Use of a bacterium of the genus blautia in the manufacture of a medicament for enhancing the sensitivity of colorectal cancer to oxaliplatin

Abstract

The application discloses application of a Blautia in preparation of a drug for enhancing sensitivity of colorectal cancer to oxaliplatin, B-CM can significantly enhance OXa cytotoxicity on drug-resistant colorectal cancer cells, inhibit proliferation, clone formation, migration and invasion ability. Combination therapy can synergistically induce apoptosis, although the expression of MDR1 in drug-resistant cells is up-regulated, but the combination of B-CM and OXa can significantly reduce the mRNA and protein levels. In vivo experiments show that B-CM enhances the tumor inhibition effect of OXa, which is related to the reduction of the expression of Ki-67 and MDR1 in tumor tissues. Further, it can be shown that the soluble factor derived from Blautia can reverse the drug resistance of colorectal cancer to oxaliplatin by down-regulating MDR1 and inhibiting epithelial-mesenchymal transition. This shows that Blautia-derived metabolites are a promising new adjuvant strategy to overcome chemotherapy resistance of colorectal cancer.

Description

Technical Field

[0001] This invention relates to the field of medical biotechnology, and more particularly to the use of a Brontë bacterium in the preparation of a drug for enhancing the sensitivity of colorectal cancer to oxaliplatin. Background Technology

[0002] Colorectal cancer (CRC) remains a major global health burden, ranking among the most common malignancies worldwide and a leading cause of cancer-related deaths globally. Despite significant advances in diagnostic techniques and treatment strategies (including surgery, radiotherapy, chemotherapy, and targeted therapy), the overall prognosis for patients with advanced or metastatic CRC remains poor, with chemotherapy resistance being a major obstacle to effective treatment. Oxaliplatin (OXa), a platinum-based chemotherapy drug, is the cornerstone of first-line combination therapy for CRC. However, inherent and acquired resistance to OXa is widespread, leading to treatment failure and disease relapse. The mechanisms of oxaliplatin resistance are multifaceted, including reduced drug accumulation, enhanced DNA repair, and evasion of apoptosis. A key factor in multidrug resistance is the overexpression of efflux transporters (such as MDR1 / P-glycoprotein), which actively pump chemotherapy drugs out of cancer cells. Therefore, overcoming this resistance is a crucial and unmet need in the treatment of CRC.

[0003] In recent years, the human gut microbiota has become a key regulator of host health and disease, including cancer. Increasing evidence suggests that the complex ecosystem of the gut microbiota can significantly influence tumorigenesis, cancer progression, and treatment response. Specific gut microbes can modulate the local tumor microenvironment, the systemic immune system, and the metabolism and efficacy of chemotherapeutic drugs. For example, studies have shown that certain bacteria can enhance the antitumor effects of immune checkpoint inhibitors and cyclophosphamide, opening new avenues for oncology research to explore the potential of improving cancer treatment outcomes through gut microbiota modulation. Among the many gut bacteria, *Blautia*, as a genus of anaerobic Gram-positive bacteria, has attracted attention due to its potential beneficial effects. This genus is considered an important symbiotic, helping to maintain intestinal barrier integrity and producing short-chain fatty acids such as acetic acid, which have anti-inflammatory and immunomodulatory properties. Notably, the relative abundance of *Blautia* is negatively correlated with various diseases, including inflammatory bowel disease, metabolic diseases, and certain cancers. However, the specific role and potential mechanisms of Blautia in regulating chemosensitivity (especially in colorectal cancer) still need to be elucidated. Summary of the Invention

[0004] The purpose of this application is to provide the use of Broutidae spp. in the preparation of drugs for enhancing the sensitivity of colorectal cancer to oxaliplatin, aiming to explore the effects of Broutidae spp. and its metabolites on the chemosensitivity of colorectal cancer cells, and to discover that Broutidae conditioned medium (B-CM) has the potential to reverse oxaliplatin resistance in colorectal cancer models.

[0005] To address the aforementioned technical problems, this application provides the use of *Brutella* spp. in the preparation of drugs for enhancing the sensitivity of colorectal cancer to oxaliplatin. The characteristic feature is that the *Brutella* conditioned medium significantly enhances the cytotoxic effect of oxaliplatin on drug-resistant colorectal cancer cells, inhibiting their proliferation, colony formation, migration, and invasion.

[0006] As a preferred embodiment, the use of *Brutella* spp. in the preparation of a drug for enhancing the sensitivity of colorectal cancer to oxaliplatin, wherein the *Brutella* conditioned medium combined with oxaliplatin can induce apoptosis in drug-resistant colorectal cancer cells.

[0007] The protocol requires detailed explanation of the use of a Broutella species in the preparation of a drug for enhancing the sensitivity of colorectal cancer to oxaliplatin, wherein the Broutella conditioned medium enhances chemotherapy sensitivity by regulating the multidrug resistance transporter MDR1.

[0008] The protocol requires further detailed explanation of the application of a Broutella species in the preparation of a drug for enhancing the sensitivity of colorectal cancer to oxaliplatin. The Broutella conditioned medium and the oxaliplatin combined treatment can significantly reduce the mRNA and protein expression levels of the multidrug resistance transporter MDR1 in drug-resistant cells.

[0009] As a preferred embodiment, the use of *Brutella* spp. in the preparation of a drug for enhancing the sensitivity of colorectal cancer to oxaliplatin, wherein the *Brutella* conditioned medium enhances the sensitivity of colorectal cancer to oxaliplatin in vivo.

[0010] The protocol requires further detailed explanation of the use of a Broutella species in the preparation of a drug for enhancing the sensitivity of colorectal cancer to oxaliplatin. The Broutella conditioned medium and the oxaliplatin combination therapy significantly reduced the expression levels of Ki-67 and the multidrug resistance transporter MDR1 in tumor tissue.

[0011] As a preferred embodiment, the use of *Brutella* spp. in the preparation of a drug for enhancing the sensitivity of colorectal cancer to oxaliplatin, wherein the invasive ability of the drug-resistant colorectal cancer cells is evaluated by a Transwell assay.

[0012] In a preferred embodiment, the use of the aforementioned *Brutella* spp. in the preparation of a drug for enhancing the sensitivity of colorectal cancer to oxaliplatin includes a method for preparing the *Brutella* conditioned medium comprising: Bronchiolus spheroidae, with accession number BNCC361193 from Guangzhou Zoko Biotechnology Development Co., Ltd., was placed in a modified minced meat medium and cultured under anaerobic conditions at 37°C for 4-5 days. Collect bacterial cells by centrifugation at 4℃ and 8000×g for 10 minutes; The supernatant was filtered through a 0.22 µm polyethersulfone membrane filter for sterilization to obtain the Brontë conditioned medium. The Broutella conditioned medium was aliquoted and stored at -80°C for later use.

[0013] The present invention provides the application of *Brutella* spp. in the preparation of drugs for enhancing the sensitivity of colorectal cancer to oxaliplatin, which includes at least the following beneficial effects: B-CM significantly enhances the cytotoxic effect of OXa on drug-resistant colorectal cancer cells, inhibiting their proliferation, colony formation, migration, and invasion. Combined treatment synergistically induces apoptosis; although MDR1 expression is upregulated in drug-resistant cells, combined treatment with B-CM and OXa significantly reduces its mRNA and protein levels. In vivo experiments show that B-CM enhances the tumor-suppressive effect of OXa, an effect associated with reduced Ki-67 and MDR1 expression in tumor tissue. This further indicates that soluble factors derived from *Brutella* spp. can reverse colorectal cancer resistance to oxaliplatin by downregulating MDR1 and inhibiting epithelial-mesenchymal transition. This suggests that *Brutella*-derived metabolites represent a promising new adjuvant strategy for overcoming chemotherapy resistance in colorectal cancer. Attached Figure Description

[0014] To more clearly illustrate the technical solution of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0015] Figure 1 This is a schematic diagram illustrating the effect of B-CM enhancing OXa in inhibiting the proliferation of colorectal cancer cells, provided in an embodiment of this application. Figure 2 A schematic diagram illustrating the role of B-CM in enhancing the anti-migration and anti-invasion properties of OXa and inhibiting EMT, as provided in the embodiments of this application; Figure 3 This is a schematic diagram illustrating the induction of apoptosis in drug-resistant colorectal cancer cells by B-CM combined with OXa, provided in an embodiment of this application. Figure 4This is a schematic diagram illustrating how B-CM enhances chemotherapy sensitivity by regulating MDR1, as provided in the embodiments of this application. Detailed Implementation

[0016] To enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings.

[0017] The core of this application is to provide the application of Broutidae spp. in the preparation of drugs for enhancing the sensitivity of colorectal cancer to oxaliplatin. The aim is to explore the effects of Broutidae spp. and its metabolites on the chemosensitivity of colorectal cancer cells and to discover that Broutidae conditioned medium (B-CM) has the potential to reverse oxaliplatin resistance in colorectal cancer models.

[0018] Figure 1 This is a schematic diagram illustrating the effect of B-CM enhancing OXa in inhibiting the proliferation of colorectal cancer cells, provided in an embodiment of this application. Figure 2 This is a schematic diagram illustrating the role of B-CM in enhancing the anti-migration and anti-invasion properties of OXa and inhibiting EMT, as provided in the embodiments of this application. Figure 3 This is a schematic diagram illustrating the induction of apoptosis in drug-resistant colorectal cancer cells by B-CM combined with OXa, as provided in an embodiment of this application. Figure 4 A schematic diagram illustrating how B-CM enhances chemosensitivity by regulating MDR1, as provided in this application embodiment, is shown below. Figures 1 to 4 As shown.

[0019] Oxaliplatin (OXa) resistance is a major obstacle in the treatment of colorectal cancer (CRC). The gut microbiota has become a key regulator of chemotherapy efficacy, but the role of specific symbionts like *Blautia* in overcoming OXa resistance remains unexplored. This application establishes oxaliplatin-resistant HCT15 and RKO colorectal cancer cell lines. The effects of *Blautia* conditioned medium (B-CM) alone or in combination with OXa on cell proliferation (CCK-8, colony formation, Ki-67), migration / invasion (scratch healing, transwell), apoptosis (flow cytometry), epithelial-mesenchymal transition (EMT), and expression of resistance proteins (Western blot, RT-qPCR) were observed. Validation was performed using an in vivo xenograft mouse model.

[0020] The application of a Broutella species in the preparation of a drug to enhance the sensitivity of colorectal cancer to oxaliplatin (B-CM) significantly enhanced the cytotoxic effect of OXa on drug-resistant colorectal cancer cells, inhibiting their proliferation, colony formation, migration, and invasion. Combined treatment synergistically induced apoptosis, upregulated E-cadherin expression, and downregulated N-cadherin and vimentin expression, indicating inhibition of the EMT process. Mechanistically, although MDR1 expression was upregulated in drug-resistant cells, combined treatment with B-CM and OXa significantly reduced its mRNA and protein levels. In vivo experiments showed that B-CM enhanced the tumor-suppressive effect of OXa, an effect associated with reduced Ki-67 and MDR1 expression in tumor tissue.

[0021] Materials and Methods 1. Cell culture and establishment of oxaliplatin-resistant cell lines Human colorectal cancer cell lines HCT15 and RKO were purchased from the American Type Culture Collection (ATCC). Cell identification was performed by short tandem repeat (STR) typing, and mycoplasma contamination was detected periodically. All cell lines were cultured in RPMI-1640 medium (Gibco) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Thermo Fisher Scientific) at 37°C under humid conditions of 5% CO2.

[0022] Oxaliplatin-resistant sublines (HCT15 / OXaR and RKO / OXaR) were established using a continuous exposure method: parental cells were induced with progressively increasing concentrations of OXa (MedChemExpress) over 18 weeks. The resistant cells were then maintained in complete medium containing 12 µM OXa to preserve the resistance phenotype. All experiments using resistant cells were performed after culturing in drug-free medium for at least 72 hours.

[0023] 2. Bacterial culture and B-CM preparation Blautia coccoides (BNCC361193) was purchased from Guangzhou Zoco Biotechnology Development Co., Ltd. This strain was cultured for 4-5 days at 37°C under anaerobic conditions (80% N2, 10% H2, 10% CO2) in modified minced meat medium (MMB, containing minced beef extract supplemented with peptone, yeast extract, L-cysteine ​​hydrochloride, heme, vitamin K, K2HPO4, and resazurite). Bacterial cells were collected by centrifugation at 8000×g for 10 minutes at 4°C. 8000×g indicates that the acceleration experienced by the sample during centrifugation is 8000 times the acceleration due to gravity (g). The supernatant was sterilized by filtration through a 0.22 µm polyethersulfone (PES) membrane filter (Millipore) to obtain Blautia coccoides conditioned medium (B-CM). The control medium was treated in the same manner but without bacterial inoculation. B-CM was aliquoted and stored at -80°C for later use.

[0024] 3. Cell viability assay Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo), with detailed operating procedures following the Kit-8 instruction manual. The simplified procedure is as follows: Cells were seeded at a density of 5 × 10³ cells per well in 96-well plates. After culturing for 24 hours, cells were treated with different concentrations of OXa, B-CM (diluted with fresh culture medium at the specified ratio), or combinations thereof. After culturing for 1, 2, 3, 4, or 5 days, 10 µL of CCK-8 solution was added to each well, and the plates were incubated for another 2 hours. The absorbance was measured at 450 nm using a BioTek microplate reader.

[0025] 4. Clonogenesis experiment Cells were seeded at a low density (500 cells / well) in 6-well plates and treated according to the experimental design. The culture medium was changed every 3 days. After culturing for 10-14 days, once visibly colonized cells were formed, the cells were washed with PBS, fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet (Sigma-Aldrich). Each experiment was independently replicated three times.

[0026] 5. Scratch healing test Cells were seeded in 6-well plates and cultured to 90-95% confluence. Scratches were made on the monolayer of cells using a sterile 200 μL pipette tip. Cells were then washed with PBS to remove debris and cultured further in serum-free medium containing the specified treatment. Scratches were imaged at 0 and 24 hours using an inverted microscope (Nikon), and relative migration distances were quantified using ImageJ software (NIH).

[0027] 6. Transwell experiment Cell invasion ability was assessed using pre-coated Matrigel (BD Biosciences) 24-well Transwell chambers (8 μm pore size; Corning). The procedure was briefly as follows: 5 × 10⁶ cells were placed in each well. 4 Cells were suspended in 200 μL of serum-free medium and seeded in the upper chamber. 600 μL of complete medium containing 10% FBS as a chemotactic agent was added to the lower chamber. After culturing under specified conditions for 48 hours, uninvaded cells from the upper chamber were wiped off with a cotton swab. Cells that migrated to the surface of the lower chamber were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and then counted under a microscope by randomly selecting five fields of view.

[0028] 7. Flow cytometry Apoptosis was detected using the Annexin V-FITC / PI apoptosis detection kit (BD Biosciences). After treatment, adherent and suspension cells were collected, washed with pre-chilled PBS, and resuspended in 1× binding buffer. Annexin V-FITC and propidium iodide (PI) were then added, and the cells were incubated at room temperature in the dark for 15 minutes. The stained cells were immediately analyzed using a BD FACS-Canto II flow cytometer, and the data were processed using FlowJo software (version 10.0).

[0029] 8. Western blot analysis Total protein was extracted from cells or homogenized tumor tissue using RIPA lysis buffer (Beyotime) containing a mixture of protease and phosphatase inhibitors (Roche). Protein concentration was determined using a BCA protein quantification kit (Thermo Fisher Scientific). An equal volume of protein (50 μg) was separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane (Millipore). After membrane blocking, the membrane was incubated overnight at 4°C with the following primary antibodies: E-cadherin, N-cadherin, vimentin, Bax, Bcl-2, MDR1, BCRP, MRP2, and GAPDH (all purchased from Cell Signaling Technology, diluted 1:1000). After incubation with the corresponding HRP-labeled secondary antibodies, the images were developed using an enhanced chemiluminescence (ECL) detection system (Bio-Rad). Band grayscale analysis was performed using ImageJ software.

[0030] 9. RNA extraction and real-time quantitative polymerase chain reaction Total RNA was extracted using TRIzol reagent (Invitrogen). 1 μg of RNA was reverse transcribed into cDNA using the PrimeScript RT kit (Takara). RT-qPCR was performed using a SYBR Green Premix Pro Taq HS (Accurate Biology) system on an Applied Biosystems 5 real-time quantitative PCR system. GAPDH was used as an internal control. -ΔΔCt The relative expression levels of MDR1, BCRP, and MRP2 mRNA were calculated using a method where Ct refers to the number of cycles required for the fluorescence signal to reach a set threshold during qPCR. ΔCt is the difference between the Ct values ​​of the target gene and the internal reference gene. ΔΔCt is the difference between the ΔCt values ​​of the treatment group and the control group.

[0031] 10. Immunohistochemistry Formalin-fixed and paraffin-embedded tumor tissue was sectioned into 4 μm thick sections. After dewaxing, hydration, and antigen retrieval, the sections were incubated overnight at 4°C with Ki-67 and MDR1 primary antibodies. Subsequently, they were incubated sequentially with biotinylated secondary antibody and streptavidin-HRP, and developed using a DAB chromogenic kit (ZSGB-BIO), followed by hematoxylin counterstaining.

[0032] 11. In vivo xenograft tumor model All animal experiments were approved by the Animal Care and Use Committee of a certain institution (Approval No.: XXX) and conducted strictly in accordance with relevant guidelines. Four-week-old female BALB / c nude mice were purchased from Jiangsu Huachuang Zhong Sheng Pharmaceutical Technology Co., Ltd. To establish a xenograft tumor model, 5 × 10⁶ mice were used. 6 HCT15 / OXaR cells were subcutaneously injected into the right back of each mouse. When the tumor volume reached approximately 100 mm³, the mice were randomly divided into four groups (n=6 per group): (1) control group (PBS), (2) B-CM group (200 μL, administered by gavage daily), (3) OXa group (5 mg / kg, administered intraperitoneally, twice weekly), and (4) B-CM+OXa combined treatment group. The long and short diameters of the tumor were measured using calipers every 3 days, and the tumor volume was calculated using the formula (volume = length × width² / 2). After 4 weeks, the mice were sacrificed, and the tumor tissue was removed, weighed, and used for subsequent immunohistochemistry and Western blot analysis.

[0033] 12. Statistical Analysis All data are expressed as mean ± standard deviation (SD) of at least three independent trials. Statistical analysis was performed using GraphPadPrism 9.0 software. Student's t-test (commonly referred to as t-test) was used for comparisons between two groups, a statistical method used to compare whether there is a significant difference between the means of two groups. One-way ANOVA combined with Tukey's post-hoc test was used for comparisons among multiple groups. A p-value less than 0.05 was considered statistically significant.

[0034] The above experiments confirm the following results. 1. B-CM can enhance the inhibitory effect of OXa on the proliferation of colorectal cancer cells. To evaluate the moderating effect of Brontë bacteria on the efficacy of oxaliplatin (OXa), this application examined the sensitivity of the control group and OXa-resistant HCT15 and RKO cell lines to OXa at different time points. CCK-8 assay results showed that the cytotoxic response of resistant cells to OXa was significantly reduced (e.g., Figure 1 AB). Subsequently, Brutella conditioned medium (B-CM) was collected and used to treat drug-resistant cells alone or in combination with OXa. The results showed that B-CM significantly enhanced the cytotoxic effect of OXa on drug-resistant cells in a dose-dependent manner (e.g., AB). Figure 1 CD). To further investigate the effect of B-CM on cell proliferation, this application examined the proportion of Ki-67 positive cells and their colony-forming ability. The results showed that combined treatment with B-CM and OXa significantly reduced the Ki-67 positivity rate (e.g., CD). Figure 1 EF) and inhibit clone formation (e.g. Figure 1 In summary, these data indicate that B-CM can effectively reverse OXa resistance in colorectal cancer cells.

[0035] 2. B-CM can enhance the anti-migration and anti-invasion effects of OXa and inhibit EMT. To investigate the effects of B-CM on the antitumor migration and invasion capabilities of OXa, this application evaluated cell migration behavior using a scratch healing assay. The results showed that treatment with OXa or B-CM alone had no significant effect on the migration ability of drug-resistant cells, while combination therapy exhibited a significant inhibitory effect on migration (e.g., Figure 2 Transwell assays further demonstrated that B-CM significantly enhanced the inhibitory effect of OXa on cell invasion (e.g., AD). Figure 2 Epithelial-mesenchymal transition (EMT) is a key step in tumor metastasis, endowing epithelial cells with the ability to migrate and invade. Western blot analysis showed that combined treatment with OXa and B-CM upregulated the epithelial marker E-cadherin while downregulating the mesenchymal markers N-cadherin and vimentin (e.g., EF). Figure 2 GH) indicates that the EMT process is significantly inhibited.

[0036] 3. B-CM combined with OXa can induce apoptosis in drug-resistant colorectal cancer cells. To further investigate the effect of B-CM on tumor cell apoptosis, this application analyzed HCT15 and RKO cells using flow cytometry. The results showed that treatment with OXa or B-CM alone failed to significantly induce apoptosis, while combination therapy significantly increased the apoptosis rate in drug-resistant colorectal cancer cells (e.g., Figure 3 AB). Western blot analysis further showed that combined treatment with OXa and B-CM upregulated the expression of the pro-apoptotic protein Bax, while downregulating the expression of the anti-apoptotic protein Bcl-2. Figure 3 These results suggest that B-CM can enhance the pro-apoptotic effect of OXa in drug-resistant colorectal cancer cells.

[0037] 4. B-CM enhances chemosensitivity by regulating MDR1. To elucidate the potential molecular mechanism by which B-CM enhances the sensitivity of colorectal cancer cells to OXa, this application investigated the expression of OXa resistance-related genes MDR1, BCRP, and MRP2 at both the protein and mRNA levels using Western blotting and RT-qPCR. The results showed that MDR1 expression was significantly upregulated in OXa-resistant cells (e.g., ...). Figure 4 Further analysis revealed that treatment with OXa or B-CM alone had no significant effect on MDR1 expression, but combined treatment with either significantly reduced the mRNA and protein expression levels of MDR1 in drug-resistant cells (e.g., AD). Figure 4 These results indicate that B-CM can partially restore the sensitivity of colorectal cancer cells to OXa by downregulating MDR1 expression.

[0038] 5. B-CM can enhance the sensitivity of colorectal cancer to OXa in vivo. To further verify the enhancing effect of B-CM on the efficacy of OXa chemotherapy in vivo, a mouse xenograft model of colorectal cancer was established. Experimental results showed that B-CM significantly enhanced the tumor-suppressive effect of OXa, with tumor growth in the combined treatment group being significantly inhibited (Figures 5A-C). Immunohistochemical (IHC) analysis indicated that, compared with monotherapy, the combination therapy of OXa and B-CM significantly reduced the expression levels of Ki-67 and MDR1 in tumor tissue (Figure 5D). Further Western blot results confirmed that the combination therapy downregulated MDR1 protein expression, inhibited EMT-related markers, and enhanced apoptosis-related signaling pathways (Figure 5E). In summary, these results demonstrate that B-CM can effectively reverse the resistance of colorectal cancer cells to OXa in vivo and enhance its anti-tumor efficacy.

[0039] Figure 1A. The proliferation rate of OXa-treated HCT15 and HCT15 / OXaR cells was determined by CCK8 assay. B. The proliferation rate of OXa-treated RKO and RKO / OXaR cells was determined by CCK8 assay. C. The proliferation rate of OXa or B-CM-treated HCT15 / OXaR cells was determined by CCK8 assay. P<0.05, P<0.01, P<0.001. D. The proliferation rate of OXa or B-CM-treated RKO / OXaR cells was determined by CCK8 assay. P<0.01, P<0.001. E.* The Ki67 positivity rate in OXa or B-CM-treated HCT15 and HCT15 / OXaR cells was detected by flow cytometry. P<0.001. F.* The Ki67 positivity rate in OXa or B-CM-treated RKO and RKO / OXaR cells was detected by flow cytometry. P<0.001. G.* Representative images and clone counts of HCT15 / OXaR cells treated with OXa or B-CM. P<0.05, P<0.001. H.** Representative images and clone counts of RKO / OXaR cells treated with OXa or B-CM. ***P<0.001.

[0040] Figure 2 A. Representative images of HCT15 / OXaR cell migration rate detected by scratch healing assay using OXa or B-CM. B. Representative images of RKO / OXaR cell migration rate detected by scratch healing assay using OXa or B-CM. C. Statistical results of HCT15 / OXaR cell migration rate detected by OXa or B-CM. P<0.001. D.* Statistical results of RKO / OXaR cell migration rate detected by OXa or B-CM. P<0.05, P<0.001. E.** Representative images and statistical results of invasive ability of HCT15 / OXaR cells detected by Transwell assay using OXa or B-CM. P<0.001. F.* Representative images and statistical results of invasive ability of RKO / OXaR cells detected by Transwell assay using OXa or B-CM. P<0.001. G.* Representative bands of EMT-related proteins in HCT15 / OXaR cells treated with OXa or B-CM via Western blot. Representative bands of EMT-related proteins in RKO / OXaR cells treated with H. OXa or B-CM via Western blot.

[0041] Figure 3A. Flow cytometry detection of apoptosis in HCT15 / OXaR cells treated with OXa or B-CM. P<0.001. B.* Flow cytometry detection of apoptosis in RKO / OXaR cells treated with OXa or B-CM. P<0.001. C.* Representative bands of apoptosis-related proteins in HCT15 / OXaR cells treated with OXa or B-CM via Western blot. D. Representative bands of apoptosis-related proteins in RKO / OXaR cells treated with OXa or B-CM via Western blot.

[0042] Figure 4 A. Representative bands of oxaliplatin resistance-related proteins in OXa-treated HCT15 cells by Western blot. B. Representative bands of oxaliplatin resistance-related proteins in OXa-treated RKO cells by Western blot. C. RT-qPCR detection of oxaliplatin resistance-related gene mRNA levels in OXa-treated RKO cells. P<0.05, P<0.001. D. RT-qPCR detection of oxaliplatin resistance-related gene mRNA levels in OXa-treated HCT15 cells. P<0.05, P<0.001. E. RT-qPCR detection of MDR1 mRNA levels in OXa or B-CM-treated HCT15 / OXaR cells. P<0.001. F. RT-qPCR detection of MDR1 mRNA levels in OXa or B-CM-treated RKO / OXaR cells. P<0.001. Representative Western blot bands of MDR1 protein in HCT15 / OXaR cells treated with G. OXa or B-CM. Representative Western blot bands of MDR1 protein in RKO / OXaR cells treated with H. OXa or B-CM.

[0043] Other embodiments of this application will readily occur to those skilled in the art upon consideration of the specification and practice of the application disclosed herein. This application is intended to cover any variations, uses, or adaptations of this application that follow the general principles of this application and incorporate common knowledge or customary techniques in the art disclosed herein. The specification and examples are to be considered exemplary only, and the true scope of this application is indicated by the claims.

[0044] It should be understood that this application is not limited to the precise structure described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope. The embodiments of this application described above do not constitute a limitation on the scope of protection of this application.

Claims

1. The use of *Brutella* spp. in the preparation of drugs for enhancing the sensitivity of colorectal cancer to oxaliplatin, characterized in that, Broutella conditioned medium significantly enhanced the cytotoxicity of oxaliplatin against drug-resistant colorectal cancer cells, inhibiting their proliferation, colony formation, migration, and invasion.

2. The use of *Brutella* spp. according to claim 1 in the preparation of drugs for enhancing the sensitivity of colorectal cancer to oxaliplatin, characterized in that, The Broutella conditioned medium combined with oxaliplatin can induce apoptosis in drug-resistant colorectal cancer cells.

3. The use of *Brutella* spp. according to claim 2 in the preparation of drugs for enhancing the sensitivity of colorectal cancer to oxaliplatin, characterized in that, The Broutella conditioned medium enhances chemosensitivity by regulating the multidrug resistance transporter MDR1.

4. The use of *Brutella* spp. according to claim 3 in the preparation of drugs for enhancing the sensitivity of colorectal cancer to oxaliplatin, characterized in that, The combined treatment with the Brouderella conditioned medium and oxaliplatin significantly reduced the mRNA and protein expression levels of the multidrug resistance transporter MDR1 in drug-resistant cells.

5. The use of *Brutella* spp. according to claim 1 in the preparation of a drug for enhancing the sensitivity of colorectal cancer to oxaliplatin, characterized in that, The Broutella conditioned medium enhances the sensitivity of colorectal cancer to oxaliplatin in vivo.

6. The use of *Brutella* spp. according to claim 5 in the preparation of a drug for enhancing the sensitivity of colorectal cancer to oxaliplatin, characterized in that, The Broutista conditioned medium and the oxaliplatin combination therapy significantly reduced the expression levels of Ki-67 and the multidrug resistance transporter MDR1 in tumor tissue.

7. The use of *Brutella* spp. according to claim 1 in the preparation of a drug for enhancing the sensitivity of colorectal cancer to oxaliplatin, characterized in that, The invasive ability of the drug-resistant colorectal cancer cells was assessed using a Transwell assay.

8. The use of *Brutella* spp. according to any one of claims 1 to 7 in the preparation of a drug for enhancing the sensitivity of colorectal cancer to oxaliplatin, characterized in that, The method for preparing the Brontë conditioned medium includes: Bronchiolus spheroidus, with accession number BNCC361193 from Guangzhou Zoko Biotechnology Development Co., Ltd., was placed in a modified minced meat medium and cultured under anaerobic conditions at 37°C for 4-5 days. Collect bacterial cells by centrifugation at 4℃ and 8000×g for 10 minutes; The supernatant was filtered through a 0.22 µm polyethersulfone membrane filter for sterilization to obtain the Brontë conditioned medium. The Broutella conditioned medium was aliquoted and stored at -80°C for later use.

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