Tumor curative effect analysis system

Abstract

The invention belongs to the field of biomedicine, and particularly relates to a tumor curative effect analysis system. The system comprises: a sample acquisition module, the sample acquisition module is used for acquiring paraffin samples of the same individual to be evaluated before and after treatment measures are applied, and the paraffin samples contain tumor tissues; the information acquisition module is used for acquiring tumor microenvironment information based on the paraffin sample; and the analysis module is used for analyzing and comparing the tumor microenvironment information to obtain cell information highly associated with the tumor treatment effect. According to the system, tumor microenvironment analysis can be truly, accurately and comprehensively carried out, related samples can be easily obtained and traced back, and good convenience is achieved.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to a tumor efficacy analysis system. Background Art

[0002] Tumor efficacy refers to the effect of medical interventions on tumor growth, spread, and improvement of patient symptoms. Evaluating tumor efficacy is of vital importance for guiding clinical treatment, predicting patient prognosis, designing clinical trials, and achieving individualized treatment. It enables doctors to quantitatively monitor the tumor's response to treatment, thereby judging the effectiveness of treatment plans and adjusting treatment strategies in a timely manner to improve treatment outcomes. At the same time, efficacy evaluation helps predict patients' long-term survival rate and quality of life, providing patients with more accurate prognostic information. In addition, in the development of new drugs or new therapies, efficacy evaluation is a key indicator for measuring their safety and effectiveness, and is crucial for drug approval and clinical application.

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

[0004] Currently, commonly used methods for evaluating tumor efficacy include imaging examinations, tumor marker detection, RECIST standards, immunohistochemistry and molecular biology testing.

[0005] Among them, the most widely used is imaging examination, which can show the size and location of the tumor in detail and monitor changes in the tumor. Among imaging tests, CT scans are relatively fast, but may have artifacts or false negative / false positive results, and there is a risk of radiation; MRI has high resolution for soft tissues, but is expensive and takes a long time to examine; ultrasound is radiation-free and low-cost, but has poor resolution for deep tumors. PET scans can evaluate the metabolic activity of tumors and help detect tiny lesions, but they are expensive and take a long time to examine.

[0006] At the same time, the advantages and disadvantages of other evaluation methods are also obvious. Specifically, tumor marker testing evaluates the efficacy of treatment by changes in marker levels in the blood or body fluids. It is simple and non-invasive, but the specificity of the marker may be insufficient and the level may be affected by other factors. The RECIST standard is an objective and standardized evaluation method that evaluates the treatment effect by measuring changes in the size of tumor lesions, but its applicability to certain tumor types is limited. Immunohistochemistry and molecular biology tests can provide molecular characteristic information of tumor cells, which helps to understand the biological characteristics of tumors, but they are usually used as supplementary methods, are complex and costly.

[0007] In addition, in translational medicine research, there is also a method of sequencing unpaired fresh or frozen samples of different patients before and after treatment. The shortcoming of this method is that the patient's tumor heterogeneity is extremely high, and the samples before and after treatment cannot truly reflect the differences in the tumor microenvironment before and after treatment, making it impossible to accurately evaluate the efficacy.

[0008] Based on this, it is necessary to provide a tumor efficacy analysis system to quickly and accurately obtain cell information that is highly correlated with tumor efficacy. Summary of the invention

[0009] The first object of the present invention is to provide a tumor efficacy analysis system, which includes: a sample acquisition module, the sample acquisition module is used to obtain paraffin samples of the same individual to be evaluated before and after the treatment measures are applied, and the paraffin samples contain tumor tissue; an information acquisition module, the information acquisition module is used to obtain tumor microenvironment information based on the paraffin samples; and an analysis module, the analysis module is used to analyze and compare the tumor microenvironment information to obtain cell information that is highly correlated with the tumor treatment efficacy.

[0010] Furthermore, the paraffin sample is a sample fixed with formalin and processed by paraffin embedding technology (FFPE).

[0011] Furthermore, the thickness of the paraffin sample is 5-100 μm, preferably 10-50 μm.

[0012] 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.

[0013] Furthermore, the tumor microenvironment information includes at least one of cell type information, cell subpopulation information and differential expression information.

[0014] Furthermore, the data acquisition unit obtains single-cell transcriptome data by the following method: after preparing the paraffin sample into a single-cell suspension, dissociating and fixing the single cell; using a reverse transcription primer to perform an in situ reverse transcription reaction on the RNA of the fixed single cell to synthesize the first chain of cDNA; adding a corresponding capture adapter to the tail end of the first chain of cDNA, and the capture adapter is complementary to the sequence of the capture adapter complementary chain on the encoding microsphere; containing the single cell and a single encoding microsphere in a single chamber to form a cell partition, and the single-stranded DNA on the encoding microsphere contains an upstream amplification primer complementary fragment, a barcode, a UMI and a capture adapter complementary chain; synthesizing the second chain of cDNA in the single chamber, and performing PCR amplification, library construction and sequencing on the double-stranded cDNA.

[0015] Furthermore, the reverse transcription primer includes a tag sequence and an RNA binding sequence, and the RNA binding sequence is a random RNA binding sequence, an RNA binding sequence designed for a target RNA sequence, or a combination thereof.

[0016] Furthermore, the method further comprises, after fixing the single cell and before performing the in situ reverse transcription reaction: permeabilizing the fixed single cell and sealing the exposed single-stranded DNA into double-stranded DNA.

[0017] Furthermore, the treatment measures include at least one of surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy, and electric field therapy.

[0018] Furthermore, the tumor tissue may be selected from pancreatic cancer, preferably advanced and unresectable pancreatic cancer.

[0019] The tumor efficacy analysis system of the present invention can quickly, truly, accurately and comprehensively obtain cell information that is highly correlated with the efficacy of treatment measures adopted by tumor patients, and is easy to obtain and trace back to relevant clinical samples, has good convenience, and does not bring additional risks to patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

[0022] Figure 2 The figure is a flow chart of tumor efficacy analysis using the tumor efficacy analysis system of the present invention.

[0023] Figure 3 The CT scan images of the same tumor site of an individual to be evaluated at different times in an embodiment of the present invention; wherein, Figure 3 A in the figure is the CT scan image of the individual to be evaluated when he is diagnosed as a tumor patient; Figure 3 B is a CT scan image of the individual to be evaluated after receiving two cycles of combined treatment with electric field therapy and chemotherapy; Figure 3 C in the figure is the CT scan image of the individual to be evaluated before the surgery; Figure 3 D in is the CT scan image of the individual to be evaluated 1 month after the surgery; Figure 3 A~ Figure 3 The red symbols in C indicate the location of the tumor.

[0024] Figure 4 for Figure 3 The pathological analysis diagram of the tumor site of the individual to be evaluated at different stages is reflected by the CA-19-9 biomarker level at different stages; among which, Figure 4 A in the figure is a pathological analysis image of a sample (FFPE sample) obtained by needle aspiration biopsy of the tumor site of the individual to be evaluated at the time of initial diagnosis and processed by formalin fixation and paraffin embedding technology; Figure 4 B is the pathological analysis of the sample (FFPE sample) obtained by needle aspiration biopsy and processed by formalin fixation and paraffin embedding technology when the individual to be evaluated meets the surgical resection criteria after 5 cycles of combined electric field therapy and chemotherapy. Figure 4 C in it is a pathological analysis image of the sample (FFPE sample) obtained by surgical biopsy of the tumor site and processed by formalin fixation and paraffin embedding technology when the individual to be evaluated reaches the surgical resectable standard after receiving the combined treatment of electric field therapy and chemotherapy.

[0025] Figure 5A This is a split UMAP projection of the data obtained from single-cell transcriptome sequencing (scRNA-seq) of FFPE samples from the tumor site of the individual to be evaluated before receiving treatment (i.e. at the time of diagnosis).

[0026] Figure 5B Split-UMAP projection of data from single-cell transcriptome sequencing (scRNA-seq) of FFPE samples from tumor sites of the evaluated individuals after treatment and surgical resection.

[0027] Figure 5CThe percentage of each cell type obtained by single-cell transcriptome sequencing (scRNA-seq) of FFPE samples of the tumor site of the evaluated individual before receiving treatment and FFPE samples of the tumor site after treatment and surgical resection.

[0028] Figure 5D UMAP projections of epithelial cell divisions obtained from single-cell transcriptome sequencing (scRNA-seq) of FFPE samples of tumor sites in the evaluated individual before treatment and FFPE samples of tumor sites after treatment and surgical resection.

[0029] Figure 5E To integrate Figure 5D UMAP projection of a mid-epithelial cell, showing the different subpopulations of epithelial cells.

[0030] Fig. 5F For Figure 5E Dot plots of the expression levels of the top five marker genes in each subpopulation of epithelial cells. DETAILED DESCRIPTION

[0031] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention. Those skilled in the art may make various modifications and changes to the present invention without departing from the scope or spirit of the present invention. For example, a feature described or illustrated as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0032] Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present invention is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0033] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0034] As used herein, the terms "comprising", "including" and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

[0035] Numerical ranges expressed as endpoints herein include all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0036] The present invention relates to concentration values, and the meaning includes fluctuations within a certain range. For example, it can fluctuate within a corresponding precision range. For example, 2%, it is allowed to fluctuate within ±0.1%. For values ​​that are large or do not require too fine control, it is also allowed to have a greater fluctuation. For example, 100mM, it is allowed to fluctuate within the range of ±1%, ±2%, ±5%, etc. Involving molecular weight, it is allowed to have a fluctuation of ±10%.

[0037] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0038] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0039] In the present invention, “preferred”, “better”, “more preferred” and “suitable” are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present invention.

[0040] In the present invention, "optionally", "optional", "optionally", "optionally", "optional", "optional", "optional" means optional, that is, it means to be selected from any one of the two parallel schemes of "yes" or "no". If multiple "optional" or "optional" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" or "optional" is independent.

[0041] In the present invention, the term "individual to be evaluated" refers to a patient whose tumor treatment effect needs to be evaluated. These individuals are receiving or have received treatment and need to evaluate the effect of treatment. In some embodiments, the individual to be evaluated is a human (Homo sapiens). In some embodiments, the individual to be evaluated is a mammal such as a dog (Canis lupusfamiliaris), a cat (Felis catus), a horse (Equus ferus caballus), a rabbit (Oryctolaguscuniculus), a sheep (Ovis aries), a mouse (Mus musculus), a rat (Rattus norvegicus), etc.

[0042] In the present 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 information: (1) Cell component information: i) Tumor cell information: including the type, characteristics and variation 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 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, etc. (3) Molecular and signaling pathway information: i) Growth factor information: such as the levels and effects 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 main signaling pathways involved (such as PI3K / Akt, MAPK / ERK, etc.). (4) Metabolic characteristic information: i) pH value information: pH changes in tumor tissue and their effects; ii) Oxygen level information: oxygen levels in tumor tissue and their effects on tumor development. (5) Spatial organization information of the microenvironment: i) Cell localization information: spatial distribution of different types of cells in tumor tissue; ii) Tissue structure information: how the spatial structure of the tumor microenvironment affects tumor behavior and treatment effects.

[0043] In the present invention, the term "Paraffin-Embedded Samples" refers to tissue samples processed by paraffin embedding technology.

[0044] In the present invention, the term "FFPE samples (Formalin-Fixed, Paraffin-Embedded Samples)" refers to tissue samples processed by formalin fixation and paraffin embedding technology.

[0045] In the present invention, the term "single cell" includes but is not limited to a single cell and a single cell nucleus.

[0046] In the present invention, the term "single-cell transcriptome data" refers to detailed data about each single-cell transcriptome obtained by single-cell RNA sequencing (scRNA-seq) technology. In some embodiments, the single-cell transcriptome data includes one or more of gene expression matrix, cell heterogeneity, and cell type.

[0047] Combination Figure 1 As shown, the tumor efficacy analysis system provided by the present invention includes a sample acquisition module, an information acquisition module and an analysis module. The sample acquisition module is used to respectively obtain paraffin samples of the same individual to be evaluated before and after the treatment measures are applied, and the paraffin samples contain tumor tissues. The information acquisition module is used to obtain tumor microenvironment information based on each paraffin sample acquired by the sample acquisition module. The analysis module analyzes and compares the tumor microenvironment information acquired according to the information acquisition module to obtain cell information that is highly correlated with the tumor treatment efficacy.

[0048] In some embodiments, the paraffin sample is a sample fixed with formalin and processed by paraffin embedding technology (FFPE sample), which has a thickness that is conducive to obtaining tumor microenvironment information. The thickness of the paraffin sample can be 5 to 100 μm, preferably 5 to 50 μm, and more preferably 10 to 50 μm. The treatment measures may include at least one of electric field therapy, chemotherapy, immunotherapy, targeted therapy, radiotherapy, and surgical treatment. Specifically, the treatment measures include at least one of electric field therapy and chemotherapy. More specifically, the treatment measures are electric field therapy combined with chemotherapy.

[0049] The paraffin sample before the treatment measure is applied can be a FFPE sample when the lesion site of the individual to be evaluated is diagnosed as a tumor or cancer and no anti-tumor or anti-cancer treatment is applied. Specifically, the lesion site can be the pancreas. The tumor tissue can be advanced and unresectable pancreatic cancer. The paraffin sample after the treatment measure is applied is a FFPE sample obtained by surgery after the lesion site of the individual to be evaluated reaches the surgical resection standard after the treatment measure is applied.

[0050] The information acquisition module obtains tumor microenvironment information by performing single-cell transcriptome sequencing on FFPE samples before and after treatment.

[0051] In some embodiments, 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.

[0052] In some embodiments, the tumor microenvironment information includes at least one of cell type information, cell subpopulation information, and differential expression information.

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

[0054] In some embodiments, the reverse transcription primer includes a tag sequence and an RNA binding sequence, and 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, after fixing the single cells and before performing the in situ reverse transcription reaction, the method further comprises: permeabilizing the fixed single cells and sealing the exposed single-stranded DNA into double-stranded DNA.

[0056] In some specific embodiments, the treatment measure is selected from one or more of surgery, chemotherapy, and electric field therapy.

[0057] In some specific embodiments, the tumor is pancreatic cancer. More specifically, the tumor is advanced and unresectable pancreatic cancer.

[0058] Combination Figure 2 As shown, the tumor efficacy analysis method provided by the present invention includes: obtaining paraffin samples of the same individual to be evaluated before and after the treatment measures are applied, wherein the paraffin samples contain tumor tissue; obtaining tumor microenvironment information based on the paraffin samples; and analyzing and comparing the tumor microenvironment information to obtain cell information that is highly correlated with the tumor treatment efficacy.

[0059] The present invention can reflect detailed information related to tumor efficacy more quickly, truly and objectively by analyzing and comparing the tumor microenvironment information in the paraffin samples of the evaluated individual before the treatment measures are applied and the tumor microenvironment information in the paraffin samples after the treatment measures are applied; and paraffin samples are a commonly used method for preserving tumor tissues in clinical practice, so it is not only easy to obtain paraffin samples after the treatment measures are applied, but also easier to trace back to obtain paraffin samples before the treatment measures are applied.

[0060] In addition, among pancreatic cancer patients, only a portion of patients (<10%) can meet the criteria for surgical resection after treatment, and most patients cannot ensure that they can meet the criteria for surgical resection after treatment. Therefore, patients cannot determine whether they can undergo surgical resection of the tumor after treatment and obtain surgically removed tumor samples at the time of initial diagnosis, and cannot confirm at the time of initial diagnosis whether they can perform tumor microenvironment comparison analysis of paired samples before and after treatment to evaluate the efficacy of tumor treatment. However, if samples removed after treatment are obtained, it is much more convenient to retrospectively search for samples at the time of initial diagnosis, and paired samples can be easily obtained.

[0061] In some embodiments, the paraffin sample is a FFPE sample.

[0062] In some embodiments, the paraffin sample has a thickness that is conducive to obtaining tumor microenvironment information, which may be 5 to 100 μm, preferably 5 to 50 μm, and more preferably 10 to 50 μm.

[0063] In some embodiments, obtaining tumor microenvironment information based on the paraffin sample further includes: obtaining single-cell transcriptome data based on the paraffin sample; and confirming the tumor microenvironment information using the single-cell transcriptome data.

[0064] In some embodiments, the tumor microenvironment information includes at least one of cell type information, cell subpopulation information, and differential expression information. By analyzing and comparing these information, cell information that affects the efficacy of tumor treatment can be efficiently and accurately confirmed.

[0065] In some embodiments, the acquisition of single-cell transcriptome data based on the paraffin sample further includes: preparing the paraffin sample into a single-cell suspension, dissociating and fixing the single cell; using a reverse transcription primer to perform an in situ reverse transcription reaction on the RNA of the fixed single cell to synthesize the first chain of cDNA; adding a corresponding capture adapter to the tail end of the first chain of cDNA, wherein the capture adapter is complementary to the sequence of the capture adapter complementary chain on the encoding microsphere; containing the single cell and a single encoding microsphere in a single chamber to form a cell partition, the single-stranded DNA on the encoding microsphere containing an upstream amplification primer complementary fragment, a barcode (Barcode), a UMI (unique multiplex index) and a capture adapter complementary chain; synthesizing the second chain of cDNA in the single chamber, and performing PCR amplification, library construction and sequencing on the double-stranded cDNA.

[0066] In some embodiments, the reverse transcription primer includes a tag sequence and an RNA binding sequence, and the RNA binding sequence is a random RNA binding sequence, an RNA binding sequence designed for a target RNA sequence, or a combination thereof.

[0067] In some embodiments, after fixing the single cell and before performing the in situ reverse transcription reaction, the method further includes: permeabilizing the fixed single cell and sealing the exposed single-stranded DNA into double-stranded DNA. This will help reduce the proportion of bases aligned to the intergenic region and increase the proportion of bases aligned to the coding region, UTR region, intron region and ribosomal region, which will help to significantly improve the utilization rate of bases obtained by sequencing and reduce the contamination of the intergenic region on the gene expression amount.

[0068] In the specific implementation, those skilled in the art can confirm the reagents and specific operations mentioned in the above method in combination with common sense. In some specific embodiments, the specific reagents and specific operations involved in the above method for obtaining single-cell transcriptome data based on the paraffin sample can be confirmed by referring to the disclosures of Chinese patent applications with application numbers 202210174619.9, 202210550351.4 and 202310156589.3.

[0069] In some embodiments, the treatment measure is selected from one or more of surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy, and electric field therapy.

[0070] In some specific embodiments, the treatment measure is selected from one or more of surgery, chemotherapy, and electric field therapy.

[0071] In some specific embodiments, the treatment measure is selected from one or both of chemotherapy and electric field therapy. For example, the treatment measure can be electric field therapy combined with chemotherapy.

[0072] In some embodiments, the tumor is selected from advanced and unresectable pancreatic cancer.

[0073] In some embodiments, some steps of the method described in the present 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 the present invention are performed by a device (such as a computer, a detection device, etc.).

[0074] Those skilled in the art can combine the above-mentioned implementation modes with common sense to obtain a preferred embodiment of the method of the present invention.

[0075] Those skilled in the art can combine the above-mentioned implementation modes with common sense to obtain a preferred embodiment of the tumor efficacy analysis system of the present invention.

[0076] Example - Analysis of the efficacy of tumor electric field therapy combined with chemotherapy in the treatment of pancreatic cancer This embodiment specifically uses the above-mentioned tumor efficacy analysis system and the above-mentioned tumor efficacy analysis method to evaluate the efficacy of a late-stage, unresectable pancreatic cancer before and after treatment. Figures 3 to 5F As shown, in this embodiment, the individual to be evaluated is a patient who is initially diagnosed with advanced, unresectable pancreatic cancer and has not received anti-tumor or anti-cancer treatment. Figure 3 A in Figure 4 As shown in A, the individual to be evaluated is a patient whose CT image shows suspected tumor lesions in the pancreas after the initial diagnosis through the CT scanning equipment, and the pathological analysis after puncture biopsy confirmed it to be pancreatic cancer. Figure 4 A in the figure shows the pathological analysis of the pancreatic FFPE sample containing tumor tissue obtained by needle aspiration biopsy when the patient was first diagnosed.

[0077] The paraffin sample of the individual to be evaluated before the treatment measure is obtained as follows: a pancreatic pathological sample containing tumor tissue is obtained by needle aspiration biopsy at the initial diagnosis; the obtained pancreatic pathological sample is processed by formalin fixation and paraffin embedding technology to obtain the paraffin sample before the treatment measure. The pathological sample in this embodiment is a pancreatic cancer sample. The paraffin sample of the individual to be evaluated before the treatment measure is a pancreatic cancer FFPE sample.

[0078] The treatment measure applied to the individual to be evaluated after the initial diagnosis is a treatment measure of tumor electric field therapy combined with chemotherapy. Tumor electric field therapy is achieved by applying an alternating current signal to the pancreatic tumor site of the individual to be evaluated by a tumor electric field therapy device. The tumor electric field therapy device applies an alternating current signal with a frequency of 100-300kHz and an electric field strength of at least 0.1V / cm to the pancreatic tumor site of the individual to be evaluated. Preferably, the frequency of the alternating current signal is 150kHz and the electric field strength is at least 1V / cm. The application time of the alternating current signal is at least 18 hours per day.

[0079] Chemotherapy is achieved by administering chemical drugs to the patient. In this embodiment, the chemical drugs administered are gemcitabine and albumin paclitaxel.

[0080] Specifically, each treatment cycle of the treatment measures of tumor electric field therapy combined with chemotherapy for the individual to be evaluated is 28 days. The tumor electric field therapy for the individual to be evaluated is applied by the tumor electric field therapy device from the first day of the first treatment cycle and continues throughout the treatment process. The chemotherapy for the individual to be evaluated is specifically injected intravenously with 125 mg / m on the first day, the eighth day and the fifteenth day of each treatment cycle. 2 Immediately after nab-paclitaxel, 1000 mg / m 2That is, the chemotherapy for the individual to be evaluated is as follows: on the first day of each treatment cycle, the individual to be evaluated is first intravenously injected with 125 mg / m 2 nab-paclitaxel, followed immediately by an intravenous injection of 1000 mg / m 2 Gemcitabine; on day 8 of each treatment cycle, the subject to be evaluated was first given an intravenous injection of 125 mg / m 2 nab-paclitaxel, followed immediately by an intravenous injection of 1000 mg / m 2 gemcitabine; and on the 15th day of each treatment cycle, the subject to be evaluated was first given an intravenous injection of 125 mg / m 2 nab-paclitaxel, followed immediately by an intravenous injection of 1000 mg / m 2 of gemcitabine.

[0081] The total dose of the drug administered to the individual to be evaluated each time by intravenous injection is determined based on the individual's body surface area, wherein the body surface area is determined by the body weight of the individual to be evaluated one day before the administration of the drug. 2 ) is calculated according to the following formula: Body surface area (m 2 )=[height (cm)+weight (kg)] / 100-0.6.

[0082] Tumor Treatment Fields can be performed simultaneously with chemotherapy, or after chemotherapy or before chemotherapy. Optionally, Tumor Treatment Fields can be performed seconds, minutes, or hours after chemotherapy; or Tumor Treatment Fields can be performed seconds, minutes, or hours before chemotherapy.

[0083] At the end of every two treatment cycles, the pancreatic tumor site of the individual to be evaluated is examined by CT scanning equipment to obtain CT images after the individual receives the above-mentioned combined treatment measures.

[0084] Based on the CT imaging data of the lesion or tumor site at each stage of the individual to be evaluated and the results of pathological sample analysis at each stage, it is confirmed whether the individual to be evaluated meets the criteria for surgical resection of the lesion.

[0085] Specifically, combined Figure 3 It can be clearly seen from the CT images of the individual to be evaluated at different disease stages or periods of pancreatic cancer shown in A, B, and C (the red marked areas in each image) that after two cycles of treatment with tumor electric field therapy combined with albumin paclitaxel and gemcitabine, the focus, lesion or tumor site of the individual to be evaluated has been greatly reduced compared with the initial diagnosis; and after five cycles of treatment with tumor electric field therapy combined with albumin paclitaxel and gemcitabine, the focus, lesion or tumor site has achieved partial remission according to the RECIST1.1 standard and can be surgically removed. Figure 3 D in the figure shows a CT scan image of the individual under evaluation 1 month after surgery, in which no tumor is visible.

[0086] Combination Figure 4 As shown in A, B, and C, the CA-19-9 biomarker levels at different stages of pancreatic cancer are used to represent the pathological analysis results of FFPE samples of pancreatic cancer. It can be clearly seen that after five cycles of treatment with tumor electric field therapy combined with albumin paclitaxel and gemcitabine (that is, before surgery), the CA-19-9 biomarker level decreased significantly, and the individual to be evaluated can undergo surgical resection.

[0087] Next, patients who received the above-mentioned tumor electric field treatment combined with albumin-paclitaxel and gemcitabine for 5 cycles and whose CT imaging examination showed that the pancreas was in a resectable area, underwent surgical resection of the tumor to obtain FFPE samples for surgical biopsy.

[0088] The pancreatic cancer FFPE samples obtained by puncture sampling at the time of initial diagnosis of the individual to be evaluated (i.e., FFPE samples before the treatment measures) and the pancreatic cancer FFPE samples obtained by surgical biopsy after the pancreatic cancer lesions were resected after receiving the above-mentioned combined treatment were also processed as follows: 2 to 3 wax roll samples containing tumor tissues with a thickness of 50 μm were obtained from the FFPE samples before the treatment measures were applied, and 2 to 3 wax roll samples containing tumor tissues with a thickness of 50 μm were obtained from the FFPE samples after the treatment measures were applied; and each wax roll sample obtained was transported under non-high temperature conditions for subsequent sample processing and sequencing. The temperature of the wax roll sample transportation was less than 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, capture adapter addition, cell separation, synthesis of the second strand of cDNA, library construction and high-throughput sequencing.

[0089] The pretreatment and single cell nucleus dissociation steps of pancreatic cancer FFPE samples can be operated with reference to the description of Example 1 in patent application number CN202310156589.3. The single cell nucleus permeabilization and single-stranded DNA blocking steps can also be operated with reference to the description of Example 2 in patent application number CN202310156589.3. The single cell nucleus after DNA blocking reaction is reverse transcribed, capture adapter is added, cell separation, second strand of cDNA is synthesized, library is constructed and high-throughput sequencing is performed with reference to the description of Example 3 in patent application number CN202310156589.3.

[0090] In this embodiment, a wax roll sample is used as an example to illustrate the specific steps of sample pretreatment to prepare a single cell nucleus as follows: Take a piece of FFPE sample wax roll and place it in a 1.5ml centrifuge tube; Add 1 ml of xylene to the centrifuge tube, treat at room temperature for 5 to 10 minutes, then remove the xylene and add 1 ml of new xylene to dewax, wherein the specific treatment time at room temperature is 5 minutes; After dewaxing, xylene was removed and gradient concentrations of 100%, 95%, 80%, 70%, 50%, and 30% ethanol were added for rehydration; After rehydration, 1 ml of single cell nucleus lysis buffer is added, wherein the single cell nucleus lysis buffer is a 2X SSC buffer containing 0.5-2% NP-20 ionic surfactant and MgCl2 (1-5mM), wherein the single cell nucleus lysis buffer is specifically a 2X SSC buffer containing 1% NP-20 ionic surfactant and 2mM MgCl2; The sample and lysate mixture was added to a Dounce homogenizer for homogenization, homogenized 10 to 30 times, and placed on ice for 5 to 15 minutes, specifically homogenized 15 times and placed on ice for 10 minutes; Add 100 μL of proteinase K (5-15 mg / ml) to a portion of the homogenized mixture, react at 25-50° C. for 5-30 minutes, and then centrifuge at room temperature. Specifically, add 100 ul of proteinase K (10 mg / ml) to a portion of the homogenized mixture, react at 37° C. for 15 minutes, and then centrifuge at room temperature; Then the supernatant was removed to obtain single cell nuclei, which were washed three times with washing solution for the next step.

[0091] In this embodiment, the specific steps of performing single cell nucleus permeabilization and single-stranded DNA blocking on the single cell obtained by the sample pretreatment to prepare a single cell nucleus are as follows: After washing, the single cell nucleus sample was permeabilized with 0.2% Triton X-100 for 10 minutes. After permeabilization, the sample was centrifuged to remove the supernatant and washed three times with detergent. A DNA blocking reaction reagent containing DNA extension enzyme (1-10 U / μL), blocking primer (5-50 mM), dNTPs (1-10 mM), and 1X ThermoPol reaction buffer was added to the permeabilized sample, and the reaction was carried out at 37°C for 20-45 minutes. Specifically, a DNA blocking reaction reagent containing DNA extension enzyme 10 U / μL, blocking primer 10 mM, dNTPs 5 mM, and 1X ThermoPol reaction buffer was added to the permeabilized sample, and the reaction was carried out at 37°C for 30 minutes; After blocking, wash three times with detergent for the next step.

[0092] In this embodiment, the specific steps of reverse transcription, adding capture adapters, cell separation, synthesizing the second chain of cDNA, constructing a library and high-throughput sequencing of a single cell nucleus after a DNA blocking reaction are as follows: add a reverse transcription reaction reagent containing reverse transcriptase, reverse transcription reaction buffer, dNTPs, and reverse transcription primers to the single cell nucleus sample after a DNA blocking reaction (i.e., after the previous step) to perform a reverse transcription reaction, and add a detergent to wash three times after the reaction; add terminal transferase, dCTP and reaction buffer to the reverse transcribed sample and incubate at 37° C. for 30 minutes; add a detergent to wash three times after the reaction; add the single cell nucleus sample after reverse transcription, an extension reaction reagent including DNA polymerase, dNTPs and reaction buffer, a coding microsphere, and an oil phase into a syringe respectively; connect the syringe to the corresponding microfluidic chip through a hose. The liquid inlet is connected and a suitable flow rate is set to form an oil-in-water single droplet containing a single single cell nucleus, a single encoding microsphere, and an extension reaction reagent; the single droplets are collected to form a cell partition containing a single cell in a single chamber; the collected single droplets are divided into different tubes and then subjected to extension reaction to synthesize a second cDNA chain with a barcode in the single droplet; after the extension reaction, the single droplet is broken and the cDNA in the extraction tube is purified by a magnetic bead method; part of the double-stranded cDNA is taken as a template for qPCR experiment to detect the total cDNA content captured; the remaining cDNA is amplified by PCR, the amplified cDNA is end-repaired and A-tailed using the TA cloning adapter library construction method, and the adapter is connected using a library construction kit to construct a library; the constructed library is subjected to single-cell transcriptome high-throughput sequencing using an Illumina sequencing platform.

[0093] The above single-cell transcriptome high-throughput sequencing (scRNA-seq) data were preprocessed to extract unique molecular identifiers (UMI, 8 nucleotides) and cell-specific barcodes (30 nucleotides) for each Read1. Gene expression matrices were generated using STARsolo (version 2.7.10a) using Read2 software. The number of nuclei for each sample was determined using a log10(genes) scatter plot for each barcode, and a threshold was set at the minimum position with the highest log10(gene) value.

[0094] After barcode filtering and removal of mitochondrial and ribosomal RNA, gene expression matrices were analyzed using the Seurat 3 toolkit in RStudio (version 4.2.1). The process included preprocessing, integration, visualization, clustering, cell type identification, and differential expression detection. Genes detected in less than three nuclei were excluded. Nuclear-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. Shared nearest neighbor (SNN) graphs were constructed using principal component analysis (PCA) and FindNeighbors with 25 principal components, and clusters were identified using FindClusters with a resolution of 0.5 and visualized by UMAP. Cell type identification was performed manually using published marker genes.

[0095] FIG. 5A to FIG. 5F Shown is a data analysis graph obtained by performing single-cell transcriptome high-throughput sequencing (scRNA-seq) on the FFPE samples of the pancreatic cancer patient at the initial diagnosis (before the treatment measures were administered) according to the above-mentioned tumor efficacy analysis method and the FFPE samples of the pancreatic cancer patient who underwent surgical resection after the above-mentioned tumor electric field therapy combined with albumin paclitaxel and gemcitabine treatment measures were administered to the pancreatic cancer patient. Figure 5A and Figure 5B A total of 10,216 nuclei were sequenced and analyzed from two FFPE samples, yielding a comprehensive landscape of various cell types, including astrocytes, T cells, macrophages, epithelial cells, B cells, endocrine cells, fibroblasts, neurons, endothelial cells, mesenchymal cells, plasma cells, adipocytes, and mast cells. Figure 5C The changes in the proportion of cell types in the two FFPE samples before and after treatment can confirm which type of cells are the dominant tumor cells. In this example, epithelial cells accounted for a higher proportion of pancreatic cancer tissue before treatment, but their percentage decreased significantly after treatment, and they can be confirmed as tumor cells. FIG. 5D to FIG. 5FAs shown, further analysis of epithelial cell populations in the comprehensive single-cell transcriptome sequencing data before and after treatment revealed that different subpopulations had unique gene expression profiles. Epithelial cells in the single-cell transcriptome sequencing data of FFPE samples before treatment mainly formed subpopulations labeled as basal-like cells, progenitor-like cells, and secretory cells, based on the most upregulated genes in each subpopulation. In contrast, epithelial cells in the single-cell transcriptome sequencing data from FFPE samples after treatment mainly formed a subpopulation labeled as inflammatory-like cells. This information indicates that the tumor microenvironment has been improved.

[0096] In summary, the method of the present invention obtains the transcriptional information of pancreatic cancer FFPE samples at the single cell level before and after combined treatment; and the information shows that the tumor microenvironment has improved, which matches the patient's physical condition, imaging data, pathological analysis results and various indicators, proving that the method can be used for tumor efficacy analysis; and by collecting and comparing the information obtained by single-cell transcription sequencing of FFPE samples before and after treatment, pancreatic adenocarcinoma can be deeply understood, and cell information highly related to the efficacy of various treatment schemes can be evaluated at the cellular and molecular levels. Compared with the traditional pathological analysis of FFPE samples using needle biopsy, the present invention collects more and more comprehensive transcriptional information after single-cell transcriptome sequencing of FFPE samples from needle biopsy, which can be used for tumor microenvironment analysis.

[0097] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A tumor efficacy analysis system, comprising: A sample acquisition module, the sample acquisition module is used to respectively acquire paraffin samples of the same individual to be evaluated before and after the treatment measures are administered, wherein the paraffin samples contain tumor tissue; An information acquisition module, the information acquisition module is used to acquire tumor microenvironment information based on the paraffin sample; as well as An analysis module is used to analyze and compare the tumor microenvironment information to obtain cell information that is highly correlated with the tumor treatment efficacy.

2. The tumor efficacy analysis system according to claim 1, wherein: The paraffin samples are samples fixed with formalin and processed by paraffin embedding technology.

3. The tumor efficacy analysis system according to claim 1 or 2, wherein: The thickness of the paraffin sample is 5-100 μm.

4. The tumor efficacy analysis system according to claim 1 or 2, wherein: The information acquisition module further comprises: A data acquisition unit, the data acquisition unit is used to acquire single-cell transcriptome data based on the paraffin sample; And, a data analysis unit, wherein the data analysis unit is used to confirm tumor microenvironment information using the single-cell transcriptome data.

5. The tumor efficacy analysis system according to claim 1, wherein: The tumor microenvironment information includes at least one of cell type information, cell subpopulation information and differential expression information.

6. The tumor efficacy analysis system according to claim 4, wherein: The data acquisition unit obtains single-cell transcriptome data by the following method: After preparing the paraffin sample into a single cell suspension, dissociating and fixing the single cells; Using reverse transcription primers, an in situ reverse transcription reaction is performed on the RNA of fixed single cells to synthesize the first strand of cDNA; Adding a corresponding capture adapter to the tail end of the first strand of the cDNA, wherein the capture adapter is complementary to the sequence of the capture adapter complementary strand on the encoding microsphere; Containing a single cell and a single encoded microsphere in a single chamber to form a cell partition, wherein the single-stranded DNA on the encoded microsphere contains an upstream amplification primer complementary fragment, a barcode, a UMI, and a capture adapter complementary strand; The second strand of cDNA is synthesized in the single chamber, and the double-stranded cDNA is PCR amplified, library constructed and sequenced.

7. The tumor efficacy analysis system according to claim 6, wherein: The reverse transcription primer includes a tag sequence and an RNA binding sequence, and the RNA binding sequence is a random RNA binding sequence, an RNA binding sequence designed for a target RNA sequence, or a combination thereof.

8. The tumor therapeutic efficacy analysis system according to claim 6 or 7, wherein: After fixing the single cell and before performing the in situ reverse transcription reaction, the method further comprises: permeabilizing the fixed single cell and sealing the exposed DNA single strand into double-stranded DNA.

9. The tumor efficacy analysis system according to claim 1, wherein: The treatment measures include at least one of surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy, and electric field therapy.

10. The tumor efficacy analysis system according to claim 1, wherein: The tumor tissue is selected from pancreatic cancer.

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