Application of flash radiotherapy in reversing radioresistance in head and neck squamous cell carcinoma
Through high-energy X-ray flash radiotherapy based on petal accelerator, the problem of radiotherapy resistance in the prior art was solved, and the reversal of radiotherapy resistance to head and neck squamous cell carcinoma is achieved, which significantly improves the treatment effect.
Patent Information
- Application Number
- CN202411813304.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-11
- Publication Date
- 2025-04-04
AI Technical Summary
The existing flash radiation therapy (FLASH-RT) based on high-energy X-rays is mainly focused on the radiotherapy effect on tumors, and no research has been found to relieve radiotherapy resistance.
X-ray flash radiotherapy based on petal accelerator was used, with a peak dose rate of 176Gy/s and an average dose rate during irradiation was 88Gy/s, which was used to reverse the radiotherapy resistance of squamous cell carcinoma in the head and neck.
By inhibiting the proliferation and invasion of radiotherapy-resistant cells, enhancing apoptosis and DNA damage, regulating the immune microenvironment, increasing the ratio of CD8+ T cells and M1/M2 macrophages, significantly alleviating radiotherapy resistance.
Smart Images
Figure CN119746281A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of radiotherapy, and in particular relates to an application of flash radiotherapy in reversing radiotherapy resistance of head and neck squamous cell carcinoma. Background Art
[0002] Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and is characterized by high mortality and morbidity. As a non-invasive and function-preserving therapy, radiotherapy (RT) is the standard treatment for HNSCC. However, due to the aggressive biological behavior of this disease, approximately half of HNSCC patients experience local control failure and subsequently develop recurrent or metastatic disease, resulting in a poor prognosis and significantly reduced quality of life in this patient subgroup. Previous studies have shown that the underlying mechanisms of RT resistance are multifaceted, including radiation-induced DNA damage repair, changes in the tumor immune microenvironment, cell cycle arrest, apoptosis escape, proliferation of cancer stem cells, regulation of exosomes and non-coding RNA, metabolic reprogramming, etc. Currently, pharmacological treatment strategies include the use of radiosensitizers such as cisplatin, cetuximab, immune checkpoint inhibitors, HfO2, and apoptosis protein inhibitors. However, there are currently few established and effective clinical strategies to reverse RT resistance, indicating a serious unmet clinical need.
[0003] Flash radiotherapy (FLASH-RT) can deliver radiation doses at ultra-high dose rates (≥40 Gy / s) within a few pulses (milliseconds) and has been shown to differently alter the response of normal tissues and tumors to radiation. Therefore, compared with Conv-RT (0.01-0.40 Gy / s), FLASH-RT not only has a significantly faster irradiation completion speed, but also allows higher irradiation doses to be applied to tumors under the same tissue protection conditions. This is expected to overcome RT resistance while greatly reducing the irradiation cycles required for Conv-RT in the clinical setting and the associated human and material costs. In addition, it reduces damage to patients' normal organs, alleviates patients' pain, improves their quality of life, and solves problems related to organ motion during RT, achieving precise RT targets. Therefore, FLASH-RT represents a very promising and potentially transformative radiotherapy technology.
[0004] High-energy X-rays are the most widely used beam type in clinical RT settings, with deep penetration and low divergence, making them highly anticipated in tumor control. However, the development of FLASH-RT based on high-energy X-rays has been very slow due to limitations in conversion efficiency and technical challenges. my country's independently developed ultra-high-power petal accelerator based on high-energy X-rays is capable of achieving a high dose rate of 100Gy / s at a distance of 0.8 meters from the target, with the characteristics of high duty cycle, high efficiency, high beam power and high beam quality. The accelerator can repeatedly accelerate electrons to near the speed of light in the same accelerating cavity, effectively generating high-power, high-energy electron beams to conduct FLASH-RT while meeting the requirements of multiple irradiation angles.
[0005] Currently, there are few studies on FLASH-RT using high-energy X-rays, and there are no reports on FLASH-RT overcoming radiotherapy resistance. Summary of the invention
[0006] The technical problem to be solved by the present invention is that the existing FLASH-RT based on high-energy X-rays is still mainly focused on the radiotherapy effect on tumors, and there is no research related to whether FLASH-RT can alleviate radiotherapy resistance.
[0007] The technical solution of the present invention to solve the above technical problem is: to provide an application of flash radiotherapy in reversing radiotherapy resistance of head and neck squamous cell carcinoma.
[0008] Wherein, in the above application, the flash radiotherapy is X-ray-based flash radiotherapy.
[0009] Furthermore, in the above application, the X-rays are accelerated using a petal accelerator.
[0010] Furthermore, in the above application, the peak dose rate of X-rays is 176 Gy / s, and the average dose rate during irradiation is 88 Gy / s.
[0011] Among them, in the above application, the specific manifestation of reversing the radiotherapy resistance of head and neck squamous cell carcinoma is: inhibiting the proliferation and invasion of radiotherapy-resistant cells, and enhancing the apoptosis and DNA damage of radiotherapy-resistant cells.
[0012] Among them, in the above application, the specific manifestation of reversing the radiotherapy resistance of head and neck squamous cell carcinoma is: the immune regulatory pathway in the radiotherapy-resistant tumor is upregulated, and the activity of the DNA repair pathway is downregulated.
[0013] Among them, in the above application, the specific manifestation of reversing the radiotherapy resistance of head and neck squamous cell carcinoma is: increasing CD8 + T cells and increased the M1 / M2 macrophage ratio.
[0014] The beneficial effects of the present invention are:
[0015] The present invention provides an application of flash radiotherapy in reversing radiotherapy resistance of head and neck squamous cell carcinoma. It is the first time to find through experiments that flash radiotherapy has a very good effect on reversing radiotherapy resistance of HNSCC. It can not only inhibit the proliferation and invasion of radiotherapy-resistant cells, enhance the apoptosis and DNA damage of radiotherapy-resistant cells, but also upregulate the immune regulation pathway in radiotherapy-resistant tumors and downregulate the activity of DNA repair pathways. At the same time, it can also increase CD8 + T cells, and at the same time increase the ratio of M1 / M2 macrophages, and work together to achieve the effect of alleviating radiotherapy resistance. The present invention provides a new application scenario for flash radiotherapy in radiotherapy resistance, which is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 The figure shows the comparison between FLASH-RT and Conv-RT using a petal accelerator; a is the petal accelerator; b is the radiation platform and the fixation method of cell samples and animal samples; c is the irradiation time structure and dose rate of FLASH-RT, d is the irradiation time structure and dose rate of Conv-RT; e, f are the dose distribution of the irradiated area of cell samples; g, h are the dose distribution of the irradiated area of mouse samples; i, j are the dose management and dose distribution of cell experiments; k, l are the dose management and dose distribution of animal experiments. Ctrl, control group; Conv-RT, conventional radiotherapy; FLASH-RT, FLASH radiotherapy; ns, no statistical significance.
[0017] Figure 2The results of the cell experiments shown in the figure. a represents the results of CCK-8 detection, showing that the proliferation inhibition effect of FLASH-RT in CAL33_R cells is significantly higher than that of Conv-RT. b represents the quantitative analysis of the degree of proliferation inhibition in the CCK-8 experiment, and the quantitative comparison of the differences between different treatment groups. c represents the image of the scratch healing experiment, showing that FLASH-RT significantly inhibits the migration ability of CAL33_R cells. d represents the quantitative analysis of the scratch healing experiment, further verifying the statistical significance of the migration inhibition. e represents the image of the Transwell invasion experiment, indicating that FLASH-RT significantly reduces the invasion ability of CAL33_R cells. f represents the quantitative results of the Transwell invasion experiment, which is used to compare the effects of different radiation treatments on invasion ability. g represents the results of γ-H2AX immunofluorescence staining, showing that the degree of DNA damage induced by FLASH-RT is significantly higher than that of Conv-RT. h represents the results of multi-parameter joint analysis, including the comprehensive display of data such as DNA damage, mitochondrial membrane potential changes and cell apoptosis. i represents the quantitative analysis of γ-H2AX-positive cells, showing the significant increase in DNA damage after FLASH-RT treatment. j represents the results of mitochondrial membrane potential measurement, reflecting the ability of FLASH-RT to reduce mitochondrial membrane potential (increased proportion of monomeric dye). k represents the quantitative analysis of cell death rate, indicating that the proportion of cell death caused by FLASH-RT is significantly higher than that of Conv-RT. l represents the results of cell apoptosis detection, and the effects of different treatments on apoptosis rate were quantitatively analyzed by flow cytometry.
[0018] Figure 3The experimental results of mice are shown in the figure. a represents the change curve of tumor volume of mice in the MOC1_R group, showing that FLASH-RT significantly reduced the tumor volume compared with Conv-RT. b represents the statistical analysis results of the final measurement of tumor volume, further verifying that the tumor control effect of FLASH-RT in the MOC1_R group was significantly enhanced compared with Conv-RT. c represents the results of Ki-67 immunohistochemistry staining, showing that FLASH-RT significantly inhibited the proliferation activity of tumors in mice in the MOC1_R group. d represents the quantitative analysis results of Ki-67 positive cells, further illustrating the significant inhibitory effect of FLASH-RT on tumor proliferation. e represents the results of Tunel staining, showing that FLASH-RT significantly increased the apoptosis level of tumor cells in the MOC1_R group. f represents the quantitative analysis results of Tunel positive cells, verifying the statistical significance of FLASH-RT-induced apoptosis. g represents the results of γ-H2AX immunofluorescence staining, indicating that FLASH-RT significantly increased the level of DNA damage in tumor tissues in the MOC1_R group. h represents the results of transcriptome PCA analysis, showing that the FLASH-RT subgroup in the MOC1_R group was significantly distinguished from the other two groups. i indicates that the volcano plot shows the differentially expressed genes between the FLASH-RT subgroup and the Conv-RT subgroup. FLASH-RT significantly upregulated the expression of immune-related genes including Cd8a, Gzmk, Ifng, Ccl17, Cxcl9, etc. j indicates the results of KEGG pathway enrichment analysis, showing that the FLASH-RT subgroup was significantly enriched in T cell activation-related pathways, including Th1 / Th2 cell differentiation, antigen processing and presentation, T cell receptor signaling pathway, and cytokine-cytokine receptor interaction pathway. k indicates that GSEA enrichment analysis showed that the FLASH-RT subgroup significantly upregulated innate and adaptive immune-related pathways. l indicates that GSEA enrichment analysis showed that the FLASH-RT subgroup significantly downregulated DNA damage repair-related pathways, which was consistent with the results of DNA damage staining. DETAILED DESCRIPTION
[0019] The present invention established radiotherapy-resistant HNSCC cell line models and animal models, and used the petal accelerator to perform FLASH-RT or Conv-RT treatment respectively. The results showed that FLASH-RT inhibited the proliferation and invasion of radioresistant cells, while enhancing apoptosis and DNA damage. Transcriptomic and proteomic analysis showed that FLASH-RT upregulated the immune regulatory pathway in radiotherapy-resistant tumors, while downregulated the activity of the DNA repair pathway. Immunofluorescence and flow cytometry showed that CD8 + T cells increased significantly, and the ratio of M1 / M2 macrophages also increased significantly. Mechanistically, FLASH-RT activated CD8 +T cells then stimulate M1 macrophages through paracrine IFN-γ signals, thereby activating the immune microenvironment and ultimately achieving the effect of reversing HNSCC radioresistance.
[0020] Finally, the present invention provides an application of flash radiotherapy in reversing radiotherapy resistance of head and neck squamous cell carcinoma.
[0021] The specific implementation modes of the present invention will be further explained below through examples, but it is not intended to limit the protection scope of the present invention to the scope described in the examples.
[0022] The equipment and materials used in the examples are all common commercially available products.
[0023] Example 1: FLASH-RT Processing Using Petal Accelerator
[0024] The layout of the petal accelerator and irradiation platform is as follows Figure 1 As shown in ab. The electron beam is guided by a stainless steel target. Three lead bricks are used as collimators to form the X-ray field. A PMMA stand is used to support the mouse or culture dish. The stand is mechanically aligned to the platform, and the position error is less than 1 mm during the experiment. The EBT4 film is placed between the sample and the stand. A TW30013 ionization chamber (PTW Farmer model) and an ammeter (PTW Unidos Romeo) are installed at the centerline position for real-time monitoring of the irradiation process. In our experiments, the method of changing the average dose rate is achieved by adjusting the irradiation time structure. For FLASH-RT, the peak dose rate was 176 Gy / s and the average dose rate during irradiation was 88 Gy / s ( Figure 1 c). For Conv-RT, the peak dose rate was 176 Gy / s and the average dose rate was approximately 3.095 Gy / min ( Figure 1 d). Figure 1 ef shows the dose distribution of the irradiated area in the cell experiment. Figure 1 gh shows the dose distribution in animal experiments. EBT4 film in cell experiments ( Figure 1 ij) and animal experiments ( Figure 1 kl) both demonstrate accurate dose management and good dose distribution.
[0025] Example 2 Effect of FLASH-RT treatment on radiotherapy-resistant cells
[0026] Radiotherapy-resistant human tongue squamous cell carcinoma cell line CAL33_R and mouse oral squamous cell carcinoma cell line MOC1_R were constructed. The specific construction method of the human tongue squamous cell carcinoma cell line CAL33_R is as follows: conventional culture (37°C, 5% CO2) CAL33 cell line, select cells in the logarithmic growth phase to simulate the radiotherapy process of HNSCC patients, radiotherapy 5 days a week, rest 2 days, irradiation 2Gy each time, until the cumulative dose of cells reaches 70Gy. Specifically, the irradiated cells grow to about 80% of the culture dish, placed in the center of the irradiation platform, and irradiated with 160kV X-rays using an RS2000 irradiator (Rad Source Technologies, USA), with a single irradiation dose of 2Gy, and the specific parameters are set as: 160KV, 25mA, 0.1mm copper alloy filter, no reflector. After irradiation, the cells were placed in an incubator for continued culture. When the cells grew to about 90% of the culture dish, they were passaged. When the cell lines to be verified were in the logarithmic growth phase, they were plated in groups. After adherence, they were irradiated with 2, 4, 6, 8, and 10 Gy of X-rays for group gradient irradiation. After irradiation, the plates were immediately re-plated for cloning experiments. 6-well plates were used for plating, with 1,000 cells plated per well. After 14 days, the cloning was evaluated. After irradiation, the plates were plated for CCK8 experiments (Japan DOJINDO Company). 96-well plates were used for plating, with 2,500 cells plated per well. The multi-function microplate reader (Synergy TM H1, BioTek) detected the absorbance (OD value) of the cells at 450nm for 48h and 72h respectively. Finally, the presence or absence of radiation resistance was determined by comparing the clone formation rate and cell survival rate of the two groups of cells under the same radiation dose. The successfully constructed human tongue squamous cell carcinoma cell line CAL33_R was deposited in the State Key Laboratory of Biotherapy, Sichuan University. Among them, the construction method of the mouse oral squamous cell carcinoma cell line MOC1_R was constructed by referring to the method in patent CN118599778A.
[0027] The invention verifies the construction of radiotherapy-resistant cell lines through CCK-8 and clone formation experiments, and determines that the radiation dose of CAL33_R cells in subsequent cell and animal experiments is 4Gy, and that of MOC1_R cells is 6Gy. When FLASH-RT is used, the radiation doses of the two cells are 4Gy and 6Gy, respectively.
[0028] The results of cell experiments are as follows Figure 2As shown, the results showed that FLASH-RT reversed radioresistance and caused more DNA damage. In CAL33_R cells, FLASH-RT could significantly inhibit their proliferation (ab), migration (cd) and invasion ability (ef), while increasing DNA damage (gi), reducing mitochondrial membrane potential (gh and j), and increasing the levels of cell death (gh and k) and apoptosis (gh and l) compared with conventional radiotherapy (Conv-RT). Ctrl, control group; Conv-RT, conventional radiotherapy; FLASH-RT, FLASH radiotherapy; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.
[0029] Example 3 Effects of FLASH-RT treatment on radiotherapy-resistant mice
[0030] SPF female C57BL / 6J mice aged 6-8 weeks were purchased from Beijing Huafukang Biotechnology Co., Ltd. The experimental mice received standard feed provided by the Experimental Animal Center of West China Hospital and were raised under strict pathogen-free conditions.
[0031] In the process of establishing the mouse HNSCC model, MOC1 or MOC1_R cells (1×106 cells) were subcutaneously injected into C57BL / 6J mice to induce tumor formation. After tumor formation was confirmed, when the tumor reached a standardized size (approximately 100±25mm 3 ) was started. Tumor volume was measured with a caliper and calculated according to the following formula: volume = L × W2 / 2, where L is the length of the tumor and W is the width. The petal accelerator of the China Academy of Engineering Physics (CAEP) was used to complete the radiation experiments of FLASH-RT and Conv-RT. The schematic diagram is attached. Figure 1 a. The layout of the accelerator and its radiation platform is shown in Figure S1b. PMMA brackets were used to fix mice or culture dishes in the experiment (see Figure 1 b).
[0032] The results of the mouse experiment are as follows Figure 3As shown. The results showed that the C57BL / 6J mouse experiment confirmed that in the MOC1_R group, the tumors in the FLASH-RT group (n=5) were better controlled than those in the conventional radiotherapy (Conv-RT) group (n=5) (ab). Ki-67 staining, Tunel staining, and DNA damage immunofluorescence (IF) of mouse tumor tissues showed that FLASH-RT significantly increased the levels of DNA damage and apoptosis, while reducing the proliferation activity of tumors in the MOC1_R group (cg). Transcriptome PCA analysis showed the subgroups within the MOC1_R group (h), the volcano plot showed the differentially expressed genes between the FLASH-RT and Conv-RT subgroups in the MOC1_R group (i), and the KEGG analysis showed the enriched signal pathways of the FLASH-RT subgroup in the MOC1_R group (j). GSEA enrichment analysis showed the upregulated (k) and downregulated (l) pathways in the FLASH-RT subgroup. Ctrl, control group; Conv-RT, conventional radiotherapy; FLASH-RT, FLASH radiotherapy; PCA, principal component analysis. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Figure 3 The results also showed that compared with Conv-RT, FLASH-RT significantly reversed radiotherapy resistance, induced more DNA damage, and significantly activated the immune microenvironment.
[0033] The above experimental results show that the flash radiotherapy used in the present invention has a very good effect on reversing HNSCC radioresistance. It can not only inhibit the proliferation and invasion of radioresistance cells, enhance the apoptosis and DNA damage of radioresistance cells, but also upregulate the immune regulation pathway in radioresistance tumors and downregulate the activity of DNA repair pathways. At the same time, it can also increase the CD8 + T cells and at the same time increase the M1 / M2 macrophage ratio, which together achieves the effect of alleviating radiotherapy resistance, thereby providing an application of flash radiotherapy in reversing radiotherapy resistance in head and neck squamous cell carcinoma.
Claims
1. Application of flash radiotherapy in reversing radiotherapy resistance in head and neck squamous cell carcinoma.
2. The use of flash radiotherapy in reversing radiotherapy resistance of head and neck squamous cell carcinoma according to claim 1, characterized in that: The flash radiotherapy is based on X-rays.
3. The use of flash radiotherapy in reversing radiotherapy resistance of head and neck squamous cell carcinoma according to claim 1, characterized in that: The X-rays are accelerated by a petal accelerator.
4. The use of flash radiotherapy in reversing radiotherapy resistance of head and neck squamous cell carcinoma according to claim 1, characterized in that: The X-ray peak dose rate is 176 Gy / s.
5. The use of flash radiotherapy in reversing radiotherapy resistance of head and neck squamous cell carcinoma according to claim 1, characterized in that: The average dose rate during the X-ray irradiation process was 88 Gy / s.
6. The use of flash radiotherapy in reversing radiotherapy resistance of head and neck squamous cell carcinoma according to claim 1, characterized in that: The specific manifestations of reversing the radioresistance of head and neck squamous cell carcinoma are: inhibiting the proliferation and invasion of radioresistance cells, and enhancing the apoptosis and DNA damage of radioresistance cells.
7. The use of flash radiotherapy in reversing radiotherapy resistance of head and neck squamous cell carcinoma according to claim 1, characterized in that: The specific manifestations of reversing the radioresistance of head and neck squamous cell carcinoma are: upregulation of immune regulatory pathways and downregulation of DNA repair pathway activity in radioresistance tumors.
8. The use of flash radiotherapy in reversing radiotherapy resistance of head and neck squamous cell carcinoma according to claim 1, characterized in that: The specific manifestation of reversing the radiotherapy resistance of head and neck squamous cell carcinoma is: increasing CD8 + T cells and increased the M1 / M2 macrophage ratio.