Cancer has emerged as a leading cause of death among residents in China with incidence and fatality rates persistently on the rise. According to data from the International Agency for Research on Cancer, China had approximately 4.57 million new cases of cancer and 3 million cancer-related deaths in 2020, accounting for 24% of new cancer cases and 30% of cancer-related deaths globally [1]. Moreover, the overall 5-year survival rate for cancer in China has a significant disparity compared to the United States (40% and 67%, respectively). This discrepancy is mainly attributed to the fact that most cancer patients in China are diagnosed in the middle-to-late stages, resulting in a lower 5-year survival rate. Treatment of stage I and II cancer greatly improves the 5-year survival rate [1]. Early detection, diagnosis, and treatment are crucial factors in improving the cancer patient survival rate. Tissue biopsy, despite drawbacks (infection, bleeding, insufficient sampling, and trauma), remains an essential method for early cancer diagnosis.
Liquid biopsy involves detecting cancer signals released into body fluids during cancer cell growth, necrosis, and apoptosis, and presents a novel technique for early cancer detection. Generally, liquid biopsy tumor biomarkers mainly include circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), exosomes, and microRNA (miRNA). Cell-free DNA (cfDNA) in peripheral blood is the most widely used analytical indicator in liquid biopsy. Analytical techniques encompass chromosome open reading, ctDNA mutations, DNA fragmentation, and DNA methylation. Cell-free DNA, representing extracellular DNA, includes ctDNA and DNA fragments from normal cells. Potential sources of cfDNA include cerebrospinal fluid, saliva, urine, pleural and peritoneal effusions, and feces. These samples, enriched with cfDNA, can be utilized for detecting various types of cancers.
On 19 January 2024 a research team from the Massachusetts Institute of Technology published a paper entitled “Priming agents transiently reduce the clearance of cfDNA to improve liquid biopsies” in Science [2]. The researchers introduced a new method to enhance the sensitivity of liquid biopsy. According to preclinical experiment results, injecting a pre-treatment agent 1–2 h in advance increases the collected ctDNA by >10-fold. Analysis of tumors using ctDNA significantly improves the sensitivity of detecting small tumors from <10% to >75% ( Figure 1 ).
Administering a pre-treatment agent 1–2 h in advance results in a >10-fold enhancement in collected ctDNA. Subsequent tumor analysis using ctDNA not only achieves heightened sensitivity, surpassing 75%, especially in detecting small tumors compared to the initial sensitivity of <10%.
Following intravenous injection of the pre-treatment agent and a waiting period of 1–2 h, the augmentation in collected ctDNA is attributed to two key mechanisms: (1) nanoparticles effectively suppress the phagocytic activity of macrophages towards cfDNA; and (2) antibodies inhibit the degradation of cfDNA by nucleases. Consequently, this strategy elevates the collected ctDNA yield by >10-fold and significantly improves the sensitivity of tumor analysis using ctDNA, pushing the capability to detect small tumors from <10% to >75%.
With respect to the origin of cfDNA, it is generally believed that the DNA fragments released by cell active release, cell apoptosis, and cell necrosis are the primary sources. In general, the circulating cfDNA levels in cancer patients are significantly elevated compared to healthy individuals. This increase in circulating cfDNA is attributed to the expansion of tumor volume, resulting in heightened cell turnover, accompanied by a proportional rise in the quantities of apoptotic and necrotic cells. Compared to tissue, cfDNA detection has three major advantages: 1. accessibility (cfDNA is easily obtainable and can be sampled repeatedly); 2. comprehensiveness (cfDNA comprehensively reflects the heterogeneity of tumor tissues); and 3. real-time monitoring (cfFNA enables real-time dynamic monitoring of disease status, such as drug resistance, making cfDNA detection increasingly favored in the field of in vitro diagnostics). However, the sensitivity of liquid biopsy is still insufficient for many applications. For example, the sensitivity of screening tests in oncology based on ctDNA is low (approximately 20%–40% in stage I cancer) [3] and liquid biopsy may yield uncertain results in up to 40% of late-stage cancer patients. Additionally, up to 75% of patients with negative minimal residual disease detection after surgery may experience a recurrence [4]. This finding is primarily due to the low content and rapid degradation of cfDNA, which leads to false-negative results. Typically, cfDNA in the circulatory system is rapidly degraded and cleared by the action of nucleases and phagocytosis by hepatic macrophages. The estimated half-life of cfDNA is only 30–120 min. In a standard 10-ml blood draw, approximately 5 ml of plasma can be separated, providing an average of 10 ng of cfDNA/ml, which is equivalent to approximately 15,000 single-copy genomic equivalents. For late-stage cancer patients, ctDNA constitutes up to 10% of cfDNA, but in middle to late-stage patients this proportion drops to 0.1–1%. In early-stage cancer patients, the proportion of ctDNA may be <0.1%. Therefore, amplifying this minute cfDNA signal is urgent, especially in cases in which the tumor is small or the location is unknown.
Scientists have devised various methods to increase the extracted sample volume. For example, plasma exchange, although requiring specialized equipment and time-consuming, is not suitable for critically ill patients. Another approach is to sample closer to the tumor area, such as collecting cfDNA from the urine of bladder cancer patients. Additionally, interventions within the body, such as focused ultrasound or radiation therapy, can increase ctDNA shedding. However, the feasibility of the latter two methods relies on knowing the tumor location, which makes ultrasound and radiation therapy unsuitable for cancer screening. To address this issue, the authors propose two novel methods to temporarily block these degradation and clearance mechanisms before extracting blood samples. First, circulating cfDNA binds to histones, forming nanoscale particles with a diameter of approximately 11 nm. The researchers designed a similar lipid nanoparticle that binds to cfDNA, which evades phagocytosis by macrophages and temporarily reduces cfDNA clearance [5]. The researchers initially investigated the nanoparticle initiation strategy and identified a liposome formulation based on succinyl phosphoethanolamine that inhibits cfDNA uptake in vitro. The researchers then tested the effectiveness of this nanoliposome in experimental animals. In a lung cancer nude mouse model, the injection of succinyl phosphoethanolamine liposomes via the vein resulted in a 60-fold increase in the amount of detectable ctDNA in mouse blood samples. For early metastatic lesions with a smaller tumor burden, researchers injected nanoliposomes intravenously 1–2 h before blood collection, revealing a sensitivity increase from <10% to >75% compared to tumor-bearing mice without pretreatment. This outcome demonstrated the potential for early cancer detection. Second, using a monoclonal antibody to bind with cfDNA prevent degradation by nucleases. The authors found that the interaction between DNA-binding monoclonal antibodies and cfDNA components prevents double-stranded DNA degradation by nucleases. In healthy mice the engineered mAb, designed to eliminate Fc-γ receptor binding, demonstrated increased circulatory persistence and facilitated the recovery of cfDNA from the bloodstream compared to natural mAb and isotype control mAb counterparts.
In summary, the authors innovatively propose liposome nanoparticles to weaken the uptake capacity of liver macrophages for cfDNA, while the DNA-binding antibody (aST3) protects cfDNA from nucleolytic degradation and plasma clearance through a new mechanism. Both drugs increase the recovery rate of ctDNA molecules in the blood by >10-fold, enabling the retrieval of more tumor genomes during blood collection and enhancing the sensitivity of ctDNA diagnostic tests. The priming agents intervenes in the intrinsic pathways for cfDNA clearance in vivo, fostering the retrieval of ctDNA and addressing the acknowledged impediments associated with low input cfDNA quantities.
This method not only allows for the early diagnosis of cancer but also enables a more sensitive detection of tumor mutations, which guide treatment decisions. The method may also aid in improving the detection of cancer recurrence. However, implementing liquid biopsy in cancer care faces challenges, such as sensitivity, standardization, cost, clinical utility, and regulatory approval. To address these challenges, collaboration, education, multidisciplinary approaches, and continuous improvement are essential. Collaborating with researchers, clinicians, industry partners, and regulatory agencies to share data, resources, and expertise can accelerate the development and validation of liquid biopsy technologies. Engaging multidisciplinary teams of experts, including oncologists, pathologists, molecular biologists, bioinformaticians, and regulatory specialists, can help address the complex challenges associated with implementing liquid biopsy in cancer care. By addressing these challenges and implementing the proposed steps, liquid biopsy techniques can advance significantly and contribute to the early detection, personalized treatment, and improved outcomes for cancer patients.