Predictive role of UCA1-containing exosomes in cetuximab-resistant colorectal cancer

Patients and sample processing

Informed consent was obtained from each patient, and the protocols were approved by the Ethics Committee of Harbin Medical University Cancer Hospital. Serum samples were collected from Harbin Medical University Cancer Hospital to investigate the feasibility of detecting exosomal UCA1. Changes in serum exosomal UCA1 levels were validated in an independent cohort of 53 CRC patients treated with cetuximab at Harbin Medical University Cancer Hospital from 2015 to 2016. Blood samples from all CRC patients were collected by vena puncture. To extract exosome from human blood, a two-step centrifugation protocol—1600g for 10 min and 16,000g for 10 min at 4 °C was used to isolate serum, and then serum was stored at − 80 °C until exosome extraction. Blood samples with evidence of hemolysis were excluded. According to the RECIST criteria for a pathological response, these patients were divided into two groups: 30 patients responded to cetuximab therapy [complete response (CR) or partial response (PR)], and 23 patients did not respond [stable disease (SD) or progressive disease (PD)].

Cell lines and culture

The human Caco2 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). We established cetuximab-resistant cell lines by chronically exposing cetuximab-sensitive Caco2 (Caco2-CS) cells to increasing cetuximab doses in medium over a period of 6 months. The final concentration of cetuximab for the cetuximab-resistant subclone Caco2-CR was 300 μg/ml. Caco2-CS and Caco2-CR cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Invitrogen, USA) containing 10% fetal bovine serum (FBS, Gibco, Invitrogen, USA) and 1% penicillin–streptomycin (Invitrogen, USA) at 37 °C in a humidified atmosphere of 95% air/5% CO₂.

Cell proliferation assay and colony formation assay

For the cell proliferation assay, cell viability was determined by Cell Counting Kit 8 (CCK8, Dojindo, Japan) according to the manufacturer’s instructions. For the colony formation assay, about 1000 cells were placed in each well of a 6-well plate in 2 ml media containing cetuximab (300 μg/ml for Caco2-CR). The media were changed every 3 day. After 12–15 days, the colonies were fixed in 80% methanol and stained with 0.1% crystal violet for 20 min. The number of colonies was counted using an inverted microscope.

Isolation of exosomes

Medium and serum were filtered through a 0.45 μm polyvinylidene fluoride filter (Millipore, Billerica, MA, USA); ExoQuick solution (System Biosciences, Mountain View, CA, USA) was added to the serum, which was then incubated for 0.5 h at room temperature, and ExoQuick-TC solution was added to the medium, which was then incubated at 4 °C for 12 h. The mixture was centrifuged at 1500g for 30 min and supernatant was removed by aspiration. Pelleted fractions were resuspended in 25 μl phosphate-buffered saline (PBS).

Transmission electron microscopy (TEM)

A sample of exosomes was diluted to a final concentration of 0.5 mg/ml in PBS, spotted onto a glow-discharged copper grid on filter paper and dried for 10 min. Exosomes were stained with 1% aqueous phosphotungstic acid (PTA) for 5 min and dried for 20 min and then examined at 100 keV with TEM (JEM-1-11 microscope, Japan).

RNA extraction

Total RNA was extracted from cells using TRIzol^® Reagent (Invitrogen, CA, USA). Exosomal RNA was extracted using the Total Exosome RNA and Protein Isolation Kit (Invitrogen, USA). The concentration and quality of RNA were measured by UV absorbance at 260 and 280 nm (260/280 nm) using a Nanodrop 2000 spectrophotometer (Thermo Scientific, USA).

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR)

Total RNA was extracted from cells and exosomes as described above. RNA templates were treated with DNase I to avoid genomic DNA contamination. The first strand of cDNA was synthesized using the SuperScript First-Strand Synthesis System (Invitrogen, CA). PCR amplification was performed using an Applied Biosystems 7500 Detection System (Applied Biosystems, CA) and primers for UCA1 (forward: 5′-ACGCTAACTGGCACCTTGTT-3′, reverse: 5′-TGGGGATTACTGGGGTAGGG-3′) and β-actin (forward, 5′-CACCTTCTACAATGAGCTGCGTGTG-3′; reverse, 5′-ATAGCACAGCCTGGATAGCAACGTAC-3′). Real-time PCR was performed on triplicate samples according to the instructions of the SYBR^® Premix Ex Taq™ Kit (Takara, Japan). The expression level of UCA1 was normalized to that of β-actin using the comparative 2^−ΔΔCt method.

Western blot analysis

Proteins were extracted from cells using RIPA lysis buffer (Biouniquer Technology, China). Exosomal proteins were extracted using the Total Exosome RNA and Protein Isolation Kit (Invitrogen, USA). Protein content was measured using a Nanodrop 2000 spectrophotometer (Thermo Scientific, USA). Equal amounts of protein from each sample were separated by electrophoresis in sodium dodecyl sulfate (SDS)-polyacrylamide gels before being transferred onto polyvinylidene difluoride (PVDF) membranes, blocked for 2 h with 5% skim milk, incubated overnight at 4 °C with primary antibodies against TSG101, Alix and CD81 (Abcam, Cambridge, UK) and then incubated for 1 h with the secondary antibody (Kangwei Ltd., China). The signals were visualized with an ECL detection system.

Immunofluorescence assay

For exosomal labeling, Dil (Beyotime Biotechnology, China) was added to the exosome suspension at 1 μM for 20 min, and the exosome suspension was then washed through Exosome Spin Columns (MW3000) (Invitrogen, USA) to remove excess dye. Dil-labeled exosomes were incubated with CRC cells and visualized by fluorescence microscopy or EPICS XL flow cytometry (Beckman Coulter) after 24 h.

RNA interference

Caco2-CR cells were transfected with UCA1-small interfering RNA (siRNA) (RNAi: forward primer: 5′-GCACCUUGUUAGCUACAUAAA-3′, reverse primer: 5′-UAUGUAGCUAACAAGGUGCCA-3′) using Lipofectamine™ 2000. After 48 h of transfection, RNAi efficiency was assessed by qRT-PCR. Another siRNA was designed as a negative control (Ctrl siRNA): forward primer: 5′-UUCUCCGAACGUGUCACGUdTdT-3′, reverse primer: 5′-ACGUGACACGUUCGGAGAAdTdT-3′.

Electroporation of UCA1 into exosomes

UCA1 was electroporated into exosomes using a GenePulser Xcell™ electroporation system (Bio-Rad, USA) as previously described [21]. Briefly, 2 μg exosomes and 400 nmol RNA were mixed in 400 μl electroporation buffer and electroporated at 350 V and 150 μF in a 4-mm cuvette. The mixture was incubated at 37 °C for 30 min to ensure that the exosome membrane fully recovered, and then, the mixture was treated with RNase to remove unincorporated RNA. Labeled RNA in exosomes was quantified by detecting fluorescence using a Fluorescence Spectrophotometer F-4600 (HITACHI, Japan).

Apoptosis assay

Twenty-four hours after 1 × 10⁵ Caco2-CS cells were seeded in six-well plates, different amounts of CR-exo (1, 5, or 10 μg) were added to the supernatants. The samples were divided into the following four groups: CS, CS + 1 μg CR-exo, CS + 5 μg CR-exo, and CS + 10 μg CR-exo. After culturing for 24 h, the cells were treated with cetuximab (300 μg/ml) for 48 h. Then, all the cells were harvested by centrifugation, washed with PBS, stained using Annexin V fluorescein isothiocyanate (APC) and propidium iodide (PI) and analyzed using a flow cytometer (BD Biosciences, San Diego, CA).

Statistical analyses

All statistical analyses in this study were performed with SPSS 16.0 software. All experiments were carried out three times independently. Data are presented as the mean ± standard deviation. p values < 0.05 by Student’s t-test or one-way ANOVA indicated statistical significance. All statistical tests were two-sided.

Upregulation of UCA1 levels in cetuximab-resistant cells and their exosomes

We generated cetuximab-resistant Caco2-CR cells from Caco2-CS CRC cells following long-term exposure to increasing doses of cetuximab. Caco2-CR cell lines could grow in culture medium containing 300 μg/ml cetuximab and displayed a robust cetuximab-resistant phenotype as compared to parental controls (Fig. 1a). Additionally, the clonogenicity of Caco2-CR cells was significantly greater than that of Caco2-CS cells in response to cetuximab treatment (Fig. 1b).

Total RNA was extracted from Caco2-CS and Caco2-CR cells, and UCA1 expression levels were assessed using qRT-PCR. UCA1 expression was markedly higher in Caco2-CR cells than in parental Caco2-CS cells (Fig. 1c). We then isolated exosomes from the conditioned medium of Caco2-CS and Caco2-CR cells. Western blot analysis demonstrated the expression of TSG101, Alix, and CD81, which are all exosome markers and are associated with exosome formation, in both exosomes and cells (Fig. 1d). In addition, TEM revealed that the predominant vesicles were of typical exosome size (30–150 nm in diameter), had a characteristic round or saucer shape, and were delineated by a lipid bilayer (Fig. 1e). Further quantitative analysis suggested that the number of exosomes expelled from both cell variants did not differ significantly (p > 0.05; Fig. 1f). Moreover, we detected UCA1 levels in exosomes derived from Caco2-CR cells (CR-exo) and Caco2-CS cells (CS-exo) and found that UCA1 levels were significantly upregulated in CR-exo compared with CS-exo (Fig. 1g). Interestingly, the increase in UCA1 levels between CR-exo and CS-exo (~ 20-fold) was much greater than that between Caco2-CR and Caco2-CS cells (~ 7-fold), indicating that UCA1 was concentrated in exosomes derived from Caco2-CR cells; moreover, UCA1 expressions may be related to cetuximab resistance in CRC cells.

Characterization of serum exosomal UCA1

Figure 2a shows the relative expression of lncRNA UCA1 acquired using the same amount of total RNA. qRT-PCR analysis indicated that the exosomal UCA1 could be reliably detected but outside exosomal UCA1 was only minimally detected. To explore the potential benefit of detecting exosomal UCA1, we skipped the exosome extraction step and extracted RNA from whole serum directly. The data presented in Fig. 2a suggested that UCA1 levels were higher in exosome samples than in whole serum samples (p < 0.05).

To ascertain whether the exosomal UCA1 can be protected by exosome membrane, exosome samples and nucleic acids isolated from exosome were subjected to harsh conditions. Samples were incubated with RNase A, a strong acid, or a strong base for 3 h at room temperature, and the results indicated that RNase A (Fig. 2b) and acid or base (Fig. 2c) treatments had minimal effects on the expression of exosomal UCA1 in the exosome group but caused complete degradation of UCA1 in the isolated nucleic acid group. In the freeze–thaw cycle test, exosomal UCA1 levels showed no significant change but decreased markedly in the isolated nucleic acid group after eight freeze–thaw cycles (Fig. 2d). The levels of exosomal UCA1 in the exosome group were not significantly changed from 0 to 36 h at room temperature, but were markedly decreased in the isolated nucleic acid group after 24 h (Fig. 2e). Moreover, as shown in Fig. 2f, exosomal UCA1 remained stable in both groups at − 80 °C. Collectively, these data suggest that exosomal UCA1 is detectable and stable in exosomes, providing a basis for its use as a predictive biomarker of the response to cetuximab.

Circulating UCA1-containing exosomes predict the clinical outcome of cetuximab therapy in CRC patients

As shown in Fig. 3a, in equal amounts of serum, the quantity of exosomes was higher (1.3-fold) in patients who did not respond to cetuximab therapy (PD/SD) than in patients who responded to treatment (PR/CR), but this difference was not significant (p > 0.05). Interestingly, serum exosomal UCA1 expression was significantly higher in the PD/SD group than in the PR/CR group (p < 0.01; Fig. 3b), suggesting that high exosomal UCA1 expression levels might predict increased resistance to cetuximab therapy.

UCA1 can be transferred from resistant cells to sensitive cells through exosomes

Previous studies have suggested that UCA1 is a resistance factor for anticancer drugs [17]. In addition, cell-secreted exosomes and capped nucleic acid cargo can be internalized by neighboring cells [22, 23]. Accordingly, we reasoned that exosomes may carry UCA1 and mediate its intercellular transport. First, CR-exo were labeled with the dye PKH26 and then added to the culture medium of Caco2-CS cells in order to investigate whether exosomes are internalized by recipient cells. Microscopic images showed that labeled exosomes were internalized by Caco2-CS cells in a time-dependent manner after coincubation (Fig. 4a). To further confirm that UCA1 could be transferred to recipient cells via exosomes, Caco2-CS cells were electroporated with fluorescein isothiocyanate (FITC)-UCA1, then exosomes were isolated and labeled with Dil. As shown in Fig. 4b, we observed that colocalization of FITC and Dil was in most recipient cells, whereas no internalization of naked FITC-UCA1 was found in recipient cells after incubation with labeled exosomes. Moreover, to evaluate whether the transport of exosomes derived from drug-resistant cells altered UCA1 levels, we coincubated Caco2-CS cells with CS-exo or CR-exo for 48 h, and UCA1 levels in the Caco2-CS, Caco2-CS + CS-exo, Caco2-CS + CR-exo and Caco2-CR groups were analyzed by qRT-PCR. The level of UCA1 increased in the Caco2-CS + CR-exo group compared with the Caco2-CS and Caco2-CS + CS-exo groups but did not increase in UCA1-knockdown-resistant cells; however, UCA1 levels did not significantly change in the Caco2-CS + CS-exo group compared with the Caco2-CS group (Fig. 4c). These results show that exosomes carrying UCA1 content from cetuximab-resistant cells can be transferred to recipient sensitive cells.

Exosomes derived from cetuximab-resistant cells can transmit drug resistance to sensitive cells and alter UCA1 expression

In the end, we investigated the role of UCA1-containing exosomes in the transfer of cetuximab resistance. We added different amounts of CR-exo to the medium of Caco2-CS cells (CS + 1/5/10 μg CR-exo, Caco2-CS cells were used as the control group). After 24 h of culture, we added serial dilutions of cetuximab to all groups. Two days later, we found a reduction in apoptosis in the groups coincubated with CR-exo (CS + 1/5/10 μg CR-exo) compared with the CS group (p < 0.05), indicating the increased resistance to cetuximab (Fig. 5a). Interestingly, the apoptosis rates of Caco2-CS cells incubated with CR-exo were inversely dependent on the CR-exo dose. Moreover, UCA1 levels significantly increased in CR-exo-supplemented cells in a dose-dependent manner (Fig. 5b). Thus, we reasoned that UCA1 might play a role in cetuximab resistance and that exosomal UCA1 might transmit cetuximab resistance in CRC cells, resulting in greater cetuximab resistance in recipient cells.