No description available.

Side population is a minor subpopulation (SP) of some cell lines, exporting staining dye Hoechst 33342 out of their cytoplasm. It is discussed as a possible source of "cancer stem cells", "tumour initiating cells" or "metastasis initiating cells". However, broad literature suggest, that stemness and other privileged properties of SP are very variable between different cell types, cell lines and stage of disease. Cell lines TRAMP are the only widely available murine models for testing of prostate cancer therapy. We noticed in literature a mention about existence of 1-2% of cells constituting side population, but detailed characteristic have not been described until now. In this diploma thesis, we worked on characterisation of SP of the TRAMP-C2 cell line in comparison to other cells (nonSP). In the first part, we compared stem properties of SP and nonSP. We started with checking the existence of SP by its verapamil sensitivity. Using mRNA analysis, we showed that neither SP nor nonSP have increased c-Kit expression and that there are no differences in Bmi-1 expression. We found that SP is heterogenic mixture of CD24-CD44-, CD24-CD44+ and CD24+CD44+ cells, while nonSP is almost solely CD24-CD44+. We documented that SP and nonSP returned back to original SP ratio during cultivation. Then we showed on...

No description available.

MHCI molecules are constitutively expressed in all nucleated cells and play a key role in antigen presentation to CD8+ T lymphocytes. One of the tumor immune evasion strategies is MHCI expression downregulation. This leads to an impaired recognition of tumor antigens by CD8+ T lymphocytes that are unable to start the immune response. Since the MHCI expression downregulation occurs in up to 90 % of some tumors it is neccesary to have a clinical relevant tumor model without a MHCI surface expression that would be used for testing of immunotherapeutic approaches. This thesis describes a production of new model cell lines of TC-1 tumor cells with irreversibly downregulated MHCI. That was achieved by an inactivation of B2m, which is a part of MHCI, by gene editing using CRISR/Cas9. The B2m inactivation was confirmed by flow cytometry, western blot and sanger sequencing of single alleles. The inactivation slowed down the cell growth for both in vitro and in vivo. The cell metastatic activity was not affected. The tumors established by cells without the B2m expression are not sensitive to DNA vaccine against HPV16 E7 oncoprotein by a pBSC/PADRE.E7GGG vaccine. The main effector function against these tumors possess the NK1.1+ cells. In a therapeutic vaccination experiment it was repeatedly achieved of...

We studied patterning mechanisms and cell fates during limb regeneration in the axolotl. 1) It is crucial to understand the earliest events of patterning. Since it is technically challenging to study early events, we established single cell PCR. This new tool will allow us to obtain novel insight into the initial steps of limb patterning. 2)We have examined the roles of different tissues regarding their fates and features of proximo- distal patterning. Our strategy was to transplant GFP+ skin, skeleton, muscle and Schwann cells from transgenic donors to limbs of wild type hosts, amputate through the graft and analyze fluorescent progeny combined with the use of molecular markers. Our results revealed that different subpopulations of blastema cells exist regarding two aspects. First, we found that progeny of skin and skeleton have some tissue specific memory since they did not give rise to muscle lineages. However, cells of the skin contributed to other mesenchymal tissues like cartilage or tendons, while the majority of skeleton- derived cells undergoes self- renewal. Second, we performed one cellular and two molecular assays to investigate what tissues generate cells that exhibit features of proximo- distal patterning. Both assays revealed that Schwann cell- derived progeny do not display such features while progeny of skin, skeleton and muscle did. Therefore, we conclude that the blastema is a heterogeneous mix of cells regarding tissue lineages and features of proximo- distal patterning.

We studied patterning mechanisms and cell fates during limb regeneration in the axolotl. 1) It is crucial to understand the earliest events of patterning. Since it is technically challenging to study early events, we established single cell PCR. This new tool will allow us to obtain novel insight into the initial steps of limb patterning. 2)We have examined the roles of different tissues regarding their fates and features of proximo- distal patterning. Our strategy was to transplant GFP+ skin, skeleton, muscle and Schwann cells from transgenic donors to limbs of wild type hosts, amputate through the graft and analyze fluorescent progeny combined with the use of molecular markers. Our results revealed that different subpopulations of blastema cells exist regarding two aspects. First, we found that progeny of skin and skeleton have some tissue specific memory since they did not give rise to muscle lineages. However, cells of the skin contributed to other mesenchymal tissues like cartilage or tendons, while the majority of skeleton- derived cells undergoes self- renewal. Second, we performed one cellular and two molecular assays to investigate what tissues generate cells that exhibit features of proximo- distal patterning. Both assays revealed that Schwann cell- derived progeny do not display such features while progeny of skin, skeleton and muscle did. Therefore, we conclude that the blastema is a heterogeneous mix of cells regarding tissue lineages and features of proximo- distal patterning.

<p>  </p> <p>Metastatic breast cancer (MBC) is an extremely recalcitrant disease capable of overcoming targeted therapies and evading immune surveillance via the engagement of complicated signaling networks. Resistance to targeted therapies and therapeutic failure of immune checkpoint blockade (ICB) are two major challenges in treating MBC. To survive in the dynamic tumor microenvironment (TME) during metastatic progression, shared signaling nodes are required for MBC cells to regulate the signaling networks efficiently, which are potential multifunctional therapeutic targets. SH2 containing protein tyrosine phosphatase-2 (SHP2) is a druggable oncogenic phosphatase that is a key shared node in both tumor cells and immune cells. How tumor-cell autonomous SHP2 manages its signaling inputs and outputs to facilitate the growth of tumor cells, drug resistance, immunosuppression, and the limited response of ICB in MBC is not fully understood. Herein, we used inducible genetic depletion and two distinct types of pharmacological inhibitors to investigate anti-tumor effects with immune reprogramming during SHP2 targeting. </p> <p>We first focus on the signaling inputs and outputs of SHP2. We find that phosphorylation of SHP2 at Y542 predicts the survival rates of breast cancer patients and their immune profiles. Phosphorylation of SHP2 at Y542 is elevated with differential activation mechanisms under a growth-factor-induced and extracellular matrix (ECM)-rich culture environment. Phosphorylation of SHP2 at Y542 is also elevated in HER2 positive MBC cells upon acquired resistance to the HER2 kinase inhibitor, neratinib. The resistant cells can be targeted by SHP2 inhibitors. SHP2 inhibitors block ERK1/2 and AKT signaling and readily prevented MBC cell growth induced by multiple growth factors. Inhibition of SHP2 also blocks these signaling events generated from the ECM signaling. In fact, the inhibitory effects of SHP2 blockade are actually enhanced in the ECM-rich culture environment. We utilize the <em>in vitro</em> T-cell killing assays and demonstrate that pretreatment of tumor cells with FGF2 and PDGF reduces the cytotoxicity of CD8+ T cells in a SHP2-dependent manner. Both growth factors and ECM-rich culture environment transcriptionally induce PD-L1 via SHP2. SHP2 inhibition balances MAPK signaling and STAT1 signaling, which prevents growth factor-mediated suppression of INF-γ-induced expression of MHC class I. </p> <p>Next, we evaluate the efficacy of SHP2 inhibitors. Blockade of SHP2 in the adjuvant setting decreased pulmonary metastasis <em>in vivo</em> and extended the survival of systemic tumor-bearing mice. Tumor-cell autonomous depletion of SHP2 reduces pulmonary metastasis and relieves exhaustion markers on CD8+ and CD4+ cells. Meanwhile, both systemic SHP2 inhibition and tumor-cell autonomous SHP2 depletion reduce tumor-infiltrated CD4+ T cells and M2-polarized tumor associated macrophages. </p> <p>Finally, we investigate potential combination therapies with SHP2 inhibitors. The combination of SHP2 inhibitors and FGFR-targeted kinase inhibitors synergistically blocks the growth of MBC cells. Pharmacological inhibition SHP2 sensitizes MBC cells growing in the lung to α-PD-L1 antibody treatment via relieving T cell exhaustion induced by ICB. </p> <p>Overall, our findings support the conclusion that MBC cells are capable of simultaneously engaging several survival pathways and immune-suppressive mechanisms via SHP2 in response to multiple growth factors and ECM signaling. Inhibition of SHP2, potentially in combination with other targeted agents and ICB, holds promise for the therapeutic management of MBC.</p>