Adoptive T-cell therapy of prostate cancer targeting the cancer stem cell antigen EpCAM
© Deng et al.; licensee BioMed Central. 2015
Received: 17 July 2014
Accepted: 23 December 2014
Published: 31 January 2015
Adoptive transfer of tumor infiltrating or circulating lymphocytes transduced with tumor antigen receptors has been examined in various clinical trials to treat human cancers. The tumor antigens targeted by transferred lymphocytes affects the efficacy of this therapeutic approach. Because cancer stem cells (CSCs) play an important role in tumor growth and metastasis, we hypothesized that adoptive transfer of T cells targeting a CSC antigen could result in dramatic anti-tumor effects.
An EpCAM-specific chimeric antigen receptor (CAR) was constructed to transduce human peripheral blood lymphocytes (PBLs) and thereby enable them to target the CSC marker EpCAM. To investigate the therapeutic capabilities of PBLs expressing EpCAM-specific CARs, we used two different tumor models, PC3, the human prostate cancer cell line, which has low expression levels of EpCAM, and PC3M, a highly metastatic clone of PC3 that has high expression levels of EpCAM. We demonstrate that CAR-expressing PBLs can kill PC3M tumor cells in vitro and in vivo. Despite the low expression of EpCAM on PC3 cells, CAR-expressing PBLs significantly inhibited tumor growth and prolonged mouse survival in a PC3 metastasis model, probably by targeting the highly proliferative and metastatic population of cancer cells.
Our data demonstrate that PBLs expressing with EpCAM-specific CARs have significant anti-tumor activity against prostate cancer. Therefore, the adoptive transfer of T cells targeting EpCAM could have great potential as a cancer treatment.
Adoptive T-cell immunotherapy involves using ex vivo isolated and expanded autologous or allogeneic tumor-reactive lymphocytes to treat cancer patients. It has been highly effective in treating patients with metastatic melanoma and objective responses have been detected in 50% of patients [1,2].
Since tumor-infiltrating lymphocytes with tumor-specific receptors can only be generated from some cancer patients, adoptive T-cell therapy has been improved by introducing antigen receptors into circulating lymphocytes. To do this, genes encoding T-cell receptors isolated from high avidity, tumor-specific T cells or chimeric antigen receptors (CAR) containing an antibody-based external receptor structure and intracellular T-cell signaling domains, such as CD3ζ, are introduced into lymphocytes by retroviral or lentiviral vectors. Because CARs can induce T cells to attack tumors in an MHC-unrestricted manner, the application of adoptive T-cell therapy in cancer treatments has expanded. Currently, multiple clinical trials investigating CARs that recognize cell surface tumor antigens are underway, including for the treatment of lymphoma, chronic lymphocytic leukemia, melanoma, and neuroblastoma [3-5].
Cancer stem cells (CSCs) enable the tumor to grow and metastasize, therefore, eradicating CSCs is expected to provide cancer patients long-term disease-free survival. However, CSCs have also been demonstrated to be more resistant to chemotherapy and radiotherapy . Currently, the research on immunotherapies targeting CSCs is limited.
In this study, we developed a new adoptive immunotherapy that targets cancer stem cell antigen, epithelial cell adhesion molecule (EpCAM). Studies have shown that EpCAM is expressed on CSCs from breast, colon, pancreas, and prostate tumors [7-11]. In breast cancer, EpCAM+ CD44+ CD24− lineage− cells are 10 times more likely to form tumors than the EpCAM− CD44+ CD24− lineage− population . In addition, our previous studies show that EpCAM+ cells of the human prostate cancer cell line PC3 display higher proliferation rates than EpCAM− or unsorted PC3 cells. Interestingly, PC3M cells, a highly metastatic clone of PC3, express much higher levels of EpCAM than PC3, which suggests that EpCAM expression is associated with the proliferation and metastasis of prostate cancer cells.
In this paper, we show that human peripheral blood lymphocytes (PBLs) expressing EpCAM-specific CARs can kill PC3M cells in vitro and in vivo. Interestingly, despite the low expression level of EpCAM on PC3 cells, lymphocytes targeting EpCAM can cause significant killing and inhibit the metastasis of PC3 cells in NOD/SCID mice. This indicates that immunotherapies targeting CSCs, a small population of cancer cells, could result in distinct anti-tumor effects. Our results suggest that adoptive T-cell therapy targeting CSCs is a promising therapeutic strategy for cancer treatment.
In this study, we constructed a CAR targeting the cancer stem cell marker EpCAM and demonstrated that human PBLs transduced with the EpCAM-specific CAR can kill the prostate cancer cells PC3M and PC3, both in vitro and in vivo. Numerous clinical trials have been performed and are underway to examine the treatment of cancer with the adoptive transfer of tumor-reactive T cells; however, few studies have investigated the therapeutic potential of adoptive T-cell immunotherapy targeting CSCs. EpCAM is a marker that has been detected on CSCs from prostate cancer. In our study, we showed that EpCAM+ PC3 cells have higher proliferation rates than EpCAM− cells. In addition, a more metastatic clone of PC3, PC3M, expresses higher levels of EpCAM (Figure 1A and B). This suggests that EpCAM expression is associated with the proliferation and metastatic potential of PC3 cells. Therefore, despite the low expression of EpCAM on PC3 cells, targeting EpCAM may cause dramatic tumor-killing effects.
PBLs transduced with retroviruses encoding EpCAM-specific CARs displayed significantly increased cytotoxicity against PC3M cells when compared with PBLs transduced with control retroviruses (Figure 3A). However, control PBLs also displayed significant cytotoxic activity against tumor cells relative to the untreated group. This is probably caused by direct recognition between alloreactive T cells from the healthy donors and human leukocyte antigen expressed by tumor cells . The recognition between control PBLs and tumor cells is also reflected by the proliferation assay (Figure 3C), where cell proliferation was detected for the control PBLs co-cultured with PC3M cells.
At an E:T ratio of 2:1, PBLs expressing EpCAM-specific CARs lysed 73% of PC3M cells, whereas only 32% of PC3 cells were lysed (Figure 3A and Figure 5). The relatively low level of cytotoxicity detected for PC3 cells is probably because of its low expression of EpCAM. However, considering the association between EpCAM+ cells and PC3 cell proliferation and metastasis, targeting this small population of cells may cause dramatic tumor-killing effects. This is supported by the success of the CAR-expressing PBLs at protecting mice against PC3 development. Metastatic PC3 tumor cells were detected in the lung, peritoneal cavity, and bone of untreated mice and in mice treated with control PBLs, whereas treatment with EpCAM-specific PBLs significantly inhibited PC3 metastasis and prolonged mouse survival (Figure 6B-D).
The side effects of targeted therapies depend on the expression levels of the target in other tissues. Like other CSC antigens, EpCAM expression is not restricted to CSCs. As a cell adhesion molecule, EpCAM is expressed in a variety of epithelial tissues, which raises the concern that targeting EpCAM may lead to toxicity. Toxicity has been observed in several CAR trials, such as when targeting Her2/neu in colorectal cancer , or CD19 in B-cell malignancies . One possible way to reduce acute toxicity by CAR transuded T cells is to administer multiple small doses of T cells rather than one large dose. Another possibility is to insert suicide genes into the CAR, which enables the T cells to be deleted when severe toxicity is observed. It is possible that with a better understanding of EpCAM and its function in cancer cells and CSCs, a component of EpCAM signaling may be identified that will provide a better and more specific target for cancer therapy.
Our data demonstrate that the adoptive transfer of human PBLs with CARs specific for EpCAM can cause PC3M tumor cell killing in vitro and in vivo. Despite the low expression of EpCAM on PC3 tumor cells, EpCAM-specific PBLs had significant anti-tumor activity against PC3, probably by targeting the CSCs of prostate cancer. Our data suggest that adoptive transfer of T cells targeting CSC antigens is a promising therapeutic approach for treating cancer.
Developing retroviruses encoding an EpCAM-specific CAR
The EpCAM-specific CAR construct is similar to the FMC63-28z CAR (Genebank identifier HM852952.1), except the anti-CD19, single-chain variable fragment sequence is replaced with an anti-EpCAM fragment (sequence corresponds to Genebank identifier AJ564232.1). The construct was synthesized and inserted into a pLNCX retroviral vector. Retroviruses encoding the EpCAM-specific CAR or an empty pLNCX vector for controls were generated using the retrovirus packaging kit, Ampho (Takara), and a 293 T packaging cell line, following the manufacturer’s protocol.
PBL preparation and retrovirus transduction
For PBL preparation, donor blood was obtained from healthy volunteers with consent from the Institutional Review Board of the Cancer Institute, Chinese Academy of Medical Sciences, and written informed consent for participation in the study was obtained from participants. After centrifugation on Ficoll-Hypaque density gradients (Sigma-Aldrich), PBMCs were plated at 2 × 106 cells/mL in cell culture for 2 h and the non-adherent cells were collected. The cells were then stimulated for 2 d on a non-tissue-culture-treated 24-well plate coated with 1 μg/mL OKT3 (Biolegend) at 1 × 106 cells/mL and in the presence of 1 μg/mL of anti-human CD28 antibody (Biolegend). For retrovirus transduction, a 24-well plate was coated with RetroNectin (Takara) at 4°C overnight, according to the manufacturer’s protocol, and then blocked with 2% BSA at room temperature for 30 min. The plate was then loaded with retrovirus supernatants at 300 μL/well and incubated at 37°C for 6 h. Next, 1 × 106 stimulated PBLs in 1 mL of medium were added to 1 mL of retrovirus supernatants before being transferred to the pre-coated wells and cultured at 37°C for 2 d. The cells were then transferred to a tissue-culture-treated plate at 1 × 106 cells/mL and cultured in the presence of 100 U/mL of recombinant human IL-2.
PC3, PC3M, Hela, and 293 T cells were obtained from ATCC and were maintained in culture with DMEM medium (Gibco) supplemented with 10% FBS. PBLs were cultured in RPMI (Gibco) supplemented with 10% FBS, 1× nonessential amino acid, L-glutamine, sodium pyruvate, penicillin-streptomycin, and 0.1% β-mercaptoethanol. To establish PC3-luc, PC3M-luc, and Hela-luc stable cell lines, the luciferase gene was cloned from pGL4.17-luc/Neo and inserted into pLNCX retroviral vectors; the retroviruses were prepared as described above. To transduce the tumor cell lines, retrovirus supernatants were mixed with cell culture medium at a ratio of 1:1, which was added to the tumor cells at 60–70% confluence with 15 μg/mL polybrene. Twenty-four hours after transduction, the cells were split into ten plates and 400 μg/mL of G418 was added to the culture. Culture medium was changed every 2–3 d, and 10–14 d later selected cells were passaged and maintained in culture medium supplemented with 200 μg/mL of G418.
Male NOD/SCID mice, 5–8 weeks of age, were purchased from Vital River Laboratories, and used in compliance with institutional animal healthcare regulations. For the PC3M in vivo model, 5 × 105 PC3M-luc cells were intraperitoneally injected into mice and 5 d later 1 × 107 PBLs transduced with the CAR or control vector were injected. For the PC3 metastasis model, PC3-luc cells were injected intravenously at 5 × 106 cells/mouse and 6 h later 5 × 106 PBLs transduced with the CAR or control vector were injected intravenously. Live animal imaging was performed as described previously , briefly, the mice were intraperitoneally injected with 15 μg/μL of luciferin (Promega) in 200 μL and 10 min later luminescence imaging was conducted with an IVIS system (Xenogen/Caliper Life Sciences). For the in vivo experiments, five mice were used per group and each experiment was repeated at least twice.
Sorted or unsorted PC3 cells in 100 μL of medium were seeded in a 96-well plate at 2,500 cells/well; control wells received 100 μL of medium only. Ten microliters of CCK-8 solution (Dojindo) was added to each well and after 4 h of incubation at 37°C, the cell number was determined by measuring the absorbance at 450 nm using a microplate reader. Cells were cultured for 24, 48, and 72 h and a CCK-8 assay was performed at each time point. The absorbance was subtracted with that of the control well and the resulting OD450 at each time point was divided by the starting value to calculate the relative proliferation ratio.
Flow cytometry and cell sorting
PBLs were stained with FITC, PE, or Percp-Cy5.5 conjugated CD3, CD4, or CD8 antibodies (eBioscience). Fluorescence was measured using a FACS Calibur flow cytometer and was analyzed using Flowjo software. To detect CAR transduced cells, PBLs were stained with an optimal concentration of biotinylated protein L (GeneScript), followed by staining with PE conjugated streptavidin (eBioscience). A PE-conjugated anti-human EpCAM antibody (eBioscience) was used to stain the tumor cells PC3 and PC3M and a FACSAria II cell sorter was used to sort EpCAM+ and EpCAM− cells.
Luciferase-expressing tumor cells were seeded in a 96-well plate at 1 × 105 cells/well and PBLs transduced with retroviruses were added at different E:T ratios. After incubation at 37°C for 24 h, luciferin (Promega) was added at a final concentration of 0.3 mg/mL and cytotoxicity was determined by luminescence imaging.
CFSE proliferation assay
Retrovirus-transduced PBLs were labeled with CFSE (Invitrogen) according to the manufacturer’s protocol and incubated with tumor cells at an E:T ratio of 2:1. Three days later, cells were collected and stained with Percp-Cy5.5 conjugated CD8 antibodies (eBioscience) and were analyzed by flow cytometry. The analysis was carried out on the CD8+ population.
Data are presented as means ± standard error of the mean. To determine the significance of differences between samples or groups, a student’s t-test or two-way analysis of variance was used as indicated in the figure legends.
We thank Tao Xu for assisting with flow cytometry and Xiao Liang for assisting with luminescence imaging. This work was supported by grants from the Natural Science Foundation of China (No. 81101554) and the Chinese Academy of Medical Sciences.
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