A method of detecting colorectal cancer tumor cell-associated microorganisms

Abstract

This invention relates to a method for detecting colorectal cancer tumor cell-associated microorganisms, specifically a method for eliminating intestinal fecal microbial contamination and detecting colorectal cancer tumor-associated microorganisms, belonging to the field of biotechnology. This invention initially eliminates fecal microbial contamination from the sample structure by coarsely shearing and enzymatically digesting the intestinal folds and villi; then, it uses a 40μm cell sieve to filter out incompletely digested tissue fragments, further eliminating intestinal fecal bacteria contamination, and collecting a single-cell suspension; subsequently, the collected single-cell suspension is repeatedly washed with 300g centrifugation to remove as many intercellular or loosely bound "transient bacteria" as possible, retaining intracellular and firmly colonized microorganisms. Ultimately, this eliminates the severe contamination from the large and complex fecal microorganisms, allowing the detection of tumor cell-associated microorganisms, which are already present in low biomass. This method is suitable for detecting microorganisms in gastrointestinal tissues that are in direct contact with the environment.

Description

Technical Field

[0001] This invention relates to a method for detecting colorectal cancer tumor cell-associated microorganisms, specifically a method for excluding intestinal fecal microbial contamination and detecting colorectal cancer tumor-associated microorganisms, belonging to the field of biotechnology. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive tract. It is known that the largest number of microorganisms in the human body reside in the gastrointestinal tract; this complex ecosystem is known as the gut microbiota. Advances in sequencing technology and bioinformatics have made it possible to conduct in-depth research on the composition, metabolism, and metagenomics of the gut microbiota. [1] Although many studies have revealed the close relationship between gut microbiota and CRC from different perspectives, most studies have focused on gut fecal microbiota and its metabolites. [2] Given the vast and complex microbiota in feces and its multifactorial regulation of interaction with the intestinal mucosal epithelium, and the fact that most bacteria are "transient" rather than "colonizing" bacteria, it has become increasingly recognized that exploring the direct evidence and biological behavior of bacterial enrichment and invasion of tumor tissues and cells will be a promising research direction in this field.

[0003] Therefore, in recent years, some studies have begun to focus on the tumor-associated microbiome. The tumor microbiome is an important component of the tumor microenvironment. [3-5] Studies have shown that the tumor microbiome is an important component of tumor tissue and plays a crucial role in shaping the tumor microenvironment. [6,7] Intratumoral bacteria are mostly found inside cells, primarily in cancer cells and immune cells, and each tumor type has a different microbial composition. [4] Intratumoral bacteria and their predicted functions are related to tumor type, subtype, and response to immunotherapy. [4] Cancer cells infected with bacteria invade their surrounding environment as single cells and recruit bone marrow cells to the bacterial region. Intratumoral microbes are primarily distributed and enriched in immune and epithelial cells that promote cancer progression. [3] In conclusion, tumor-associated microorganisms play a more important and direct role in inducing tumorigenesis, altering cell metabolism, promoting tumor metastasis, and preventing immune surveillance. [8] The regulation and in-depth study of tumor-associated microbiota may pave the way for the discovery of new treatment options for cancer patients.

[0004] However, unlike solid tumors such as breast cancer that are not directly connected to the external environment, CRC tumor microbes are severely contaminated by fecal microbes due to the intestinal folds and villous structure. Figure 1The handling of contamination and the detection of tumor-associated microbiota have become technical challenges limiting the detection of such tumor microbiota.

[0005] [1].Bastiaanssen TFS, Cowan CSM, Claesson MJ, et al. Making Sense of the Microbiome in Psychiatry[J].Int J Neuropsychopharmacol, 2019.22(1):37-52.

[0006] [2].Dougherty MW, Jobin C. Intestinal bacteria and colorectal cancer: etiology and treatment. Gut Microbes. 2023.15(1):2185028.

[0007] [3]. Galeano JL, Wu H, LaCourse KD, et al. Effect of the intratumoralmicrobiota on spatial and cellular heterogeneity in cancer. Nature. 2022.611(7937):810-817.

[0008] [4]. Nejman D, Livyatan I, Fuks G, et al. The human tumor microbiome iscomposed of tumor type-specific intracellular bacteria. Science. 2020.368(6494):973-980.

[0009] [5].Sepich-Poore GD,Zitvogel L,Straussman R,Hasty J,Wargo JA,KnightR.The microbiome and human cancer.Science.2021.371(6536):eabc4552.

[0010] [6].Tumors Appear Rife with Bacterial Lodgers.Cancer Discov.2020.10(8):1085-1086.

[0011] [7]. Fu A, Yao B, Dong T, Cai S. Emerging roles of intratumor microbiotain cancer metastasis. Trends Cell Biol. 2023.33(7):583-593.

[0012] [8]. Liu Z, Hong LL, Ling ZQ. Potential role of intratumor bacteria outside the gastrointestinal tract: More than passengers. Cancer Med. 2023.12(16):16756-16773. Summary of the Invention

[0013] The problem the invention aims to solve

[0014] Tumor-associated microbiota (MAM) plays a crucial role in the development and progression of colorectal cancer. However, unlike solid tumors such as breast cancer, which are not directly connected to the external environment, colorectal cancer is heavily contaminated by fecal microorganisms due to its folded and villi-like structure. This contamination masks the already low biomass of MAM-associated microorganisms, making them difficult to detect. Eliminating interference from fecal flora and accurately detecting MAM-associated microorganisms remains a challenge.

[0015] Solution for solving the problem

[0016] In this invention, the inventors, by subjecting colorectal cancer tissue to different treatments and detecting colorectal tumor-associated microorganisms in the treated samples, ultimately discovered a novel method for isolating colorectal cancer tumor cell-associated microbiota. The microbiota and potential functions detected by different methods were analyzed and compared, resulting in the identification of colorectal cancer tumor cell-associated microorganisms.

[0017] The intestinal folds and villi are disrupted through coarse shearing and enzymatic digestion, and a single-cell suspension is prepared to initially remove fecal microbial contamination from the intestinal structure. Undigested tissue fragments are then removed using a cell sieve to further eliminate fecal bacterial contamination, and the single-cell suspension is collected. The suspension is then repeatedly washed using 300g centrifugation to retain only microorganisms firmly colonized on or within the cells. Finally, the suspension is sent for sequencing.

[0018] This invention provides the following technical solution:

[0019] [1]. A method for detecting colorectal cancer cell-associated microorganisms, wherein the method comprises:

[0020] The steps to preliminarily exclude fecal microbial contamination from intestinal folds and villi in the sample include coarse cutting and enzymatic digestion of the sample;

[0021] Further steps to eliminate intestinal fecal microbial contamination include filtering the cell suspension obtained from enzymatic digestion through a cell sieve to collect single-cell suspensions; and,

[0022] The step of removing contaminating and transient bacteria involves washing and centrifuging the single-cell suspension to obtain single cells.

[0023] [2]. According to the method described in [1], the enzymatic digestion includes enzymatically separating the coarsely cut sample into a cell suspension under the action of an enzyme catalyst.

[0024] [3]. According to the method of [2], wherein the enzyme catalyst comprises neutral protease and collagenase.

[0025] [4]. According to the method described in [3], wherein the neutral protease is neutral protease II and the collagenase is collagenase IV.

[0026] [5]. The method according to any one of [1] to [4], wherein the pore size of the cell sieve is 40 μm.

[0027] [6]. The method according to any one of [1] to [5], wherein the single-cell suspension is washed with phosphate buffer solution and collected by centrifugation at 280 to 300 g.

[0028] [7]. The method according to any one of [1] to [6], wherein the sample comprises colorectal tissue, the colorectal tissue comprising colorectal cancer tissue and normal tissue.

[0029] [8]. The method according to any one of [1] to [7], wherein the sample is washed three times with physiological saline before coarse cutting of the sample.

[0030] [9]. The method according to any one of [1] to [8], wherein the method further comprises: a detection step, which includes using a single cell obtained in the step of removing contaminating bacteria and transient bacteria as a detection sample to detect colorectal cancer cell-associated microorganisms.

[0031]

[10] . According to the method of [9], the detection includes detecting the sample by 5R 16S rRNA gene sequencing technology.

[0032] The effects of the invention

[0033] The method for detecting tumor-associated microorganisms in colorectal cancer provided by this invention is simple and efficient, and can effectively eliminate the interference of fecal microorganisms on the detection results of tumor-associated microorganisms in colorectal cancer cells. It is applicable to the detection of related microorganisms in gastrointestinal tissues that are in direct contact with the environment. Attached Figure Description

[0034] Figure 1 A schematic diagram of the intestinal structure (folds and villi) and microorganisms: The colorectal tissue is heavily contaminated by fecal microorganisms due to the close contact between the intestinal folds and villi structure and feces.

[0035] Figure 2 The distribution of LPS in colorectal tissue is shown: LPS staining is mainly distributed in feces on the surface of intestinal tissue as well as in epithelial cells and macrophages within the tissue (LPS is a component of the outer wall of Gram-negative bacterial cell walls, mainly composed of lipids and polysaccharides, and can characterize the distribution of Gram-negative bacteria, such as Escherichia coli).

[0036] Figure 3 The alpha diversity analysis results are shown in the figure. The alpha dilution curve indicates a reasonable sequencing depth, and the Goods value of 1 indicates complete sequencing coverage of the microbial community. Chao1 and observed_species are mainly used to estimate the number of community species, showing that the number of species measured after processing by the method of this invention is significantly lower than that of traditional detection methods. Simpson / Shannon mainly reflects the richness and evenness of species. Wherein, PO: normal tissue; TO: tumor tissue; PC: normal cells; TC: tumor cells.

[0037] Figure 4 Figure showing the results of beta diversity analysis: Beta diversity reflects species differences between groups. Figure 4 In the figure, A represents PCA analysis; the more similar the species composition of the samples, the closer they are in the graph. Figure 4 In the B section, PCoA analysis shows that the closer the two points are, the smaller the difference in community composition. Figure 4 In the analysis, C represents UPGMA analysis, where the shorter the branch length between samples, the more similar the two samples are; where PO: normal tissue; TO: tumor tissue; PC: normal cells; TC: tumor cells.

[0038] Figure 5 The diagram shows the number of species at the phylum and genus level in the four groups detected by the detection method of the present invention and conventional detection methods (left side), as well as the dominant bacterial groups (right side); wherein, PO: normal tissue; TO: tumor tissue; PC: normal cells; TC: tumor cells.

[0039] Figure 6The diagram shows the results of differential bacterial communities detected by the detection method of the present invention and the conventional detection method; the differential bacterial communities between the groups before and after treatment were obtained by LEfSe analysis; wherein, PO: normal tissue; TO: tumor tissue; PC: normal cells; TC: tumor cells.

[0040] Figure 7 The method of this invention is used in conjunction with traditional tissue testing to screen four groups of differentially expressed bacterial groups. Detailed Implementation

[0041] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0042] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0043] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0044] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0045] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0046] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0047] In this article, "colorectal cancer" refers to colon cancer, which is a malignant tumor that occurs in the mucosal epithelium and glands of the colon. When tumor tissue in the colon and rectum breaks through the muscularis mucosa and reaches the submucosa, it is called "colorectal cancer," which includes colon cancer and rectal cancer.

[0048] In this document, "normal tissue" refers to normal tissue adjacent to cancerous tissue that is more than 5 cm from the edge of cancerous tissue. In some specific embodiments of the present invention, "normal tissue" includes "normal intestinal tissue," which refers to normal tissue adjacent to colorectal cancerous tissue that is more than 5 cm from the edge of colorectal cancerous tissue in the colon and rectum.

[0049] In this article, "normal cell" refers to a single cell obtained by processing normal intestinal tissue through the steps of coarse cutting, enzymatic digestion, cell sieve filtration, washing, and centrifugation of this invention.

[0050] In this article, "neutral protease," also referred to as dispersase, is derived from Bacillus subtilis and is an endonuclease used in various protein hydrolysis processes. It exhibits relatively mild proteolytic activity, causes minimal cell damage, and maintains cell membrane integrity, making it suitable for isolating primary and subcultured cells. Neutral protease can also be used to eliminate cell aggregation that occurs during suspension cell culture.

[0051] The technical solution of the present invention will be described in detail below:

[0052] The inventors previously detected the distribution of lipopolysaccharide (LPS) in the colorectal tissue of colorectal cancer patients, such as... Figure 2 As shown, LPS is mainly distributed on the surface of intestinal tissue and within the epithelial cells and macrophages of the tissue. (LPS is a component of the outer wall of Gram-negative bacterial cell walls, mainly composed of lipids and polysaccharides, and can characterize the distribution of Gram-negative bacteria, such as Escherichia coli). This suggests that intestinal tissue microbiota is affected by intestinal feces, and microorganisms adhere to the intestinal epithelial mucosa on the luminal side in contact with feces. These microorganisms can interfere with the detection of microorganisms within tumor tissue.

[0053] The inventors initially attempted to reduce interference from fecal intestinal microorganisms in the detection of microorganisms within tumor tissue by using different sampling sites and multiple washing processes. They observed that intestinal flora contamination significantly impacts the detection of microbiota in colorectal cancer tissue. Whether the submitted colorectal cancer tissue contains intestinal mucosa, and its proportion, directly affects the detection results. Different washing processes also have an impact, but it is much smaller than the influence of whether mucosa is present, and can be avoided by maintaining consistent washing conditions during tissue processing. Normal tissue serves as a control group. Since the epithelial cells in the mucosa are the origin of colorectal cancer, mucosa cannot be removed during sampling. Therefore, this method of removing mucosa during sampling cannot control uniform conditions. Furthermore, fecal intestinal microorganism contamination is far greater than the microorganisms within the tissue itself. Previous conventional methods involved directly submitting colorectal cancer tissue blocks or colonoscopy biopsies, which mostly detected fecal microorganisms within the tissue structure, making it difficult to accurately detect tissue-related microorganisms.

[0054] There is a lack of accurate, effective, and standardized detection methods for colorectal tissue, especially for microorganisms associated with colorectal cancer and normal intestinal tissue.

[0055] Methods for detecting microorganisms associated with colorectal cancer cells.

[0056] In view of the existing problems, according to some aspects of the present invention, a method for detecting colorectal cancer cell-associated microorganisms is provided, the method comprising:

[0057] The steps include: initially excluding fecal microbial contamination from intestinal folds and villi in the sample; further excluding fecal microbial contamination; and removing contaminating and transient bacteria.

[0058] In some implementations, single cells obtained after steps to remove contaminating and transient bacteria can be used as test samples.

[0059] According to other aspects of the present invention, a method for isolating colorectal cancer tumor cell-associated microorganisms is provided, the method comprising: a step of initially excluding contamination from fecal microorganisms in the intestinal folds and villous structures of a sample; a step of further excluding contamination from fecal microorganisms; and a step of removing contaminating bacteria and transient bacteria. The method for isolating colorectal cancer tumor cell-associated microorganisms effectively separates microorganisms colonizing or within colorectal cancer cells (i.e., colorectal cancer cell-associated microorganisms) from fecal microorganisms, contaminating bacteria, and transient bacteria.

[0060] According to other aspects of the present invention, a sample pretreatment method for detecting colorectal cancer cell-associated microorganisms is provided. The method includes: a step of initially excluding contamination from fecal microorganisms in the intestinal folds and villous structures of the sample; a step of further excluding contamination from fecal microorganisms; and a step of removing contaminating bacteria and transient bacteria. Single cells to be tested are obtained through this pretreatment method. Using the single cells to be tested obtained by this pretreatment method as a test sample, tumor cell-associated microorganisms of colorectal cancer can be effectively obtained.

[0061] In this invention, "colorectal cancer cell-associated microorganisms" refers to microorganisms that invade colorectal cancer cells or firmly colonize the cell surface, and are closely related to the occurrence and development of colorectal cancer.

[0062] In this invention, the "contaminating bacteria" or "transit bacteria" are microorganisms that exist in the intestines but do not colonize them. They remain in the host body for several hours, days or weeks, are mainly distributed in feces on the surface of intestinal tissues, and do not invade colorectal tumor tissues and tumor cells or have difficulty attaching to the surface of tumor cells.

[0063] In some embodiments, the sample includes colorectal tissue, and further, the colorectal tissue includes colorectal cancer tissue and normal intestinal tissue.

[0064] (Steps to initially rule out microbial contamination in the sample)

[0065] In some implementations, the step of initially excluding fecal microorganisms from the intestinal folds and villi of the sample involves breaking down the intestinal structure through steps such as coarse shearing and enzymatic digestion, and preparing and collecting a cell suspension to initially exclude fecal microorganisms from the intestinal structure.

[0066] Rough cut

[0067] In some implementations, the preliminary step of excluding microbial contamination from the sample includes a step of coarsely cutting the sample (e.g., colorectal cancer tissue / normal intestinal tissue). This coarse cutting step involves arbitrarily trimming the sample into small pieces to facilitate more thorough enzymatic digestion into single cells later. For example, the sample (e.g., colorectal cancer tissue / normal intestinal tissue) can be cut into pieces of 1–2 mm. 3 Small pieces of various sizes.

[0068] In some specific implementations, after removing surrounding fat, blood vessels, and blood from the sample (e.g., colorectal cancer tissue / normal intestinal tissue), it is cut into 1–2 mm pieces. 3 Small pieces of various sizes.

[0069] In some embodiments of the present invention, the colorectal tissue is washed at least once before coarsely cutting the sample. To remove as much contamination as possible from the surface of the colorectal tissue, the number of washes can be appropriately increased, for example, 2, 3, 4, 5, 6, 7, 8, or 9 washes. To save time and cost while achieving the desired cleaning effect, three washes are performed. There are no special restrictions on the water used during the washing process, such as deionized water, physiological saline, or phosphate buffer. To maintain cell morphology, sterile physiological saline, especially 0.9% (m / v) physiological saline, is preferred.

[0070] Enzymatic digestion

[0071] In some embodiments of the present invention, the coarsely shredded tissue blocks are enzymatically digested into cells using enzymatic digestion technology. Any enzyme capable of breaking down tissue into individual cells without damaging the cells can be used in this invention. In some embodiments, the enzymatic digestion includes enzymatically digesting the coarsely shredded sample under the action of an enzyme catalyst to obtain a cell suspension. In some preferred embodiments, in order to disrupt intestinal folds and villi (to exclude contamination by fecal microorganisms) while preserving cell integrity, neutral proteases and collagenases are used to enzymatically digest the colorectal tissue, instead of pancreatic enzymes. In some exemplary embodiments, the neutral protease is neutral protease II, and the collagenase is collagenase IV.

[0072] In some specific implementations, enzymatic hydrolysis is carried out in an enzymatic hydrolysis system containing neutral protease and collagenase in equal mass ratios; the hydrolysis temperature is 37°C; and the hydrolysis time is 20–40 minutes, preferably 30 minutes.

[0073] (Further steps to eliminate fecal microbial contamination)

[0074] Based on enzymatic digestion, the cell suspension obtained from enzymatic digestion is screened. Specifically, it is filtered through a cell filter to remove incompletely digested tissue blocks. In order to further eliminate contamination from intestinal fecal bacteria, the pore size of the cell filter is 40μm. After filtering the cell suspension obtained from enzymatic digestion to remove incompletely digested tissue blocks, a single-cell suspension is collected.

[0075] (Steps to remove contaminating and transient bacteria)

[0076] In some implementations, the steps of removing contaminating and transient bacteria involve centrifugation and repeated washing of the single-cell suspension to ultimately retain only microorganisms firmly colonized on or inside the cells.

[0077] In some specific implementations, the single cells obtained after sieving are washed and centrifuged multiple times. Specifically, the single cells are collected by centrifugation after washing. In some exemplary embodiments, in order to retain as many intracellular or firmly colonized microorganisms as possible, while separating intercellular or loosely adhered contaminants or transient bacteria from the cells in the supernatant, the centrifugation force is maintained at 280–300 g, preferably 300 g. There are no particular limitations on the solution used for washing the single cells; any solution used for washing cells in the art is acceptable, with phosphate-buffered saline (PBS) being preferred, especially phosphate-buffered saline with a pH of 7.2–7.4.

[0078] (Sample testing)

[0079] In some embodiments of methods for detecting colorectal cancer cell-associated microorganisms, the detection of a test sample includes the detection of colorectal cancer cell-associated microorganisms in the test sample, which includes any method capable of sequencing the microorganisms, including the detection of sequences of bacterial 16S rRNA, fungal 18S rRNA, and / or fungal ITS. Exemplarily, the method includes using 5R 16S sequencing technology (i.e., 5R 16S rRNA gene sequencing technology) on the test sample.

[0080] Example

[0081] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0082] sample

[0083] From September 2022 to December 2022, postoperative pathological tissues for colorectal cancer were obtained from patients in the Department of Gastrointestinal Surgery at the Affiliated Hospital of Chengde Medical College, with informed consent and signature. Inclusion criteria: (1) patients diagnosed with colorectal cancer before surgery; (2) no history of colorectal surgery; (3) no use of antibiotics, corticosteroids, or probiotics within one month prior to specimen collection; (4) no familial adenomatous polyposis or hereditary nonpolyposis colorectal cancer; no inflammatory bowel disease (IBD), metabolic diseases (such as diabetes, obesity, hyperlipidemia), or infectious diseases; no severe liver or kidney disease; no immunodeficiency; (5) no special dietary habits; (6) patients who voluntarily joined the study. Exclusion criteria: (1) pregnant or lactating women; (2) patients with a previous diagnosis of cancer; (3) patients who had undergone neoadjuvant chemoradiotherapy; (4) patients who did not wish to join the study.

[0084] Reagents and consumables

[0085] Collagenase IV (C8160, Solarbio), neutral proteinase II (D6430, Solarbio, 0.5 U / mg), fetal bovine serum, Hanks' solution, PBS, physiological saline, DMEM medium (containing 20% ​​FBS, purchased from GIBCO), serum-free medium (purchased from GIBCO); culture dishes, cryovials, 15ml centrifuge tubes, 50ml centrifuge tubes, 40um cell filters (BD FALCON, 352340 cell filters), Pasteur tubes, ophthalmic scissors, ophthalmic forceps, 50ml centrifuge tube rack, alcohol lamp, 95% alcohol, 75% alcohol; all of the above were sterile.

[0086] Example 1

[0087] 1. Sampling

[0088] After obtaining informed consent and signing from the patient, normal intestinal tissue (located more than 5 cm from the tumor) and colorectal cancer tissue were harvested separately using sterile scissors and a scalpel after colorectal cancer surgery, with each sample containing no less than 300 mg.

[0089] 2. Cleaning

[0090] The collected samples were washed three times with physiological saline. The entire process was carried out under aseptic conditions. The washed colorectal cancer tissue and normal intestinal tissue were then divided into two portions.

[0091] 3. Enzymatic digestion of tissues into single cells

[0092] Take 300mg of cleaned colorectal cancer tissue and 300mg of normal intestinal tissue, respectively. Remove surrounding fat, blood vessels and blood in a laminar flow hood, place in a 1.5ml EP tube, and quickly cut into small pieces (approximately 1-2mm in size) with ophthalmic scissors. 3 The cells were transferred into a 15ml centrifuge tube containing neutral protease II (1mg / ml) and collagenase IV (1mg / ml), with a final enzyme volume of 4ml (2ml of each enzyme). The tissue block was digested at 37℃, with continuous pipetting during digestion. After 30 minutes, 20% DMEM medium was added to terminate the digestion. A 40µm cell sieve (i.e., a 40µm cell filter) was placed on a 50ml centrifuge tube to collect the single-cell suspension. The single-cell suspension was centrifuged at 300g for 5 minutes, the supernatant was discarded, and the cells were resuspended in 5ml PBS, washed, and centrifuged repeatedly for 3 times. Finally, the cells were resuspended in 500μl of serum-free medium and stored in liquid nitrogen for testing.

[0093] Environmental samples were taken at the same time as each experiment.

[0094] Finally, the tissue blocks that had been directly taken and washed only three times, the enzymatically digested cell suspension, and the environmental samples were sent together for testing.

[0095] 4. Testing and Results

[0096] The samples obtained above were tested using 5R 16S sequencing technology, and the results were analyzed.

[0097] result:

[0098] (1) Distribution of microorganisms in samples that have not undergone enzymatic digestion and those that have undergone enzymatic digestion

[0099] ① Quality control of sample sequencing data

[0100] Excluding environmental controls, a total of 4,606,353 raw reads (greater than 100,000 reads / sample) and 4,589,247 clean reads (104,301.0682 reads / sample) were obtained from 44 samples from 11 patients. The Q20 values ​​were all greater than 95%, and the Q30 values ​​were greater than 92%, with an average Q20 of 98.71% and a Q30 of 96.19% (Table 1). In Table 1, TC and PC represent tumor cells obtained after treatment, and TO and PO represent directly sampled tumor tissue and normal tissue, respectively.

[0101] Table 1

[0102]

[0103] ②Alpha diversity analysis

[0104] Alpha diversity analysis reflects species richness, evenness, and sequencing depth. Among them, Chao1 and Observed_species indices mainly reflect the number of species in the sample community; Goods_coverage reflects the bacterial community coverage of the sample; and Simpson / Shannon mainly reflects the overall species richness and evenness.

[0105] The results are shown in Table 2 and Figure 3 As shown, a Goods value (i.e., Goods_coverage) of 1 for each group indicates complete microbial coverage of the sample. The observed_species index and Chao1 abundance index showed that the number of species in the treated tumor cells (TC) and normal cells (PC) was significantly lower than that in the directly sampled tumor tissue (TO) and normal tissue (PO). The Simpson and Shannon indices also showed that the species diversity of the cell group (TC / PC) was lower than that of the tissue group (TO / PO). This indicates that by enzymatically digesting the tissue into single cells, the types and numbers of microorganisms are significantly reduced, suggesting that the method of this invention can significantly reduce interference from the gut microbiota and more accurately detect relevant microorganisms in the sample.

[0106] Table 2: Alpha Diversity Analysis

[0107]

[0108] (2) Beta diversity analysis

[0109] Beta diversity analysis reflects species differences between groups, mainly through methods such as principal component analysis (PCA), principal coordinates analysis (PCoA), analysis of similarities (ANOSIM), and unweighted pair-group method with arithmetic mean (UPGMA) to observe differences between samples.

[0110] like Figure 4 As shown, PCA and PCoA analyses revealed small differences in microbial distribution between tumor tissue (TO) and normal tissue (PO) groups, and small differences in microbial distribution between treated tumor cells (TC) and treated normal cells (PC) groups, but significant differences existed between cells and tissues. UPGMA analysis showed that the microbiome of tumor tissue samples from 8 out of 11 patients was similar to that of normal tissue samples. However, only 3 patients showed similarities between the microbiome of tumor cell samples and normal cell samples after enzymatic digestion; significant differences existed between tumor cell and normal cell samples in most patients. Figure 5 This indicates that the data from each group are consistent, and the enzymatic cell digestion procedure is effective.

[0111] The ANOSIM results (Table 3) showed significant differences between the enzymatically treated cell groups and tissue groups (PC_vs_PO, TC_vs_TO) (R>0, p<0), but no significant differences were observed within either the cell or tissue groups (TC_vs_PC, TO_vs_PO) (R<0, p>0). These results indicate a significant difference in microbial distribution between the enzymatically treated cell groups and the untreated tissue groups.

[0112] Table 3: ANOSIM Results

[0113]

[0114] The above results demonstrate that by treating the tissue according to the method of the invention before testing, the interference of fecal microorganisms is significantly removed.

[0115] (3) Results of microbial community testing

[0116] The results showed differences in the distribution and quantity of microbial species between conventional tissue samples and cell samples treated with the invented technology. For example... Figure 5 As shown:

[0117] A total of 22 phyla and 518 genera were detected in tumor tissue samples, 24 phyla and 594 genera in normal tissue samples, 21 phyla and 367 genera in tumor cell samples, and 24 phyla and 357 genera in normal cell samples.

[0118] As can be seen from the stacked bar chart of bacterial species distribution: at the phylum level ( Figure 5 The most abundant phyla in the tissue samples were Firmicutes and Bacteroidetes, consistent with reports from other studies. Proteobacteria were the most abundant phylum in the treated cell samples. The abundance of Firmicutes in tumor tissue was lower than in normal tissue, while the abundance of Firmicutes in the cell group was higher than in normal tissue. There was no significant difference in the number of actinomycetes between the tumor tissue group and the normal tissue group, but after treatment, the number of actinomycetes in the tumor cell group was significantly lower than that in the normal cell group.

[0119] At the genus level, the TC group was dominated by Pseudomonas, Bacteroides, Brevundimonas, and Lactobacillus. The PC group was dominated by Pseudomonas, Brevundimonas, Bacteroides, and Streptococcus, while the TO group was dominated by Bacteroides, Prevotella, and Fusobacterium. The PO group was dominated by Bacteroides, Prevotella, and Faecalibacterium. Figure 5 ).

[0120] Differences between groups were analyzed using LEfSe (Linear Discriminant Analysis Effect Size), and the results are as follows: Figure 6 As shown, there are a large number of differentially expressed microbiota between the treated cell samples and the untreated tissue samples.

[0121] Furthermore, combined cell and tissue analysis can predict bacterial motility. For example, a comparison of four groups revealed that, at both the cellular and tissue levels, the number of certain bacteria in tumors was significantly higher than in the normal group. For instance, *Fusobacterium*, *Eikenella*, *Shewanella*, *Listeria*, and *Akkermansia* were lower in tumors than in the normal group, suggesting a link between carcinogenesis and these bacteria. The proportions of certain bacteria in cells and tissues also changed significantly. For example, at the tissue level, *Gemella* was lower in the tumor tissue group than in the normal tissue group, but at the cellular level, tumor cells were significantly higher than normal cells, suggesting a significant increase in intracellular *Gemella* or its migration from extracellular to intracellular spaces in colorectal cancer. *Escherichia shigella* was higher in both tumor cells and tissues than in normal tissues. *Blautia* was higher in tumors at the tissue level than in normal tissues, but significantly lower at the cellular level than in normal tissues, suggesting a possible decrease or migration of this bacterium to extracellular spaces in colorectal cancer. Figure 7 ).

[0122] The distribution of microorganisms in cell groups treated by the method of this invention differs significantly from that in conventionally treated tissue groups.

[0123] In summary, the cell samples treated by the method of this invention show lower species and quantities of microorganisms than tissue samples, and significant differences in bacterial communities. This indicates that, on the one hand, the method of this invention significantly reduces fecal microbial contamination, allowing the differences in tumor-associated microorganisms, which are already at low biomass levels, to be revealed, thus avoiding masking by contaminating bacteria. On the other hand, combined with tissue detection results, bacterial movement can be predicted. For example, when the biomass of a certain species is high in both tumor tissue and tumor cells, it suggests bacterial enrichment and invasion into tumor cells; if there is no difference in bacterial biomass between tumor tissue and normal tissue, but the intracellular biomass increases, it suggests bacterial invasion into the tumor. Conventional detection methods can only detect bacterial enrichment in tissues, and it is difficult to detect bacterial movement.

[0124] In addition, cell enzymatic digestion can be combined with microbial culture technology to accurately detect tumor-related microorganisms. This method is more accurate than tissue block culture, but the number of microbial species is also reduced, thus narrowing the scope of research.

[0125] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0126] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for isolating microorganisms associated with the detection of colorectal cancer cells, characterized in that, The method includes: The steps to preliminarily exclude contamination from fecal microorganisms in the intestinal folds and villi of the sample include coarse cutting and enzymatic digestion of the sample; Further steps to eliminate fecal microbial contamination include filtering the cell suspension obtained from enzymatic digestion through a cell sieve to collect single-cell suspensions; and, The steps to remove contaminating and transient bacteria involved washing the single-cell suspension with phosphate buffer solution and centrifuging at 300g to obtain single cells. The enzymatic digestion includes enzymatically digesting and separating the coarsely shredded sample into a cell suspension under the action of an enzyme catalyst; The enzyme catalyst includes a neutral protease and a collagenase; the neutral protease is neutral protease II, and the collagenase is collagenase IV; The pore size of the cell sieve is 40 μm; The sample included colorectal tissue; Before coarsely cutting the sample, wash the sample three times with physiological saline.

2. The method according to claim 1, characterized in that, The colorectal tissue includes colorectal cancer tissue and normal tissue.

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