CTC Typing Kit Using Epithelial and Mesenchymal RNA Probes
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Solution Overview
Problem
Current methods for detecting circulating tumor cells (CTCs) are prone to false positives and false negatives due to the presence of non-neoplastic epithelial cells and loss of epithelial antigens during epithelial-mesenchymal transition, leading to inaccurate identification and typing of CTCs.
Innovation Solution
A circulating tumor cell typing and identification kit that uses a combination of capture, amplification, and labeled probes specific to epithelial and mesenchymal marker genes, including EPCAM, E-cadherin, VIMENTIN, and CD45, to accurately identify and type CTCs by hybridization and signal amplification, reducing non-specific binding and improving sensitivity and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If epithelial markers (EPCAM, CKs) are used as target spots to capture CTCs, then CTC detection can be performed using CellSearch system, but false positive or false-negative detection results occur due to presence of non-neoplastic epithelial cells and loss of epithelial antigens during EMT
Solution Approach 1:
The patent segments the detection approach by using multiple independent marker genes (EPCAM, E-cadherin, VIMENTIN, CD45) instead of relying on a single marker. This segmentation allows the system to detect CTCs through multiple pathways, reducing the impact of antigen loss on any single marker during EMT and eliminating false positives from non-neoplastic cells that don't express the specific gene combinations.
Solution Approach 2:
The patent employs a multi-functional detection system that can identify both epithelial and mesenchymal phenotypes using a unified probe set. The kit is designed to detect various CTC states (epithelial, mesenchymal, and transitional) through universal markers that remain stable during EMT, making the system applicable to all CTC populations regardless of their differentiation state.
2Measurement precision
If RT-PCR process is used for CTC separation and identification, then CTC detection can be performed, but mRNA is prone to degradation which disables CTC cell typing and high requirements for environments and operation are needed
Solution Approach 1:
The patent replaces the complex RT-PCR mechanical and chemical process with a simpler in situ hybridization method using fluorescently labeled probes. This substitution eliminates the need for reverse transcription, PCR amplification, and stringent temperature cycling, while directly visualizing mRNA in intact cells through fluorescence microscopy, thus preserving mRNA integrity and simplifying the operational requirements.
Solution Approach 2:
The patent introduces fluorescently labeled RNA probes as intermediaries that directly bind to target mRNA sequences within CTCs. These probes serve as mediators between the detection system and the target mRNA, allowing specific identification of CTC subtypes without requiring mRNA extraction or amplification, thereby preventing degradation and reducing environmental controls.
3Ease of operation
If single marker gene detection is used, then detection process is simplified, but false-negative results occur due to variation in expression levels of certain marker genes among individual circulating tumour cells
Solution Approach 1:
The patent merges multiple marker gene detections (EPCAM, E-cadherin, VIMENTIN, CD45) into a single integrated in situ hybridization assay. By combining these markers in one detection system with distinct fluorescent labels, the method maintains operational simplicity while significantly improving sensitivity through the ability to detect CTCs expressing any combination of these markers, eliminating false negatives from variable expression patterns.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The kit achieves high specificity and sensitivity in detecting and typing CTCs, reducing false-negative results and improving detection accuracy by using multiple RNA probes and a signal amplification system, allowing for precise identification of epithelial, mesenchymal, and mixed epithelial-mesenchymal phenotypes.
Implementation Method 1
the capture probe binds the marker gene mRNA to the amplification probe
Implementation Method 2
the amplification probe has the following base composition from 5'-terminal to 3'-terminal in sequence: a P3 sequence that is able to complementarily pair with the P2 sequence of the corresponding capture probe
Data Source
AI summary
This disclosure relates to a circulating tumour cell typing and identification kit, comprising a capture probe, an amplification probe, and a labeled probe for each marker gene mRNA, wherein the marker gene mRNA comprises the following two types: at least two epithelial cell marker gene mRNAs selected from the group consisting of EPCAM, E-cadherin, CEA, KRT5, KRT7, KRT17, and KRT20 mRNAs; and, at least two mesenchymal cell marker gene mRNAs selected from the group consisting of VIMENTIN, N-cadherin, TWIST1, AKT2, ZEB2, ZEB1, FOXC1, FOXC2, SNAI1 and SNAI2 mRNAs. This disclosure prevents false-positive results caused by, for example, possible presence of a number of non-neoplastic epithelial cells in peripheral blood, introduction of normal epithelial cells during blood sampling, and the like. Accordingly, it may be assured that cells detected with epithelial cell marker genes and/or mesenchymal cell marker genes are indeed circulating tumour cells, further improving accuracy and reliability of the detection results.