX9 linked to diminished breast tumor growth and lung metastasis in animal models. These insights highlight the critical role of SOX9 in TNBC pathogenesis and its potential as a therapeutic target. OCT4’s overexpression in TNBC is linked to poorer clinical outcomes, highlighting its significance in the disease’s pathology.[5] Interestingly, research by Jin et al.[45] revealed that OCT4 might play a dual role by suppressing EMT in both TNBC and luminal cancers, suggesting a complex function in regulating stemness and cell renewal. Similarly, NANOG has been implicated in enhancing stemness and correlating with adverse outcomes in TNBC. However, a subsequent study by Nagata et al.[46] presented a paradox, indicating that NANOG might actually serve as a favorable prognostic marker for TNBC. This contradiction suggests that NANOG’s reliability as a biomarker for TNBC is questionable, likely due to poorly understood mechanisms in chemoresistance induction. Further complicating the landscape, Jha et al.[47] demonstrated that silencing the LGR5 gene does not alter NANOG expression, adding another layer to the complex interaction between these markers and TNBC.
In recent years, research in nanomedicine has been directed toward the use of combined treatments (chemotherapy and gene therapy) in a single nanoparticle such as an exosome as an important cancer treatment option.[4849] The simultaneous use of drugs and siRNAs effectively interferes with the activity of nuclear DNA, thus significantly inhibiting the growth of tumors and reducing side effects, and can produce synergistic effects against cancer.[50] Given these findings, our study proposes assessing the synergistic effect of combining chemotherapy drugs with LGR5 siRNA as a strategy to enhance treatment efficacy in 3D multicellular spheroids of the MDA-MB-231 TNBC cell line. This approach aims to target both the LGR5 pathway and chemoresistance mechanisms simultaneously, potentially providing a more effective therapeutic option for TNBC patients.

CONCLUSION
Our study showed that the use of LGR5 siRNA is an effective method to downregulate LGR5 expression. Cisplatin also increased the expression of genes involved in stemness on the 3D multicellular spheroids model of the TNBC cell line, while inhibition of LGR5 expression resulted in inhibition of genes that were involved in stemness and drug resistance. Therefore, LGR5 knockdown can be considered as a possible strategy to combat chemoresistance. In addition, our results showed that exosome is a suitable carrier for siRNA delivery. These findings suggest that LGR5 can be used as a promising molecular target for the development of TNBC therapies.

Ethics approval and consent to participate
The experimental procedures had already been confirmed by the Isfahan University Ethics Committee (IR.MUI.MED.REC.1399.979).

Conflicts of interest
There are no conflicts of interest.