Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2021 Aug 1.
Published in final edited form as: Phys Med. 2020 Jul 27;76:236–242. doi: 10.1016/j.ejmp.2020.06.024

Increased carcinoembryonic antigen expression on the surface of lung cancer cells using gold nanoparticles during radiotherapy

Romy Mueller a,b,c, Sayeda Yasmin-Karim c,d, Kaylie DeCosmo c,e, Ana Vazquez-Pagan f, Srinivas Sridhar g, David Kozono c, Juergen Hesser a,b,h,i, Wilfred Ngwa c,d,j
PMCID: PMC7500560  NIHMSID: NIHMS1617008  PMID: 32731132

Abstract

Purpose:

Tumor-associated antigens are a promising target of immunotherapy approaches for cancer treatments but rely on sufficient expression of the target antigen. This study investigates the expression of the carcinoembryonic antigen (CEA) on the surface of irradiated lung cancer cells in vitro using gold nanoparticles as radio-enhancer.

Methods:

Human lung carcinoma cells A549 were irradiated and expression of CEA on the cell surface measured by flow cytometry 3 hours, 24 hours and 72 hours after irradiation to doses of 2 Gy, 6 Gy, 10 Gy, and 20 Gy in the presence or absence of 0.1 mg/ml or 0.5 mg/ml gold nanoparticles. CEA expression was measured as median fluorescent intensity and percentage of CEA-positive cells.

Results:

An increase in CEA expression was observed with both increasing radiation dose and time. There was doubling in median fluorescent intensity 24 hours after 20 Gy irradiation and 72 hours after 6 Gy irradiation. Use of gold nanoparticles resulted in additional significant increases in CEA expression. Change in cell morphology included swelling of cells and increased internal complexity in accordance with change in CEA expression.

Conclusions:

This study showed an increase in CEA expression on human lung carcinoma cells following irradiation. Increase in expression was observed with increasing radiation dose and in a time dependent manner up to 72 hours post irradiation. The results also showed that gold nanoparticles can significantly increase CEA expression during radiotherapy.

Keywords: radiotherapy, nanoparticles, carcinoembryonic antigen, neoantigens

1. Introduction

The carcinoembryonic antigen (CEA), also known as CD66, is an onco-fetal antigen expressed on some tissues during fetal development. After birth, CEA levels drop to very low concentrations but circulates in high concentrations in adult patients with certain malignancies [1]. In its first description in 1965, CEA was assumed to be a tumor-specific antigen for colon cancer [2]. Today it is known for being a tumor-associated antigen in a variety of other cancers including lung [3], breast [3], colorectal [4], and cervical [5]; which are predominantly epithelial tumors.

The level of circulating CEA below 2.5 to 5.0 ng/ml is considered a normal value and consequently observed in healthy patients [1]. However, elevated levels of CEA even in healthy patients have been reported, for example, in smokers [6] and the elderly [7]. These increases in CEA caused by non-cancer related conditions renders CEA unsuited as a standalone cancer screening biomarker. Nevertheless, CEA still remains as a valuable prognostic marker for various cancers of the colon, stomach, breast, and lung, where CEA levels at the time of diagnosis relates to the stage of disease. In addition, CEA levels can be used to monitor treatment response and cancer recurrence. In 90 patients undergoing complete surgical resection of colorectal cancer, previously elevated CEA level dropped to normal range within 4 months post surgery for 84 patients. All remaining 6 patients for whom CEA levels did not drop had subsequent tumor recurrence [4].

Besides its applications as a prognostic and monitoring marker, CEA has been targeted for immunotherapy, but these strategies rely on sufficient expression of the target antigen. Research for therapeutic use is still ongoing. Most clinical trials targeting CEA require elevated CEA levels of more than 10 ng/ml as inclusion criteria for recruiting patients (NCT00923806, NCT00004085, NCT03682744, NCT02850536). Previous studies showed elevated levels of CEA following irradiation [8], making it a potential target for immunotherapy following radiotherapy. Besides using CEA-specific agents in radioimmunotherapy [9], use of T-cell bispecific antibodies [10,11], or CEA-targeting immunocytokines [12], CEA as a neoantigen could improve systemic immune responses by antitumor CD8-positive T-cells. T-cell responses are generated by antigen presenting cells, which reveal tumor-associated antigens to the T-cells. CEA and other neoantigens overexpressed after irradiation in combination with immunoadjuvants (which enhance activation of antigen presenting cells) have the potential to increase tumor control.

Besides adjuvant therapy approaches of radiotherapy with immunotherapy, recent advances have been made by combining radiotherapy with radio-enhancer such as nanoparticles (NPs) [13-18]. These nanoparticles consist of a high-atomic number material; which increase the locally deposited dose under ionizing radiation. The main mechanism for this dose enhancement is the higher photoelectric cross-section and corresponding emitted photo-/Auger electrons. The short range of this electron radiation leads to a nanoscopic deposition of energy. Recent studies highlight dose inhomogeneities around the NPs, suggesting sub-cellular localization of NPs might be an important factor for radio-enhancement [19]. Evidence of dose enhancement during radiotherapy by gold NPs (GNPs) was shown in vitro and in vivo. Hainfeld et al. [20] demonstrated in tumor-bearing mice a significant tumor volume reduction and prolonged survival in the cohort combining radiotherapy with GNPs. While gold is the most commonly studied high-atomic material (Z = 79) for NPs as radio-enhancer, other materials serve similar purposes, partially while offering additional features. Gadolinium for example, has an atomic number of 64 and provides, besides radio-enhancing properties, contrast during Magnetic Resonance Imaging [21]. These offer the possibility of theranostics, in which NPs serve a dual purpose of image-guidance for identifying the tumor as well as its radio-enhancing properties. However, choice of NP material is not only given by their physical properties, but in addition by their biocompatibility. Hafnium Oxide NPs for example have not only shown to have high energy deposition capacity, but their chemically inert behavior in cellular and subcellular systems has been demonstrated [22]. Biocompatibility and biological response can generally be achieved by surface coating but depend as well on size of NPs and their shape, known together as the three ‘S’ of NPs [23].

However, according to recent studies observed response can only be partially explained by the physical processes of radiosensitizing using GNPs [24-27]. Other biological and chemical impacts of GNPs on the cell cycle [28-30], induction of reactive-oxygen species [31,32], DNA damage and repair [33], as well its contribution to the bystander effects [34] are observed and discussed as supplementary mechanisms of GNP radiosensitization. Gaining a deeper understanding of these biological effects is an important step in applying GNP in clinical applications.

The study in this paper focuses on the expression of CEA on lung cancer cells in vitro. A human lung cancer cell line was chosen as it is one of the most common cancers worldwide with a high prevalence of forming metastases with a 5-year survival rate of 17% in the United states [35]. Furthermore, the cell line chosen, A549 is known for its radio resistance [36,37]. For the first time, CEA expression is quantified both in a radiation dose and time dependent manner, and potential effects of gold nanoparticles as a radio-enhancer on such expression are studied.

2. Materials and Methods

Tumor Cell Line

Human epithelial lung carcinoma cell line A549 was obtained from American Type Culture Collection (ATCC) and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific), supplemented with 10% Fetal Bovine Serum (FBS, Thermo Fisher Scientific) and 1% Penicillin/Streptomycin (Thermo Fisher Scientific). Throughout this study cells with a passage number between 12 and 37 were used. Cells were incubated at 37°C and 5% CO2.

Irradiation

Irradiation was performed on a Small Radiation Research Platform (SARRP, XStrahl) operated at 220kV, 13 mA. The first half value layer was measured to be 0.64 mm in copper. Cells were irradiated at the isocenter at a distance of 35 cm from the source at dose rate of 3.59 Gy/min as shown in figure 1. The field size at this depth is 11.5 cm x 10.0 cm. Dosimetry was performed using radiochromic films calibrated at the SARRP using an ionization chamber following the Task Group 61 report by the American Association of Physicists in Medicine (AAPM) [38]. Calibration was performed under same conditions and energy range as the irradiation as described in previous studies [39]. Dose rate delivered on the SARRP is verified on a monthly basis.

Figure 1:

Figure 1:

Open in a new tab

Setup for cell irradiation at the Small Animal Radiation Research Platform (SARRP).

Cells were pre-seeded to ensure they are in log-growth phase. On day of irradiation, cells were put in suspension following the protocol by Garnett et al. [8] at 2 · 106 cells per T-25 flask filled with 5ml media and irradiated using the SARRP at 2 Gy, 6 Gy, 10 Gy, and 20 Gy. For the analysis 24 hours and 72 hours post irradiation cells were washed in media and resuspended for further incubation. Cells for analysis after 3 hours were immediately processed for staining.

Flow Cytometric Analysis

Cells were harvested (24 and 72 hours) and washed twice in Phosphate Buffered Saline (PBS, Thermo Fisher Scientific) and resuspended in PBS supplemented with 1% Bovine Serum Albumin (BSA). Cells were diluted towards a final amount of 100,000 cells per sample. The antibody used for staining is FITC Mouse Anti-Human CD66 (BD Pharmingen), clone: B1.1/CD66. The corresponding isotype control is mouse IgG2a, K, clone: G155-178. Mouse Anti-human CEA antibody was used after determining the optimal titration to be one quarter of the recommended volume (5 μl). After excess antibody was washed out, Propidium Iodide (PI) in optimized titration was added as viability stain. Stained cells were acquired on a BD LSRFortessa analyzer (BD Bioscience) and analyzed using FCS Express 6 software (DeNovo).

Only viable cells were analyzed regarding CEA expression and all gates were concluded from a negative sample of the same measurement without staining. To diminish influence of the gating for CEA-positive cells, the CEA-positive signal was determined as inverse of CEA-negative signal for further analysis. Specificity of staining was confirmed using the corresponding isotype control.

Irradiation and staining in presence of Gold Nanoparticles

Spherical GNPs (GNPs) used in this study were provided following procedure described in Kumar et al. [40]. These are ultrasmall NPs with a core diameter of 2-3 nm, which were further PEGylated by a ligand exchange process and covalently conjugated with the fluorochrome AF 647. This increased the hydrodynamic diameter to an overall size of 12 nm as has been previously determined using dynamic light scattering [40]. These NPs have shown a robust uptake into cancerous cells with a strong cellular staining in the endoplasmatic reticulum, while no morphological damage to the cells were observed, indicating low toxicities of the GNPs [40].

Cells were seeded at 50,000 cells per well in a 6-well plate and treated after 24 hours with GNPs. NP concentrations were used in a concentration of 0.1 mg/ml and 0.5 mg/ml following previously published results [40], in which toxicity of this NP type was determined negligible for concentrations not exceeding 0.5 mg/ml. The final GNP solution is sonicated and mixed with the cell culture media. After an additional 24 hours cells post GNP incubation plates are irradiated with a total volume of 2 ml media including GNPs per well. 72 hours post irradiation cells are harvested and stained for viability using the Near-IR fixable viability stain (Thermo Fisher Scientific) at optimized titration, followed by staining for CEA and subsequent fixation in 10% formalin (Sigma).

Clonogenic Assay

Cell survival following irradiation was determined by the clonogenic assay following standard protocol [41] in duplication. Briefly, cells were seeded at 300 cells per well in a 6-well plate and after adherence, 24 hours post seeding, irradiated at 2 Gy, 6 Gy, 10 Gy, and 20 Gy or mock irradiated. Cells were maintained until colonies formed about 1.5 weeks after irradiation. At this point cells were fixed in 75% Ethanol 25% Water mixture and stained with Crystal Violet at a final concentration of 0.5%. After wells have been washed, colonies were counted, and survival fraction calculated.

Statistical Analysis

All experiments were performed in at least three independent experiments and are represented as mean ± SEM unless otherwise noted. Statistical significance was measured as a two-sided paired t test with a confidence interval of 95%.

3. Results

The CEA/FITC median fluorescent intensity for three independent experiments as a function of radiation dose up to 20 Gy for three time points post irradiation (3 hours, 24 hours, and 72 hours) are shown in figure 2A. The value observed after three hours post irradiation remains unchanged, whereas an increase with both increasing radiation dose and increasing time up to three days are observed. A large variability is observed, especially for the 72 hours sample, caused by a spread, which is potentiated with increasing radiation dose. However, the trend of increase with both increasing radiation dose and time post irradiation is clearly visible and exceeds the signal measured for the 24 hours sample. This spread is reduced while investigating the fold increase per repeat experiment before averaging, as shown in figure 2B. A two-fold increase is highlighted. Increased expression of CEA is observed 24 hours after 20 Gy irradiation and 72 hours after at least 6 Gy of ionizing radiation, whereas no change is observed for any radiation dose immediately after irradiation (three hours). This constant state of the cells short times after irradiation (three hours) is observed as well for the viability, which is unchanged up to 24 hours at a level of 85% (figure 3A). Only after 72 hours, is a drop in viability observed but remains at a high level of more than 60% even after radical irradiation up to 20Gy. This aggressiveness of the used human lung cancer cell line A549 is further confirmed by clonogenic survival, as shown in figure 3B.

Figure 2:

Figure 2:

Open in a new tab

CEA/FITC median fluorescent intensity. CEA/FITC median fluorescent intensity is shown for three independent experiments as function of radiation dose and time post treatment (A). The average fold increase (mean ± SEM) shows up to 6-fold higher median fluorescent intensity (B). The level of median intensity doubling is highlighted.

Figure 3:

Figure 3:

Open in a new tab

Viability and Survival of lung cancer cells following irradiation. Viability of irradiated cells at the moment of flow cytometric analysis (3, 24, and 72 hours post irradiation) as function of radiation dose (A). Survival of cells following irradiation is determined by clonogenic assay. Some error bars are not visible due to magnitude.

This increase in expression of CEA was continuously observed with increasing time and radiation dose as shown in figure 4, in which the change in CEA-positive cells is given relative to the cells undergoing mockirradiation. Whereas no change is observed early after irradiation (three hours), CEA expression increases with time and radiation dose in the evaluated regimens. A significant increase is observed from 3 hours to 24 hours at 10 Gy and 20 Gy and from 3 hours to 72 hours, respectively.

Figure 4:

Figure 4:

Open in a new tab

Increased expression of CEA with both increasing radiation dose and time up to 72 hours. Values are shown as mean ± SEM for three independent experiments.

The impact of irradiation on cell morphology is shown in figure 5. The histogram of the Forward Scatter (FSC), commonly associated to be proportional to cell size [42] shows no change immediately after irradiation. However, with increasing time and radiation dose the histogram shifts to higher forward scatter, suggesting cell swelling. A similar shift was observed for the Side Scatter (SSC), which is commonly associated with internal complexity [43] suggesting the disruption of internal cellular compartments by the radiation. As observed earlier, both shifts are only distinct beyond the 3 hours time point.

Figure 5:

Figure 5:

Open in a new tab

Scatter characteristic. Histogram of Forward Scatter (FSC) (A) and Side Scatter (SSC) (B) shows shift of cells toward higher amplitude with increasing radiation dose and time post irradiation.

Overexpression of CEA was further increased in presence of GNPs during irradiation (figure 6). Cells were incubated 24 hours prior to irradiation and analyzed 72 hours post irradiation according to previously acquired data. In addition to an elevation in CEA expression in absence of GNPs (figure 4 red bars and figure 6 black bars), significantly increased expression was shown for 0.5 mg/ml GNPs with 6 Gy and 10 Gy irradiation as well as for 0.1 mg/ml for 10 Gy irradiation. No significant increase could be seen with 20 Gy irradiation. Furthermore, this increased CEA expression was only observed with the combination of GNPs and radiation, but not for GNPs alone.

Figure 6:

Figure 6:

Open in a new tab

Increased expression of CEA following irradiation in presence of GNPs. Increased expression of CEA with and without 0.1 mg/ml or 0.5 mg/ml of GNPs 72 hours post irradiation. GNP = Gold Nanoparticle.

4. Discussion

In this study the expression of the carcinoembryonic antigen (CEA) on the human epithelial lung cancer cell line A549 was tested in vitro. Experiments show an increase in CEA expression with increasing radiation dose in a time dependent manner up to 72 hours post irradiation. This overexpression was evidenced by an increase in CEA/FITC median fluorescent intensity and in the percentage of CEA-positive cells. Change in cell morphology was observed as shift towards higher forward scatter and higher side scatter signal suggesting radiation-induced cell swelling and organelle disruption. These changes were observed in correlation with CEA expression on the cell surface. CEA expression was further elevated utilizing GNPs acting as radio-enhancer due to their high-atomic number and corresponding higher photoelectric effect and emission of Auger-electrons under irradiation.

The human lung cancer cell line A549 employed in this study is known for its radio resistance [36,37]. Survival rates observed in this study are in accordance with previously published results of about 10% survival after 10 Gy of irradiation [44,45].

Overexpression of CEA in vitro following irradiation has been shown previously on various cancer cell lines including breast [46], colon [8], gastric [47], prostate [8,46], and lung [8,46]. These studies focused on overexpression of CEA three or four days post irradiation following one radiation dose of 10 Gy or 20 Gy. Overexpression after this radiation treatment was confirmed in this study. Whereas most studies only investigated one radiation dose, the study on investigating expression of CEA on the gastric adenocarcinoma cells MKN45 indicated an increase in CEA expression in proportion to radiation dose (0 Gy, 5 Gy, 10 Gy, and 15 Gy) [47]. Results presented in this study support this finding and, for the first time, quantifies these results and investigates expression not only in a radiation dose, but in addition in a time dependent manner and in combination with a known radio-enhancer, GNPs.

Results presented in this study showed an increase in CEA expression following irradiation in a time-dependent manner (figure 2 and 4), with highest expression 72 hours post irradiation. During this course, higher expression for one time point was observed with increasing time post irradiation besides for the analysis 3 hours post irradiation. At this short time no overexpression of CEA was observed with any radiation dose. Based on this result, GNPs were investigated as radio-enhancer and CEA expression studied 72 hours after irradiation (figure 6). A significant boost compared to irradiation in absence of GNPs was observed, which was further increased with higher GNP concentration. However, in absence of radiation but presence of GNPs, the change in CEA-positive cells is negligible and in particular showing to be negative, instead of positive (compare figure 6, 0Gy). With 0.1 mg/ml and 0.5 mg/ml of GNPs this change was −0.39 and −6.87 percent points, respectively. This observation supports it is the radio-enhancing property of the GNPs that is increasing CEA expression. While irradiation in absence of GNPs showed increasing CEA expression with increasing radiation dose up to 20Gy, a drop in CEA overexpression is observed for irradiations higher than 10Gy in presence of GNPs. This might be an effect in which CEA-expression reaches a maximum, which, due to the radio-enhancing properties of the GNPs, is reached after a lower nominal radiation dose applied to the cells. Further studies will be needed to elaborate this conclusively.

Nanoparticle dependent uptake into cells has been observed early on with the emerging field of nanoparticle use in radiation therapy. Besides the well-known size and shape dependent intracellular uptake [48] many more factors and parameters have influence on localization and NP excretion. PEG coating of NPs, as used in this study, is commonly used to prevent NP interaction with its microenvironment extending NP circulation in-vivo. However, PEG density and length influence cellular NP uptake with a higher uptake for the lower PEG density, for which a shorter circulation time is reported [49]. Furthermore, recent studies showed not only influence of NPs itself and their incubation time, but in addition a cell line dependence [50]. In their study five cancer and one fibroblast cell line were investigated indicating strong cell line dependence as result of different cell internalization pathways. As a result, even the impact of functionalization on NP uptake is cell line dependent [51]. Taken together, further studies focusing on NP cellular internalization, localization, excretion, and pathways involved will allow to improve intracellular NP localization with the potential to increase CEA expression even further. These investigations should be performed under consideration of in-vivo applications, as shown for PEGylation requiring trade-off between circulation time and cellular uptake. Similar considerations must be made for the incubation times, for which longer incubation (on the time scale of a few hours) is generally favorable for NP inclusion, but in-vivo will be limited by NP redistribution.

In this study, significant CEA enhancement was observed for higher doses of radiation (6-20 Gy) typical used during stereotactic body radiotherapy. It is worth mentioning during typical conventional fractionated radiotherapy for lung cancer, lower doses of about 2 Gy of radiation are delivered per fraction over an extended time, adding to a total dose between 55 Gy to 75 Gy [52]. This impact of fractionation on CEA expression will need to be investigated.

The SARRP used for irradiations in this study operates at 220 kVp, which differs from conventional clinical LINACs commonly operated at 6 MV. In particular, the photoelectric effect is known to be less distinct at higher beam energies. However, significant photoelectric interactions have been reported including dose enhancements when using nanoparticles with LINACs including flattening filter free beams (FFF) [13,14,53] which are actively being considered for treatment [54]. Furthermore, clinical applications do not only include external irradiation by the use of LINACs, but internal radiations as employed by brachytherapy sources with lower energies. Brachytherapy is particularly an option for patients, for which dose escalation with external-beam RT is limited by the tolerance of surrounding normal tissues [55].

These findings could have significance in ongoing efforts combining radiotherapy and immunotherapy, by providing useful insights regarding timing as a factor for consideration when combining radiotherapy with immunoadjuvants. This supports the perspective in recent studies that sustained delivery of immunoadjuvants using nanoparticles or Smart Radiotherapy Biomaterials (SRBs) might be more effective in priming the abscopal effect [56] resulting in greater local and metastatic tumor eradication. The use of SRBs for sustained delivery of immunoadjuvants allows for contemporaneous presence of the adjuvant over time including the time when CEA and other neoantigens are at optimal levels for generating an immune response to boost local and metastatic cell eradication.

This study also shows that GNPs can enhance generation of neoantigens like CEA during radiotherapy. If generation of these antigens can engender an immune response as expected, then these results further support the notion that observed dose enhancement may only partially be explained by purely physical dose enhancement and that other mechanisms may contribute to observed therapy outcomes in presence of GNPs. More studies are needed to establish this conclusively.

5. Conclusions

The results presented in this study indicate that CEA expression during radiotherapy is dose and time dependent. This is important in considering potential applications in the timing of immunotherapy targeting CEA expression during radiotherapy. This study suggests that combining nanoparticles as radio-enhancer with radiotherapy has the potential to make the tumor more immunogenic by increasing expression of neo-antigens such as CEA.

  • Quantifying overexpression of Carcinoembryonic antigen following irradiation

  • Carcinoembryonic antigen expression during radiotherapy is dose and time dependent

  • Expression can be significantly boosted by employing gold nanoparticles

  • Results have significance in efforts to combine radiotherapy and immunotherapy

Funding and Acknowledgement:

Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number R01CA239042 and 1 R25 CA174650-01. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We also acknowledge funding support from the Dana-Farber/Harvard Cancer Spore in Lung Cancer.

We would like to thank Suzan Lazo and the Dana-Farber Flow Cytometry Core Facility for their technical support.

Footnotes

Conflict of interests:

Declarations of interest: none

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Bibliography

  • [1].Fletcher RH. Carcinoembryonic Antigen. Ann Intern Med 1986;104:66–73. [DOI] [PubMed] [Google Scholar]
  • [2].Gold P, Freedman SO. Demonstration of tumor-specific antigens in human colonic carcinomata by immunological tolerance and absorption techniques. J Exp Med 1965;121:439–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [3].Robbins PF, Eggensperger D, Qi C-F, Schlom J. Definition of the expression of the human carcinoembryonic antigen and non-specific cross-reacting antigen in human breast and lung carcinomas. Int J Cancer 1993;53:892–7. [DOI] [PubMed] [Google Scholar]
  • [4].Arnaud J, Koehl C, Adloff M. Carcinoembryonic antigen (CEA) in diagosis and prognosis of colorectal carcinoma. Dis Colon Rectum 1980;23:141–4. [DOI] [PubMed] [Google Scholar]
  • [5].Tendler A, Kaufman HL, Kadish AS. Increased carcinoembryonic antigen expression in cervical intraepithelial neoplasia grade 3 and in cervical squamous cell carcinoma. Hum Pathol 2000;31:1357–62. [PubMed] [Google Scholar]
  • [6].Clarke C, Hine K, Dykes P, Whitehead T, Whitfield A. Carcinoembryonic Antigen and Smoking. J R Coll Physicians Lond 1980;14:227–8. [PMC free article] [PubMed] [Google Scholar]
  • [7].Berardi R, Ruiz R, Becknell JW, Keonin Y. Does advance age limit the usefulness of CEA assays? Geriatr (Basel, Switzerland) 1977;32:86–8. [PubMed] [Google Scholar]
  • [8].Garnett CT, Palena C, Chakarborty M, Tsang K, Schlom J, Hodge JW. Sublethal Irradiation of Human Tumor Cells Modulates Phenotype Resulting in Enhanced Killing by Cytotoxic T Lymphocytes. Cancer Res 2004;64:7985–94. [DOI] [PubMed] [Google Scholar]
  • [9].Sahlmann C-O, Homayounfar K, Niessner M, Dyczkowski J, Conradi L-C, Braulke F, et al. Repeated Adjuvant Anti-CEA Radioimmunotherapy After Resection of Colorectal Liver Metastases: Safety, Feasibility, and Long-Term Efficacy Results of a Prospective Phase 2 Study. Cancer 2017;123:638–49. 10.1002/cncr.30390. [DOI] [PubMed] [Google Scholar]
  • [10].Bacac M, Fauti T, Sam J, Colombetti S, Weinzierl T, Ouaret D, et al. A Novel Carcinoembryonic Antigen T-Cell Bispecific Antibody (CEA TCB) for the Treatment of Solid Tumors. Clin Cancer Res 2016;22:3286–97. 10.1158/1078-0432.CCR-15-1696. [DOI] [PubMed] [Google Scholar]
  • [11].Osada T, Patel SP, Hammond SA, Osada K, Morse MA, Lyerly HK. CEA/CD3-bispecific T cell-engaging (BiTE) antibody-mediated T lymphocyte cytotoxicity maximized by inhibition of both PD1 and PD-L1. Cancer Immunol Immunother 2015;64:677–88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].Klein C, Waldhauer I, Nicolini VG, Freimoser A, Nayak T, Vugts DJ, et al. Cergutuzumab amunaleukin (CEA-IL2v), a CEA- targeted IL-2 variant-based immunocytokine for combination cancer immunotherapy: Overcoming limitations of aldesleukin and conventional IL-2- based immunocytokines. Oncoimmunology 2017;6:e1277306 10.1080/2162402X.2016.1277306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [13].Detappe A, Tsiamas P, Ngwa W, Zygmanski P, Makrigiorgos M, Berbeco R. The effect of flattening filter free delivery on endothelial dose enhancement with gold nanoparticles. Med Phys 2013;40 10.1118/1.4791671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [14].Berbeco RI, Korideck H, Ngwa W, Kumar R, Patel J, Sridhar S, et al. DNA damage enhancement from gold nanoparticles for clinical MV photon beams. Radiat Res 2012;178:604–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Ngwa W, Kumar R, Sridhar S, Korideck H, Zygmanski P, Cormack RA, et al. Targeted radiotherapy with gold nanoparticles: current status and future perspectives. Nanomedicine 2014;9:1063–82. 10.2217/nnm.14.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [16].Hao Y, Altundal Y, Moreau M, Sajo E, Kumar R, Ngwa W. Potential for enhancing external beam radiotherapy for lung cancer using high-Z nanoparticles administered via inhalation. Phys Med Biol 2015;60:7035–43. 10.1088/0031-9155/60/18/7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [17].Kunjachan S, Detappe A, Kumar R, Ireland T, Cameron L, Biancur DE, et al. Nanoparticle mediated tumor vascular disruption: a novel strategy in radiation therapy. Nano Lett 2015;15:7488–96. 10.1021/acs.nanolett.5b03073.Materials. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • [18].Ngwa W, Irabor OC, Schoenfeld JD, Hesser J, Demaria S, Formenti SC. Using immunotherapy to boost the abscopal effect. Nat Rev Cancer 2018;18:313–22. 10.1038/nrc.2018.6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].McMahon SJ, Hyland WB, Muir MF, Coulter JA, Jain S, Butterworth KT, et al. Nanodosimetric effects of gold nanoparticles in megavoltage radiation therapy. Radiother Oncol 2011;100:412–6. [DOI] [PubMed] [Google Scholar]
  • [20].Hainfeld JF, Slatkin DN, Smilowitz HM. The use of gold nanoparticles to enhance radiotherapy in mice. Phys Med Biol 2004;49:N309. [DOI] [PubMed] [Google Scholar]
  • [21].Sancey L, Lux F, Kotb S, Roux S, Dufort S, Bianchi A, et al. The use of theranostic gadolinium-based nanoprobes to improve radiotherapy efficacy. Br J Radiol 2014;87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Maggiorella L, Barouch G, Devaux C, Pottier A, Deutsch E, Bourhis J, et al. Nanoscale radiotherapy with hafnium oxide nanoparticles. Futur Oncol 2012;8:1167–81. [DOI] [PubMed] [Google Scholar]
  • [23].Burda C, Chen X, Narayanan R, El-Sayed MA. Chemistry and properties of nanocrystals of different shapes. Chem Rev 2005;105:1025–102. [DOI] [PubMed] [Google Scholar]
  • [24].Butterworth KT, McMahon SJ, Currell FJ, Prise KM. Physical basis and biological mechanisms of gold nanoparticle radiosensitization. Nanoscale 2012;4:4830–8. [DOI] [PubMed] [Google Scholar]
  • [25].Ionita P, Gilbert BC, Chechik V. Radical Mechanism of a Place-Exchange Reaction of Au Nanoparticles. Angew Chemie Int Ed 2005;44:3720–2. [DOI] [PubMed] [Google Scholar]
  • [26].Jain S, Coulter JA, Hounsell AR, Butterworth KT, McMahon SJ, Hyland WB, et al. Cell-specific radiosensitization by gold nanoparticles at megavoltage radiation energies. Int J Radiat Oncol Biol Phys 2011;79:531–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Rosa S, Connolly C, Schettino G, Butterworth KT, Prise KM. Biological mechanisms of gold nanoparticle radiosensitization. Cancer Nanotechnol 2017;8 10.1186/s12645-017-0026-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [28].Kang B, Mackey MA, El-Sayed MA. Nuclear targeting of gold nanoparticles in cancer cells induces DNA damage, causing cytokinesis arrest and apoptosis. J Am Chem Soc 2010;132. [DOI] [PubMed] [Google Scholar]
  • [29].Roa W, Zhang X, Guo L, Shaw A, Hu X, Xiong Y, et al. Gold nanoparticle sensitize radiotherapy of prostate cancer cells by regulation of the cell cycle. Nanotechnology 2009;20 10.1088/0957-4484/20/37/375101. [DOI] [PubMed] [Google Scholar]
  • [30].Muller X, Engelbrecht M, Slabbert J. P7. Effect on cell cycle proliferation rates of cell lines using gold nanoparticles. Phys Medica 2016;32. [Google Scholar]
  • [31].Zhang Z, Berg A, Levanon H, Fessenden RW, Meisel D. On the Interactions of Free Radicals with Gold Nanoparticles. J Am Chem Soc 2003;125:7959–63. 10.1021/ja034830z. [DOI] [PubMed] [Google Scholar]
  • [32].Nel A, Xia T, Mädler L, Li N. Toxic Potential of Materials at the Nanolevel. Science (80-) 2006;311:622–8. [DOI] [PubMed] [Google Scholar]
  • [33].Choudhury A, Cuddihy A, Bristow RG. Radiation and new molecular agents part I: targeting ATM-ATR checkpoints, DNA repair, and the proteasome. Semin Radiat Oncol 2006;16:51–8. https://doi.Org/10.1016/j.semradonc.2005.08.007. [DOI] [PubMed] [Google Scholar]
  • [34].Prise KM, O’sullivan JM. Radiation-induced bystander signalling in cancer therapy. Nat Rev Cancer 2009;9:351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [35].American Cancer Society: Cancer Facts & Figures 2016. Atlanta: 2016. [Google Scholar]
  • [36].Yang HJ, Kim N, Seong KM, Youn H, Youn B. Investigation of radiation-induced transcriptome profile of radioresistant non-small cell lung cancer A549 cells using RNA-seq. PLoS One 2013;8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [37].Wang Y, He J, Zhang S, Yang Q. Intracellular calcium promotes radioresistance of non--small cell lung cancer A549 cells through activating Akt signaling. Tumor Biol 2017;39. [DOI] [PubMed] [Google Scholar]
  • [38].Ma C-M, Coffey C, DeWerd L, Liu C, Nath R, Seltzer S, et al. AAPM protocol for 40–300 kV x-ray beam dosimetry in radiotherapy and radiobiology. Med Phys 2001;28:868–93. [DOI] [PubMed] [Google Scholar]
  • [39].Jermoumi M, Korideck H, Bhagwat M, Zygmanski P, Makrigiogos G, Berbeco R, et al. Comprehensive quality assurance phantom for the small animal radiation research platform (SARRP). Phys Medica 2015;31:529–35. 10.1158/1078-0432.CCR-15-0428.Bioactivity. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [40].Kumar R, Korideck H, Ngwa W, Berbeco RI, Makrigiorgos GM, Sridhar S. Third generation gold nanoplatform optimized for radiation therapy. Transl Cancer Res 2013;2 https://doi.Org/10.3978/j.issn.2218-676X.2013.07.02. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [41].Franken NA, Rodermond HM, Stap J, Haveman J, Van Bree C. Clonogenic assay of cells in vitro. Nat Protoc 2006;1. [DOI] [PubMed] [Google Scholar]
  • [42].Koch AL, Robertson BR, Button DK. Deduction of the cell volume and mass from forward scatter intensity of bacteria analyzed by flow cytometry. J Microbiol Methods 1996;27:49–61. [Google Scholar]
  • [43].Gant VA, Phillips I, Savidge G. The application of flow cytometry to the study of bacterial responses to antibiotics. J Med Microbiol 1993;39:147–54. [DOI] [PubMed] [Google Scholar]
  • [44].Xu Z, Yan Y, Xiao L, Dai S, Zeng S, Qian L, et al. Radiosensitizing effect of diosmetin on radioresistant lung cancer cells via Akt signaling pathway. PLoS One 2017;12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [45].Toomeh D, Gadoue S, Yasmin-Karim S, Singh M, Shanker R, Pal SS, et al. Minimizing the potential of cancer recurrence and metastasis by the use of graphene oxide nano-flakes released from smart fiducials during image-guided radiation therapy. Phys Medica 2018;55. [DOI] [PubMed] [Google Scholar]
  • [46].Gameiro SR, Jammeh ML, Wattenberg MM, Tsang KY, Ferrone S, Hodge JW. Radiation-induced immunogenic modulation of tumor enhances antigen processing and calreticulin exposure, resulting in enhanced T-cell killing. Oncotarget 2014;5:403–16. https://doi.org/1719 [pii]. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [47].Hareyama M, Imai K, Kubo K, Takahashi H, Koshiba H, Hinoda Y, et al. Effect of radiation on the expression of carcinoembryonic antigen of human gastric adenocarcinoma cells. Cancer 1991;67:2269–74. [DOI] [PubMed] [Google Scholar]
  • [48].Chithrani BD, Ghazani AA, Chan WC. Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Lett 2006;6:662–8. [DOI] [PubMed] [Google Scholar]
  • [49].Cruje C, Chithrani D. Polyethylene glycol density and length affects nanoparticle uptake by cancer cells. J Nanomed Res 2014;1. [Google Scholar]
  • [50].Ivošev V, Sánchez GJ, Stefancikova L, Abi Haidar D, Vargas CRG, Yang X, et al. Uptake and excretion dynamics of gold nanoparticles in cancer cells and fibroblasts. Nanotechnology 2020;31. [DOI] [PubMed] [Google Scholar]
  • [51].Jiménez Sánchez G, Maury P, Stefancikova L, Campion O, Laurent G, Chateau A, et al. Fluorescent radiosensitizing gold nanoparticles. Int J Mol Sci 2019;20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [52].Jeter MD, Komaki R, Cox JD. Radiation time, dose, and fractionation in the treatment of lung cancer. Adv. Radiat. Oncol. Lung Cancer, 2005, p. 67–75. [Google Scholar]
  • [53].Tsiamas P, Liu B, Cifter F, Ngwa WF, Berbeco RI, Kappas C, et al. Impact of beam quality on megavoltage radiotherapy treatment techniques utilizing gold nanoparticles for dose enhancement. Phys Med Biol 2013;58:451. [DOI] [PubMed] [Google Scholar]
  • [54].Ma C, Chen M, Long T, Parsons D, Gu X, Jiang S, et al. Flattening filter free in intensity-modulated radiotherapy (IMRT)--Theoretical modeling with delivery efficiency analysis. Med Phys 2019;46:34–44. [DOI] [PubMed] [Google Scholar]
  • [55].Nag S, Kelly JF, Horton JL, Komaki R, Nori D. Brachytherapy for carcinoma of the lung. Brachytherapy 2001;15. [PubMed] [Google Scholar]
  • [56].Moreau M, Yasmin-Karim S, Kunjachan S, Sinha N, Gremse F, Kumar R, et al. Priming the abscopal effect using multifunctional smart radiotherapy biomaterials loaded with immunoadjuvants. Front Oncol 2018;8:56 10.3389/fonc.2018.00056. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES