Synthetic Biomarkers for Early Cancer Detection

Introduction

Early detection when the cancer is still localized can improve the response of patients with most cancer types to medical intervention. The success of screening tools such as cervical cytology in reducing mortality has led to much interest in new methods for early detection, such as non-invasive blood or biofluid biomarkers. However, biomarkers produced from early lesions are limited by basic biological and transport barriers, e.g. short circulation times and dilution in the blood, requiring highly sensitive methods to detect very low signal levels. In addition, individual biomarkers often lack specificity, they may be elevated in noncancerous conditions, or present in multiple cancers, demanding the identification of multiple analyte combinations to assess the disease.

These experiences and lessons for the sensor based on biological engineering, such as molecular probes or the design of gene encoding vectors emerging diagnosis provides the basis for the sensors using the imbalance characteristics of the early tumor or its precursor, generate a signal amplification, these exogenous sensors using tumor dependency activation mechanism, such as enzyme amplification, to drive the synthesis of biomarkers and magnified. Cancer can also be detected by imaging systems that may have the essential features of synthetic biomarker approaches, such as reporter gene imaging. These emerging technologies have promoted the development of early cancer detection, and synthetic biomarkers may become the future method of early cancer detection.

Synthetic biomarkers based on active molecules

Systemic administration of exogenous drugs to assess biological function in vivo has a long clinical history. Activity-based synthetic biomarkers are based on this pattern, including sensors and small-molecule probes activated by enzymes in the tumor or its microenvironment to provide molecular amplification mechanisms for tumor biomarkers.

• Synthetic biomarkers activated by the protease

Synthetic biomarkers of protease activation include peptide substrates bound to the surface of an inert carrier that releases a reporter for detection in blood or urine after being digested by tumor protease. In addition to molecular signal amplification, another key strategy to achieve the required limit of detection (LOD) for early detection is to use human physiological features to increase the concentration of synthetic biomarkers in biological fluids. One approach is to use the kidney to filter by selecting a carrier with a fluid dynamics radius larger than the glomerular filtration barrier (~5nm) to prevent the clearance of the surface-bound peptide into the urine.

Another key strategy is to enhance passive delivery to the tumor site. For example, the use of polyethylene glycol (PEG) polymers plus iron oxide nanoparticles (IONPs) has a higher passive diffusion rate and can increase tumor delivery. An alternative approach is to functionalize sensors using tumor-penetrating ligands that are involved in active transport pathways in the tumor microenvironment.

Proteases are hybrid enzymes capable of cleaving multiple substrate sequences, which limits the detection specificity of individual sensors. Therefore, another key design principle is to design a multisensor library to detect cancer through feature analysis. This approach requires that each synthetic biomarker in the sensor library be labeled with a unique molecule.

• Small molecule probes

Given the growing number of tumor-specific antigens, cell-surface markers, and metabolic pathways available as targets for small molecules, some research has focused on engineering molecular probes to generate synthetic biomarkers for cancer detection.

Stable isotope-labeled small molecules have been widely used as diagnostic probes in research laboratories for more than 30 years. The advantages of stable isotope labeling include no radiation risk to the patient, no metabolic difference compared with their unmodified counterparts, and a high signal-to-noise ratio. The FDA has approved several isotopically labeled probes, including ¹³C-methacetin and ¹³C-cholate, which measure hepatocellular cytochrome P450 activity and liver shunt, respectively, to measure liver dysfunction in the context of liver fibrosis, an important risk factor for hepatocellular carcinoma.

Synthetic biomarkers encoded by genes

In addition to activity-based probes, gene-encoded structures form another major set of strategies that use engineered components or cells to amplify the release of synthetic biomarkers. These approaches focus on strategies that drive resident or infiltrating cells in the tumor microenvironment to produce or secrete bioorthogonal reporters. The main advantage of these approaches is the ability to transcribe the production of synthetic biomarkers into cells of a specific phenotype, thereby potentially reducing the number of false positives caused by background production in healthy tissues. Currently, there are three broad categories of gene-coding systems for the generation of synthetic biomarkers, including vector-based systems, mammalian cell-based systems, and bacterial cell-based systems.

• Synthetic biomarkers based on vectors

Vector-based systems rely on two key design components: a tissue-selective or cancer-selective promoter to drive transcription, and a synthetic biomarker designed to be secreted into blood or urine for detection. Tissue-selective promoters provide the first level of specificity, such as the normally silent promoter of human telomerase reverse transcriptase (TERT), which encodes telomerase, which is frequently activated in cancer cells to achieve proliferative immortalization, one of the hallmarks of cancer. Because TERT is expressed at high levels in about 90% of human cancers but is silenced in almost all somatic cells, TERT promoters have been used to drive gene expression in a variety of tumor cells.

The second component of the vector-based strategy is a secreted reporter that acts as a synthetic biomarker and can be detected in blood or urine. Secreted embryonic alkaline phosphatase (SEAP) was one of the first reporters designed for use in vivo. SEAP is an engineered form of human placental alkaline phosphatase that contains a stop codon in the membrane-anchored domain, converting it to a truncated but fully active secretory reporter. In xenograft tumor models, SEAP levels are directly related to tumor size and cell number. Another commonly used reporter is luciferase.

• Synthetic biomarkers based on mammalian cells

The recent clinical success of adoptive cell therapies has motivated attempts to engineer mammalian cells as biosensors in vivo. A clear advantage of cells as diagnostic carriers is that some cells are able to localize and infiltrate cancer sites compared to molecular probes, which rely on passive diffusion of the vascularity to accumulate in the tumor and are therefore limited.

Mesenchymal stem cells (MSCs) are adult pluripotent stem cells with regenerative and immunomodulatory properties, and Liu et al. demonstrated that engineered MSC can be used to detect cancer metastasis in blood using a mouse model. First, MSCs were engineered to secrete humanized glutamate, and after intravenous administration, engineered MSC persisted longer in mice with lung metastases from breast cancer compared with tumor-free mice, resulting in higher blood levels of humanized Gluc.

Aalipour et al. further developed cell-based diagnostics using engineered macrophages as live-cell sensors. It was found that M2-type reprogramming of tumor-associated macrophages resulted in significant changes in arginase 1 (encoded by ARG1) levels, and that adoptively transferred macrophages in solid tumors upregulated arginase 1 up to 200-fold. Based on this finding, they used the ARG1 promoter to drive glutamate production in macrophage M2 polarization. This study lays the foundation for the concept of cellular immunodiagnosis, and given that many other immune cells similarly regulate the expression of metabolic genes in the tumor microenvironment, this approach can also be extended to T cells, B cells, and natural killer cells.

• Synthetic biomarkers based on bacteria

Certain types of bacteria can infiltrate and selectively grow in tumors, which is attributed to the suppression of immune surveillance and increased levels of nutrients released from necrotic cells within the core of solid tumors. This has led to the use of engineered tumor-targeting bacteria as programmable vectors for cancer detection.

Panteli et al. genetically engineered an attenuated strain of Salmonella enterica to be 10,000 times less virulent than the wild-type strain and to release ZsGreen as a fluorescent biomarker or "fluorescent marker". After intravenous administration to tumor-bearing mice, the level of fluorescent markers in serum depends on tumor mass, and its ability to detect tumors can be predicted by mathematical modeling.

Although engineered strains, including Clostridium, E. coli, and Salmonella, are non-pathogenic to animals and humans, the inherent virulence of the bacterial components and the possibility of reverting virulence pose safety concerns. Advances in synthetic biology could provide solutions to these challenges, while also providing opportunities to design "smart" microbes with specific and controlled behaviors. For example, bacteria designed using quorum-sensing biological circuits could be used for bacterial communication to synchronize activity and generate emergent behaviors, such as the timed release of therapeutic agents to kill tumors or promote systemic antitumor immunity after a threshold population density has been reached.

Conclusion

As the field moves toward human testing, many of the components and vectors that constitute synthetic biomarkers are under clinical evaluation, have a proven safety record in humans, or have been approved by the FDA. For example, proteinase-activated substrates are used in imaging probes for intraoperative detection of tumor margins. Similarly, for genetically encoded synthetic biomarkers, many clinical trials have highlighted the safety and utility of attenuated bacteria as vectors to target tumors and deliver therapy. These precedents provide a broader understanding of embodiments of synthetic biomarkers that will be safe and well tolerated in humans.

However, although the emerging field of synthetic biology markers is exciting and full of hope, the researchers’ understanding of cancer pathogenesis still has some gaps, especially in biology and precursor lesions’ early pathological changes when and how to translate into the understanding of malignant tumor is limited, which is synthesis field of biomarkers of cancer early detection's challenges. Moreover, many questions need to be answered. For example, what early tumors or precancerous lesions could drive sensor engineering? How can machine learning support the identification of key features in complex biological datasets to achieve the predictive power required for synthetic biomarkers?

Reference

1.Synthetic biomarkers: a twenty-first century path to early cancer detection. Nat Rev Cancer. 2021 Oct; 21(10):655–668.