Molecular probe for targeting tumor-associated fibroblasts as well as preparation method and application of molecular probe

Abstract

The invention belongs to the field of tumor detection, and relates to a molecular probe targeting tumor-associated fibroblasts as well as a preparation method and application thereof, the preparation method comprises the following steps: mixing < 68 > Ga with sodium acetate, then adding an FAPI precursor, and uniformly mixing and reacting at constant temperature to obtain the < 68 > Ga-FAPI molecular probe. The < 68 > Ga-FAPI can visualize a tumor microenvironment, provides a protective micronest for tumor cells, and helps to evaluate the promotion effect of the tumor microenvironment on tumor chemotherapy drug resistance.

Description

Technical Field

[0001] The present invention belongs to the field of tumor detection, and relates to a molecular probe targeting tumor-associated fibroblasts, a preparation method thereof, and an application thereof. Background Art

[0002] The American Society of Clinical Oncology (ASCO) guidelines recommend that platinum-based combination chemotherapy is the first-line treatment for patients with advanced lung cancer. However, acquired resistance has always been a difficult problem in clinical practice during cisplatin treatment and has become a major obstacle to extending the survival of lung cancer patients. According to the Response Evaluation Criteria in Solid Tumors (RECIST 1.1) for lung cancer, imaging methods based on anatomical structure changes, such as X-rays or CT, have lag and limitations in evaluating the activity of tumor cells after chemotherapy. PET / CT molecular imaging equipment can provide tissue functional and anatomical information simultaneously through non-invasive multimodal imaging technology. Currently, 18 PET / CT metabolic imaging represented by the 18 F-fluorodeoxyglucose (FDG) molecular probe can not only provide intuitive anatomical images, but also semi-quantitatively measure the standardized uptake value (SUV) of tumors and evaluate the biochemical metabolic state of tissue cells. However, it is found in clinical practice that 18 the false negatives of

[0003] More than 90% of solid tumors are composed of tumor stroma. Tumor stromal cells and the extracellular matrix together constitute the tumor microenvironment (TME). TME can provide a protective micro-nest for tumor cells, help them avoid drug attacks, lead to treatment resistance, and promote tumor recurrence. Among the stromal cells that make up TME, tumor-associated fibroblasts (CAFs) are the most important cell subset. Fibroblast activation protein (FAP) is a cell-specific biomarker of CAFs. Imaging studies targeting FAP involve structural modification of FAPI series small molecule inhibitors to facilitate the connection of chelates with radionuclides, and then PET / CT, PET / MRI or SPECT / CT imaging.

[0004] 18 18 F-FDG PET / CT has been included in the lung cancer diagnosis and staging guidelines, but its non-specificity and false positives make its clinical application efficacy not reach the best, especially 18 ​F-FDG PET / CT showed no meningeal metastasis, but 68 Ga-FAPI showed a large amount of 68 Ga-FAPI uptake in the meningeal region corresponding to the enhanced area of MRI. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a molecular probe targeting tumor-associated fibroblasts, its preparation method and application, a protective micro-niche provided for tumor cells, and helps to evaluate the promoting effect of the tumor microenvironment on tumor chemotherapy resistance.

[0006] The technical solution provided by the present invention is as follows:

[0007] A preparation method of a molecular probe targeting tumor-associated fibroblasts, the preparation method comprising: mixing 68 Ga with sodium acetate, then adding the FAPI precursor, and reacting at a constant temperature with mixing to obtain 68 the Ga-FAPI molecular probe.

[0008] Further, the concentration of the sodium acetate is 0.8 - 1.2 mol / L.

[0009] Further, 68 the ratio of

[0010] Ga to the FAPI precursor is 925 - 1110 MBq: 25 μg.

[0011] Further, the constant temperature reaction is carried out at 95 - 105 °C for 12 - 18 min. 68 After obtaining the Ga-FAPI molecular probe, high performance liquid chromatography is used to detect the radiochemical purity.

[0012] The present invention also provides a molecular probe targeting tumor-associated fibroblasts, which is prepared according to the above preparation method.

[0013] The present invention also provides an imaging agent targeting tumor-associated fibroblasts, which contains the above-mentioned molecular probe targeting tumor-associated fibroblasts.

[0014] The present invention also provides the application of the above-mentioned molecular probe targeting tumor-associated fibroblasts in the preparation of preparations for tumor diagnosis or treatment.

[0015] Further, the tumor is a lung cancer tumor.

[0016] Further, the tumor is a cisplatin chemotherapy-resistant tumor.

[0017] Further, the preparation is used to inhibit the tumor volume.

[0018] Beneficial Effects

[0019] As a PET / CT tumor imaging agent, FAPI has a high tumor uptake ratio and retention rate. However, there is currently no relevant report on the integrated diagnosis and treatment of patients with lung cancer resistant to cisplatin chemotherapy using FAPI. This application 68 evaluates cisplatin chemotherapy resistance in NSCLC using 68Ga-FAPI imaging and gives 177 innovative and clinically applicable prospects for the study of targeted therapy with 177Lu-FAPI on drug-resistant models positive for the FAP target.

[0020] Before cisplatin treatment, there were differences in the SUVmax values of 68Ga-FAPI imaging between the A549 sensitive strain and the A549-DDP cisplatin-resistant strain 68 indicating that preliminary evaluation is feasible before cisplatin-resistant treatment in NSCLC. After cisplatin treatment, there were statistically significant differences in the SUVmax values between the co-culture of A549 and CAFs cells and the single A549 group, indicating that A549+CAFs could significantly promote cisplatin resistance in NSCLC xenografts. The tumor ΔSUVmax of the A549 group before and after treatment was 15.59%, and the tumor volume inhibition rate was 30.85%; the tumor ΔSUVmax of the A549 / DDP group before and after treatment was 60.33%, and the tumor volume inhibition rate was 18.17%, indicating that the drug-resistant group had a higher initial uptake value of 68Ga-FAPI, and the uptake of 68Ga-FAPI continued to increase during the treatment process, making it more difficult to be inhibited by cisplatin chemotherapy, resulting in more vigorous tumor growth. 68Ga-FAPI PET / CT imaging can visually visualize the heterogeneity of the tumor microenvironment in vitro and is a key means to evaluate tumor efficacy and drug resistance before and during treatment.

[0021] Compared with 18 18F-FDG, the FAPI tracer shows good advantages, such as rapid renal clearance rate, better tumor-background contrast, exclusion of the influence of blood glucose level, and faster image acquisition. The molecular probe 68 68Ga-FAPI targeting tumor-associated fibroblasts can provide a lot of additional information that traditional 18 18F-FDG scans cannot provide. 68 68Ga-FAPI can visualize the protective micro-nests provided by the tumor microenvironment for tumor cells and help evaluate the promoting effect of the tumor microenvironment on tumor chemotherapy resistance. At the same time, 177 177Lu-FAPI can play an internal radiotherapy role on drug-resistant lesions by labeling the therapeutic radionuclide 177 177Lu, truly achieving the purpose of integrated diagnosis and treatment.

[0022] This invention will be applied 68The Ga-FAPI molecular probe is used as a visualization tool for evaluating the chemoresistance of lung cancer through PET / CT imaging, and the positive lesions of drug resistance are integrally treated by labeling therapeutic radionuclides 177 Lu to achieve the purpose of integrated diagnosis and treatment. Brief Description of the Drawings

[0023] Figure 1 For the experimental grouping, chemotherapy regimen of tumor-bearing mice and 68 Ga-FAPI Micro PET / CT parameter imaging process;

[0024] Figure 2 For 68 Preparation of Ga-FAPI molecular probe and PET / CT imaging, where (A) is LC-MS detection 68 Labeling rate of Ga-FAPI probe; (B) is micro-PET / CT imaging of A549 group and A549 / DDP group; (C) is fluorescence staining maps of A549 group and A549 / DDP group;

[0025] Figure 3 For the effect of A549+CAFs co-culture on drug resistance, where (A) is the comparison of tumor volumes in each group after completing the drug cycle chemotherapy; (B) is Ga-FAPI micro-PET / CT imaging of tumor-bearing nude mice after cisplatin treatment 68 ;

[0026] Figure 4 For the comparative analysis of imaging before and after treatment in A549 group and A549-DDP group; where (A) is the change of SUVmax at the tumor site before and after treatment; (B) is the change of tumor volumes in each group after completing the drug cycle chemotherapy; (C) is the positive cell staining results of A549 group and A549 / DDP group. Detailed Implementation Manner

[0027] Glossary of Related Technical Terms:

[0028] PET / CT: Positron Emission Tomography / Computed Tomography;

[0029] Micro-PET / CT: Micro Positron Emission Tomography / Computed Tomography;

[0030] FAPI precursor: Fibroblast Activation Protein Inhibitor precursor (purchased from MCE, catalog number HY-128643);

[0031] 68 Ga: Positron radionuclide gallium-68;

[0032] 177 Lu: Nuclide lutetium-177.

[0033] Example 1

[0034] Construction of a xenograft tumor model of lung cancer in nude mice with different cisplatin chemotherapy sensitivity states

[0035] Figure 1 Experimental grouping, chemotherapy regimen for tumor-bearing mice, and 68Ga-FAPI Micro PET / CT parameter imaging process;

[0036] Experimental steps: BALB / c male nude mice, 3 - 4 weeks old, body weight 18 - 20 g, specific pathogen free. Digest the amplified cisplatin-sensitive A549 cells and cisplatin-resistant A549-DDP cells of lung cancer, pipette the cells with pre-cooled normal saline to make a uniform suspension, and take an appropriate amount of cell suspension for cell counting; take 100 μL of cell suspension with a total cell count of 1×10 7 into a 1.5 mL centrifuge tube, label it and place it on ice; keep the cells on ice during the operation to reduce cell metabolism. Select the right axilla for inoculation. Before inoculating each nude mouse, resuspend the cells, draw 0.1 mL of cell suspension with a 1 mL sterile syringe connected to an insulin needle tip, and expel the air; when inoculating, place the nude mouse on its side, hold the skin of the back of the nude mouse with the left hand, expose and disinfect the right anterior axilla of the nude mouse to the maximum extent, hold the needle with the right hand, pierce the skin and then continue to insert it forward about 1 cm and slowly inject, quickly withdraw the needle, and try to avoid residual cell fluid or overflow in the pore canal. A round skin papule can be seen at the inoculation site. Try to complete the whole inoculation process within half an hour. Mark the cell type injected on each side. After inoculation, continue to raise the nude mice in the cage under the original conditions, observe them daily and measure and record the tumor growth with a vernier caliper. About 1 - 3 days later, the skin papule roughly absorbs and disappears, and a hard mass about the size of a rice grain can be felt in the axilla after about 7 days.

[0037] About 2 weeks later, a visible mass can be observed in the axilla of the nude mice, and then record the size of the subcutaneous tumor and the body weight of the nude mice every day. Randomly divide the nude mice in the two groups. The treatment group is treated with intraperitoneal injection of cisplatin (dose 3 mg / kg), and the control group is injected with the same volume of normal saline as a control. Place the cisplatin powder in normal saline and dissolve it into a cisplatin solution by heating in a 50°C water bath. Using the intraperitoneal injection method, after weighing the nude mice, administer cisplatin at a dose of 3 mg / Kg, once every 4 days, for a total of 4 chemotherapy treatments.

[0038] Experimental results: About 2 weeks after A549 cell inoculation, subcutaneous soft tissue nodules can be felt in the right forelimb of nude mice, with a diameter of about 0.8~1 cm, hard texture, good mobility, and no signs of metastasis. A total of 24 subcutaneous tumors were transplanted, 22 of which were tumor-forming, with a modeling success rate of 91.7%; nude mice were divided into 4 groups according to the transplanted cell lines: A549 wild type (n = 6); A549 wild type + CAFs co-culture (n = 5); A549 / DDP cisplatin-resistant type (n = 6); A549 / DDP cisplatin-resistant type + CAFs co-culture (n = 5). Some larger tumors were soft in texture, and dark red necrotic areas were visible after the tumors were peeled off in some tumors with a diameter of more than 120 mm.

[0039] The tumor volume of each group was measured 3 weeks after tumor inoculation. It was found that the tumor volume of the mixed group after A549+CAFs co-culture was larger than that of the A549 group. The average volumes were: A549 group (137.87 ± 36.08 mm3), A549+CAFs group (274.27 ± 68.05 mm3), and the volume comparison was statistically significant (P = 0.021). According to the volume monitoring data, during the entire experimental process, the tumor growth rate and volume of the mixed group after A549+CAFs co-culture were faster than those of the A549 group alone. The average tumor volume of the resistant strain group before administration was: A549 / DDP group: 227.49 ± 10.73 mm3, A549 / DDP+CAFs group: 339.43 ± 8.21 mm3, and the volume comparison was statistically significant (P = 0.037).

[0040] This study successfully established a nude mouse transplant tumor model of non-small cell lung cancer, with a tumor formation rate of more than 90%, a stable tumor growth curve, and pathological characteristics that were highly consistent with the primary tumor. This study showed that the tumor volume of the mixed group was significantly larger than that of the single group, suggesting that CAFs promote tumor growth through tumor-stroma interaction. The co-culture model of A549 or A549 / DDP cells and CAFs cells successfully verified the interaction between the tumor microenvironment and tumor formation and its effect on drug sensitivity, which provided a basis for subsequent 68 Ga-FAPI imaging provides a reliable model guarantee for the study of chemotherapy efficacy.

[0041] Example 2

[0042] 68 Synthesis and labeling of Ga-FAPI imaging probe

[0043] Experimental steps: Use 0.1 M HCl from 68 Ge / 68 Elution on Ga generator 68Ga, adjust the pH to 4.0 - 4.5 by adding 1 M sodium acetate (pH = 5), according to 925 - 1110 MBq 68 Add 25 μg (17.0 nmol) of FAPI precursor to Ga, place it in a 100 °C constant temperature mixer for reaction for 15 minutes, and obtain 68 Ga-FAPI molecular probe. The quality control of radiochemical synthesis is detected by high performance liquid chromatography (HPLC) for radiochemical purity (generally the purity is higher than 95%). The developing agent is 1 M methanol∶ammonium acetate = 1∶1. Add 1 M Na2CO3 to adjust the pH to 7.0; then aspirate 2 mL of the reaction solution and normal saline, rinse the C18 column, and then dry it. Rinse the column with 1 mL of ethanol, take the highest one for dilution and preparation before administration.

[0044] Experimental results: In this study, 68 Ga-FAPI molecular probe was successfully synthesized. First, by adjusting the pH to 4.0 - 4.5, the acidic environment was used to promote 68 the stable coordination of Ga³⁺ with the FAPI precursor, combined with heating reaction at 100 °C for 15 minutes, significantly improving the labeling efficiency; second, the use of C18 column purification effectively removed free 68 Ga and impurities. Finally, the labeling rate was verified to be as high as 99% by LC-MS, and the radiochemical purity detected by HPLC > 95%, indicating that the synthesis process is stable and reliable.

[0045] 68 Ga-FAPI molecular probe, the key lies in the optimized labeling conditions and efficient purification process. The buffering effect of sodium acetate and precise pH control in the reaction system are the keys to avoiding 68 the hydrolysis of Ga. The selection of methanol-ammonium acetate developing agent ensures the separation effect of HPLC analysis. The establishment of this method provides a technical basis for the application of FAPI probes in tumor diagnosis. Subsequently, the diagnostic efficacy can be evaluated by combining with PET / CT imaging, and the potential of combination with therapeutic radionuclides (such as 177 Lu) can be explored.

[0046] Example 3

[0047] Tumor-bearing nude mice 68 Ga-FAPI micro-PET / CT imaging.

[0048] Experimental procedure: Perform micro-PET / CT imaging on the established tumor-bearing nude mouse model before treatment and 21 days after treatment respectively.

[0049] No special preparation is required for the nude mice before the examination. Weigh the nude mice and prepare the corresponding dose of anesthetic and imaging agent. According to 50 μCi per nude mouse 68The Ga-FAPI injection volume was dissolved in 100 μL of normal saline. First, the nude mice were placed in a pre-anesthesia box for anesthesia. During the process, isoflurane was used for anesthesia, with an induction anesthesia of 3% and a maintenance anesthesia of 1.5 - 2%, and an air flow rate of 2.5 L / minute. After the righting reflex of the animals disappeared, they were placed on a mouse tail vein injection fixator, and a heat radiation lamp tube was used to irradiate the tails of the nude mice to promote the dilation of the tail veins. After the tail veins could be clearly distinguished with the naked eye, the prepared 68 Ga-FAPI imaging agent solution; about 40 μCi per mouse, and then an equal volume of normal saline was injected to rinse the syringe; the radioactivity of the syringe before and after injection and the hemostatic cotton ball after injection, as well as the injection time, were recorded. After injection, the mice were allowed to move freely in the cage. Before the 60-minute time point after injection, anesthesia before scanning was carried out in advance. The anesthetic drug and method were the same as pre-anesthesia. After the righting reflex disappeared, the animals were induced to urinate before imaging, and then the animals were fixed with their four limbs on a flat plate and placed prone on the scanning bed for imaging. Isoflurane inhalation was continuously given throughout the acquisition process.

[0050] Set the scanning parameters of micro-PET / CT (Inveon Siemens). After determining the scanning range, CT scanning was first performed. Low-dose CT scanning (tube current 500 μA, tube voltage 80 kV) was carried out for attenuation correction and providing anatomical localization; the PET acquisition energy window was set to 350 - 650 keV, the coincidence time was set to 3.438 ns, the axial length: 127 mm, and the slice thickness: 0.796 mm; the scanning lasted for 18 minutes in total. First, CT scanning was carried out for 8 minutes, and then PET scanning was carried out for 10 minutes. After the scanning was completed, fused images were reconstructed, and the regions of interest (ROIs) of the tumor site and the right upper limb muscle were outlined, and the maximum SUV value of the tumor body was measured.

[0051] Experimental results: When the long diameter of the tumor tissue in the right anterior axilla of the tumor-bearing mice was about 7 - 8 mm, whole-body 68 Ga-FAPI Micro PET / CT imaging was performed. The radioactive uptake at the tumor site was relatively uniform. Whole-body imaging showed that except for the tumor site, 68Ga-FAPI is mainly distributed in the kidneys, liver, and bladder, and this probe is mainly excreted through the urinary system. There was a statistically significant difference in the pre-treatment tumor SUVmax values between the A549 group and the A549 / DDP group. The SUVmax values were (1.86 ± 0.675, 2.150 ± 0.478, 2.883 ± 0.515; 4.460 ± 0.080; P < 0.05), indicating that there were differences in the affinity of the A549 sensitive strain and the drug-resistant strain for this new imaging agent. There was a statistically significant difference in the SUVmax values between A549 and the A549+CAFs group before treatment (1.86 ± 0.675, 2.883 ± 0.515; P < 0.05). There was a statistically significant difference in the tumor SUVmax values between the A549 / DDP group and the A549 / DDP+CAFs group. The SUVmax values were (2.150 ± 0.478, 4.460 ± 0.080; P = 0.003). Due to the high affinity of FAP specifically expressed by CAFs for this imaging agent, after co-culturing NSCLC cells with CAFs cells, for 68 the sensitivity of the Ga-FAPI imaging agent increased.

[0052] 68 Ga-FAPI Micro PET / CT imaging revealed the uptake differences and mechanisms of different NSCLC models for the FAP-targeted probe. The characteristics of uniform radioactive uptake in tumor tissues and the excretion of the probe through the urinary system suggest 68 that Ga-FAPI has stable targeting and a typical renal metabolic pathway, which is consistent with the known pharmacokinetic properties of the FAPI probe. The SUVmax of the A549 / DDP drug-resistant group was significantly higher than that of the A549 sensitive group, indicating that the drug-resistant strain may be accompanied by an increase in FAP expression or aggravated matrix fibrosis in the tumor microenvironment, suggesting that FAPI-PET may be a potential imaging biomarker for evaluating tumor drug resistance. More importantly, whether it is the sensitive strain or the drug-resistant strain, the SUVmax increased significantly after co-culturing with CAFs, confirming that CAFs significantly enhanced probe uptake by highly expressing FAP, and the pro-fibrotic effect of CAFs in the drug-resistant strain was more prominent. This result not only verified the core role of FAP in tumor stromal remodeling but also provided an experimental basis for non-invasively evaluating tumor drug resistance using FAPI and guiding combined therapy targeting CAFs.

[0053] Example 4

[0054] 177 Targeted radiotherapy study of Lu-FAPI on chemotherapy-resistant nude mice

[0055] Experimental steps: 177 Labeling method of the Lu-labeled radioactive drug. (1) Manual labeling. Take 0.05 mol / L hour CI solution, and177 Dilute the Lu solution (5550 - 7400 MBq) to 4 mL in a reaction flask. Separately, dissolve 100 - 150 μg of FAPI or DOTA-TOC precursor in 1 mL of 0.25 mol / L sodium acetate solution and add it to the reaction flask (pH value 4 - 5); react at 90 - 100 °C for 30 minutes, then collect the liquid in the reaction flask through an activated C18 column into a waste liquid bottle; separately, rinse the reaction flask with 5 mL of normal saline and collect the rinse through the C18 column into the waste liquid bottle; finally, inject 1 mL of 60% (volume fraction) ethanol through the C18 column and a sterile filter membrane into the product bottle, and dilute with normal saline to about 20 mL for standby; measure the activities of the C18 column, waste liquid bottle, and product bottle respectively, and calculate the labeling yield. (2) Dissolve the FAPI or DOTA-TOC precursor (100 - 150 μg) in pure water (2 mL) sufficiently and then transfer it to an anhydrous sodium ascorbate buffer solution and shake well. Transfer the 177 LuCl solution to the reaction flask, transfer it to the reaction flask together with the precursor solution, the pH value of the reaction system is 4 - 5, after reacting the reaction mixture at 95 °C for 30 minutes, transfer it to the product bottle through a sterile filter; rinse the reaction flask with normal saline and then transfer it to the product bottle, and the final product is about 20 mL; measure the activities of the waste liquid bottle and the product bottle respectively, and calculate the labeling yield.

[0056] 177 Quality control of Lu-labeled radiopharmaceuticals. Refer to the Pharmacopoeia of the People's Republic of China (2020 Edition) to determine the clarity, pH value, sterility, and bacterial endotoxin of the above-mentioned labeled products. Take an appropriate amount of the sample and add it to PBS and FBS, and measure the radiochemical purity of the sample using a high-performance liquid chromatograph at 0, 12, 24, and 48 hours respectively to examine its in vitro stability. High-performance liquid chromatograph analysis conditions: Rax-C18 column, mobile phase A is an aqueous solution containing 0.05% (volume fraction) trifluoroacetic acid; B is an acetonitrile solution containing 0.05% trifluoroacetic acid. 177 Gradient elution of Lu-DOTA-TOC: 0 - 5 minutes is 100% - 90% A, 0 - 10% B; 5.01 - 20 minutes is 90% - 20% A, 10% - 80% B; 20.01 - 30 minutes is 100% B; 177 Gradient elution of Lu-FAPI: within 15 minutes, mobile phase B is 0 - 100%, and the flow rate is 1 mL / minute.

[0057] 68 After the Ga-FAPI imaging, randomly divide the nude mice in the cisplatin-resistant group into two groups, and the experimental group is injected with 30 MBq 177For Lu-FAPI, the control group was injected with the same volume of normal saline. SPECT / CT imaging was performed 24 and 48 hours after administration. The planar imaging matrix was 256x1024, and the scanning speed was 15 cm / min; for SPECT / CT imaging, the matrix was 128x128, the acquisition radius remained fixed, the magnification was 1, the stepwise rotation mode was used, 10° / frame, and 30 seconds / frame. The CT scan parameters were: matrix 512x512, tube voltage 120 kV, tube current 200 mA, and slice thickness 2.5 mm.

[0058] The size of the subcutaneous tumor and the body weight of the nude mice were recorded every day. After 21 days, 68 Ga-FAPI micro-PET / CT imaging was performed again. After the imaging was completed, the nude mice bearing tumors were sacrificed by cervical dislocation, the tumor tissues were removed for paraffin embedding, and after sectioning, HE staining, Ki-67, tunel, and FAP immunohistochemical staining were performed.

[0059] Experimental results: After completing the drug cycle chemotherapy, the tumor volumes of each group were monitored. It was found that the tumor growth in the A549 + cisplatin treatment group was significantly inhibited. In the A549 group: 260.88 ± 44.06 mm 3 , and the tumor volume inhibition rate was: 60.52%; in the A549 + CAFs group: 356.53 ± 205.52 mm 3 , and the tumor volume inhibition rate was: 30.85%; after administration, the average tumor volumes of the drug-resistant strain groups were: in the A549 / DDP group: 882.94 ± 48.66 mm 3 , and the tumor volume inhibition rate was: 21.11%; in the A549 / DDP + CAFs group: 1080.95 ± 126.71 mm 3 , and the tumor volume inhibition rate was: 11.17%; it was shown that in the co-culture environment, the tumor grew more rapidly and was more difficult to be inhibited by cisplatin chemotherapy.

[0060] After cisplatin treatment of nude mice bearing tumors 68 Ga-FAPI micro-PET / CT imaging showed that radioactive uptake of Ga-FAPI was concentrated at the tumor site before and after treatment, and inside the tumor mass 68 68Ga-FAPI uptake was uneven, with obvious radioactive concentration areas locally. The degree of SUVmax decline in transplanted tumors varied among groups after treatment; when comparing before and after chemotherapy, there was a statistically significant difference in the tumor SUVmax values of the A549 group before and after treatment (1.860 ± 0.080, 2.150 ± 0.478; P = 0.046), and ΔSUVmax was 15.59%; there was a statistically significant difference in the tumor SUVmax values of the A549 / DDP group before and after treatment (2.110 ± 0.921, 3.883 ± 0.515; P = 0.044), and ΔSUVmax was 60.33%. The drug-resistant group was less sensitive to cisplatin, and the change in SUVmax value was more obvious after cisplatin chemotherapy.

[0061] The above results revealed the dynamic association between the efficacy of cisplatin chemotherapy and the tumor microenvironment and drug resistance. After co-culturing A549 or A549 / DDP with CAFs, the inhibition rates of the two groups further decreased, indicating that CAFs synergistically promoted chemotherapy resistance by remodeling the microenvironment. 68 Ga-FAPI imaging showed that the increase in SUVmax in the drug-resistant group was more significant after cisplatin treatment, which might be related to chemotherapy-induced fibrotic reactions or upregulation of FAP expression: the drug-resistant strains activated CAFs under drug pressure, resulting in increased FAP-dependent matrix remodeling, and the residual tumor cells formed a "pro-survival niche" with activated CAFs, manifested as local radioactive concentration. The highly metabolically active area might represent the "surviving foci" of treatment resistance, which was visualized by 68 Ga-FAPI imaging in vitro.

[0062] Example 5

[0063] Pathological detection

[0064] After all scans were completed, the nude mice were sacrificed by cervical dislocation, and the excised tumor masses were immersed in paraformaldehyde solution for fixation; after paraffin sectioning, they were dewaxed to water, washed with gradient alcohol (absolute, 95%, 75%), and then washed with TBS; EDTA antigen retrieval solution was added to the pressure cooker, timed for 90 s after jetting and then closed, and washed with TBS after natural cooling to room temperature; the sections were immersed in 3% H2O2 for 30 minutes, then circled with an immunohistochemistry pen and placed in TBST, and incubated with 10% goat serum for 30 minutes at room temperature; the serum was discarded, 100 μL of primary antibody dilution was added dropwise to each section, and incubated overnight in a 4°C refrigerator; the next day, the sections were rewarmed for 15 minutes, washed with TBST, 100 μL of secondary antibody dilution was added dropwise to each section, and incubated at 37°C for 45 minutes, then washed with TBST; 100 μL of DAB was added dropwise to each section and observed under the microscope, and the reaction was terminated immediately when there was a color change; hematoxylin for 1 minute, hydrochloric acid alcohol for 1 - 2 s, and bluing solution for bluing for several seconds, and then the nuclear staining was observed under the microscope; sealed with a mounting medium and photographed under a white light microscope.

[0065] Analysis method of pathological results: The staining results were determined by two methods: the percentage of positive cells and the intensity of cell staining. The standards were: cell staining intensity score, no positive staining (0 point), light yellow (1 point), brown (2 points), and brown (3 points); positive cell percentage score, ≤25% (1 point), 26%-50% (2 points), 51%-75% (3 points), and >75% (4 points). The final result was obtained by multiplying the two scores.

[0066] Experimental results: HE staining after treatment showed that there were more tumor-associated fibroblasts and necrotic cells inside the tumor of the A549-DDP group, and HE staining of the co-culture group showed that the lower part of the tumor 68 There were many tumor-associated fibroblasts and necrotic cells in the area of ​​abnormal Ga-FAPI uptake. Ki-67 positive cells were widely expressed in the CAFs co-culture group, and the Ki-67 positive area was mainly located in the nucleus, mainly yellow or brown, and there was also a small amount of expression in the cytoplasm. The results showed that the number of positive stained cells in the single-species group was lower than that in the co-culture mixed group. The score results of the number of positive stained cells in the A549, A549+CAFs, A549 / DDP and A549 / DDP+CAFs groups were (32.20±7.60, 124.87±16.80, 42.40±10.00, 151.80±39.26), respectively. The expression of Ki-67 in the tumor cell single-species group was significantly lower than that in the CAFs cell co-culture mixed group, and the difference was statistically significant. Immunohistochemistry results showed that FAP was mainly concentrated in the cytoplasm and cell membrane, and was diffuse or flaky brown-yellow in cells. The positive cell staining intensity scores of A549 group, A549+CAFs group, A549 / DDP group and A549 / DDP+CAFs group were (2.33±0.58, 9.33±2.31, 3.50±0.50, 8.67±3.06), respectively. The FAP expression in the tumor cell single-species group was significantly lower than that in the CAFs cell co-culture mixed-species group, and the difference was statistically significant, indicating that the A549 / DDP group could recruit and form more CAFs cells, leading to vigorous tumor proliferation and resistance to chemotherapeutic drugs.

[0067] HE staining showed that there were more CAFs and necrotic cells in the tumor of A549-DDP group, and this phenomenon was also confirmed in the co-culture group, especially 68Ga-FAPI abnormal uptake area. The expression levels of Ki-67 positive cells were widely distributed in the co-culture group of CAFs, indicating a high proliferative ability of tumor cells. By scoring the number of positive stained cells in different experimental groups, it was found that the Ki-67 expression in the single tumor cell group was significantly lower than that in the CAFs co-culture group, further indicating that CAFs played a promoting role in tumor cell proliferation. The immunohistochemical results showed positive expression of the fibroblast activation marker FAP in the cytoplasm and cell membrane, suggesting that CAFs played an important role in regulating the tumor microenvironment and promoting tumor development. In particular, the A549 / DDP group recruited more CAFs cells, resulting in increased tumor proliferation and resistance of tumor cells to chemotherapeutic drugs. Therefore, these results emphasized that CAFs promoted the proliferation and drug resistance of NSCLC tumor cells. 68 Ga-FAPI Micro PET / CT imaging has certain value for tumor imaging of NSCLC-bearing nude mice and evaluation of the therapeutic effect after cisplatin chemotherapy.

Claims

1. A method for preparing a molecular probe targeting tumor-associated fibroblasts, characterized in that, The preparation method includes: mixing 68 Ga with sodium acetate, adding the FAPI precursor, and reacting at a constant temperature with uniform mixing to obtain 68 the Ga-FAPI molecular probe.

2. The preparation method of the molecular probe targeting tumor-associated fibroblasts according to claim 1, characterized in that, The concentration of the sodium acetate is 0.8 to 1.2 mol / L.

3. The preparation method of the molecular probe targeting tumor-associated fibroblasts according to claim 1, characterized in that, 68 The ratio of Ga to the FAPI precursor is 925 - 1110 MBq: 25 μg.

4. The preparation method of the molecular probe targeting tumor-associated fibroblasts according to claim 1, wherein, The constant temperature reaction is carried out at 95 to 105 °C for 12 to 18 minutes.

5. The preparation method of the molecular probe targeting tumor-associated fibroblasts according to claim 1, wherein, Obtained 68 After the Ga-FAPI molecular probe was obtained, high performance liquid chromatography was used to detect the radiochemical purity.

6. A molecular probe targeting tumor-associated fibroblasts, characterized in that, Prepared by the preparation method according to any one of claims 1 to 5.

7. A imaging agent targeting tumor-associated fibroblasts, characterized in that, Comprising a molecular probe targeting tumor-associated fibroblasts as described in claim 6.

8. Use of the molecular probe targeting tumor-associated fibroblasts as described in claim 6 in the preparation of a preparation for tumor diagnosis or treatment.

9. The application according to claim 8, wherein The tumor is a cisplatin chemotherapy-resistant tumor.

10. The application according to claim 8, wherein The preparation is used to inhibit tumor volume.

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