Karakterizacija mišjih tumorskih modelov z večbarvnim imunofluorescenčnim barvanjem

Delač Žagar, Neja (author), Čemažar, Maja (mentor), Markelc, Boštjan (comentor)

Pri preučevanju razvoja tumorja moramo upoštevati tudi njegovo mikrookolje, saj to pomembno vpliva na njegov razvoj. Za karakterizacijo tumorja in njegovega mikrookolja je zelo uporabna metoda večbarvna imunohistokemija v kombinaciji s konfokalno mikroskopijo. Ta nam omogoči spremljanje več različnih tipov celic na eni tkivni rezini. Postavili smo protokol za večbarvno imunohistokemijsko barvanje, s katerim smo okarakterizirali štiri različne tumorske modele, in sicer mišji melanom B16F10, mišji karcinom debelega črevesja in danke CT26, mišji karcinom debelega črevesja in danke MC38 ter mišji karcinom dojke 4T1. Barvali smo krvne žile v kombinaciji s celicami CD4, celicami CD8, makrofagi, naravnimi celicami ubijalkami in celicami, ki so v procesu delitve. Protokol je najprimernejši za uporabo na zamrznjenih rezinah, na katerih smo pokazali najvišjo vsebnost imunskih celic v tumorju MC38, kjer so bile prisotne vse barvane imunske celice. Sledi tumor CT26 z nižjo vsebnostjo imunskih celic in nato tumorja 4T1 in B16F10, ki sta vsebovala zgolj makrofage. Z barvanjem krvnih žil smo videli, da so te v tumorju bolj neurejene in redkejše kot v zdravem tkivu. Opazili smo tudi, da so celične delitve izrazitejše ob mestih, kjer so krvne žile številčnejše in večje. Krvne žile so tudi vstopna točka imunskih celic v tumor, kot se to lepo vidi na primeru celic ubijalk v tumorju MC38. Na osnovi rezultatov naše naloge smo postavili protokol večbarvnega imunohistokemijskega barvanja, primernega za uporabo na zamrzlih tkivnih rezinah, s katerim smo opredelili mišje tumorske modele glede na vsebnost imunskih celic, sestavo krvnih žil ter hitrost celičnih delitev.

• Language: Slovenian
• Keywords: biologija, rak, IHC, tumorsko mikrookolje, imunski sistem, tumorski modeli
• Work type: Master's thesis/paper
• Organization: BF - Biotechnical Faculty
• Year: 2020
• PID: 20.500.12556/RUL-121228
• COBISS.SI-ID: 35346691
• Publication date in RUL: 02.10.2020

Secondary language

• Language: English
• Title: Characterisation of mice tumor models with multicolor imunofluoresence staining

Abstract

When studying tumors we have to take into account the tumor microenvironment as well since its effect on tumor development is significant. Multiplexed immunohistochemistry combined with confocal microscopy is a useful method for tumor characterization that enables us to visualize multiple cell types on one tumor tissue section. We have prepared a staining protocol with which we have characterized four different murine tumor models. These tumor models were skin melanoma B16F10, colon carcinoma MC38, colon carcinoma CT26 and mammary carcinoma 4T1. We stained blood vessels in combination with helper T cells CD4, cytotoxic T cells CD8, macrophages, natural killer cells and cells in process of division. Our protocol is best suited for frozen sections on which we showed that the tumor model most infiltrated with immune cells is MC38. It contained all of the immune cells that we have stained. Second most infiltrated tumor model was CT26, whereas the 4T1 and B16F10 tumor models were infiltrated only with macrophages. By staining blood vessels, we have seen that they are disorganized and fewer compared to surrounding healthy tissue. We noticed a greater number of cell divisions occurring in areas where blood vessels are bigger and their density is higher. In the MC38 tumor tissue sections we have also observed the importance of blood vessels for the entry of cytotoxic T cells into the tumor. Based on the results of our study we have prepared a protocol for multiplexed immunohistochemistry that is suitable for frozen tissue sections and used it to characterize tumor models according to the immune cell infiltration, blood vessel organization and rate of cell proliferation.

• Keywords: biology, cancer, IHC, tumor microenvironment, tumor model