Tumor response evaluation system and application of single-cell transcriptome sequencing in tumor response evaluation

Abstract

This invention relates to a tumor efficacy evaluation system and the application of single-cell transcriptome sequencing in tumor efficacy evaluation. The tumor efficacy evaluation system includes: a sample acquisition module for acquiring paraffin-embedded samples of lesions at different stages from the same patient being evaluated, the paraffin samples containing tumor tissue; an information acquisition module for acquiring corresponding tumor microenvironment information based on each paraffin sample, the tumor microenvironment information including cell type-related information; and an analysis module for analyzing and comparing the tumor microenvironment information to obtain cellular information for tumor efficacy evaluation. The tumor efficacy evaluation is realistic, accurate, and comprehensive. This invention also discloses the application of single-cell transcriptome sequencing in tumor efficacy evaluation.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a tumor efficacy evaluation system and the application of single-cell transcriptome sequencing in tumor efficacy evaluation. Background Technology

[0002] Cancer efficacy refers to the effect of medical interventions on tumor growth, spread, and improvement of patient symptoms. Assessing cancer efficacy is crucial for guiding clinical treatment, predicting patient prognosis, designing clinical trials, and achieving personalized treatment. It enables physicians to quantitatively monitor the tumor's response to treatment, thereby determining the effectiveness of treatment plans and adjusting treatment strategies in a timely manner to improve outcomes. Simultaneously, efficacy assessment helps predict long-term survival and quality of life, providing patients with more accurate prognostic information. Furthermore, in the development of new drugs or therapies, efficacy assessment is a key indicator for measuring their safety and effectiveness, and is essential for drug approval and clinical application.

[0003] With the development of medical imaging technology, molecular biology, and information technology, methods for evaluating the efficacy of tumor treatment are becoming increasingly diversified and precise. This plays an important role in improving the targeting and effectiveness of treatment and achieving precision medicine.

[0004] Currently, commonly used methods for evaluating the efficacy of tumor treatment include imaging examinations, tumor marker detection, RECIST criteria, and immunohistochemical and molecular biological tests.

[0005] Among these, imaging examinations are the most widely used, as they can display the size and location of tumors in detail and monitor changes in the tumor. CT scans are relatively quick, but may produce artifacts or false negative / false positive results, and carry radiation risks; MRI offers high resolution for soft tissues, but is expensive and time-consuming; ultrasound is radiation-free and inexpensive, but has poor resolution for deep tumors. PET scans can assess the metabolic activity of tumors and help detect small lesions, but are expensive and time-consuming.

[0006] Meanwhile, the advantages and disadvantages of other assessment methods are also quite apparent. Specifically, tumor marker testing assesses treatment efficacy by measuring changes in marker levels in blood or body fluids; it is simple and non-invasive, but the specificity of the markers may be insufficient, and their levels may be affected by other factors. The RECIST criteria are an objective and standardized assessment method that evaluates treatment effectiveness by measuring changes in the size of tumor lesions, but its applicability to certain tumor types is limited. Immunohistochemistry and molecular biology testing can provide molecular characterization information of tumor cells, helping to understand the biological characteristics of tumors, but they are usually used as supplementary methods, and are complex and costly.

[0007] In addition, in translational medicine research, there are also methods that use unpaired fresh or frozen samples from different patients before and after treatment for sequencing. The drawback of this method is that the tumors of patients are highly heterogeneous, and the samples before and after treatment cannot truly reflect the differences in the tumor microenvironment before and after treatment, thus making it impossible to accurately evaluate the efficacy.

[0008] Therefore, it is necessary to provide a tumor efficacy evaluation system to quickly and accurately assess tumor efficacy. Summary of the Invention

[0009] The first objective of this invention is to provide a tumor efficacy evaluation system, comprising: a sample acquisition module for acquiring paraffin samples of lesions at different stages of the same subject to be evaluated, wherein the paraffin samples contain tumor tissue; an information acquisition module for acquiring corresponding tumor microenvironment information based on each paraffin sample, wherein the tumor microenvironment information includes cell type-related information; and an analysis module for analyzing and comparing the tumor microenvironment information to obtain cell information for tumor efficacy evaluation.

[0010] Furthermore, the lesions are lesions in the same location, the paraffin samples are samples fixed in formalin and processed by paraffin embedding technology, and include paraffin samples when the lesions are initially diagnosed as cancer or tumors and paraffin samples after treatment. The cell type related information includes cell types and the percentage of each cell type.

[0011] Furthermore, the paraffin samples include paraffin samples of thyroid cancer that were initially diagnosed as thyroid cancer and surgically removed, and paraffin samples of thyroid cancer that recurred and were surgically removed.

[0012] Furthermore, the lesion is papillary thyroid carcinoma, and the thickness of the paraffin sample is 5-100 μm, preferably 10-20 μm.

[0013] Furthermore, the paraffin samples include paraffin samples obtained by needle aspiration biopsy after the lesion is initially diagnosed as pancreatic cancer and the lesion is surgically removed after treatment measures have been implemented and the lesion has reached the criteria for resectable surgery. The tumor microenvironment information also includes at least one of cell subpopulation information and differential expression information.

[0014] Furthermore, the lesion is an advanced and unresectable pancreatic cancer initially diagnosed, and the treatment measures include at least one of surgery, electric field therapy, and chemotherapy. The thickness of the paraffin sample is 5-100 μm, preferably 10-50 μm.

[0015] Furthermore, the treatment measure is a combined treatment measure of electric field therapy by applying an alternating current signal through a tumor electric field therapy device and chemotherapy by administering chemical drugs. The alternating current signal has a frequency of 150 kHz and an electric field strength of at least 1 V / cm. The chemical drugs include albumin-bound paclitaxel and gemcitabine. The chemical drugs are administered on day 1, day 8 and day 15 of each treatment cycle, respectively. The gemcitabine is administered immediately after the albumin-bound paclitaxel is administered.

[0016] Furthermore, the information acquisition module further includes: a data acquisition unit, which is used to acquire single-cell transcriptome data based on the paraffin sample; and a data analysis unit, which is used to confirm tumor microenvironment information using the single-cell transcriptome data.

[0017] Furthermore, the data acquisition unit obtains single-cell transcriptome data through the following method: after preparing the paraffin sample into a single-cell suspension, the single cells are dissociated and fixed; an in situ reverse transcription reaction is performed on the RNA of the fixed single cells using reverse transcription primers to synthesize the first strand of cDNA; a corresponding capture adapter is added to the tail of the first strand of cDNA, the capture adapter being sequence-complementary to the capture adapter complementary strand on the coding microsphere; the single cell and a single coding microsphere are contained in a single chamber to form a cell separator, the single-stranded DNA on the coding microsphere containing the upstream amplification primer complementary fragment, barcode, UMI, and capture adapter complementary strand; a second strand of cDNA is synthesized in the single chamber, and the double-stranded cDNA is subjected to PCR amplification, library construction, and sequencing.

[0018] This invention also provides an application of single-cell transcriptome sequencing in tumor efficacy evaluation, wherein tumor efficacy is evaluated using the aforementioned tumor efficacy evaluation system.

[0019] The tumor efficacy evaluation system of this invention uses single-cell transcriptome sequencing to analyze paraffin samples of lesions from subjects to be evaluated, which are easily obtained and traced back. It can obtain tumor microenvironment information of lesions in a real, accurate and comprehensive manner, thereby completing the tumor efficacy evaluation. It is convenient and does not bring additional risks to the subjects to be evaluated compared with the current method of evaluating efficacy through imaging examinations. It can also be applied to the efficacy evaluation of different types of cancer. Attached Figure Description

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

[0021] Figure 1 This is a schematic diagram of the framework of the tumor efficacy evaluation system of the present invention.

[0022] Figure 2 This is a schematic diagram of the process for evaluating the efficacy of tumor treatment using the tumor efficacy evaluation system of the present invention.

[0023] Figure 3 These are CT scan images of pancreatic cancer at different stages in one embodiment of the present invention; wherein, Figure 3 In the image, A represents the CT scan image of the patient initially diagnosed with advanced and unresectable pancreatic cancer. Figure 3 B in the image is a CT scan image of the subject under evaluation after two treatment cycles of electric field therapy combined with chemotherapy. Figure 3 In the image, C represents the CT scan image of the subject before surgery. Figure 3 In the image, D represents the CT scan image of the subject being evaluated one month after surgery; Figure 3 A in Figure 3 The red symbol in the C indicates the location of the tumor, and Figure 3 The CT scan image in C before surgery is also the CT scan image of the subject under evaluation after 5 treatment cycles of electric field therapy combined with chemotherapy.

[0024] Figure 4 for Figure 3 Pathological analysis images of pancreatic cancer at different stages in subjects to be evaluated, reflecting the levels of the CA-19-9 biomarker at different stages; among them, Figure 4 In the figure, A is the pathological analysis diagram of the pancreatic cancer sample obtained by needle aspiration biopsy of the subject at the initial diagnosis and processed by formalin fixation and paraffin embedding (FFPE sample); Figure 4 B in the figure represents the pathological analysis of the pancreatic cancer sample obtained by needle aspiration biopsy and processed by formalin fixation and paraffin embedding (FFPE sample) of the subject under evaluation after 5 treatment cycles of combined electric field therapy and chemotherapy to achieve the criteria for surgical resection. Figure 4 C in the figure represents the pathological analysis of a pancreatic cancer biopsy obtained from the subject under evaluation after receiving combined electric field therapy and chemotherapy and meeting the criteria for surgical resection (FFPE sample), which was then processed using formalin fixation and paraffin embedding techniques.

[0025] Figure 5A This is a split UMAP projection map of data obtained from single-cell transcriptome sequencing (scRNA-seq) of FFPE pancreatic cancer samples from subjects to be evaluated before receiving treatment.

[0026] Figure 5B This is a split UMAP projection map obtained from single-cell transcriptome sequencing (scRNA-seq) of FFPE samples of pancreatic cancer from subjects who underwent treatment and surgical resection.

[0027] Figure 5C This is a graph showing the percentage of each cell type obtained from single-cell transcriptome sequencing (scRNA-seq) of FFPE samples of pancreatic cancer in the subjects under evaluation before treatment and FFPE samples of pancreatic cancer after treatment and surgical resection.

[0028] Figure 5D The divisional UMAP projections of epithelial cells were obtained by single-cell transcriptome sequencing (scRNA-seq) of FFPE samples of pancreatic cancer before treatment and FFPE samples of pancreatic cancer after treatment and surgical resection.

[0029] Figure 5E To integrate Figure 5D The UMAP projection of the mid-epithelial cells shows the different subpopulations of epithelial cells.

[0030] Figure 5F In response to Figure 5E A dot plot showing the expression levels of the top 5 marker genes in each subpopulation of epithelial cells.

[0031] Figure 6A This is a split UMAP projection map of data obtained from single-cell transcriptome sequencing (scRNA-seq) of FFPE samples from subjects initially diagnosed with thyroid cancer and who underwent surgical removal of the thyroid cancer.

[0032] Figure 6B This is a split UMAP projection map of data obtained from single-cell transcriptome sequencing (scRNA-seq) of FFPE samples of thyroid cancer in the subject of evaluation after recurrence and surgical resection.

[0033] Figure 6C This is a graph showing the percentage of each cell type obtained from single-cell transcriptome sequencing (scRNA-seq) of FFPE samples of thyroid cancer initially diagnosed and surgically removed, and FFPE samples of thyroid cancer after recurrence and surgical removal. Detailed Implementation

[0034] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the invention. Those skilled in the art can make various modifications and variations to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment can be used in another embodiment to produce further embodiments.

[0035] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0036] In this invention, the term "subject to evaluation" refers to a patient whose tumor treatment efficacy needs to be evaluated. These individuals are receiving or have received treatment, and the effectiveness of the treatment needs to be assessed. In some embodiments, the subject to evaluation is a human (Homo sapiens). In other embodiments, the subject to evaluation is a mammal such as a dog (Canis lupus familiaris), cat (Felis catus), horse (Equus ferus caballus), rabbit (Oryctolagus cuniculus), sheep (Ovis aries), mouse (Mus musculus), or rat (Rattus norvegicus).

[0037] In this invention, "tumor microenvironment information" refers to information about the tumor microenvironment (TME), which involves various components in the microenvironment and their interactions. In some embodiments, the tumor microenvironment information includes one or more of the following aspects: (1) Cell component information: i) Tumor cell information: including the type, characteristics, and variations of the main tumor cells; ii) Immune cell information: the number, distribution, and functional status of different types of immune cells (such as T cells, B cells, macrophages, etc.) in the tumor tissue; iii) Fibroblast and myofibroblast information: their role and distribution in the tumor matrix; iv) Vascular endothelial cell information: the characteristics and status of tumor angiogenesis; etc. (2) Extracellular matrix (ECM) information: such as the components of the extracellular matrix. (3) Molecular and signaling pathway information: i) Growth factor information: such as the level and function of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF); ii) Cytokine and chemokine information: such as the expression and function of tumor necrosis factor (TNF) and interleukin (IL); iii) Signaling pathway information: the activation status of the major signaling pathways involved (such as PI3K / Akt, MAPK / ERK, etc.). (4) Metabolic characteristic information: i) pH information: pH changes in tumor tissue and their effects; ii) Oxygen level information: oxygen level in tumor tissue and its effect on tumor development. (5) Spatial organization information of the microenvironment: i) Cell localization information: spatial distribution of different cell types in tumor tissue; ii) Tissue structure information: how the spatial structure of the tumor microenvironment affects tumor behavior and treatment efficacy.

[0038] In this invention, the term "paraffin-embedded samples" refers to tissue samples processed using paraffin embedding technology.

[0039] In this invention, the term "FFPE sample (Formalin-Fixed, Paraffin-Embedded Samples)" refers to tissue samples processed by formalin fixation and paraffin embedding techniques.

[0040] In this invention, the term "single cell" includes, but is not limited to, a single cell and a single cell nucleus.

[0041] In this invention, the term "single-cell transcriptome data" refers to detailed data about each single-cell transcriptome obtained through single-cell RNA sequencing (scRNA-seq) technology. In some embodiments, the single-cell transcriptome data includes one or more of gene expression matrices, cellular heterogeneity, and cell types.

[0042] Combination Figure 1 As shown, the tumor efficacy evaluation system provided by this invention includes a sample acquisition module for acquiring paraffin samples of lesions at different stages of the same subject to be evaluated, an information acquisition module for obtaining corresponding tumor microenvironment information based on each paraffin sample acquired by the sample acquisition module, and an analysis module for analyzing and comparing the tumor microenvironment information acquired by the information acquisition module to obtain cellular information for tumor efficacy evaluation. The paraffin samples contain tumor tissue and are samples fixed in formalin and treated with paraffin embedding technology (FFPE samples).

[0043] In some implementations, the lesions are located in the same site. Paraffin samples include those taken when the lesion is initially diagnosed as cancer or a tumor, as well as those taken after treatment.

[0044] In some embodiments, the lesion is located in the thyroid gland, and the paraffin sample includes paraffin samples of lesions initially diagnosed as thyroid cancer and surgically removed, and paraffin samples of thyroid cancer that has recurred and been surgically removed. More specifically, the lesion is papillary thyroid carcinoma, and the thickness of the paraffin sample is 5-100 μm, preferably 10-20 μm, and more preferably 20 μm. Cell type-related information includes cell types and the percentage of each cell type.

[0045] In some embodiments, the lesion is located in the pancreas, and the paraffin sample includes a paraffin sample obtained by needle aspiration biopsy of the lesion after initial diagnosis of pancreatic cancer, and a paraffin sample obtained by surgical resection biopsy of the lesion after treatment to meet the criteria for resectable surgery. Specifically, the lesion is initially diagnosed as advanced and unresectable pancreatic cancer. The thickness of the paraffin sample is 5-100 μm, preferably 10-50 μm, and more preferably 50 μm.

[0046] In some embodiments, the treatment includes at least one of surgery, electric field therapy, and chemotherapy. More specifically, the treatment is a combination of electric field therapy (applying an alternating current signal via a tumor electric field therapy device) and chemotherapy with the administration of a chemical drug. The alternating current signal has a frequency of 150 kHz and an electric field strength of at least 1 V / cm. The chemical drug includes albumin-bound paclitaxel and gemcitabine. The chemical drug is administered on days 1, 8, and 15 of each treatment cycle, respectively, with gemcitabine administered immediately after albumin-bound paclitaxel.

[0047] In some implementations, tumor microenvironment information also includes at least one of cell subpopulation information and differential expression information.

[0048] In some implementations, the treatment may include at least one of electric field therapy, chemotherapy, immunotherapy, targeted therapy, radiotherapy, surgical treatment, stem cell transplantation, and vaccine therapy. Specifically, the treatment may include at least one of electric field therapy and chemotherapy. More specifically, the treatment may be electric field therapy combined with chemotherapy.

[0049] In some implementations, the paraffin sample taken when the lesion is initially diagnosed as cancer or tumor is a paraffin sample obtained after surgical removal of the lesion that was initially diagnosed as cancer or tumor.

[0050] In some implementations, the paraffin sample at the initial diagnosis of cancer or tumor specifically refers to a paraffin sample where the lesion was initially diagnosed as cancer or tumor and had not received any anticancer or antitumor treatment; that is, a paraffin sample where the lesion was before treatment was initiated. Specifically, the lesion is located in the pancreas. The tumor tissue may be advanced and unresectable pancreatic cancer.

[0051] In some implementations, the information acquisition module obtains tumor microenvironment information by processing FFPE samples of lesions before and after treatment using single-cell transcriptome sequencing technology.

[0052] In some implementations, the information acquisition module further includes: a data acquisition unit for acquiring corresponding single-cell transcriptome data based on each paraffin sample; and a data analysis unit for using the acquired single-cell transcriptome data to confirm tumor microenvironment information.

[0053] In some embodiments, the data acquisition unit obtains single-cell transcriptome data by: preparing a paraffin sample into a single-cell suspension, dissociating and fixing the single cells; using reverse transcription primers to perform in situ reverse transcription on the RNA of the fixed single cells to synthesize a first strand of cDNA; adding a corresponding capture adapter to the tail of the first strand of cDNA, the capture adapter being sequence-complementary to the capture adapter complementary strand on the coding microsphere; containing the single cell and a single coding microsphere in a single chamber to form a cell separator, the single-stranded DNA on the coding microsphere containing an upstream amplification primer complementary fragment, a barcode, a unique multiplex index (UMI), and a capture adapter complementary strand; synthesizing a second strand of cDNA in the single chamber, and performing PCR amplification, library construction, and sequencing on the double-stranded cDNA.

[0054] In some implementations, the reverse transcription primer includes a tag sequence and an RNA-binding sequence, wherein the RNA-binding sequence is a random RNA-binding sequence, an RNA-binding sequence designed for a target RNA sequence, or a combination thereof.

[0055] In some embodiments, the method further includes permeabilizing the fixed single cells and sealing the exposed single-stranded DNA into double-stranded DNA after fixing the single cells and before performing the in situ reverse transcription reaction.

[0056] In some specific implementations, the treatment measures are selected from one or more of surgery, radiotherapy, chemotherapy, and electric field therapy.

[0057] In some implementations, the tumors mentioned in the tumor efficacy evaluation system are selected from one or more of pancreatic cancer, thyroid cancer, melanoma, non-small cell lung cancer (NSCLC), breast cancer, colorectal cancer, ovarian cancer, prostate cancer, gastric cancer, hepatocellular carcinoma, bladder cancer, soft tissue sarcoma, multiple myeloma, glioma, and cholangiocarcinoma.

[0058] Combination Figure 2 As shown, the tumor efficacy evaluation method provided by the present invention includes: obtaining paraffin samples of lesions of the same subject at different stages, wherein the paraffin samples contain tumor tissue; obtaining corresponding tumor microenvironment information based on each paraffin sample, wherein the tumor microenvironment information includes cell type-related information; and analyzing and comparing the tumor microenvironment information to obtain cell information for tumor efficacy evaluation.

[0059] This invention analyzes and compares tumor microenvironment information in paraffin samples of lesions at different stages of the subject being evaluated, which can reflect the cellular information related to tumor efficacy in a more realistic, objective and comprehensive way. The samples used for analysis are also relatively easy to obtain, and the evaluation does not involve radiation operations, so it will not bring additional risks to the subject being evaluated. It is also applicable to the efficacy evaluation of various types of tumors.

[0060] In some embodiments, the paraffin sample is an FFPE sample.

[0061] In some embodiments, the paraffin sample has a thickness that facilitates the acquisition of tumor microenvironment information, which can be 5–100 μm, preferably 10–50 μm or 10–20 μm. Specifically, the paraffin sample contains pancreatic cancer and has a thickness of 50 μm, or the paraffin sample contains pancreatic cancer and has a thickness of 20 μm.

[0062] In some embodiments, obtaining the corresponding tumor microenvironment information based on each paraffin sample further includes: obtaining single-cell transcriptome data based on the paraffin sample; and using the single-cell transcriptome data to confirm the tumor microenvironment information.

[0063] In some embodiments, the method further includes judging the efficacy of tumor treatment based on tumor microenvironment information.

[0064] In some implementations, the tumor microenvironment information includes at least cell type-related information. Analysis and comparison of this information allows for efficient and accurate confirmation of tumor treatment efficacy.

[0065] Specifically, cell type-related information includes cell type information and percentage information of each cell type.

[0066] In some embodiments, the tumor microenvironment information further includes at least one of cell subpopulation information and differential expression information.

[0067] In some embodiments, obtaining single-cell transcriptome data from paraffin samples further includes: preparing the paraffin sample into a single-cell suspension, dissociating and fixing the single cells; using reverse transcription primers to perform in situ reverse transcription on the RNA of the fixed single cells to synthesize a first strand of cDNA; adding a corresponding capture adapter to the tail of the first strand of cDNA, the capture adapter being sequence-complementary to the capture adapter complementary strand on the coding microsphere; containing the single cell and a single coding microsphere in a single chamber to form a cell separator, the single-stranded DNA on the coding microsphere containing an upstream amplification primer complementary fragment, a barcode, a unique multiplex index (UMI), and a capture adapter complementary strand; synthesizing a second strand of cDNA in the single chamber, and performing PCR amplification, library construction, and sequencing on the double-stranded cDNA.

[0068] In some implementations, the reverse transcription primer includes a tag sequence and an RNA-binding sequence, wherein the RNA-binding sequence is a random RNA-binding sequence, an RNA-binding sequence designed for a target RNA sequence, or a combination thereof.

[0069] In some embodiments, the method further includes permeabilizing the fixed single cells and sealing the exposed single-stranded DNA into double-stranded DNA after fixation and before in situ reverse transcription. This helps reduce the proportion of bases aligning to intergenic regions and increases the proportion of bases aligning to coding regions, UTR regions, intron regions, and ribosomal regions, thereby significantly improving the utilization rate of sequencing bases and reducing contamination of gene expression levels by intergenic regions.

[0070] In practice, those skilled in the art can use common sense to identify the reagents and specific operations mentioned in the above methods. In some specific embodiments, the specific reagents and specific operations involved in the above methods for obtaining single-cell transcriptome data from various paraffin samples can be identified by referring to the disclosures of Chinese patent applications with application numbers 202210174619.9, 202210550351.4, and 202310156589.3.

[0071] In some implementations, paraffin samples of lesions at different stages include paraffin samples of lesions before treatment and paraffin samples of lesions after treatment.

[0072] In some implementations, the treatment is selected from one or more of the following: surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy, hormone therapy, stem cell transplantation, vaccine therapy, and electric field therapy.

[0073] In some specific implementations, the treatment measures are selected from one or more of surgery, radiotherapy, chemotherapy, and electric field therapy.

[0074] In some specific implementations, the treatment is selected from one or two of surgery, chemotherapy, and electric field therapy. More specifically, the treatment is a combination of surgery or electric field therapy and chemotherapy.

[0075] In some embodiments, the tumor is selected from one or more of pancreatic cancer, thyroid cancer, melanoma, non-small cell lung cancer (NSCLC), breast cancer, colorectal cancer, ovarian cancer, prostate cancer, gastric cancer, hepatocellular carcinoma, bladder cancer, soft tissue sarcoma, multiple myeloma, glioma, and cholangiocarcinoma.

[0076] In some specific embodiments, the tumor is pancreatic cancer or thyroid cancer. Specifically, the tumor is advanced and unresectable pancreatic cancer or papillary thyroid carcinoma.

[0077] In some embodiments, certain steps of the method described in this invention (such as the step of obtaining single-cell transcriptome data based on the paraffin sample) are performed manually. In some embodiments, all steps of the method described in this invention are performed by a device (such as a computer, a detection device, etc.).

[0078] Those skilled in the art can combine the above-mentioned embodiments with common sense to obtain preferred embodiments of the method of the present invention.

[0079] Those skilled in the art can combine the above-mentioned embodiments with common sense to obtain preferred embodiments of the tumor efficacy evaluation system of the present invention.

[0080] This invention also discloses the application of single-cell transcriptome sequencing in tumor efficacy evaluation, wherein the tumor efficacy is evaluated by comparing and analyzing the data obtained from paraffin-embedded samples of lesions at different stages obtained by the above-mentioned tumor efficacy evaluation system after single-cell transcriptome sequencing.

[0081] The present invention also provides a non-transitory computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the tumor efficacy evaluation method as described above.

[0082] The present invention also provides an electronic device, which includes a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor executes the computer program to implement the tumor efficacy evaluation method as described above.

[0083] The present invention also provides a computer program product comprising a computer program that, when read and executed by a computing device, causes the computing device to perform: the tumor efficacy evaluation method as described above.

[0084] Example

[0085] Example 1 - Efficacy evaluation of tumor electric field therapy combined with chemotherapy for pancreatic cancer

[0086] This embodiment specifically describes the evaluation of the pre- and post-treatment efficacy of a patient with advanced, unresectable pancreatic cancer using the aforementioned tumor efficacy evaluation system and method. Please refer to [the above-described tumor efficacy evaluation system]. Figures 3 to 5F As shown, in this embodiment, the subject to be evaluated is a patient newly diagnosed with advanced, unresectable pancreatic cancer who has not received any anti-tumor or anti-cancer treatment. Specifically, in conjunction with Figure 3 A in Figure 4 As shown in A, the patient to be evaluated is a patient whose initial CT scan showed a suspected tumor lesion in the pancreas, and whose pathological analysis after puncture biopsy confirmed it to be pancreatic cancer. Figure 4 Figure A shows the pathological analysis of a pancreatic FFPE sample containing tumor tissue obtained by needle aspiration biopsy at the time of initial diagnosis.

[0087] The paraffin-embedded sample of the subject to evaluation before treatment (i.e., initially diagnosed with advanced, unresectable pancreatic cancer and not receiving anti-tumor therapy) was obtained as follows: a pancreatic pathological sample containing tumor tissue was obtained through needle aspiration biopsy at the initial diagnosis; the obtained pancreatic pathological sample was then processed using formalin fixation and paraffin embedding techniques to obtain the paraffin-embedded sample before treatment. In this embodiment, the pathological sample is a pancreatic cancer sample. The paraffin-embedded sample of the subject to evaluation before the application of treatment was a pancreatic cancer FFPE sample.

[0088] The treatment administered to the subject after initial diagnosis was tumor electric field therapy combined with chemotherapy. Tumor electric field therapy was achieved by applying an alternating current (AC) signal to the pancreatic tumor site of the subject using a tumor electric field therapy device. The AC signal applied to the pancreatic tumor site by the tumor electric field therapy device had a frequency of 100-300 kHz and an electric field strength of at least 0.1 V / cm. Preferably, the AC signal frequency was 150 kHz and the electric field strength was at least 1 V / cm. The alternating current signal was applied for at least 18 hours per day.

[0089] Chemotherapy is achieved by administering chemical drugs to the patient. In this example, the chemical drugs administered are gemcitabine and albumin-bound paclitaxel.

[0090] Specifically, each treatment cycle of the subject receiving tumor-treating fields (TTFields) combined with chemotherapy was 28 days. TTFields therapy was initiated on day 1 of the first treatment cycle using a TTFields device and continued throughout the entire treatment. Chemotherapy was administered intravenously at a dose of 125 mg / m² on days 1, 8, and 15 of each treatment cycle. 2 Immediately after administration of albumin-bound paclitaxel, administer 1000 mg / m² intravenously. 2 Gemcitabine. Specifically, the chemotherapy administered to the subject of evaluation involved an intravenous injection of 125 mg / m² on day 1 of each treatment cycle. 2 The albumin-bound paclitaxel was administered, followed immediately by an intravenous injection of 1000 mg / m² into the subject to be evaluated. 2 Gemcitabine; 125 mg / m² was administered intravenously to the subject of evaluation on day 8 of each treatment cycle. 2 The albumin-bound paclitaxel was administered, followed immediately by an intravenous injection of 1000 mg / m² into the subject to be evaluated. 2 Gemcitabine; and on day 15 of each treatment cycle, administer 125 mg / m² intravenously to the subject to be evaluated. 2 The albumin-bound paclitaxel was administered, followed immediately by an intravenous injection of 1000 mg / m² into the subject to be evaluated. 2 Gisitabin.

[0091] The total dose of the chemical drug administered intravenously to the subject of evaluation was determined based on their body surface area, which was determined by the subject's weight the day before drug administration. Specifically, body surface area (m²) 2 Calculate the body surface area (m²) using the following formula: 2 = [height (cm) + weight (kg)] / 100 - 0.6.

[0092] Tumor-treating fields (TTFields) therapy can be performed concurrently with chemotherapy, or after or before chemotherapy. Optionally, TTFields therapy can be performed seconds, minutes, or hours after chemotherapy; or seconds, minutes, or hours before chemotherapy.

[0093] At the end of every two treatment cycles, the pancreatic tumor site of the subject to be evaluated was subjected to CT scanning imaging to obtain CT images of the pancreatic tumor after receiving the above-mentioned combined treatment measures.

[0094] Based on the CT imaging data of the lesion or tumor site at each stage and the analysis results of the pathological samples at each stage, it is determined whether the subject meets the criteria for surgically resectable lesions.

[0095] Specifically, in combination Figure 3 The CT images of the subject under evaluation in Figures A, B, and C, showing different stages or phases of pancreatic cancer (marked in red in each figure), clearly demonstrate that after two cycles of treatment with tumor-treating fields combined with albumin-bound paclitaxel and gemcitabine, the lesions or tumors of the subject under evaluation showed a significant reduction in size compared to the initial diagnosis. Furthermore, after five cycles of treatment with tumor-treating fields combined with albumin-bound paclitaxel and gemcitabine, the lesions or tumors of the subject achieved partial remission and met the criteria for surgical resection according to RECIST 1.1. Figure 3 D in the image shows a CT scan of the individual being evaluated one month after surgery, in which no tumor is visible.

[0096] Combination Figure 4 As shown in Figures A, B, and C, which represent the pathological sample analysis results of pancreatic cancer at different stages using CA-19-9 biomarker levels, it is evident that after five treatment cycles of tumor electric field therapy combined with albumin-bound paclitaxel and gemcitabine (i.e., before surgery), the CA-19-9 biomarker level significantly decreased, indicating that the patient was suitable for surgical resection.

[0097] Next, for pancreatic sites that had received the above-mentioned tumor electric field therapy combined with albumin-bound paclitaxel and gemcitabine for 5 treatment cycles and whose CT imaging showed that they were ready for surgical resection, FFPE samples were obtained for surgical biopsy.

[0098] The pancreatic cancer FFPE samples obtained from the initial diagnosis of the subject (i.e., FFPE samples before treatment) and the pancreatic cancer FFPE samples obtained after surgical biopsy of the pancreatic cancer lesions following the aforementioned combined treatment were further processed as follows: 2-3 50μm thick paraffin-embedded samples containing tumor tissue were obtained from the FFPE samples before treatment, and 2-3 50μm thick paraffin-embedded samples containing tumor tissue were obtained from the FFPE samples after treatment; and all obtained paraffin-embedded samples were transported under non-high-temperature conditions for subsequent sample processing and sequencing. The temperature during paraffin-embedded sample transport was below 50 degrees Celsius. Subsequent sample processing and sequencing included sample pretreatment, single-cell nucleus dissociation, single-cell nucleus preparation, single-cell nucleus fixation and permeabilization, single-stranded DNA blocking, reverse transcription, addition of capture adapters, cell septation, synthesis of the second strand of cDNA, library construction, and high-throughput sequencing.

[0099] The pretreatment and single-cell nucleus dissociation steps for pancreatic cancer FFPE samples can be performed according to the description in Example 1 of patent application number CN202310156589.3. The single-cell nucleus permeation and single-stranded DNA blocking steps can also be performed according to the description in Example 2 of patent application number CN202310156589.3. The reverse transcription, addition of capture adapters, cell septation, synthesis of the second strand of cDNA, library construction, and high-throughput sequencing of the single-cell nuclei after the DNA blocking reaction are performed according to the description in Example 3 of patent application number CN202310156589.3.

[0100] This embodiment uses a wax roll sample as an example to illustrate the specific steps of sample pretreatment to single-cell nucleus preparation as follows:

[0101] Place one FFPE sample wax roll into a 1.5ml centrifuge tube;

[0102] Add 1 ml of xylene to the centrifuge tube and treat at room temperature for 5-10 minutes. Then remove the xylene and add 1 ml of new xylene to dewax the tube. The treatment time at room temperature is 5 minutes.

[0103] After dewaxing, xylene was removed, and ethanol at gradient concentrations of 100%, 95%, 80%, 70%, 50%, and 30% was added for rehydration.

[0104] After rehydration, add 1 ml of single-cell nuclear lysis buffer, which is a 2X SSC buffer containing 0.5-2% NP-20 ionic surfactant and MgCl2 (1-5 mM). Specifically, the single-cell nuclear lysis buffer is a 2X SSC buffer containing 1% NP-20 ionic surfactant and 2 mM MgCl2.

[0105] Add the sample and lysis buffer mixture to a Durns homogenizer for homogenization, homogenize 10 to 30 times, and then incubate on ice for 5 to 15 minutes; specifically, homogenize 15 times and incubate on ice for 10 minutes.

[0106] Add 100 μL of proteinase K (5-15 mg / ml) to a portion of the homogenized mixture, react at 25-50℃ for 5-30 minutes, and then centrifuge at room temperature. Specifically, add 100 μL of proteinase K (10 mg / ml) to a portion of the homogenized mixture, react at 37℃ for 15 minutes, and then centrifuge at room temperature.

[0107] Then remove the supernatant to obtain single cell nuclei, wash three times with washing solution, and use them for the next step.

[0108] In this embodiment, the specific steps for single-cell nucleus permeation and single-stranded DNA blocking of the single cells obtained from the above-mentioned sample pretreatment to single-cell nucleus preparation are as follows:

[0109] After washing, add a permeabilizing agent containing 0.2% Triton X-100 to the single-cell nucleus sample and permeabilize for 10 minutes. After permeabilization, centrifuge to remove the supernatant and wash three times with detergent.

[0110] After permeabilization, add DNA blocking reagent containing DNA elongase (1-10 U / μL), blocking primer (5-50 mM), dNTPs (1-10 mM), and 1X ThermoPol reaction buffer to the sample and react at 37°C for 20-45 minutes. Specifically, add DNA blocking reagent containing 10 U / μL DNA elongase, 10 mM blocking primer, 5 mM dNTPs, and 1X ThermoPol reaction buffer to the permeabilized sample and react at 37°C for 30 minutes.

[0111] After sealing, wash three times with detergent before proceeding to the next step.

[0112] In this embodiment, the specific steps for reverse transcription, adding capture adapters, cell septation, synthesis of the second strand of cDNA, library construction, and high-throughput sequencing of single-cell nuclei after DNA blocking are as follows: A reverse transcription reaction reagent containing reverse transcriptase, reverse transcription buffer, dNTPs, and reverse transcription primers is added to the single-cell nucleus sample after DNA blocking (i.e., after the previous step). After the reaction, the sample is washed three times with detergent. Terminal transferase, dCTP, and reaction buffer are added to the reverse-transcribed sample and incubated at 37°C for 30 minutes. After the reaction, the sample is washed three times with detergent. The reverse-transcribed single-cell nucleus sample, extension reaction reagent including DNA polymerase, dNTPs, and reaction buffer, coding microspheres, and oil phase are added to syringes. The syringes are then connected to the corresponding microfluidic chips via tubing. The inlet was connected and a suitable flow rate was set to form a water-in-oil single droplet containing a single cell nucleus, a single coding microsphere, and extension reaction reagent. The single droplets were collected to form a single-chamber cell separator containing a single cell. The collected single droplets were aliquoted into different tubes and extended to synthesize a second strand of barcoded cDNA in the single droplet. After the extension reaction, the single droplets were broken and the cDNA in the extraction tube was purified by magnetic bead extraction. A portion of the double-stranded cDNA was used as a template for qPCR to detect the total cDNA content. The remaining cDNA was amplified by PCR. The amplified cDNA was used for end repair and A-tailing by TA cloning and adapter ligation library construction. Adapters were ligated using a library construction kit to construct a library. The constructed library was then used for high-throughput single-cell transcriptome sequencing on the Illumina sequencing platform.

[0113] The single-cell transcriptome high-throughput sequencing (scRNA-seq) data were preprocessed, and a unique molecular identifier (UMI, 8 nucleotides) and a cell-specific barcode (30 nucleotides) were extracted from each Read1. Gene expression matrices were generated using STARsolo (version 2.7.10a) software on Read2. The number of cell nuclei for each sample was determined using a log10 (genes) scatter plot for each barcode, and a threshold was set at the minimum position where the log10 (gene) value was highest.

[0114] After barcode filtering and removal of mitochondrial and ribosomal RNA, the gene expression matrix was analyzed using the Seurat 3 toolkit in RStudio (version 4.2.1). This process included preprocessing, integration, visualization, clustering, cell type identification, and differential expression detection. Genes detected in fewer than three nuclei were excluded. Nucleus-level filtering used thresholds: GeneThresh = mean(nFeature_RNA) + 2*sd(nFeature_RNA), CountThresh = mean(nCount_RNA) + 2*sd(nCount_RNA). Counts were normalized and scaled using the SCTransform function, and dataset integration was performed using the Harmony package. A shared nearest neighbor (SNN) graph was constructed using principal component analysis (PCA) and FindNeighbors with 25 principal components. Clusters were identified using FindClusters at a resolution of 0.5 and visualized using UMAP. Cell type identification was performed manually using published marker genes.

[0115] Figures 5A to 5F The figure shows the data analysis obtained by single-cell transcriptome high-throughput sequencing (scRNA-seq) of FFPE samples from the pancreatic cancer patient at the initial diagnosis (before the treatment) and FFPE samples from the pancreatic cancer patient after surgical resection following the treatment with the above-mentioned tumor electric field therapy combined with albumin-bound paclitaxel and gemcitabine. Figure 5A and Figure 5B The analysis revealed a total of 10,216 cell nuclei sequenced and analyzed from two FFPE samples. Comprehensive single-cell transcriptome sequencing analysis of these samples provided a comprehensive landscape of each cell type, including stellate cells, T cells, macrophages, epithelial cells, B cells, endocrine cells, fibroblasts, neurons, endothelial cells, mesenchymal cells, plasma cells, adipocytes, and mast cells. Figure 5CThe changes in the proportion of cell types in the two FFPE samples before and after treatment can identify which cell type is the dominant tumor cell. In this example, epithelial cells accounted for a high proportion of pancreatic cancer tissue before treatment, but their percentage decreased significantly after treatment, confirming them as tumor cells. Figures 5D to 5F As shown, further analysis of the epithelial cell populations in the combined single-cell transcriptome sequencing data before and after treatment revealed that different subpopulations possessed unique gene expression profiles. In the pre-treatment FFPE sample single-cell transcriptome sequencing data, epithelial cells primarily formed subpopulations, labeled as basal-like cells, progenitor-like cells, and secretory cells, based on the most upregulated genes in each subpopulation. In contrast, the epithelial cells in the post-treatment FFPE sample single-cell transcriptome sequencing data primarily formed a single subpopulation, labeled as inflammatory-like cells. This information suggests an improvement in the tumor microenvironment.

[0116] In summary, the method of this invention obtained single-cell transcriptional information of pancreatic cancer FFPE samples before and after combined treatment. This information indicates an improved tumor microenvironment, consistent with the patient's physical condition, pathological results, and various indicators, confirming the method's applicability for tumor efficacy evaluation. Furthermore, by collecting and comparing information obtained from single-cell transcriptome sequencing of FFPE samples before and after treatment, a deeper understanding of pancreatic adenocarcinoma can be gained, allowing for the evaluation of various treatment regimens at the cellular and molecular levels. Compared to traditional pathological analysis using needle aspiration biopsy FFPE samples, this invention, through single-cell transcriptome sequencing of needle aspiration biopsy FFPE samples, collects more comprehensive and abundant transcriptional information, which can be used for tumor microenvironment analysis.

[0117] Example 2 - Efficacy evaluation of tumor electric field therapy combined with chemotherapy for thyroid cancer

[0118] Combination Figure 1 , Figure 2 as well as Figures 6A to 6C As shown, this embodiment specifically employs the aforementioned tumor efficacy evaluation system and method to perform single-cell transcriptome sequencing on an FFPE sample initially diagnosed with papillary thyroid carcinoma and surgically removed, and a recurrent FFPE sample that has also undergone surgical removal, to evaluate the efficacy of the treatment for papillary thyroid carcinoma in the patient. In this embodiment, the patient being evaluated is a patient initially diagnosed with papillary thyroid carcinoma who underwent surgical removal and subsequently experienced recurrence. When the patient was initially diagnosed with papillary thyroid carcinoma, surgical removal was performed to obtain a sample containing papillary thyroid carcinoma, which was then formalin-fixed and embedded in paraffin (FFPE sample). After the patient's recurrence, surgical removal was performed to obtain a sample containing the recurrent papillary thyroid carcinoma, which was then formalin-fixed and embedded in paraffin (FFPE sample).

[0119] Paraffin-embedded samples from both the initial diagnosis and recurrence of the patient were obtained through surgical resection. The FFPE samples from the patient at the initial diagnosis of papillary thyroid carcinoma and the FFPE samples from the patient diagnosed with recurrent papillary thyroid carcinoma underwent the following processing: three 20μm thick paraffin-embedded samples containing tumor tissue were obtained from the FFPE samples from the initially diagnosed and surgically resected papillary thyroid carcinoma, and three 20μm thick paraffin-embedded samples containing tumor tissue were obtained from the FFPE samples from the patient diagnosed with recurrent and surgically resected papillary thyroid carcinoma. All obtained paraffin-embedded samples were transported under non-high-temperature conditions for subsequent sample processing and sequencing. The temperature during transport was below 50 degrees Celsius. Subsequent sample processing and sequencing included sample pretreatment, single-cell nucleus dissociation, single-cell nucleus preparation, single-cell nucleus fixation and permeabilization, single-stranded DNA blocking, reverse transcription, addition of capture adapters, cell septation, synthesis of the second strand of cDNA, library construction, and high-throughput sequencing.

[0120] In this embodiment, the processing and analysis procedures for FFPE samples of papillary thyroid carcinoma were performed in accordance with the methods described in Example 1, including FFPE sample pretreatment and single-cell nucleus dissociation, single-cell nucleus permeation and single-stranded DNA blocking, reverse transcription, addition of capture adapters, cell separation, synthesis of the second strand of cDNA, library construction and high-throughput sequencing, scRNA-seq data analysis, cell type identification, cluster analysis and comparative analysis of cell ratio changes.

[0121] Figures 6A to 6C The figure shown is a data analysis diagram obtained from single-cell transcriptome high-throughput sequencing of FFPE samples at initial diagnosis and at recurrence in this patient with papillary thyroid carcinoma, according to the above-mentioned tumor efficacy evaluation method. From Figure 6A As can be seen, in newly diagnosed papillary thyroid carcinoma samples, thyroid follicular cells are the main component of the tumor tissue, accounting for nearly 50%. The proportion of fibroblasts and immune cells (such as T cells and macrophages) is low, reflecting that the tumor tissue has less stromal components and a less significant immune response at initial diagnosis. This is consistent with the pathological diagnosis, which shows that the tumor is mainly composed of thyroid follicular cells, accompanied by a low degree of immune cell infiltration and fibrosis.

[0122] from Figure 6BAs can be seen, in FFPE samples of recurrent papillary thyroid carcinoma, the proportion of thyroid follicular cells decreased significantly to less than 20%, while the proportions of fibroblasts and immune cells increased significantly. The proportion of fibroblasts approached 40%, indicating significant fibrosis in the recurrent lesions. This finding is consistent with the histiocytic proliferation and multinucleated giant cell response described in the pathological results, suggesting that fibrosis plays an important role in recurrent tumors. Simultaneously, the proportion of immune cells, including T cells, B lymphocytes, and macrophages, increased significantly. This increase in immune cells suggests enhanced interaction between tumor tissue and the immune system during recurrence, possibly accompanied by inflammatory responses and tissue remodeling. The elevated proportions of macrophages and multinucleated giant cells further confirm enhanced immune responses and phagocytic activity in the recurrent tissue.

[0123] like Figure 6C As shown, by comparing the cell type ratios of newly diagnosed and recurrent papillary thyroid carcinoma (FFPE) samples, a decrease in the proportion of tumor-associated cells and a significant increase in stromal cells and immune cells can be observed in recurrent FFPE samples. This change reflects the enhanced immune remodeling and fibrosis that occurs in recurrent tumor tissue after surgery and tumor cell stimulation. This change in cell composition not only reveals the pathological features of recurrent lesions but also suggests the dynamic adjustment mechanism of the microenvironment during tumor recurrence.

[0124] The results of this embodiment demonstrate that single-cell transcriptome sequencing analysis can accurately resolve the differences in cellular composition at different stages of papillary thyroid carcinoma. The significant fibrosis and enhanced immune response in recurrent samples provide important insights for further exploring recurrence mechanisms and personalized treatment strategies.

[0125] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A tumor treatment efficacy assessment system, comprising: The sample acquisition module is used to acquire paraffin samples of lesions at different stages of the same subject to be evaluated, and the paraffin samples contain tumor tissue. An information acquisition module is used to acquire tumor microenvironment information at corresponding time periods based on each paraffin sample, the tumor microenvironment information including cell type-related information; and, An analysis module is used to analyze and compare the tumor microenvironment information to obtain cellular information for tumor efficacy evaluation.

2. The tumor efficacy evaluation system according to claim 1, wherein, The lesions are located in the same area, and the paraffin samples are samples that have been fixed in formalin and processed by paraffin embedding technology. The paraffin samples include paraffin samples when the lesions are initially diagnosed as cancer or tumors and paraffin samples after treatment. The cell type information includes cell types and the percentage of each cell type.

3. The tumor efficacy evaluation system according to claim 2, wherein, The paraffin samples include paraffin samples of thyroid cancer that were initially diagnosed as thyroid cancer and surgically removed, and paraffin samples of thyroid cancer that recurred and were surgically removed.

4. The tumor efficacy evaluation system according to claim 3, wherein, The lesion is papillary thyroid carcinoma, and the thickness of the paraffin sample is 5-100 μm, preferably 10-20 μm.

5. The tumor efficacy evaluation system according to claim 2, wherein, The paraffin samples include paraffin samples obtained by needle aspiration biopsy after the lesion is initially diagnosed as pancreatic cancer and the lesion is surgically removed and biopsyed after the lesion meets the criteria for resectable surgery following treatment. The tumor microenvironment information also includes at least one of cell subpopulation information and differential expression information.

6. The tumor efficacy evaluation system according to claim 5, wherein, The lesion is an advanced and unresectable pancreatic cancer initially diagnosed. The treatment measures include at least one of surgery, electric field therapy, and chemotherapy. The thickness of the paraffin sample is 5-100 μm, preferably 10-50 μm.

7. The tumor efficacy evaluation system according to claim 6, wherein, The treatment is a combination of electric field therapy, which applies an alternating current signal via a tumor electric field therapy device, and chemotherapy, which administers chemical drugs. The alternating current signal has a frequency of 150 kHz and an electric field strength of at least 1 V / cm. The chemical drugs include albumin-bound paclitaxel and gemcitabine. The chemical drugs are administered on days 1, 8, and 15 of each treatment cycle, respectively. The gemcitabine is administered immediately after the albumin-bound paclitaxel is administered.

8. The tumor efficacy evaluation system according to any one of claims 1 to 7, wherein, The information acquisition module further includes: A data acquisition unit, configured to acquire corresponding single-cell transcriptome data based on each of the paraffin samples; and, A data analysis unit is used to confirm tumor microenvironment information using the single-cell transcriptome data.

9. The tumor efficacy evaluation system according to claim 7, wherein, The data acquisition unit obtains single-cell transcriptome data using the following method: After preparing the paraffin sample into a single-cell suspension, the single cells were dissociated and fixed. The first strand of cDNA was synthesized by in situ reverse transcription on RNA in fixed single cells using reverse transcription primers. A corresponding capture adapter is added to the end of the first strand of the cDNA, the capture adapter being complementary to the sequence of the capture adapter complementary strand on the microsphere; A single cell and a single coding microsphere are contained within a single chamber to form a cell separator, wherein the single-stranded DNA on the coding microsphere contains complementary fragments of upstream amplification primers, barcodes, UMIs, and capture adapters; and The second strand of cDNA is synthesized in the single chamber, and the double-stranded cDNA is then subjected to PCR amplification, library construction, and sequencing.

10. An application of single-cell transcriptome sequencing in tumor treatment efficacy evaluation, wherein, The tumor efficacy is evaluated using the tumor efficacy evaluation system described in any one of claims 1-9.

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