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The Often Neglected Red Blood Cell Offers a Breakthrough in Liquid Biopsy Technology for Early Stage Cancer Detection

February 5, 2021

Liquid Biopsy Bottleneck

Liquid biopsy can capture tumor-related information from the blood. With the advantages of high sample accessibility, avoidance of tissue heterogeneity, and multiple sampling, it can be used to guide cancer treatment, recurrence monitoring, early screening and diagnosis. At present, liquid biopsy mainly detects free circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), exosomes or other biomarkers derived from serum or plasma in the blood, but there are two major bottlenecks: (1) the amount of biomarkers found in serum or plasma is low and difficult to enrich, (2) the detection sensitivity or specificity is not high.

Can Red Blood Cells Carry Tumor Markers

The current liquid biopsy practice removes red blood cells in the first step of sample processing, because it is conventionally believed that there is no DNA in red blood cells (RBCs), but is RBC really useless? RBCs are the most abundant cell type in blood, accounting for up to 45% blood volume and have a survival cycle of up to 120 days. RBCs are generally considered to have frequent molecular exchange with metabolically active cancer cells, providing oxygen and nutrients for the growth and development of cancer cells, and also quickly taking carbon dioxide and metabolic by-products.

Based on the above characteristics of red blood cells, our research team put forward the hypothesis: in the early stages of solid tumor formation, due to vascular proliferation, RBCs may have increased chances of absorbing tumor-derived substances such as nucleic acids from rapidly growing cancer cells. It is known that tumor cells release nucleic acid through exosomes, and also known that RBCs can interact directly with cancer cells and exosomes, and can directly absorb nucleic acids from their environment. Therefore, it is possible for RBCs to absorb tumor-derived nucleic acids through these interactions.

Our researchers went on prove this hypothesis and published the preliminary results in the preprint platform medRxiv, under the title “New Role of Red Blood Cells in Absorption of DNA Bearing Tumorigenic Mutations from Lung Cancer Tissue”.

From literature: Schematic diagram of material exchange between early tumor cells and red blood cells

New scientific discoveries in research

The research team first used the density gradient method to separate RBCs from the whole blood sample, and verified that the purity of the separated RBCs were close to 100% through direct microscope observation and flow cytometry detection. Then it was verified from in vitro experiments that RBCs can absorb tumor-derived nucleic acid. Fresh RBCs were co-cultured with lung cancer cell lines (A549 and H1975), and it was found that the same mutations (A549: KRAS mutation; H1975: EGFR mutation) could be detected in the RBCs within 24hours.

In vitro test: cancer cell mutation detected in red blood cells

The clinical significance of this phenomenon was further explored through a pilot clinical study to determine if DNA mutations are detected in RBCs obtained directly from patients. The first pilot study involved 28 lung cancer patients from Nanjing Gulou Hospital and Peking Union Medical College Hospital. Out of 28 patients, 21 were stage I patients, including 4 patients with stage IA1 with tumor nodules less than 10 mm. NGS sequencing was performed on DNA derived from the tissues and RBCs of these patients and found that mutations including EGFR 19del, EGFR L858R, and KRAS G12 could be detected in both tissue and RBCs.

DNA mutations in tissues and red blood cells of 28 patients

Interestingly, there was a patient with multiple primary nodules sites who underwent two operations between two months, and first removing the upper left lobe and then upper right lobe respectively. The researchers collected RBC and tumor tissue from the second operation. The initial tissue L858R test was negative, while the red blood cell test was positive; however, when the first operation tissue sample was test, it came back as L858R positive. This example showed in this case, the false positive results of seen in RBCs may be caused by the heterogeneity of tumor tissue, and it also revealed that DNA fragments derived from tumors may remain in RBC circulation for at least two months, which is very different from ctDNA in biology terms.

In order to further confirm that red blood cells carry tumor-derived DNA, the research team used ddPCR technology, which is more sensitive for detecting low-frequency mutations, and analyzed 12 red blood cell samples from Gulou Hospital. The researchers directly used DNA as a template for 6 samples, and the other 6 used PCR products after DNA amplification as templates. The ddPCR detection results of the two were highly consistent with the tissue results. It proves once again that red blood cells absorb tumor-specific DNA fragments, and it is not caused by PCR amplification artifacts.

Breakthrough research may usher in a new era of liquid biopsy

While this is only a small sample of data, and further verification is needed in a larger sample, our research team has confirmed for the first time that red blood cells can absorb tumor-derived nucleic acid fragments and can be used as a source for tumor-specific biomarkers. This study shows that the sensitivity of red blood cells to detect tumor-derived DNA is far superior to the current ctDNA technology platform, especially in very early tumors. Furthermore, it is much easier to isolate DNA from red blood cells than from serum, and more samples can be obtained with a lower cost and simpler method. In addition to DNA, other molecules such as proteins, lipids, sugars and RNA may be exchanged between cancer cells and RBCs, and may be diagnostic markers for subsequent research.

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