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.
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