All cell lines (SW620, SW480, HCT116, LoVo, HT-29 and 293T; preserved by the Department of Digestive Tumor Microenvironment of the First Affiliated Hospital, Sichuan, China) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS; Gibco; Thermo Fisher Scientific, Inc.).

In total, 20 STR loci, plus the gender determining locus amelogenin, were amplified using the commercially available PowerPlex^® 21 System from Promega Corporation. The amplified products were processed using the Applied Biosystems 3730×l DNA Analyzer and data were analyzed using GeneMapper 5.0 software (Applied Biosystems; Thermo Fisher Scientific, Inc.). Appropriate positive and negative controls were run and confirmed for each sample submitted.

For all experiments, blood samples were collected with informed consent from healthy volunteers using a protocol approved by the Ethics Committee of the First Affiliated Hospital of Chengdu Medical College (Chengdu, China). Between March and May 2017, peripheral blood samples were collected from 3 healthy volunteers (2 males and 1 female), aged 20–35 years. The lymphocyte density gradient centrifugation kit (GE Healthcare Life Sciences) was used to isolate peripheral blood mononuclear cells (PBMCs) from blood. Briefly, peripheral blood was slowly added to the upper layer of the equal volume lymphocyte liquid and centrifuged at 400 × g for 30 min at room temperature. Then, the mononuclear cell layer was collected and washed with PBS buffer.

The EpCAM scFv was cloned from the vector pET-26b-EpCAM (Novagen, Inc.), which contained the sequence for the scFv antibody for EpCAM. This vector was established in our laboratory as previously described (17). Then, the EpCAM-scFv was linked the CD8 α hinge-transmembrane region with 4-1BB co-stimulatory domain and CD3 ζ chain, and the DNA sequence encoding this cassette was digested by HindIII and XhoI, and cloned into the lentiviral backbone pCLK-EF-1 (Invitrogen; Thermo Fisher Scientific, Inc.) vector as previously described (18). The plasmids pCLK-EpCAM-CAR, psPAX-2 (Invitrogen; Thermo Fisher Scientific, Inc.) and pMD2.G (Addgene, Inc.) were transfected into Escherichia coli HD5a (cat. no. CD201-01; Beijing Transgen Biotech Co., Ltd.); 1 µl plasmids (1 µg/µl) were incubated with E. coli DH5a on ice for 30 min, heated for 90 sec in a water bath at 42°C and incubated on ice for 2 min. DH5a were cultured in 900 µl LB liquid medium (cat. no. 12795027; Invitrogen; Thermo Fisher Scientific, Inc.) on 37°C for 1 h at 200 RPM. Then, the mixture was coated on the surface of LB solid medium (cat. no. 22700025; Invitrogen; Thermo Fisher Scientific, Inc.) plates and static-cultured at 37°C for 16 h. Mono-bacteria were selected and cultured for plasmid extraction. The plasmid extraction kit (cat. no. 12381; Qiagen, Inc.) was used to extracted these three plasmids for detection and lentiviral packaging. Then, a 0.5% agarose gel was used to detect the size of plasmid (1 µl DNA/lane). Goldview was used as the visualization reagent.

A lentiviral supernatant was generated from 293T cells transfected with PCLK-EF-1-CAR, pMD2.G and psPAX-2. 293T cells were cultured and used for packaging lentivirus at 70–80% confluence. The vectors (9 µg PCLK-EF-1-CAR; 9 µg pMD2.G; 4.5 µg psPAX-2) were transfected into 293T cells using the calcium phosphate method. Following transfection, 293T cells were cultured at 37°C with 5% CO2 for 72 h. The lentivirus suspension was collected and filtered with a 0.22-µm filter. Then, the lentivirus suspension was ultracentrifuged at 70,000 × g at 20°C for 2 h to concentrate the virus. Reverse transcription-quantitative PCR (RT-qPCR) was used to determine the titer of concentrated virus.

Human PBMCs were cultured in RPMI 1640 medium with 10% FCS, and activated with CD3 antibodies (cat. no. MA1-10175; 50 ng/ml; Invitrogen; Thermo Fisher Scientific, Inc.) and interleukin (IL)-2 (cat. no. 0208AF12; 300 U/ml; PeproTech, Inc.) for 24 h (19). Then, T cells were transduced with the concentrated lentiviral at a multiplicity of infection of 4 on RetroNectin-coated plates (Takara Bio, Inc.). Transduced cells were cultured with IL-2 (300 U/ml) for 14 days before subsequent analysis. Non-transduced T cells were used as negative controls and were cultured under the same conditions. In all trials, the functions of transduced and non-transduced T cells obtained from the same donor were compared.

In order to detect the expression of EpCAM on the cell surface, the colon cancer cell lines SW620, SW480, HCT116 and LoVo, and the colorectal cancer cell line HT-29, were incubated with EpCAM antibody (1:500; cat. no. 2929; Cell Signaling Technology, Inc.) at 37°C for 30 min. Then, cells were washed and incubated with a Cy3-conjugated fluorescent secondary antibody (1:100; cat. no. SA00009-1; ProteinTech Group, Inc.) at 37°C for 30 min in the dark. In order to detect the expression of CAR on the surface of T cells, T cells were incubated with an antigen-binding fragment 2 [F(ab)2] antibody (1:200; cat. no. NBP1-51900; Novus Biologicals, Ltd.) at 37°C for 30 min. Cells were then washed, and incubated with a Cy3-conjugated fluorescent secondary antibody (1:100; cat. no. SA00009-4; ProteinTech Group, Inc.) at 37°C for 30 min in the dark. Cells were washed with PBS and detected using a flow cytometer. BD Accuri™ C6 software was used for data analysis (BD Biosciences).

Total proteins were obtained using RIPA buffer (Beyotime Institute of Biotechnology) and quantified using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Proteins (30 µg/lane) were then transferred to PVDF membranes (Beyotime Institute of Biotechnology) after separation by 10% SDS-PAGE. Membranes were blocked using 5% nonfat milk for 1 h at 26°C. In order to detect the expression of EpCAM, membrane-bound proteins from colon cancer cells were incubated with an anti-EpCAM primary antibody (1:1,000; cat. no. 2929; Cell Signaling Technology, Inc.) at 4°C overnight. Membranes were reacted with secondary horseradish peroxidase-conjugated antibody (1:2,000; cat. no. 7076; Cell Signaling Technology, Inc.) for 2 h at 37°C. Membranes containing T cell-derived proteins were instead incubated with an anti-F(ab)2 antibody primary (1:5,000; cat. no. NBP1-51900; Novus Biologicals, Ltd.) at 4°C overnight, and then with secondary horseradish peroxidase-conjugated antibody (1:1,000; cat. no. HAF109; R&D Systems, Inc.) for 2 h at 37°C. After washing, protein bands were measured using an enhanced chemiluminescence assay kit (EMD Millipore) and imaged with a chemiluminescence detection system (Bio-Rad Laboratories, Inc.). The relative expression of a target protein was determined as the ratio of the grayscale value of the target protein to that of β-actin (1:1,000; cat. no. 58169; Cell Signaling Technology, Inc.) by Image Lab (version 4.0; Bio-Rad Laboratories, Inc.). All experiments were repeated three times.

Total RNA was isolated from lentiviruses or EpCAM-CAR-T cells and untransfected T cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The primers used for viral titer determination were: Lentiviral Rev response element forward, 5′-TTTGTTCCTTGGGTTCTTGGG-3′ and reverse, 5′-GATTCTTGCCTGGAGCTGCTT-3′. For CAR mRNA expression analysis, the primers were: Forward, 5′-CAAGATTACACTCAGGAGTCCC-3′ and reverse, 5′-GTGGGTATTACTGGATGGTGGG-3′. The primers used to measure GAPDH were: Forward, 5′-TGACTTCAACAGCGACACCCA-3′ and reverse, 5′-CACCCTGTTGCTGTAGCCAAA-3′. RNA was reverse transcribed using a PrimeScript™ RT reagent kit (cat. no. RR047A; Takara Bio, Inc.) to obtain cDNA as follows: 37°C for 15 min, 85°C for 5 sec and 4°C for 1 h. qPCR was performed using a TB Green^® Premix Ex Taq™ II kit (cat. no. RR820A; Takara Bio, Inc.). The thermocycling conditions were as follows: 95°C for 30 sec, then 39 cycles of 95°C for 5 sec and 60°C for 30 sec, followed by 95°C for 10 sec, then 65°C for 5 sec, and finally 95°C for 0.5 sec. The Cq values of the target genes were normalized to that of GAPDH (20).

The antitumor effect of EpCAM-CAR-T cells on colon cancer cells was measured using a LDH-Glo™ Cytotoxicity Assay (J2380, Promega). Briefly, EpCAM-CAR-T cells and untransfected T cells were added as effector cells to each well, followed by the addition of the target colon cancer cells (105; SW620, SW480, HCT116, LoVo or HT-29). The final Effector: Target (E:T) ratios were 0.5:1, 1:1, 2:1, 4:1, 8:1 or 16:1. The cell mixtures were incubated at 37°C under 5% CO2 for 4 h. Collecting 50 µl culture supernatant, mixture with 50 µl LDH Detection Reagent, then transferred to fresh 96-well flat-bottom plates. Record luminescence after incubate for 60 min at room temperature. The percentage of cell lysis was calculated as: Specific lysis (%)=(Effector spontaneous release-Target spontaneous release)/(Target maximum release-Target spontaneous release) ×100. Each assay was performed in triplicate.

To measure cytokine production in vitro, colon and colorectal cancer cells were co-cultured with EpCAM-CAR-T cells or untransfected T cells at the ratios of 0.5:1, 1:1, 2:1, 4:1, 8:1 or 16:1 at 37°C under 5% CO2. After 24 h, the supernatant was collected, and the levels of IL-2 (cat. no. EK0397), IL-6 (cat. no. EK0410) and IFN-γ (cat. no. EK0373) were analyzed by ELISA (all Boster Biological Technology).

Data are expressed as the mean ± standard deviation from at least three independent experiments. Differences between different treatment groups were analyzed using one-way ANOVA analysis. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS 16.0 (SPSS, Inc.).

Firstly, the primary plasmid EpCAM-CAR for lentiviral packaging, as well as the helper plasmids psPAX-2 and pMD2.G, were constructed. The structure of the main plasmid EpCAM-CAR is shown in Fig. 1A and B. Then, the plasmids were identified by agarose gel electrophoresis. As shown in Fig. 1C, the sizes of the plasmids EpCAM-CAR, psPAX-2, and pMD2.G were 10.8, 11.0 and 5.8 kb, respectively.

The viral-packaged master plasmid (EpCAM-CAR) and the helper plasmids (psPAX-2 and pMD2.G) were transfected into 293T cells using the calcium phosphate method to obtain a recombinant plasmid carrying the EpCAM-CAR gene expression cassette. Cells were also transfected with a construct whereby the main plasmid was replaced with a fluorescent plasmid with the same fragment size and skeleton for use as a positive control. After 48 h, the fluorescence of the positive control 293T cells was ~90% (Fig. 2A), indicating that the lentivirus was successfully packaged. Then, virus supernatants were concentrated by ultracentrifugation. The concentrated lentiviral titer was assessed by RT-qPCR, and a standard curve was drawn according to the Cq value and the copy number. The lentiviral titers before and after concentration were 4.3×106 infection function units (IFU)/ml and 1.8×108 IFU/ml, respectively (Fig. 2B). Based on these results, the recombinant lentivirus carrying the EpCAM-CAR gene was considered to have been successfully packaged and concentrated.

The present study used the colon cancer cell lines SW620, SW480, HCT116 and LoVo, and the colorectal cancer cell line HT-29, as target cells to detect the antitumoral effects of EpCAM-CAR-T cells. All cell lines were authenticated using STR profiling, and all cells had a matching degree >95%. Western blotting and flow cytometry were used to detect EpCAM expression in these cancer cell lines. The results of the western blotting indicated that EpCAM protein expression was observed in all five cancer cell lines, and the molecular weight of the protein was 40 kDa. Among the lines, SW620 exhibited the highest expression, while HT-29 exhibited the lowest expression of EpCAM (Fig. 3A and B). The results of the flow cytometry showed that the expression rates of EpCAM on the surfaces of HCT116, HT-29, LoVo, SW620 and SW480 cells were 78.3, 67.3, 75.4, 97.5 and 85.4%, respectively (Fig. 3C).

To construct T cells that stably expressed EpCAM-CAR, T cells were transfected with recombinant lentivirus. The co-stimulatory domain 9/4-1BB and CD3 chain in the CAR can activate T cells and promote the proliferation of T cells following transfection of CAR into T cells. The results indicated that the number of cells increased over time, and T cells tended to become activated (Fig. 4A). On the 14th day following viral transfection, the expression of CAR mRNA in T cells was detected by RT-qPCR, confirming that CAR expression levels were higher in transfected cells than in control cells (Fig. 4B). In addition, the expression of CAR in T cells evaluated by protein immunoblotting confirmed that CAR was only expressed in transfected T cells, and its molecular weight was 53 kDa (Fig. 4C), which was consistent with the theoretical molecular weight (21). The expression of CAR on the surface of T cells facilitates recognition of and binding to antigens on the surface of tumor cells. Therefore, the expression of CAR on the surface of T cells was evaluated by flow cytometry. The results showed that CAR was expressed on the surface of 50.4% of transfected T cells (Fig. 4D), indicating that the transfected T cells stably expressed EpCAM-CAR.

In order to evaluate the antitumoral effect of EpCAM-CAR-T cells on cancer cells, SW620, SW480, HCT116, LoVo and the colorectal cancer cell line HT-29, all of which exhibit, different EpCAM expression rates, were used as target cells for EpCAM-CAR-T cells and untransfected T cells (effector cells). First, T cells were co-cultured with colon cancer SW620 cells, which exhibited the highest EpCAM expression, at E:T ratios of 0.5:1, 1:1, 2:1, 4:1, 8:1, and 16:1 for 4 h. The supernatant was then extracted for analysis of the cytotoxic effects of T cells at different effector ratios using an LDH release assay kit. The results showed that as the E:T ratio increased, the LDH release of the EpCAM-CAR-T cell group also increased, indicating that the antitumor effect of EpCAM-CAR-T cells was dependent on the number of EpCAM-CAR-T cells (Fig. 5A). Studies have shown that IL-2, IFN-γ and IL-6 have antitumor roles in tumor immunotherapy by regulating immune responses (22–24). Therefore, levels of the inflammatory cytokines IL-2, IL-6 and IFN-γ in the co-culture supernatants of EpCAM-CAR-T cells and SW620 cells were evaluated by ELISA. The results showed that the levels of these inflammatory cytokines released by EpCAM-CAR-T cell co-culture were significantly higher than those in the control group. In addition, cytokine levels increased as the E:T ratio increased (Fig. 5B-D).

The results indicated that the antitumor effects of EpCAM-CAR-T cells were strongest, and the release of cytokines was highest at an E:T ratio of 16:1 when T cells were co-cultured with colon cancer SW620 cells. Therefore, the remaining cell lines (SW480, HCT116, LoVo and HT-29) were co-cultured with T cells at an E:T ratio of 16:1 for 4 h. Compared with the control T cells, EpCAM-CAR-T cells exhibited a stronger antitumor effect on the four cancer cell lines (Fig. 6A). Furthermore, the levels of IL-2, IL-6 and IFN-γ released by EpCAM-CAR-T cells were also significantly higher than those released by control T cells (Fig. 6B-D). Moreover, the antitumor effect and inflammatory cytokine release may also be associated with the expression of EpCAM on the surface of cancer cells, as cell lines with higher expression rates of EpCAM appeared to exhibit more potent antitumor effects and to release higher levels of inflammatory cytokines (Fig. 6).