Despite tremendous advances in both clinical and basic research, cancer remains one of the leading causes of mortality and a major public health problem worldwide [1
]. Many tumors continue to have a poor prognosis even after conventional therapies such as surgical treatment, radiation, and chemotherapy. Thus, biological therapy has become a novel and promising strategy for cancer treatment. The definition of biological therapy is the use of living organisms, substances derived from living organisms, or laboratory-produced versions of such substances to treat disease [2
]. Current biological therapy for cancer includes gene therapy, targeted therapy, and immunotherapy such as vaccines, cytokines, and antibodies. Nonetheless, one of the major obstacles encountered in cancer biological therapy is the inefficient delivery of therapeutic agents into the tumor lesions, especially the small metastatic or relapsed tumors. Previously, we have demonstrated that, after systemic delivery, bone marrow-derived mesenchymal stem cells (BMSCs) could migrate to microscopic tumor lesions and engraft into tumor stroma [3
]. The tumor tropism of mesenchymal stem cells (MSCs) has been established in various cancer types [4
], which, thus, made MSCs potential vehicles for delivering anti-cancer agents [9
Taking advantage of the tumor-homing and infiltrating abilities of MSCs, we developed a unique cancer immunotherapeutic platform by combining MSCs with an antigen-specific protein vaccine. We utilized human papillomavirus type 16 (HPV-16) E6/E7-immortalized BMSCs as an E7 antigen-delivering vehicle to mediate the antitumor effect of the E7 antigen-specific protein vaccine on non-E7 expressing fibrosarcoma cells [13
]. However, there are some limitations of the previous platform for future clinical application. First, the MSCs used in the previous platform were immortalized by HPV-16 E6/E7 genes, which raises safety concerns in clinical utilization. Although the immortalized MSCs have been characterized to be non-tumorigenic in advance [14
], such immortalized MSCs are inappropriate for clinical application due to the unforeseen outcome of long-term usage. Hence, in the current study, we aim to establish the antigen-delivering vehicle with a modified-E7 antigen, which is a non-oncogenic protein, and freshly prepared primary MSCs. Moreover, we used adipose tissue as the cell source of MSCs in this study. The main advantage of adipose-derived stem cells (ADSCs), over MSCs derived from other sources (e.g., from bone marrow), is that they can be easily and repeatable harvested by utilizing minimally invasive techniques with low morbidity [15
Second, by using MSCs, we successfully expanded the therapeutic spectrum of the E7-specific protein vaccine in non-E7 expressing tumors in the previous study [13
]. To prove that the combination of antigen-delivering MSCs with an antigen-specific protein vaccine has the potential to serve as a universal treatment for different cancer types, we aim to expand the therapeutic spectrum of this unique cancer immunotherapeutic platform in colon cancer and lung cancer, which are two of the most common cancers worldwide [16
]. Furthermore, the immunological mechanisms underlying tumor inhibition are unclear. Hence, in the current study, we also aim to investigate the role of different immune cells involved in the anti-tumor responses.
Mesenchymal stem cell (MSC)-based therapies offer a promising strategy that provides alternative therapeutic solutions, for various diseases, to repair and regenerate tissues and organs [18
]. In addition, MSCs are emerging as potential vehicles for delivering anti-cancer agents due to their inherent tumor homing capacity [3
]. The most common strategy of MSC-based therapies for cancer is to genetically modify them with tumor suppressor genes, anti-angiogenic agents, and immunomodulating cytokines [19
]. Other therapeutic approaches include MSC-mediated gene directed enzyme prodrugs, or their loading with anticancer drugs/ nanoparticles [20
]. Previously, we have developed a unique cancer immunotherapeutic platform by combining tumor-targeting MSCs with a protein vaccine [13
]. We utilized E6/E7-immortalized MSCs as an E7 antigen-delivering vehicle to expand the therapeutic spectrum of the E7 antigen-based protein vaccine. However, such immortalized MSCs are associated with many safety concerns and are inappropriate for clinical application. Therefore, in this study, we re-established the cancer immunotherapeutic platform by using freshly prepared primary ADSCs and a syngeneic tumor model to mimic the future clinical application. Additionally, we modified the E7 antigen of ADSCs (indicated as E7’), as a non-oncogenic protein with mutation, in the R6 binding site. Either subcutaneously co-inoculated with cancer cells or systemically administered after tumor growth, the ADSC-E7’-eGFP showed significant antitumor activity when combined with the protein vaccine.
MSCs are multipotent stem cells that can self-renew and differentiate into a variety of cell lineages, and were first isolated and identified from bone marrow [21
]. Currently, MSCs can routinely be isolated from several tissue niches in the body, such as fat, muscle, tendon, umbilical cord blood and amniotic fluid [22
]. MSCs from distinct origins have revealed variable growth potentials; however, they share a similar surface marker profile and multilineage differentiation capacity. It is well documented that adipose tissue is a rich source of MSCs, and, compared to other sources, the clinical procedure to harvest them is minimally invasive and less painful [23
]. Hence, due to their convenient acquisition and innate tumor tropism, ADSCs have attracted much recent attention as a promising vehicle for delivering anti-cancer molecules. Studies have shown that the genetic modification of ADSCs with interferon β (IFN-β) inhibits the growth of melanoma cells in vitro as well as in vivo. Moreover, the antitumor activity of IFN-β-expressed ADSCs was increased when combined with low-dose cisplatin [24
]. The antitumor effect of ADSCs modified with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), one of the most promising therapeutic pro-apoptotic cytokines, has also been demonstrated in several types of tumors [25
]. In addition to IFN-β or TRAIL, there are several other tumor-suppressor genes and cytokines with anticancer activities, such as CXCL10 [29
], IL-12 [30
], and pigment epithelium-derived factor (PEDF) [31
], which are utilized for the genetic modification of ADSCs. These ADSCs have also been employed as delivery vehicles in prodrug cancer gene therapy, including herpes simplex virus type 1 thymidine kinase (HSV1-TK)/ganciclovir (GCV) [32
], cytosine deaminase (CD)/5-fluorocytosine (5-FC) [33
], cytosine deaminase-uracil phosphoribosyl transferase (CD/UPRT)/5-FC [34
], and rabbit carboxylesterase(rCE)/irinotecan-7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) [35
]. Based on previous studies, demonstrating the therapeutic potential of modified MSCs, we isolated primary ADSCs and transfected them with the E7’ vector in order to utilize it as an antigen-delivering vehicle in our cancer immunotherapeutic platform.
An ideal cancer treatment should be capable to specifically discriminate normal and cancer cells and systemically eradicate multiple tumor lesions in the body. Hence, due to the induction of tumor-specific reactions, antigen-specific immunotherapy is a potentially attractive cancer treatment modality. However, such a strategy was limited by tumor variants that lacked the target antigen and resisted the specific immune response. To overcome the main obstacles encountered in cancer immunosurveillance, we previously utilized immortalized BMSCs as an E7 antigen-delivering vehicle to mediate the antitumor effect of the E7 antigen-based protein vaccine on non-E7 expressing fibrosarcoma cells [13
]. In this study, we further expanded the therapeutic spectrum of the E7 antigen-based protein vaccine in colon and lung cancer cells by using E7’-expressing ADSCs. In a study by Liao et al., the PE(ΔIII)-E7-KDEL3 protein vaccine was proved to elicit its antitumor effect through all venues of immunological responses, including CD4+ T, CD8+ T, and natural killer (NK) cells [17
]. We also previously showed that the combined treatment of MSCs and PE(ΔIII)-E7-KDEL3 induced CD4+ T cell activation via major histocompatibility complex (MHC) class II molecules in vitro [13
]. Nonetheless, here we showed that CD4+ T, CD8+ T, and NK cells contributed to the antitumor activity of the combined treatment of ADSCs and the protein vaccine by in vivo antibody depletion. We also suggest that the antitumor effect of the combined treatment might be achieved by CD8+ T cells. Although CD4+ T cells and MHC class II molecules are associated with the tumor antigen-specific immune response [36
], the tumor antigens have been shown to present predominantly in association with MHC class I molecules, and to be recognized by tumor-specific CD8+ T cells [40
]. In addition, MSCs have been demonstrated to exert antigen-presenting properties to activate CD4+ T cells via MHC class II molecules upon interferon gamma (IFN-γ) stimulation [42
]. François et al. further identified that MSCs could cross-present exogenous antigens and induce an effective CD8+ T-cell immune response both in vitro and in vivo [44
]. These data strongly suggest that MSCs could behave as conditional antigen-presenting cells (APCs) to activate antigen-specific immune responses. This unique property of MSCs render them as a potential cell-based immune biopharmaceutic for cancer treatment.
Taken together, we demonstrated that the freshly prepared primary ADSCs could be genetically modified as an antigen-delivering vehicle to expand the therapeutic spectrum of the antigen-specific protein vaccine. Further, the combination of ADSC-E7’-eGFP cells with the PE(ΔIII)-E7-KDEL3 protein vaccine significantly inhibited the tumor growth of CT26 and LLC1 cells, through apoptotic activity in addition to the reduction of tumor angiogenesis. The activated immune system was mediated by CD4+ T and NK cells, whereas the antitumor activity was mainly contributed to by CD8+ T cells. These results provide strong evidences that support this promising immunotherapeutic platform for future clinical application in cancer therapy.
4. Materials and Methods
4.1. Cell Lines
LLC1 (ATCC CRL-1642, ATCC, Taipei, Taiwan) Lewis lung carcinoma cells and CT26 (ATCC CRL-2638, Taipei, Taiwan) colon cancer cells were infected with FUW-Luc-mCh-puro lentiviral particles and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco, Mexico City, Mexico), 100 units/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin B, and 2 μg/mL puromycin in a humidified atmosphere, with 5% CO2 at 37 °C, to stably express firefly luciferase and mCherry fluorescent protein.
4.2. Isolation, Culture, and Lentiviral Transduction of ADSCs
ADSCs were isolated and cultured as previously described [45
]. The E7’ antigen and enhanced green fluorescent protein were expressed in ADSCs by using lentiviral transduction. The lentiviral vector (pLL3.7-E7’-eGFP) and the packaging constructs (pMDL g/p RRE, p RSV-REV, and pMD2.G) were transfected into 293FT cells (Invitrogen, Fisher Scientific, Carlsbad, CA, USA) with Lipofectamine 2000 Transfection Reagent (Invitrogen). Infectious viral particles were collected 48 h after transfection. ADSCs were infected with appropriate virus titers in media containing 8 μg/mL polybrene. After 24 h of infection, the medium of ADSCs were replaced with complete growth medium.
4.3. Preparation and Vaccination of Protein Vaccines
The preparation of the PE(ΔIII)-E7-KDEL3 protein vaccine and the vaccination in mice were conducted as previously described [13
]. Briefly, mice were immunized with 0.1 mg/mouse PE(ΔIII)-E7-KDEL3, mixed with 10% ISA206 adjuvant, by subcutaneous injection into the backs of the mice. These animals were then boosted, subcutaneously, 1 and 2 weeks later using the same regimen.
4.4. Animal Studies
All animal studies were approved by The Institutional Animal Care and Use Committee (IUCAC) of Taipei Medical University (Approval no. LAC-2014-004; 16 January 2014). Six-week-old female BALB/c and female C57BL/6 mice were purchased from the National Laboratory Animal Center and the National Applied Research Laboratories (Taipei, Taiwan). The mice were housed under pathogen-free conditions and fed autoclaved food and water. Tumor xenografts were established by subcutaneous injection of 2 × 105 LLC1 cells or 2 × 105 CT26 cells. ADSCs were administered by subcutaneous co-inoculation with cancer cells at the same time, or by intravenous injection 3 days later. Mice were first immunized with the PE(ΔIII)-E7-KDEL3 protein vaccine 7 days after tumor inoculation, and received boost shots 1 and 2 weeks later.
4.5. Bioluminescence Imaging (BLI)
BLI of animals was performed with an IVIS Imaging System 200 Series (PerkinElmer, Waltham, MA, USA) and quantitated with Living Image® software by measuring photon flux (photons/s/cm2/steradian) in regions of interest drawn around appropriate signals. For in vivo BLI, anesthetized mice (n = 5) were injected intraperitoneally with 75 mg/kg of D-Luciferin, and images were acquired 2 to 5 min after injection. Acquisition times were 2 min initially, and were reduced in accordance with signal intensity to avoid saturation.
Tumor tissues were fixed in 10% neutral buffered formalin, processed, and embedded in paraffin. For immunohistochemistry, tissue sections were incubated overnight with an anti-GFP antibody. The primary antibodies were detected using ABC and DAB substrate kits (Vector, Burlingame, CA, USA) and the sections were counterstained with hematoxylin. For immunofluorescence, issue sections were incubated overnight with anti-CD31 or anti-VEGF PE-conjugated antibodies.
4.7. TUNEL Assay
After 28 days of CT26 and LLC1 inoculation, a TUNEL assay was performed using DeadEnd™ Fluorometric TUNEL System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, subcutaneous tumor sections from the mice of the combined-treatment group or the stem cells only group were made permeable with 20 μg/mL of proteinase K for 10 minutes at room temperature, and the fragmented DNA was labeled using the TdT (terminal deoxynucleotidyl transferase) reaction mixture, containing fluorescein-12-dUTP, for 1 hour at 37 °C, according to supplier recommendations. The slides were mounted in VECTASHIELD Antifade Mounting Medium with DAPI (Vector). The results were expressed quantitatively by the number of apoptotic cells per field of view.
4.8. In Vivo Antibody Depletion
For in vivo antibody depletion, mice were treated via intraperitoneal injection with 500 μg/day of anti-CD4 (clone GK1.5), anti-CD8 (clone 53-6.7), or anti-NK1.1 (clone PK136) antibodies for 3 consecutive days.
4.9. Statistical Analysis and Replicates
The sizes of the sample groups in all data were at least n
= 5, unless otherwise indicated. All data presented were representative of at least three independent experiments that yielded similar results. Statistical analyses were performed using GraphPad Prism 5 (https://www.graphpad.com/support/prism-5-updates/