Construction method of human marrow paraffin tissue space transcriptome sequencing map

Abstract

The invention discloses a method for constructing a human marrow paraffin tissue space transcriptome sequencing map, which comprises the following steps of: obtaining high-quality space transcriptome data from a marrow paraffin section of an AML (acute myeloid leukemia) patient, and then integrating the space transcriptome with scRNA-seq and histopathology. Relatively complete transcriptome information is still preserved after the bone marrow specimen is subjected to better decalcification treatment, and meanwhile, higher-quality slicing treatment can be performed on the embedded paraffin tissue, so that very complete bone marrow space transcriptome sequencing data is obtained. Then, the cell type of each detection site in the bone marrow space transcriptome map can be accurately defined by integrating bone marrow single cell sequencing data and pathological morphology annotations, and subsequent analysis is carried out.

Description

Technical Field

[0001] The present invention relates to the field of spatial transcriptome sequencing, and in particular to a method for constructing a spatial transcriptome sequencing map of human bone marrow paraffin tissue. Background Art

[0002] Acute myeloid leukemia (AML) is a highly aggressive and heterogeneous hematological malignancy with a very poor prognosis, mainly due to resistance to first-line chemotherapy and high relapse rates in patients in remission, indicating that conventional chemotherapy is insufficient to cure most AML patients. Although new chemotherapy regimens and immune-based treatments have been approved or are undergoing clinical trials, the differences in patient responses to these interventions indicate that AML patients still face challenges in terms of treatment efficacy. Researchers have been working hard to link AML tumor heterogeneity to the complex bone marrow microenvironment. In response to this, the emergence of single cell RNA sequencing (scRNA-seq) has confirmed the heterogeneity of AML with high resolution. However, although scRNA-seq provides the existence of cell subpopulations in the bone marrow of AML patients, it cannot present the spatial distribution of various tumor cells and their surrounding cells in the bone marrow. In addition to single cell technology and next-generation sequencing, other technologies are needed to fully elucidate the complex interactions in the bone marrow microenvironment of AML patients.

[0003] In order to deepen the understanding of the interaction between tumor cells and their microenvironment, it is necessary to explore the occurrence of tumors in the spatial dimension. Spatial transcriptomics (ST) is a technology that analyzes gene expression while retaining tissue spatial information. This method enables the visualization and quantification of transcriptome profiles in single or multiple tissue sections. Spatial transcriptomics technology has obvious advantages in showing the spatial relationship between cancer cells and neighboring cells, thus helping to develop more effective clinical treatment targets and methods.

[0004] Currently, spatial transcriptome technology is commonly used in soft tissues, such as brain tissue or solid tissue tumors such as gastric cancer, but its application in bone or bone marrow tissue has not been realized until now. The main obstacle to spatial transcriptome sequencing in existing bone marrow is the preservation of intact mRNA during decalcification. In the standard processing method of bone marrow tissue, in order to achieve the best section quality, decalcification usually takes at least 72 hours, and in some cases up to 2 weeks, which will inevitably lead to the loss of transcriptome information. The currently recommended method for spatial transcriptome cell annotation is to follow the analysis process of the Seurat package, however, the relatively complex environment of the bone marrow cannot be used for cell annotation using the Seurat package.

[0005] To solve this problem, the present invention proposes a new bone marrow spatial transcriptome specimen processing and analysis technology. The present invention obtains high-quality spatial transcriptome data from bone marrow paraffin sections of AML patients for the first time, and then integrates spatial transcriptome with scRNA-seq and histopathology, so that we can obtain a complete bone marrow map of AML patients. The present invention helps to fill the gap in spatial transcriptomics in the field of hematological malignancies and high calcium tissue research. Summary of the invention

[0006] The purpose of the present invention is to propose a method for constructing a spatial transcriptome sequencing map of human bone marrow paraffin tissue in view of the deficiencies of the prior art, and to explore a series of processes for processing bone marrow paraffin specimens, so that the processed bone marrow paraffin specimens can be sliced ​​with high quality and complete bone marrow spatial transcriptome sequencing data can be obtained. Secondly, after obtaining the data, the analysis method of the present invention can fully display the bone marrow microenvironment of AML patients, that is, it is consistent with the distribution of pathological morphology and accurately presents the expression level of the cell transcriptome.

[0007] The object of the present invention is achieved through the following technical scheme: a method for constructing a spatial transcriptome sequencing map of human bone marrow paraffin tissue, the method comprising the following steps:

[0008] (1) Obtain bone marrow tissue from patients with acute myeloid leukemia (AML), dehydrate it, embed it in paraffin, and store it for later use;

[0009] (2) The paraffin tissues embedded with the bone marrow tissues of AML patients were treated with EDTA solution and sliced ​​to obtain paraffin sections for construction and sequencing of spatial transcriptomics libraries. After data statistics, the gene count values ​​of the paraffin tissues were processed and the results were compared with the human genome to merge duplicate genes;

[0010] (3) The RCTD deconvolution algorithm was used to map the genes of bone marrow single cell subsets to each detection site of the bone marrow spatial transcriptome. The chief pathologist performed pathological morphological analysis of the cell tissues covered by each detection site, combined with RCTD annotation and pathological morphological annotation, to obtain the final cell annotation of the bone marrow spatial transcriptome of AML patients.

[0011] Furthermore, the bone marrow tissue needs to be fixed with 4g / 100mL formaldehyde solution at 4°C for 48 hours before dehydration.

[0012] Furthermore, in step (2), the paraffin tissue embedding the bone marrow tissue of the AML patient is immersed in a 0.5 mol / L EDTA solution at pH 8.0 for 6 hours, then sliced ​​into 5 μm sections and developed in double distilled water at 58° C. to obtain paraffin sections.

[0013] Furthermore, paraffin sections need to be processed for hematoxylin and eosin (H&E) staining to assess cell abundance and cell types within the bone marrow, and paraffin-embedded bone marrow tissues of AML patients that meet the requirements will be sent for spatial transcriptome analysis.

[0014] Furthermore, RNA was extracted from paraffin sections, and DV200 was tested to evaluate the RNA integrity of bone marrow paraffin tissue. Paraffin tissue sections of AML patient bone marrow tissue that met the requirements were used to construct and sequence spatial transcriptomics libraries.

[0015] Furthermore, the construction and sequencing process of the spatial transcriptomics library is as follows: the paraffin sections are placed on Visium spatial transcriptome capture slides, and after drying overnight, the slides are incubated at 60°C for 2 hours and covered with human whole-genome transcriptome probes. After the probes are hybridized with the target genes and connected to each other, the connection products are released after RNase treatment and permeabilization, and the spatial position oligonucleotides of the connection probes and the capture area are hybridized to construct and sequence the spatial transcriptomics library.

[0016] Furthermore, in step (3), using the bone marrow single cell RNA-seq data of AML patients before and after treatment, firstly a dimensionality reduction cluster analysis is performed and each cell population is annotated to obtain marker genes of tumor cell populations and non-tumor cell populations.

[0017] Furthermore, based on the spatial transcriptome cell annotation of the bone marrow of AML patients and combined with the bone marrow histopathology annotation, the status of the bone marrow tumor microenvironment of AML patients was accurately portrayed.

[0018] Beneficial effects of the present invention: The bone marrow specimens of the present invention can still preserve relatively complete transcriptome information after good decalcification, and can also perform high-quality sectioning of the embedded paraffin tissue, thereby obtaining very complete bone marrow spatial transcriptome sequencing data. Then, by integrating bone marrow single cell sequencing data and pathological morphological annotations, the cell type of each detection site in the bone marrow is very accurately defined for subsequent analysis. The data obtained by the method of the present invention is much higher than the published mouse bone marrow spatial transcriptome data. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0020] Figure 1This is a schematic diagram of using a disposable bone marrow biopsy needle to obtain bone marrow tissue from the posterior superior iliac spine of an AML patient and performing specimen processing in an embodiment of the present invention;

[0021] Figure 2 This is a schematic diagram of performing dimensionality reduction clustering on bone marrow single cell data of AML patients in an embodiment of the present invention, and obtaining a total of 15 groups of cells;

[0022] Figure 3 Schematic diagram of characteristic expression genes of each cell subpopulation in the embodiment of the present invention;

[0023] Figure 4 A schematic diagram of using RCTD to map bone marrow single cell sequencing data to the spatial transcriptome to obtain the cell type enrichment score of each detection site in an embodiment of the present invention;

[0024] Figure 5 This is a schematic diagram of cell annotation of pathological morphology of each detection site by H&E tissue staining in an embodiment of the present invention;

[0025] Figure 6 A schematic diagram of integrating the cell types mapped by RCTD and the pathological morphology cell annotations to obtain the final AML patient bone marrow cell annotations in an embodiment of the present invention;

[0026] Figure 7 Schematic diagram of the total number of genes (nCount) and the number of genes after integration (nFeature) at each detection site of the bone marrow spatial transcriptome of AML patients detected in the embodiment of the present invention;

[0027] Figure 8 Schematic diagram of the spatial transcriptomic information of the bone marrow of AML patients detected in the embodiments of the present invention. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with the accompanying drawings and implementation examples. It should be understood that the specific implementation examples described herein are only used to explain the present invention and are not used to limit the present invention.

[0029] The present invention provides a method for constructing a spatial transcriptome sequencing map of human bone marrow paraffin tissue, the method comprising the following steps:

[0030] (1) Preparation of paraffin-embedded bone marrow tissue from AML patients, such as Figure 1 As shown, a disposable bone marrow biopsy needle was used to obtain bone marrow tissue of about 1 cm from the posterior superior iliac spine of an AML patient, fixed with 4g / 100mL formaldehyde at 4°C for 48 hours, dehydrated and embedded in 0.5mol / LEDTA solution for 6 hours, and the sections were stained with H&E. The specific process is as follows:

[0031] 1) With the patient's signed informed consent, a one-time biopsy was performed on the posterior superior iliac spine of the AML patient to obtain bone marrow tissue about 1 cm long;

[0032] 2) Immediately place the bone marrow biopsy tissue in 10 times the volume of 4g / 100mL formaldehyde solution for fixation for 48 hours at a temperature of 4°C;

[0033] 3) Use Sakura VIP-6 fully enclosed tissue dehydrator to dehydrate the fixed bone marrow tissue and embed it in paraffin. If short-term storage is required, store it at 4°C;

[0034] 4) The bone marrow paraffin tissue was immersed in 0.5 mol / L pH 8.0 EDTA solution for 6 hours, then sliced ​​into 5 μm sections using a Leica RM2125RTS manual wheel slicer, developed with 58°C double distilled water, and the tissue sections were attached to anti-shedding glass slides;

[0035] 5) Bake in oven at 90°C for 30 minutes, dewax in xylene for 15 minutes, repeat twice;

[0036] 6) Dehydrate the bone marrow paraffin sections with gradient ethanol solutions of 70%, 80%, 90% and anhydrous ethanol solutions, stain with hematoxylin for 5 minutes, and wash with double distilled water for 10 minutes;

[0037] 7) Soak in 1% hydrochloric acid ethanol for 30 seconds and wash with double distilled water for 30 seconds;

[0038] 8) Eosin staining for 3 minutes, and washing with double distilled water for 30 seconds;

[0039] 9) Wash with 80% ethanol solution for 30 seconds, then wash with 95% ethanol solution for 1 minute, repeat twice;

[0040] 10) Wash with anhydrous ethanol for 3 minutes, repeat twice;

[0041] 11) Soak in xylene for 3 minutes, repeat twice;

[0042] 12) Seal the slides with neutral resin, observe and photograph the bone marrow of AML patients under a microscope and perform hematoxylin and eosin (H&E) staining to assess cell abundance and cell types in the bone marrow and decide whether to send the samples for spatial transcriptome analysis.

[0043] (2) Quality control and spatial transcriptome sequencing of paraffin-embedded bone marrow samples from AML patients

[0044] 1) Take out the paraffin tissue of AML patient bone marrow stored at 4°C, immerse it in 0.5 mol / L pH 8.0 EDTA solution for 6 hours, and then slice it into 5 μm slices using a Leica RM2125RTS manual wheel slicer. Collect about 5-8 pieces of scattered paraffin slices and place them in 1.5 mL EP tubes;

[0045] 2) RNA was extracted from the collected paraffin sections using the RNeasy FFPE Kit (No. 73504, Qiagen), and the RNA integrity of the bone marrow paraffin tissue was assessed by measuring the ratio of RNA molecules with a length greater than 200 nt to the total number of molecules (DV200). The higher the percentage value of DV200, the more complete the extracted RNA sample;

[0046] 3) After the tissue adhesion test, the bone marrow paraffin tissue of AML patients soaked in EDTA solution was sliced ​​into 5 μm sections using a Leica RM2125RTS manual wheel slicer, and the sections were developed with 58°C double distilled water and placed on Visium spatial transcriptome capture slides;

[0047] 4) After drying overnight, the sections were incubated at 60°C for 2 hours;

[0048] 5) Transfer the bone marrow paraffin tissue sections to the capture area of ​​the 10x Visium spatial transcriptome chip, then perform tissue permeabilization to destroy the cells, release the mRNA, allow it to bind to the probes in the capture area, and then reverse transcribe the mRNA into cDNA.

[0049] 6) The complex of the probe and cDNA was eluted, and the spatial transcriptomics library of the probe was constructed and sequenced using the Illumina NovaSeq 6000 system;

[0050] 7) Perform statistics on the raw sequencing data and evaluate the data quality and GC content during the sequencing cycle;

[0051] 8) The generated Fastq files and histological images were processed for gene counts of paraffin tissues using the long probe alignment algorithm of Space Ranger (V1.3.0) from 10× Genomics, and the results were aligned with the human genome (GRCh38) and duplicate genes were merged.

[0052] (3) Spatial transcriptome annotation of bone marrow in AML patients

[0053] 1) Using the existing bone marrow single-cell RNA-seq data of AML patients before and after treatment, we first performed dimensionality reduction cluster analysis and annotated each cell population ( Figure 2 ), the marker genes of tumor cell populations and non-tumor cell populations can be obtained ( Figure 3 );

[0054] 2) Use the RCTD deconvolution algorithm to map the marker genes of each cell subset to each detection site of the bone marrow spatial transcriptome in the subsequent detection, and each detection site can obtain the corresponding cell type enrichment score ( Figure 4 );

[0055] 3) Through the morphological analysis of the cells and tissues covered by each detection site by three chief pathologists, the pathological morphological annotation of each site can be obtained ( Figure 5 );

[0056] 4) Combining RCTD annotation and pathological morphology annotation, the final AML patient bone marrow spatial transcriptome cell annotation can be obtained ( Figure 6 ), can very accurately depict the state of the bone marrow tumor microenvironment in AML patients.

[0057] The method of the present invention was used to detect and analyze the spatial transcriptome of the bone marrow of an AML patient, and it was found that the count value and feature value of the specimen were both at a high level ( Figure 7 ), the data obtained by the present invention are much higher than the published mouse bone marrow spatial transcriptome data ( Figure 8 ), the published mouse bone marrow spatial transcriptome detection is the existing technology used. The bone marrow spatial transcriptome of AML patients in the present invention was annotated using Seurat, and it was found that there were few cell types and the cell types in the bone marrow could not be accurately defined. The cell types annotated by the method of the present invention can accurately distinguish the cell types in the trabeculae and the bone marrow ( Figure 6 ).

[0058] The above embodiments are used to illustrate the present invention rather than to limit the present invention. Any modification and change made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A method for constructing a spatial transcriptome sequencing map of human bone marrow paraffin tissue, characterized in that: The method comprises the following steps: (1) Obtain bone marrow tissue from patients with acute myeloid leukemia (AML), dehydrate it, embed it in paraffin, and store it for later use; (2) The paraffin tissues embedded with the bone marrow tissues of AML patients were treated with EDTA solution and sliced ​​to obtain paraffin sections for construction and sequencing of spatial transcriptomics libraries. After data statistics, the gene count values ​​of the paraffin tissues were processed and the results were compared with the human genome to merge duplicate genes; (3) The RCTD deconvolution algorithm was used to map the genes of bone marrow single cell subsets to each detection site of the bone marrow spatial transcriptome. The chief pathologist performed pathological morphological analysis of the cell tissues covered by each detection site, combined with RCTD annotation and pathological morphological annotation, to obtain the final cell annotation of the bone marrow spatial transcriptome of AML patients.

2. The method for constructing a spatial transcriptome sequencing map of paraffin tissue of human bone marrow according to claim 1, characterized in that: Bone marrow tissue needs to be fixed with 4g / 100mL formaldehyde solution at 4℃ for 48 hours before dehydration.

3. The method for constructing a spatial transcriptome sequencing map of paraffin tissue of human bone marrow according to claim 1, characterized in that: In step (2), the paraffin tissue embedding the bone marrow tissue of the AML patient is immersed in a 0.5 mol / L EDTA solution with a pH of 8.0 for 6 hours, then sliced ​​to 5 μm, and developed in double distilled water at 58° C. to obtain paraffin sections.

4. The method for constructing a spatial transcriptome sequencing map of paraffin tissue of human bone marrow according to claim 1, characterized in that: Paraffin sections need to be processed for hematoxylin and eosin (H&E) staining to evaluate cell abundance and cell types within the bone marrow, and paraffin tissues embedded in the bone marrow tissues of AML patients that meet the requirements will be sent for spatial transcriptome examination.

5. The method for constructing a spatial transcriptome sequencing map of paraffin tissue of human bone marrow according to claim 1, characterized in that: RNA was extracted from paraffin sections, and DV200 was tested to evaluate the RNA integrity of bone marrow paraffin tissue. Paraffin tissue sections of AML patient bone marrow tissue that met the requirements were used to construct and sequence spatial transcriptomics libraries.

6. The method for constructing a spatial transcriptome sequencing map of paraffin tissue of human bone marrow according to claim 1, characterized in that: The construction and sequencing process of the spatial transcriptomics library is as follows: paraffin sections are placed on Visium spatial transcriptome capture slides. After drying overnight, the slides are incubated at 60°C for 2 hours and covered with human whole-genome transcriptome probes. After the probes are hybridized with the target genes and connected to each other, the connection products are released after RNase treatment and permeabilization. The connection probes and the spatial position oligonucleotides of the capture area are hybridized to construct and sequence the spatial transcriptomics library.

7. The method for constructing a spatial transcriptome sequencing map of paraffin tissue of human bone marrow according to claim 1, characterized in that: In step (3), using the bone marrow single-cell RNA-seq data of AML patients before and after treatment, first perform dimensionality reduction cluster analysis and annotate each cell population to obtain marker genes for tumor cell populations and non-tumor cell populations.

8. The method for constructing a spatial transcriptome sequencing map of paraffin tissue of human bone marrow according to claim 1, characterized in that: Based on the spatial transcriptome cell annotation of the bone marrow of AML patients and combined with the bone marrow histopathology annotation, the status of the bone marrow tumor microenvironment of AML patients is accurately depicted.

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