Engineering autologous tumor tissue scaffold and application thereof

Abstract

The invention discloses an engineered autologous tumor tissue scaffold and application thereof, and the engineered autologous tumor tissue scaffold is prepared by performing specific treatment on autologous tumor tissue to induce immunogenic cell death of the tumor tissue, and then sequentially performing freezing and drying steps; the engineered autologous tumor tissue scaffold can be used for loading dendritic cells and constructing personalized tumor vaccines. According to the stent, autologous tumor tissue is adopted, so that a specific and comprehensive tumor antigen pedigree is reserved, and multi-epitope immunostimulation is provided; and meanwhile, excellent biocompatibility is ensured, the immunological rejection risk is reduced, and relatively high safety is ensured.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to an engineered autologous tumor tissue scaffold and applications thereof. Background Art

[0002] Surgical resection is currently the preferred option for the clinical treatment of early-stage, non-metastatic solid tumors. However, residual microscopic lesions or circulating tumor cells after surgery often lead to fatal tumor recurrence and metastasis within months or years. Immunotherapy, as an emerging treatment modality, has shown great promise in anti-tumor research. In particular, the successful application of immune checkpoint inhibitors (such as anti-PD-1 monoclonal antibodies) has provided a new direction for tumor treatment. In addition to immune checkpoint inhibitors, cancer vaccines are also gaining attention. Cancer vaccines activate the patient's immune system by injecting anti-tumor antigens into the body, thereby inducing systemic and tumor-specific immune protective responses, and are expected to effectively inhibit tumor recurrence after surgery.

[0003] Sipuleucel-T (dendritic cell vaccine), the first therapeutic cancer vaccine approved for the treatment of metastatic castration-resistant prostate cancer, achieves treatment by loading dendritic cells (DCs) with antigens in vitro and inducing their maturation before reinfusion into the patient. However, its complex preparation process limits cell viability. Specifically, DCs tend to lose viability during in vitro culture; mature DCs have a short lifespan in vivo, typically surviving only a few days, requiring multiple reinfusions to maintain efficacy, increasing the treatment burden; furthermore, maintaining high activity and functional stability of DCs during cell preparation and transportation presents considerable technical challenges.

[0004] To overcome the limitations of traditional DC vaccines, researchers have begun exploring the potential of loading DCs onto bioactive scaffolds to enhance their activity and prolong their in vivo retention. For example, Rao's team used porous biomaterials as scaffolds to load DCs for anti-tumor immunotherapy (Xu, Yangtao, et al. Adv. Funct. Mater. 2024, 34, 2400507); David J. Mooney's team indirectly loaded DCs via hydrogels encapsulating chemokines (Wang, Hua, et al. Nat. Mater. 2020, 19, 1244–1252). These scaffolds mimic the extracellular matrix in the body, providing a favorable microenvironment for DCs to survive, maintain their high activity and functional stability, and enable controlled localized release of cells.

[0005] However, current bioactive scaffolds used to load immune cells are typically made of synthetic materials, require additional loading of tumor antigens and immune stimulatory factors, and struggle to fully mimic the tumor's antigenic spectrum. Due to tumor heterogeneity and individual differences in antigen expression, the use of universal antigens can lead to unstable therapeutic effects, limiting their clinical application.

[0006] In response to the above problems, there is an urgent need for a new delivery platform that can simultaneously provide individual-specific antigens and a DCs-supporting microenvironment. Based on this, the present invention proposes a strategy for delivering DCs using autologous tumor tissue as a natural bioactive scaffold. Autologous tumor tissue is derived from the patient himself and naturally carries a complete and individual-specific antigen spectrum, which can achieve precise immune activation without the need for exogenous antigen loading. After engineering treatments such as freeze-drying, the tissue can form a porous three-dimensional structure, retaining key extracellular matrix components, which is conducive to the attachment, survival and function of DCs. At the same time, the damage-associated molecular patterns (DAMPs) released during the processing can serve as endogenous immune stimulation signals to further promote DCs maturation and antigen presentation. This material has a stable source, good biocompatibility, low immunogenicity and low manufacturing cost, and has good clinical translation prospects. It provides a new technical path for the immune prevention and control of postoperative tumor recurrence. Summary of the Invention

[0007] One of the purposes of the present invention is to provide an engineered autologous tumor tissue scaffold, characterized in that it is prepared from engineered autologous tumor tissue through a freezing and drying process; The engineering method is to process autologous tumor tissue to induce immunogenic cell death in the tumor tissue.

[0008] Furthermore, the engineering methods include but are not limited to photothermal therapy, photodynamic therapy, radiotherapy, ICD-inducible chemical drug treatment, genetic engineering treatment, immune stimulation treatment, or a combination of any two or more of the above methods.

[0009] The photothermal treatment is a method of irradiating tumor tissue with a laser of a specific wavelength (650 nm-1064 nm) containing a photothermal agent, wherein the photothermal agent (which can be IR780 iodine salt, gold nanorods, black phosphorus nanoparticles, polydopamine nanoparticles, indocyanine green ICG, etc.) absorbs light energy and converts it into thermal energy, raising the temperature of the tumor tissue to 42-55°C and maintaining it for at least 3 minutes.

[0010] Photodynamic therapy is a method that uses a light source with a specific wavelength (630 nm-808 nm) to irradiate tumor tissue that has been pre-incubated with a photosensitizer. The photosensitizer (Ce6, porphyrins, aluminum chloride phthalocyanine, or ICG-PEG, etc.) is excited to produce reactive oxygen molecules such as singlet oxygen, which causes oxidative damage to tumor cells. The power density is 50-100 mW / cm 2 , the irradiation time is 3-10 min.

[0011] Radiation therapy is a type of therapy that uses X-rays of specific energy (4-15 MeV) or 60 A method of Co γ-ray irradiation of tumor tissue, wherein ionizing radiation acts on tumor tissue at a dose rate of 200-600 cGy / min, with a single dose of 2-8 Gy (preferably 4 Gy) and a total dose of 4-20 Gy, inducing immunogenic cell death and tumor antigen release by directly or indirectly damaging DNA and cell structures. High atomic number sensitizers (such as Ta2O5, HfO2 or Bi2S3) can be optionally added to enhance the therapeutic effect.

[0012] In the present invention, there is no specific limitation on the engineering method, and any method known to those skilled in the art can be used to achieve the effect of immunogenic cell death in tumor tissue.

[0013] Furthermore, the freezing is to freeze the engineered autologous tumor tissue in an environment of -20°C to -196°C, preferably at -80°C or in liquid nitrogen; the drying process can be conventional tissue drying methods such as freeze drying.

[0014] Furthermore, the autologous tumor tissue is derived from primary or metastatic tumor tissue surgically removed from the same treatment subject, preferably from a solid tumor, such as melanoma, pancreatic cancer, breast cancer, lung cancer, colorectal cancer or glioma.

[0015] A second object of the present invention is to provide a method for preparing the above-mentioned engineered autologous tumor tissue scaffold, characterized in that it comprises the following steps: a) Obtaining autologous tumor tissue after surgical resection of the tumor; b) subjecting the autologous tumor tissue to immunogenic cell death treatment; c) freezing the autologous tumor tissue treated in step b); d) drying the autologous tumor tissue after the freezing treatment in step c) to obtain an engineered autologous tumor tissue scaffold.

[0016] A third object of the present invention is to provide the use of the above-mentioned engineered autologous tumor tissue scaffold in the preparation of drugs for tumor recurrence, metastasis control, postoperative adjuvant therapy or tumor vaccine development, preferably personalized tumor vaccines.

[0017] A fourth object of the present invention is to provide a tumor immunotherapy composition comprising the engineered autologous tumor tissue scaffold and dendritic cells.

[0018] Furthermore, the usage ratio of the engineered autologous tumor tissue scaffold and DCs is 1g of engineered autologous tumor tissue scaffold loading 1×10 5 to 1×10 8 Dendritic cells, preferably 1 g of engineered autologous tumor tissue scaffold loaded with 1×10 6 to 1×10 7 dendritic cells.

[0019] In a specific embodiment of the present invention, freshly excised tumor tissue is subjected to photothermal therapy and then sequentially frozen and dried to obtain a porous scaffold material, which is then used to load DCs to construct a personalized tumor vaccine.

[0020] The beneficial effects of the present invention are as follows: (1) The autologous tumor scaffold vaccine of the present invention provides more comprehensive tumor antigen information and an optimized immune microenvironment by embedding DCs into freeze-dried tumor tissue with complete tumor antigens and immune stimulatory factors, which helps to overcome the problem of unstable efficacy caused by tumor heterogeneity and differences in antigen expression.

[0021] (2) The porous structure of the freeze-dried tumor tissue scaffold helps maintain the high activity and functional stability of DCs, significantly extending their retention time in the body.

[0022] (3) The scaffold is made of autologous tumor tissue, which has good biocompatibility, reduces the risk of immune rejection, and ensures high safety. In addition, the freeze-drying process ensures the complete inactivation of tumor cells, avoiding the potential risk of tumor cell residue and recurrence.

[0023] (4) The process of combining freeze-dried scaffolds with DCs is simple, which reduces the preparation cost and improves the operability of DCs vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are images of hematoxylin-eosin (HE)-stained sections of pancreatic tumor tissue and melanoma tissue after freeze-drying in Example 1.

[0025] Figure 2 This is a scanning electron microscope (SEM) image of the freeze-dried tumor tissue loaded with DCs in Example 1.

[0026] Figure 3 This is a confocal laser microscope image of the freeze-dried tumor tissue loaded with DCs in Example 1.

[0027] Figure 4 This is a Western Blot image of the detection of calreticulin (CRT) expression level in Example 2.

[0028] Figure 5 This is an immunofluorescence section staining image for detecting the CRT expression level in Example 2.

[0029] Figure 6 These are the in vivo imaging results of freeze-dried tumor tissue before and after loading with DCs in Example 3.

[0030] Figure 7 This is a statistical graph showing the number of DCs released from freeze-dried tumor tissue in Example 3.

[0031] Figure 8 This is a fluorescence microscopy image of tumor cells released from freeze-dried tumor tissue in Example 4.

[0032] Figure 9 These are in vivo imaging images of tumor cells released or proliferating from freeze-dried tumor tissue in Example 4.

[0033] Figure 10 This is the in vivo imaging result of the retention of exogenous DCs in Example 5.

[0034] Figure 11 The figure shows the distribution of exogenous DCs in lymph nodes at different time points in Example 6. DETAILED DESCRIPTION

[0035] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Example 1

[0038] 1. Preparation of engineered autologous freeze-dried tumor tissue scaffolds 1.1 Obtaining autologous tumor tissue Melanoma (B16F10 cell line) and pancreatic cancer (PANC02 cell line) tumor cells were inoculated subcutaneously in C57BL / 6 mice. 3 Tumor resection surgery was performed under sterile conditions. After surgery, the obtained tumor tissue was thoroughly rinsed in sterile, ice-cold PBS buffer to remove blood and impurities, ensuring tissue sterility and purity. Fresh pancreatic tumor tissue and fresh melanoma tissue were designated as PFTT and BFTT, respectively.

[0039] 1.2 Engineering treatment In the present invention, engineering induction methods may include various types, such as photothermal therapy, photodynamic therapy, radiotherapy, ICD-inducible chemical drug treatment, genetic engineering treatment, or immune stimulation treatment. This embodiment uses photothermal treatment, photodynamic treatment, and radiotherapy as representative methods for illustration, and other engineering methods can also be performed using similar steps.

[0040] Photothermal therapy treatment: fresh tumor tissue is placed in a sterile environment, and 10 micrograms (μg / g) of IR780 solution is injected into the tissue evenly. After the injection is completed, a wavelength of 808 nm and a power density of 1.0 W / cm 2 Laser is used to irradiate tumor tissue to induce immunogenic cell death in the tissue.

[0041] Photodynamic therapy treatment: fresh tumor tissue was placed in a 10 μg / mL Ce6 solution and incubated at 37°C for 30 min to allow the photosensitizer to fully penetrate the tissue. Then, a 630 nm wavelength laser was used at 100 mW / cm 2 Power density irradiation for 5 minutes, excitation Ce6 produces singlet oxygen ( 1 O2) and other reactive oxygen species, which induce oxidative stress and death of tumor cells.

[0042] Radiotherapy treatment: Fresh tumor tissue is spread flat on an irradiation tray and irradiated with 60Co γ rays at a dose rate of 300 cGy / min and a single irradiation dose of 6 Gy, which directly induces DNA double-strand breaks and oxidative damage, inducing the release of ICD-related signals.

[0043] The engineered tissues (denoted as iFTT) were obtained, among which the irradiated tissues derived from pancreatic cancer were denoted as iPFTT, and those derived from melanoma were denoted as iBFTT.

[0044] 1.3 Freeze-drying After photothermal engineering, the tissue was immediately transferred to a −80°C freezer and frozen overnight. Once completely frozen, the tissue was placed in a freeze dryer and lyophilized under vacuum (less than 20 Pa) for 12–24 hours to obtain a photoengineered, freeze-dried tumor tissue scaffold. The pancreatic cancer-derived scaffold was designated iPLTT, and the melanoma-derived scaffold was designated iBLTT, collectively referred to as iLTT.

[0045] Fresh tumor tissue without light-induced treatment was directly frozen at -80 °C overnight and then freeze-dried under the same conditions. The resulting scaffold was designated as LTT and used as a control group.

[0046] Observation through HE sections, such as Figure 1 As shown in the figure, compared with fresh tumor tissue, both photoengineered freeze-dried pancreatic tumor tissue (iPLTT) and photoengineered freeze-dried melanoma tissue (iBLTT) exhibited a porous structure, which was more conducive to the subsequent loading of DCs.

[0047] 2. Preparation and loading of DCs 2.1 Acquisition and culture of DCs To isolate and culture DCs, 6–8-week-old C57BL / 6 mice were euthanized by cervical dislocation and rapidly dissected to obtain the femur and tibia. RPMI-1640 medium supplemented with 2% FBS, 100 U / mL penicillin G sodium, and 100 μg / mL streptomycin was injected into the bone marrow cavity using a syringe to flush and collect bone marrow cells. A total of 40 mL of medium was used for each femur and tibia.

[0048] The harvested bone marrow cells were centrifuged at 800 g for 5 minutes at room temperature. The supernatant was discarded and the cells were resuspended in 1 mL of ACK erythrocyte lysis buffer and incubated at room temperature for 90 seconds to lyse the erythrocytes. Subsequently, 3 mL of sterile PBS was added to terminate the lysis reaction, and the cell suspension was filtered through a 70 μm cell strainer. The cells were centrifuged again at 800 g for 5 minutes, the supernatant discarded, and the cells were resuspended in 12 mL of RPMI-1640 medium containing 10% FBS, 100 U / mL penicillin G sodium, 100 μg / mL streptomycin, 1% HEPES buffer, 0.05 mM β-mercaptoethanol (β-ME), 20 ng / mL interleukin-4 (IL-4), and 20 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF). The cells were then aliquoted into six wells of a six-well plate.

[0049] During culture, replace half of the culture medium with fresh medium every 2–3 days to maintain optimal cell growth. On day 6, collect the non-adherent cells (i.e., mature DCs) from the culture supernatant by centrifugation at 800 g for 5 minutes at room temperature. Discard the supernatant and resuspend the DCs in 10 mL of RPMI 1640 medium supplemented with 10% FBS, 100 U / mL penicillin G sodium, and 100 μg / mL streptomycin.

[0050] 2.2 Loading of DCs Under sterile conditions, DCs were collected by centrifugation and accurately counted using a cell counter. 5 × 10 6 Each DC was resuspended in 100 μL of PBS to prepare a DC suspension. Using a syringe, 20 μL of the cell suspension was evenly injected into approximately 200 mg of iPLTT and iBLTT, respectively, to create DC-loaded photoengineered freeze-dried tumor tissues (DC@iPLTT and DC@iBLTT). The entire procedure was performed on ice to maintain high cell viability and functional stability.

[0051] like Figure 2 and Figure 3 As shown, biological scanning electron microscopy and confocal microscopy observations together demonstrated the successful loading of DCs by photoengineered freeze-dried tumor tissues (iPLTT and iBLTT). Example 2

[0052] Evaluation of the effect of freeze-dried tumor tissue engineering Fresh pancreatic tumor tissue (PFTT) and photoengineered fresh pancreatic tumor tissue (iPFTT) were sheared, ground, and centrifuged to create single-cell suspensions. Cell membrane components were then extracted from the single-cell suspensions using sonication combined with gradient centrifugation. Simultaneously, protein was extracted from photoengineered freeze-dried pancreatic cancer tissue (iPLTT) using tissue grinding.

[0053] Western blot analysis was then performed to examine the expression levels of CRT in the PFTT, iPFTT, and iPLTT samples, with ATP1A1 used as an internal reference protein for quantitative analysis. Furthermore, immunofluorescence staining was performed to co-stain CRT (green fluorescence) and ATP1A1 (red fluorescence) in the PFTT, iPFTT, and iPLTT samples to visually assess CRT expression and localization.

[0054] like Figure 4 and Figure 5As shown in the results, CRT was significantly translocated to the cell membrane surface in tumor tissues treated with light. This phenomenon is particularly important in the process of immunogenic cell death, as the membrane epitope of CRT is a key marker of ICD, effectively promoting the recognition and uptake of tumor antigens by DCs. Furthermore, Western blotting and immunofluorescence staining experiments further verified that the overall expression level of CRT in tumor tissues did not change significantly before and after freeze-drying, indicating that the freeze-drying process has good stability in retaining CRT expression and its membrane localization. Example 3

[0055] Efficiency and in vitro release assay of DCs loaded with freeze-dried tumor tissue The loading efficiency of DCs was analyzed by in vivo imaging experiments. The specific steps were as follows: First, 1×10 6 DCs were labeled to track and quantify the amount of DCs in subsequent experiments. 6 DCs were uniformly injected into 200 mg of photoengineered freeze-dried tumor tissue (iPLTT and iBLTT) placed in a 48-well plate and incubated at room temperature for 5 minutes to allow the DCs to fully bind to the freeze-dried tumor scaffold. After incubation, DC@iPLTT and DC@iBLTT were obtained. Subsequently, fluorescence intensity in the original wells was measured using an in vivo imaging system. By comparing the changes in fluorescence intensity in each well before and after transfer, the number of DCs leaking from the freeze-dried tumor tissue could be quantified.

[0056] like Figure 6 As shown, quantitative analysis of fluorescence intensity revealed that the DC loading efficiency of photoengineered freeze-dried melanoma tissue was 58.0%, while the DC loading efficiency of photoengineered freeze-dried pancreatic cancer tissue reached 74.7%. This finding suggests that different types of tumor tissues exhibit significant differences in their ability to adsorb and retain DCs during the freeze-drying process. This may be due to the unique tissue structure and extracellular matrix composition of pancreatic cancer tissue, which enables more efficient embedding and retention of DCs, resulting in higher loading efficiency. In contrast, the relatively low loading efficiency of melanoma tissue may be related to its cell density, matrix composition, or the pore structure of the freeze-dried scaffold.

[0057] 1×10 6DCs were seeded into 200 mg of photoengineered, freeze-dried tumor tissue to create DC@iPLTT and DC@iBLTT. These cells were then inserted into 70 μm pore cell strainers. The cell strainers were then used as transwell inserts in 6-well plates, with complete DC culture medium at the bottom. At designated time points, the cell strainers containing freeze-dried tumor tissue were transferred to new wells containing fresh culture medium, and the number of cells in the lower chamber was accurately counted using an automated cell counter.

[0058] like Figure 7 As shown, DCs in DC@iBLTT release faster than those in DC@iPLTT. This may be due to the larger pore size of the photoengineered freeze-dried melanoma tissue. The larger pore size facilitates a more uniform distribution of DCs within the scaffold and reduces physical barriers between cells, thereby accelerating cell migration and release. In contrast, the smaller pore size of the pancreatic cancer tissue scaffold may limit the free migration and release rate of DCs, causing them to remain within the scaffold for a longer period of time. This pore size difference has a significant impact on the release kinetics of DCs. Example 4

[0059] Safety testing of autologous tumor stent vaccines Fresh tumor tissue was sequentially photoengineered and lyophilized to obtain photoengineered tumor tissue (iPLTT, iBLTT) and photoengineered lyophilized tumor tissue (iPLTT, iBLTT). These tissues were then inserted into 70 μm pore-size cell strainers. The cell strainers were then placed as transwell inserts in the upper chamber of a six-well plate. Complete tumor cell culture medium was pre-filled into the lower chamber of the six-well plate. At designated time points, the cell strainers loaded with tumor tissue (iPFTT, iBFTT, iPLTT, iBLTT) were transferred to new wells containing fresh culture medium. Cells in the lower chamber were fluorescently stained with calcein and imaged using fluorescence microscopy to assess the residual tumor cells in the lyophilized tumor tissue.

[0060] like Figure 8 As shown, fluorescence microscopy results revealed no tumor cells in the freeze-dried tumor tissue groups (iPLTT and iBLTT) on days 0, 1, 2, and 4. Specifically, at the initial freeze-drying stage (day 0), the day after treatment, no active tumor cells remained in the freeze-dried tumor tissue, demonstrating the instantaneous effectiveness of the freeze-drying process in killing tumor cells. Continuous fluorescence microscopy observations on days 1, 2, and 4 further verified the durability of this inactivation effect, ensuring the safety and stability of the tumor tissue in subsequent applications.

[0061] At the same time, pancreatic cancer (PANC02-luc) and melanoma (B16F10-luc) tumor cells expressing luciferase (luc) were used to construct tumor models. The specific steps are as follows: First, PANC02-luc or B16F10-luc tumor cells were injected subcutaneously into mice to induce tumor formation. When the tumor reached 150-200 mm 3 After that, surgical resection was performed, and the resected tumor tissue was photoengineered and freeze-dried according to the conditions. The treated tumor tissue was divided into two groups: photoengineered fresh tumor tissue (iPFTT and iBFTT) and photoengineered freeze-dried tumor tissue (iPLTT and iBLTT). Subsequently, iPFTT, iBFTT, iPLTT and iBLTT were inoculated back into the subcutaneous tissue of mice to evaluate the effect of freeze-drying on the viability of tumor cells. After inoculation, the luc fluorescence expression in the mice was regularly detected by the in vivo imaging system to monitor the presence or absence of living tumor cells in real time. On the 0th, 5th and 10th day after inoculation, the mice were subjected to fluorescence detection using an in vivo imager, and the changes in fluorescence intensity at each time point were recorded. As Figure 9 As shown, no significant luc fluorescence signals were detected in the photoengineered freeze-dried tumor tissue groups (iPLTT and iBLTT) at each time point (day 0, day 5, and day 10), indicating that the freeze-drying treatment effectively inactivated tumor cells and prevented their recurrence and metastasis in the body; in contrast, the photoengineered fresh tumor tissue group was able to continuously express luc fluorescence after inoculation, verifying the effectiveness of freeze-drying in inactivating tumor cells.

[0062] These results not only validate the reliability of freeze-drying technology in inactivating tumor cells but also provide a solid foundation for the subsequent use of freeze-dried tumor tissue as a bioactive scaffold carrier. Because freeze-drying effectively eliminates active tumor cells, the prepared freeze-dried tumor tissue can be safely used to load DCs without worrying about potential tumor cell reactivation or spread. Example 5

[0063] Detection of the in vivo retention time of DCs in freeze-dried tumor tissue In vivo DC retention was visualized and its average retention half-life was quantified by in vivo imaging. The specific steps were as follows: First, DCs were labeled with CMTPX fluorescent dye to track their distribution and migration in real time during in vivo imaging. 6Each labeled DC was inoculated into 200 mg of photoengineered, freeze-dried pancreatic tumor tissue and photoengineered, freeze-dried melanoma tissue, respectively, to create DC-loaded freeze-dried tumor scaffolds (DC@iPLTT and DC@iBLTT). Following inoculation, the DC-loaded freeze-dried tumor tissue was implanted into experimental animal models. Fluorescence images and quantitative analysis were performed using an in vivo imaging device on days 0, 1, 4, 7, and 14 after inoculation. The average in vivo retention half-life of the DCs was calculated by measuring changes in fluorescence intensity at each time point.

[0064] like Figure 10 As shown, the fluorescence signal in the free DCs group was almost undetectable on day 7 after inoculation, indicating a short in vivo retention time. In contrast, significant fluorescence signals were detected in the photoengineered freeze-dried pancreatic tumor (DC@iPLTT) and photoengineered freeze-dried melanoma (DC@iBLTT) groups at all time points (days 0, 1, 4, 7, and 14), demonstrating that the freeze-dried scaffolds significantly prolonged the retention time of DCs. Furthermore, the fluorescence intensity of the DC@iPLTT group was significantly higher than that of the DC@iBLTT group, indicating that the former was more efficient in supporting DC retention. Analysis of the fluorescence intensity data revealed that the average retention half-life of each group was 2.19 days for the free DCs group, 4.65 days for the freeze-dried melanoma tissue group, and 6.24 days for the freeze-dried pancreatic cancer tissue group. These results demonstrate that freeze-drying significantly prolongs the in vivo retention time of DCs, particularly in the pancreatic cancer tissue scaffolds, where DC retention efficiency was higher. Example 6

[0065] Lymph node migration ability assay Three treatment groups were implanted subcutaneously near the lymph nodes of C57BL / 6J mice: free DCs group (DC+Lysate pre + LPS pre ), freeze-dried pancreatic tumor tissue group loaded with DCs (DC@PLTT), and photo-engineered freeze-dried pancreatic tumor tissue group loaded with DCs (DC@iPLTT). Among them, the DCs in the free DCs group were incubated in vitro with cell lysate (1 mg / mL) extracted from freeze-thawed pancreatic tumor tissue and lipopolysaccharide (LPS, 100 ng / mL) in advance to induce their maturation. DCs were fluorescently labeled with CMTPX. The free DCs group only injected pre-treated free DCs subcutaneously; while the DC@PLTT group and DC@iPLTT group injected 1×10 6DCs were injected into 200 mg of freeze-dried pancreatic tumor tissue. The inguinal lymph nodes of the mice were collected on days 3, 7, and 14 after implantation, and fluorescence imaging was performed using a live imaging system to characterize the distribution of DCs in the lymph nodes at different time points. like Figure 11 As shown, the fluorescence signal of the free DCs group in the lymph nodes was weak and significantly weakened over time, suggesting that free DCs have a short residence time in vivo. DC@PLTT exhibited strong fluorescence signals on days 3 and 7, but the signal significantly weakened by day 14. In contrast, DC@iPLTT displayed a strong and sustained fluorescence signal throughout the observation period, reaching a peak on day 7 and remaining at a high level on day 14. This indicates that DCs loaded on freeze-dried tumor tissue scaffolds have superior migration properties in vivo compared to free DCs.

Claims

1. An engineered autologous tumor tissue scaffold, characterized in that: Prepared from engineered autologous tumor tissue through freezing and drying; The engineering method is to process autologous tumor tissue to induce immunogenic cell death in the tumor tissue.

2. The engineered autologous tumor tissue scaffold according to claim 1, characterized in that: The engineering treatment methods include but are not limited to photothermal therapy, photodynamic therapy, radiotherapy, ICD-inducible chemical drug treatment, genetic engineering treatment, immune stimulation treatment, or a combination of any two or more of the above methods.

3. The engineered autologous tumor tissue scaffold according to claim 1, characterized in that: The freezing process is performed by placing the engineered autologous tumor tissue in a low-temperature environment, and the temperature is preferably -20°C to -196°C.

4. The engineered autologous tumor tissue scaffold according to claim 1, characterized in that: The autologous tumor tissue is derived from primary tumor tissue or metastatic tumor tissue surgically removed from the same treatment subject.

5. The engineered autologous tumor tissue scaffold according to claim 1, characterized in that: The tumor tissue is selected from solid tumors, including but not limited to melanoma, pancreatic cancer, breast cancer, lung cancer, colorectal cancer or glioma.

6. A method for preparing the engineered autologous tumor tissue scaffold according to any one of claims 1 to 5, characterized in that: The following steps are involved: a) obtaining autologous tumor tissue after surgical resection of the tumor; b) subjecting the autologous tumor tissue to immunogenic cell death treatment; c) freezing the autologous tumor tissue treated in step b); d) drying the autologous tumor tissue after the freezing treatment in step c) to obtain an engineered autologous tumor tissue scaffold.

7. Use of the engineered autologous tumor tissue scaffold according to any one of claims 1 to 5 in the preparation of a drug for tumor recurrence, metastasis control, postoperative adjuvant therapy or tumor vaccine development.

8. A tumor immunotherapy composition, characterized in that: The invention comprises the engineered autologous tumor tissue scaffold and dendritic cells according to any one of claims 1 to 5.

9. The tumor immunotherapy composition according to claim 8, characterized in that The usage ratio of the engineered autologous tumor tissue scaffold to the dendritic cells is 1g of the engineered autologous tumor tissue scaffold loading 1×10 5 to 1×10 8 Dendritic cells.

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