Capture efficiency up to 73%! "Nature" sub-issue blockbuster: CTC high purity, comprehensive analysis, great breakthrough in liquid biopsy!


We know that circulating tumor cells (CTCs) are cancer cells that "fall" into the peripheral blood. These cells spread with the blood to form new metastases in other parts of the body, so they are closely related to the metastasis and recurrence of cancer.

However, circulating tumor cells are very rare in peripheral blood, which brings great difficulty to the development of related research. About 1 circulating tumor cell for every 1 billion blood cells!

A new type of circulating tumor cell detection method developed by scientists from the University of Michigan in the United States surpasses current technology and studies each circulating tumor cell with ultra-high purity for the first time, comprehensively analyzes genetic information, and reveals its "mystery" of metastasis "!


Hydro-Seq techniques capture circulating tumor cells

The study was recently published in the top international journal Nature Communications. The study describes a fluid barcoding technology (Hydro-Seq) that can capture and isolate every circulating tumor cell in the blood with extremely high efficiency for high-throughput analysis.


It has been proved that using this technology in a 10ml blood sample containing 100 tumor cells, it can capture 90.43±6.08% circulating tumor cells!

Using this technology, after "severely beating" 666 circulating tumor cells from 21 breast cancer patients, the researchers finally confirmed that although tumor stem cells account for a small proportion of all tumor cells, about 30%-50% of tumor cells captured in the blood show the characteristics of tumor stem cells!

Previously, scientists lacked an effective means of detecting information about circulating tumor cells. To collect information from circulating tumor cells, there are two major challenges.

First of all, the number of circulating tumor cells is small, diluted to a very small concentration in a large amount of blood, the capture efficiency of the existing technology is low, and it is easy to miss some important types of tumor cells.

Secondly, it is highly difficult to purify, and the presence of a large number of blood cells, including red and white blood cells, will contaminate circulating tumor cells and interfere with their analytical results.

With this latest non-invasive detection technology, researchers can fully describe the genetic characteristics of different circulating tumor cells in a single patient with only a small amount of blood.

First, the researchers achieved a preliminary extraction of tumor cells based on cell size. In general, tumor cells will be much larger than normal cells. After crude extraction, the ratio of blood cells to circulating tumor cells will be concentrated from 1 billion: 1 to 95:1.


Concentration of tumor cells

Although the purity of circulating tumor cells is significantly improved, the contamination is still too severe for detailed genetic analysis.

Then, the key technology Hydro-Seq is applied to filter all the remaining blood cells.

Hydro-Seq chip consists of a chamber and a channel system. The specific structure is shown in the figure below, and the core unit circulating tumor cell capture chamber is shown in e in the figure below. After the blood sample has passed through the unit chamber, the circulating tumor cells therein will bind and plug in the position 6.


Specific purification steps

First load cells: at this time, the valve at the end of the barcode bead is closed, the tumor cells bind to the capture site, the valve at the end of the capture site is blocked by the tumor cells, and the rest of the cells can only turn to the downstream chamber, so there will be at most one tumor cell in each chamber.


The green ones are tumor cells, the red ones are blood cells.

After the tumor cells are loaded, the valve at the end of the barcode bead is opened, and other blood cells in the chamber can be washed away.


The tumor cells get stuck at the capture site and block the valve here.

Subsequently, the researchers used barcoded beads to capture the transcriptome of cancer cells: after the barcoded beads entered each chamber, all the valves in the chamber were closed and the cell membrane of the tumor cell was destroyed, and the tumor cell released RNA hybridized with the genetic code on the surface of the barcoded beads.


Here come the gray barcoded beads


Close all valves and "detonate" tumor cells

Finally, by opening the valve, the researchers obtained the barcoded beads and analyzed the genetic information of each tumor cell separately to reveal its active genes.


Barcoded beads carrying tumor information are "freshly baked"!

The researchers tested this technology: in 10ml blood samples of healthy subjects, about 100 tumor cells were added for analysis. After verification, the technology can finally effectively capture 90.43±6.08% circulating tumor cells.

And counting the cells lost during loading, elution, and barcode bead hybridization, the final circulating tumor cells used for analysis still reached 72.85±2.64%!

Based on this technology, the researchers conducted an in-depth analysis of 666 circulating tumor cells from 21 breast cancer patients. The results showed that even in the same patient, the performance of different cancer cells in the body usually varies greatly.


Gene analysis results

Previous studies have confirmed that the metastasis of malignant tumors is induced by tumor cells with stem cell characteristics, that is, tumor stem cells. It should be noted that the previous technology captures tumor cells in the blood of patients by identifying cell surface proteins, but it is particularly easy to miss tumor stem cells because the surface of tumor stem cells does not have consistent protein markers.


Comparison of repeatability test results of the same patient

The analysis results of this study show that although cancer stem cells account for a small proportion of all tumor cells, they account for a large proportion of circulating tumor cells, accounting for 30%-50%!

In the push a few days ago, the medical world elephant introduced the detailed progress of cancer stem cells from other angles. It is worth emphasizing that Professor Max Wich, one of the authors of the current study, has the same deep origin with cancer stem cells. He is the first discoverer of human solid tumor stem cells and one of the most influential researchers in the field of international cancer stem cells.

With the constant changes of gene expression in malignant tumor cells, the target of tumor treatment may also be in a state of constant change, and therapeutic drugs can only kill some of the tumor cells. This technology is very powerful and can monitor the treatment of tumors at the cellular level.

The researchers said that they will use this latest technology to track the progress of patients' tumors and monitor treatment in an upcoming drug clinical trial.


  Hydro-Seq enables contamination-free high-throughput single-cell RNA-sequencing for circulating tumor cells

Change: 10.1038/s41467-019-10122-2


2019 ASCO | 循环肿瘤细胞CTCs在肿瘤免疫治疗中的研究进展

China CTC Industry Alliance is an industry alliance jointly initiated by 10 domestic CTC R & D enterprises and formally established on September 30, 2016. The purpose of the alliance is to "promote the academic exchange of CTC, promote the clinical application of CTC, and establish the industry standard of CTC", so as to contribute to the prevention and treatment of chronic diseases in cancer patients and cancer high-risk groups in China. The companies forming the alliance are Beijing Boao Jingdian Biotechnology Co., Ltd., Beijing Zhongkonatai Biotechnology Co., Ltd., Hangzhou Huadesen Biotechnology Co., Ltd., Yishan Biotechnology Co., Ltd., Zhuhai Lizhu Shengmei Medical Diagnostic Technology Co., Ltd., Shanghai Baihuikang Biotechnology Co., Ltd., Yichang Meiguang Silicon Valley Life Technology Co., Ltd., and Guangzhou Anfang Biotechnology Co., Ltd.

China CTC Industry Alliance relies on domestic academic organizations such as China Society of Clinical Oncology, Laboratory Medicine Branch of Chinese Medical Association, Pathology Branch of Chinese Medical Association, and connects relevant medical institutions, universities, scientific research institutes and technical enterprises to better develop liquid biopsy technology, promote the liquid biopsy market more greatly, promote the status of liquid biopsy more highly, amplify the sound of liquid biopsy more strongly, and realize the clinical transformation of liquid biopsy more quickly, more accurate for the Chinese people to carry out cancer prevention and treatment services.