Listeria monocytogenes promotes breast cancer proliferation and enhances the survival rate of circulating breast cancer cells

doi:10.21203/rs.3.rs-2783293/v1

Mammary gland microbiota is closely related to the progression of breast cancer (BC). Recent studies have shown that Listeria is differentially enriched in breast cancer and normal paracancerous breast tissue, and associated with epithelial-mesenchymal transition (EMT) in breast cancer. However, studies have rarely reported the relationship between Listeria monocytogenes (L. monocytogenes) enrichment and clinical parameters and possible mechanisms in breast cancer. In this study, we found that L. monocytogenes was highly enriched in the cytoplasm of cells, especially in tumor tissues. By analyzing the relationship between L. monocytogenesenrichment and the clinicopathological indicators and prognostic survival of patients, we found that L. monocytogenes is positively correlated with BC proliferation, metastasis, and poor recurrence-free survival (RFS) on patients. Moreover, we performed cell proliferation and invasion assays on breast cancer cells bearing L. monocytogenes, and proved that L. monocytogenes was able to promote the proliferation and invasion of breast cancer cells, their proliferation and invasion capacity was significantly reduced after antibiotic treatment. Mechanistically, L. monocytogenes was identified to increase the survival rate of circulating breast cancer cells by implementing the fluid shear stress model. In conclusion, our study demonstrated that L. monocytogenes could promote breast cancer development by increasing survival rate of circulating cancer cells.

Listeria monocytogenes

Breast cancer

Proliferation

Fluid shear stress

Cytoskeletal reorganization

Survival rate

Female BC has surpassed lung cancer as the most common cancer, with about 2.3 million new cases (11.7%)^[1]. It is the leading cause of cancer-related deaths in women worldwide^[2]. Despite significant advances in diagnostic and treatment strategies in recent years, approximately 15.4% of women still die from BC ^[1]. Breast cancer genome enrichment analysis suggests that Listeria genus is associated with the expression profile of genes involved in EMT^[3]. A review of the virulence factor database (VFDB) revealed that L. monocytogenes in Listeria genus, a Gram-positive, rod-shaped, non-enveloped, non-sportulating bacterium, has a unique mode of invasion into host cells, and whether there is an association with the malignant biological behavior of breast cancer should be of interest. L. monocytogenes is a typical parthenogenic intracellular parasitic bacterium that enters tumor cells through a zipper mechanism^[4-6] and survives and multiplies in the cells. The zipper mechanism refers to the zipper of the host cell membrane being pulled open when bacteria invade the host cell. The bacterial ligand interacts with a surface molecule on the host cell, which are usually proteins involved in the activation of cell adhesion or cytoskeletal components. Ligand-receptor interactions induce actin cytoskeleton reorganization and other signals, ultimately causing the bacteria being tightly wrapped by the plasma membrane. The invasion of L. monocytogenes into host cells consists mainly of the following intracellular cycle: (i) invasion of the host cell (ii ) survival within the phagocytic vesicle (iii) disruption of the phagosomal membrane and escape into the cytoplasm (iv) replication in the cytoplasmic lysis (v) actin-based motility (vi) direct intercellular spread, in which the cycle is restarted ^[7].

The presence of bacterial flora in mammary gland has been confirmed ^{[8, 9]} . The bacterial profile is different in healthy women compared to breast cancer tissue, and dysbiosis increases the risk of breast cancer^{[8, 10-12]} . There are multiple interactions between the breast cancer host and the microbiome^{[8, 13]}, and the main pathway of microbiome-to-host signaling is the secretion of bacterial metabolites that enter the circulation and reach their target cells^{[8, 14]}. For instance, bacteria, such as Collinsella, Edwardsiella, Alistipes, Bifidobacterium, etc, can stimulate estrogen deconjugate and reuptake through the expression of β-glucuronidases^{[15, 16]}, and the reactivated estrogens bond with estrogen receptor, thereby promoting ER + breast cancer progression^[17-20]. Lachnospiraceae, Ruminococcus obeum and Roseburia inulinivorans, on the other hand, are capable of producing short-chain fatty acids ^[21] that regulate breast cancer cell proliferation, apoptosis, cell invasion, endocrine resistance, cachexia and metabolism^[22-26].

Molecularly, the surface protein ActA is indispensable for L. monocytogenes pathogenicity. ActA is encoded by a major pathogenic gene actA, and is responsible for actin-based motility and intercellular spread ^[27-30]. Actin-based bacterial motility depends on the activation of the Arp2/3 complex nucleation activity by ActA, which contains functional domains that simulate the actions of the Arp2/3 complex-activating proteins WASP/Scar family, which are downstream effectors of signaling pathways involved in dynamic reorganization of the actin cytoskeleton^[31-35]. The invasive front of the tumor is often abnormally regulated by cytoskeletal actin, such as elevated expression of Arp2/3 and WAVE2 complexes, and in breast cancer Arp2/3 expression is positively correlated with the invasive phenotype^[36].

At present, the relationship between L. monocytogenes and breast cancer has not been confirmed. Our research proved that L. monocytogenes was enriched in tumor microenvironment (TME) of breast cancer, and was a positive regulator of cancer cell proliferation and invasion. Our findings provide a novel mechanism of breast cancer development, it can be used as a marker for early diagnosis and prognosis assessment of breast cancer, which is expected to provide a new reference for clinical diagnosis and treatment.

Patients and tissue specimens

101 BC and 43 normal paracancerous breast tissues samples were obtained from the Department of Breast Surgery, The Second Affiliated Hospital of Harbin Medical University (246 Xuefu Street, Nangang District, Harbin, China). The 101 patients were Asian women, aged from 33 to 77 years (median age of 49 years). Fresh BC tissues and adjacent normal tissues were frozen in liquid nitrogen immediately after surgical excision and stored at -80° C. Hematoxylin & eosin (H&E) staining of excised tissues was used for histologic validation of tissue types. This research was approved by the Ethic Committee of Harbin Medical University.

Inclusion criteria: 1) pathological diagnosis of invasive cancer; 2) operable breast cancer; 3) no preoperative treatment; 4) overall survival time ≥ 3 months; 5) complete clinical data and postoperative follow-up.

Exclusion criteria: 1) combined immune system diseases; 2) distant organ metastases; 3) surgical complications or infections; 4) history of antibiotic application within three weeks.

Ultrasound imaging assessment

Enrolled breast cancer patients were routinely examined by ultrasonographers before surgery. The patient is placed in a supine position with arms elevated. If necessary, the patient will be adjusted to the appropriate lateral position to allow scanning of the lateral and lower portions of the breast. The procedure of ultrasound scanning was as follows: firstly, the ultrasonic probe targets the lesion and surrounding breast tissue in the same quadrant, then the rest of the ipsilateral breast, finally, the contralateral breast. During the examination, the lesion site is evaluated in detail, and two-dimensional images of the mass, blood flow signal images, and elasticity score images are recorded, along with attention to the presence of spiculation after recording whether the boundaries of the mass are clear. The above ultrasound imaging evaluation of all patients was recorded using ultrasound machine (SuperSonic, Aixplorer) of Harbin Medical University Second Hospital.

Immunohistochemistry (IHC)

The tissue microarrays were made by the production company (SHANGHAI OUTDO BIOTECH CO.,LTD.) using 101 breast cancer and 43 normal paracancerous tissue wax blocks. After de-paraffinization and rehydration, tissue sections are incubated in antigen retrieval buffer and heated in a steamer at above 97°C for 20 min. All IHC staining was performed using Ventana Discovery XT Automated Slide Stainer (Ventana Medical Systems, Inc., Tucson, AZ, USA). Deparaffinization, antigen retrieval, blocking, DAB detection, counterstain, post-counterstain, and slide cleaning steps were automated on the Discovery XT. Primary antibodies and secondary antibodies were manually applied at programmed steps. Anti-Listeria monocytogenes (Genex, GTX11439, Mouse IgG) is applied to tissue sections overnight in a humidity chamber at a dilution of 1:800 at 4°C. After being washed in TBS, antigen-antibody binding is detected with the Envision+ system and DAB+ chromogen (DAKO). Tissue sections were briefly immersed in hematoxylin for counterstaining, washed with water and covered with coverslips. Slides were assessed by two pathologists blindly on a multi-headed microscope.

Bacteria culture

The species of L. monocytogenes (GOYJ13727) was obtained from (GuYan Biotech Co.,Ltd.(Shanghai,china)). Glycerol stock solutions with L. monocytogenes was generated. During this study, stock cultures were maintained in cryovials at a concentration of about 8.5 x 10⁸ colony forming units/mL (cfu/mL) and at a temperature of 80° C. Before each experiment, bacterial broth subcultures from stock cultures were prepared by inoculating 100 mL of L. monocytogenes in a sterile flask containing 10 mL of Tryptone Soya Broth (TSB; Scharlab Chemie S.A., Barcelona, Spain), and incubated at 37 ° C for 12 h with agitation. Then an aliquot of 100 mL was again inoculated in 10 mL of TSB, and incubated at 37 ° C for 12 h under agitation. Bacterial suspension with a cell concentration of 10⁸ cfu/mL was confirmed by plate counts on Tryptone Soya Agar (TSA; Scharlab Chemie S.A., Barcelona, Spain). Those cells were used in subsequent experiments.

Cell Lines and Cell infection

Human BC cell lines (MCF-7 and MDA-MB-231) were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM (Thermo Fisher Scientific) containing 10% FBS at 37°C with 5% CO₂ in humidified gas conditions. BC cells (MCF-7 and MDA-MB-231) were incubated in 1 mL complete culture medium with the L. monocytogenes strain for 1 h while control BC cells were incubated with complete culture medium only. After infection, cells were centrifuged at 900 rpm/min for 5 min, and extracellular bacteria were removed by washing with DPBS twice.

Cell viability test

To test the cell viability after bacteria invasion, we cocultured tumor cells with CFSE-labeled L. monocytogenes strains and added the bacteria-invaded tumor cells (1.2×10⁴cells/well) to a 24 well-plate in colony forming medium without Y27632(Sigma). After eight hours, tumors cells both in the supernatant and adhering on the plate were stained with Annexin V (Biolegend, #640912) following manufacturer’s instructions. Cells were then analyzed by FACS using BC CytoFLEX LX(Beckman) and quantified by FlowJo software (Tree Star, Inc., Ashland, OR).

MTT Assay

First, the cell suspension was prepared by trypsin digestion of logarithmic phase cells, aborted by serum digestion, pipetting until the cells were suspended, and the cell suspension was centrifuged at 1000 rpm/min for 5 min, the supernatant was discarded, and the cell suspension was made by resuspending the precipitate. After the cell suspension was prepared, the cells were inoculated and implanted into 96-well plates (Corning Life Sciences), with one column (6 multiple wells) for each experimental group, and the amount of cells implanted in each well was 3 × 10³ cells/well. Place the inoculated plates in an incubator and incubate to the appropriate cell density. Remove the inoculated plates from the incubator, add 10 μL of MTT (5 mg/mL) to each well, incubate for 4 h and then terminate the incubation, and add 150 μl of dimethyl sulfoxide to dissolve the precipitate. The absorbance was then measured at 560 nm using a microplate reader, and the background value of 650nm was subtracted. Cell proliferation is represented as a percentage of the value obtained for cells incubated with the vehicle (DMSO). The experiment was performed independently in triplicate.

Soft Agar Assay

MCF-7 and MDA-MB-231 cells were divided into three groups: NC group, L.moco group and L.mono+Clarithromycin group. After 48 h of transfection, the cells were harvested, and the soft agar assay was performed to assess the ability of cancer cells to undergo anchorage-independent growth. Briefly, 1 ml culture media with 0.8% agar solution was plated in six-well plates, solidified, and then overlaid with 1 ml culture medium containing 0.3% agar solution and 2000 cells, and the cells were allowed to grow. To prevent drying, 0.5 ml culture medium was added to each well. After 14 days, 0.5 ml iodonitrotetrazolium violet (INT, 0.5 mg/ml in PBS) was added to stain the cells overnight. Cell Colonies were counted from nine high powered fields. Results are reported as means ±SEM from at least three independent experiment, each containing duplicate measurements. Representative images (20x) were taken using an EVOS 5000 Imaging System (Thermo Fisher Scientific).

Transwell Assay

Cell invasion assay was performed on 6-well Trans well plate (polycarbonate membrane with 8 µm pore size, Costar, USA) precoated with 10 µg/mL Matrigel (SIGMA, USA). 5 × 10⁵ MCF-7 or MDA-MB-231 cells suspension was inoculated into the upper chamber with serum-free L-15 medium, and L-15 medium containing 10% FBS was added to the lower chamber, then placed at 37°C, 5% CO2 incubator. After 24 h, the Trans well chamber was taken out and washed with PBS. The cells on the upper layer of the membrane were wiped off with a cotton swab, and fixed at lower layer with 4% paraformaldehyde for 10 min, then stained with 0.25% crystal violet. they were then photographed using fluorescence microscopy (NIKON Eclipse 80i, Ku, Tokyo, Japan). We counted 8 fields randomly to determine the number of invaded cells. The number of invading cells was analyzed using image j software, and the data from all three independent replicates were analyzed for statistical significance and plotted in a bar graph.

Fluid Shear Stress Model

The microfluidic circulation system with peristaltic pump in the study was assembled according to published literature (Regmi et al., 2017). The microfluidic circulation system with a peristaltic pump (SHENCHEN #LabS3) was used to control the flow rate of the system. Our microfluidic system is based on 4 rollers (Figure 4a), which pumps fluid in a pulsatile manner, mimicking blood flow in the human body. DMEM or RPMI 1640 (PS, Gibco, USA) containing 10% FBS, 1% penicillin and streptomycin, pH 7.4 was used as the fluid component of the circulation system. The fluid mechanical contraction force encountered by cells in circulation can be calculated using the Poiseuille equation, τ = 4Qη/πR³, where Q is the flow rate in ml/sec, η is the dynamic viscosity of the fluid, equivalent to 0.012dynes.sec/cm², and R is the inner radius of the circulation tube (R2). Specifically, the shear stress is about 14 dynes/cm² when Q is equal to 0.859 ml/60s and about 20 dynes/cm² when Q is equal to 1.227 ml/60s. We have tested various cell densities and found that 2×10⁵ cells/ml was the best condition. This concentration allows the collection of sufficient cells for analysis while they can circulate in the system without conjugation or precipitation. The length of the silica-based cycle is 1.5 m; R1 has a size of 0.5 mm, while R2 has a fixed size of 0.25 mm (Figure 4a). The tubes were sterilized before applying to the model by washing with 70% ethanol (4 ml) and then rinsing with high-pressure distilled water (4 ml). Before introducing the cells into the microfluidic system, the morphology of the cells was checked to ensure that only healthy cells were used. Cells were digested by trypsin and suspended in fresh DMEM at a density of 2 × 10⁵/ml. 1 ml of cell suspension (2 × 10⁵ cells) was injected into the circulation system and treated with fluid shear stress at different times at 37°C in a 5% CO2 incubator. 100μl of cell suspension was then collected at each indicated time points.

Immunofluorescence (IF)

For frozen sections, tissues were fixed in 4% paraformaldehyde (BBI Life Sciences #E672002-0500) for 2 hours and then soaked in 30% sucrose to infiltrate overnight. The fixed tissues were embedded in O.C.T. (Tissue-Tek) compound and frozen at -80°C. For formalin-fixed paraffin-embedded tissues (FFPE), tissues were fixed overnight in 4% paraformaldehyde at 4°C. Antigen retrieval was carried out by boiling in slightly citrate buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0) in a microwave oven at medium-low power for 15 minutes. The samples were blocked with TBS + 2% BSA + 5% donkey serum + 0.1% Triton X-100 in a humid chamber at room temperature for 1 h. Subsequently, samples were incubated with primary antibodies: Mouse Anti-Gram Positive Bacteria antibody (1:100, Abcam, ab20344), overnight at 4°C followed by staining with fluorophore-conjugated secondary antibodies: Alexa Fluor 488-phalloidin (1:500, Invitrogen #A12379) in blocking buffer for 1 h at room temperature protected from light. Slides were then stained with DAPI (1ug/mL (1x), sigma) for 5min, and mounted with Antifade Mounting Medium (Beyotime #P0126). Fluorescence microscope was used for observation and acquisition of fluorescence images.

Statistical Analysis

The enrichment levels of L. monocytogenes in breast cancer tissues and normal paracancerous control tissues were analyzed by unpaired t test (Fig.1b, c) (Fig.4c). The chi-square and t tests were performed to assess the relationship between L. monocytogenes enrichment levels and clinicopathological features (Table 1). The ANOVA summary was performed to assess the enrichment of L. monocytogenes in different tumor grades, lymph node metastasis status and various molecular subtypes of breast cancer (Fig.2a, b, c) (Fig.3c, e, g, i) (Fig.4b), Overall survival and disease-free survival were analyzed according to the Log-rank (Mantel-Cox) test method (Fig.2e, f). Univariate analysis was applied by log-rank test. Cox multivariate proportional hazards model was applied to analyze the survival variables (Table 2). P values less than 0.05 were considered statistically significant. All statistical analysis was performed using SPSS version 27.0. and Graph-Pad Prism version 9 Software. The results are expressed as the mean±s.e.m. Immunohistochemical staining intensity was calculated as a percentage of stained area by ImageJ software using cytoplasmic staining statistics, and histochemistry score (H-Score) were calculated using the following formula: score= strongly positive (+++) area percentage*3+ moderately positive (++) area percentage*2+ weakly positive (+) area percentage*1+negative (-) area percentage*0.

L. monocytogenes is highly enriched in breast cancer cells and localized in the breast cancer cytoplasm.

Previous studies have demonstrated that L. fleischmannii in the genus Listeria is differentially enriched in breast cancer and normal paracancerous breast tissue and may be associated with the EMT process. In this study, breast cancer and normal paracancerous tissues were collected and tissue microarrays were prepared, and IHC staining and H-Score of L. monocytogenes were performed on the tissue microarrays. In Figure 1a, different intensity (0-3+) of IHC staining on tissue microarrays were shown. We found that L. monocytogenes mainly located in the cytoplasm of cancer cells, but in the nucleus of normal cells. Thus, we performed H-Score analysis of the tissue microarray to compare the enrichment of L. monocytogenes in cancerous and normal paracancerous tissues. As shown in figure 1b, L. monocytogenes was highly presented in breast cancer tissues but lowly in normal paracancerous tissues (Unpaired t test, ****P<0.0001, Fig.1b). As for the nuclear enrichment of L. monocytogenes, no significance was found between tumor and normal groups (Unpaired t test, ns P= 0.1007, Fig.1c). Thus, we revealed the fact that L. monocytogenes was highly presented in the cytoplasm of breast cancer cells, with significant differences compared to normal paracancerous breast tissues.

L. monocytogenes has a defined correlation with the clinicopathological indicators of breast cancer.

It was recently established that some bacteria might present in the cytoplasm of cells and play a pro-carcinogenic role in cancer. We next explore the clinical significance of L. monocytogenes in breast cancer patients. The clinicopathological indicators of the patients in tissue microarray are shown in Table 1. We categorized cytoplasmic L. monocytogenes enrichment levels into high and low levels according to the median value, and assessed the association between L. monocytogenes enrichment and clinicopathological indicators. Statistical results indicated that high L. monocytogenes enrichment was associated with Tumor grade(P=0.018), Lymph node metastasis (LNM) (P=0.004), P53 level(P=0.031), Ultrasonic elasticity score(P<0.001) and Spiculation (P<0.001) (Chi-Square Test, Table 1). The enrichment of L. monocytogenes in each stage of tumor grade and LNM was plotted as box plots (Fig.2a, b), where 13 (25%) patients in the L. monocytogenes high enrichment group showed tumor grade III and LNM (N2/3), and 15 (29%) patients in L. monocytogenes high enrichment group had P53 ≥15% (Table 1). The high enrichment group is characterized by higher ultrasonic elasticity score and presence of spiculation, with 37(73%) of the patients in the high enrichment group had an elasticity score of 4-5 and 41(80%) had spiculation.

High enrichment of L. monocytogenes in the cytoplasm is a poor prognostic factor in breast cancer patients.

Log-rank (Mantel-Cox) test results showed that high enrichment of L. monocytogenes in breast cancer patients indicated worse DFS. (**P=0.0018, Fig. 2e). Univariate and multivariate analyses were performed to investigate the indicators responsible for DFS. Univariate analysis of DFS showed that LNM (P=0.010), Tumor grade (P<0.001), Tumor size (P=0.006), ER (P=0.002), PR (P=0.001), Her-2 status (P=0.010) and L. monocytogenes (P=0.004) were statistically significant prognostic indicators (Table 2). Moreover, multivariate analysis revealed that high enrichment levels of L. monocytogenes showed significantly shorter DFS, and it was a poor independent prognostic factor for disease-free survival (P=0.012; Table 2) in breast cancer patients.

L. monocytogenes promotes proliferation and invasion of breast cancer cells

After clarifying the clinical significance of L. monocytogenes enrichment in breast cancer, the impact on breast cancer cell function was next explored. MTT and Soft Agar assays confirmed that the proliferation capacity of breast cancer cells infected with L. monocytogenes was significantly higher than that of control breast cancer cells; the proliferation capacity of breast cancer cells infected with L. monocytogenes was significantly reduced after treatment with clarithromycin (an intracellular bacterial antibiotic). Statistical results between groups are presented in histograms (Figure 3a-e). Transwell invasion assay also confirmed that the invasive ability of breast cancer cells infected with L. monocytogenes was significantly higher than that of control breast cancer cells; the invasive ability of breast cancer cells infected with L. monocytogenes was significantly reduced after the addition of clarithromycin treatment (Figure 3f-i). Statistical results between groups are presented in histograms. The above functional recovery assay confirmed the ability of L. monocytogenes to promote malignant phenotypes such as proliferation and invasion of breast cancer cells.

L. monocytogenes enhances the survival rate of circulating breast cancer cells under mechanical stress.

To simulate fluid stress generated by blood circulation on breast cancer cells, we set up an in vitro fluid shear stress model using a peristaltic pump to simulate blood flow. (Fig.4a). Tumor cells were placed in the model and were applied with fluid stresses of 0,14,20 Dyn cm^-2, and the survival rate of BC cells was found to decrease with increasing stress (P <0.0001, Fig.4b). BC cells with or without L. monocytogenes in the cytoplasm were subjected to 14 Dyn cm^-2 fluid stress. A significantly higher survival rate of BC cells with L. monocytogenes in the cytoplasm was noticed (P=0.0188, Fig.4c). Immunofluorescence staining of surviving cells with L. monocytogenes altered the cytoskeleton and showed a much larger size than the control. This phenomenon indicated a possible role for L. monocytogenes in the reorganization of the actin cytoskeleton. Consistent with this notion, phalloidin staining of the cells showed a significantly reduced stress fiber intensity after the invasion of L. monocytogenes in vitro in the culture dish, suggesting that the mechanical stress induced contractile forces can be relieved by the cytoplasmic L. monocytogenes (Fig.4d). These results indicate that L. monocytogenes enhances the survival rate of circulating breast cancer cells under fluid stress, may promotes the metastasis of circulating breast cancer cells to distant organs. (Fig.5).

In this study, 101 breast cancer patients were selected from the Second Hospital of Harbin Medical University, and pathological specimens, clinicopathological indicators, survival time, and imaging data were collected from the patients. Tissue microarrays were made from breast cancer tissues and normal paracancerous breast tissues, and IHC staining was performed to detect the enrichment level of L. monocytogenes. BC tissue was found to have higher levels of L. monocytogenes enrichment than normal paracancerous breast tissue. On this basis, the level of L. monocytogenes enrichment in breast cancer patients was analyzed in conjunction with clinicopathological indicators, and the results showed that the level of L. monocytogenes enrichment was positively correlated with the degree of LNM, P53 expression, and the appearance of higher elasticity scores and spiculation. Tumor tissue sclerosis and spiculation showed in imaging examination could be caused by tumor cell overproliferation and cytoskeletal reorganization, this finding is consistent with existing studies in which L. monocytogenes has a structural domain to regulate cytoskeletal actin^[36]. The enrichment of L. monocytogenes greatly shortened the DFS of BC hosts. In addition, this study verified the role of L. monocytogenes in promoting the proliferation and invasion of tumor cells using MTT, Soft Agar, and Transwell functional assays. Our study established an in vitro model that showed L. monocytogenes in the cell cytoplasm enhanced the survival of circulating tumor cells by promoting actin cytoskeletal reorganization, which may promote metastasis of circulating breast cancer cells to distant organs.

Previous studies focusing on the intra-tumor flora as a group have found that breast cancer in situ flora can contribute to the development and progression of breast cancer metastasis by affecting cytoskeletal rearrangements and other mechanisms ^[37]. Notably, the study by Fu A et al. demonstrated that bacterial alteration of the cytoskeleton is one of the reasons that help circulating tumor cells to resist blood flow shear, and the process of L. monocytogenes invasion of host cells also involves cytoskeletal alteration. Thus, L. monocytogenes promotes breast cancer metastasis through cytoskeletal reorganization has a more solid argument. This study focused on whether L. monocytogenes, as a component of the breast cancer flora, is associated with cancer metastasis. It was found that L. monocytogenes was significantly correlated with the spiculation of breast cancer in ultrasound images (P<0.001), which is an imaging sign of cancer invasion to surrounding normal paracancerous tissues, and whether L. monocytogenes can enhance tumor invasion through cytoskeleton protein-related pathways also needs to be further explored^[38]. P53 protein, an oncogenic protein encoded by the TP53 gene. However, TP53 mutations often result in increased P53 expression in IHC staining of breast cancer specimens, an alteration that suggests a poor prognosis for patients and possible early recurrence^[39]. Kadosh et al. found that intestinal flora can disrupt the WNT pathway by producing gallic acid to convert the oncogenic P53 mutant phenotype to the pro-oncogenic phenotype^[40]. In the patient cohort of this study, the expression of P53 increased with the enrichment of L. monocytogenes, and it is also urgent to further investigate whether L. monocytogenes can alter the function of P53 protein through metabolites.

The key pathway regulating cytoskeleton dynamics is the RhoA-ROCK pathway. ROCK is involved in the control of several processes, including actomyosin (actin monomer) contractility, intermediate filament assembly, microtubule dynamics, and integration of membrane proteins in the actin cytoskeleton^[41-45].Overall, our study established the viable hypothesis that there may be a competitive inhibitory relationship between L. monocytogenes invasion of host tumor cells and the circulating hydrodynamically activated RhoA-ROCK pathway. As a result of cytoplasmic actin recruitment caused by L. monocytogenes invasion into tumor cells and intercellular dissemination, the amount of actin activating the RhoA-ROCK pathway in circulating tumor cells in response to fluid stress was reduced, thereby attenuating the cytoskeletal contractility of circulating tumor cells and promoting L. monocytogenes-bearing cytoskeletal reorganization in breast cancer cells, allowing circulating tumor cells to avoid apoptosis triggered by mechanical forces. This discovery fills in the mechanism of action between breast in situ flora and breast cancer disease development, and provides a new feasible direction for breast cancer treatment.

Cancer flora as an emerging target for the diagnosis and treatment of malignancies offers new possibilities for reducing the disease burden in oncology patients. In breast cancer, a variety of in situ flora can be used as prognostic factors, such as Porphyromonas, Azomonas, etc^[46]. In this study, L. monocytogenes was found to be significantly enriched in breast cancer tissue and may be a potential diagnostic marker for breast cancer. This study is the first to suggest that L. monocytogenes is associated with breast cancer prognosis, and further studies are needed to investigate whether L. monocytogenes has an impact on the treatment of breast cancer. Mechanistic studies suggest that as one of the components of the tumor microenvironment, tumorigenic flora can interact with immune cells, thereby modulating the tumor immune microenvironment and regulating tumor growth. In breast cancer, Fusobacterium has been shown to be a key regulatory molecule in the growth of lymphatic lineage cells, disrupting tumor immunity and promoting breast cancer metastasis^[47]. L. monocytogenes, on the other hand, is considered as a potential vector for cancer immunotherapy. Since L. monocytogenes induces low levels of humoral immunity, can be reused multiple times and can induce both innate and acquired immunity, it can be used as a cancer vaccine to induce the proliferation of killer lymphocytes^[48]. This study demonstrated that L. monocytogenes in breast cancer is detrimental to patient survival and promotes cancer cell invasion and metastasis through cellular-level studies, but whether L. monocytogenes can be used as a target for cancer treatment in vivo trials is debatable.

Studies on in situ flora, intestinal flora and breast cancer have been partially reported, and more comprehensive and deeper studies are needed to reveal the role of flora in the progression of breast cancer and through which pathways the malignant phenotype is regulated. There are some limitations of this study: (1) The results found no significant difference in the enrichment level of L. monocytogenes among the molecular subtypes of breast cancer, which may be related to the small number of enrolled patients, and whether there is a difference in the distribution of L. monocytogenes among the different molecular subtypes needs to be verified in a large sample. (2) In addition, in this study, only two breast cancer cell lines, MCF7 and MDA-MB-231, were used to verify the biological effects of L. monocytogenes on breast cancer, including in vitro proliferation and invasion, and there were no other breast cancer cell lines for multiple validation and in vivo experiments on experimental animals. (3) The lack of more validation experiments on the cytoskeletal contractility of L. monocytogenes in inhibiting the RhoA-ROCK pathway. This part of the experiment is in progress and more additional information on the results will be provided in the future.

In conclusion, this study verified through clinical data that the degree of L. monocytogenes enrichment was positively correlated with lymph node metastasis and malignant signs under ultrasound, and that patients with higher enrichment had a worse prognosis. Cellular studies have confirmed the role of L. monocytogenes in breast cancer in promoting tumor proliferation, invasion and metastasis. These results suggest that detection of L. monocytogenes may be useful for early diagnosis and prognostic assessment of breast cancer, while the prospect of its application in the field of oncology treatment and the specific molecular mechanisms by which it promotes breast cancer progression need to be explored in more experiments in the future.

Funding

This research was funded by the National Natural Science Foundation of China (81872135 and 82002791) and the Distinguished Young Scientist Fund of the Second Affiliated Hospital of Harbin Medical University.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Author Contributions

Yunqiang Duan, Fei Ma and Baoliang Guo devised the study. Yunqiang Duan and Ziang Chen should be considered joint first author. Fei Ma and Baoliang Guo should be considered joint corresponding author. Yunqiang Duan, Yansong Liu, Xiaohui Jiang,Yu Ding, Zhiyuan Rong, Yiyun Zou, Hanyu Zhang, Ting Wang and Yuhang Shang completed the data collection and statistical analysis. Ziang Chen, Jianyuan Feng, Anbang Hu, Weilun Cheng, Tianshui Yu and Mingcui Li performed the experiments. Yunqiang Duan, Yanling Li and Jiarui Zhang conceptualized the initial manuscript collectively. Baoliang Guo and Fei Ma edited the manuscript and provided revisions. All authors have read and agreed to the final version of the manuscript.

Data Availability Statement

Privacy/ethical restrictions: The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

Included in article: The data that support the findings of this study are available in the methods and/or supplementary material of this article.

Code Availability Statement

The software application or custom code that support the findings of this study are available in the methods and/or supplementary material of this article.

Ethical Approval

This study was approved by the Ethics Committee of Harbin Medical University. The study was conducted in accordance with the ethical standards of the Declaration of Helsinki.

Consent to participate

Informed consent for the use of tissues was obtained from the patient prior to the initiation of the study.

Consent to publish

The authors affirm that human research participants provided informed consent for publication of the images in Figure(s) 1a, 2d , 3b, 3d, 3f, 3h and 4d.

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Table 1. Correlation between L. monocytogenes enrichment and clinicopathological indicators

indicators	Number of cases(%)	L. monocytogenes enrichment		P value
		low	high
Age(y)
<49	46(45.5)	22	24	0.758
≥49	55(54.5)	28	27
Tumor grade
I	39(38.6)	26	13	0.018
II	43(42.6)	18	25
III	19(18.8)	6	13
Tumor size
pT1	67(66.3)	35	32	0.197
pT2	32(31.7)	13	19
pT3	2(2)	2	0
Lymph node metastasis
pN0	58(57.4)	37	21	0.004
pN1	24(23.8)	7	17
pN2/3	19(18.8)	6	13
Pathological grade
I	17(16.8)	8	9	0.664
II	46(45.5)	21	25
III	38(37.6)	21	17
ER
Negative	31(30.7)	13	18	0.311
Positive	70(69.3)	37	33
PR
Negative	46(45.5)	18	28	0.057
Positive	55(54.5)	32	23
Her-2 status
Negative	76(75.2)	40	36	0.273
Positive	25(24.8)	10	15
Ki67
<30%	45(44.6)	22	23	0.912
≥30%	56(55.4)	28	28
P53
<15%	80(79.2)	44	36	0.031
≥15%	21(20.8)	6	15
Ultrasonic elasticity score
2	11(10.9)	11	0	<0.001
3	36(35.6)	22	14
4	46(45.5)	13	33
5	8(7.9)	4	4
spiculation
No	38(37.6)	28	10	<0.001
Yes	63(62.4)	22	41

Table 2. Cox proportional hazards model of DFS in breast cancer patients

Indicators	Univariate			Multivariate
	HR	95 % CI	P	HR	95 % CI	P
Age(y)	1.177	0.540-2.564	0.682
Tumor grade	3.291	1.880-5.762	<0.001	4.091	1.642-10.188	0.002
Tumor size	2.371	1.283-4.381	0.006	1.937	0.896-4.187	0.093
Lymph node metastasis	1.853	1.160-2.960	0.010
Pathological grade	1.349	0.766-2.376	0.299
ER	0.286	0.131-0.626	0.002
PR	0.238	0.100-0.569	0.001	0.223	0.045-1.099	0.065
Her-2 status	2.763	1.275-5.987	0.010
Ki67	2.097	0.907-4.851	0.083
P53	1.428	0.618-3.301	0.404
Ultrasonic elasticity score	1.172	0.727-1.887	0.515
spiculation	1.427	0.620-3.285	0.403
*L. monocytogenes*	0.261	0.105-0.651	0.004	0.235	0.076-0.729	0.012

No competing interests reported.

Listeria monocytogenes promotes breast cancer proliferation and enhances the survival rate of circulating breast cancer cells

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Abstract

Figures

Introduction

Materials and methods

Results

Discussion

Declarations

References

Tables

Additional Declarations

Status:

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