<p>The role of platelet-derived growth factor (PDGF) in malignancies involves both autocrine and paracrine stimulation of cells within the tumor. The interstitial fluid pressure (IFP) is one of the forces that govern the transvascular flow of fluids. In both experimental and clinical cancers, the IFP is elevated and is thought to act as a barrier for delivery of drugs. Increasing evidence points to PDGF as a positive regulator of the interstitial fluid pressure in loose connective tissue. In this thesis, the effect of PDGF receptor inhibition on the tumor IFP, transvascular transport and efficacy of anti-cancer drugs is investigated.</p><p>All studies were performed using tumor models that display extensive tumor stroma and PDGF receptor expression restricted to stroma cells. Blocking of PDGF receptor signaling significantly reduced the tumor IFP in various tumor models. In parallel, pre-treatment with PDGF antagonists increased the tumor content of cytotoxic agents without affecting the uptake in other organs. Moreover, combination treatment with PDGF receptor inhibitors and chemotherapeutic agents dramatically enhanced the anti-tumor effects of the cytotoxic drugs, whereas treatment with only PDGF receptor inhibitors did not affect the growth of the tumors. Beneficial effects on the tumor reponse to radioimmunotherapy were also produced after concomitant administration of PDGF antagonists. Importantly, anti-angiogenic effects, changes in cell composition and increased tumor cell sensitivity to cytotoxic agents were ruled out as the cause for the synergistic effects. </p><p>Studies with different temporal scheduling of PDGF receptor inhibitors demonstrated a perfect correlation between a reduced IFP, an increased transvascular transport and an enhanced therapeutic effect of cytotoxic drugs, strongly suggesting that the phenomena are causally linked.</p><p>The studies presented herein illustrate for the first time the potential of cells in the stroma compartment as a target for efforts to treat cancer. In conclusion, a novel, possibly general, strategy to enhance the effects of conventional anti-cancer drugs has been identified.</p>

The role of platelet-derived growth factor (PDGF) in malignancies involves both autocrine and paracrine stimulation of cells within the tumor. The interstitial fluid pressure (IFP) is one of the forces that govern the transvascular flow of fluids. In both experimental and clinical cancers, the IFP is elevated and is thought to act as a barrier for delivery of drugs. Increasing evidence points to PDGF as a positive regulator of the interstitial fluid pressure in loose connective tissue. In this thesis, the effect of PDGF receptor inhibition on the tumor IFP, transvascular transport and efficacy of anti-cancer drugs is investigated. All studies were performed using tumor models that display extensive tumor stroma and PDGF receptor expression restricted to stroma cells. Blocking of PDGF receptor signaling significantly reduced the tumor IFP in various tumor models. In parallel, pre-treatment with PDGF antagonists increased the tumor content of cytotoxic agents without affecting the uptake in other organs. Moreover, combination treatment with PDGF receptor inhibitors and chemotherapeutic agents dramatically enhanced the anti-tumor effects of the cytotoxic drugs, whereas treatment with only PDGF receptor inhibitors did not affect the growth of the tumors. Beneficial effects on the tumor reponse to radioimmunotherapy were also produced after concomitant administration of PDGF antagonists. Importantly, anti-angiogenic effects, changes in cell composition and increased tumor cell sensitivity to cytotoxic agents were ruled out as the cause for the synergistic effects. Studies with different temporal scheduling of PDGF receptor inhibitors demonstrated a perfect correlation between a reduced IFP, an increased transvascular transport and an enhanced therapeutic effect of cytotoxic drugs, strongly suggesting that the phenomena are causally linked. The studies presented herein illustrate for the first time the potential of cells in the stroma compartment as a target for efforts to treat cancer. In conclusion, a novel, possibly general, strategy to enhance the effects of conventional anti-cancer drugs has been identified.

The ErbB family of receptors are key regulators of growth, differentiation, migration and survival of epithelial cells. CI-1033 is an irreversible pan-ErbB tyrosine kinase inhibitor that has the ability to inhibit EGFR function but has shown limited therapeutic efficacy. Lovastatin targets the activity of HMG-CoA reductase, the rate-limiting step in the mevalonate pathway. In this study, the ability of lovastatin to potentiate the cytotoxic effects of CI-1033 was evaluated. The combination of lovastatin and CI-1033 exhibited some cooperative cytotoxic activity in a squamous cell carcinoma–derived cell line. This combination resulted in enhanced cell death by induction of a potent apoptotic response. Furthermore, this drug combination inhibited EGF-induced EGFR autophosphorylation and activation of the downstream signaling effectors, ERK and AKT. These findings suggest that combining lovastatin and tyrosine kinase inhibitors may represent a novel combinational therapeutic approach in squamous cell carcinoma of the head and neck.

The ErbB family of receptors are key regulators of growth, differentiation, migration and survival of epithelial cells. CI-1033 is an irreversible pan-ErbB tyrosine kinase inhibitor that has the ability to inhibit EGFR function but has shown limited therapeutic efficacy. Lovastatin targets the activity of HMG-CoA reductase, the rate-limiting step in the mevalonate pathway. In this study, the ability of lovastatin to potentiate the cytotoxic effects of CI-1033 was evaluated. The combination of lovastatin and CI-1033 exhibited some cooperative cytotoxic activity in a squamous cell carcinoma–derived cell line. This combination resulted in enhanced cell death by induction of a potent apoptotic response. Furthermore, this drug combination inhibited EGF-induced EGFR autophosphorylation and activation of the downstream signaling effectors, ERK and AKT. These findings suggest that combining lovastatin and tyrosine kinase inhibitors may represent a novel combinational therapeutic approach in squamous cell carcinoma of the head and neck.

Efficacy of tyrosine kinase inhibitor (TKI) therapy depends on epidermal growth factor receptor (EGFR) mutation status in patients with non-small cell lung cancer (NSCLC). There has been an increasing interest in studying mutation-specific rabbit monoclonal antibodies of delE746-A750 mutation in exon 19 and L858R point mutation in exon 21 for detecting EGFR mutants. These two mutations account for approximately 90% of all EGFR mutations. We evaluated the two mutation-specific monoclonal antibodies for the detection of EGFR mutations by immunohistochemistry (IHC) and the relationship with treatment outcome and survival. Twenty-five patients (58.1%) harbored EGFR mutations. These mutations include delE746-A750 mutation for seven patients, L858R point mutation for in eighteen patients. IHC showed, for the delE746-A750 and L858R mutations, sensitivity (57.1% and 66.7%), specificity (97.3% and 100%), positive predictive value (80.0% and 100%), and negative predictive value (94.7% and 80.6%). Analysis for progression-free survival was not correlated to IHC staining, but the overall survival was correlated to IHC staining. These mutation-specific antibodies for delE746-A750 and L858R mutations have high positive predictive value and specificity for predefined EGFR mutations and may be suitable for screening for these predefined mutations. However, negative IHC results required further mutation analyses before excluding EGFR TKI therapy.

Sarcomas are rare mesenchymally-derived tumors with limited treatment options. Tyrosine kinases may serve as potential targets for sarcoma therapy because many are mutated or overexpressed in sarcomas and cell lines. One potential molecular target for sarcoma treatment is the Src tyrosine kinase. Three independently synthesized Src kinase inhibitors were evaluated in human sarcoma cell lines. Of the three, dasatinib, provided promising results as a potential sarcoma therapy. Until this study, dasatinib activity had not been characterized in sarcoma cells. Based on our previous findings of Src activation in human sarcomas, we evaluated the effects of dasatinib in twelve sarcoma cell lines. Dasatinib inhibited Src activity and downstream signaling at nanomolar concentrations. Inhibition of Src signaling was accompanied by blockade of cell migration and invasion. Moreover, apoptosis was induced in a subset of bone sarcomas at nanomolar concentrations of dasatinib. Inhibition of Src protein expression by siRNA also induced apoptosis, indicating that these bone sarcoma cell lines are dependent on Src activity for survival. These results demonstrate that dasatinib inhibits migration and invasion of diverse sarcoma cell types, and selectively blocks the survival of bone sarcoma cells. Therefore dasatinib may provide therapeutic benefit by preventing the growth and metastasis of sarcomas. Microarray analysis of the sarcoma cell lines lead to the identification of a molecular signature that successfully predicts response to dasatinib by induction of apoptosis. Components of this molecular signature are expressed in primary human sarcomas. Furthermore, expression of the molecular signature in sarcomas can be utilized to cluster tumors based on theoretical response to dasatinib. While the prediction of response in tumors is theoretical, there is encouraging evidence to support further endeavors into validating the potential of this molecular signature to predict response in patients.Together, these studies reveal that, in cell lines, both constitutive Src activation and the presence of a molecular signature that predicts response to dasatinib are important parameters to consider when selecting dasatinib as a treatment for. Furthermore, novel therapeutic approaches that inhibit Src signaling may selectively induce apoptosis in tumor cells and sensitize to chemotherapy those tumors that contain the relevant molecular signature.

The ErbB family of receptors are key regulators of growth, differentiation, migration and survival of epithelial cells. CI-1033 is an irreversible pan-ErbB tyrosine kinase inhibitor that has the ability to inhibit EGFR function but has shown limited therapeutic efficacy. Lovastatin targets the activity of HMG-CoA reductase, the rate-limiting step in the mevalonate pathway. In this study, the ability of lovastatin to potentiate the cytotoxic effects of CI-1033 was evaluated. The combination of lovastatin and CI-1033 exhibited some cooperative cytotoxic activity in a squamous cell carcinoma–derived cell line. This combination resulted in enhanced cell death by induction of a potent apoptotic response. Furthermore, this drug combination inhibited EGF-induced EGFR autophosphorylation and activation of the downstream signaling effectors, ERK and AKT. These findings suggest that combining lovastatin and tyrosine kinase inhibitors may represent a novel combinational therapeutic approach in squamous cell carcinoma of the head and neck.

Le but des travaux est de caractériser l’influence de la signalisation VEGF, en particulier la signalisation autocrine VEGF, sur la biologie et la sensibilité/résistance aux médicaments anticancéreux de cellules de cancer colorectal. Nous avons souhaité caractériser l’impact de la signalisation autocrine VEGF dans des modèles de CRC avec une résistance naturelle au bevacizumab, un anticorps anti-VEGF. Bien que ce composé soit actif dans le CRC, une sous population de patients ne répondent pas au traitement. Nos résultats montrent une sur régulation de la voie autocrine HIF-VEGF-VEGFR en réponse à une exposition prolongée au bevacizumab dans les cellules bevacizumab résistantes. Si la résistance à cet anticorps est bien établie, d’autres inhibiteur de la voie VEGF restent actifs (comme la petite molécule ciblée nintedanib) et peuvent inhiber la voie mTOR. La signalisation VEGF autocrine joue un rôle dans la survie de cellules de CRC. Chez les sujets résistants au bevacizumab, il serait intéressant d’introduire le nintedanib seul ou en combinaison pour accentuer l’inhibition angiogénique. Une autre combinaison d’agents (anti VEGF(R) et anti EGFR) a montré une efficacité dans des modèles de CRC en préclinique. La combinaison du bevacizumab et d’une petite molécule ciblant EGFR (erlotinib) a montré une plus grande efficacité que le bevacizumab seul dans des modèles de CRC indépendamment du statut KRAS. Le bevacizumab induit une activation de la voie de survie EGFR dans les cellules tumorales et dans les cellules endothéliales associées à la tumeur. Cette activation se trouve diminuée avec l’introduction de l’erlotinib. Les résultats indiquent que la combinaison du bevacizumab et de l’erlotinib sont plus actif en thérapie de maintenance que le bevacizumab seul, même pour les patients mutés KRAS. Ces résultats ont mené à l’étude clinique positive GERCOR phase III DREAM dans le cancer du côlon métastatique. / The aim of the work is to characterize the influence of VEGF signaling , especially autocrine VEGF signaling , the biology and susceptibility / resistance to anticancer drugs of colorectal cancer cells. We wished to characterize the impact of the autocrine VEGF signaling in CRC models with natural resistance to bevacizumab , an anti -VEGF antibody. Although this compound is active in the CRC, a subpopulation of patients do not respond to treatment. Our results show an autocrine regulatory pathway HIF- VEGF- VEGFR in response to prolonged exposure to bevacizumab in bevacizumab resistant cells. If the resistance to the antibody is established, other inhibitos of VEGF pathway remain active (such as small molecule nintedanib ) and can inhibit the mTOR pathway. Autocrine VEGF signaling plays a role in CRC cell survival. In subjects resistant to bevacizumab, it would be interesting to introduce the nintedanib alone or in combination to enhance the angiogenic inhibition. Another combination of targeted agents ( anti-VEGF (R) and anti EGFR) has shown efficacy in preclinical models of CRC. The combination of bevacizumab and a small molecule targeting EGFR (erlotinib) showed greater efficacy than bevacizumab alone in CRC models regardless of KRAS status. Bevacizumab induces activation of the EGFR survival pathway in tumor cells and in endothelial cells associated with the tumor. This activation is decreased with the introduction of erlotinib. The results indicate that the combination of bevacizumab and erlotinib are more active in maintenance therapy than bevacizumab alone, even for patients mutated KRAS . These findings led to the positive Phase III clinical study GERCOR DREAM in metastatic colon cancer.

The ErbB family of receptors are key regulators of growth, differentiation, migration and survival of epithelial cells. CI-1033 is an irreversible pan-ErbB tyrosine kinase inhibitor that has the ability to inhibit EGFR function but has shown limited therapeutic efficacy. Lovastatin targets the activity of HMG-CoA reductase, the rate-limiting step in the mevalonate pathway. In this study, the ability of lovastatin to potentiate the cytotoxic effects of CI-1033 was evaluated. The combination of lovastatin and CI-1033 exhibited some cooperative cytotoxic activity in a squamous cell carcinoma–derived cell line. This combination resulted in enhanced cell death by induction of a potent apoptotic response. Furthermore, this drug combination inhibited EGF-induced EGFR autophosphorylation and activation of the downstream signaling effectors, ERK and AKT. These findings suggest that combining lovastatin and tyrosine kinase inhibitors may represent a novel combinational therapeutic approach in squamous cell carcinoma of the head and neck.

The submitted thesis is devoted to the quantitative analysis of tyrosine kinase inhibitors, specifically imatinib and nilotinib, by liquid chromatography-mass spectrometry method. The main purpose of developing this new method of analysis at the Department of Clinical Biochemistry and Diagnostics at the University Hospital in Hradec Králové was measuring and monitoring serum or plasma concentration levels of these drugs in patients with chronic myeloid leukemia, less often in patients with gastrointestinal stromal tumour. The main task during the elaboration of the thesis was to fully optimize and validate the method. Previously, this method for the analysis of tyrosine kinase inhibitors was routinely performed here by high-performance liquid chromatography with spectrophotometric (UV) detection. As part of the modernization of laboratory technology, they started to use high-performance liquid chromatography with mass spectrometry at the workplace. The analytes with their internal standards were obtained by a liquid-liquid extraction process. Then, samples were separated on a C18 reverse phase column using isocratic elution. Subsequently, both analytes were detected by a triple quadrupole tandem mass spectrometer with ESI ion source in a positive mode. As a part of the method validation was to...