| As its incidence is growing, breast cancer has become a serious threat ofwomen’s health. Numerous studies on the pathogenesis, therapy and prevention ofbreast cancer have been extensively carried out. Mouse models offer an importantplatform for investigation into the mechanisms and the evaluation the efficacy ofanti-cancer drugs/treatments. Several mouse models of mammary cancer have beendeveloped. These models have their own advantages and disadvantages. Notably, theyfailed to provide the normal human mammary microenvironment. A few years ago,we developed a novel human breast tissue-derived orthotopic and metastatic (BOM)mouse model of breast cancer, in which the normal human breast tissues wereimplanted subcutaneously, providing a normal human mammary microenvironment.After the “breast to breast” one, our BOM model extented to “breast to bone”.Compared to other models, the advantage of our model is providing themicroenvironment of the breast and target organs in situ. We also well mimiced themetastasis process of breast cancer. In order to further understand and interpret theadvantage of our humanized models, to know well of the parameters of our models,so as to improve and promote our models, a variety of techniques were used. Weobserved the adaption, the rejuvenation, the stable period and the aging process ofhuman xenograft in mice. The changes in their structure and function in differentperiod are our most concern.Small pieces of human breast tissues were implanted into mice subcutaneouslyas described before. Each week, one of the mice was sacrificed and dissected. Theimplanted human tissues and the mouse breast fat pad were observed grossly and thensent to histological examination. Sections of human breast tissue and mice fat padwere stained with hematoxylin and eosin, and examined under a microscope. Afterantigen retrieval and blocking, sections mounted on slides were incubated with antibodies against human Calponin, P63, Ki67, Aromatase and Steroid sulfatase.Transmission electron microscopy was used for examining the cell ultrastructuremodification of human breast tissue after implanted into mice. Fresh tumor tissueswere implanted into another group of mice as a comparison.A capsule which coating the xenograft was formed within one week after theimplant surgery, and blood vessels could be found on surface of the capsule. As timepassed, the implanted tissue was gradually narrowing and became tough.There’s a lot of imagery of human breast epithelial cells swelling anddeformation and glandular structures blur in the first few weeks after implantation.This change became increasingly obvious in the first three weeks. Gland morphologyreached the worst at the2nd-3rdweeks and started to improve from the4thweek. Fromthe5thweek, the nearly normal ductal structures looked similar to thosepre-implantation and remained resemble until the13thweek. After implantation,hyaline degeneration and nucleus deformation of epithelial cells and fattydegeneration could be observed at microscopy high power (10×40). Thesedegeneration phenomena became increasingly distinct during the first three weeksafter surgery. Epithelial cell morphology reached the worst at the2nd-3rdweeks andstarted to improve from the4thweek. The5thweek’s gland morphology was similar tothat pre-implantation. Then cell swelling began to release. These slices alwayspresented interstitial fibrosis and hyaline degeneration under light microscope. Thedegeneration of stroma had no trends over time.We used TEM to study the specimens of each week. In the first week, epithelialcells morphology especially the nuclear morphology was widely different from thoseof normal mammary. Steatosis was quite obvious during the2ndweek. Visibleintracellular fat vacuoles, intranuclear lipid inclusion could also be seen while cellorganelle structure was not obvious. Mitochondrion swelling was obvious in the third week after implantation. Normal mitochondria with clear mitochondrial cristae couldbe watched in the4thweek. From the5thweek, intercellular space could be clearlyseen. What’s more, abundant organelles and intercellular junctions such asdesmosomes were visible. After then, epithelial cells maintained normal morphologyuntil the13thweek. Interestingly, we observed some cells looked similar to thosemammary epithelial cells at late stage of pregnancy in the sixth-week specimens.These cells had rich organelles and secretory vesicles. There were two differentelectron density (higher and lower) vesicles.Vascular structures were visible on the HE slides and under TEM. What’s more,red blood cells could be found in these vasculars suggesting that these blood vesselswere functional.Immunohistochemistry (IHC) was used to analyze the expression of Calponin(Cal) and P63of weekly samples so as to measure the stability of the myoepithelialcells surrounding the ductal structures. Normal breast specimens presented that theducts were well surrounded by myoepithelial cells. As what mentioned before, the1st-2ndweeks’ specimens had the poorest morphology. Although there wasn’t clearvariation of P63expression, the loss expression of Cal indicated a part of the duct lostthe surrounding of normal myoepithelial cells. We selected Ki67as a marker of cellproliferation. Ki67was scattered expressed in the mammary glands before and afterimplantation. There wasn’t obvious increasing or loss of expression over time. Wespeculated that the implanted glands may be able to locally synthetic estrogen.Aromatase and steroid sulfatase (STS) were the rate-limiting enzymes of two ways oflocal estrogen synthesis. Two antibodies showed negative staining results in breasttissue before and after transplantation.All the mice used in our experiment were virgin, so their breast tissue presentedthe performance of inactive mammary gland. The may part of MFP are lipid composition mixed with ducts and mesenchymal cells. The MFP didn’t appearsignificant time-related changes.We implanted human breast cancer tissue into six mice. After eight weeks,cancer tissue implanted all failed to grow in situ. As time goes by, the volume of thetumor tissue reduced significantly. The tumor sizes were shrink when removed fromhosts than that pre-implantation. Blood vessels could be seen around and within thetumor tissue implanted into mice. There were red blood cells in these vascularstructures. The growth of the tumor is confined to the capsule. Necrosis could be seenin the center area of tumors.Normal human breast tissues went through the adaptation period (1-3w)-therejuvenation period (4w)-the stable period (5w-13w)-aging period aftertransplantation into mice. After the rejuvenation period, normal human mammaryglandular structure can be maintained in quite a long time. The cell structure,subcellular structures, vascular structures all correspond to normal situation in humanbody.The implanted breast tissue reserved the function of normal breast tissue that isthe ability of lactation. The time-relate trend over time of the stability of basementmembrane-myoepithelial is similar to luminal epithelial cells trend. No significantchange of Ki67expression over time also reflects the stablity of graft proliferation inthe host environment. The negative staining of Aromatase and STS suggested thathuman breast tissue did not synthesize estrogen.The structure of mouse MFP and the failure of tumorigenesis both suggest thatthe environment of mouse MFP and mouse subcutaneous tissue are different fromhuman breast environment. They are not suitable for the growth and metastasis ofhuman breast cancer cells. Normal human breast tissue can survive subcutaneously inmice. It has normal structure and function so as to provide the microenvironment required by human breast cancer. |
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