1. Introduction
In recent years, cancer studies have involved the consideration of cancer stem cell (CSC) theory. CSCs are a subpopulation of cells in tumors that have self-renewal, differentiation, and tumorigenicity abilities [
1]. These cells are related to therapy drug resistance, metastasis, and recurrent cancer [
2]. The identification of CSCs is based on typical cellular surface markers, such as Cluster of Differentiation 133 (CD133), CD44, CD24, and Aldehyde dehydrogenases (ALDH), of which CD133 appears in various types of cancer cells in solid tumors. This glycoprotein is among the most popular markers for isolation of CSCs [
3]. CD133, also known as prominin-1, is a cross-membrane glycoprotein. Evidence has shown that CD133 might be related to metastasis, tumorigenesis, and drug resistance. Therefore, CD133 is used not only as a specific surface antigen to detect and isolate CSCs, but also in therapeutic strategies [
4].
Another typical feature of CSCs is immunosurveillance resistance [
5]. Programmed death-ligand 1 (PD-L1) is also reported as a CSC surface marker which blocks PD-1 on the surface of T cells. Thus, PD-L1 limits the response of T cells, helping CSCs to escape the immune system for their growth and metastasis [
6]. Since 2014, PD-L1 monoclonal antibody has been clinically approved for anticancer immunotherapy worldwide.
CD133 and PD-L1 antibodies are reported to have the ability to detect and treat cancers, especially when combined with nanomaterials. Nanomaterials have improved the therapeutic index of clinical drugs by enhancing circulation time and increasing permeability and retention [
7]. Nanomaterials help with probing, tracking, homing, and studying CSCs’ behavior. In this area, rare-earth nanomaterials such as Terbium (Tb), a lanthanide, have attracted considerable attention. The advantages of lanthanide compounds include long luminescence lifetime, large stock displacement, and narrow spectral width, which are useful for fluorescent marking, probes, and sensors for use in tests and human body imaging [
8]. Nanoscale lanthanides are highly stable, and it is easy to fabricate and functionalize their surfaces using biological substances such as antigens, monoclonal antibodies, enzymes, and aptamers. These molecules can be used to improve therapeutic efficacy or for locating nanoparticles in vivo. Therefore, in this study, Tb
3+ was used to produce nanomaterials to double conjugate with the monoclonal antibodies against CD133 and PD-L1 for the purpose of biolabeling and growth inhibition of cancer stem cells, which were NTERA-2 pluripotent human embryonic carcinoma cells.
2. Materials and Methods
2.1. Materials
Cultured Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), Trypsin-EDTA, and antibiotics (antibiotics/antimycotics) were received from Invitrogen (Carlsbad, CA, USA). Human CD133 monoclonal antibody, PD-L1 monoclonal antibody, and CD133 antibody conjugated with FITC (CD133-FITC) were sourced from Thermo Fisher (Invitrogen; Carlsbad, CA, USA). Other chemicals were provided by Sigma Aldrich (St. Louis, MO, USA).
2.2. Preparation of TbPO4·H2O@silica-NH2 Nanomaterials
Terbium orthophosphate monohydrate (TbPO4·H2O): Tb(NO3)3·5H2O (Sigma, 99.9 %) was added to NH4H2PO4 solution (Merck) in the presence of polyethylene glycol 2000 (PEG-2000) and stirred for 3–12 h. The pH of the obtained solution was adjusted in the range of 4–12 by adding 10% NaOH solution before incubating at 200 °C for 24 h. The product (TbPO4·H2O) was centrifuged at 5900 rpm and washed with ddH2O before drying at 60 °C for 5–10 h. The nanomaterial was then coated with silica through a hydrolysis reaction with tetra ethyl orthosilicate (TEOS) (Aldrich, 99.99%). Briefly, TbPO4·H2O was added to a mixture solution containing TEOS, ethanol, acetic acid, and water and stirred for 15 min (TbPO4/TEOS molar ratio of 1:0.2). The solution was then centrifuged and washed three times with 33% ethanol solution. Glycerol solution (0.5 mL) was added to a hydrous mixture of ethanol containing TbPO4·H2O coated silica (TbPO4·H2O@silica) and stirred for 30 min. 3-aminopropyl trimethoxy silane (APTMS) was dispersed in ethanol before being mixed with TbPO4·H2O@silica solution (TbPO4·H2O@silica/APTMS molar ratio of 1:0.2) and stirred for 15 min to functionalize the surface with -NH2. The TbPO4·H2O@silica-NH2 (TM) materials were washed two times with ethanol, two times with ddH2O, and finally dispersed in phosphate buffer saline (PBS) (1X, pH 7).
2.3. Conjugation of TbPO4·H2O@silica-NH2 Nanomaterials with CD133 Monoclonal Antibody and PD-L1 Monoclonal Antibody (mAb)
The TbPO4·H2O@silica-NH2 nanomaterial in sodium phosphate solution was gently vortexed before adding 0.5% glutaraldehyde solution in a ratio of 1:0.5 (v/v) and mixed for 1 h at room temperature (RT) to disperse completely. The mixture was centrifuged and washed three times with PBS solution to remove glutaraldehyde. Then, 40 μg of CD133 antibodies (Thermo Fisher, Invitrogen, Carlsbad, CA, USA) was added into the 400 μL glutaraldehyde pre-activated TbPO4·H2O@silica-NH2 and incubated at 37 °C for 30 min. After incubation, the suspension was centrifuged at 6000 rpm for 5 min at 4 °C; the supernatant was retained to determine the amount of unconjugated antibodies in the combined efficiency study. The TbPO4·H2O@silica-NH2-mAb^CD133 residue after rinsing with PBS three times was continuously conjugated with mAb^PD-L1 by adding 40 μg of this PD-L1 mAb at 37 °C for a further 30 min. After a centrifuge step at 6000 rpm for 5 min at 4 °C, the supernatant solution was retained to determine the conjugation efficiency. The PBS washing residue of TbPO4·H2O@silica-NH2-mAb^CD133-mAb^PD-L1 (TMC) nanocomplex was reconstituted in PBS and stored at 4 °C before being used for further experiments.
Conjugation efficiency was measured through the indirect detemination of unbound IgG in the supernatant after combining mAb with nanomaterials using a NANOPHOTOMETER P300 system (IMPLEN.INC., USA). The conjugated efficiency was calculated using the following fomula:
2.4. Characterization of the Obtained Nanocomplex
The morphology of nanomaterials was observed by field emission scanning electron microscopy (FESEM, Hitachi). The structure of the material was determined using an X-ray diffraction measuring system (Siemens D5000 with λ = 1.5406 Å, diffraction angle in the range of 15° ≤ 2θ ≤ 75°). Infrared spectra of the samples were measured on a NICOLET impact 410 Fourier transform infrared spectrometer (FTIR). The fluorescence spectrum of the product was measured at a wavelength of 355 nm by using the Horiba Jobin Yvon IHR 320 (USA) system at Hanoi Polytechnic University, and some samples were measured on the Horiba Jobin Yvon IHR 550 system (USA) at the Institute of Materials Science, Vietnam Academy of Science and Technology (VAST).
2.5. Cell Culture
In this study, the NTERA-2 cell line, which is a pluripotent human embryonic carcinoma cell line, served as CSCs and CCD-18Co cells (the human colon normal) were used as healthy cells. These cell lines were kindly provided by Dr. P. Wongtrakoongate, Mahidol University, Thailand and Prof. Chi-Ying Huang, National Yang-Ming University, Taiwan. Cells were maintained in DMEM medium supplement with 10% fetal bovine serum and 1% antibiotics (antibiotics/antimycotics solution, Invitrogen, Carlsbad, CA, USA) in incubator at 37 °C, 5% CO2, and 100% humidity.
2.6. Observing and Imaging TMC-Nanocomplex-Labeled Cells
Cells at log phase were seeded into 96-well plates with a concentration of 10,000 cells/well and incubated at 37 °C, 5% CO2 for 24 h. The culture medium was removed, then cells were fixed with 10% formaldehyde for 10 min at RT. TMC nanocomplex (10 µL) was dilluted in 190 µL of PBS before it was added into each well and incubated at 4 °C for 1 h. The unbound TMC were removed and washed with PBS three times. At the end of the process, PBS was added to the wells before the cells were observed under an Olympus Scan ^R fluorescence microscope (Olympus Europa SE & Co.KG, Hamburg, DE).
2.7. Detecting the TMC-Nanocomplex-Labeled Cells by Flow Cytometry
NTERA-2 cells and CCD-18Co cells at log phase were harvested with trypsin-EDTA and collected into a Falcon tube. Cells were re-suspended with DMEM medium containing 2% FBS and separated into several tubes, then TMC nanocomplex was added to the cells and incubated at 4 °C for at least 15 min and protected from light. After that, cells were washed three time with PBS to remove the unbound TMC. The cells incubated with CD133 mAb conjugated FITC (Thermo Fisher, Invitrogen; Carlsbad, CA, USA) served as a reference control. The numbers of labeled and luminescent cells in 10.000–12.000 counting cells were measured and analyzed using the Novocyte flow cytometry system and NovoExpress software (ACEA Bioscience Inc.).
2.8. TMC Cytotoxicity Determination
MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ) assay was used for measurement of the cytotoxic activity of the TMC nanocomplex. This method is based on the formation of formazan by MTT relating to the effectiveness of enzymatic activities in viable cells [
9]. Briefly, cells were seeded in 96-well plates (10,000 cells/well) and treated with
TMC at various concentrations of corresponding 0.08, 0.4, 2, or 10 µg/mL of PD-L1 mAb amounts, for 72 h at 37 °C, 5% CO
2. The experiments were performed in triplicate to ensure accuracy. Then, 10 µL fresh MTT (5 mg/mL) was added to the each well of the experimental plate and incubated at 37 °C. After 4 h, all medium was discarded and the formazan crystal formations were dissolved by adding 50 µL/well DMSO 100%. The OD values were measured at 540 nm using a spectrophotometer (BioTek, ELx800). The number of surviving cells was calculated by the formula:
2.9. Effective of TMC on the Growth of Tumor Spheroids Co-Cultured with Macrophages
Macrophages were isolated from the peritoneum of healthy BALB/c mice using a Macrophage mouse Isolation Kit (Peritoneum) (Miltenyi Biotech., Bergisch Gladbach, Germany). The isolation cells were cultured in DMEM containing 10% FBS, 1% antibiotics and incubated at 37 °C and 5% CO2.
In order to form 3D tumor spheroids, the hanging drop method was used. NTERA-2 cells (1500 cells) in 20 µL medium were dropped onto the underside of the lid of a 60 mm tissue culture dish. The lids were then inverted onto 5 mL medium-filled bottom dishes and incubated at 37 °C, 5% CO2, 95% humidity. After 3 days of incubation, cell aggregates were formed.
The obtained spheroids were then co-cultured with macrophages in 96-well plates. Wells were covered by 1% agarose before spheroids were transferred to the wells. The macrophage cells were then co-cultured with the spheroids in the wells. The TMC treatment was executed by directly adding TMC into the co-culture wells and further incubating for 3 days. The growth of spheroids was observed under microscopy. The images were analyzed using ImageJ software to determine the growth area of the spheroids and to compare with the negative control.
2.10. Statistical Analysis
The data are reported as mean ± standard deviation (SD), which were analyzed using GraphPad Prism 7 software and unpaired t-tests. p < 0.05 was considered to indicate statistical significance.
4. Discussion
Lanthanides have now been widely applied in medicine. Their applications include therapy and imaging. Unlike other metals, lanthanides are luminescent, stable, and biosafe [
14]. Among the lanthanides, Tb is a typical lanthanide with strong green fluorescence and has potential for biomedical labeling or imaging. This material has also been studied for use as a carrier for drugs such as a measles virus antibody [
15] or cobra venom antigens [
10]. Due to the advantages of Tb, we chose this material to produce a nanocomplex,
TMC, which is Tb
3+ nanorods double conjugated with CD133 and PD-L1 mAb for the purpose of CSC labeling and therapeutic solution. TbPO
4·H
2O formed nanorods 30–50 nm in diameter and 300–800 nm in length. After surface functionalization, the nanorods were successfully double conjugated with CD133 and PD-L1 mAb with high efficiency (60%–100%). The labeling ability of
TMC to detect CSCs is equivalent to that of the reference CD133-FITC. However, CD133 is also expressed in stemlike cells throughout the body. Thus, mAb against other CSC-specific markers such as EpCAM, CD44, CD24, etc., will be double conjugated with our TbPO
4·H
2O@silica-NH
2-mAb^CD133 (instead of PD-L1 mAb) in order to improve the specific targeting activity of the nanocomplex for fundamental CSC research or for future clinical applications.
Together with CSC probing,
TMC with PD-L1 mAb was produced in a structure selected for the purpose of cancer treatment. PD-L1 is a ligand of PD-1, and the interaction of PD-L1 and PD-1 in immune cells may cause inactivation of these cells [
16]. However, PD-L1 acts as an antiapoptotic receptor in response to Fas ligation. Therefore, PD-L1 is closely related to cancer stem cell proliferation [
17]. PD-L1 antibodies are commercially available to clinically treat several types of cancer [
18]. Herein, although
TMC only slightly inhibited the growth of NTERA-2 cells in vitro, the nanocomplex strongly inhibited the growth of these 3D NTERA-2 spheroids when co-cultured with macrophages. According to Genevieve, PD-L1 monoclonal antibodies enhance the ability of macrophages to proliferate and activate, leading to increased numbers of TAM (tumor-associated macrophages) and thereby inhibiting the growth of tumor tissues [
17].