Efficient circulating tumor cell detection integrated system

Abstract

The invention belongs to the field of biomedical engineering, and relates to detection of tumor circulating cells, in particular to a magnetic engineered cell, a construction method thereof and application of the magnetic engineered cell in detection of the tumor circulating cells. The engineering cell is obtained by transfecting a lentivirus of an anti-epithelial cell adhesion factor (anti-EpCAM) gene with an EGFP (enhanced green fluorescent protein) tag and a lentivirus of an anti-Vimentin (anti-EpCAM) gene with an mCherry tag to a RAW 264.7 cell. The engineered cell provided by the invention can highly express an anti-EpCAM antibody with an EGFP (Enhanced Green Fluorescent Protein) tag and an anti-Vimentin antibody with an mCherry tag, and can realize cell level screening.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering and relates to the detection of circulating tumor cells. Specifically, it relates to a genetically modified magnetic engineered cell, a construction method thereof, and its application in detecting circulating tumor cells. The invention is suitable for early diagnosis and dynamic monitoring of cancer. Background Art

[0002] Circulating tumor cells (CTCs) are intact cancer cells, either singly or in clusters, that shed from the primary or metastatic site of a malignant tumor into the bloodstream. They circulate throughout the body and cause tumor metastasis. Blood CTCs carry a wealth of important information about tumor development and progression, making them crucial for diagnosis and prognosis. Compared to currently commonly used tumor tissue pathology tests, CTCs, as a key marker for liquid biopsy, can effectively avoid the surgical risks associated with invasive methods.

[0003] Currently, the main methods for detecting CTCs include physical separation, immunoseparation, and chemical capture. Physical separation primarily relies on size screening, which can easily cause mechanical damage to cells and cannot distinguish CTCs from similarly sized blood cells. Immunoseparation methods mostly rely on EpCAM antibodies for positive enrichment, but have a capture efficiency of less than 20% for epithelial-mesenchymal transition (EMT) CTCs (such as triple-negative breast cancer), and the antibodies used are relatively expensive. Chemical capture methods have weak binding to low-expression targets and low capture rates.

[0004] However, CTCs are rare, with only 1-100 per 10mL of peripheral blood. Furthermore, CTCs are heterogeneous, with different CTCs expressing varying tumor markers and exhibiting vastly different metastatic potentials. Currently, most CTC detection methods rely on EpCAM antibodies, but EMT leads to downregulation of EpCAM, resulting in a high miss rate of 30-60% with existing antibody-based methods.

[0005] Based on the above, multi-target capture can be used as a method for detecting CTCs. However, current research in this area is very limited, and the use of antibodies is expensive. Therefore, the development of engineered cells targeting multiple targets is of great significance. Summary of the Invention

[0006] To address the above challenges, the present invention aims to provide a method for constructing magnetic engineered cells and their application in detecting circulating tumor cells. The engineered cells provided herein can express an EGFP-tagged anti-EpCAM single-chain antibody, an mCherry-tagged anti-Vimentin single-chain antibody, and a co-expressed anti-EpCAM / Vimentin single-chain antibody. These engineered cells can then engulf Fe₃O₄ nanoparticles and, in combination with an S-shaped microfluidic chip, can screen circulating tumor cells at the cellular level using fluorescence detection, thereby establishing a platform for detecting circulating tumor cells.

[0007] 1. The present invention provides a method for constructing magnetic engineered cells, comprising the following steps:

[0008] Lentivirus containing the anti-EpCAM gene with an EGFP tag was transfected into RAW 264.7 cells to obtain transformed cells;

[0009] transfecting a lentivirus containing an anti-Vimentin gene with an mCherry tag into the transformed cells;

[0010] The magnetic nanoparticles Fe3O4 nanoparticles are internalized into the above cells.

[0011] In certain embodiments, the transfection step can involve simultaneous transfection of two lentiviruses or sequential transfection of two lentiviruses. This sequential transfection method avoids the reduced transfection efficiency associated with excessively long gene sequences. Transfected RAW 264.7 cells are in their logarithmic growth phase, during which they are actively dividing and have high membrane permeability, making them more receptive to exogenous nucleic acid uptake.

[0012] In certain embodiments, a screening process is included, wherein the culture medium used in the screening process contains puromycin and hygromycin B.

[0013] Specifically, the concentration of puromycin in the culture medium was 1-20 μg / mL;

[0014] Specifically, the concentration of hygromycin B in the culture medium is 200-2000 μg / mL.

[0015] 2. The present invention provides an application of the above-mentioned engineered cells in detecting circulating tumor cells.

[0016] Specifically, the detection method is: incubating the sample to be tested with the engineered cells, and performing fluorescence imaging detection on the target after magnetic adsorption enrichment.

[0017] In certain embodiments, fluorescence detection is performed using a laser confocal microscope, wherein the excitation wavelength for fluorescence detection is 470–490 nm or 540–610 nm, and the emission wavelength for fluorescence detection is 500–550 nm or 580–650 nm.

[0018] In certain embodiments, the incubation time is 0.5-3 h.

[0019] 3. Detection of circulating tumor cells in the above-mentioned engineered cells combined with S-type microfluidic chip

[0020] Specifically, the detection method is to pass the engineered cells into an S-shaped microfluidic chip. After the engineered magnetic cells are stably distributed, the sample to be tested (stained circulating tumor cells) is passed into the microfluidic chip for incubation, and the target is detected by fluorescence imaging.

[0021] In certain embodiments, fluorescence microscopy provides real-time fluorescence detection.

[0022] In certain embodiments, the incubation time is 0.5-3 h.

[0023] 4. Compared with the prior art, the present invention has the following advantages:

[0024] The present invention provides a new magnetic engineered cell model and a construction method thereof. By lentiviral transfection of anti-EpCAM genes and anti-Vimentin genes into RAW264.7 cells, RAW264.7 cells that stably express anti-EpCAM proteins and anti-Vimentin proteins are obtained. The obtained cell model has been verified to be able to stably exist and express the corresponding proteins.

[0025] Compared with the detection of existing technologies, the cell model of the present invention can capture CTCs at multiple targets at the same time, reducing the missed detection rate, and is a low-cost and efficient way to detect CTCs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a lentiviral gene map constructed according to an embodiment of the present invention.

[0027] Figure 2 This is a graph showing the results of stably transfected cell lines after lentivirus was transfected into RAW 264.7 cells under a laser confocal microscope in an embodiment of the present invention;

[0028] Figure 3 This is a result diagram of the cells of RAW264.7 after internalization of Fe3O4 magnetic nanoparticles under a laser confocal microscope in an embodiment of the present invention;

[0029] Figure 4This is a graph showing the binding of engineered cells to CTCs under a laser confocal microscope in an embodiment of the present invention;

[0030] Figure 5 Schematic diagram of S-type microfluidic chip

[0031] Figure 6 Fluorescence microscopy imaging of circulating tumor cells

[0032] Figure 7 Circulating tumor cell capture rate detected DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below with reference to the accompanying drawings and the best embodiments.

[0034] Example 1

[0035] Lentiviral construction

[0036] The anti-EpCAM gene sequence was cloned using human cDNA templates via PCR. The resulting sequence fragments were ligated to the plasmid CMV-MCS-EGFP-PGK-Puro-WPRE using double enzyme digestion. Ligation was performed overnight using ligase to form CMV-anti-EpCAM-EGFP-PGK-Puro-WPRE (anti-EpCAM lentiviral sequence). The anti-Vimentin and mCherry gene sequences were cloned, and the resulting sequence fragments were ligated to the plasmid CMV-MCS-EGFP-PGK-HygR-WPRE using double enzyme digestion. Ligation was performed overnight using ligase to form CMV-anti-Vimentin-mCherry-PGK-HygR-WPRE (anti-Vimentin lentiviral sequence). See the map for details. Figure 1 shown.

[0037] Example 2

[0038] Lentiviral infection

[0039] RAW 264.7 cells were passaged into six-well plates at a density of 50%. Once fully adhered, the old culture medium was aspirated in a biosafety cabinet. 500 μL of fresh culture medium was added to each well, along with polybrene to a final concentration of 8 μg / mL. One aliquot of anti-EpCAM lentivirus or anti-Vimentin lentivirus was added to each well, mixed thoroughly, and returned to the incubator for continued culture. After 72 hours, cells were observed under a fluorescence microscope to determine successful expression by fluorescence intensity.

[0040] The transfected cells expressing red fluorescence were then passaged into new six-well plates, and hygromycin B at concentrations of 0, 50, 100, 200, 400, 600, 800, 1000, 1500, and 2000 μg / mL was added for drug resistance screening. The expression of anti-Vimentin antibodies in the cells was determined by fluorescence intensity. After several generations of screening, the RAW 264.7 cell line that stably expressed anti-Vimentin antibodies was finally obtained.

[0041] The cells expressing green fluorescence after transfection were passaged into six-well plates at a density of 50%. After they were completely attached, the old culture medium was aspirated in the biosafety cabinet, 500 μL of fresh culture medium was added to each well, and polybrene was added to make the final concentration 8 μg / mL. One portion of anti-Vimentin lentivirus was added to each well, mixed well, and returned to the incubator for continued culture. After 72 hours, the cells were observed under a fluorescence microscope, and the fluorescence intensity was used to determine whether the expression was successful. Puromycin was added at concentrations of 0, 1, 2, 3, 4, 5, 8, and 10 μg / mL for drug resistance screening, and the expression of anti-EpCAM antibodies in the cells was determined by fluorescence intensity. After several generations of screening, the RAW 264.7 cell line that can stably express anti-EpCAM / Vimentin antibodies was finally obtained. The results are shown in Figure 2 shown.

[0042] Example 3

[0043] Magnetization of transfected cells

[0044] After lentiviral infection, the cell lines stably expressing the virus were cultured at a density of 60-80%. Stable Fe3O4 magnetic nanoparticles were added to the culture medium and co-cultured with the cells. After 12 hours, the cells that had internalized the Fe3O4 magnetic nanoparticles were collected by magnetic attraction. Figure 2 shown.

[0045] Example 4

[0046] Engineered cell testing

[0047] The sample to be tested was incubated with the engineered cells for 2 h in a shaking incubator at 37° C., and the incubated cells were then placed in a confocal microplate for fluorescence imaging detection.

[0048] Example 5

[0049] Cell detection effect

[0050] The engineered cells were introduced into an S-shaped microfluidic chip, and the samples to be tested (circulating tumor cells with cell nuclei staining, and control J774 and Jurkat) were introduced for incubation. The introduction time was 3 minutes, and the captured CTCs were detected by fluorescence microscopy. Fluorescence imaging detection was as follows: Figure 6 As shown, the detection rate is Figure 7 shown.

[0051] The above contents are only preferred embodiments of the present invention. For ordinary technicians in this field, according to the concept of the present invention, there may be changes in the specific implementation methods and application scopes. The contents of this specification should not be understood as limiting the present invention.

Claims

1. An efficient integrated system for detecting circulating tumor cells. Features: These magnetic engineered cells are constructed by transfecting RAW 264.7 cells with a lentivirus containing the EGFP-tagged anti-epithelial cell adhesion molecule (anti-EpCAM) gene and a lentivirus containing the mCherry-tagged anti-vimentin gene. The resulting RAW264.7 magnetic cells are capable of simultaneously and highly expressing an EGFP-tagged anti-EpCAM single-chain antibody and an mCherry-tagged anti-Vimentin single-chain antibody. Feature 2: The RAW 264.7 magnetic cells expressing the two single-chain antibodies are combined with a designed S-channel microfluidic chip to form a highly efficient circulating tumor cell detection system.

2. A method for constructing the engineered cell according to claim 1, characterized in that: The steps include: Lentivirus containing the anti-EpCAM gene with an EGFP tag was transfected into RAW 264.7 cells to obtain transformed cells; transfecting a lentivirus containing an anti-Vimentin gene with an mCherry tag into the transformed cells; The magnetic nanoparticles Fe3O4 nanoparticles are internalized into the above cells.

3. The construction method according to claim 2, wherein: The method comprises a screening process, wherein the culture medium used in the screening process contains puromycin and hygromycin B.

4. The construction method according to claim 3, wherein the culture medium The concentration of puromycin in the culture medium is 1-20 μg / mL; or the concentration of hygromycin B in the culture medium is 200-2000 μg / mL.

5. Use of the engineered cell according to any one of claims 1 to 4 in detecting or screening circulating tumor cells.

6. The use according to claim 5, characterized in that: The detection or screening method is: incubating the sample to be tested with the engineered cells, passing the sample into a microfluidic chip with an S-shaped channel, and then performing fluorescence imaging detection.

7. The use according to claim 6, characterized in that: The incubation time is 0.5-3h.

Images