Karakterizacija imunskega odziva mišjih tumorjev na obsevanje z večbarvnim imunofluorescenčnim barvanjem : magistrsko delo

Kozjek-Mencinger, Lucija (avtor), Serša, Gregor (mentor), Markelc, Boštjan (komentor), Čemažar, Maja (recenzent)

Uvod: Rak je bolezen oziroma skupek bolezni za katere je značilna nenadzorovana rast in delitev celic. V zadnjem času se velik pomen v razvoju tumorja pripisuje tumorskemu mikrookolju. Stopnja ter vrsta infiltracije imunskih celic v tumorsko tkivo lahko vpliva na potek bolezni in učinkovitost terapij, zato je pomembno dobro poznavanje sestave tumorskega mikrookolja ter kako se le to spreminja zaradi delovanja terapij. Namen: Namen magistrske naloge je bil z uporabo večbarvnega imunofluorescenčnega barvanja ugotoviti, do kakšnih sprememb pride v tumorskem mikrookolju mišjih tumorskih modelov raka debelega črevesa CT26 in raka dojke 4T1 po obsevanju in kako obsevanje vpliva na zaostanek v rasti tumorja v primerjavi z ne-obsevanimi tumorji. Metode dela: V magistrski nalogi smo uporabili metodo večbarvnega imunofluorescenčnega barvanja v kombinaciji s konfokalno mikroskopijo, ki se je izkazala kot učinkovita metoda za preučevanje tumorskega mikrookolja in odziva na različne terapije. Z njo lahko sočasno opazujemo različne populacije celic, ki so prisotne v tumorskem mikrookolju. Uporabili smo dva tumorska modela: CT26 (mišji tumorski model raka debelega črevesa) in 4T1 (mišji tumorski model raka dojke). Eksperiment je vključeval obsevanje tumorjev z enkratno dozo 10 Gy ali frakcionirano dozo 3 x 5 Gy. Z barvanjem smo prikazali krvne žile, imunske celice, natančneje limfocite T CD4 in CD8 (celice CD4 oz. celice pomagalke, celice CD8 oz. celice ubijalke), makrofage in celična jedra, ki smo jih obarvali z barvilom Hoechst 33342. Protitumorski učinek obsevanja so določali s spremljanjem zaostanka v rasti podkožnih tumorjev po terapiji, in sicer z merjenjem prostornine tumorja z Vernierjevim merilnikom. Rezultati: Rezultat eksperimenta so slike in grafi (za vsako terapijo posebej). Slike smo zajemali s konfokalnim mikroskopom ZEISS LSM 800 in analizirali s programom za analizo slik Imaris. Analizo ter statistično obdelavo pridobljenih podatkov smo izvedli v programu GraphPad Prism. Uporabili smo enosmerno analizo variance ANOVA, neparni t test in Mann-Whitney U test. Obsevanje z 10 Gy in 3 x 5 Gy je povzročila spremembe v tumorskem mikrookolju in vplivala na zaostanek v rasti tumorja v primerjavi z ne-obsevanimi tumorji. Število celic CD4 in CD8 se je tretji dan po terapiji z 10 Gy pri tumorskem modelu 4T1 in sedmi dan po terapiji s 3 x 5 Gy pri tumorskem modelu CT26 povečalo. Do povečanja števila celic CD8 je prišlo tudi sedmi dan po terapiji z 10 Gy pri tumorju 4T1. Število žil se je pri tumorskem modelu 4T1 tretji dan po terapiji z 10 Gy povečalo. Pri vseh ostalih primerih tako pri CT26 kot 4T1 pa se je število celic CD4 in CD8 zmanjšalo, prav tako tudi število krvnih žil. Razprava in zaključek: V raziskovalni nalogi smo prikazali, da je metoda večbarvnega imunofluorescenčnega barvanja primerna za pridobitev informacij o sestavi tumorskega mikrookolja. Pokazali smo, da obsevanje povzroči spremembe v tumorskem mikrookolju saj se je po obsevanju spremenilo število preiskovanih imunskih celic v tumorju, s čimer smo potrdili našo drugo hipotezo. Obsevanje tumorjev je tudi povzročilo zaostanek v njihovi rasti v primerjavi z ne-obsevanimi tumorji s čimer smo potrdili našo prvo hipotezo. Poznavanje dinamike infiltracije imunskih celic v tumorsko tkivo bo pomagalo pri razumevanju mehanizmov odziva tumorja na terapijo ter pri nadaljnji optimizaciji kombiniranih terapij.

• Jezik: Slovenski jezik
• Ključne besede: magistrska dela, radiološka tehnologija, rak, mišji tumorski modeli, tumorsko mikrookolje, imunski sistem, obsevanje, večbarvno imunofluorescenčno barvanje
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: ZF - Zdravstvena fakulteta
• Kraj izida: Ljubljana
• Založnik: [L. Kozjek Mencinger]
• Leto izida: 2022
• Št. strani: 92 str.
• PID: 20.500.12556/RUL-141688
• UDK: 616-07
• COBISS.SI-ID: 124436483
• Datum objave v RUL: 05.10.2022

KOZJEK-MENCINGER, Lucija, 2022, Karakterizacija imunskega odziva mišjih tumorjev na obsevanje z večbarvnim imunofluorescenčnim barvanjem : magistrsko delo [na spletu]. Magistrsko delo. Ljubljana : L. Kozjek Mencinger. [Dostopano 21 april 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=141688

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Characterization of immune response of murine tumors to irradiation with multicolor immunofluorescence staining : master thesis

Izvleček

Introduction: Cancer is a disease or set of diseases characterized by uncontrolled growth and cell division. Recently, great importance in tumor development has been attributed to the tumor microenvironment. The degree and type of infiltration of immune cells into the tumor tissue can affect the course of the disease and the effectiveness of the therapy, therefore; it is important to know the composition of the tumor microenvironment and how it changes due to the therapy. Purpose: The purpose of the master's thesis was to determine the changes in the tumor microenvironment of mouse tumor models of colon cancer CT26 and breast cancer 4T1 after irradiation using multicolor immunofluorescence staining and how irradiation affects tumor growth compared to non-irradiated tumors. Methods: In the master's thesis we used the method of multicolor immunofluorescence staining in combination with confocal microscopy, which proved to be an effective method for studying the tumor microenvironment and the response to therapy. With it, we can simultaneously observe different populations of cells present in the tumor microenvironment. Two tumor models were used: CT26 (mouse tumor model of colon cancer) and 4T1 (mouse tumor model of breast cancer). The experiment involved irradiating tumors with a single dose of 10 Gy or a fractionated dose of 3 x 5 Gy. With the staining we showed tumor blood vessels, immune cells, more specifically T lymphocytes CD4 and CD8 (CD4 cells or helper cells, CD8 cells or killer cells), macrophages, and cell nuclei stained with Hoechst 33342 dye. The antitumor effect of irradiation was determined by determining the tumor growth delay after the therapy, namely by measuring the volume of the tumor with a Vernier caliper. Results: The result of the experiment are images (for each therapy separately). They were captured with a ZEISS LSM 800 confocal microscope and analysed with Imaris image analysis software. The analysis and statistical processing of the obtained data was carried out in the GraphPad Prism program. We used One-way analysis of variance ANOVA, unpaired t test, and Mann-Whitney U test. Irradiation with 10 Gy and 3 x 5 Gy induced changes in the tumor microenvironment and affected tumor growth delay compared to non-irradiated tumors. The number of CD4 and CD8 cells increased on the third day after 10 Gy therapy in the 4T1 tumor model and on the seventh day after 3 x 5 Gy therapy in the CT26 tumor model. An increase in the number of CD8 cells also occurred on the seventh day after 10 Gy therapy in the 4T1 tumor. The number of vessels increased in the 4T1 tumor model on the third day after 10 Gy therapy. In all other cases, in both CT26 and 4T1, the number of CD4 and CD8 cells decreased, as did the number of blood vessels. Discussion and conclusion: In our research, we showed that the method of multicolor immunofluorescence staining is suitable for obtaining information about the composition of the tumor microenvironment. We have shown that irradiation causes changes in the tumor microenvironment in the populations of the examined immune cells, thus we have confirmed our second hypothesis. Irradiation of tumors also led to tumor growth compared to non-irradiated tumors, which confirmed our first hypothesis. Knowledge of the dynamics of immune cell infiltration into tumor tissue will help in understanding the mechanisms of tumor response to therapy and in the further optimization of combined therapies.

• Ključne besede: master's theses, radiologic technology, cancer, mouse tumor models, tumor microenvironment, immune system, irradiation, multicolor immunofluorescence staining