A microfluidic chip for sorting circulating tumor cells and analyzing multi-dimensional heterogeneity at single cell level and application thereof

Abstract

The application relates to a microfluidic chip for sorting circulating tumor cells and analyzing multi-dimensional heterogeneity at a single cell level and application, and belongs to the technical field of microfluidic chips. Specifically, the application relates to a cascade microfluidic chip system based on the combination of a spiral structure and a single cell array structure. The chip comprises two-stage structures in sequence, namely a first-stage spiral structure and a second-stage single cell array structure. The first-stage structure is used for quickly removing a large number of blood cells from blood according to cell size, and the second-stage structure is used for capturing circulating tumor cells in the form of single cells and performing heterogeneity analysis of the circulating tumor cells based on the single cell level. The chip system realizes the efficient capture of circulating tumor cells in the form of single cells and further realizes the in-situ molecular and functional heterogeneity analysis of the circulating tumor cells at the single cell level.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic chip technology, specifically involving a cascaded microfluidic chip system based on a combination of helical structure and single-cell array structure, which can efficiently capture circulating tumor cells in the form of single cells and enable in-situ molecular and functional heterogeneity analysis. Background Technology

[0002] Metastasis is one of the fundamental biological characteristics of malignant tumors and a major cause of death in cancer patients. Therefore, it is necessary to explore novel diagnostic methods to provide real-time, accurate, and effective information for predicting metastasis, evaluating treatment, and assessing prognosis. Circulating tumor cells (CTCs) are tumor cells that detach from the primary tumor site or metastatic lesions and enter the peripheral blood circulation. They can invade other tissues and proliferate to form new tumors, playing a crucial role in tumor recurrence and metastasis. CTCs also have significant clinical value in prognostic assessment, treatment selection, and efficacy monitoring for cancer patients.

[0003] However, it is noteworthy that only a very small number of CTCs with high metastatic potential can form metastatic lesions. Therefore, effectively enriching and analyzing such CTCs is a key issue in predicting tumor metastasis. Simultaneously, CTCs exhibit heterogeneity, and the specific characteristics that determine their metastatic activity remain unknown. Therefore, discovering and clarifying these characteristics is crucial for accurately predicting tumor metastasis. Furthermore, given the heterogeneous nature of CTCs, it is necessary to study their multidimensional heterogeneity at the single-cell level. Currently, single-cell level heterogeneity analysis of CTCs mainly focuses on two key technical aspects: firstly, high-speed, high-recovery, and high-purity enrichment of CTCs; and secondly, multidimensional analysis of CTC heterogeneity from molecular phenotype, functional phenotype, and morphology. In addition, real-time recording of CTC dynamic changes will provide complete and comprehensive information for CTC heterogeneity analysis, but there are still few reports on real-time dynamic analysis of single-cell level CTC behavioral changes and on multidimensional heterogeneity analysis of single-cell level CTCs.

[0004] Microfluidic chips, with their advantages of simple operation, high throughput, low cost, ease of automation, miniaturization, and integration, are widely recognized as an effective platform for enriching and analyzing cytotoxic tumor cells (CTCs). However, enriching CTCs and performing multidimensional heterogeneity analysis at the single-cell level remains challenging. Therefore, establishing a microfluidic research platform to efficiently achieve CTC enrichment and multidimensional heterogeneity analysis of CTCs at the single-cell level, and exploring the correlation between CTC heterogeneity characteristics and tumor metastasis, will be of great significance to tumor metastasis research. Summary of the Invention

[0005] To address the aforementioned issues, the purpose of this invention is to create a cascaded microfluidic chip system based on a combination of helical and single-cell array structures, enabling efficient capture of CTCs in the form of single cells, further satisfying the need for in-situ single-cell level multidimensional heterogeneity analysis of CTCs, and providing applications for this microfluidic chip.

[0006] The technical problem this invention aims to solve is that single-cell level heterogeneity analysis of cytokines (CTCs) presents two key technical challenges: firstly, high-speed, high-recovery, and high-purity enrichment of CTCs; and secondly, multi-dimensional heterogeneity analysis of CTCs from molecular phenotype, functional phenotype, and morphology. Furthermore, real-time recording of CTC dynamic changes provides comprehensive information for CTC heterogeneity analysis, but real-time dynamic analysis of single-cell level CTC behavior and multi-dimensional heterogeneity analysis at the single-cell level have not yet been reported. This cascaded microfluidic chip system achieves efficient single-cell capture of CTCs and simultaneously enables in-situ multi-dimensional heterogeneity analysis at the single-cell level by cascading a primary helical structure for rapid enrichment of CTCs from blood and a secondary single-cell array structure for capturing individual CTCs.

[0007] To achieve the above objectives, the present invention provides the following technical solution.

[0008] This invention provides a microfluidic chip system for sorting circulating tumor cells and performing multidimensional heterogeneity analysis at the single-cell level, characterized in that the chip system ( Figure 1 ) is a cascaded microfluidic chip system based on the combination of helical sorting structure and single-cell array structure; the first-level helical sorting structure includes: an injection hole (1), a helical channel with a periodic expansion structure (2), two blood cell outlets (3) and a tumor cell outlet (4); the second-level single-cell array structure includes: a triangular expansion structure (5), a single-cell array (6) composed of horseshoe-shaped structures with microslits, and an open region (7).

[0009] Furthermore, the first-stage spiral sorting structure is used to quickly remove a large number of blood cells; the second-stage single-cell array structure is used to capture CTCs with high purity in single-cell form; cascading the above two-stage structures can improve CTC sorting performance.

[0010] Furthermore, in the spiral channel structure of the periodic expansion structure in the first-stage spiral sorting structure, the width of the expansion structure is 100~500μm and the length is 500~2000μm, the width of the non-expansion structure is 50~250μm, the distance between the two expansion structures is 250~1000μm, and the distance between the expansion structure in the previous spiral channel and the next spiral channel is 200~500μm.

[0011] Furthermore, the triangular expansion structure (5) in the single-cell array structure significantly reduces the flow rate of fluid after it flows out of the tumor cell outlet (4), thereby ensuring that the tumor cells are captured in the single-cell array (6).

[0012] Furthermore, the single-cell array structure includes horseshoe-shaped single-cell micro-slit structures with 10-150 columns horizontally and 10-150 rows vertically; each horseshoe-shaped micro-slit structure consists of a pair of symmetrical boundaries, with a 20-40 μm opening at the top to ensure that tumor cells enter the structure in single-cell form, and a 5-12 μm micro-slit at the bottom to ensure that a single tumor is captured in the structure and to provide a channel for cell migration and invasion. The horizontal and vertical gaps of the array structure are 20-60 μm to ensure that fluid flows stably through the chip.

[0013] Furthermore, the single-cell array (6) and the open region (7) in the single-cell array structure are connected; when the chip is placed in a cell culture dish containing cell culture medium, the liquid outside the chip can diffuse into the chip through the open region (7); when different concentrations of chemokines or nutrient conditions are applied inside and outside the chip, a concentration gradient from outside the chip to inside the chip can be formed.

[0014] Furthermore, in the single-cell array structure, tumor cells are captured in the single-cell array (6) in the form of single cells, which can perform in situ analysis of the heterogeneity of tumor cell molecular phenotype and functional phenotype, as well as real-time monitoring of the in situ dynamic changes of single cells.

[0015] Furthermore, the chip is packaged by bonding a base layer and a flow channel layer together. The material of the flow channel layer is polydimethylsiloxane, cyclic olefin copolymer, polycarbonate, or polymethyl methacrylate. The material of the base layer is silicon dioxide, silicon wafer, polydimethylsiloxane, siliconized glass, polycarbonate, or polymethyl methacrylate.

[0016] This invention also provides a method for using a microfluidic chip system for sorting circulating tumor cells and performing multidimensional heterogeneity analysis at the single-cell level, characterized by the following steps:

[0017] a. Peripheral blood from tumor patients is collected, diluted with PBS, and pumped into the chip through the injection port (1). Tumor cells flow through the spiral channel (2), tumor cell outlet (4), and triangular expansion structure (5) into the single-cell array (6) and are captured as single cells. Blood cells flow through the spiral channel (2) and out through the blood cell outlet (3) and open area (7). After all the blood sample has been pumped into the chip, PBS is introduced until no blood cells remain in the chip. A schematic diagram of the CTC sorting process is shown below. Figure 2 As shown;

[0018] b. Immunofluorescence staining of captured CTCs with relevant biomarkers was performed to determine the number of captured CTCs and analyze the molecular phenotypic heterogeneity of CTCs at the single-cell level. A schematic diagram of CTC molecular phenotypic heterogeneity analysis is shown below. Figure 3 As shown in A;

[0019] c. Add matrix gel containing a low concentration of FBS cell culture medium diluted into the chip, place the chip in a cell culture dish, and add a high concentration of FBS cell culture medium to the dish to create a serum concentration gradient within the chip that drives CTC migration and invasion. Incubate the chip in a cell culture incubator and periodically photograph the cell status at the same location within the chip to record changes in cell behavior. Additionally, place the culture dish in a live cell dynamic monitoring system to continuously photograph the cells and record real-time behavioral changes of different tumor cells within the same field of view. These steps achieve functional phenotypic heterogeneity analysis of CTCs at the single-cell level. A schematic diagram of CTC functional phenotypic heterogeneity analysis is shown below. Figure 3 As shown in B.

[0020] Further, the relevant biomarkers mentioned in step b include at least one of EpCAM, E-cadherin, CK8, CK18 and CK19 as epithelial cell type marker proteins, and / or at least one of N-cadherin, Vimentin, Fibronectin, ZEB1 and Snail1 as mesenchymal cell type marker proteins, and the leukocyte marker CD45.

[0021] The beneficial effects of the present invention compared with the prior art.

[0022] This invention provides a cascaded microfluidic chip system based on a combination of helical and single-cell array structures. This cascaded chip system can leverage the strengths of both helical and single-cell array structures, enabling the chip system to achieve high-throughput CTC sorting based on the helical structure and high-purity CTC sorting based on the single-cell array structure. This overcomes the shortcomings of low purity when using the helical structure alone and low throughput when using the single-cell array structure alone, thereby achieving efficient sorting of CTCs in the peripheral blood of tumor patients.

[0023] This invention creates a cascaded microfluidic chip based on a combination of helical and single-cell array structures, achieving seamless integration of CTC sorting and single-cell analysis. This improves analysis speed and reduces sample loss and analytical errors caused by sample transfer and human manipulation. After CTCs are captured, multi-dimensional heterogeneity analysis at the single-cell level can be performed in situ within the chip, including molecular phenotypic heterogeneity analysis, functional heterogeneity analysis such as proliferation, invasion, migration, and drug uptake, as well as real-time dynamic monitoring of these functional phenotypes. This analysis of multi-dimensional tumor cell heterogeneity will provide richer information for evaluating the correlation between CTC heterogeneity and tumor metastasis. Attached Figure Description

[0024] Figure 1 This describes the structure of the cascaded microfluidic chip of the present invention.

[0025] Figure 2 This is a schematic diagram of the process of CTC sorting and capture using cascaded microfluidic chips.

[0026] Figure 3 This is a schematic diagram illustrating the molecular phenotypic and functional phenotypic heterogeneity analysis of single-cell CTCs achieved in situ within a chip.

[0027] Figure 4 This is a diagram showing the results of sorting and capturing tumor cells in blood using the cascaded microfluidic chip in Example 1.

[0028] Figure 5 This is a diagram showing the results of sorting different types and quantities of tumor cells in the blood using the cascaded microfluidic chip in Example 2, along with an analysis of its capture efficiency.

[0029] Figure 6 This is a diagram showing the results of molecular phenotypic heterogeneity of SW620 cells within the microfluidic chip in Example 3.

[0030] Figure 7 This is a diagram showing the results of real-time observation of functional heterogeneity of A549 cells within a microfluidic chip in Example 4.

[0031] Figure 8 This is a diagram showing the results of the capture and molecular heterogeneity analysis of CTCs in the peripheral blood of tumor patients using the cascaded microfluidic chip in Example 5. Detailed Implementation

[0032] The present invention will now be described in detail with reference to specific embodiments. The following embodiments will help to understand the present invention, but these embodiments are only for illustrative purposes, and the present invention is not limited thereto. The operating methods in the embodiments are all conventional operating methods in this technical field.

[0033] Experiment 1 uses a cascaded microfluidic chip to sort and capture tumor cells in blood.

[0034] LoVo cells were fluorescently labeled with the live cell dye Cell Tracker. The cells were then incorporated into rabbit blood diluted in PBS to simulate CTCs in the blood of cancer patients. The cell suspension was pumped into the chip at a rate of 200 μL / min using a microinjection pump, and the sorting of blood cells and tumor cells at the spiral structure outlet was observed. Figure 4A) Non-fluorescent blood cells and fluorescent tumor cells are focused into bundles. Blood cells mainly flow out from the outer exit, while tumor cells flow out from the central exit and enter downstream structures. Observation of the single-cell array shows that fluorescent tumor cells are captured by the micro-slit units in the form of single cells, while a small number of residual blood cells pass through the micro-slit units and micro-slits and flow out. Figure 4 B). After blood sorting is completed, PBS is introduced into the chip to remove any remaining blood cells. Figure 4 C). The above results demonstrate that this cascaded microfluidic chip can effectively sort tumor cells in the blood and capture them as single cells in a single-cell array structure.

[0035] Example 2: Evaluation of the enrichment efficiency of cascaded microfluidic chips for different numbers and types of tumor cells in blood.

[0036] Different amounts of SGC-7901 gastric cancer cells labeled with the live-cell dye Cell Tracker were infused into 1 mL of rabbit whole blood at concentrations of 50, 100, and 150 cells / mL, respectively, to simulate peripheral blood samples from clinical patients for cell sorting. The blood was diluted 5-fold with PBS, and the cell suspension was pumped into the array using a microinjection pump at a rate of 300 μL / min. The capture of SGC-7901 gastric cancer cells at the single-cell array was recorded using an inverted fluorescence microscope, and the number of SGC-7901 cells captured in each experiment was recorded. The capture efficiency was calculated as (number of captured tumor cells / total number of injected tumor cells) × 100%. The results showed that the labeled tumor cells could be captured in the single-cell array. Figure 5 A), and has satisfactory capture efficiency ( Figure 5 B).

[0037] Gastric cancer cells (SGC-7901), lung cancer cells (A549), and colorectal cancer cells (LoVo) were infused into 1 mL of rabbit whole blood at a concentration of 100 cells / mL to simulate peripheral blood samples from clinical patients for cell sorting. The blood was diluted 5-fold with PBS, and the cell suspension was pumped into the chip using a microinjection pump at a rate of 300 μL / min. The number of tumor cells captured at the single-cell array was recorded using an inverted fluorescence microscope. The capture efficiency was calculated as (number of captured tumor cells / total number of injected tumor cells) × 100%. Results showed that the capture efficiency for all cell types exceeded 85%. Figure 5 C).

[0038] Example 3: Analysis of molecular phenotypic heterogeneity of SW620 cells within a microfluidic chip.

[0039] Colorectal cancer cells SW620 were captured in a single-cell array using a microarray system, and molecular phenotypic heterogeneity was analyzed according to the following experimental procedure. First, cells were fixed at room temperature for 15 min by aeration with 4% paraformaldehyde, followed by a 5 min rinse with PBS. Cells were then blocked for 60 min by aeration with 400 μL of BSA blocking buffer. Fluorescently labeled mouse anti-human EpCAM antibody was introduced into the microarray, and the cells were incubated at 37°C in the dark for 40 min, followed by a 5 min rinse with PBS to stain for the epithelial cell marker protein EpCAM. The microarray was then observed and recorded under an inverted fluorescence microscope to record EpCAM expression. Next, cells were permeated with 0.5% Triton X-100 for 15 min, followed by a 5 min rinse with PBS. Rabbit anti-human Vimentin antibody was introduced into the microarray, and the cells were incubated at room temperature for 2 h, followed by a 5 min rinse with PBS. Finally, fluorescently labeled goat anti-rabbit secondary antibody was introduced into the microarray, and the cells were incubated at 37°C in the dark for 40 min, followed by a 5 min rinse with PBS to stain for the mesenchymal cell marker protein Vimentin. DAPI dilution buffer was introduced into the chip, and the cells were incubated at room temperature in the dark for 5 min, followed by rinsing with PBS for 5 min. The chip was then placed under an inverted fluorescence microscope to observe and record the expression of Vimentin in the cells. The fluorescence intensity of single cells within the chip was analyzed using ImageJ image analysis software to evaluate protein expression levels. The results showed that the molecular expression of different SW620 cells exhibited high heterogeneity. Figure 6 Cells 1, 4, 6, and 7 were EpCAM-positive; cells 2 and 3 were Vimentin-positive; and cells 5 and 8 were double-positive for both EpCAM and Vimentin. ImageJ numerical analysis also showed significant heterogeneity in the expression of EpCAM and Vimentin molecules among different SW620 cells.

[0040] Example 4: Real-time observation of functional heterogeneity of A549 cells within a microfluidic chip.

[0041] Lung cancer cells A549 were captured in a single-cell array using a microarray system. The matrix gel was diluted 1:9 with cell culture medium containing 10% FBS, and the dilution was then passed into the microarray. The microarray was placed in a cell culture dish, and cell culture medium containing 20% ​​FBS was added. The cell culture dish was placed in a live-cell dynamic monitoring system and cultured in a 37°C, 5% CO2 incubator. Continuous video recording was performed to record the real-time behavioral changes of different tumor cells in the same field of view, thereby analyzing their functional heterogeneity. 29 hours of continuous video recording showed that different cells exhibited real-time heterogeneity in proliferation, invasion, and even cell-cell interaction behaviors. A screenshot of cell position at 0:00 is shown below. Figure 7 A, representative screenshots of real-time cell behavior can be found here. Figure 7 B. The results show that... Figure 7 B(a) and Figure 7The two single cells in B(b) remained viable during continuous observation for 29 hours, but no obvious positional movement was observed. Figure 7 The single cells in B(c) exhibited a migration-division-division behavior change during continuous observation; Figure 7 In cell B(d), cell 1 remains active without moving, while cell 2 exhibits a migration-division-migration behavior. Figure 7 In cell B(e), cell 1 remained active without relocation, while cell 2 exhibited a behavioral change of migration (2h) - division (13h) - migration (15h) - division (25h). Notably, cell 3, originally located in row 6 and produced by the division of cell 2, migrated to row 8 and fused with the cells in its current location (21h-29h), while simultaneously exhibiting the behavior of secreting substances. These results demonstrate that this chip allows for real-time observation of the heterogeneous behavior of individual tumor cells across multiple functional phenotypes, including proliferative capacity, migration and invasion capabilities, and cell-cell interaction capabilities. The multidimensional real-time information reflected provides an important foundation for accurately evaluating the tumor metastasis predictive value of CTCs.

[0042] Example 5: Capture of CTCs in peripheral blood of tumor patients using a cascaded microfluidic chip.

[0043] CTCs from peripheral blood of clinical cancer patients were isolated and enriched using a cascaded microfluidic chip. One mL of peripheral blood from a lung cancer patient was diluted 5-fold with PBS and pumped into the chip at a rate of 300 μL / min using a microinfusion pump. CTCs were captured as single cells in a single-cell array. PBS was then purged until no blood cells remained in the chip. Following the basic procedure in Example 3, the captured cells were stained with EpCAM and CD45 immunofluorescence. CD45+ / DAPI+ / EpCAM- were identified as leukocytes, and CD45- / DAPI+ were identified as CTCs. The results showed ( Figure 8 A) Three CTCs were captured from the peripheral blood of patient 1, one of which was EpCAM+ and two were EpCAM-, indicating that the present invention can be successfully used for CTC capture and that the EpCAM expression of CTCs is highly heterogeneous.

[0044] Peripheral blood was also collected from one colorectal cancer patient, and the captured cells were subjected to dual-fluorescent immunostaining with EpCAM and Vimentin. The results are shown in the figure. Figure 8(B) Two single-celled tumors (CTCs) and one CTC cluster composed of multiple cells were captured from the peripheral blood of patient 2. Both CTCs were EpCAM- / Vimentin+, and the CTC cluster was EpCAM+ / Vimentin+. These results suggest that this system can effectively capture CTCs from the peripheral blood of patients with different types of tumors and can analyze various molecular phenotypic heterogeneities of CTCs at the single-cell level. In particular, it can enrich CTC clusters while capturing single CTCs, thus expanding the dimensions of CTC analysis.

Claims

1. A microfluidic chip system for sorting circulating tumor cells and performing multidimensional heterogeneity analysis at the single-cell level, characterized in that, The chip system is a cascaded microfluidic chip system based on a combination of a first-level helical sorting structure and a second-level single-cell array structure. The first-level helical sorting structure includes: an injection port (1), a helical channel (2) with a periodic expansion structure, two blood cell outlets (3), and a tumor cell outlet (4). In the helical channel structure of the periodic expansion structure in the first-level helical sorting structure, the width of the expansion structure is 100~500μm, the length is 500~2000μm, the width of the non-expansion structure is 50~250μm, the spacing between the two expansion structures is 250~1000μm, and the spacing between the expansion structure in the previous helical channel and the next helical channel is 200~500μm. The second-level single-cell array structure includes: a triangular expansion structure (5), a single-cell array (6) composed of horseshoe-shaped structures with microslits, and an open region (7). The single-cell array structure includes 10~150 columns horizontally and 10~150 rows vertically of horseshoe-shaped single-cell microslit structures with microslits. Each horseshoe-shaped microslit... The slit structure consists of a pair of symmetrical boundaries. The top has a 20-40 μm opening to ensure that tumor cells enter the structure in the form of single cells, and the bottom has a 5-12 μm micro-slit to ensure that a single tumor cell is captured in the structure and to provide a channel for cell migration and invasion. The transverse and longitudinal gaps of the array structure are 20-60 μm to ensure that the fluid flow passes stably in the chip. The first-level spiral sorting structure is used to quickly remove a large number of blood cells. The second-level single-cell array structure is used to capture CTCs in the form of single cells with high purity. The cascade of the first-level spiral sorting structure and the second-level single-cell array structure can improve the CTC sorting performance. The triangular expansion structure (5) in the single-cell array structure significantly reduces the flow rate of the fluid after it flows out of the tumor cell outlet (4), thereby ensuring that the tumor cells are captured in the single-cell array (6). In the single-cell array structure, the tumor cells are captured in the single-cell array (6) in the form of single cells, which can perform in situ analysis of the heterogeneity of tumor cell molecular phenotype and functional phenotype, as well as real-time monitoring of the dynamic changes of single cells in situ.

2. The microfluidic chip system according to claim 1, characterized in that, In the single-cell array structure, the single-cell array (6) and the open region (7) are connected; when the chip is placed in a cell culture dish containing cell culture medium, the liquid outside the chip can diffuse into the chip through the open region (7); when different concentrations of chemokines or nutrient conditions are applied inside and outside the chip, a concentration gradient from outside the chip to inside the chip can be formed.

3. The microfluidic chip system according to claim 1, characterized in that, The chip is encapsulated by bonding a base layer and a flow channel layer together. The material of the flow channel layer is polydimethylsiloxane, cyclic olefin copolymer, polycarbonate or polymethyl methacrylate, and the material of the base layer is silicon dioxide, silicon wafer, polydimethylsiloxane or siliconized glass.

4. A method for using a microfluidic chip system for sorting circulating tumor cells and performing multidimensional heterogeneity analysis at the single-cell level, characterized in that, Using the microfluidic chip system according to any one of claims 1-3 includes the following steps: a. Take peripheral blood from a tumor patient, dilute it with PBS, and pump the peripheral blood dilution into the chip through the injection hole (1). Tumor cells flow through the spiral channel (2), tumor cell outlet (4), and triangular expansion structure (5) into the single cell array (6) and are captured in single cell form. Blood cells flow through the spiral channel (2) and out from the blood cell outlet (3) and open area (7). After all the blood sample has been pumped into the chip, PBS is introduced until there are no blood cells left in the chip. b. Perform immunofluorescence staining of the captured CTCs with relevant markers to determine the number of captured CTCs and analyze the molecular phenotypic heterogeneity of CTCs at the single-cell level; c. Add matrix gel containing a low concentration of FBS cell culture medium diluted into the chip, place the chip in a cell culture dish, and add a high concentration of FBS cell culture medium to the culture dish to create a serum concentration gradient inside the chip that drives CTC migration and invasion. Incubate the chip in a cell culture incubator and periodically photograph the cell status at the same location within the chip to record changes in cell behavior. In addition, place the culture dish in a live cell dynamic monitoring system to continuously photograph the cells and record the real-time behavioral changes of different tumor cells in the same field of view. The above steps achieve functional phenotypic heterogeneity analysis of CTCs at the single-cell level.

5. The method of use according to claim 4, characterized in that, The relevant biomarkers mentioned in step b include at least one of EpCAM, E-cadherin, CK8, CK18 and CK19 as epithelial cell type marker proteins, and / or at least one of N-cadherin, Vimentin, Fibronectin, ZEB1 and Snail as mesenchymal cell type marker proteins, and the leukocyte marker CD45.

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