The Relationship Between Intratumoral Flora And Gastrointestinal Flora In Canine Mammary Tumors And Its Influence On Tumor Development

Mammary gland tumor is one of the most common tumors in dogs,about 50% of which are malignant,and the high incidence rate and mortality rate pose a serious threat to the life and health of dogs.In recent years,some researchers have confirmed that tumors contain microorganisms and proposed that local microorganisms may directly promote DNA damage and increase mutagenesis,activate carcinogenic signaling pathways,and influence tumor progression through interactions with the host immune system.The occurrence and development of tumors are related to microbiota,but the exact mechanism of action is not fully understood.At present,the correlation between these microbiota and diseases has become a cutting-edge hot topic in current research.In veterinary clinical practice,research on animal tumor microbiota is in a blank space.As a common type of tumor in dogs,the presence of bacteria in canine mammary tumors has not been reported to date.This study is based on the theoretical hypothesis that there are specific microorganisms present in tumor tissue,and that there is a correlation between the microbiota in canine mammary tumors and the microbiota in the digestive tract,which affects tumor development.We carried out the following research:1)Epidemiological analysis: we collected the information of tumor-infected dogs recorded by many animal hospitals in different regions of China from 2017 to 2021,including age,sex,breed,body type,sterilization or not,living area,past medical history,tumor location,tumor number,tumor size,and tumor pathology.The statistical results show that in the past five years,the top three types of canine tumors are breast tumors,skin tumors and perianal tumors.Canine mammary tumors often occur in 9-12 years old female dogs without sterilization.The benign and malignant tumors are 48.41%and 51.59% respectively.Generally,the proportion of single tumors can reach 55.36%.The last two pairs of mammary glands in dogs are more frequent.These results preliminarily confirmed that canine mammary tumor is a common tumor type in veterinary clinic,so the clinical prevention and treatment of this disease is particularly important.2)Identification of dogs with mammary tumors: samples of mammary gland tumor tissue and blood were collected from infected dogs,and the dogs with mammary gland tumors were identified through visual examination,hematology,imaging,histopathology,serum ELISA,and molecular biology tests.The results showed that there were obvious lumps,solid texture,and even ulceration in the mammary gland of the affected dog;Hematology examination showed that there was inflammatory reaction in the infected dog;Imaging examination showed that the mammary gland mass was prominent,uneven in texture,and rich in blood supply;Histopathology confirmed 68 cases of malignant tumors and 56 cases of benign tumors;The results of q PCR showed that the relative expression of tumor markers HER-2,CA15-3,CEA,SF,Vimentin,N-cadherin in canine mammary tumor tissue was significantly higher than that in control tissue,while E-cadherin was significantly lower(P<0.05).In addition,there was no significant difference in tumor markers ER and PR between the two groups,and the results of ELISA were consistent with them.These combined clinical and laboratory results confirm the diagnosis of mammary tumors in dogs for subsequent research.3)Analyze the characteristics of intratumoral microbiota in dogs with mammary tumors: in order to analyze the characteristics of intratumoral microflora in dogs with mammary gland tumors,we carried out 16 S r RNA microbiological high-throughput sequencing on canine mammary tumor tissue and mammary gland tissue samples adjacent to the tumor.The results showed that there was no sterile environment in the canine mammary tumor tissue and there was a specific microbial flora.At the phylum level,Proteobacteria,Firmicutes and Bacteroidota are the dominant bacteria;At the genus level,the dominant species are Ralstonia,Allorhozobium,Pseudomonas,Bacteroides,Lactobacillus,Methrorubrum and Acetobacter.4)Analyze the gut microbiota characteristics of dogs with mammary tumors: in order to analyze the intestinal microflora characteristics of dogs with mammary tumors,we conducted 16 S r RNA high-throughput sequencing on fecal samples of dogs with mammary tumors and healthy dogs.The results showed that there were specific microbial communities in the fecal samples of dogs with mammary tumors and healthy dogs,and there were certain differences in the distribution and abundance of intestinal microflora between the two groups;At the genus level,the dominant intestinal flora of dogs with mammary tumors include Ralstonia,Allorhozobium,Pseudomonas,Bacteroides,Lactobacillus,Methrorubrum and Acetobacter,among which,the abundance of Bacteroides,Helicobacter and Cetobacterium in the intestinal flora of dogs with mammary tumors is significantly increased,suggesting that these intestinal differential flora may play an important role in the occurrence and development of canine breast cancer.5)Analyze the oral microbiota characteristics of dogs with mammary tumors: in order to analyze the characteristics of oral flora in dogs with mammary tumors,we conducted 16 S r RNA high-throughput sequencing on oral swab samples from dogs with mammary tumors and healthy dogs.The results showed that there were specific microbial communities in the oral swab samples of dogs with mammary tumors and healthy dogs,and there were certain differences in the distribution and abundance of oral microbial communities between the two groups;At the generic level,the dominant oral flora of dogs with mammary tumors include Porphyromonas,Fusobacterium,Moraxella,Haemophilus,Corynebacterium,Streptococcus,etc;Compared with normal dogs,the relative abundance of Treponema,Bacteroide,Acholeplasma,Pseudomonas and Mycoplasma in the oral flora of dogs with mammary tumors was significantly increased,while Fusobacterium was the opposite,suggesting that these oral differential flora might play an important role in the occurrence and development of canine mammary tumors.6)Correlation analysis between canine mammry tumors,gut,and oral microbiota:by analyzing the correlation between the canine mammary tumors tissue,intestinal tract and oral flora in dogs with mammary tumors,the intratumoral flora was isolated by culture-group method,and inoculated into different mammary tumor cells CHMp,CHMm,IMC-118,and then the intratumoral flora was counted,laser confocal scanning of bacterial invasion was performed,and the effect of intratumoral bacteria on tumor cells was preliminarily determined using CCK-8 reagent.The results showed that there was a common flora among the three,namely Streptococcus,Staphylococcus,Corynebacterium and Bacteroides;Five bacteria were successfully isolated from canine mammary tumor tissues,and identified by Gram staining,scanning electron microscopy and 16 S r RNA as Cutibacterium acnes,Streptococcus anginosus,Staphylococcus simulans,Staphylococcus epidermidis and Corynebacterium canis respectively;The colony counting method showed that the supernatant coating plate did not grow a single colony,and there were bacteria growing in the cell lysate,which means that each bacteria successfully invaded the canine mammary tumor cells;Laser confocal can more intuitively observe the invasion of bacteria into canine mammary tumor cells;CCK-8 test results showed that the cell survival rate was proportional to the concentration of bacteria.To sum up,the epidemiological investigation confirmed that canine mammry tumor is a type of tumor that frequently occurs in small animals in clinical practice.Through 16 S r RNA high-throughput sequencing analysis,it was determined that there was a specific flora in canine mammary tumor tissue,and it was confirmed that there was a common flora in canine mammary tumor tissue,intestinal tract,and oral flora.It was verified in vitro that the bacteria in the tumor had a promoting effect on tumor cell proliferation.This study preliminarily revealed the correlation between microbiota and the occurrence and development of canine mammary tumors,laying a good foundation for subsequent research on the role and mechanism of microbiota in the occurrence and development of canine mammary tumors.