The ability of cancer cells to adopt various invasive modes (the plasticity of cancer cell invasiveness) represents a significant obstacle in the treatment of cancer metastasis. Cancer invasiveness involves various modes of migration. Cells can move together (with the preserved intercellular junctions; collective invasiveness) or individually. Within individual invasiveness, we distinguish two principal invasive modes - mesenchymal and amoeboid. The mesenchymal mode of migration is characterized by an elongated shape, proteolytic degradation of the fibres of the extracellular matrix, and the formation of strong contacts with the extracellular matrix. The amoeboid mode of migration is not dependent on proteolytic activity, the cells are characterized by a round shape and increased contractility, which they use to squeeze themselves through the pores of the extracellular matrix. This thesis deals with the analysis of the plasticity of cancer cell invasiveness, specifically the transitions between individual amoeboid and mesenchymal migration modes, in the 3D environment of the collagen gel as a model of extracellular matrix. The work presents models of mesenchymal-to-amoeboid transition (MAT), which include BLM, HT1080 and MDA-MB-231 cell lines, in which MAT is induced by the expression of...

Pyrrolo [2,1-c] [1,4] benzodiazepine (PBD) is a class of natural products obtained from various actinomycetes which have both anti-tumor and antibiotic activities and can bind to specific sequences of DNA. PBD-dilactam was initially produced using isatoic anhydride and (L)-proline which was then converted to the PBD-thiolactam using Lawesson's reagent. Reaction of thiolactam with hydrazine in ethanol afforded PBD-11-hydrazinyl. Condensation of 11-hydrazinyl PBD with aldehydes possessing various substitutions was performed to obtain (S,E)-11-[2-(arylmethylene) hydrazono] pyrrolo [2,1-c] [1,4] benzodiazepine derivatives. 1HNMR, 13C-NMR, DEPT, IR, GC-MS and X-ray crystallography were used for the characterization. Inhibition activity of the products were carried out using TEM-1, AmpC and P99 β-lactamases. A minimal inhibition growth of 25% was observed for one of the selected PBDs on cancer cell line. A promising result was observed on preliminary cannabinoid binding activity test on one of the compounds.

<p>  </p> <p>Adoptive cell transfer of chimeric antigen receptors (CAR) T cells has successfully targeted hematological malignancies in human patients. However, unpredicted side effects experienced after injection of the CAR T cells suggests the need for an optimal predictive preclinical animal model. Dogs have intact immune systems and develop solid tumors spontaneously with similar morphology and genetics to humans. I hypothesize that generating CAR T cells for dogs will closely mimic human patients' outcomes, thus providing new understandings of the safety of this immunotherapy. In addition to the dog as a preclinical model, we propose using a universal CAR T cell to overcome various tumor-related immunosuppressive challenges and control the killing of the target cells. To achieve this, we established methods for activating and expanding canine T cells to a clinically relevant scale. Then, we expressed a second-generation anti-FITC-8-41BB-ζ CAR T cell via lentiviral transduction. In the presence of the correct low-molecular-weight bispecific adapter, we showed <em>in-vitro</em> CAR-mediated function. Our results proved that it is feasible to generate functional canine anti-FITC-8-BB-ζ CAR T cells for therapy.</p>

<p>Oncogenic mutations in KRAS are present in approximately 95% of patients diagnosed with pancreatic ductal adenocarcinoma (<b>PDAC</b>) and are considered the initiating event during the development of pancreatic intraepithelial neoplasia (<b>PanIN</b>) precursor lesions. While it is well established that KRAS mutations can drive the initiation of pancreatic oncogenesis, the effects of oncogenic KRAS signaling on regulation of phosphatases during this process is not fully appreciated. Protein Phosphatase 2A (<b>PP2A</b>) has been implicated in suppressing KRAS-driven cellular transformation. However, low PP2A activity is observed in PDAC cells compared to non-transformed cells, suggesting that suppression of PP2A activity is an important step in the overall development of PDAC. In the current study, we demonstrate that KRASG12D induces the expression of both Cancerous Inhibitor of PP2A (<b>CIP2A</b>), an endogenous inhibitor of PP2A activity, and the PP2A target, c-MYC. Consistent with these findings, KRASG12D sequestered the specific PP2A subunit responsible for c-MYC degradation, B56a, away from the active PP2A holoenzyme in a CIP2A-dependent manner. During PDAC initiation <i>in vivo</i>, knockout of B56a promoted KRASG12D tumorigenesis by accelerating acinar-to-ductal metaplasia (<b>ADM</b>) and the formation of PanIN lesions. The process of ADM was attenuated <i>ex vivo</i> in response to pharmacological re-activation of PP2A utilizing direct small molecule activators of PP2A (<b>SMAP</b>s). Together, the results of this study suggest that suppression of PP2A-B56a through KRAS signaling can promote Myc-driven initiation of pancreatic tumorigenesis.</p>

The radiosensitivity of tumour cells depends mainly on their capacity to maintain genomic integrity. This requires efficient repair of radiation-induced DNA double strand breaks, a process governed by the cell cycle. Based on their functions in cell cycle regulation and DNA damage repair, we hypothesised that targeting of CDK2 and CDK9 modifies cancer cell response to radiotherapy. Therefore, we evaluated the significance of CDK2 and CDK9 for the cellular radiation response in a panel of human head and neck squamous cell carcinoma (HNSCC) cell lines. In order to achieve our goal, we performed a series of experiments to measure several key parameters such as clonogenic radiation survival, cell cycling, DNA damage repair and apoptosis. We found that loss of CDK2 radiosensitises mouse embryonic fibroblasts (MEFs) as well as HNSCC two dimensional (2D) cell cultures. However, under more physiological three dimensional (3D) growth conditions in laminin-rich extracellular matrix, targeting of CDK2 failed to modulate the radiosensitivity of HNSCC cells. Moreover, CDK2 attenuated the repair of radiogenic double strand breaks (DSBs) in MEFs as well as SAS and FaDu HNSCC cells indicating a possible role of CDK2 in DNA damage repair. However, we found that CDK2 is dispensable for cell cycle and checkpoint regulation in response to irradiation in SAS and FaDu cells. Taken together, our results suggest that targeting of CDK2 may not provide a therapeutic benefit to overcome HNSCC cell resistance to radiotherapy. We also showed that depletion of CDK9 clearly enhances the radiosensitivity of HNSCC cultures. In addition, the ectopic expression of CDK9 has a radioprotective effect. These findings suggest a potential role of CDK9 in the radiation response of HNSCC cells. Moreover, our study indicates a possible role of CDK9 in the DNA damage repair response and cell cycling of HNSCC cells. Conclusively, on the basis of these data, targeting of CDK9 in addition to conventional radiotherapy might be a viable strategy to overcome cancer cell resistance.

During the epithelial-to-mesenchymal transition, the intracellular cytoskeleton undergoes severe reorganization which allows epithelial cells to transition into a motile mesenchymal phenotype. Among the different cytoskeletal elements, the intermediate filaments keratin (in epithelial cells) and vimentin (in mesenchymal cells) have been demonstrated to be useful and reliable histological markers. In this study, we assess the potential invasiveness of six human breast carcinoma cell lines and two mouse fibroblasts cells lines through single cell migration assays in confinement. We find that the keratin and vimentin networks behave mechanically the same when cells crawl through narrow channels and that vimentin protein expression does not strongly correlate to single cells invasiveness. Instead, we find that what determines successful migration through confining spaces is the ability of cells to mechanically switch from a substrate-dependent stress fibers based contractility to a substrate-independent cortical contractility, which is not linked to their tumor phenotype.

Despite significant advances in cancer treatment, clinical response remains suboptimal and there is a continued requirement for improved chemotherapeutics. The attrition rate for new therapies is high, due principally to lack of in vivo efficacy and poor pharmacodynamics. Consequently better systems are required to determine in vivo preclinical efficiency and drug-target interactions. Engineering of cancer cells to express fluorescent or bioluminescent proteins, either endogenously or under the control of specific gene promoters, and their detection by noninvasive optical imaging has the potential to improve preclinical drug development. In this study, a panel of colorectal cancer cell lines were engineered to express fluorescent and luminescent proteins either constitutively or under control of gene-promoters for the DNA damage response gene p53 or the cell cycle regulator p21, both important pharmacodynamic sensors. These cell lines were characterised for their potential as in vivo models of primary and metastatic tumour therapy response, several showing significant potential. In addition to the development of these models, this study also addressed the pharmacokinetics of different luciferase substrates and identified optimal temporal and dose characteristics for each. Furthermore, a new application for bioluminescent imaging was developed and validated for use in preclinical evaluation of vascular disrupting agents, a new generation of cancer therapeutic. This study demonstrates that despite the dynamic and variable nature of fluorescent and bioluminescent imaging, reproducible results can be obtained if appropriate precautions are taken. The models developed herein will expedite cancer drug development whilst reducing and refining the use of animals in research.

Cell migration and invasion play a role in many physiological and pathological processes and are therefore subject of intensive research efforts. Despite of the intensively investigated biochemical processes associated with the migration and invasion of cells, such as cancer cells, the contribution ofmechanobiological processes to themigratory capacity of cells as well as the role of physical polymeric phase transitions is not yet clearly understood. Unfortunately, these experiments are not very informative because they completely disregard the influence of the three-dimensional cell environment. Despite this data situation, it was possible to adequately demonstrate that there exists a direct mechanical interplay between cells and theirmicroenvironment in both directions, where both elements can bemechanically altered by one another. In line with these results, it has turned out that the mechanobiological molecular processes through which cells interact with each other and additionally sense their nearby microenvironment have an impact on cellular functions such as cellular motility. The mechanotransduction processes have become the major focus of biophysical research and thereby, diverse biophysical approaches have been developed and improved to analyze the mechanical properties of individual cells and extracellular matrix environments. Both, the cell mechanics and matrix environmentmechanics regulate the cellmigration types in confined microenvironments and hence it seems to be suitable to identify and subsequently present a common bidirectional interplay between cells and their matrix environment. Moreover, hallmarks of the mechanophenotype of invasive cells and extracellular matrices can be defined. This review will point out how on the one hand the intracellular cytoskeletal architecture and on the other hand the matrix architecture contribute to cellular stiffness or contractility and thereby determines the migratory phenotype and subsequently the emergence of a distinct migration mode. Finally, in this review it is discussed whether universal hallmarks of the migratory phenotype can be defined.

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