Nanotechnology in Radiation Oncology

Introduction

Clinical medicine has been a continually progressing branch of science which incorporates breakthrough technologies from multiple disciplines. Nanotechnology, the manipulation of matter on atomic and molecular scales, has made remarkable contributions in clinical oncology over the past decades. Nanoscale materials bear unique physical and chemical properties that distinguish them from bulk materials and small molecules. Therapeutics and diagnostic tools made with nanomaterials inherit those distinctive properties, including versatile surface functionalization, controlled release, enhanced tumor accumulation, tunable biodistribution, metabolism, and excretion. The surface of nanoparticles (NPs) enables functionalization to achieve tumor targeting effects and better stability in the bloodstream. NPs can be engineered with specific structures and dimensions such that they can encapsulate a variety of poorly water-soluble molecules and also control the in-vivo release of payloads. Due to their larger size, NPs preferentially accumulate in tumor tissues with permeable vasculature accompanied by poor lymphatics, and are mostly cleared from systemic circulation via hepatic or renal excretion and mononuclear phagocytosis, whereas small molecules are widely distributed to many organs. These characteristics grant NPs superior abilities to deliver therapeutics or imaging agents for radiation oncology.

The development of minimally invasive techniques to detect and monitor cancers remains a major challenge in different stages of cancer management. The study of liquid biopsies has shown great promise in the era of personalized medicine. Compared with tissue biopsies and radiographic imaging approaches, the analysis of circulating tumor cells (CTCs) is a much less invasive method to monitor the dynamic molecular profiles of tumors. Advancing biotechnology has enabled the analysis of CTCs at a higher sensitivity and specificity than before. The translational potential of CTCs as a novel cancer biomarker has been demonstrated in a number of clinically relevant studies. In this article, the authors will summarize the clinical applications of nanomaterials in radiation oncology and discuss the implications of CTCs in cancer detection and monitoring.

Nanotechnology in Imaging

The field of medical imaging has been reshaped by advances in nanotechnology. A wide range of materials have been engineered such that they can be incorporated into NPs to deliver or serve as contrast agents for various imaging paradigms. For example, carbon nanotubes (CNTs) have been studied as a versatile nanoplatform for imaging and drug delivery.^1,2 CNTs have been utilized as vehicles for contrast agents such as chelated paramagnetic metal ions (Gd³⁺, Mn²⁺, etc), iron oxide NPs, high Z materials (I⁻, Bi³⁺), and radionuclides (⁶⁴Cu²⁺, ^99mTc) for different imaging techniques. This has mainly been in preclinical settings due to the in vivo toxicity of CNTs which still requires further investigation before significant clinical translation can take place.^2–5 In the past two decades, several iron oxide NP products (one of the most extensively investigated contrast agents for magnetic resonance imaging (MRI) and multimodal imaging)⁶ have been approved by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) for imaging of bowel, liver lesions, and lymph node metastases and for therapy of adult anemia with chronic kidney disease. These early compounds are rarely encountered in current practice as most were discontinued due to regulatory and marketing issues after 2005.^6,7 However, there has been a recent resurgence of interest in utilizing iron oxide NPs in ongoing clinical trials attempting to improve the imaging of primary or metastatic tumors with MRI or combined with other imaging techniques (Table 1).

Table 1.

Clinical trials using inorganic nanoparticles

Name	Material/functionality	Application/indication	Clinical Trial ID
Feraheme®; Rienso®; Ferumoxytol	Iron oxide NPs coated with polyglucose sorbitol carboxylmethyether.	Childhood brain neoplasm, Brain tumors or cerebral metastases;	, , ;
	Magnetic-field responsive nanoparticles for MRI and multimodal imaging.	Bone sarcomas and osteomyelitis;	;
		Triple negative breast;	;
		Head and neck cancer;	;
		lymph nodes;	;
		lymph node metastases in prostate, bladder and kidney cancers;	;
		Thyroid cancer;	;
		Esophageal cancer;	;
		Whole body imaging for cancer staging;	;
Ferumoxtran-10; Combidex®; Sineren®	Iron oxide nanoparticles coated with dextran. Magnetic-field responsive for MRI imaging	Lymph node metastasis in advanced cervical cancer or endometrial cancer
AuroLase®	Silica-gold nanoshells coated with PEG. Laser responsive	Thermal ablation of solid tumors: head/neck cancer. Primary and /or metastatic lung tumors	,
Sienna+®	Iron oxide particles coated with carboxydextran. Magnetic responsive particles use with SentiMag® device	Mark and locate cancerous lymph nodes prior to surgery	,
Magnablate	Iron nanoparticles. Magnetic-field responsive for thermal ablation	Prostate cancer
Sensors functionalized with gold nanoparticles	Organic functionalized gold nanoparticles	Detection of gastric lesions

Adapted from Anselmo AC, Mitragotri S. A Review of Clinical Translation of Inorganic Nanoparticles. AAPS J 2015;17(5):1044; with permission.

Metastases to lymph nodes are one of the most important prognostic indicators for patients with melanoma. Early staging of lymphatic metastases would be critical for the determination of therapeutic strategies and patients’ outcomes. However, several drawbacks are associated with current techniques of mapping sentinel lymph nodes (SLNs). Non-invasive imaging techniques, such as computed tomography (CT), MRI, single photon emission computed tomography (SPECT), and positron emission tomography (PET) are commonly utilized to locate lymph nodes with abnormal dimensions or increased metabolic activity prior to surgery.^8,9 However, these imaging methods cannot provide precise 3-dimensional locations of lymph nodes for surgical procedures or differentiate real metastases from abnormalities caused by infection or inflammation. Radionuclide-labeled sulfur colloid and isosulfan blue have also been injected locally to visualize SLNs intraoperatively but they fail to provide adequate visualization due to their inefficient tissue distribution. Therefore, a new imaging platform for SLN mapping is highly needed.

A silica-based nanoplatform has been developed for cancer imaging with positron-emission tomography(PET).¹⁰ Silica cores were coated with polyethylene glycol (PEG) chains which were partially end-capped with cyclo-(Arg-Gly-Asp-Tyr) peptides (cRGDY) that target integrin α_vβ₃ expressed by various types of tumors. A positron-emitting radionuclide ¹²⁴I was further conjugated to the peptide ligand for quantitative imaging by PET.¹¹ The clearance half-life of the particles was longer than 30 hours in tumors and major organs in a M21 tumor xenograft murine model. Due to the small size (~7 nm), nearly 50% of the particles were excreted in the first 24 hours post intravenous (IV) injection via renal clearance and up to 72% was excreted in the next 3 days. Particle accumulation in tumors reached a maximum ~4 hours post injection and the concentration in tumors was 3-fold higher than with non-targeting control NPs. More particles remained in tumors than muscles 1–4 days post injection. When utilized in a spontaneous melanoma miniswine model, ¹²⁴I-cRGDY-silica NPs showed a higher sensitivity in detecting metastatic nodules and draining lymphatic channels after subdermal injection than ¹⁸F-FDG.⁹ In addition, ¹²⁴I-cRGDY-silica NPs were able to discriminate metastatic tumor burdens from inflammatory and other metabolically active tissues identified by ¹⁸F-FDG in miniswines. These preclinical findings underscored the specific targeting effect of the ¹²⁴I-cRGDY-silica NPs in staging metastatic tumors.

The silica-based nanoplatform was further assessed in a microdosing study in patients with metastatic melanoma and malignant brain tumors ().¹⁰ The systemic clearance half-lives ranged from 13 to 21 hours without notable accumulation in the reticuloendothelial system. Consistent with the preclinical study, a large fraction of the particles was cleared via renal excretion over 72 hours. A liver metastasis was seen 4 hours after IV injection of ¹²⁴I-cRGDY-silica NPs in patient #1, which was consistent with the ¹⁸F-FDG PET image (Figure 1). A cystic lesion in the pituitary gland was located in the brain of patient #2, confirming the finding on previous MRI scans (Figure 2). The toxicity of these particle tracers was assessed based on clinical symptoms and laboratory analysis of urine and blood samples. No substantial changes in liver and renal functions were found related to particle injection over 2 weeks of study. The data in this trial warranted further evaluation in the context of image-guided surgeries and interventions. Currently, these targeted silica NPs loaded with the fluorescent probe Cy5.5 are being evaluated in a Phase I/II trial for real-time imaging of sentinel lymph nodes during surgical procedures in patients with breast or colorectal cancer or melanoma ().

Figure 1.

Whole-body PET-CT imaging of particle biodistribution and tumor uptake after systemic injection of ¹²⁴I-cRGDY–PEG–C dots. (A) Coronal CT in patient #1 shows hepatic metastasis (arrowhead). (B) Coronal PET image at 4 hours after injection demonstrates particle activity along the peripheral aspect of the tumor (arrowhead). Coregistered PET-CT at (C) 4 hours and (D) 24 hours after injection. (E) Corresponding ¹⁸F-FDG PET-CT image showing the hepatic metastasis in (A) (arrowhead).

From Phillips E, Penate-Medina O, Zanzonico PB, et al. Clinical translation of an ultrasmall inorganic optical-PET imaging nanoparticle probe. Sci Transl Med 2014;6(260):149–260; with permission.

Figure 2.

Multimodal imaging of particle uptake in a pituitary lesion. (A) Multiplanar contrast-enhanced MR axial and sagittal images of patient #2 at 72 hours after injection demonstrate a subcentimeter cystic focus (arrows) within the right aspect of the anterior pituitary gland. (B) Co-registered axial and sagittal MRI-PET images reveal increased focal activity (red, 124I-cRGDY–PEG–C dots) localized to the lesion site. (C) Axial and sagittal PET-CT images localize activity to the right aspect of the sella. (D) Axial PET images of ¹²⁴I-cRGDY–PEG–C dots in the brain at 3, 24, and 72 hours after injection demonstrate progressive accumulation of activity within the sellar region.

Adapted from Phillips E, Penate-Medina O, Zanzonico PB, et al. Clinical translation of an ultrasmall inorganic optical-PET imaging nanoparticle probe. Sci Transl Med 2014;6(260):149–260; with permission.

Nanotherapeutics to Improve Chemoradiotherapy

Chemoradiotherapy (CRT) has played a significant role in the battle against many human cancers but does have limitations. Nanotherapeutics have demonstrated great potential to overcome the limitations in conventional CRT. For example, Abraxane® is nanoparticle albumin-bound paclitaxel (nab-PTX) which was developed to improve the antitumor efficacy of PTX by utilizing enhanced permeability and retention (EPR) effects in tumor tissue while avoiding the excipient-related toxicities of Taxol. Nab-PTX has been used concurrently with cisplatin and radiotherapy (RT) in a Phase II trial (ChiCTR-ONC-12002615) in patients with locally advanced nasopharyngeal carcinoma.¹² 30 complete responses and 6 partial responses were observed among 36 patients after 2 cycles of concurrent CRT using nab-PTX. The progression-free survival and cancer-specific survival rates were 86% and 92% respectively at the median follow-up time of 45 months. The combination of nab-PTX and CRT showed encouraging antitumor efficacy with manageable toxicities, warranting Phase III trials of this regimen. nab-PTX plus gemcitabine has also been evaluated in an early Phase I trial to determine the recommended dose with concurrent RT for patients with borderline resectable pancreatic cancer.¹³ Carboplatin was administered together with nab-PTX weekly for 6 weeks to patients with advanced non-small cell lung cancer (NSCLC) in a Phase I trial.¹⁴ Thoracic RT was given concurrently at 2 Gy every weekday for the same period of time. 10 out of 14 patients achieved partial response with tolerable toxicity and 12 patients survived to the median follow-up time of 13 months. Due to these promising outcomes, recent clinical trials have been focused on the treatment paradigm of CRT using nab-PTX (Table 2).

Table 2.

Clinical trials of nab-PTX combined with radiotherapy

Trial ID	Status	Study Phase	Study Design	Condition	Intervention and Arms
	Recruiting	Early Phase 1	Non-Randomized, Parallel assignment	Locally advanced pancreatic cancer, Borderline pancreatic inoperable cancer, Pancreatic cancer	Folfirinox followed by Stereotactic Body Radiotherapy (SBRT) Gemcitabine nab-paclitaxel followed by SBRT
	Recruiting	Phase 1	Single group assignment	Pancreatic adenocarcinoma, Stage III pancreatic cancer AJCC v6 & v7	Capecitabine, nab-paclitaxel, radiation therapy
	Recruiting	Phase 4	Randomized, Parallel assignment	Locally advanced pancreatic cancer	Radiotherapy followed by intra-arterial gemcitabine Radiotherapy followed by intravenous gemcitabine and nab-paclitaxel
	Recruiting	Phase 2	Single group assignment	Pancreatic adenocarcinoma Resectable pancreatic carcinoma	Pre-operative chemotherapy: nab-paclitaxel, gemcitabine HCl, Image-guided intensity-modulated radiation therapy, fluorouracil Surgical resection Post-operative chemotherapy: nab-paclitaxel, gemcitabine HCl
	Recruiting	Phase 1	Single group assignment	Locally advanced unresectable pancreatic cancer treated with chemoradiotherapy Borderline resectable pancreatic cancer treated with chemoradiotherapy	Radiotherapy, gemcitabine, nab-paclitaxel
	Recruiting	Phase 1	Single group assignment	Recurrent head and neck cancer	nab-paclitaxel combined with 5-fluorouracil and hydroxyurea and radiation for good induction responders nab-paclitaxel and hypofractionated radiotherapy for poor responders
	Active, not recruiting	Not Applicable	Single group assignment	Resectable pancreatic cancer	Gemcitabine, nab-paclitaxel, hypofractionated image-guided intensity-modulated radiation therapy, surgical resection
	Recruiting	Phase 2	Non-Randomized, Parallel assignment	Pancreatic cancer Pancreatic adenocarcinoma Pancreas ductal adenocarcinoma	nab-paclitaxel and gemcitabine followed by SBRT for resectable pancreatic ductal adenocarcinoma nab-paclitaxel and gemcitabine followed by SBRT for borderline-resectable pancreatic ductal adenocarcinoma

Other nano-formulations of anticancer drugs have also been evaluated for CRT. A lipid-mitomycin C (MMC) prodrug was encapsulated in PEGylated liposomes as a radiation-responsive formulation (Promitil) for use in CRT.¹⁵ MMC could be liberated from liposomes after cleavage of a lipid linker in the prodrug by radiation-induced increase in reducing agents in tumors, such as dithiothreitol or cysteine. In a preclinical study using human rectal tumor xenografts, Promitil was combined with RT and 5-fluorouracil (5-FU) and showed improved antitumor effects than an equivalent dose of free MMC. All mice treated with Promitil and RT survived to the end of this study while all of those treated with the combination of free MMC and RT did not, indicating the attenuated toxicity of liposomal MCC prodrug. The improved tolerance of Promitil was affirmed by a phase I clinical study () in solid tumor patients.¹⁶ A Phase I clinical study () is recruiting cancer patients for combined therapy with Promitil and external beam RT. Nanotherapeutics, such as liposomal doxorubicin and polymer-PTX conjugates, were assessed in clinical trials of CRT, however they were not adopted in CRT regimens due to a lack of significantly improved outcomes compared to standard therapy and dose-limiting toxicities respectively.¹⁷

The importance of radiosensitizers has been widely recognized in the field of RT. Some potent radiosensitizers, such as the anticancer drug camptothecin (CPT)¹⁸ and DNA repair inhibitors KU55933¹⁵ and wortmannin (Wtmn)¹⁹, are very toxic when delivered systemically in their free forms. Therefore, NP platforms were developed for these molecules, aiming to evaluate their synergisms with CRT against tumors. Wtmn has been encapsulated in biodegradable NPs together with docetaxel (DTX) or cisplatin to facilitate CRT against lung and prostate cancer.²⁰ DTX/Wtmn co-encapsulated NPs remarkably prolonged the survival time of mice with PC3 xenografts that received RT simultaneously, in contrast to other groups treated with single-drug-loaded NPs or a mixture of two single-drug-loaded NPs. In a similar study, Wtmn-loaded NPs reversed Pt resistance and inhibited tumor growth of A2780 cis xenograft tumors without inducing any significant off-target toxicity.²¹ CPT nanoparticle-drug conjugates (CRLX101) have also been evaluated as radiosensitizers in murine models grafted with human colorectal cancer cells HT-29 or SW480.¹⁸ When combined with RT, CRLX101 inhibited tumor progress better than 5-FU plus RT in SW480 xenografts. As expected, the combination of CRLX101, 5-FU, and RT showed a higher therapeutic index compared to CRLX101 plus RT in HT-29 xenografts. Additional results suggested that CRLX101 was synergistic with 5-FU and outperformed the combination of oxaliplatin and 5-FU in suppressing tumor growth in both HT-29 and SW480 murine xenograft models undergoing RT. Furthermore, CRLX101 had decreased hair and gastrointestinal toxicity in mice compared to CPT. Encouraged by the preclinical study, a Phase Ib/II clinical study () was carried out to evaluate the toxicity of CRLX101 and its ability to improve therapeutic responses when combined with capecitabine and RT for locally advanced rectal cancer.²² The results showed that capecitabine-based CRT combined with CRLX101 at 15 mg/m² per week was well tolerated with moderate to complete therapeutic responses in 24/32 patients, justifying this regimen for a larger Phase II trial.²³

With the advance of immunotherapy, antibody-based approaches have also been combined with RT against cancers. A novel two-step radioimmunotherapy paradigm has been evaluated preclinically for the treatment of Non-Hodgkin lymphoma.²⁴ In the pre-targeting step, dibenzylcyclooctyne (DBCO) functionalized anti-CD20 antibodies (Rituximab) were used to specifically bind Raji B-cell lymphoma cells overexpressing CD20 antigens. Azide-functionalized dendrimers conjugated with radionuclide ⁹⁰Y were administered to tag those pre-labeled lymphoma cells via click chemistry between DBCO and azide in vivo. This pre-targeted system achieved a highly specific delivery of radionuclides to tumor cells and concomitantly enhanced the complement-dependent cytotoxicity of antibodies, which inhibited tumor progress in both xenograft and disseminated Non-Hodgkin lymphoma xenotransplant murine models. This two-step pre-targeting strategy overcame the challenges in conventional radioimmunotherapy and bears a great potential for clinical translation.

Inorganic NPs as Radiosensitizers

Another strategy to improve CRT is to deliver inorganic NPs made of materials with high atomic numbers (Z) into tumor tissues as radiosensitizers. Gold NPs (Au, Z=79) have been extensively evaluated as a multi-modal imaging agent and radiosensitizer with photon, proton, and carbon RT.²⁵ Despite promising results from preclinical studies, the path towards clinical translation remains a challenge and the clinical evaluation of gold NPs as radiosensitizers has not begun.

Hafnium NPs (Hf, Z=72), have also been investigated as radiosensitizers and show promise for clinical translation. Hafnium oxide NPs (NBTXR3) have are potent radiosensitizers in murine models of sarcoma and colorectal cancers.²⁶ Nanobiotix has finished a Phase I clinical trial of NBTXR3 crystalline NPs (), in which all the patients well tolerated NBTXR3 and subsequent RT, demonstrating a good safety profile and encouraging antitumor effect in patients with locally advanced soft tissue sarcoma (STS).²⁷ Currently, NBTXR3 NPs are being evaluated in a Phase II/III clinical trial in advanced STS (). Several Phase I/II clinical trials of NBTXR3 have been conducted for different cancers, such as a combination of NBTXR3 with RT and chemotherapy for patients with rectal cancer and head and neck cancer (, ), NBTXR3 and RT for liver cancers (), and NBTXR3 combined with brachytherapy for prostate cancer ().

Gadolinium (Gd, Z=64)-based NPs (AGuIX) have been constructed with a polysiloxane core and Gd chelated on the surface, aiming to facilitate MRI-guided RT on multiple brain melanoma metastases.²⁸ These ~3 nm particles were mainly eliminated via renal clearance and a single bolus injection of AGuIX accumulated quickly in mouse kidneys and was retained in kidneys for less than a week. Weekly IV injections for 3 consecutive weeks only caused transient perturbation on renal function and insignificant tissue alterations in mice. Similar safety profiles were observed in rats and monkeys after weekly IV injections for 2 weeks, suggesting a promising clinical translation.^29–31 When combined with RT, AGuIX demonstrated strong radiosensitizing effects in preclinical animal models with pancreatic cancer, brain melanoma metastases, glioblastoma, head and neck cancer, and lung cancer.^{28,30,32–34} Currently, AGuIX NPs are being evaluated in Phase I and II trials for their safety and radiosensitizing effects with whole brain RT for patients with multiple brain melanoma metastases (, ).³⁵

Radiation-Enhanced Drug Delivery Using NPs

Radiation therapy has frequently been utilized in combination with both chemotherapy and immunotherapeutic NPs. However, several challenges have hindered the transport of NPs through tumor microenvironment (TME), such as long transport distances in tumor extracellular matrix, heterogenous tumor vasculature, and elevated interstitial fluid pressure.^36,37 Local tumor RT has the potential to facilitate the delivery of nanotherapeutics by changing the endothelial architecture, increasing the vascular permeability, and reducing the interstitial fluid pressure in tumor tissues, which collectively enhance the EPR effect.³⁸ In addition to the primed EPR effect, Miller et al have demonstrated that irradiation of tumors could recruit tumor associated macrophages (TAMs) which would serve as reservoirs of NPs.^39,40 Radiation-induced TAM enrichment was found near microvasculature in tumor xenografts, eliciting vascular burst and particle uptake in the neighboring tumor cells. When combined with the DNA alkylating agent cyclophosphamide, radiation improved the tumor accumulation of PLGA-BODIPY630, liposomal doxorubicin, and liposomal irinotecan in 4T1 orthotopic tumor models. Improved accumulation was translated into remarkable synergistic inhibiting effects in HT1080 human fibrosarcoma murine models treated with liposomal irinotecan and a 5 Gy dose of radiation compared to each single treatment. However, radiation-enhanced nanoparticle delivery would be attenuated if TAMs were depleted with liposomal clodronate from tumor tissues. This study demonstrated that radiation altered the TME and facilitated nanoparticle delivery in a TAM-dependent approach. However, the possibility of radioresistance in tumors under fractionated radiation has not been fully evaluated in this study. Furthermore, polarization of TAMs and their interactions with surviving tumor cells, stroma cells, and other immune cells after radiation therapy still needs further investigation.⁴¹

Nanotechnology in the Development of Cancer Biomarkers

Cancer biomarkers are generally defined as biomolecules generated either by tumor cells or other tissues as a response to cancers, and even more broadly they can be biological processes including angiogenesis, proliferation, and apoptosis.^42,43 Cancer-related biomarkers encompass a broad-spectrum of molecules such as peptides, proteins, metabolites, nucleotides, and lipids which could be detected in blood, secretions, or biological fluids produced by many organs.^43,44 Even though a large number of candidate biomarkers have been identified and evaluated preclinically, the FDA has only approved approximately 20 cancer biomarkers for clinical use. Most of these are proteins that can be detected in serum, plasma, urine, or feces, with only a small number requiring solid tissue biopsies.⁴² Among cancer biomarkers, the tumor circulome is a collection of tumor-derived elements found in the blood circulation which includes proteins, nucleic acids (DNA and RNA), exosomes, tumor-related platelets, and CTCs.^45,46 The significance of the tumor circulome has been widely recognized in recent decades because it could provide valuable information about primary and metastatic tumors, resistance-related mutations, and therapeutic responses from blood in cancer patients. Here, we will highlight the significant applications of circulome in clinical oncology.

Circulating Tumor DNA and Exosomes

Circulating tumor DNA (ctDNA) is fragmented DNA originated from tumor cells and its level in the blood is directly correlated with the presence of malignant diseases.⁴⁵ ctDNA could be actively secreted by tumor cells or simply liberated from dead tumor cells as free nucleic acids or being encapsulated in extracellular vesicles. CTCs were believed to be another source of ctDNA.⁴⁷ Similar to CTCs, ctDNA can be obtained in less invasive methods comparing to tissue biopsies, which allows longitudinal monitoring of tumor heterogeneity and multiclonality with high specificity. In addition, ctDNA yields high detection rate and can be found when detectable CTCs are absent in patients.^48,49 Tumor-derived cell-free DNA could also be detected in various bodily fluids other than blood, such as cerebrospinal fluid, saliva, sputum, pleural effusions, urine and stool,⁵⁰ suggesting the presence of local or distant tumors. Since tumor-associated cell free DNA has multiple sources, pre-analytical processing of the sample could be critical for an accurate and sensitive detection. Prior to sequencing, ctDNA needs enrichment or extraction which could be accomplished with various commercial kits.⁵¹ The detection of ctDNA usually aims at the alterations in DNA sequence, DNA methylation and variations of DNA copy number.⁵² ctDNA sequencing technologies^51,53 and recent clinical studies⁴⁵ have been summarized in the literature.

Tumor-derived exosomes are nanovesicles composed of phospholipid, proteins and encapsulated nucleic acids.⁵⁴ They are secreted by dividing cancer cells and hence carry abundant proteomic and genetic information on primary tumor and TME.⁵⁵ Similar to ctDNA, exosomes can be found in large quantities in various bodily fluids and even from tumors which release sparse amount of CTCs, such as those in the central nervous system.^56–58 The conventional methods of exosome isolation include ultracentrifugation and density-gradient separation which require extensive processing, making them impractical for clinical implications. Novel techniques can be categorized into size-dependent microfluidic platforms⁵⁹ and immunoaffinity-based systems which isolate exosomes by their specific surface markers.⁶⁰ Further characterization and analysis of biomolecules in exosomes were thoroughly discussed in recent reviews.^54,61

Circulating Tumor Cells (CTCs)

Around 90% of the mortality among patients with solid tumors is caused by the formation of metastases rather than primary tumors.⁶² The metastatic process starts with the journey of CTCs. After detaching from a primary tumor mass, CTCs migrate toward blood vasculature and intravasate into the circulation where they have to evade immune detection on their way to distant organs. Only surviving CTCs can settle within a favorable tissue and become micro/macro-metastases (Figure 3).⁶³ Preclinical studies suggested that less than 0.01% of CTCs had the chance to survive and eventually form secondary tumors.^64,65 Accumulating evidence has proven a good correlation between CTC counts in blood samples and the disease progression of cancer patients,^66–68 and therefore demonstrated the clinical value of CTCs as a significant biomarker for diagnosis and prognosis of various cancers including breast,⁶⁹ prostate,⁷⁰ lung,^71,72 colorectal,^73,74 liver,⁷⁵ and pancreatic cancers⁷⁶ as well as melanoma⁷⁷.

Figure 3.

Diagram of the metastatic progression of a malignant tumor. From Divoli A, Mendonça EA, Evans JA, et al. Conflicting Biomedical Assumptions for Mathematical Modeling: The Case of Cancer Metastasis. PLOS Comput Biol 2011;7(10): e1002132; with permission.

Liquid biopsies of cancer patients’ blood for CTCs or ctDNA have remarkable advantages over medical imaging techniques and solid tissue biopsies for cancer diagnosis and prognosis.^47,78 The analysis of the blood sample can be conducted more frequently for cancer patients than imaging techniques.^79–81 Liquid biopsy samples are collected with a less invasive method from patients and at a lower cost than solid biopsies which usually generate discomfort, pain, and the possibility of bleeding and infection. Furthermore, CTC analysis can better represent the tumor heterogeneity and real-time information from primary and metastatic tumors than solid biopsies from a single location.^82–84

However, detection of CTCs is much easier said than done due to their rarity among blood cells. The number of CTCs could be as low as 1–10 cells per 10 mL of whole blood from most cancer patients, which demands detection technologies with high sensitivity and specificity.^85–87 Enrichment of CTCs is a preceding step of detection and CTCs can be enriched on the basis of their biological and physical properties (Figure 4).⁸⁸ CTCs from epithelial tumors can be distinguished from normal blood cells by their surface markers, such as epithelial cell adhesion molecule (EpCAM) and proteins that appear after epithelial-to-mesenchymal transition.^89,90 Anti-epithelial (E) and anti-mesenchymal (M) antibodies are widely used to positively select CTCs from blood cells (Figure 4A). In addition, CTCs from certain tumors or tissues can be captured by antibodies targeting highly expressed tumor or tissue-specific markers. For example, antibody-dendrimer conjugates were utilized for the capture of CTCs expressing human epidermal growth factor receptor 2 (HER2) or prostate specific antigen (PSA) in human blood spiked with breast cancer and prostate cancer cells.⁹¹ A capture efficiency of 82% and purity of 50–90% of captured cells had been achieved from blood samples with 1×10⁵ tumor cells per milliliter. An alternative approach to select CTCs is based on their physical properties such as size, deformability, density, and surface charge in specific media. CTCs are usually larger and stiffer than normal blood cells, allowing them to be trapped in microfilters (Figure 4C and 4D).⁹² Another size-based cell separation was achieved by flowing samples through a spiral microchannel where larger CTCs were gradually separated from blood cells by inertial lift and the Dean drag force (Figure 4G).⁹³ Furthermore, hematological cells such as leukocytes and granulocytes could be separated from CTCs by immunomagnetic beads against leukocyte antigen CD45 or granulocyte marker CD15 (Figure 4B) after red blood cells have been removed by density gradient centrifugation (Figure 4E).⁹⁴ Due to the higher surface area and larger capacitance of CTCs in contrast to normal blood cells, CTCs usually exhibit negative dielectrophoresis (DEP) and hence could be isolated under a given electric field frequency (Figure 4F).⁹⁵ CTC enrichment technologies and their pros and cons have been summarized previously in literature.⁹⁶

Figure 4.

(A–G) CTC enrichment approaches. From Alix-Panabières C, Pantel K. Challenges in circulating tumour cell research. Nat Rev Cancer 2014;14(9):623–631; with permission.

An efficient enrichment of CTCs can be achieved by a combination of biological and physical property-based techniques. Ozkumur et al have developed a microfluidic CTC capture device (CTC-iChip) (Figure 5B), integrating hydrodynamic cell sorting, inertial focusing, and magnetophoresis (Figure 5A).⁹⁷ A whole blood sample was first incubated with anti-EpCAM (aEpCAM), anti-CD45, or anti-CD15 antibody conjugated magnetic beads. Red blood cells and platelets were then separated using deterministic lateral displacement based on their size (Figure 5C). The remaining CTCs and white blood cells were aligned after flowing through a microchannel by inertial focusing and entered the channel under magnetic field for further sorting (Figure 5D). Two immunomagnetic sorting options were available for the final isolation which included positive selection of CTCs and depletion of leukocytes and granulocytes from the presorted blood sample. CTC-iChip outperformed the CellSearch™ system when used to capture CTCs in patient specimens with low CTC burdens (<4 CTCs per mL).

Figure 5.

CTC-iChip. (A) Three microfluidic components of the CTC-iChip are shown schematically. (B) Integrated microfluidic system. The debulking array sits in a custom polycarbonate manifold that enables fluidic connections to the inputs, waste line, and second-stage microfluidic channels. The inertial focusing and magnetophoresis chip is placed in an aluminum manifold that houses the quadrupole magnetic circuit. Magnetically deflected cells are collected in a vial. (C) Hydrodynamic size–based sorting. A mixture of 2-mm (red) and 10-mm (green) beads enters the channel (i). Whereas the 2-mm beads remain in laminar flow and follow the fluid stream-lines, the 10-mm spheres interact with the post-array (ii and iii) as shown in the scanning electron microscope (SEM) image (right panel). Larger beads are fully deflected into the coincident running buffer stream by the end of the array (iv). Scale bars, 100 mm. (D) Cell focusing and magnetophoretic sorting. Magnetically labeled SKBR3 (red) and unlabeled PC3–9 (green) cell populations are mixed and enter the channel in random distribution (i). After passing through 60 asymmetric focusing units (pictured in the SEM, right panel), the cells align in a single central stream (ii). Magnetically tagged cells are then deflected (iii) using an external magnetic field, and separation is achieved by the end of the channel (iv). Scale bars, 100 mm. From Ozkumur E, Shah AM, Ciciliano JC, et al. Inertial Focusing for Tumor Antigen-Dependent and -Independent Sorting of Rare Circulating Tumor Cells. Sci Transl Med 2013;5(179):179ra147; with permission.

Another new CTC detection platform was built by integrating biomimetic cell rolling and multivalent binding via antibody-dendrimer conjugates.⁹⁸ E-selectin is a single-chain transmembrane glycoprotein on the vascular endothelium which plays a significant role in cell rolling and adhesion with surface ligands on various tumor cells (Figure 6A).^99,100 aEpCAM, a widely used CTC capturing antibody, was immobilized together with recombinant human E-selectin Fc chimera proteins on the capture surface to recruit CTCs and hematological cells from the bulk flow. The addition of E-selectin improved the ability of aEpCAM to recognize and bind tumor cells by >3 fold compared to aEpCAM alone, with hematological cells rolling on the E-selectin surface but not binding aEpCAM. To strengthen the binding and stability between CTCs and aEpCAM or other tumor specific antibodies such as anti-PSA (aPSA) and anti-HER2 (aHER2), antibodies were conjugated to nano-scale poly(amidoamine) dendrimers anchored on the capture surface instead of direct immobilization on the surface via conventional PEGylation (Figure 6B). To validate this combined technology, MDA-PCa-2b, MCF-7 (low HER2 expression), and MDA-MB-361 (high HER2 expression) cells were added in human blood samples and injected to the flow chamber (Figure 6C). More than 80% capture efficiency and nearly 90% purity were achieved, approximately 10 fold higher than a surface without E-selectin and dendrimers (Figure 6D and 6E).⁹¹ The result demonstrated a highly efficient CTC enrichment technique with clinical translatability. This CTC capture device (named as CapioCyte), equipped with aEpCAM, aHER2, and aEGFR has already been used in a clinical study aiming to monitor CTC changes during and after therapy.¹⁰¹ CapioCyte detected CTCs in all 24 patients and showed a significant decline of median CTC counts in 18 patients, from 113 counts per milliliter before treatment to 32 counts per milliliter at the completion of RT, which demonstrated the sensitivity, specificity, and reliability of CapioCyte. In addition to therapeutic outcomes monitoring, captured CTCs could be expanded and analyzed in vitro, facilitating the discovery of CTC biomarkers and the establishment of in vitro CTC models. CapioCyte is currently a prototype undergoing optimization. This biomimetic platform needs to be validated with more types of cancer and their therapeutic responses. Diverse combinations of capturing agents should be established to tackle tumor heterogeneity and even other biomarkers including nucleic acids, exosomes, and proteins from patients’ specimens.

Figure 6.

(A) Cell rolling and (B) multivalent binding in the CapioCyte system. (C) Schematic illustration of the surface marker- dependent cell capture using aPSA, aHER-2, and aEpCAM. (D) The capture patterns of the three cell lines labeled with three different fluorescent colors. The lower capture efficiency of MCF-7 cells for aHER-2 due to low HER-2 expression of MCF-7 cells. (E) A combination of dendrimers and E-selectin (a cell rolling inducing agent), along with multiple antibodies achieved highly sensitive differential detection of tumor cells (up to 82%, Error bars: standard error (n = 4)).

Figure 6A–C: From Myung JH, Park SJ, Wang AZ, et al. Integration of biomimicry and nanotechnology for significantly improved detection of circulating tumor cells (CTCs).

Advanced Drug Delivery Reviews 2018;125:36–47; with permission.

Figure 6D–E: From Myung JH, Gajjar KA, Chen J, et al. Differential detection of tumor cells using a combination of cell rolling, multivalent binding, and multiple antibodies. Anal Chem 2014;86(12):6091; with permission.

Clinical Utility of CTCs

CTCs can be exploited in many clinical contexts. A direct correlation has been established between the number of CTCs in peripheral blood and the survival of patients with advanced cancers.^66,67,102 FDA has approved the CellSearch™ system for the detection of CTCs in clinical specimens from patients with colorectal, prostate and metastatic breast cancer.¹⁰³ Regardless of therapies that patients received, decreased progression-free and overall survival were associated with the relatively high level of CTCs in patients’ blood samples prior to treatment. The potential utility of CTCs was expanded to detection and monitoring of melanoma¹⁰⁴ and lung cancer lately.^105–107 In addition, molecular profiling of CTCs can serve as a noninvasive method to genotype tumor during treatment and hence guide the future selection of therapy.¹⁰⁸ In an initial clinical study, DNA analysis of EGFR mutation was performed on CTCs from patients with NSCLC.¹⁰⁹ EGFR activating mutation was detected in CTCs from 92% of patients, among whom drug-resistant mutation T790M became detectable after treatment with gefitinib or erlotinib. The presence of T790M mutation was associated with the reduction of progression-free survival from 16.5 months to 7.7 months. CTC enumeration was positively correlated with tumor progression and emerging EGFR mutations. In addition, recent study has demonstrated the correlation between programmed death-ligand 1 (PD-L1) expression in CTCs and tumor tissue from NSCLC patients, indicating the potential implication of CTCs as a noninvasive and real-time biopsy to monitor the dynamic change of PD-L1 in cancer patients.¹¹⁰ In an effort to identify responders to immune checkpoint regulators, CTCs were collected from patients with HER2-negative breast cancer and analyzed for PD-L1 expression on the CTCs.¹¹¹ PD-L1-positive CTCs were found in 11 out of 16 patients and the percentage of PD-L1 expressing CTCs could range from 0.2 to 100% in different patients. This study demonstrated the existence of PD-L1 on CTCs which could benefit patient selection and monitoring for immune checkpoint blockade therapy. In another study, CTCs were isolated from patients with non-metastatic NSCLC and PD-L1 expression in CTCs were monitored before, during and after RT or CRT.¹¹² The fraction of PD-L1-positive CTCs was elevated after RT, suggesting the up-regulated expression of PD-L1 in tumor cells. Similar to other clinical reports, higher level of PD-L1 in CTCs was associated with shorter progression-free survival.¹¹³ This study did not only demonstrate the prognostic value of CTCs but also provide a rationale for combined therapies with immune checkpoint blockade and RT.

Future Perspectives

Emerging more than three decades ago, nanotechnology has made a great impact on the development of diagnostics and therapeutics for cancer patients. The clinical development of many novel nanotechnologies is still in their infancy. Clinical gains with first-generation NPs that main relied on passive tumor targeting via the EPR effect (which is dynamic and heterogenous due to different tumor types and stages) have been modest and inconsistent compared to those predicted by preclinical studies.¹¹⁴ This issue could be resolved through a better understanding of tumor biology and the development of novel technologies to address these shortcomings. In addition, most NPs used for imaging and radiosensitization are produced with heavy metals or other inorganic materials which could pose health risks. Although no evidence of immediate liver injury was found in the aforementioned studies in this review, long-term exposure of inorganic NPs to the liver could pose risks due to their lack of biodegradability, non-specific interactions with endogenous proteins, and hepatic oxidative damage.¹¹⁵ Further efforts should be directed to improve colloidal stability in systemic circulation, penetration/accumulation at target tissues, and most importantly biocompatibility by manipulating the properties of inorganic NPs.^25,115

Liquid biopsies are expected to play a vital role in precision medicine and personalized therapy against various cancers. CTCs have been validated as a prognostic biomarker in metastatic and non-metastatic cancers.⁴⁸ Other applications of CTCs are being extensively studied, aiming to benefit the current practice in screening for malignancy, therapeutic outcomes monitoring, and surveillance after treatment. With the advance of CTC isolation techniques, more CTC detection platforms will enter clinical trials and expand the applications of CTCs in the clinic in the foreseeable future.

KEY POINTS.

• Nanoparticles can facilitate the delivery of contrast agents and thus obtain better radiographic imaging with high sensitivity and specificity.

• Nanotherapeutics serve as a complement to conventional radiotherapy by providing high antitumor efficacy with manageable toxicities.

• Nanoparticles made with high Z materials can serve as radiosensitizers and improve the therapeutic outcomes of radiotherapy.

• CapioCyte is a system that integrates nanotechnology and biomimicry, which significantly improves the sensitivity and specificity of the capture of circulating tumor cells in clinical pilot studies.

• Analysis of circulating tumor cells can have a profound impact on conventional cancer management and its clinical translation as an indispensable assay to current methodologies is rapidly approaching.

SYNOPSIS.

In recent decades, nanotechnology has made remarkable contributions to clinical oncology. Nanotherapeutics and diagnostic tools have distinctive characteristics, such as versatile surface properties, controlled release behaviors, enhanced tumor accumulation, and unique biodistribution and pharmacokinetics. These properties have allowed them superior abilities to deliver therapeutics and imaging agents for radiation oncology. Even with other advances in the detection and monitoring of cancer, it has remained a challenge to do so throughout different stages of cancer with robust and minimally invasive techniques. Compared to solid biopsies and imaging, the analysis of circulating tumor cells (CTCs) offers a more rapid, real-time, and less invasive method to monitor the dynamic molecular profiles of tumors. The potential of CTCs to be translated as a novel cancer biomarker has been demonstrated in numerous clinical studies. This review will discuss clinical applications of nanomaterials in radiation oncology and the implication of CTCs in cancer detection and monitoring.