Selenol-alkynyl click chemistry mediated cell surface rapid functional modification method
The rapid functional modification of cell surfaces through selenol-alkyne click chemistry solves the problems of long reaction time, low efficiency and insufficient biocompatibility in existing technologies, achieves rapid and efficient cell surface modification, and expands biomedical applications.
Patent Information
- Application Number
- CN202510785206.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-12
- Publication Date
- 2025-09-12
AI Technical Summary
Existing cell surface modification methods have long reaction times, low efficiency, and significant interference with cell functions. In particular, it is difficult to achieve rapid and efficient functional modification in living cell systems, and they also lack biocompatibility.
Selenol-alkynyl click chemistry is used to introduce selenol-functionalized molecules onto the cell surface under physiological conditions, and then covalently couple them with biocompatible materials carrying alkynyl functional groups through the selenol-alkynyl click reaction to achieve rapid functional modification.
Without significantly affecting cell activity, cell surface functionalization is achieved, coupling efficiency and selectivity are improved, the scope of biomedical applications is expanded, and cell-to-cell interactions and immune responses are enhanced.
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Figure CN120624362A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a method for rapid functional modification of cell surfaces mediated by selenol-yne click (SYC) chemistry. Background Art
[0002] The cell surface is a biological interface composed of a phospholipid bilayer, carbohydrates, and proteins. It plays an important role in maintaining a stable intracellular metabolic environment and regulating the exchange of substances inside and outside the cell, among many other life processes. For example, cells' responses to the external biochemical microenvironment are primarily mediated by specific molecules on the cell membrane, such as polysaccharides. These molecules can influence cell behavior by controlling intercellular interactions and selective adhesion.
[0003] Cell surface engineering technology selectively modifies the surface of living cells with natural or synthetic materials (such as nanoparticles), enabling targeted cell delivery, enhanced imaging signals, and regulation of intercellular interactions, thereby effectively enhancing immune responses and providing technical support for the construction of advanced biological functional systems. Currently, common cell surface modification methods include strategies based on bioconjugation chemistry, such as the use of metabolic engineering to introduce non-natural functional groups (such as azide), followed by coupling through a click reaction between azide and alkyne, or by breaking disulfide bonds in cell membrane proteins with reducing agents, followed by reaction with maleimide or other sulfophilic groups to achieve modification.
[0004] However, these existing methods generally have problems such as long reaction time, low modification efficiency, and high non-specificity. Some strategies, such as breaking disulfide bonds, may damage the structure and function of membrane proteins, while other methods (such as NHS esters and amino-alkyne clicks) often require continuous reactions for several hours, which increases the risk of cell stress response, especially for primary cells and other cell types that are sensitive to external interference. In in vivo application scenarios, reaction rate becomes a key factor in determining coupling efficiency and therapeutic specificity. Rapid coupling not only helps to improve target recognition accuracy and shorten the in vivo circulation time of unbound reagents, but also reduces non-target side effects. Therefore, the ability to quickly and efficiently complete cell surface functionalization under physiological conditions with excellent biocompatibility is the research direction of new coupling strategies in the future.
[0005] Click chemistry has been widely used in biological systems due to its advantages such as mild reaction conditions, high selectivity, and good yield. However, due to the complex structure of the cell membrane and its dynamic environment, achieving sub-minute or even second-level coupling on the cell surface remains a major challenge. In recent years, the selenol-alkyne click reaction has attracted attention due to its extremely fast kinetics and can theoretically complete efficient coupling within minutes. Despite this, the feasibility and biocompatibility of this reaction system in the living cell surface environment have not been fully verified. Therefore, there is an urgent need to develop a new cell surface coupling method suitable for living cell systems with faster reaction speed, stronger selectivity, and no obvious interference with cell function, so as to overcome the limitations of existing technologies and promote the further application of cell surface engineering in the biomedical field. Summary of the Invention
[0006] In response to the problems existing in the prior art, the present invention provides a method for rapid functional modification of cell surfaces mediated by selenol-yne click (SYC) chemistry, so as to achieve rapid and convenient multiple functional modifications of cell surfaces under biocompatible conditions, which is beneficial to the development of living cell-mediated drug delivery systems and the regulation of specific cell-cell interactions.
[0007] In order to solve the above technical problems and achieve the above technical effects, the present invention is implemented through the following technical solutions:
[0008] A method for rapid cell surface functionalization modification mediated by selenol-alkyne click chemistry comprises the following steps:
[0009] First, selenol-functionalized molecules are introduced into the surface of living cells to modify the surface of living cells with active selenol groups to form a selenolated cell surface. Then, a biocompatible material carrying an alkynyl functional group is brought into contact with the selenolated cell surface. Under physiologically compatible conditions, covalent coupling of the biocompatible material and the selenolated cell surface is achieved through a selenol-alkynyl click reaction, thereby giving the cells new properties or functions.
[0010] Furthermore, the selenol functionalized molecule is a selenol molecule with membrane insertion function.
[0011] Furthermore, the structure of the selenol molecule with membrane insertion function is that cholesterol or lipid molecules are covalently bound to the selenol group (-SeH) via a connecting arm, and the connecting arm is selected from a carbon chain, polyethylene glycol (PEG) or its derivatives.
[0012] Furthermore, the concentration of the selenol functionalized molecules is 0.1-10 mM, and the incubation time after the selenol functionalized molecules are introduced into the living cell surface is 1-30 minutes.
[0013] Furthermore, the biocompatible material carrying an alkyne functional group includes one or more of a small molecule polymer, a macromolecular polymer, a polypeptide, a protein, a nucleic acid, a nanobody, a micron / nanoparticle, a glycosylated modification, or an immunomodulatory molecule.
[0014] Furthermore, the biocompatible material carrying the alkyne functional group is covalently coupled to the cell surface through a click reaction between the alkyne group and the selenol group, and the cell survival rate after coupling is ≥90%.
[0015] Furthermore, the conditions for the selenol-alkyne click reaction are: pH = 6.0-8.0, temperature 0-40° C., and reaction time 10 seconds to 30 minutes.
[0016] Furthermore, the living cells are mammalian cells including primary cells, continuous cell lines or engineered cells.
[0017] The present invention also provides a functionalized cell prepared by the above-mentioned selenol-alkyne click chemistry-mediated rapid functionalization modification method of the cell surface. The cell surface of the prepared functionalized cell is coupled with a biocompatible material, and the coupling process does not significantly affect the cell activity and function.
[0018] The present invention also provides a variety of new applications of the above-mentioned selenol-alkyne click chemistry-mediated rapid functionalization modification method for cell surfaces, which can be specifically used for cell surface labeling and tracing, cell-material complex construction, regulation of intercellular interactions or signal transduction, preparation of functional cell therapy preparations or enhancement of the immunogenicity of cell vaccines. These new applications are achieved by coupling different functional materials.
[0019] The present invention also provides a method for preparing a whole tumor vaccine, which uses the above-mentioned selenol-alkyne click chemistry-mediated rapid cell surface functionalization modification method, and the specific steps include:
[0020] First, inactivated tumor cells are incubated with selenol-functionalized cholesterol to introduce selenol groups. Subsequently, alkyne-functionalized immune-activating molecules or targeting ligands are coupled to the tumor cell surface through a selenol-alkyne click reaction. Finally, the modified tumor cells are used to activate immune cells and induce specific anti-tumor immune responses.
[0021] The beneficial effects of the present invention are:
[0022] The present invention introduces reactive selenol groups onto the cell surface, thereby enabling the rapid coupling of functional materials carrying alkynyl functional groups to the cell surface under biocompatible conditions, providing a new method for the functionalization of cell surfaces and the regulation of specific cell-to-cell interactions. The coupling process of the method of the present invention does not significantly affect cell activity and function, and the cell survival rate after coupling is ≥90%. The present invention not only expands the application scope of selenol-alkynyl click chemistry in the biomedical field, but also further enriches the existing toolbox for cell surface chemical modification, providing new tools for clinical diagnosis, tumor immunotherapy, and basic cell biology research.
[0023] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the invention and to implement it according to the contents of the description, the following preferred embodiments of the present invention are described in detail with reference to the accompanying drawings. The specific implementation methods of the present invention are given in detail by the following embodiments and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:
[0025] Figure 1 Figure 1 is a schematic diagram of the process for the multifunctional modification of cell surfaces mediated by selenol-alkynyl click chemistry of the present invention; Figure A is a schematic diagram of the process when the selenol-functionalized molecule is selenol-functionalized cholesterol, and Figure B is a schematic diagram of the chemical structure of selenol-functionalized cholesterol (Chol-SeH).
[0026] Figure 2 These are confocal images of HeLa cells in Experimental Example 1 of the present invention, incubated with Chol-SeH (Groups A and C) or cholesterol (Group B) for 10 minutes and then stained with alkynyl-functionalized fluorescent molecules (Groups A and B) or unfunctionalized (Group C) for 15 minutes.
[0027] Figure 3 Figure 2 is a microscopic tracking result of the interaction between the SBA material and cells in Experimental Example 2 of the present invention; Figure A shows the loading process of SBA particles from the solution to the cell surface, and Figure B shows the cells after being treated with the SBA material.
[0028] Figure 4 The chemical structure of the alkynylated glycopolymer in Experimental Example 3 of the present invention and the results of the interaction between glycosylated T cells and non-glycosylated T cells on the surface of SYC and Hela cells are shown.
[0029] Figure 5Figure 4 shows the activation results of dendritic cells by the glycosylated whole tumor vaccine constructed by the method of the present invention in Experimental Example 4 of the present invention; Figure A shows the flow cytometry analysis results of dendritic cell activation, and Figure B shows the statistical analysis results of the relative CD80 / CD86 expression in the flow cytometry analysis results. DETAILED DESCRIPTION
[0030] The following will be described in detail with reference to the accompanying drawings to better understand the purpose, features and advantages of the invention. It should be understood that the embodiments shown in the accompanying drawings are not intended to limit the scope of the invention, but are only intended to illustrate the essential spirit of the technical solution of the invention.
[0031] In the following description, for the purpose of illustrating the various disclosed embodiments, certain specific details are set forth in order to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the relevant art will recognize that the embodiments may be practiced without one or more of these specific details. In other cases, well-known devices, structures, and techniques associated with this application may not be shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.
[0032] Unless the context requires otherwise, throughout the specification and claims, the word "comprise" and variations such as "include" and "have" should be construed in an open, inclusive sense, that is, should be interpreted to mean "including, but not limited to."
[0033] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any manner in one or more embodiments.
[0034] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be noted that the term "or" is generally employed in its sense including "and / or" unless the context clearly dictates otherwise.
[0035] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. Unless otherwise specified, the reagents and materials used in the present invention are commercially available.
[0036] The present invention mainly provides a method for rapid functional modification of cell surfaces mediated by selenol-alkyne click chemistry, comprising the following steps:
[0037] Step 1) mammalian cells such as primary cells, continuous cell lines or engineered cells are selected as living cells for rapid functional modification of the cell surface.
[0038] Step 2) Selecting a selenol molecule with a membrane-insertion function as the selenol functionalized molecule. The structure of the selenol molecule with a membrane-insertion function is that cholesterol or a lipid molecule is covalently bound to a selenol group (-SeH) via a linker, and the linker is selected from a carbon chain, polyethylene glycol (PEG) or a derivative thereof.
[0039] Step 3) selecting one or more of small molecule polymers, macromolecular polymers, polypeptides, proteins, nucleic acids, nanobodies, micro / nanoparticles, glycosylated modifiers or immunomodulatory molecules as the biocompatible material carrying the alkyne functional group.
[0040] Step 4) incubating the living cells with the selenol molecules with membrane insertion function at a concentration of 0.1-10 mM for 1-30 minutes, thereby introducing selenol groups into the living cell surface, modifying the living cell surface with active selenol groups, and forming a selenolized cell surface.
[0041] Step 5) contacting the biocompatible material carrying the alkynyl functional group with the surface of the selenolated cell. Under physiologically compatible conditions, the biocompatible material carrying the alkynyl functional group is covalently coupled to the cell surface through a selenol-alkynyl click reaction, thereby achieving covalent coupling of the biocompatible material to the selenolated cell surface, thereby imparting new properties or functions to the cells, and the cell survival rate after coupling is ≥90%.
[0042] The conditions for the selenol-alkyne click reaction are: pH = 6.0-8.0, temperature 0-40° C., and reaction time 10 seconds to 30 minutes.
[0043] Chemical structure such as Figure 1 As shown in B, selenol-functionalized cholesterol (Chol-SeH) is used as an example. Figure 1 As shown in A, active selenol groups are introduced as click reaction sites on the cell surface by selenol-functionalized cholesterol (Chol-SeH). By utilizing its rapid and specific reaction mechanism with alkynyl groups, different functional materials containing alkynyl chemical functional groups are successfully connected to the cell surface using vinyl double bonds as linkers, thus providing a new method for giving cells new properties or functions.
[0044] The present invention also provides a functionalized cell prepared by the above-mentioned selenol-alkyne click chemistry-mediated rapid functionalization modification method of the cell surface. The cell surface of the prepared functionalized cell is coupled with a biocompatible material, and the coupling process does not significantly affect the cell activity and function.
[0045] The present invention also provides a variety of new applications of the above-mentioned selenol-alkyne click chemistry-mediated rapid functionalization modification method for cell surfaces, which can be specifically used for cell surface labeling and tracing, cell-material complex construction, regulation of intercellular interactions or signal transduction, preparation of functional cell therapy preparations or enhancement of the immunogenicity of cell vaccines. These new applications are achieved by coupling different functional materials.
[0046] The present invention also provides a method for preparing a whole tumor vaccine, which uses the above-mentioned selenol-alkyne click chemistry-mediated rapid cell surface functionalization modification method, and the specific steps include:
[0047] First, inactivated tumor cells are incubated with selenol-functionalized cholesterol to introduce selenol groups. Subsequently, alkyne-functionalized immune-activating molecules or targeting ligands are coupled to the tumor cell surface through a selenol-alkyne click reaction. Finally, the modified tumor cells are used to activate immune cells and induce specific anti-tumor immune responses.
[0048] The following multiple related test experiments are conducted to verify the authenticity and feasibility of the rapid cell surface functional modification method of the present invention.
[0049] Experimental Example 1: Fluorescence labeling of cells using the method of the present invention
[0050] In order to verify the feasibility and specificity of the method of the present invention in cell surface modification, Hela cells were selected as a research model for cell surface fluorescent molecule modification. The specific method is as follows:
[0051] 90,000 Hela cells were transferred to a confocal dish and cultured for 24 hours using complete high-glucose DMEM culture medium. The culture medium was removed and 1 mL of high-glucose DMEM culture medium was added, which was preheated in an incubator for 30 minutes. The cells were divided into three groups, one of which was numbered A as the experimental group, and the remaining two groups were numbered B and C as the control groups. Chol-SeH solution (2.6 mM, dissolved in DMF) was added to groups A and C, and cholesterol (Chol) solution (2.6 mM, dissolved in DMF) was added to group B. After being placed in a cell culture incubator for 10 minutes, the cells were washed twice with PBS. Alkyne-modified fluorescent molecules FITC-PEG-A were added to groups A and B, and alkynyl-free fluorescent molecules FITC-PEG-NH2 were added to the C sample as a control. After incubation at room temperature for 15 minutes, the samples were washed twice with PBS and 1 mL of PBS was added. The differences in cell surface fluorescence intensity were analyzed using a confocal microscope, and confocal microscopy was used to take pictures. Figure 2 shown.
[0052] from Figure 2It can be seen that the cell surface modification method based on selenol-alkyne click chemistry is selective. Selenolated cells do not react with non-alkyne-functionalized fluorescent molecules, and alkyne-functionalized fluorescent molecules do not react with non-selenolated cells. Surface modification of cells can only be achieved when the cells and fluorescent molecules are correctly selenolated and alkyne-functionalized.
[0053] Example 2: Using the method of the present invention to load cell backpacks
[0054] In order to make the method of the present invention universal in modifying materials, mesoporous silica (SBA) particles were coupled to the cell surface. The specific method is as follows:
[0055] Hela cells were seeded in 48-well plates at 18,000 cells per well and cultured for 24 hours. The medium was then replaced with 250 μL of DMEM culture medium, and the culture plate was preheated in a 37°C incubator for 30 minutes. The cells were divided into three groups. Groups A and C were added with 12 μL of Chol-SeH solution (2.6 mM, dissolved in DMF), and group B was added with Chol solution (2.6 mM, dissolved in DMF). After thorough mixing, the cells were incubated at 37°C for 10 minutes. The culture medium was discarded and the cells were washed twice with PBS. Then 1 mL of SBA-A suspension (concentration of 165 particles / mL in PBS) was added to groups A and B, and SBA-15 suspension was added to group C. An optical microscope was then immediately used to microscopically trace the cells and SBA materials. The loading process of SBA particles from the solution to the cell surface is shown in the figure. Figure 3 As shown in A, cells treated with SBA materials are Figure 3 As shown in B.
[0056] from Figure 3 A shows that selenolized cells can quickly couple to alkynylated mesoporous silica particles SBA-A, and the coupling process is completed within one minute. The coupling is also selective as described in Experimental Example 1. Figure 3 The SEM image shown in B shows that only when the cells are correctly selenolized and the SBA particles are correctly alkynylated can the SBA material couple to the cell surface.
[0057] Experimental Example 3: Modifying glycopolymers on the surface of T cells using the method of the present invention to enhance their interaction with Hela cancer cells
[0058] To illustrate the applicability of the method of the present invention in different cell types and application scenarios, a glycopolymer layer was constructed on the cell surface through cell surface engineering to effectively regulate cell-cell interactions, thereby enhancing immune responses, such as T cell activation and tumor killing. The specific method is:
[0059] Using the rapid SYC (selenol-alkyne click) reaction, we first explored its application in the modification of T cell surface glycopolymers to enhance their interaction with cancer cells. We synthesized a glucose-based polymer with an alkyne end (pM-A) and attached it to the surface of selenolated T cells via the SYC reaction using the same method as in Experimental Example 1 to obtain T-pM cells. Figure 4 As shown, T-pM cells exhibited significantly stronger interactions with HeLa cancer cells that highly expressed Glut1 compared to unmodified T cells.
[0060] Experimental Example 4: Surface glycosylation of B16-OVA (B16O) using the method of the present invention to prepare a whole tumor vaccine
[0061] This study aims to verify the application of the SYC (selenol-alkyne click) method in the construction of surface glycosylated whole tumor vaccines (WTCVs) and explore its effect on enhancing the immune response. The specific methods are:
[0062] First, B16-OVA melanoma cells (2,000,000) were seeded in a 35 mm culture dish and irradiated with ultraviolet light (253 nm, 21 μW / cm 2 ) for 30 minutes to inactivate the cells. Subsequently, 500,000 inactivated B16O cells were suspended in 3 mL of culture medium and incubated at 37°C for 10 minutes with 15 μL of a 2.6 mM Chol-SeH solution (dissolved in DMF) or an equal concentration of Chol solution as a control. Selenol groups were introduced to the cell surface.
[0063] After removing the culture medium, 0.6 mL of a 2.6 mM solution of the glucosyl acetylated glycopolymer pG-A in PBS or PBS was added to the cells. The cells were incubated at room temperature for 15 minutes to allow for glycosylation. The culture medium was then replaced with 1 mL of fresh medium to produce whole-tumor vaccines with different modification states: B16O-pM, B16O-SeH, B16O / pM, and unmodified B16O. Each vaccine was then co-incubated with DC2.4 cells for 48 hours, and the expression levels of CD80 and CD86 costimulatory molecules were measured by flow cytometry.
[0064] See also Figure 5 A and Figure 5 As shown in Figure 2, the B16O-pM group significantly promoted DC maturation, and its CD80 / CD86 expression levels increased by about 3.8 times compared with the unmodified group, and were significantly higher than the B16O-SeH and B16O-she groups treated with only Chol-SeH or pM-A, indicating that glycopolymers modified by SYC reaction can effectively enhance the immunogenicity of tumor vaccines and have good application prospects in tumor immunotherapy.
[0065] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.
Claims
1. A method for rapid cell surface functionalization modification mediated by selenol-alkyne click chemistry, characterized in that: The following steps are involved: First, selenol-functionalized molecules are introduced into the surface of living cells to modify the surface of living cells with active selenol groups to form a selenolated cell surface. Then, a biocompatible material carrying an alkynyl functional group is brought into contact with the selenolated cell surface. Under physiologically compatible conditions, covalent coupling of the biocompatible material and the selenolated cell surface is achieved through a selenol-alkynyl click reaction, thereby giving the cells new properties or functions.
2. The method for rapid cell surface functionalization modification mediated by selenol-alkynyl click chemistry according to claim 1, characterized in that: The selenol functionalized molecule is a selenol molecule with membrane insertion function.
3. The method for rapid cell surface functionalization modification mediated by selenol-alkynyl click chemistry according to claim 2, characterized in that: The structure of the selenol molecule with membrane insertion function is that cholesterol or lipid molecule is covalently bound to the selenol group via a connecting arm, and the connecting arm is selected from a carbon chain, polyethylene glycol or its derivatives.
4. The method for rapid cell surface functionalization modification mediated by selenol-alkynyl click chemistry according to claim 1, characterized in that: The concentration of the selenol functionalized molecules is 0.1-10 mM, and the incubation time after the selenol functionalized molecules are introduced into the living cell surface is 1-30 minutes.
5. The method for rapid cell surface functionalization modification mediated by selenol-alkynyl click chemistry according to claim 1, characterized in that: The biocompatible material carrying an alkyne functional group includes one or more of a small molecule polymer, a macromolecular polymer, a polypeptide, a protein, a nucleic acid, a nanobody, a micron / nanoparticle, a glycosylation modification or an immunomodulatory molecule.
6. The method for rapid cell surface functionalization modification mediated by selenol-alkynyl click chemistry according to claim 1, characterized in that: The conditions for the selenol-alkyne click reaction are: pH = 6.0-8.0, temperature 0-40° C., and reaction time 10 seconds to 30 minutes.
7. The method for rapid cell surface functionalization modification mediated by selenol-alkynyl click chemistry according to claim 1, characterized in that: The living cells are mammalian cells including primary cells, continuous cell lines or engineered cells.
8. A functionalized cell prepared by the method for rapid cell surface functionalization mediated by selenol-alkynyl click chemistry according to any one of claims 1 to 7, characterized in that: The cell surface of the functionalized cells is coupled with a biocompatible material, and the coupling process does not significantly affect the cell activity and function.
9. Application of the selenol-alkynyl click chemistry-mediated rapid cell surface functionalization modification method as described in any one of claims 1 to 7 in cell surface labeling and tracing, cell-material complex construction, regulation of intercellular interactions or signal transduction, preparation of functional cell therapy preparations, or enhancement of the immunogenicity of cell vaccines.
10. A method for preparing a whole tumor vaccine, characterized in that: The method for rapid cell surface functionalization modification mediated by selenol-alkyne click chemistry according to any one of claims 1 to 7 comprises the following steps: First, inactivated tumor cells are incubated with selenol-functionalized cholesterol to introduce selenol groups. Subsequently, alkyne-functionalized immune-activating molecules or targeting ligands are coupled to the tumor cell surface through a selenol-alkyne click reaction. Finally, the modified tumor cells are used to activate immune cells and induce specific anti-tumor immune responses.