Fluid shear stress-mediated upregulated exosomal-IGF2 promotes hepatocellular carcinoma cell migration and metastasis

Abstract

Exosomes, as important intercellular message transporters, can be secreted by hepatocellular carcinoma (HCC) cells and transported to adjacent cells, thus promoting their migration and invasion in turn. However, whether the exosomes secreted by HCC are affected by physical abnormalities, such as fluid shear stress (FSS), is still largely unknown. Here, we observed that 1.4 dyn/cm² FSS could significantly increase the release of exosomes by up-regulating Rab27a and down-regulating Rab7 in HCC cells. Exosomes from FSS-induced HCC cells were more effective at encouraging recipient cell migration and invasion. Exosomes produced by static or FSS-stimulated cells were thoroughly analyzed using quantitative proteomics, and more than 1000 exosome proteins were found. Based on the differentially expressed proteins, IGF2, a potential migration-related protein, was discovered to be strongly expressed in FSS-stimulated cells, HCC tissues, as well as HCC patient-derived exosomes. Furthermore, we verified that exosomal IGF2 aggravated HCC migration and invasion via activating Ras/Raf/Erk signaling in recipient cells. Collectively, our data demonstrated that exosomes from FSS-stimulated HCC cells promote recipient cell migration through IGF2-Ras/Raf/Erk signaling, which might serve as potential targets for both cancer treatment and cancer prevention.

Introduction

Hepatocellular carcinoma (HCC) is an aggressive malignancy that poses a nonnegligible threat to human health and life, with the second mortality ranking [1]. Therefore, exploring the migration and metastasis mechanism of HCC, restraining its further development, and finding effective intervention targets have become the key to halting HCC. Existing studies have shown that tumor development is a precise process mediated by multiple factors, in which the tumor microenvironment (TME) plays a key role [2]. The TME exhibits several biological and physical anomalies, which can be generally regarded as an indicator of cancer occurrence [3]. Elevated interstitial fluid pressure from the leaky blood arteries and lack of functional lymphatic capillaries in the tumor’s biomechanical environment leads to aberrant interstitial flow and increased fluid shear stress (FSS) [4]. Studies have shown that cancer cells in the tumor microenvironment are often exposed to FSS ranging from 0.01 to 0.2 Pa (0.1 dyn/cm² to 2 dyn/cm²) [5]. 2 dyn/cm² FSS markedly upregulated the expression of matrix metalloproteinase-12 (MMP-12) and promoted the invasion of chondrosarcoma [6]. Our prior research showed that 0.1-2.0 dyn/cm² FSS stimulates EMT or induces autophagy, which encourages HCC cell migration and invasion [7,8]. However, whether FSS can affect the intercellular communication in the microenvironment of HCC thus altering cancer cells’ behavior still needs further investigation.

Exosomes are extracellular vesicles with diameters from 30 to 100 nm that are produced in multivesicular bodies, followed by fusion with cytomembranes and thus secretion to the extracellular milieu [9]. A variety of types of cells can create exosomes, which carry cargoes such proteins, lipids, and nucleic acids as messengers in intercellular communication [10]. TSG101, Hsp70, and CD9 are specific proteins that are substantially abundant in exosomes and are typically regarded as markers to identify exosomes [11]. The formation and secretion of exosomes involve a series of elaborate biological processes, of which the Rab family plays an important role, including Rab27a/b, Rab7, and Rab5a/c. Rab family, a member of small GTPases, has been identified as regulating the transport and movement of intracellular vesicles. Additionally, to attaching to the cell’s plasma membrane and releasing exosomes into the extracellular area [12,13]. Recently, scholars have investigated that cancer cell-released exosomes could absorbed by nearby recipient cells, thus reshaping their biological function [14,15]. FSS, as an important component of the tumor mechanical microenvironment, is widely present in solid tumors [3], but there is still limited research regarding their impact on exosomes.

IGF2, a widely recognized factor that controls cell growth and movement in solid tumors, has been discovered to be increased in many types of human cancers and is linked to a negative prognosis [16]. In cases of liver cancer, the upregulation of IGF2 in cancer may be partially attributed to the loss of imprinting, or re-activation of IGF2 transcription [17]. The specific roles and processes of IGF2 release in the physical tumor microenvironment are still not understood. It is necessary to clarify if the exosomes release the abnormal expression of IGF2 under FSS and if it can impact cancer cells in this particular setting.

Here, we hypothesized that FSS could regulate the secretion of exosomes in HCC reshape the protein contents in exosomes, and further promote the migration ability of HCC cells through transporting the biofunctional contents in exosomes. Thus, we investigated the secretion of exosomes under FSS condition and their roles in HCC cell migration and invasion. Proteomics using mass spectrometry was subsequently conducted to uncover the crucial proteins that exhibited differential expression. Furthermore, we explored the molecular mechanisms of exosomal-IGF2-mediated cancer cell migration and invasion.

Materials and methods

Cell cultures

HepG2 and QGY-7703 cell lines derived from human hepatocellular carcinoma (HCC) were procured from the Institute of Cell Biology and Biochemistry (Shanghai, China) and culture at RPMI-1640 medium (Gibco, USA), with 10% fetal bovine serum supplementary (FBS, Gibco, USA) and 100 units/mL each of penicillin and 100 g/mL streptomycin (Hyclone, USA). Both cell lines were maintained in a controlled incubation environment at 37°C with a 5% CO₂ atmosphere. Prior to experiments, their identities were validated through short tandem repeats (STR) profiling.

HCC patients’ tissue and plasma samples

HCC tumors, and paired adjacent normal tissues, as well as normal subjects and liver cancer patients’ plasma samples, were gathered from West China Hospital, Sichuan University, with the participants’ full comprehension and written consent, and the medical review board gave the study their approval (no.2016120). The whole procedure followed the guidelines outlined in the Helsinki Declaration.

FSS loading

0.25% trypsin (Hyclone, USA) was used to enzymatically separate HepG2 and QGY-7703 cells, and they were subsequently resuspended in culture media containing 10% FBS. In the polystyrene tissue culture plates, cells were planted on the sterilized glass slide (24 mm × 75 mm) at a density of 1.0 × 10⁵ cells/mL. Cells were subjected using a parallel flow chamber to 1.4 dyn/cm² FSS up until 90% confluence on the glass slides [18,19]. As a control, static-grown cells without FSS activation were used.

Isolation and purification of exosomes

The method of exosome isolation and purification was described previously [20]. Exosomes secreted by HCC cells under static culture conditions, which served as a baseline control, and a group without exosome treatment (PBS only). In brief, in the appropriate complete exosome-free media, cells were grown in monolayers for 48 hours. Cells were then pretreated either with or without FSS for 1 h, 4 h, and 8 h, respectively, and the circulation medium was collected for exosome isolation. To separate dead cells and other cell debris, exosomes were isolated by a differential centrifugation method, which is 300×g (Centrifuge 5804R; Eppendorf, Hamburg, Germany) for 20 min, and then 3000×g for 20 min. This was followed 10,000×g for 30 min. After that, a 0.22 μm filter (Millipore, Billerica, MA, USA) was used to filter the supernatant. Exosomes were finally in the supernatant and get pelleted by ultracentrifuge (Optima L-80 XP, Beckman Coulter, Brea, CA, USA) at 100,000×g for 2 hours.

Transmission electron microscopy (TEM)

Exosomes, resuspended in PBS, were thrown onto the golden grid, and then air-dried for 15 min after being negatively stained with 2% phosphotungstic acid for 3 min. To view the exosomes, TEM (H-600IV, Japan) operating at 80 kV was used.

Nanoparticle tracking analysis

Exosome quantity and size were determined using ZetaView (Particle Metrix GmbH, Germany). Exosomes that had been purified were reconstituted, diluted in PBS, and then injected into the sample chamber. In triplicate, each sample was measured.

Antibodies and reagents

Table 1 provides comprehensive information on the antibodies used for immunofluorescence staining and western blot analysis.

Table 1.

Detailed information of antibodies and reagents

Antibody	Manufacturer	Catalog Number	Country	Isotype
anti-GAPDH	Signalway Antibody	#21612	USA	Rabbit polyclonal
anti-TSG101	HuaAn Biotechnology	ET1701-59	China	Rabbit monoclonal
anti-CD9	Abcam	Ab236630	UK	Rabbit monoclonal
anti-Hsp70	Signalway Antibody	#48597	USA	Rabbit monoclonal
anti-Rab27a	HuaAn Biotechnology	EM1706-32	China	Mouse monoclonal
anti-Rab7	HuaAn Biotechnology	China	China	Rabbit monoclonal
anti-IGF2	Abcam	Ab177467	UK	Rabbit monoclonal
anti-Ras	HuaAn Biotechnology	ET1601-16	China	Rabbit monoclonal
anti-Raf	HuaAn Biotechnology	ET1701-21	China	Rabbit monoclonal
anti-ERK	Cell Signaling Technology	#4370	USA	Rabbit monoclonal
anti-p-ERK	Cell Signaling Technology	#4695	USA	Rabbit monoclonal
anti-FAP	HuaAn Biotechnology	ER1706-84	China	Rabbit polyclonal
anti-α-SMA	Abcam	Ab32575	UK	Rabbit monoclonal
anti-CD63	HuaAn Biotechnology	ET1607-2	China	Rabbit monoclonal
anti-Alix	Cell Signaling Technology	#2171	USA	Mouse monoclonal
anti-PI3K	Cell Signaling Technology	#4292	USA	Rabbit monoclonal
anti-p-PI3K	Cell Signaling Technology	#17366	USA	Rabbit monoclonal
anti-AKT	Cell Signaling Technology	#9272	USA	Rabbit polyclonal
anti-p-AKT	Cell Signaling Technology	#4060	USA	Rabbit monoclonal
IGF2	Peprotech	100-12	USA	-
Xentuzumab	Wuhan Chemstan Biotechnology	CSD00418	China	-
PKH26	Sigma-aldrich	-	USA	-

Western blot analysis

SDS-PAGE was used for protein electrophoresis. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes using a transmembrane. The membranes were then blocked for a further three hours at room temperature in TBST buffer. Subsequently, the membranes underwent an overnight incubation at 4°C with primary antibodies, followed by a two-hour incubation at room temperature (RT) with secondary antibodies. Band visualization was achieved using the ChemiDocTM XRS+ system, while quantification was performed using Image Lab. Data analysis and plotting were executed using GraphPad Prism. Each western blot analysis was independently conducted in triplicate.

Quantitative RT-PCR (qRT-PCR)

The process for performing qRT-PCR was previously disclosed [21]. The sequences of the primers employed were as follows: Rab27a, 5’-CAAACAGCTTCCAGCTAAGGAC-3’ (forward) and 5’-GAGAACTCTGTGCCTCACCTCA-3’ (reverse); Rab7, 5’-ATGACCTCTAGGAAGAAAG-3’ (forward) and 5’-TCAGCAACTGCAGCTTTCT-3’ (reverse); Rab5a, 5’-ATACACTCTCATCCTACGGG-3’ (forward) and 5’-GAACTTCCAGAATTCAAGGG-3’ (reverse); Rab5c, 5’-AATTGAATTCATGGCGGGTCGAGGAGGTGC-3’ (forward) and 5’-GGCCGAATTCTCAGTTGCTGCAGCACTGGC-3’ (reverse); IGF2, 5’-AGTGGGCCATTCGGAACATT-3’ (forward) and 5’-AGGTAATTTGGGGTGCCTCG-3’ (reverse) and GAPDH, 5’-GGATGCAGGGATGATGTTC-3’ (forward) and 5’-TGCACCACCAACTGCTTAG-3’ (reverse). Using the 2-ΔΔCt method, GAPDH normalized the level of gene expression.

Exosome uptake assay

A type of red lipophilic dye called PKH26 (Thermo Fisher Scientific, Waltham, USA) might be incorporated into the membrane of the exosome. In this experiment, exosomes were dyed in the dark with a PKH26 and PKH linker combination. Following a PBS wash, the tagged exosomes were isolated in accordance with the step ‘Isolation and purification of exosome’. Exosome uptake was then confirmed by incubating cells with tagged exosomes.

Immunofluorescence staining

As previously stated [21], cellular fixation was achieved by immersing the cells in a 4% paraformaldehyde solution for a duration of 15 minutes at RT. Following fixation, a thorough triple wash with PBS was carried out. Subsequently, cell permeabilization was accomplished by exposing them to a 0.1% Triton X-100 solution. To prevent non-specific binding, a 5% goat serum diluted in PBS was employed for a 30-minute blocking step. The cells were rinsed after being treated with primary antibodies for an entire night. The secondary antibodies were then diluted in PBS containing 5% goat serum and added to the washed samples. DAPI was used to stain the nuclei for 10 min at room temperature. Images were captured using an Olympus confocal microscope (FV1000, Tokyo, Japan). To track the expression of the target proteins, five random perspectives were chosen.

Cell migration and invasion assay

Briefly, the cells were grown on a glass slide and developed into a confluent monolayer before being used in a wound healing assay as described [8]. A pipette tip was used to create a scratch wound. ImageJ was used to examine images that were collected at various times. Cells (5 × 10⁴ cells/well) were seeded in the top chambers of the Transwell plates in FBS-free medium with membrane inserts covered in Matrigel, as described in the instructions for the Transwell invasion assays [22]. The cells that had infiltrated the lower surface of the membrane were fixed, stained with crystal violet, and examined under an inverted microscope after a 24-hour incubation period.

Label-free quantitative proteomics

Exosomes derived from static or with 8 h FSS loading HepG2 cells were dissolved in PBS, each sample with 3 duplicates, and they were subsequently given SDT buffer (4% SDS, 100 mM Tris-HCl, pH 7.6). The lysate was then sonicated and heated for 15 minutes after that. After measuring the supernatant after centrifuging at 14,000×g for 40 min, protein denaturation by cooking for 5 min. The 12.5% SDS-PAGE gel was used to separate the proteins. Coomassie Blue R-250 staining was used to see the protein bands. The FASP method was then used to digest exosome proteins [23]. Differentially expressed proteins included those with a fold changed > 2 or < 0.5 and a p-value (Student’s t-test) of 0.05. For differentially expressed proteins, the GO functional analysis was conducted.

Bioinformatics analysis

Protein clustering analysis: Quantitative data of the target protein set were first normalized. Subsequently, relative expression levels were visualized along both sample and protein dimensions using matplotlib (distance metric: Euclidean; linkage method: average linkage). A clustered heatmap was generated to display the results.

Enrichment analysis: Pathway annotation and enrichment analysis of the target protein set were conducted using the GO and KEGG databases. Fisher’s exact test was employed to compare the distribution of GO terms or KEGG pathways between the target protein set and the background whole proteome. Significantly enriched terms or pathways (P < 0.05) were selected and visualized using ggplot2.

Immunohistochemical staining

Using the previously reported conventional techniques, tumor tissues were embedded in paraffin blocks and preserved in formalin [18]. To find out whether the target proteins were expressed, immunohistochemistry was performed. The secondary antibodies were HRP-conjugated. Using the Dako Envision System with DAB substrate (Agilent, Santa Clara, CA, USA), blocking and chromogenic detection were carried out in accordance with the manufacturer’s instructions. Table 1 contains a list of the primary antibodies utilized in this investigation.

Experimental animal model

The male BALB/c nude mice utilized in this investigation were acquired from Gempharmatech (Nanjing, China), and the animal experiment was approved by Sichuan University’s Medical Ethics Committee (no. K2021015). HepG2 cells stably transfected with Luciferase (Luci-HepG2) were constructed by our lab [19]. For cell preparation, Luci-HepG2 cells co-culture with exosomes (10 μg/mL) from FSS-induced HepG2 cells for 2 weeks. The medium with exosomes was replaced once in the middle to maintain the exosomes’ concentration.

Approximate 2 × 10⁶ Luci-HepG2 or Luci-HepG2 co-culture with FSS-Exo were suspended in 30 μl PBS for each nude mouse. Each naked mouse (six in each group, 6-week-old) was orthotopically injected with a microsyringe into the left hepatic lobe through a 1 cm transverse incision in the upper abdomen while under anesthesia. Tumor growth was tracked using an in vivo imaging system (IVIS Lumina III, PerkinElmer, USA).

Statistical analysis

In this study, the data were given as the mean plus standard error of the mean (SEM) or mean plus standard deviation (SD). GraphPad Prism 6 (La Jolla, California, USA) was used for all statistical analysis. One-way ANOVA and Tukey’s test were used to compare multiple groups. Statistical significance was defined as P < 0.05.

Results

FSS induces the secretion of exosomes via altering Rabs in HCC cells

1.4 dyn/cm² FSS was loaded onto HepG2 and QGY-7703 cells for 0, 1, 4, and 8 h, respectively. Using the standard exosome separation technique of ultracentrifugation, the exosomes were extracted from the conditioned media and isolated and purified. Then, using TEM, the exosomes’ cup-shaped structure was observed. Exosome markers were identified by western blot. It is recognized that exosomes derived from various cell types and bodily fluids commonly share specific marker proteins, such as TSG101, Hsp70, and CD9 [11]. In our study, we used Western blot to confirm the presence of three classic exosomal markers. The results demonstrated that exosomes were successfully isolated, and 8h FSS stimulation to the cells was chosen for the following study (Figure 1A and 1B).

Figure 1.

HCC cells secreted exosomes and Rabs expression changes with FSS stimulation. A. The typical TEM images of exosomes in HepG2 and QGY-7703 cells when exposed to FSS for 1 h, 4 h, and 8 h. Scale bar = 100 μm. B. Western blot analysis of exosome markers TSG101, CD9, and Hsp70 when exposed to FSS for 1 h, 4 h, and 8 h (n = 3). C. Nanoparticle tracking analysis (NTA) of exosomes derived from HepG2 and QGY-7703 cells with 8 h FSS stimulation. D. NTA analysis of exosomes concentration, and BCA analysis of exosomes protein concentration when HepG2 and QGY-7703 cells with 8 h FSS stimulation. E. Western blot analysis of Rab27a and Rab7 with 8 h FSS stimulation (n = 3). F. q-PCR analysis of Rab27a and Rab7 with 8 h FSS stimulation (n = 3).

Further, the concentration and size of exosomes were quantified by NTA with normalization to total cell count. The isolated exosomes were within the expected size range (30 to 160 nm) in both static and FSS-treated exosomes (Static-Exo and FSS-Exo), and 8 h FSS loading was used in the later study (Figure 1C). Interestingly, exosome concentration was significantly higher in the FSS group than in the static group in both HepG2 and QGY-7703 cells observed by NTA analyses (Figure 1C), and further quantification validated this result (Figure 1D).

Rab7, a crucial protein in endolysosomal fusion, and Rab27a, a regulator of late endosome docking with the plasma membrane, were investigated in light of the crucial role of Rab-GTPases in regulating several phases of vesicle trafficking. With FSS stimulation, the expression of protein and mRNA levels of Rab7 and Rab27 exhibit precisely opposite patterns (Figure 1E and 1F).

Exosomes secreted from FSS-induced HCC cells promote the migration of recipient cells

To evaluate the effect of exosomes on the migration ability of recipient cells, we first investigated whether the cancer cells can take up exosomes after cells were incubated with PKH26-labeled static exosomes (Static-Exo) and FSS exosomes (FSS-Exo) for 6 h. Observation by confocal microscopy demonstrated that both HepG2 and QGY-7703 cells could take up exosomes (Figure 2A).

Figure 2.

FSS-Exo promoted HCC migration and invasion. A. Images showing the absorption of PKH26-labeled exosomes (red) from static or FSS condition HepG2 and QGY-7703 cells by HepG2 and QGY-7703 cells. B. Representative images of scratch in HepG2 and QGY-7703 cells when treated with static exosomes or FSS-induced exosomes derived from HepG2 or QGY-7703 cells. C. The statistical results of the distance of cell migration. D. Representative images of invasion cells in HepG2 and QGY-7703 cells when treated with static exosomes or FSS-induced exosomes derived from HepG2 or QGY-7703 cells. E. The quantification of the numbers of invasive cells. Scale bar = 100 μm.

We questioned whether oncogenic phenotypes may be transferred from highly migratory cells to passive poorly migratory cells because exosomes can be easily ingested by destination cells. We carried out in vitro research by incubating static HepG2 and QGY-7703 with exosomes derived from FSS-stimulated cancer cells. Compared with PBS control, Static-Exo slightly improved the migration ability of cancer cells while FSS-Exo significantly enhanced their migration ability (Figure 2B and 2C); meanwhile, FSS-Exo increased the invasive cells number than other groups (Figure 2D and 2E). These results show that FSS-Exo could promote HCC migration and invasion.

IGF2 is significantly upregulated in FSS-Exo, HCC patients’ tissue and plasma exosomes

To further explore the potential mechanisms regulating exosome-mediated intercellular transfer of phenotype and cancer cell migration. Comparing the exosome protein contents of highly migratory (with FSS) and weakly migratory (static) HCC cells was done using quantitative proteomics. The proteome samples from three biological replicates displayed certified reproducibility, according to SDS-PAGE results (Figure S1). Here, 1705 proteins were identified, among them, 615 proteins were filtered out when data analyzing because these proteins were detected in only one sample in 3 biological replicates. A volcano plot illustrated the differentially expressed proteins in FSS-Exo compared with Static-Exo (Figure 3A). Moreover, GO and KEGG pathway analyses suggested that MAPK and PI3K signaling is associated with cell migration activity and HCC (Figure S2). Further, 32 upregulated proteins with more than 2-fold change were identified in FSS-Exo compared to Static-Exo.

Figure 3.

IGF2 enriched in Exosomes derived from FSS-induced HCC cells and upregulated in HCC. A. Proteins expression profiles of Static-Exo and FSS-Exo are visualized in volcano plots. Here, compares the protein expression of FSS-Exo to Static-Exo, blue: down-regulated proteins; red: up-regulated proteins; gray: no significant difference. B. Heatmap showing the proteins that exhibit 3-fold higher expression levels in FSS-Exo than in Static-Exo. C. Western blot images show IGF2 expression from exosomes derived from static or FSS-induced HCC cells. D. Western blot images show IGF2 expression from exosomes derived from HCC patients and normal people. E. HE and IHC staining images show IGF2 expression in HCC patient tissue. F. IF staining images show IGF2 expression in HCC patient tissue. G. q-RT PCR showed the expression of IGF2 in HCC patient tissue. Scale bar = 100 μm.

Figure 3B showed the proteins that had over 3-fold change. Among them, the high expression of Insulin-like Growth Factor 2 (IGF2) attracted our attention. IGF2 is a member of the IGF system which is composed of two IGFs (IGF1, IGF2), two IGF receptors (IGFR1, IGFR2), and seven IGF-binding proteins (IGFBPs) composition [24]. Interestingly, our mass spectrometry results found that IGF2 was highly enriched in FSS-Exo samples, while other members of the IGF system were not detected, so our further exploration focused on IGF2.

Subsequently, we validated IGF2 expression in exosomes both derived from HepG2 and QGY-7703 cell lines. As shown in Figure 3C, IGF2 was highly expressed in HCC cells with FSS stimulation. Further, we also investigated IGF2 highly expressed in exosomes derived from HCC cancer patients’ serum (Figure 3D). Additionally, to show the IGF2 expression profile in HCC, we then conducted H&E, IF, and qRT-PCR assays, the results (Figure 3E-G) indicated IGF2 expression was markedly elevated in HCC.

Exosomal-IGF2-induced HCC migration and invasion in vitro

To further explore if IGF2 acted as the bioactive factor in FSS-Exo to promote recipient HCC cells gained a more migratory phenotype, we used Xentuzumab (BI 836845), a monoclonal antibody that can neutralize IGF2 to abolish its signaling [25]. It could be observed that the expression of IGF2 was significantly upregulated in HepG2 and QGY-7703 cells when treated with FSS-Exo while Xentuzumab treatment could significantly weaken this effect (Figure 4A). We further detected the changes in cell migration and invasion ability after different treatments by scratch experiment and Transwell respectively. As shown in Figure 4B and 4C, treatment of FSS-Exo increased the migration distance of the cells. In contrast to the FSS-Exo group, the migratory distance of cells was decreased after applying Xentuzumab. Additionally, Figure 4D, 4E showed FSS-Exo increased the invasive cells number than the control group, and the invasive number reduced after applying Xentuzumab. These findings imply that IGF2 is a mediator of HepG2 and QGY-7703 cell migration and invasion caused by FSS-Exo.

Figure 4.

IGF2 mediated FSS-Exo induced HCC migration and invasion. A. Western blot detection of the expression of IGF2 in HepG2 and QGY-7703 cells after treatment with FSS-Exo and IGF2 antagonist. B. 24 h migration pictures of HepG2 and QGY-7703 cells under FSS-Exo and IGF2 antagonist treatments. C. The quantitative analysis of would area. D. Typical images of HepG2 and QGY-7703 cells invasion stimulated by FSS-Exo and IGF2 antagonist. E. The quantification of invaded cells. Scale bar = 100 μm.

FSS-Exo activated Ras/Raf/Erk signaling of HCC cells

Our previous bioinformatic analysis indicated that MAPK and PI3K/Akt signaling pathways were critical in FSS-Exo and HCC (Figure S2). Due to the common pathway of IGF2 and HCC is the Ras signaling, and studies found that the activation of the classic Ras/Raf/ERK signaling axis could efficiently enhance the growth and development of renal cancer cells migration [26], can also promote the migration ability of human synovial fibroblasts [27]. Therefore, we consider this signaling axis as a target to study whether FSS-Exo activates Ras signaling to promote the migration ability of HCC cells, and the role of IGF2 in it. In our results, the expression of both mRNA and protein level of Ras, Raf, and p-ERK could be up-regulated after the IGF2 human recombinant protein treatment was applied to HCC cells (Figure 5A and 5B). Subsequent application of FSS-Exo to HepG2 and QGY-7703 cells also caused a significant upregulation of Ras, Raf, and p-ERK expressions also at mRNA and protein level, indicating that FSS-Exo can activate the Ras/Raf/ERK signaling axis of target cells (Figure 5C and 5D).

Figure 5.

FSS-Exo activated Ras/Raf/Erk signaling of HCC cell. A. Western blot analysis of Ras/Raf/ERK expression in HCC cells after IGF2 recombinant protein treatment. B. q-RT PCR analysis of Ras/Raf/ERK expression in HCC cells after IGF2 recombinant protein treatment. C. Western blot analysis of Ras/Raf/ERK expression in HCC cells after FSS-Exo treatment. D. q-RT PCR analysis of Ras/Raf/ERK expression in HCC cells after FSS-Exo treatment. E. Western blot analysis of Ras/Raf/ERK expression in HCC cells after co-culture with FSS-Exo and treated with Xentuzumab. F. q-RT PCR analysis of Ras/Raf/ERK expression in HCC cells after co-culture with FSS-Exo and treated with Xentuzumab. G. 24 h migration images of HepG2 and QGY-7703 cells treated with FSS-Exo or PD98059 and the quantitative analysis of wound area, at least 10 random areas were selected to be photographed (n = 3). Scale bar: 100 µm.

Subsequently, two kinds of HCC cells were co-cultured with FSS-Exo, with or without adding IGF2 antagonist Xentuzumab. The results showed that the expression of crucial proteins of the Ras/Raf/ERK signaling axis in the two cells was down-regulated when Xentuzumab was presented (Figure 5E and 5F). After that, we further studied whether the activation of this pathway regulated the migration ability of HCC cells, so we used the ERK inhibitor PD98059, and then a wound healing assay was performed on HCC cells. As shown in Figure 5G, the migration ability of HCC cells induced by FSS-Exo was weakened when pretreated with ERK inhibitors. Based on the previous results, it was shown that the FSS-Exo could activate the Ras/Raf/ERK signaling axis in HCC cells, thereby enhancing the migration ability of HCC cells.

Exosomal-IGF2 induced HCC metastasis in vivo

We finally studied the effect of FSS-Exo on the growth and metastasis of liver cancer tumors in vivo, and the role of IGF2 in it. Live imaging was performed to observe intrahepatic tumor metastasis in nude mice. According to the treatment method adopted in the literature [28]. We co-incubated FSS-Exo and Luci-HepG2 cells for 2 weeks (maintaining the concentration of exosomes at 10 μg/mL), and then the exosome-pretreated cells and control cells implanted in the left lobe of mouse liver. Xentuzumab was injected into the Xentuzumab group after tumor formation.

As shown in Figure 6A, consistent with in vitro experiments, FSS-Exo can significantly induce liver metastasis in nude mice, and after blocking IGF2, liver metastasis in nude mice was significantly weakened. Likewise, the number of intrahepatic tumors was significantly increased in the FSS-Exo group, and this increase was reduced with IGF2 blockade (Figure 6B and 6C). At the same time, there was no significant difference in the body weight of the nude mice among the groups (Figure 6D).

Figure 6.

IGF2 mediated FSS-Exo induced HCC migration and invasion in vivo. A. Luciferase live imaging shows intrahepatic metastasis in the liver of nude mice after FSS-Exo and IGF2 antagonist treatments. B. Intrahepatic metastasis of nude mice after FSS-Exo and IGF2 antagonist treatments. C. Number of liver tumors in nude mice. D. Record of body weight of nude mice.

Discussion

Since the development of new solid tumor treatment methods has been made possible by the connections between cancer biology and physics, the physical characteristics of cancer have drawn increasing amounts of interest. Recent research showed that stiff ECM encourages the release of exosomes from Huh7, Panc1, and MCF-7 cells and identified a biochemical route connecting stiff ECM to Akt activation [29]. FSS, as a critical force factor in solid tumors, could induce changes in the biological behavior of tumor cells, such as FSS promotes the migration of HCC cells; FSS activates the mechanical-chemical signal mediated by Integrin in HCC cells, thus enhancing migration and invasion ability of HCC cells in our previous studies [7,19]. In the present study, we applied 1.4 dyn/cm² FSS to HCC cells HepG2 and QGY-7703, finding that the number of secreted exosomes of HCC cells after FSS stimulation was significantly elevated. Previous studies have indicated that Rab27a acts as a positive regulator of exosome release by promoting the docking of late endosomes/MVBs to the plasma membrane, thereby enhancing exocytosis [30]. In contrast, Rab7 is often considered a negative regulator, as it facilitates the fusion of MVBs with lysosomes, leading to degradation of the contents and thus reducing exosomal release [31]. In our study, FSS induced the up-regulation of Rab27a in HCC cells, and promoted the anchoring of exosomes on the membrane, thus promoting their release from the extracellular space. Meanwhile, it induces the down-regulation of Rab7, that is, the down-regulation of the pathway of MVB transfer to lysosomes, making more exosomes released to extracellular space (Figure 1). The behavior of Rab proteins is consistent with previous observations [11,28].

There’s emerging evidence that exosomes, depending on their diverse origins, can promote tumor progression in various ways [32,33]. Here, we investigated the functional impact of exosomes produced under FSS on cancer cell migration. According to our findings, FSS-Exo significantly increased HCC cell migration and invasion when compared to Static-Exo (Figure 2). This suggests that FSS affects contacting tumor cells not only through intrinsic oncogenic pathways but also through the creation of exosomes that promote migration.

The various impacts we found are most likely the result of the differing compositions between the two types of exosomes as the identical amounts of FSS-Exo and Static-Exo were utilized in our tests. To address this, we conducted proteomics analysis and biochemistry experiments to find the potential factors that influence the ability of FSS-Exo. Using mass spectrometry-based proteomic analysis of exosomes derived from HepG2 cells, we identified a total of 249 differentially expressed proteins between FSS Exo and Static Exo groups, with a fold-change threshold of 2.0. Among these, 32 proteins were significantly up-regulated and 217 were down-regulated in FSS Exo compared to Static Exo. We then ranked the 32 up-regulated proteins based on their fold-change values. IGF2 attracted our particular attention as it was among the most highly up-regulated proteins. IGF2 is a well-recognized polypeptide with potent growth-promoting functions and is known to play important roles in development, cardiovascular diseases, and tumorigenesis. IGF2 has been linked to a bad prognosis and has been discovered to be overexpressed in several malignancies [17,34]. Our result of IGF2 expression in HCC patients’ serum and tissue sample also confirm that IGF2 is highly expression in HCC (Figure 3). This outcome supports our earlier finding that IGF2 is overexpressed in FSS condition [18]. Further, the neutralizing antibody was used to abolish IGF2 signaling, and the migration and invasion ability of HCC cells significantly decreased accordingly (Figure 4), indicating that IGF2 is a key functional content in FSS-Exo.

Further bioinformatic analysis implicated in HCC all converged on the MAPK and PI3K/Akt signaling pathways (Figure S2), In particular, pathways shared between IGF2 and HCC highlighted the involvement of the Ras signaling cascade. Previous studies have reported that activation of the canonical Ras/Raf/ERK axis within the MAPK pathway can promote growth and migration in renal cell carcinoma, and enhance migratory in human synovial fibroblasts [35,36]. These findings led us to hypothesize that FSS Exo may enhance recipient cell migration by activating this specific signaling axis, and that IGF2 might play a key role in this process. So, we detected the expression of the major proteins of the Ras/Raf/ERK signaling axis in two types of HCC cells after FSS-Exo treatment and found that Ras, Raf proteins and p-ERK were up-regulated after FSS-Exo treatment. Further, to find out whether IGF2 mediates the activation of this signaling axis caused by FSS-Exo, we used IGF2 recombinant protein and IGF2 antagonist to treat two kinds of HCC cells respectively (Figure 5). The results showed that IGF2 recombinant protein could significantly upregulate the expression of Ras, Raf protein, and p-ERK, while the activation of this pathway caused by FSS-Exo was weakened after using antagonists to inhibit the effect of IGF. This demonstrates that IGF2 facilitates the stimulation of the Ras/Raf/ERK signaling axis brought on by FSS-Exo, which in turn results in the improvement of HCC cells’ capacity for migration and invasion. Finally, an in vivo investigation showed that IGF2 was crucial to the migration of HCC cells and liver metastases that FSS-Exo produced in naked mice (Figure 6).

In summary, we found that FSS induces exosome secretion in HCC cells, and Rab family proteins participate in the regulation of FSS-mediated exosome secretion, and FSS reshaped proteins contained in exosomes of HCC cells, IGF2 was enriched in FSS-induced exosomes, and mediated FSS-Exo induced-Ras/Raf/ERK signaling activation, causing the enhancement of HCC cells migration ability (Figure 7).

Figure 7.

Schematic overview of FSS-mediated upregulated exosomal-IGF2 promoting HCC cell migration and metastasis.

Disclosure of conflict of interest

None.