A tumor cell hypoxia-sensitive fluorescent probe, its preparation method and application

Abstract

This invention discloses a tumor cell hypoxia-sensitive fluorescent probe, its preparation method, and its application, belonging to the field of biomedical application technology. The tumor cell hypoxia-sensitive fluorescent probe provided by this invention uses triazine as the response unit, responding to the cellular microenvironment and being sensitive to hypoxia. It exhibits a significant specific activation effect in hypoxic tumor cells, enabling precise and efficient identification of hypoxic tumor cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical application technology, specifically to a hypoxia-sensitive fluorescent probe for tumor cells containing a triazine responsive unit, its preparation method, and its application. Background Technology

[0002] Fluorescent probes are widely used in tumor biology, particularly in cancer screening, diagnosis, and treatment. They can be broadly categorized into chemifluorescent probes and biomolecular fluorescent probes. Chemifluorescent probes mainly include fluorescent thiols and fluorescent azo compounds, while biomolecular fluorescent probes mainly include fluorescein and fluorescent dyes. Fluorescent probes are characterized by high sensitivity, rapid response, wide detection range, non-toxicity, and strong reproducibility, thus finding widespread application in tumor biology research.

[0003] Hypoxic tumors refer to hypoxic regions within solid tumors caused by insufficient oxygen supply and heterogeneity of the vascular network. This hypoxic state affects the tumor's biological behavior and response to treatment, contributing to poor prognosis. Hypoxia-activated fluorescent probes primarily transform the hypoxic tumor microenvironment into a visualized fluorescent signal, constructing a systematic logical closed loop from diagnosis to treatment. First, at the diagnostic level, it utilizes reductases highly expressed in the hypoxic tumor microenvironment to trigger fluorescence "on," enabling spatial localization and assessment of the degree of hypoxia in the hypoxic region, transforming physiological characteristics that are difficult to perceive using traditional methods into real-time measurable imaging indicators. Second, at the treatment guidance level, based on the intensity and distribution of this fluorescent signal, doctors can predict the patient's potential resistance to radiotherapy and chemotherapy, thereby adjusting the treatment plan in advance or combining hypoxia modifiers for personalized treatment decisions. Finally, at the interventional treatment level, by designing the probe as a hypoxia-activated prodrug or a therapeutic platform, the fluorescently activated region can simultaneously become a site for drug release or reactive oxygen species generation, and even allow for fluorescent-guided resection of easily recurring hypoxic residual lesions during surgery. The three progressively layered approach—from "seeing hypoxia" to "adjusting strategies based on hypoxia" and finally to "direct intervention at hypoxic sites"—jointly empowers the entire process of hypoxia-activated fluorescent probes for tumor hypoxia-related treatment. Its "therapeutic integration" design strategy is mainly divided into two categories. The first category is hypoxia-activated prodrugs: the probe itself is inactive, but after being reduced in the hypoxic region, it not only emits fluorescence but also releases cytotoxic drugs (such as nitrogen mustard and autophagy inducers). Thus, the imaged fluorescent area is precisely the area where the drug is activated, achieving targeted chemotherapy and avoiding systemic toxicity. The second category is a combination of photodynamic and acoustic dynamics: after activation in the hypoxic region, the probe can absorb light or ultrasound to generate singlet oxygen. Although hypoxia limits the efficacy of traditional photodynamic therapy, some novel probes utilize a type I photochemical pathway (generating free radicals, independent of oxygen) and are actually more effective in hypoxic regions, with fluorescence imaging allowing for simultaneous monitoring of efficacy. Summary of the Invention

[0004] This invention proposes a tumor cell hypoxia-sensitive fluorescent probe, its preparation method, and its application. This probe has photodynamic therapy capabilities and can specifically kill refractory hypoxic tumor cells that lead to recurrence and metastasis, while circumventing existing drug resistance mechanisms, thereby providing a safer and more efficient tumor diagnosis and treatment strategy.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution: Including general structural formula I or general formula II: In general formulas I and II, R represents a fluorescent molecule; in general formula I, R1 represents a ligand or an antitumor drug; R2 represents a hydrogen atom or an antitumor drug; in general formula II, R3 represents a ligand or an antitumor drug, and X represents a cycloalkane.

[0006] Furthermore, R is a saturated or unsaturated fluorescent molecule containing sulfur and / or oxygen; R2 is a ligand / tumor drug of hydrogen atom or chain / benzene ring-containing saturated / unsaturated alkyl group; R1 and R3 are ligands or tumor drugs of chain or benzene ring-containing saturated or unsaturated alkyl group; X is selected from cycloalkanes with saturated / unsaturated single or combined four-, five-, or six-membered rings.

[0007] Furthermore, the fluorescent probe is selected from Formula I and Formula II; The structural formula of Formula I is as follows: ; The structural formula of Formula II is as follows: .

[0008] The second objective of this invention is to provide a method for preparing a hypoxia-sensitive fluorescent probe for tumor cells, comprising the following steps: (1) Add hydrochloric acid dropwise to ice water, add reactant A dropwise while stirring, and then add sodium nitrite and react for 30 minutes; (2) Add acetonitrile solution of reactant B dropwise to the reaction solution in step (1), heat the reaction system to room temperature, and react for 10-12 hours until the reaction ends. (3) Add saturated sodium bicarbonate aqueous solution to the reaction system after the reaction in step (2), then extract with ethyl acetate, collect and combine the organic phases, remove the solvent to obtain crude product, and purify the crude product to obtain tumor cell hypoxia-sensitive fluorescent probe.

[0009] Furthermore, reactant A in step (1) is tetrahydroisoquinoline.

[0010] Further, reactant B in step (2) is (E)-2-{2-[2-(5-(4-aminophenyl)thiophene-2-yl)vinyl]-6-methyl-4H-thiochromene-4-ylidene}malononitrile or (E)-2-{2-[2-(5-(4-aminophenyl)thiophene-2-yl)vinyl]-4H-benzopyran-4-ylidene}malononitrile, with the following structural formulas: or .

[0011] Furthermore, the mass ratio of reactant A to sodium nitrite in step (1) is 1-2:1-2. Preferably, it is 1.2-1.5:1.

[0012] Furthermore, the mass ratio of reactant A to reactant B is 1:1-3, preferably 1:2.0-2.1.

[0013] The third objective of this invention is to provide an application of a tumor cell hypoxia-sensitive fluorescent probe in the preparation of detection or diagnostic products for hypoxia-specifically activated tumor cells.

[0014] Furthermore, the detection or diagnostic product is selected from detection reagents, detection kits, diagnostic reagents, and diagnostic kits.

[0015] The fourth objective of this invention is to provide the application of a tumor cell hypoxia-sensitive fluorescent probe in the preparation of a prodrug for treating hypoxia-specific activation of tumor cells.

[0016] Furthermore, the hypoxia-activating prodrug mainly involves attaching a therapeutic drug to a tumor cell hypoxia-sensitive fluorescent probe of the present invention, delivering the therapeutic drug to the tumor site locally or in a targeted manner to achieve a therapeutic effect. Alternatively, it may also attach a radiotherapy drug to detect and track the effect of radiotherapy, improving the precision of radiotherapy.

[0017] The present invention relates to a tumor cell hypoxia-sensitive fluorescent probe, its preparation method, and its application, the beneficial effects of which are as follows: (1) The tumor cell hypoxia-sensitive fluorescent probe provided by the present invention uses triazine as the response unit. It responds to the cellular microenvironment, is sensitive to hypoxia, and has a significant specific activation effect in hypoxic tumor cells, enabling it to accurately and efficiently identify hypoxic tumor cells.

[0018] (2) Since normal cells or tumor cells are normoxic, they have almost no photodynamic response. However, the fluorescent probe of the present invention can achieve fluorescence imaging under laser induction in a hypoxic environment, thus achieving photodynamic therapy on tumor cells.

[0019] (3) The fluorescent probe of the present invention can specifically kill refractory hypoxic tumor cells that cause recurrence and metastasis, while avoiding existing drug resistance mechanisms, thus achieving a safer and more efficient tumor diagnosis and treatment strategy. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The fluorescence spectra of cpd 5 and cpd 7 (10 μM) in response to hypoxia are shown.

[0022] Figure 2 The results of cytotoxicity tests and phototoxicity tests for cpd 5 and cpd 7.

[0023] Figure 3 Fluorescence imaging of cpd 5 (20 μM) in hypoxic and normoxic cells.

[0024] Figure 4 Fluorescence imaging of cpd 7 (20 μM) in hypoxic and normoxic cells.

[0025] Figure 5 In vivo imaging of cpd 5 and cpd 7 (500 μM, 20 μL, 0.5–48 h) in tumor-bearing (HepG2) nude mice; Figure 6 The hydrogen spectrum of compound I (cpd 5) is shown below. Figure 7 The hydrogen spectrum of compound II (cpd 7) is shown below. in addition, Figures 1-5 In the formula, cpd 5 is compound I, and cpd 7 is compound II. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0027] Example 1 A method for preparing a tumor cell hypoxia-sensitive fluorescent probe includes the following steps: (1) Add hydrochloric acid (179.33 mg, 1.77 mmol) dropwise to ice water, and add reactant A, namely tetrahydroisoquinoline (120.33 mg, 0.885 mmol), dropwise while stirring. Then add sodium nitrite (82.27 mg, 1.18 mmol) and react for 30 minutes. (2) Add an acetonitrile solution of reactant B (250.00 mg, 0.59 mmol) dropwise to the reaction solution in step (1), heat the reaction system to room temperature, and react for 12 h to end the reaction; (3) Add saturated sodium bicarbonate aqueous solution to the reaction system after the reaction in step (2), then extract with ethyl acetate, collect and combine the organic phases, remove the solvent under reduced pressure to obtain crude product, and purify the crude product by silica gel column chromatography (ethyl acetate / petroleum ether = 1:1, v / v) to obtain red solid, which is the formula I tumor cell hypoxia sensitive fluorescent probe. In this embodiment, compound B is (E)-2-{2-[2-(5-(4-aminophenyl)thiophene-2-yl)vinyl]-6-methyl-4H-thiochromene-4-ylidene}malononitrile, and its structural formula is: .

[0028] The synthesis pathway of the tumor cell hypoxia-sensitive fluorescent probe in this embodiment is as follows: .

[0029] The chemical name of the tumor cell hypoxia-sensitive fluorescent probe of Formula I in this embodiment is: 2-{2-[(E)-2-{5-[4-[(E)-(3,4-dihydroisoquinoline-2(1H)-yl)azo]phenyl]thiophene-2-yl}vinyl]-6-methyl-4H-thiochromene-4-yl}malononitrile, HR-MS: m / z 568.1622 [M+H] + C 34 H 25 N5S2, calculate for 567.16; 1 H NMR(600MHz, Chloroform-d) δ 8.92 (d, J = 8.5 Hz, 1H), 7.75 – 7.69 (m, 2H), 7.63(d, J = 8.3 Hz, 3H), 7.53 (p, J = 7.5 Hz, 5H), 7.45 (t, J = 7.8 Hz, 2H), 7.37(d, J = 16.7 Hz, 2H), 6.83 (s, 1H), 5.00 (s, 3H), 4.15 (s, 3H), 3.13 – 3.07(m, 3H).

[0030] Example 2 A method for preparing a tumor cell hypoxia-sensitive fluorescent probe includes the following steps: (1) Add hydrochloric acid (231.65 mg, 2.29 mmol) dropwise to ice water, and add reactant A, namely tetrahydroisoquinoline (152.33 mg, 1.14 mmol), dropwise while stirring. Then add sodium nitrite (106.27 mg, 1.52 mmol) and react for 30 minutes. (2) Add acetonitrile solution of reactant B (300.00 mg, 0.77 mmol) dropwise to the reaction solution in step (1), heat the reaction system to room temperature, and react for 12 h to end the reaction; (3) Add saturated sodium bicarbonate aqueous solution to the reaction system after the reaction in step (2), then extract with ethyl acetate, collect and combine the organic phases, remove the solvent under reduced pressure to obtain crude product, and purify the crude product by silica gel column chromatography (ethyl acetate / petroleum ether = 1:1, v / v) to obtain red solid, which is the formula II tumor cell hypoxia sensitive fluorescent probe. In this embodiment, compound B is (E)-2-{2-[2-(5-(4-aminophenyl)thiophene-2-yl)vinyl]-4H-benzopyran-4-ylidene}malononitrile, with the following structural formula: .

[0031] The synthesis pathway of the tumor cell hypoxia-sensitive fluorescent probe in this embodiment is as follows: The chemical name of the hypoxia-sensitive fluorescent probe for tumor cells in this embodiment (Formula II) is: 2-{2-[(E)-2-{5-[4-[(E)-(3,4-dihydroisoquinoline-2(1H)-yl)azo]phenyl]thiophene-2-yl}vinyl]-4H-chromene-4-ylidene}malononitrile. HR-MS: m / z 538.1694 [M+H] + C 33 H 23 N5OS, calculate for 537.16; 1H NMR (600MHz,Chloroform-d) δ 8.91(d, 1H), 7.72(dd, 2H), 7.63(d, 2H),7.53(t, 3H), 7.47-7.41(m, 1H), 7.37(d, 1H), 7.30(d,2H), 7.23(d, 2H), 6.82(s, 1H), 6.57(m, 1H), 4.99(s, 2H), 4.15(s, 3H), 3.10(t, 2H).

[0032] Example 3 Phototoxicity tests of compound I in Example 1 and compound II in Example 2 The cytotoxicity of compounds of formula I and II under 620 nm light and dark conditions for 48 hours in human hepatocellular carcinoma cells (HepG2), human hepatocellular carcinoma cells (SMMC-7721), and normal human liver immortalized cells (THLE-2) was tested using the CCK8 assay.

[0033] 1. Cell Culture: Human hepatocellular carcinoma cells (HepG2), human hepatocellular carcinoma cells (SMMC-7721), and normal human liver immortalized cells (THLE-2) were seeded at a rate of 5000-12000 cells per well into commercially available sterile 96-well plates. Cell suspensions were prepared using the corresponding 10% fetal bovine serum culture medium, and the volume per well was fixed at 100 μL. It is important to note that the cells need to be pre-cultured for half a day to a day before a toxicity test. Two types of cells were selected: tumor cells (human hepatocellular carcinoma cells: HepG2, SMMC-7721) and normal cells (human liver immortalized cells: THLE-2).

[0034] 2. Add and incubate the solutions of compounds I and II: Dissolve the probes, namely compounds I and II, in dimethyl sulfoxide as solvent to obtain probe solutions. Then, screen the solutions at concentrations of 0, 1, 2, 5, 10, 25, 50, and 100 μM. Add the probe solutions of the above gradients to the cells in the pre-incubated 96-well plates. The volume added to each well is 100 μL, and each treatment has 3 replicates.

[0035] 3. Color development: After culturing in a 37°C, 5% CO2 incubator for 48 hours, add 10 μL of CCK8 reagent to each well; set up 3 blank replicates (containing only 100 μL of culture medium + 100 μL of solvent + 20 μL of CCK8 reagent, cell-free); continue incubation in a 37°C, 5% CO2 incubator for 4 hours.

[0036] 4. Colorimetric analysis: Select a wavelength of 450 nm, use a multi-functional microplate reader to read the absorbance values ​​of each well, record the results, and after data processing, plot a cell viability graph with the compound number as the x-axis and cell viability as the y-axis.

[0037] from Figure 2 As can be seen, compounds of formula I and formula II were not activated in THLE-2 cells and therefore did not exhibit phototoxicity; however, in HepG2 and SMMC-7721 cells, compounds of formula I and formula II were activated and therefore showed significant phototoxicity after exposure to 620 nm light.

[0038] Example 4 Tumor Imaging Test of a Hypoxia-Sensitive Fluorescent Probe for Tumor Cells in This Invention Cell samples: THLE-2, HepG2 and SMMC-7721 cells; Group setup: normoxic group and hypoxic group; Specific handling methods: Normoa group: THLE-2, HepG2, and SMMC-7721 cells with a density of over 80% and intact morphology and high viability were used at 1×10⁻⁶ cells per cell line. 5 Cells were seeded at a concentration of [number] cells / well in 24-well plates and cultured under normal aerobic conditions at 37°C for 48 h. The culture medium was then discarded, the cells were washed twice with PBS, digested with trypsin for 3 min, and then 2 mL of PBS was added. Cells were collected, centrifuged at 1200 rpm for 3 min, the supernatant was discarded, and the cells were diluted with PBS to 1 × 10⁶ cells / well. 7 / mL. Add cell lysis buffer and incubate on a shaker at 4℃ for 0.5h to allow cells to fully lyse. Then centrifuge for 15min (4℃, 14000×g), and collect the supernatant as cell response substance stock solution, which is stored at -80℃ for later use. Place the cell response substance stock solution in a centrifuge tube, add 10μL of the stock solution of compound I (cpd5) and compound II (cpd7) to the centrifuge tube respectively, and add 4980μL of PBS buffer (pH=7.4) to each tube. Add 10μL of cell response substance stock solution to each tube, and incubate the above reaction solution at 37℃ for 60min. Use a 5nm slit width to obtain fluorescence spectra and fluorescence microscopy for imaging tests at λex=490nm (cpd5) and λex=485nm (cpd7). Hypoxia group: THLE-2, HepG2, and SMMC-7721 cells with a density of over 80% and intact morphology and high viability were used at 1×10⁻⁶ cells per cell line. 5Cells were seeded at a concentration of [missing value] in 24-well plates and incubated in incubators with oxygen concentrations of 0.5%, 1%, 5%, and 21% respectively, and induced at 37°C for 48 h. The culture medium was then discarded, and the cells were washed twice with PBS, digested with trypsin for 3 min, and then 2 mL of PBS was added. Cells were collected, centrifuged at 1200 rpm for 3 min, the supernatant was discarded, and the cells were diluted with PBS to 1×10⁶ cells / well. 7 / mL. Add cell lysis buffer and incubate on a shaker at 4℃ for 0.5h to allow for complete cell lysis. Then centrifuge for 15min (4℃, 14000×g), and collect the supernatant as a cell response stock solution, storing at -80℃ for later use. Take eight 5mL centrifuge tubes and accurately measure 10μL of the stock solutions of compound I (cpd5) and compound II (cpd7) using a 100μL pipette, adding them to each centrifuge tube (4 tubes in total). Simultaneously, add 4980μL of PBS buffer (pH=7.4) to each tube, and then add 10 μL of cell response stock solution at different oxygen concentrations to prepare the test solution (final concentration 1×10⁻⁶). -5 The above reaction solution was incubated at 37°C for 60 min, and fluorescence spectra and fluorescence microscopy were obtained using a 5 nm slit width at λ ex = 490 nm (cpd5) and λ ex = 485 nm (cpd7) for imaging tests.

[0039] The results obtained are as follows Figure 1 , Figure 3 and Figure 4 As shown. From Figure 1 It can be seen that compounds of formula I and formula II exhibit different fluorescence results under different oxygen concentrations. Under severe hypoxia (0.5%), the probe shows good fluorescence performance; under moderate hypoxia (1%), the probe shows weak fluorescence performance; and under physiological hypoxia (5%) and normoxic conditions (21%), the probe shows almost no fluorescence. This indicates that the fluorescent probe of the present invention possesses hypoxia-sensitive properties, enabling photodynamic therapy in hypoxic environments.

[0040] from Figure 3 It can be seen that compound I is significantly activated in hypoxic HepG2 and SMMC-7721 cells, exhibiting obvious red fluorescence, while no fluorescence is produced in normoxic HepG2 and SMMC-7721 cells, proving that it cannot be activated under normoxic conditions. In THLE-2 cells, no fluorescence is produced under either normoxic or hypoxic conditions, proving that it is not activated by THLE-2 cells.

[0041] from Figure 4It can be seen that compound II is significantly activated in hypoxic HepG2 and SMMC-7721 cells, exhibiting obvious red fluorescence, while no fluorescence is produced in normoxic HepG2 and SMMC-7721 cells, proving that it cannot be activated under normoxic conditions. In THLE-2 cells, no fluorescence is produced under either normoxic or hypoxic conditions, proving that it is not activated by THLE-2 cells.

[0042] Example 5 animal experiments Animal model establishment: Balb / c-Nude nude mice were acclimatized for one week, and HepG2 cells (1×10⁻⁶) were selected. 7 (1 cell) was transplanted into the axilla of male nude mice weighing approximately 18-20 g, and the transplantation was performed 7 days later.

[0043] After successful modeling, mice were intravenously injected with compounds of formula I and II (500 μM, 20 μL, 0.5–48 h) for in vivo imaging. In vivo (live mouse) imaging was performed using a PerkinElmer IVIS Lumina III imaging system. The results are as follows: Figure 5 As shown.

[0044] from Figure 5 As can be seen, significant near-infrared (NIR) fluorescence was observed at the tumor site 30 minutes after tail vein injection of compounds I and II. The NIR fluorescence initially increased over time, then gradually decreased. The fluorescence intensity in the tumor region reached its peak approximately after 6 hours, and a relatively significant fluorescence signal remained at the tumor site even after 48 hours. This indicates that the fluorescent probes of this invention can specifically recognize tumor cells.

[0045] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A fluorescent probe sensitive to hypoxia in tumor cells, characterized in that: Including general structural formula I or general formula II: ； In general formulas I and II, R represents a fluorescent molecule; in general formula I, R1 represents a ligand or an antitumor drug; R2 represents a hydrogen atom or an antitumor drug; in general formula II, R3 represents a ligand or an antitumor drug, and X represents a cycloalkane.

2. The tumor cell hypoxia-sensitive fluorescent probe according to claim 1, characterized in that: R is a saturated or unsaturated fluorescent molecule containing sulfur and / or oxygen; R2 is a ligand / tumor drug of hydrogen atom or chain / benzene ring-containing saturated / unsaturated alkyl group; R1 and R3 are ligands or tumor drugs of chain or benzene ring-containing saturated or unsaturated alkyl group; X is selected from cycloalkanes with saturated / unsaturated single or combined four-, five-, or six-membered rings.

3. The tumor cell hypoxia-sensitive fluorescent probe according to claim 1, characterized in that: The fluorescent probe is selected from Formula I and Formula II; The structural formula of Formula I is as follows: ； The structural formula of Formula II is as follows: 。 4. A method for preparing a tumor cell hypoxia-sensitive fluorescent probe according to any one of claims 1-3, characterized in that: Includes the following steps: (1) Add hydrochloric acid dropwise to ice water, add reactant A dropwise while stirring, and then add sodium nitrite and react for 30 minutes; (2) Add acetonitrile solution of reactant B dropwise to the reaction solution in step (1), heat the reaction system to room temperature, and react for 10-12 hours until the reaction ends. (3) Add saturated sodium bicarbonate aqueous solution to the reaction system after the reaction in step (2), then extract with ethyl acetate, collect and combine the organic phases, remove the solvent to obtain crude product, and purify the crude product to obtain tumor cell hypoxia-sensitive fluorescent probe.

5. The method for preparing the tumor cell hypoxia-sensitive fluorescent probe according to claim 4, characterized in that: The reactant A in step (1) is tetrahydroisoquinoline.

6. The method for preparing the tumor cell hypoxia-sensitive fluorescent probe according to claim 4, characterized in that: The structural formula of reactant B in step (2) is: or .

7. The method for preparing the tumor cell hypoxia-sensitive fluorescent probe according to claim 4, characterized in that: The mass ratio of reactant A to sodium nitrite in step (1) is 1-2:1-2.

8. The method for preparing a tumor cell hypoxia-sensitive fluorescent probe according to claim 4, characterized in that: The mass ratio of reactant A to reactant B is 1:1-3.

9. The use of the tumor cell hypoxia-sensitive fluorescent probe according to claim 1 in the preparation of a detection or diagnostic product for hypoxia-specifically activated tumor cells.

10. The use of the tumor cell hypoxia-sensitive fluorescent probe according to claim 1 in the preparation of a prodrug for treating hypoxia-specific activation of tumor cells.

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