A system for rapid detection of tumor cell invasiveness and risk grade reference based on matrix remodeling fluorescent reporter

Abstract

This invention discloses a system for rapid detection of tumor cell invasiveness and risk level reference based on matrix remodeling fluorescence reporter assay. After staining, test cells are seeded on a matrix material formed by cell adhesion ligands and fluorescent dye-modified polyisocyanate peptides for 2D culture, or the two components are thoroughly mixed and gelled to form a 3D culture environment, resulting in a cell-induced matrix remodeling fluorescence detection system. Fluorescence images of the area surrounding the sample are acquired, and cell invasiveness risk is analyzed based on changes in the intensity of the fluorescence signal in the surrounding area and the matrix remodeling index. This invention provides a more direct and rapid assessment of tumor cell invasiveness by observing the matrix remodeling characteristics induced during cell invasion in real time in vitro, providing a risk level reference for tumor malignancy.

Description

Technical Field

[0001] This invention belongs to the field of tumor cell detection, specifically relating to a system for rapid detection of tumor cell invasiveness and risk level reference based on matrix remodeling fluorescence reporter assay. Background Technology

[0002] Tumor cell invasiveness is a crucial biological characteristic determining their local infiltration ability, distant metastasis, and poor clinical prognosis. Tumor cell invasive behavior is not only regulated by their intrinsic genetic background and phenotypic state but also highly dependent on the composition and mechanical properties of the extracellular matrix (ECM) within the tumor microenvironment. During invasion and migration, tumor cells typically breach the stromal barrier and spread to surrounding tissues through cytoskeleton contraction to generate traction stress, dynamic adhesion and deadhesion cycles, and remodeling of the surrounding ECM (including densification, directional alignment, or local degradation). Therefore, establishing a method based on cell-induced matrix remodeling that can rapidly and accurately assess the invasive potential of tumor samples and its associated malignant progression is of significant value for clinical prognosis, personalized treatment strategy development, and efficacy monitoring.

[0003] In current clinical practice, the traditional diagnosis of tumors and the assessment of their invasive potential mainly rely on the clinicopathological and histological characteristics of biopsy tissues, such as differentiation degree and mitotic index, combined with other morphological observations for comprehensive judgment. While these methods are widely used clinically, they typically involve steps such as tissue sampling, fixation, embedding, sectioning, staining, and pathological examination, making the process relatively complex and time-consuming. Furthermore, the assessment results are influenced by factors such as sample representativeness (e.g., whether the sampling site covers the invasion front), section quality, processing conditions, and the examiner's experience, resulting in a degree of subjectivity and variability. More importantly, traditional histological characteristics represent static morphological endpoints and may not directly reflect the dynamic invasive behavior of tumor cells in the three-dimensional microenvironment within the early time window, thus limiting the rapid and direct prediction and quantitative grading of the "individual sample invasive potential."

[0004] Recent biomechanical and mechanobiological studies have demonstrated the inseparable link between tumor invasion and migration and cell-matrix mechanical interactions. More aggressive tumor cells often exert more significant traction forces and induce stronger matrix remodeling behavior, leading to measurable changes in the structure and mechanical state of the pericellular ECM (e.g., localized compaction, fiber orientation, or rearrangement). Simultaneously, the development of fluorescent and biomaterials with mechanically responsive properties has made it possible to transform cellular force exertion and matrix rearrangement processes into visualized and quantifiable fluorescence readouts, providing a new technical pathway for establishing "rapid invasion characterization based on cell-matrix interactions."

[0005] However, current technologies still lack a method to easily, rapidly, quantitatively, and standardly translate the matrix remodeling signals induced during tumor cell invasion into characterizations of invasiveness and pathological deterioration. In particular, a solution to output reproducible invasion scores / risk stratification results within an early time window (e.g., within hours) to meet the needs of rapid screening, sample difference assessment, and potential clinical decision support remains to be developed.

[0006] Therefore, it is necessary to provide a new detection and characterization strategy: using the cell-matrix interaction of tumor cells in the stromal microenvironment and the induced stromal mechanical remodeling as a functional readout, and using the fluorescence signal of cell-induced stromal remodeling response to achieve rapid imaging and quantification of tumor cell invasiveness and risk level, and outputting results that can be used for grading or risk stratification. Summary of the Invention

[0007] This invention addresses the shortcomings of existing technologies by providing a matrix material comprising a cell-induced matrix remodeling responsive fluorescent reporter unit and its application in the preparation of reagents for reference on tumor cell invasiveness and risk levels.

[0008] The matrix material containing the cell-induced matrix remodeling response fluorescent reporter unit is a polyisocyanate polypeptide co-modified with cell adhesion ligands and fluorescent dyes.

[0009] The matrix material containing the cell-induced matrix remodeling responsive fluorescent reporter unit is prepared by a method comprising the following steps:

[0010] (1) Preparation of cell adhesion functionalized modified polyisocyanate peptides By modifying at least one of the cell adhesion ligands RGD, cRGD, HAVID and the invasin into the polyisocyanate peptide through covalent or non-covalent means, cell adhesion functionalized polyisocyanate peptides are obtained. (2) Linkage of polyisocyanate peptides with fluorescent dyes Weigh the cell adhesion functionalized modified polyisocyanate peptide and dissolve it in buffer solution. Mix it thoroughly with fluorescent dye 1 in a certain proportion to obtain a polyisocyanate peptide co-modified by cell adhesion ligand and fluorescent dye, which is a matrix material containing a cell-induced matrix remodeling response fluorescent reporter unit.

[0011] In step (1) of the above method, the helical polypeptide is covalently linked or assembled with RGD to obtain RGD-functionalized polyisocyanate polypeptide; More specifically, azide-containing helical polypeptides can be reacted with DBCO-PEG4-RGD via click chemistry to obtain RGD-functionalized polyisocyanate polypeptides. The structural formula of the azide-bearing helical polypeptide is shown below:

[0012] Where n represents the polymer, n = 50 - 50000; The ratio of helical polypeptide to RGD can be 10-100mg:1-2mg, specifically 35mg:1mg.

[0013] The click chemical reaction was carried out at room temperature for 18-28 hours.

[0014] In step (2), the buffer solution is 1×PBS; The concentration of polyisocyanate peptide dissolved in 1×PBS is 0.5 mg / mL-10 mg / mL, specifically 2 mg / mL; The dissolution is carried out at 2-6℃ for 6-24 hours. The fluorescent dye 1 can be any dye suitable for fluorescence microscopy imaging, specifically TAMRA (tetramethylrhodamine). The concentration range of the fluorescent dye 1 is 50 μM-150 μM, specifically 100 μM; The mixing was carried out at 2-6°C for 15-30 minutes.

[0015] In the application described, the matrix material containing the cell-induced matrix remodeling response fluorescent reporter unit is dissolved in PBS or culture medium; the tumor cells to be tested are incubated with fluorescent dye 2 to stain the cells, and after digestion and centrifugation, a cell suspension is obtained; the stained cells are seeded on the matrix material for 2D culture, or the two components are thoroughly mixed and gelled to form a 3D culture environment, thereby obtaining a fluorescent reporter detection system for matrix remodeling.

[0016] The matrix material containing the cell-induced matrix remodeling response fluorescent reporter unit, namely the cell adhesion ligand and the fluorescently modified polyisocyanate peptide, is dissolved in 1×PBS at a concentration of 0.5 mg / mL-10 mg / mL. The dissolution process is carried out at 2-6℃ for 6-24 h. The fluorescent dye 2 is CeLLTracker (live cell tracer), and the concentration of dye 2 is 5μM-20μM, specifically 10μM. The staining process is carried out at room temperature.

[0017] The cells to be tested can be tumor cells or tissue samples, specifically 231 (MBA). MB 231) Triple-negative human breast cancer cells and MCF-7 (Michigan Cancer Foundation-7) human breast cancer cells.

[0018] The final cell concentration was 1×10⁻⁶. 4-1×10 6 Cells / mL; the volume ratio of the stained cell suspension to the polyisocyanate peptide is 50-100 μL: 50-100 μL, specifically 1:1; The gelation time can be 15-30 minutes, specifically 20 minutes.

[0019] The culture conditions are 37℃, 5% CO2, and the culture time is 0-2 days, specifically 1 day.

[0020] In this application, fluorescence images of the area surrounding the sample are acquired, and the invasiveness of the tumor cells to be tested is determined based on the intensity changes of the fluorescence signal in the area surrounding the sample and the matrix remodeling index. The matrix remodeling index is calculated as follows: after the cells have been cultured in the matrix material for a period of time (1-24 hours), imaging is performed using a fluorescence microscope to obtain a fluorescence image of the cell matrix remodeling, and the matrix remodeling index is analyzed; specifically: 1) The fluorescence intensity of materials without cells was used as the benchmark (A0); 2) With the cell as the center, observe the fluorescence intensity (A1) of the matrix in the area surrounding the cell. If A1 is greater than A0, it is calculated as the matrix remodeling volume. 3) Observe and calculate the number of cells with cellular matrix remodeling characteristics.

[0021] This invention also provides an in vitro fluorescence detection method for detecting the invasiveness of test cells based on cell-induced matrix remodeling, the method comprising: a) Provide a matrix material containing a cell-induced matrix remodeling response fluorescent reporter unit; b) Place the cells to be tested or cell samples in the matrix material for two-dimensional (2D) or three-dimensional (3D) culture, so that the cells come into contact with the matrix material; c) Obtain fluorescence images of the region surrounding the cells; d) Analyze cell invasiveness based on changes in the intensity of fluorescence signals in the pericellular region and the matrix remodeling index; The matrix material comprising the cell-induced matrix remodeling response fluorescent reporter unit is a polyisocyanate polypeptide co-modified with cell adhesion ligands and fluorescent dyes.

[0022] The cell adhesion ligand is selected from at least one of RGD, cRGD, HAVID, and the invasin. The fluorescent dye can be any dye suitable for fluorescence microscopy imaging, specifically TAMRA (tetramethylrhodamine).

[0023] The matrix remodeling index is calculated as follows: after cells have been cultured in the matrix material for a period of time (1-24 hours), fluorescence microscopy is used to image the cells and obtain a fluorescence image of the cell matrix remodeling, and the matrix remodeling index is analyzed; specifically: 1) The fluorescence intensity of materials without cells was used as the benchmark (A0); 2) With the cell as the center, observe the fluorescence intensity (A1) of the matrix in the area surrounding the cell. If A1 is greater than A0, it is calculated as the matrix remodeling volume. 3) Observe and calculate the number of cells with cellular matrix remodeling characteristics.

[0024] Polyisocyanate peptides possess the porous network structure characteristic of natural collagen and fibrin gels. When tumor cells are encapsulated within this network, they exert their own mechanical forces, directly driving changes in the spatial arrangement and structural remodeling of the fibers within the polyisocyanate peptides. In other words, the cells, through the pulling force of their own mechanical forces, cause adaptive morphological and structural reshaping of the original fibrous matrix. By conjugating fluorescent dyes to polyisocyanate peptides, structural changes in the matrix environment can be directly observed by detecting fluorescence changes. This helps us quickly identify the invasiveness of tumor cells and allows for in vitro analysis of tumor samples.

[0025] This invention constructs a system for rapid detection of tumor cell invasiveness and risk level reference based on matrix remodeling fluorescence reporter assay. By timely observing the mechanical interaction characteristics between cells and the matrix and matrix remodeling phenomena, the invasive ability of tumor cells can be assessed more directly and rapidly, providing a certain reference for determining the risk level of tumor cells. This system not only helps to deepen the understanding of the mechanical regulation mechanism of tumor progression, but also provides a rapid, objective, and quantifiable basis for risk level assessment, thereby assisting in disease staging, prognosis, and individualized treatment selection, and has an important impact on promoting the development of precision oncology. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the reference system for rapid fluorescence detection of tumor cell invasiveness and risk level based on matrix remodeling, as described in this invention.

[0027] Figure 2 This is a schematic diagram of the connection between the polyisocyanate polypeptide and the cell adhesion ligand / fluorescent dye in Example 2 of the present invention, showing the matrix structure after fluorescent labeling.

[0028] Figure 3 The rapid fluorescence detection system prepared in Example 3 of this invention shows the fluorescence contrast between invasive and non-invasive cell lines of pericytes.

[0029] Figure 4 A bar chart comparing the fluorescence intensity of invasive and non-invasive cell lines.

[0030] Figure 5 Reference assessment of the invasiveness and risk level of tumor tissue samples. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0033] The DBCO-PEG4-RGD used in the following examples was purchased from Shanghai Angbo Biotechnology Co., Ltd.

[0034] The implementation process of a rapid fluorescence detection reference system for tumor cell invasiveness and risk level based on matrix remodeling is as follows: Figure 1 As shown, after culturing tumor cells in a material containing polyisocyanate peptides for a period of time, the invasiveness of the cells can be rapidly determined within hours by observing the matrix fluorescence intensity under a microscope, thus assessing the risk level of the tumor cells.

[0035] Example 1: Preparation of RGD-functionalized polyisocyanate peptides Weigh 35 mg of polyisocyanate polypeptide (azid helical polypeptide) into 14 mL of anhydrous acetonitrile and stir overnight on a magnetic stirrer until fully dissolved. Weigh 1 mg of DBCO-PEG4-RGD into 4.5 μL of DMSO, gently pipette to dissolve, and then add it to the dissolved polyisocyanate polypeptide solution. Re-stir on a magnetic stirrer for 24 h. After the reaction, precipitate the mixture with isopropyl ether, centrifuge the precipitated reaction solution, discard the supernatant, and freeze the remaining reaction mixture at -80°C. Then, transfer it to a lyophilizer and freeze-dry to obtain RGD-functionalized polyisocyanate polypeptide.

[0036] The structural formula of the helical polypeptide is shown below:

[0037] n represents the degree of aggregation, n = 50-50000.

[0038] Example 2: Structural characterization of polyisocyanate peptide networks Weigh 2 mg of RGD-functionalized polyisocyanate peptide into 1 mL of 1×PBS solution and incubate at 4°C for 24 h. Every 8 h, remove the peptide from the refrigerator and gently pipette it onto an ice box to ensure thorough mixing. Mix 10 μL of the fluorescent dye TAMRA with 190 μL of the RGD-functionalized polyisocyanate peptide solution and incubate on an ice box for 20 min to obtain the dye-linked polyisocyanate peptide. Transfer 100 μL of the mixture to a cell culture plate and incubate at 37°C for 20 min until gelation. Observe the gel using a confocal microscope.

[0039] Figure 2 This is a schematic diagram showing the binding of polyisocyanate peptides to cell adhesion ligands / fluorescent dyes, displaying a representative fluorescence image of the fluorescently labeled polyisocyanate peptide hydrogel. Scale bar: 50 μm.

[0040] Example 3: Construction of a rapid fluorescence detection system based on matrix remodeling Weigh 2 mg of the RGD-functionalized polyisocyanate peptide prepared in Example 1 and dissolve it in 1 mL of 1×PBS (dissolve at 4°C for 24 h) to achieve a concentration of 2 mg / mL. Incubate 10 μL of the fluorescent dye TAMRA and 190 μL of the RGD-functionalized polyisocyanate peptide on an ice box for 20 min. Remove well-adhered 231 cells (invasive cells) or MCF-7 cells (non-invasive cells) from the 6-well plates, discard the surface culture medium, wash with 1×PBS, and then add CeLLTracker dye to the surface and incubate for 30 min to stain the cells (CeLLTracker dye concentration: 10 μM). Digest, centrifuge, and dilute the stained cells to a cell concentration of 2×10⁻⁶. 5 Cells / mL. 50 μL of stained cell suspension and polyisocyanate peptide solution were respectively pipetted into cell culture plates at a 1:1 volume ratio, placed in a 37°C incubator, and allowed to gel for 20 min. 100 μL of LMEM medium was added to each well for three-dimensional culture. Confocal imaging was performed after three days of culture.

[0041] The fluorescence images after 3 days of three-dimensional culture were processed, and the matrix remodeling volume around invasive and non-invasive cell lines was statistically analyzed. GraphPaD was used to generate the statistical results.

[0042] Figure 3 The confocal images show that green fluorescence represents 231 cells (or MCF-7 cells), and red fluorescence represents polyisocyanate polypeptide hydrogel. The bright red area in the left image indicates that 231 cells remodeled the surrounding gel matrix during invasion, while non-invasive cells did not cause matrix remodeling. The scale bar is 50 μm.

[0043] Figure 4Statistical results showed that there was a significant difference in the matrix remodeling volume around invasive and non-invasive cell lines. Using the fluorescence intensity of materials without cells as a baseline (A0), the fluorescence intensity of the matrix around the cell was observed (A1). If A1 was greater than A0, it was calculated as the matrix remodeling volume. The results showed that the matrix remodeling volume around invasive cells was much larger than that around non-invasive cells.

[0044] Example 4: Reference for assessing the invasiveness and risk level of tumor tissue samples using a matrix remodeling-based fluorescence rapid detection system. Tumor tissue fragments were collected, and single-cell suspensions were obtained through enzymatic digestion. These suspensions were then inoculated into RGD-functionalized polyisocyanate peptides linked to the fluorescent dye TAMRA, prepared in Example 2, and cultured for 1-12 hours under suitable conditions (37°C, 5% CO2). Subsequently, the number of invasive cells and the volume of matrix remodeling were quantified using microscopy and image analysis software. Combined with dynamic data on cell behavior, the invasive ability and risk level of tumor cells could be further assessed.

[0045] Figure 5 The results showed that the more invasive cells there were, the larger the volume of matrix remodeling, and the higher the risk level of the tumor cells.

[0046] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application of matrix materials containing cell-induced matrix remodeling responsive fluorescent reporter units in the preparation of reagents for reference on tumor cell invasiveness and risk levels. The matrix material containing the cell-induced matrix remodeling response fluorescent reporter unit is a polyisocyanate polypeptide co-modified with cell adhesion ligands and fluorescent dyes.

2. Use according to claim 1, characterized in that, The matrix material containing the cell-induced matrix remodeling responsive fluorescent reporter unit is prepared by a method comprising the following steps: (1) Preparation of cell adhesion functionalized modified polyisocyanate peptides By modifying at least one of the cell adhesion ligands RGD, cRGD, HAVID and the invasin into the polyisocyanate peptide through covalent or non-covalent means, cell adhesion functionalized polyisocyanate peptides are obtained. (2) Linkage of polyisocyanate peptides with fluorescent dyes Weigh the cell adhesion functionalized modified polyisocyanate peptide and dissolve it in buffer solution. Mix it thoroughly with fluorescent dye 1 in a certain proportion to obtain a polyisocyanate peptide co-modified by cell adhesion ligand and fluorescent dye, which is a matrix material containing a cell-induced matrix remodeling response fluorescent reporter unit.

3. Use according to claim 1, characterized in that, In the application described, the matrix material containing the cell-induced matrix remodeling response fluorescent reporter unit is dissolved in a buffer or culture medium; the tumor cells to be tested are incubated with fluorescent dye 2 to stain the cells, and after digestion and centrifugation, a cell suspension is obtained; the stained cells are seeded on the matrix material for 2D culture, or the two components are thoroughly mixed and gelled to form a 3D culture environment, thereby obtaining a matrix remodeling fluorescent reporter detection system.

4. The application according to claim 1, characterized in that, In this application, fluorescence images of the area surrounding the sample are acquired, and the invasiveness of the tumor cells to be tested is determined based on the intensity changes of the fluorescence signal in the area surrounding the sample and the matrix remodeling index.

5. The application according to claim 4, characterized in that, The matrix remodeling index is calculated as follows: after cells have been cultured in the matrix material for a period of time (1-24 hours), fluorescence microscopy is used to image the cells and obtain a fluorescence image of the cell matrix remodeling, and the matrix remodeling index is analyzed; specifically: 1) The fluorescence intensity of materials without cells was used as the benchmark (A0); 2) With the cell as the center, observe the fluorescence intensity (A1) of the matrix surrounding the cell. If A1 is greater than A0, it is calculated as the matrix remodeling volume. 3) Observe and calculate the number of cells with cellular matrix remodeling characteristics.

6. The application according to claim 2, characterized in that, The fluorescent dye 1 is any dye suitable for fluorescence microscopy imaging.

7. The application according to claim 3, characterized in that, The fluorescent dye 2 is a live cell tracer.

8. An in vitro fluorescence detection method for detecting the invasiveness of test cells based on cell-induced matrix remodeling, the method comprising: a) Provide a matrix material containing a cell-induced matrix remodeling response fluorescent reporter unit; b) Place the cells to be tested or cell samples in the matrix material for two-dimensional (2D) or three-dimensional (3D) culture, so that the cells come into contact with the matrix material; c) Obtain fluorescence images of the region surrounding the cells; d) Analyze cell invasiveness based on changes in the intensity of fluorescence signals in the pericellular region and the matrix remodeling index; The matrix material comprising the cell-induced matrix remodeling response fluorescent reporter unit is a polyisocyanate polypeptide co-modified with cell adhesion ligands and fluorescent dyes.

9. The method according to claim 7, characterized in that, The cell adhesion ligand is selected from at least one of RGD, cRGD, HAVID, and invaginin.

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