Application of rhodobacter harzicola in colorectal cancer diagnosis or prognosis

Abstract

The invention discloses application of rhodobryum harzianum in colorectal cancer diagnosis or prognosis, and belongs to the technical field of biomedicine.16SrRNA genome sequencing analysis finds that the content of rhodobryum harzianum in colorectal cancer tissue of a colorectal cancer patient is remarkably increased, the cancer promoting effect of the rhodobryum harzianum is further proved through a mouse AOM / DSS spontaneous tumor formation model, and the application of the rhodobryum harzianum in colorectal cancer diagnosis or prognosis is achieved. Compared with the prior art, the method provided by the invention has the advantages that the rhodobryum harzicola in a clinical sample is detected and a corresponding diagnosis model is established, so that the method has relatively high diagnosis efficiency and shows that the rhodobryum harzicola has important potential in clinical auxiliary diagnosis, treatment or prognosis evaluation of the colorectal cancer, a new way is provided for diagnosis, treatment or prognosis of the colorectal cancer, and the clinical application prospect is wide.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Rhodotorula haematobium in the diagnosis or prognosis of colorectal cancer. Background Technology

[0002] According to GLOBOCAN 2020 estimates of cancer incidence and mortality, colorectal cancer ranked third among new cancer cases in 2020, accounting for 10.0%. It was the second leading cause of cancer-related deaths, accounting for 9.4% (Sung. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021 May;71(3):209-249). The diagnosis and treatment of colorectal cancer remain challenging. Colorectal cancer is closely linked to gut microbiota dysbiosis, and intratumoral flora is closely related to the occurrence and development of colorectal cancer.

[0003] Increasing evidence suggests the importance of the tumor microbiota (Garrett WS. Cancer and the microbiota. Science. 2015 Apr 3;348(6230):80-6), and exploring the roles of different bacteria in colorectal cancer has potentially significant implications for its clinical treatment. Therefore, discovering new bacteria with functional responses to colorectal cancer at the microbial level holds promise for guiding the clinical diagnosis and treatment of colorectal cancer, providing new avenues and strategies for its diagnosis and treatment. Currently, there are no studies or reports on the use of *Rhodotorula harzianum* in the diagnosis, prognosis prediction, or treatment of colorectal cancer, and even fewer studies or reports on the use of intracellular antibiotics to treat colorectal cancer by killing intracellular *Rhodotorula harzianum*. Summary of the Invention

[0004] To overcome the aforementioned technical problems in this field, the present invention aims to provide the application of *Rhodotorula harzianum* in the diagnosis or prognosis of colorectal cancer. This invention is the first to discover that the abundance of *Rhodotorula harzianum* in tumor tissue of colorectal cancer patients is significantly increased compared to normal intestinal tissue, and that it is located intracellularly. Killing intracellular bacteria can restore the promoting effect of bacteria (*Rhodotorula harzianum*) on colorectal cancer. This invention provides a new approach for the research of novel therapeutic targets for colorectal cancer.

[0005] The present invention achieves the above-mentioned objectives by adopting the following technical solution: The first aspect of the present invention provides the use of a reagent for detecting the content of Rhodotorula harzianum in the preparation of products for the diagnosis or prognosis prediction of colorectal cancer.

[0006] Furthermore, the product includes testing reagents, testing kits, test strips, or testing chips; Optionally, the reagents include those for detecting the content of Rhodotorula harzianum in tissue samples using 16S rRNA sequencing, whole genome sequencing, quantitative polymerase chain reaction, PCR-pyrosequencing, fluorescence in situ hybridization, microarray and / or PCR-ELISA. Optionally, the content of *Rhodotorula harzianum* in the tissues of colorectal cancer patients is higher than that in the intestinal tissues of healthy individuals.

[0007] Furthermore, the detection reagent includes primers, probes, antisense oligonucleotides, aptamers, and / or antibodies that are specific to the *Rhodotorula haematobium*. Optionally, the test kit includes reagents for detecting the content of Rhodotorula harzianum in tissue samples; Optionally, the test strip includes a substrate material, a test line and a control line fixed on the substrate, wherein the test line is coated with an antibody or nucleic acid probe that can specifically bind to Rhodotorula harzianum; Optionally, the detection chip includes a solid support and a reagent fixed on the solid support for detecting the content of Rhodotorula harzianum in a tissue sample.

[0008] In this invention, *Hungatella hathewayi* (H. hathewayi for short) is a strictly anaerobic bacterium that was formally identified and named in 2001. This bacterium is known to colonize the intestinal systems of normal humans and mice, and participates in the synthesis and degradation of metabolites such as butyrate, ethanol, and carbon dioxide. Because this bacterium is a relatively new species, current research on its function is very limited. To date, there are no studies or reports suggesting that this bacterium may have a protective or pathogenic effect against colorectal cancer.

[0009] In this invention, the detection of *Rhodotorula harzianum* content refers to the process of qualitative and / or quantitative analysis of *Rhodotorula harzianum* present in tissue samples obtained from human subjects (such as colorectal cancer patients or healthy individuals) using specific molecular biological or immunological techniques. Its core objective is to assess its correlation with the occurrence, development, or prognosis of colorectal cancer by accurately measuring the quantity (absolute copy number) or relative abundance (proportion compared to other bacteria) of this bacterium in the sample.

[0010] In some embodiments, the detection reagents are a collective term for a class of chemical or biological substances capable of specifically recognizing and binding to unique biomarkers of *Rhodotorula harzianum*. These reagents are designed for *Rhodotorula harzianum*-specific gene sequences (such as specific segments of the 16S rRNA gene) or proteins, and include, but are not limited to: specific primers (for PCR amplification), probes (for fluorescence signal detection in fluorescence in situ hybridization (FISH) or qPCR), antisense oligonucleotides, aptamers (a novel type of nucleic acid antibody), and antibodies (for recognizing bacterial surface antigens). They are fundamental tools for achieving highly specific and sensitive detection.

[0011] In this invention, the primer is a short single-stranded DNA or RNA fragment that serves as the starting point for DNA replication. When detecting *Rhodotorula harzianum*, specific primers targeting the species-specific gene sequences (such as specific variable regions of the 16S rRNA gene) need to be designed. Typically, a pair of primers (forward and reverse) is used, which specifically bind to both ends of the *Rhodotorula harzianum* DNA template. During polymerase chain reaction (PCR) or qPCR, DNA polymerase uses the primer as a starting point to synthesize a new strand complementary to the template, thereby achieving exponential amplification of the target gene fragment.

[0012] In this invention, the probe is an oligonucleotide sequence labeled with a reporter gene (such as a fluorescent group). In detection methods such as real-time quantitative PCR (qPCR), the probe specifically hybridizes to the target DNA fragment of *Rhodotorula harzianum* amplified by primers. When the probe is intact, its fluorescent group is inhibited by a quenching group; during PCR amplification, the 5'→3' exonuclease activity of Taq polymerase degrades the probe bound to the template, releasing the fluorescent group and causing it to fluoresce. The system accurately quantifies *Rhodotorula harzianum* DNA by monitoring the fluorescence signal intensity in real time. The probe provides higher specificity than simply using primers.

[0013] In this invention, the antisense oligonucleotide is a single-stranded DNA or RNA molecule whose sequence is complementary to a specific target RNA (such as the mRNA or rRNA of *Rhodotorula harzianum*). By designing antisense oligonucleotides targeting essential gene mRNAs of *Rhodotorula harzianum*, specific binding can be achieved. This binding can directly block the translation process of the target RNA, thereby inhibiting bacterial protein synthesis; or it can recruit intracellular ribonuclease H to degrade the target RNA. In detection, the labeled antisense oligonucleotide can be used to directly detect and trace the presence of specific RNAs within bacteria.

[0014] In this invention, the aptamer is a short single-stranded DNA or RNA oligonucleotide obtained through in vitro screening technology (SELEX), capable of binding to target molecules (such as specific proteins on the surface of *Rhodotorula harzianum*) with high affinity and high specificity, much like an antibody. Compared to antibodies, aptamers have advantages such as small molecular weight, ease of in vitro synthesis and modification, good stability, and low immunogenicity. In detection, the aptamer can be labeled with a fluorescent molecule or a reporter group such as biotin to develop sensitive ELISA-like detection methods or biosensors, enabling rapid and low-cost detection of *Rhodotorula harzianum*.

[0015] In some embodiments, the antibody is a Y-shaped protein produced by the immune system that specifically recognizes and binds to a specific antigen, such as the characteristic polysaccharide, lipoprotein, or flagellin on the surface of *Rhodotorula harzianum*. Polyclonal or monoclonal antibodies against *Rhodotorula harzianum* can be prepared by immunizing animals with purified *Rhodotorula harzianum* or specific components thereof. These antibodies can be used in various immunological assays, such as enzyme-linked immunosorbent assay (ELISA), Western blotting, immunohistochemistry (IHC), or immunofluorescence (IF). The advantage of antibody-based detection methods is that they can directly detect intact bacteria or their specific antigens, are relatively simple to operate, and are suitable for the development of rapid clinical test strips or kits.

[0016] In some implementations, the detection kit refers to a commercially available product that standardizes and packages all the reagents, consumables, and instructions required for detecting *Rhodotorula harzianum*. It typically includes core detection reagents (such as the aforementioned specific primers and probes), buffer solutions for sample processing, necessary enzymes (such as DNA polymerase), a positive control (a sample containing the *Rhodotorula harzianum* sequence), and a negative control (a sample not containing the sequence). The advantage of this detection kit is that all components are optimized and pre-prepared; users only need to follow standard procedures, greatly improving the convenience, standardization, and comparability of results, facilitating its widespread application in clinical or research laboratories.

[0017] In some implementations, the test strip refers to a rapid detection device based on immunochromatography or similar principles, typically presented in strip form. Its structure includes a substrate material (such as a nitrocellulose membrane) with two key lines pre-set on the membrane: a detection line (coated with an antibody or nucleic acid probe capable of capturing Rhodotorula harzianum) and a control line (indicating whether the test strip is functioning correctly). When a processed sample is dropped onto one end of the test strip, the sample chromatographically crosses the membrane. If Rhodotorula harzianum is present, it binds to the marker and a color reaction occurs at the detection line. The advantages of this method are its simplicity, speed (results typically within minutes), and lack of complex equipment, making it suitable for rapid on-site screening.

[0018] In some implementations, the detection chip refers to a miniature analytical system in which thousands of different detection probes are densely immobilized on a solid support (such as a glass slide or silicon wafer). For a detection chip targeting *Rhodotorula harzianum*, a large number of nucleic acid probes or antibodies capable of specifically recognizing different characteristic sequences of this bacterium are immobilized on its surface. When a labeled sample flows through the chip, a large number of parallel detections can be performed simultaneously in a single reaction, achieving high-throughput analysis. This method is extremely efficient, not only confirming the presence of *Rhodotorula harzianum* but also enabling analysis of its strain typing or interactions with other bacterial groups.

[0019] In some embodiments, any method well known to those skilled in the art can be used to detect or determine the level of the microbial marker (i.e., *Rhodotorula harzianum*). These methods include, but are not limited to, methods utilizing primer sequence amplification and immunological methods using antigen-antibody reactions. Primer sequence amplification methods can include, for example, polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), multiplex PCR, landing PCR, hot-start PCR, nested PCR, enhancement PCR, real-time PCR, differential PCR, rapid amplification of cDNA ends, reverse polymerase chain reaction, vector-mediated PCR, thermal asymmetric interleaved PCR, ligase chain reaction, repair chain reaction, transcription-mediated amplification, autonomous sequence replication, and selective amplification of target base sequences. Immunological methods using antigen-antibody reactions can include, for example, Western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, Eustachian immunodiffusion, tissue immunostaining, immunoprecipitation, complement fixation, fluorescently activated cell sorters, protein chips, etc., but the scope of the invention is not limited thereto.

[0020] In some embodiments, the tissue sample is derived from the subject to be tested, which refers to any animal subject, including but not limited to humans, laboratory animals, livestock, and domestic pets. The subject may harbor a variety of microorganisms. The subject may have different microbiomes in various habitats on and within their body. The subject may be diagnosed with a disease or suspected of having a disease. The subject may have a disease-causing microbiome state (ecological imbalance). In other embodiments, the subject is preferably a human.

[0021] In some implementations, the diagnosis refers to using the significantly higher levels of *Rhodotorula harzianum* in the tissues of colorectal cancer patients compared to healthy individuals as a biomarker to assist physicians in determining whether a subject has colorectal cancer. Its core application lies in the early detection and identification of the disease. Specifically, the levels of *Rhodotorula harzianum* in clinical samples (such as intestinal tissue) are detected and compared to thresholds established through large-scale clinical studies. If the detected value is significantly higher than that of healthy controls, it suggests a high probability that the subject has colorectal cancer. This provides a novel, microbiome-based objective indicator for the early screening and diagnosis of colorectal cancer, particularly suitable for assisting traditional methods such as endoscopy, improving the accuracy and convenience of diagnosis.

[0022] In some implementations, prognostic prediction refers to assessing the future progression of a patient's disease, risk of recurrence, or potential response to treatment by detecting the levels of *Rhodotorula harzianum* in the tumor tissue of a diagnosed colorectal cancer patient, thereby providing a basis for developing individualized treatment plans. Its core objective is to predict disease outcomes and guide treatment decisions. For example, this invention has demonstrated through animal models that *Rhodotorula harzianum* promotes cancer progression. Therefore, detecting high levels of *Rhodotorula harzianum* in a patient's tumor may predict faster disease progression, a higher risk of recurrence, or a worse survival outcome. This information can help clinicians determine the patient's risk stratification; for high-risk patients, more aggressive or cutting-edge treatment strategies may be preferred (such as considering antibiotics targeting this bacterium), thus achieving precision medicine.

[0023] A second aspect of the present invention provides a product for the diagnosis or prognosis prediction of colorectal cancer.

[0024] Furthermore, the product includes reagents for detecting the content of Rhodotorula haematobium in tissue samples.

[0025] In some embodiments, the reagent is a reagent for detecting the content of Rhodotorula harzianum in the tissue sample using any one or more of the following detection methods: 16S sequencing, whole genome sequencing, quantitative polymerase chain reaction, PCR-pyrosequencing, fluorescence in situ hybridization, microarray and / or PCR-ELISA.

[0026] In some implementations, the product is a testing reagent, a testing kit, a test strip, or a testing chip.

[0027] A third aspect of the present invention provides a system or apparatus for the diagnosis or prognosis prediction of colorectal cancer.

[0028] Furthermore, the system or apparatus includes: (1) Tissue sample DNA extraction unit: used to extract microbial DNA from subject tissue samples; (2) DNA sample screening and pretreatment unit: used to screen and pretreatment DNA samples obtained from the tissue sample DNA extraction unit, quantitatively detect bacterial 16S rRNA fragments in DNA samples using qPCR technology, remove samples that are negative by qPCR, and enrich 16S rRNA gene V4 fragments in qualified samples. (3) Sequencing unit: used to sequence the enriched and amplified fragments obtained from the DNA sample screening and pretreatment unit to obtain sequencing results; (4) Data processing unit: used to detect and analyze the relative abundance of Rhodotorula harzianum in tissue sample flora based on the sequencing results obtained by the sequencing unit, and obtain the relative abundance value of Rhodotorula harzianum; (5) Result determination unit: used to compare the relative abundance value of Rhodotorula harzianum obtained by the data processing unit with the set diagnostic threshold, obtain the colorectal cancer diagnosis result, and store the result in the data carrier; Optionally, the data carrier is a computer-readable storage medium; Optionally, if the relative abundance value of Rhodotorula harzianum in the subject's tissue sample is higher than the diagnostic threshold, it is judged as a suspected positive for colorectal cancer; otherwise, it is judged as negative.

[0029] A fourth aspect of the present invention provides the use of *Rhodotorula haematobium* in the preparation of reagents that promote animal models of colorectal cancer; Optionally, the bacterial concentration of *Rhodotorula haematobium* is 1-5 × 10⁻⁶. 9 CFU; Optionally, the colorectal cancer animal model is an AOM / DSS-induced colorectal cancer animal model; Optionally, the *Rhodotorula haematobacterium* enters the cells and promotes the progression of colorectal cancer.

[0030] In a specific embodiment of the present invention, colorectal cancer is induced in mice by gavage with *Rhodotorula harzianum*, intraperitoneal injection with AOM, and drinking DSS. Mice treated with *Rhodotorula harzianum* showed significantly more and larger colorectal tumors than those in the PBS group and the control bacterial group, and the progression of colorectal cancer was more rapid, indicating that *Rhodotorula harzianum* is a type of bacteria that can promote the occurrence and development of colorectal cancer.

[0031] In a specific embodiment of the present invention, colorectal cancer is induced in mice by subcutaneously injecting MC38 colon cancer cells containing *Rhodotorula harzianum*. Mice treated with cells containing *Rhodotorula harzianum* were significantly larger than those in the bacterial-killed group and the control group, and their colorectal cancer progressed more rapidly, indicating that *Rhodotorula harzianum* is a type of bacteria that can survive within colorectal cancer cells and promote colorectal cancer progression.

[0032] The fifth aspect of the invention provides the use of intracellular bacterial antibiotics in the preparation of medicaments for the treatment of colorectal cancer, wherein the intracellular bacterial antibiotics are tetracycline, doxycycline, minocycline, erythromycin, azithromycin, clarithromycin, roxithromycin, levofloxacin, moxifloxacin, ciprofloxacin, or gatifloxacin.

[0033] Furthermore, the intracellular bacterial antibiotic is tetracycline.

[0034] Furthermore, the intracellular antibiotic treats colorectal cancer by killing intracellular Rhodotorula harzianum.

[0035] This invention provides a novel treatment strategy for colorectal cancer by using antibiotics capable of penetrating cell membranes to target and kill *Rhodotorula harzianum* parasitizing tumor cells, thereby achieving therapeutic goals. These antibiotics are known as intracellular antibiotics, including tetracyclines, macrolides, and fluoroquinolones, all of which share the characteristic of effectively entering host cells and acting on intracellular bacteria. This invention particularly favors tetracycline as a representative drug, which has been demonstrated in animal model experiments described in this application to significantly inhibit tumor growth by eliminating intracellular *Rhodotorula harzianum*.

[0036] This invention is the first to discover and confirm that *Rhodotorula harzianum* can exist as an intracellular bacterium in colorectal cancer cells and directly promote the malignant progression of cancer. Therefore, traditional antibiotics that primarily target extracellular bacteria (such as penicillin and gentamicin) are ineffective against this, while antibiotics that can enter cells can precisely eliminate this cancer-driving factor. In a specific embodiment of this invention (animal experiments), the use of tetracycline to kill intracellular bacteria significantly inhibited tumor growth, providing direct principle verification and experimental evidence for the use of "antibacterial therapy" to treat cancers driven by specific bacteria.

[0037] The present invention also provides a method for diagnosing colorectal cancer or predicting the prognosis of colorectal cancer patients, the method comprising the following steps: detecting the content of Rhodotorula harzianum in a tissue sample from a subject, and based on this, determining whether the subject has colorectal cancer or its prognosis.

[0038] In some implementations, the method determines whether a subject has colorectal cancer (diagnosis) or its disease progression (prognosis) by quantitatively detecting the amount of Rhodotorula harzianum in a subject's tissue sample and comparing that amount with known standards (such as baseline levels in healthy individuals or thresholds related to clinical outcomes).

[0039] In some implementation schemes, various molecular biology techniques (such as qPCR or 16S rRNA sequencing) can be used to perform absolute or relative quantification of *Rhodotorula harzianum* DNA in tissue samples from subjects, obtaining accurate bacterial load data. The key to this step is using highly specific primers and probes targeting *Rhodotorula harzianum*-specific gene sequences (such as the 16S rRNA variable region) to ensure that the detection results accurately reflect the true abundance of this bacterium in the tumor microenvironment, providing a reliable experimental basis for subsequent judgment.

[0040] In diagnostic applications, the judgment process relies on comparing the subject's test results with diagnostic thresholds established in advance through large-scale clinical studies. For example, ROC curve analysis can be used to determine the optimal threshold for distinguishing colorectal cancer tissue from normal tissue. If a subject's test value is significantly higher than this threshold, it suggests a very high probability of having colorectal cancer; conversely, the risk is lower. This method provides a novel and objective microbiological indicator for the early screening and auxiliary diagnosis of colorectal cancer, and is particularly suitable for supplementing traditional examination methods such as endoscopy.

[0041] In terms of prognostic prediction, the judgment logic focuses on correlating the *Rhodotorula harzianum* content in confirmed patients with their clinical outcomes. Based on animal experiments in this application, *Rhodotorula harzianum* can directly promote tumor progression, and high bacterial loads are considered a marker of highly aggressive disease. By analyzing patient population data to establish a prognostic risk threshold, if a patient's bacterial load exceeds this threshold, a higher risk of recurrence and a poorer prognosis are predicted, potentially requiring more aggressive treatment strategies and close follow-up. This not only achieves biological prediction of disease progression trends but also provides crucial decision-making basis for individualized precision treatment of colorectal cancer.

[0042] The sixth aspect of the present invention provides for any of the following applications: (1) Application of Rhodotorula harzianum in the study of colorectal cancer microecology, wherein the application is related to the diagnosis or treatment of colorectal cancer; (2) The use of reagents for detecting the abundance or content of Rhodotorula harzianum in tissue samples in the preparation of systems or devices for the diagnosis or prognosis prediction of colorectal cancer.

[0043] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: This invention is the first to discover that *Rhodotorula harzianum* has a significantly increased abundance in tumor tissue of colorectal cancer patients compared to normal intestinal tissue. *Rhodotorula harzianum* is located intracellularly, and its promotion of colorectal cancer can be reversed by using intracellular antibiotics to kill the intracellular bacteria. Furthermore, through dual validation using a screening set and a validation set composed of real clinical samples, this invention demonstrates that *Rhodotorula harzianum* exhibits excellent diagnostic efficacy against colorectal cancer, with high accuracy, sensitivity, and specificity. This invention provides a key tool for the early diagnosis and accurate assessment of colorectal cancer, and also offers novel ideas and strategies for the development of biopsy diagnostic products and new therapeutic targets for colorectal cancer, showing broad prospects for clinical application. Attached Figure Description

[0044] Figure 1 The content of Rhodotorula harzianum was significantly increased in the intestinal cancer tissue of colorectal cancer patients. Figure A shows the relative content of Rhodotorula harzianum in the control group and colorectal cancer patient tissues by 16S rRNA sequencing analysis; Figure B shows that the abundance of Rhodotorula harzianum was significantly higher in colorectal cancer tissues by fluorescence in situ hybridization staining of colorectal cancer tissues and normal intestinal tissues. Figure 2 Figure 1 shows the results of *Rhodotorula harzianum* promoting the progression of colorectal cancer in mice. Figure A illustrates the process of AOM / DSS-induced spontaneous tumor formation in mice. Intestinal flora was cleared with antibiotics 10 days prior to treatment. Following this, mice were induced to develop colorectal cancer through intraperitoneal injection of AOM and DSS via drinking water. During the DSS-drinking interval, the experimental group received daily gavage with *Rhodotorula harzianum*. After three cycles of induction, mice were sacrificed and photographs of the colorectal cancer burden were taken. Figure B shows the tumor-bearing status of the colorectal region in mice. Figure C shows the proportion of colorectal tumors in mice. Control represents the control group, Fn represents the *Fusobacterium nucleatum* group, and Hh represents the *Rhodotorula harzianum* treatment group. Figure 3 The study investigated the effect of antibiotics on the inhibition of subcutaneous tumor progression in mouse intestinal cancer by killing Rhodotorula harzianum. Figure A shows the effect of antibiotics on Rhodotorula harzianum on mouse subcutaneous tumors; Figure B shows the statistical analysis of subcutaneous tumor volume in different groups of mice; Figure C shows the statistical analysis of subcutaneous tumor weight in different groups of mice. PBS was the control group; Bac-ABX represented the group treated with tetracycline to kill Rhodotorula harzianum; Fn represented the group treated with Fusobacterium nucleatum; and Hh represented the Rhodotorula harzianum treatment group. Figure 4The predictive power of *Rhodotorula harzianum* in the screening and validation sets is shown in Figure A: *Rhodotorula harzianum* abundance box plot, showing that the abundance of *Rhodotorula harzianum* in colorectal cancer tissues was significantly higher than that in normal tissues in the screening set; Figure B: ROC curve of *Rhodotorula harzianum* used to diagnose colorectal cancer in the screening set; Figure C: *Rhodotorula harzianum* abundance box plot, showing that the abundance of *Rhodotorula harzianum* in colorectal cancer tissues was significantly higher than that in normal tissues in the validation set; Figure D: ROC curve of *Rhodotorula harzianum* used to diagnose colorectal cancer in the validation set. Detailed Implementation

[0045] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. The experimental consumables, reagents, and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following embodiments are for illustrative purposes only and should not limit the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0046] Example 1: The content of Rhodotorula harzianum was significantly increased in the colorectal cancer tissue of patients with colorectal cancer. 1. Sample Source In this study, the applicant collected 710 colorectal cancer tissue samples and 227 normal tissue samples (normal mucosal tissue samples), and extracted DNA from the tissues using the QIAamp PowerFecal (pro) DNA kit (QIAGEN #51804) in conjunction with a tissue disruptor.

[0047] The colorectal cancer tissue samples and normal tissue samples included in this study were all from the outpatient and inpatient populations of Sir Run Run Shaw Hospital, affiliated with Zhejiang University School of Medicine. The inclusion and exclusion criteria for colorectal cancer patients are as follows: (1) Inclusion criteria Histopathological examination confirmed the diagnosis as colorectal cancer. The patient's age at the time of surgery or procedure is between 18 and 90 years old; No history of anti-cancer treatment prior to sample acquisition; The biobank contains high-quality frozen tissue specimens that can be used for DNA extraction and 16S rRNA sequencing; Electronic medical records contain complete and essential clinical and pathological data.

[0048] (2) Exclusion criteria Antibiotics, prebiotics, or probiotics were used within one month prior to the procedure; Combined diagnosis of other gastrointestinal diseases; Previous history of colorectal resection; The organization is of substandard quality or lacks key clinical data.

[0049] 2. Experimental Methods To ensure the success rate of subsequent sequencing, the 16S fragment (bacterial content) in the sample DNA was first quantitatively detected by qPCR. Samples with negative qPCR results were discarded, and the remaining samples underwent 16S V4 fragment enrichment and amplification, as follows: (1) Synthetic biotin-labeled 16S V4 fragment primers (containing sequencing adapter sequences), see the table below:

[0050] (2) Perform PCR amplification for 25-30 cycles using the kit required by the sequencer (the number of cycles needs to be determined based on the bacterial content in the tissue); (3) The biotin-labeled amplified fragments were enriched and precipitated using Dynabeads MyOne Streptavidin C1 Beads (Thermo 65002).

[0051] The enriched and precipitated fragments (without eluting the magnetic beads) are then used to construct sequencing libraries.

[0052] 3. Experimental Results The experimental results are shown in Figure 1 ,like Figure 1 As shown in Figure A, compared to the normal intestinal tissue of the control group, the content of *Rhodotorula harzianum* was significantly increased in tissue samples derived from colorectal cancer tissue. Figure 1 As shown in Figure B, FISH staining of colorectal cancer tissue and normal intestinal tissue sections revealed that the abundance of Rhodotorula harzianum was significantly higher in colorectal cancer tissue.

[0053] Example 2: Rhodotorula haematobium promotes the progression of colorectal cancer in mice 1. Anaerobic culture of Rhodotorula haematobium in vitro Experimental method: Rhodotorula haematobium was inoculated into PYG medium and cultured in an anaerobic incubator at 37°C for 2 days. 3 days, concentration 1.0×10 9 CFU / mL, collected by centrifugation of Rhodotorula harzianum.

[0054] 2. Rhodotorula haematobacterium promotes the progression of colorectal cancer in mice. Experimental Methods: Ten days prior to treatment, the intestinal flora of mice was depleted with antibiotics. Following this, mice were intraperitoneally injected with AOM (10 mg / kg) and induced to develop intestinal cancer in drinking water with 1.5% DSS for 7 days, then switched to regular drinking water for 14 days. During the DSS drinking water interval, the experimental group mice were administered Rhodotorula harzianum via gavage daily (1.0 × 10⁻⁶). 8 CFU). After three cycles of induction, the mice were sacrificed and photographs of their colon and rectum were taken.

[0055] The method for constructing the AOM / DSS-induced colorectal cancer animal model includes the following steps: eliminating intestinal flora by gavage and drinking water with antibiotics; (1) injecting azomethane AOM (10 mg / kg) into the peritoneum of experimental mice; (2) replacing drinking water with 2.5% DSS for one week on the second day; (3) feeding with regular drinking water for 2 weeks; (4) repeating steps (2) and (3) 3 times.

[0056] 3. Experimental Results The results are as follows Figure 2 As shown, Figure 2 A describes the AOM / DSS-induced spontaneous tumorigenesis model in mice. Ten days prior to treatment, intestinal flora were cleared with antibiotics, followed by intraperitoneal injection of AOM and DSS via drinking water to induce intestinal cancer in mice. During the DSS-water interval, the experimental group received daily gavage with *Rhodotorula harzianum*. After three cycles of induction, mice were sacrificed and their colon and rectum were photographed. Figure 2 B represents the tumor-bearing status of the mouse's colorectal region; Figure 2 Figure C shows the proportion of colorectal tumors in mice. The results indicate that after colorectal cancer was induced in mice by gavage with Rhodotorula harzianum, the cancer progressed more rapidly, suggesting that Rhodotorula harzianum is a type of bacteria that promotes colorectal cancer.

[0057] Example 3: Antibiotics inhibit the progression of colorectal cancer in mice by killing Rhodotorula harzianum. 1. Experimental Methods Rhodotorula haematobium was labeled with CFSE at a multiplicity of infection (MOI) of 50 and co-cultured with MC38 cells for 5-6 hours. Cells containing CFSE fluorescence (i.e., cells with intracellular bacteria) were sorted by flow cytometry. The experimental group was treated with tetracycline (10 μg / mL) to kill both intracellular and extracellular bacteria; the control group was treated with penicillin (200 μg / mL) and gentamicin (200 μg / mL) to kill extracellular bacteria, and cultured overnight. Each mouse was injected with 5 × 10⁵ CFSE via unilateral groin injection. 5 Cells. After 4 weeks, mice were sacrificed and subcutaneous tumors were collected for size analysis.

[0058] 2. Experimental Results The results are as follows Figure 3As shown, the results indicate that the subcutaneous tumor size in the group where intracellular bacteria antibiotic (tetracycline) killed Rhodotorula harzianum was significantly smaller than that in the group where it was not killed, meaning that intracellular bacteria antibiotic can inhibit the progression of colorectal cancer by killing Rhodotorula harzianum.

[0059] Example 4: Predictive power of Rhodotorula haematobium on the screening and validation sets. 1. Experimental Methods The collected clinical samples (710 colorectal cancer tissue samples and 227 normal tissue samples) were randomly divided into a screening set and a validation set. In the screening set, the ratio of colorectal cancer tissue samples to normal tissue samples was 213:69, while in the validation set, the ratio was 497:158. The abundance difference of *Rhodotorula harzianum* in the screening and validation sets was validated, and the abundance difference results for the screening and validation sets were plotted. Furthermore, ROC curves for *Rhodotorula harzianum* in the screening and validation sets were plotted to verify its diagnostic efficacy.

[0060] The method for analyzing the abundance differences of *Rhodotorula harzianum* in the screening and validation sets is as follows: First, DNA was extracted from tissue samples using the QIAamp PowerFecal (pro) DNA kit (QIAGEN #51804) combined with a tissue disruptor. Then, the 16S fragment in the sample DNA was quantitatively detected by qPCR to remove negative samples. Subsequently, the remaining samples were enriched and amplified with 16S V4 fragments using biotin-labeled primers containing sequencing adapter sequences (specific sequences are shown in Example 1) (25-30 cycles). Then, the biotin-labeled amplified fragments were enriched and precipitated using Dynabeads MyOne Streptavidin C1 Beads (Thermo 65002). After that, a sequencing library was constructed, and finally, the abundance of *Rhodotorula harzianum* was determined by sequencing analysis. The core principle of using 16S rRNA sequencing technology to detect the abundance of Rhodotorula harzianum is to universally amplify conserved regions of bacterial 16S rRNA genes (such as the V4 region), and then compare the sequenced data (matching the sequenced fragments with a database of known Rhodotorula harzianum 16S rRNA gene sequences) to screen out the corresponding sequences of Rhodotorula harzianum and calculate their abundance. In this process, universal primers can meet the requirements of obtaining 16S fragments from all bacteria. Subsequently, bioinformatics analysis can be used to achieve specific identification and abundance statistics of Rhodotorula harzianum.

[0061] The method for analyzing the diagnostic efficacy of *Rhodotorula harzianum* in the screening and validation sets is as follows: For *Rhodotorula harzianum*, a biomarker obtained from Example 1 that shows significant differences between healthy tissues of healthy controls and colorectal cancer tissues of colorectal cancer patients, receiver operating function (ROC) curves were plotted using the R package "pROC". The accuracy, sensitivity, specificity, and AUC value of this biomarker for differentiating colorectal cancer patients from healthy controls were analyzed to determine its diagnostic efficacy in the screening and validation sets.

[0062] In assessing the diagnostic efficacy of the biomarker *Rhodotorula harzianum* in the screening and validation sets, the relative abundance level of *Rhodotorula harzianum* was used for analysis, and the level corresponding to the point with the largest Youden index was selected as its cutoff value. The resulting AUC value is the area under the ROC curve and the coordinate axis, ranging from 0.5 to 1. A higher AUC value indicates greater accuracy of the biomarker in differentiating colorectal cancer patients from healthy controls.

[0063] 2. Experimental Results In the screening set, the abundance of Rhodotorula harzianum differed significantly between colorectal cancer tissue samples and normal tissue samples. Compared with normal tissue samples, the content of Rhodotorula harzianum in colorectal cancer tissue samples was significantly increased and had a higher AUC value (AUC=0.709, sensitivity 67.6%, specificity 72.3%).

[0064] In the validation set, the abundance of Rhodotorula harzianum differed significantly between colorectal cancer tissue samples and normal tissue samples. Compared with normal tissue samples, the content of Rhodotorula harzianum in colorectal cancer tissue samples was significantly increased and had a higher AUC value (AUC=0.711, sensitivity 68.0%, specificity 73.0%).

[0065] Based on the above experimental results, it can be seen that Rhodotorula harzianum exhibits significant abundance differences in both the screening and validation sets composed of real clinical samples collected by this invention. Furthermore, Rhodotorula harzianum demonstrates high accuracy (AUC value), sensitivity, and specificity in both the screening and validation sets, making it suitable for accurate and effective diagnosis of colorectal cancer.

Claims

1. Application of reagents for detecting Rhodotorula haematobium content in the preparation of products for colorectal cancer diagnosis or prognosis prediction.

2. The application as described in claim 1, characterized in that, The products include testing reagents, testing kits, testing strips, or testing chips; Optionally, the reagents include those for detecting the content of Rhodotorula harzianum in tissue samples using 16S rRNA sequencing, whole genome sequencing, quantitative polymerase chain reaction, PCR-pyrosequencing, fluorescence in situ hybridization, microarray and / or PCR-ELISA. Optionally, the content of *Rhodotorula harzianum* in the tissues of colorectal cancer patients is higher than that in the intestinal tissues of healthy individuals.

3. The application as described in claim 2, characterized in that, The detection reagent includes primers, probes, antisense oligonucleotides, aptamers and / or antibodies that are specific to the Rhodotorula haematobium. Optionally, the test kit includes reagents for detecting the content of Rhodotorula harzianum in tissue samples; Optionally, the test strip includes a substrate material, a test line and a control line fixed on the substrate, wherein the test line is coated with an antibody or nucleic acid probe that can specifically bind to Rhodotorula harzianum; Optionally, the detection chip includes a solid support and a reagent fixed on the solid support for detecting the content of Rhodotorula harzianum in a tissue sample.

4. A product for the diagnosis or prognosis prediction of colorectal cancer, characterized in that, The product includes reagents for detecting the content of Rhodotorula harzianum in tissue samples.

5. A system or device for the diagnosis or prognosis prediction of colorectal cancer, characterized in that, The system or apparatus includes: (1) Tissue sample DNA extraction unit: used to extract microbial DNA from subject tissue samples; (2) DNA sample screening and pretreatment unit: used to screen and pretreatment DNA samples obtained from the tissue sample DNA extraction unit, quantitatively detect bacterial 16S rRNA fragments in DNA samples using qPCR technology, remove samples that are negative by qPCR, and enrich 16S rRNA gene V4 fragments in qualified samples. (3) Sequencing unit: used to sequence the enriched and amplified fragments obtained from the DNA sample screening and pretreatment unit to obtain sequencing results; (4) Data processing unit: used to detect and analyze the relative abundance of Rhodotorula harzianum in tissue sample flora based on the sequencing results obtained by the sequencing unit, and obtain the relative abundance value of Rhodotorula harzianum; (5) Result determination unit: used to compare the relative abundance value of Rhodotorula harzianum obtained by the data processing unit with the set diagnostic threshold, obtain the colorectal cancer diagnosis result, and store the result in the data carrier; Optionally, the data carrier is a computer-readable storage medium; Optionally, if the relative abundance value of Rhodotorula harzianum in the subject's tissue sample is higher than the diagnostic threshold, it is judged as a suspected positive for colorectal cancer; otherwise, it is judged as negative.

6. Application of Rhodotorula haematobium in the preparation of reagents for promoting animal models of colorectal cancer; Optionally, the bacterial concentration of *Rhodotorula haematobium* is 1-5 × 10⁻⁶. 9 CFU; Optionally, the colorectal cancer animal model is an AOM / DSS-induced colorectal cancer animal model; Optionally, the *Rhodotorula haematobacterium* enters the cells and promotes the progression of colorectal cancer.

7. The use of intracellular bacterial antibiotics in the preparation of drugs for treating colorectal cancer, characterized in that, The intracellular antibiotics are tetracycline, doxycycline, minocycline, erythromycin, azithromycin, clarithromycin, roxithromycin, levofloxacin, moxifloxacin, ciprofloxacin, or gatifloxacin.

8. The application as described in claim 7, characterized in that, The intracellular antibiotic is tetracycline.

9. The application as described in claim 7, characterized in that, The intracellular antibiotic treats colorectal cancer by killing intracellular Rhodotorula harzianum.

10. Applied to any of the following aspects: (1) Application of Rhodotorula harzianum in the study of colorectal cancer microecology, wherein the application is related to the diagnosis or treatment of colorectal cancer; (2) The use of reagents for detecting the abundance or content of Rhodotorula harzianum in tissue samples in the preparation of systems or devices for the diagnosis or prognosis prediction of colorectal cancer.

Images