To Construct And Authenticate The Triple Negative Breast Cancer Animal Model With High Expression Of PD-L1 And EGFR From Human Breast Cancer

ObjectiveThe paper constructs and identifies humanized TNBC animal models with high expression PD-L1 and EGFR to find new treatments and targets for TNBC with high expression PD-L1 and EGFR,build more Chinese breast cancer models and provide reliable and effective technical guidance.Methods1.Buy 4 Balb/c nude mice with good physical condition,body weight 16g-21g and 6-8 weeks;use MDA-MB-231 cells where Trypsin digestion shows logarithmic growth to make cell suspension whose concentration is about 1X10⁶/ml;inoculate it into milk pad area of Balb/c nude mice;observe and record in detail the tumor growth,infiltration and metastasis after tumor formation.At the end of the 8th-week anesthesia,kill the tumor-bearing mice to collect fresh cancer tissues and divide them into two.A cancer tissue is soaked and fixed by 10%formalin solution and embedded by paraffin to make about 4um paraffin sections,utilizing He staining and immunohistochemistry to detect the expression of ER,PR,Her-2,Ki-67,EGFR and PD-L1.The other is cleaned up blood clot and fat and other impurities by double anti-Hank’s solution and cut into small pieces of size about 1-2mm³ by ophthalmic scissors.The tissue fragments with appropriate amount of collagenase is placed into constant temperature incubator with 37℃、5%CO₂ at night to make it smal er microparticles and even single cell suspensions.Filter it with 100 mesh and gradient centrifugal filtrate to obtain cell suspension with different types of cells and cell precipitation with appropriate amount of culture medium.Adjust the cell suspension with about 1X10⁶/ml concentration and transfer it to the T-25 flask.Place into constant temperature incubator with 37℃、5%CO₂to incubate,change its fluid and passage.To purify the breast cancer cel s by differential adherence method and trypsin digestion method.Al ow it to passage until the cell covers about 80%of the bottom of the culture bottle and then frozen it with liquid nitrogen.2.We collect the fresh breast cancer tissue from the operation room of The First Affiliated Hospital of Hainan Medical University,used double antibiotic Hank’s solution to clean up the surrounding blood clots and adipose tissues and other impurities.（The following methods of operation are same as above）Results1 Specimen identification:Cell suspension was inoculation on the 6th day touched a induration;measuring was carried out on the 9th days;the tumor size was about 0.97±0.073 cm³ at week 4,3.28±0.145cm³at week 8;tumor was more uniform in growth,rich in blood vessels,less necrosis in the central and not with ulcers,see Figure1-Figure2.HE staining:invasive ductal carcinoma;immunohistochemistry:ER（-）,PR（-）,Her-2（-）,Ki-67（+,85%）,EGFR（+90%）and PD-L1（+80%）,its molecular classification and the original cell line all belong to TNBC,see Figure 4–Figure10.2 Cells primary cultured:（1）single cancer cell suspension:inoculation cel s have been grown stably against the wall on the 2nd day when inoculated shown in Figure 11 and covered with more than 80%on the 4th day as shown in Figure 12,give passage and store with being frozen;（2）cancer tissue particles:cancer cel s crawled out from the small particles on the 3rd day and grew around it as shown in Figure 13;the cel s were filled with about 80%of the bottom on the 10th day,as shown in Figure14,give passage and store with being frozen;（3）Cancer cells grows stably until the 6th generation with being good status and high purity as shown in Figure 15-Figure16.（4）Chinese cel s primary cultured:（1）a patient with Luminal-A:cancer cel s crawled out from the small particles of cancer tissue and grew around it on the 7th day when culture as shown in Figure 18-Figure 19.（2）another patient with Luminal-A:cancer cel s crawled out from the small particles of cancer tissue and grew around it on the 4th day as shown in Figure 20-Figure 21.;it grew more robust on the 8th day as shown in Figure 22-Figure 23 and developed slowly on the 15th day as shown in Figure 24-Figure 25.They did not reflect the infinite proliferation like tumor cel s,implement digestion and passage;With the passage of time,the cells gradually break away from the bottom of culture bottle and apoptosis,unable to pass on.Conclusions1.The paper constructed and identified humanized TNBC animal models with high expression PD-L1 and EGFR to find new treatments and targets for TNBC with high expression PD-L1 and EGFR and provide reliable and effective technical guidance for the TNBC model.2.The author complete cancer cell original cultured of tumor-bearing mice （MDA-MB-231 cells）to provide the corresponding research model and guidance for further study on TNBC,especially,its immune therapy,chemotherapy and technical experience for the construction of new Chinese breast cancer cell lines.