Cancer Stem Cell Target Labeling and Efficient Growth Inhibition of CD133 and PD-L1 Monoclonal Antibodies Double Conjugated with Luminescent Rare-Earth Tb³⁺ Nanorods

Abstract

Rare-earth nanomaterials are being widely applied in medicine as cytotoxicity agents, in radiation and photodynamic therapy, as drug carriers, and in biosensing and bioimaging technology. Terbium (Tb), a rare-earth element belonging to the lanthanides, has a long luminescent lifetime, large stock displacement, narrow spectral width, and biofriendly probes. In cancer therapy, cancer stem cell (CSC)-targeted treatment is receiving considerable attention due to these cells’ harmful characteristics. However, CSCs remain barely understood. Therefore, to effectively label and inhibit the growth of CSCs, we produced a nanocomplex in which TbPO₄·H₂O nanorods were double conjugated with CD133 and PD-L1 monoclonal antibodies. The Tb³⁺ nanomaterials were created in the presence of a soft template (polyethylene glycol 2000). The obtained nanomaterial TbPO₄·H₂O was hexagonal crystal and nanorod in shape, 40–80 nm in diameter, and 300–800 nm in length. The nanorods were further surfaced through tetraethyl orthosilicate hydrolysis and functionalized with amino silane. Finally, the glutaraldehyde-activated Tb³⁺ nanorods were conjugated with CD133 monoclonal antibody and PD-L1 monoclonal antibody on the surface to obtain the nanocomplex TbPO₄·H₂O@silica-NH₂+mAb^CD133+mAb^PD-L1 (TMC). The formed nanocomplex was able to efficiently and specifically label NTERA-2 cells, a highly expressed CD133 and PD-L1 CSC cell line. The conjugate also demonstrated promising anti-CSC activity by significant inhibition (58.50%) of the growth of 3D tumor spheres of NTERA-2 cells (p < 0.05).

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, Tb³⁺ 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:

$$\mathrm{CE}\% = 100\% - \frac{\mathrm{amount} \mathrm{IgG} \mathrm{in} \mathrm{supernatant} \mathrm{solution}}{\mathrm{amount} \mathrm{total} \mathrm{input} \mathrm{IgG} } \times 100.$$

(1)

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% CO_2, 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% CO₂. 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:

$$\% \mathrm{survival} = \frac{OD\left(reagent\right)-OD\left(blank\right)}{OD\left(control\right)-OD \left(blank\right)}\times 100.$$

(2)

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.

3. Results

3.1. Characteristics of the Synthesiszed Nanomaterials

The morphologies of nano TbPO₄·H₂O and TMC are presented in Figure 1. From the FESEM images, TbPO₄·H₂O formed nanorods with diameter 30–40 nm and length 300–800 nm. After coating with silica, surfacing with –NH₂, and conjugating with mAb, the diameter of the complex slightly increased to 40–80 nm, but the length remained at 300–800 nm.

The designed TbPO₄·H₂O nanorods were also typical hexagonal crystals, as proven by the X-ray diagram (Figure 2).

To be suitable for biological labeling, created nanomaterials must be strongly luminescent after design. Therefore, the fluorescence of the obtained nano TbPO₄·H₂O and TMC was measured. The results in Figure 3 show that strong fluorescence levels of TbPO4·H₂O and TMC were excited at 355 nm and emitted at 545 nm. They also exhibit that the main emission peaks of the TbPO₄·H₂O product were at 488, 545, 586, and 620 nm, which correspond to the ⁵D₄−⁷F_j (J = 6, 5, 4, 3) transitions of Tb³⁺ ions. These results are consistent with the data reported by Lien et al. [10].

The FTIR spectra of TbPO₄·H₂O, TbPO₄·H₂O@silica-NH₂, TbPO₄·H₂O@silica-NH₂-mAb are shown in Figure 4. Curve (a) is the FTIR spectrum of TbPO₄·H₂O. Curves (b) and (c) (corresponding to the products TbPO₄·H₂O@silica-NH₂ and TbPO₄·H₂O@silica-NH₂-mAb, respectively) have the same profile as curve (a), which indicates strong absorption in the region 770–600 cm⁻¹. The two peaks at 660 and 600 cm⁻¹ are typically attributed to the Tb-O and PO₄^3- vibrations, respectively. In Figure 4, curve (a), we can also observe oscillations of the O-H bond at around 1600 cm⁻¹ and near 3600 cm⁻¹. In curves (b) and (c), the unique absorption peaks from internal vibration of the amino bands (1642 cm⁻¹) and the strong absorption band (3443 cm⁻¹) from symmetric and asymmetric N–H stretching vibration can be observed, which demonstrate the appearance of APTMS and mAb on the obtained TbPO₄·H₂O@silica-NH₂ and TbPO₄·H₂O@silica-NH₂-mAb. The strong absorption band in the region 1000–950 cm⁻¹ (two peaks at 1008 and 950 cm⁻¹) arises from Si-O-Si asymmetric vibration. Thus, it can be suggested that conjugation (linkage) between luminescent nanorods and mAb was formed in the TbPO₄·H₂O@silica-NH₂-mAb nanocomplex.

3.2. Probing NTERA-2 and CCD-18Co Cells with TMC Nanocomplex

As presented in

Table 1. The conjugated efficiency of TbPO₄·H₂O@silica-NH₂ nanorods with mAb against CD133 and PD-L1.

No.	Samples	Input mAb	Free mAb in Supernatant	CE
1	TbPO4·H2O@silica-NH2 -CD133 mAb	40 µg	0	100%
2	TbPO4·H2O@silica-NH2-mAb^CD133-mAb^PD-L1	40 µg	8–16 µg	60–80%

, the CE index, which reports the conjugation efficiency of TbPO₄·H₂O@silica-NH₂ nanorods with mAb against CD133 and PD-L1, was high, ranging from 60% to 100%.

To evaluate the binding ability of TMC to cells, cells were observed under a fluorescence microscope. According to Feng et al., NTERA-2 highly express CD133, so this cell line was chosen for this experiment [11]. As shown in Figure 5, NTERA-2 cells labeled with luminescent nanocomplex TMC expressed strong luminescence under fluorescence microscopy compared with the negative control (Figure 5). Although TbPO₄·H₂O@silica-NH₂ could bond with the cells, the bonds were weak; therefore, the fluorescence image was not bright.

The flow cytometry results also provided the percentage of probed cells using the TMC nanocomplex. CD133-FITC (Invitrogen; Carlsbad, CA, USA) served as a reference control. As shown in Figure 6, CD133 expression was found in about 95.83% ± 7.31% of NTERA-2 cells when stained with CD133-FITC. A similar result (97.77% ± 5.69%) was found in NTERA-2 cells that were incubated with TMC (p > 0.05). The percentage of CD133-positive cells was only 1.11% ± 0.06% for NTERA-2 cells incubated with unconjugated TbPO₄·H₂O nanorods.

CCD-18Co cells were also incubated with the nanorods conjugated with mAb under the same conditions. However, luminescent expression of CCD-18Co cells bound with nanomaterials was not noticed under the fluorescent microscopic observation. The results from flow cytometry analysis also showed a very low percentage of CD133-positive cells in the CCD-18Co cell population (0.85% ± 0.07%) which had been incubated with TMC (Figure 7). Lodi reported that the expression of CD133 in CCD-18Co cells is hardly noticeable [12]. Thus, this cell line served as the negative control for CD133 markers. These results prove that the TMC nanocomplex could specifically bind to CSCs.

3.3. Effect of TMC on the Proliferation of NTERA-2 and CCD-18Co Cells

The proliferation of NTERA-2 and CCD-18Co cells treated with TMC was assessed using the MTT assay. TMC showed the ability to inhibit the growth of NTERA-2 cells by up to 14.12% at the highest concentration of 10 µg/mL (

Table 2. The effects of TMC on the proliferation of NTERA-2 and CCD-18Co cells

Samples	% Proliferation
	NTERA-2	CCD-18Co
TM (TbPO4·H2O @silica-NH2)	92.23 ± 3.68	93.41 ± 2.19
TMC (TbPO4·H2O @silica -NH2 -mAb^CD133-mAb^PD-L1)	85.88 ± 5.76	89.67 ± 4.36
CD133-FITC (ThermoFisher)	87.63 ± 7.08	90.33 ± 2.41
Negative control	100	100

). The antiproliferative activity of TMC on CCD-18Co cells was slightly lower than that on NTERA-2 cells (P > 0.05). TbPO₄·H₂O@silica-NH₂ did not show any cytotoxicity on either NTERA-2 or CCD-18Co cells.

3.4. Effect of TMC on NTERA-2 Spheroids

One of the key features of CSCs in tumors is adaptive immune resistance. This unique characteristic helps CSCs escape destruction by immune cells such as lympho T and NK cells or macrophages, resulting in tumor progression and metastasis. This phenomenon is thought to be related to programmed cell death ligand 1 (PD-L1). PD-L1 is expressed on tumors and binds to programmed cell death 1 (PD1) on immune cells, leading to the inhibition of tumor-infiltrating lymphocytes (TILs) [13]. Therefore, PD-L1 blocking decreases the growth of tumors. In this study, we measured tumor growth inhibition due to the activity of TMC using the research model, which was 3D NTERA-2 spheroids co-cultured with macrophages. TMC showed the ability to inhibit the growth of 3D tumor spheroids (Figure 8). As a result of TMC application, the areas of the 3D spheroids were reduced by 58.50% ± 1.60%, which is a significant reduction in comparison with the negative control after three days of treatment (p < 0.05).

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 Tb³⁺ nanorods double conjugated with CD133 and PD-L1 mAb for the purpose of CSC labeling and therapeutic solution. TbPO₄·H₂O 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 TbPO₄·H₂O@silica-NH₂-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].

5. Conclusions

Tb³⁺ nanomaterials were produced using polyethylene glycol 2000 (PEG-2000) as a soft template. The obtained TbPO₄·H₂O nanomaterial was a hexagonal crystal and nanorod in shape, 40–80 nm in diameter, and 300–800 nm in length. These Tb³⁺ nanorods were further silica surfaced using tetraethyl orthosilicate (TEOS) hydrolysis and functionalized with amino silane to obtain TbPO₄·H₂O@silica-NH_{2 (}TM) materials. The glutaraldehyde-activated TM was double conjugated with CD133 and PL-D1 monoclonal antibodies to produce the nanocomplex TbPO₄·H₂O@silica-NH₂+mAb^CD133+mAb^PD-L1 (TMC). The formed nanocomplex presented highly efficient and specific labeling of NTERA-2 cells, a CSC cell line strongly expressing CD133 and PD-L1. The nanoconjugate also exhibited promising anti-CSC properties, including 58.50% inhibition of the growth of 3D tumor spheres of NTERA-2 cells, and its anti-tumor properties should be further tested in in vivo experiments.