Oliver Thews

Treatment resistance of solid tumors

Hypoxia is a characteristic property of locally advanced solid tumors, resulting from an imbalance between the supply and consumption of oxygen. Major pathogenetic mechanisms for the development of hypoxia are (1) structural and functional abnormalities of the tumor microvasculature, (2) increased diffusion distances, and (3) tumor-associated and therapy-induced anemia. The oxygenation status is independent of clinical tumor size, stage, grade, and histopathological type, but is affected by the hemoglobin level. Hypoxia is intensified in anemic patients, especially in tumors with low perfusion rates. Hypoxia and anemia (most probably via worsening of tumor hypoxia) can lead to therapeutic problems, as they make solid tumors resistant to sparsely ionizing radiation and some forms of chemotherapy. In addition to more direct mechanisms involved in the development of therapeutic resistance, there are also indirect machineries that can cause barriers to therapies. These include hypoxia-driven proteome and genome changes and clonal selection. These, in turn, can drive subsequent events that are known to further increase resistance to therapy (in addition to critically affecting long-term prognosis). Treatment resistance in anemic patients can be, at least partially, prevented or overcome by anemia correction, resulting in better locoregional tumor control and overall survival of patients.

Enhanced radiosensitivity in experimental tumours following erythropoietin treatment of chemotherapy-induced anaemia.

The radiosensitivity of solid tumours in anaemic rats treated with recombinant human erythropoietin (rhEPO, epoetin beta) was studied. Anaemia was induced by a single dose of carboplatin (45 mg kg(-1) i.v.), resulting in a reduction in the haemoglobin concentration by 30%. In a second group, the development of anaemia was prevented by rhEPO (1000 IU kg(-1)) administered s.c. three times per week starting 6 days before the carboplatin application. Three days after carboplatin treatment, DS-sarcomas were implanted subcutaneously onto the hind foot dorsum. Neither carboplatin nor rhEPO treatment influenced tumour growth rate. Five days after implantation, tumours were irradiated with a single non-curative dose (10 Gy), resulting in a growth delay with a subsequent regrowth of the tumours. In the rhEPO-treated group, the growth delay lasted significantly longer (9.5 days vs. 4.5 days) and the regrowth was slower (6.0 days vs. 4.1 days) compared with the anaemic group. These data suggest that the correction of chemotherapy-induced anaemia by rhEPO (epoetin beta) treatment increases tumour radiosensitivity, presumably as a result of an improved oxygen supply to tumour tissue.

Oxygenation status of gynecologic tumors: what is the optimal hemoglobin level?

Background: Tumor hypoxia has been linked to the development of treatment resistance, tumor progression, and poor prognosis. Since anemia is a major causative factor for the development of hypoxia, the association between blood hemoglobin concentration (cHb) and tumor oxygenation was examined in this study. Patients and Methods: Pretreatment O2 tension (pO2) measurements were performed in 59 primary carcinomas of the uterine cervix in which a stringent histopathologic examination of the electrode tracks was mandatory in order to exclude measurements in necrotic, stromal or normal cervical tissue. In addition, pO2 readings in twelve primary cancers and 17 local recurrences of vulvar cancers were included in this study. cHb was determined at the time of pO2 measurements. Results: Data presented clearly show that an optimal Hb level with regard to the median pO2 values of cervical and vulvar cancers should prevail at cHb values of between 12 and 14 g/dl (7.45–8.60 mmol/l). In anemic patients (cHb < 12 g/dl), the deterioration of the tumor oxygenation status can be explained by a reduced O2 transport capacity. At cHb values > 14 g/dl, a worsening of the tumor oxygenation is apparent, most probably due to a drop in perfusion following a drastic increase in viscous resistance to flow. This patogenetic mechanism is thought to counteract and finally to abrogate the high O2 transport capacity in this cHb range. Conclusions: This study suggests that cHb values of between 12 and 14 g/dl are optimal with regard to the oxygenation status in the tumor entities investigated, a finding which may have far-reaching implications in the clinical setting.Hintergrund: Hypoxie trägt ursächlich zur Therapieresistenz und Tumorprogression bei und ist ein aussagekräftiger, unabhängiger Prognoseparameter in lokal fortgeschrittenen Zervixkarzinomen. Ein wesentlicher pathogenetischer Faktor für das Auftreten einer Hypoxie ist die tumorassoziierte und/oder therapieinduzierte Anämie. Deshalb wurde die Abhängigkeit der Tumoroxygenierung von der Hämoglobin-(Hb-)Kozentration untersucht. Patienten und Methodik: In 59 Zervixkarzinomen wurden prätherapeutisch O2-Drücke (pO2) in histologisch gesichertem, vitalem Tumorgewebe gemessen. Darüber hinaus konnte der Oxygenierungsstatus von Vulvakarzinomen (zwölf Primärtumoren, 17 Lokalrezidive) erfasst werden. Die Bestimmung der Hb-Konzentration erfolgte zur Zeit der pO2-Messung. Ergebnisse: Die Studie zeigt eindeutig, dass bei Hb-Konzentrationen zwischen 12 und 14 g/dl (7,45–8,69 mmol/l) sowohl in Zervix- als auch in Vulvakarzinomen die höchsten medianen pO2-Werte auftreten. In anämischen Patientinnen (cHb < 12 g/dl) verschlechtert sich aufgrund der eingeschränkten O2-Transportkapazität der Oxygenierungsstatus in beiden Tumorentitäten. Bei Hb-Konzentrationen > 14 g/dl tritt ebenfalls eine Verschlechterung der O2-Versorgung auf, die im Wesentlichen auf einer Perfusionsstörung infolge eines extrem gesteigerten viskösen Strömungswiderstandes des Blutes beruht. Diese Perfusionseinschränkung kann demnach nicht durch die mit höheren Hb-Konzentrationen einhergehende Steigerung der O2-Transportkapazität kompensiert werden. Schlussfolgerungen: Hb-Konzentrationen zwischen 12 und 14 d/gl scheinen für den Oxygenierungsstatus von lokal fortgeschrittenen Zervix- und Vulvakarzinomen “optimal” zu sein. Sowohl höhere als auch niedrigere Hb-Werte haben eine Verschlechterung der O2-Versorgung zur Folge. Aufgrund dieses Befundes ergibt sich eine Reihe von klinisch relevanten Konsequenzen.

Radioactive Labeling of Defined HPMA-Based Polymeric Structures Using [18F]FETos for In Vivo Imaging by Positron Emission Tomography

During the last decades polymer-based nanomedicine has turned out to be a promising tool in modern pharmaceutics. The following article describes the synthesis of well-defined random and block copolymers by RAFT polymerization with potential medical application. The polymers have been labeled with the positron-emitting nuclide fluorine-18. The polymeric structures are based on the biocompatible N-(2-hydroxypropyl)-methacrylamide (HPMA). To achieve these structures, functional reactive ester polymers with a molecular weight within the range of 25,000-110,000 g/mol were aminolyzed by 2-hydroxypropylamine and tyramine (3%) to form (18)F-labelable HPMA-polymer precursors. The labeling procedure of the phenolic tyramine moieties via the secondary labeling synthon 2-[(18)F]fluoroethyl-1-tosylate ([(18)F]FETos) provided radiochemical fluoroalkylation yields of ∼80% for block copolymers and >50% for random polymer architectures within a synthesis time of 10 min and a reaction temperature of 120 °C. Total synthesis time including synthon synthesis, (18)F-labeling, and final purification via size exclusion chromatography took less than 90 min and yielded stable (18)F-labeled HPMA structures in isotonic buffer solution. Any decomposition could be detected within 2 h. To determine the in vivo fate of (18)F-labeled HPMA polymers, preliminary small animal positron emission tomography (PET) experiments were performed in healthy rats, demonstrating the renal clearance of low molecular weight polymers. Furthermore, low metabolism rates could be detected in urine as well as in the blood. Thus, we expect this new strategy for radioactive labeling of polymers as a promising approach for in vivo PET studies.

Erythropoietin restores the anemia-induced reduction in radiosensitivity of experimental human tumors in nude mice

PURPOSE The effect of recombinant human erythropoietin (rhEPO) on the radiosensitivity of human tumor xenografts growing in anemic and nonanemic nude mice was studied. METHODS AND MATERIALS Anemia was induced by total body irradiation ([TBI], 2 x 4 Gy) of mice before tumor implantation into the subcutis of the hind leg. The development of anemia was prevented by rhEPO (750 U/kg s.c.) given 3 times weekly starting 2 weeks before TBI. Fourteen days after fractionated TBI (tumor volume of approx. 40 mm(3)), single-dose irradiation of the tumor with varying doses was performed so that in full dose-response relationship for the probability of tumor cure was obtained. RESULTS Radiation-induced anemia (hemoglobin concentration [cHb] = 9.9 g/dl) led to a reduced radiosensitivity compared to controls [49.4 vs. 40.1 Gy radiation dose to control 50% of the tumors (TCD50)]. Upon rhEPO treatment for anemia prevention (cHb = 13.3 g/dl), the TCD50 was 39.8 Gy, illustrating restored radiosensitivity compared to anemic mice. CONCLUSION These data provide further experimental evidence for restored radiosensitivity upon prevention of anemia with rhEPO.

Acidic Environment Leads to ROS-Induced MAPK Signaling in Cancer Cells

Tumor micromilieu often shows pronounced acidosis forcing cells to adapt their phenotype towards enhanced tumorigenesis induced by altered cellular signalling and transcriptional regulation. In the presents study mechanisms and potential consequences of the crosstalk between extra- and intracellular pH (pHe, pHi) and mitogen-activated-protein-kinases (ERK1/2, p38) was analyzed. Data were obtained mainly in AT1 R-3327 prostate carcinoma cells, but the principle importance was confirmed in 5 other cell types. Extracellular acidosis leads to a rapid and sustained decrease of pHi in parallel to p38 phosphorylation in all cell types and to ERK1/2 phosphorylation in 3 of 6 cell types. Furthermore, p38 phosphorylation was elicited by sole intracellular lactacidosis at normal pHe. Inhibition of ERK1/2 phosphorylation during acidosis led to necrotic cell death. No evidence for the involvement of the kinases c-SRC, PKC, PKA, PI3K or EGFR nor changes in cell volume in acidosis-induced MAPK activation was obtained. However, our data reveal that acidosis enhances the formation of reactive oxygen species (ROS), probably originating from mitochondria, which subsequently trigger MAPK phosphorylation. Scavenging of ROS prevented acidosis-induced MAPK phosphorylation whereas addition of H2O2 enhanced it. Finally, acidosis increased phosphorylation of the transcription factor CREB via p38, leading to increased transcriptional activity of a CRE-reporter even 24 h after switching the cells back to a normal environmental milieu. Thus, an acidic tumor microenvironment can induce a longer lasting p38-CREB-medited change in the transcriptional program, which may maintain the altered phenotype even when the cells leave the tumor environment.

Acidosis induces multi-drug resistance in rat prostate cancer cells (AT1) in vitro and in vivo by increasing the activity of the p-glycoprotein via activation of p38

Because solid growing tumors often show hypoxia and pronounced extracellular acidosis, the aim of this study was to analyze the impact of an acidotic environment on the activity of the p‐glycoprotein (pGP) and on the cellular content and cytotoxicity of the chemotherapeutic drug daunorubicin in the AT1 R‐3327 Dunning prostate carcinoma cell line cultured in vitro and in vivo. In vitro, extracellular acidosis (pH 6.6) activated p38 and ERK1/2 and thereby induced daunorubicin resistance via a pronounced activation of pGP. De‐novo protein synthesis was not necessary and analysis of transport kinetics indicated a fast and persistent pGP activation at pH 6.6 (when compared with 7.4). Intracellular acidification also induced daunorubicin resistance via activation of pGP, which was mediated by activation of p38 alone. In vivo, tumors were implanted subcutaneously, and the tumor pH was artificially lowered by forcing anaerobic metabolism. In vivo, the reduced extracellular pH of 6.6 was also able to induce daunorubicin resistance, which was abolished by inhibition of p38. These results suggest that pGP activity is dependent on extracellular pH in vitro and in vivo. Moreover, there is strong indication that this effect is mediated via activation of p38 in vivo. Activation of ERK is also suitable to induce pGP activity. Therefore, inhibition of p38 (and ERK) may be used to prevent acidosis induced increase in pGP activity and thereby attenuate multidrug resistance. In addition, supportive treatments reducing tumor acidosis may improve the cytotoxic effect of chemotherapeutic drugs.

Modifying the Body Distribution of HPMA-Based Copolymers by Molecular Weight and Aggregate Formation

There is a recognized need to create well-defined polymer probes for in vivo and clinical positron emission tomography (PET) imaging to guide the development of new generation polymer therapeutics. Using the RAFT polymerization technique in combination with the reactive ester approach, here we have synthesized well-defined and narrowly distributed N-(2-hydroxypropyl)methacrylamide homopolymers (pHPMA) (P1* and P2*) and random HPMA copolymers consisting of hydrophilic HPMA and hydrophobic lauryl methacrylate comonomers (P3* and P4*). The polymers had molecular weights below (P1* and P3*) and above the renal threshold (P2* and P4*). Whereas the homopolymers dissolve in isotonic solution as individual coils, the random copolymers form larger aggregates above their critical micelle concentration (∼ 40 nm), as determined by fluorescence correlation spectroscopy. Structure-property relationships of the pharmacokinetics and biodistribution of the different polymer architectures were monitored in the living organism following radiolabeling with the positron emitter (18)F via fluoroethylation within a few hours. Ex vivo organ biodistribution and in vivo μPET imaging studies in male Copenhagen rats revealed that both size and the nature of the aggregate formation (hydrophobically modified copolymers) played a major role in blood clearance and biodistribution, especially concerning liver and kidney accumulation. The high-molecular-weight random copolymer P4* (hydrophobically modified), in particular, combines low liver uptake with enhanced blood circulation properties, showing the potential of hydrophobic interactions, as seen for the represented model system, that are valuable for future drug carrier design.

Modulation of tumor oxygenation

There is a large body of evidence suggesting that deficiencies in the O2 supply of tumors exist due to restrictions (i) in the O2 delivery by perfusion and/or diffusion, and (ii) in the O2 transport capacity. Whereas the former are mostly based on inadequate and heterogeneous microcirculatory functions, the latter are predominantly due to tumor-associated anemia. Possible uses and limitations of measures are discussed which can increase the microvascular O2 content and thus may preferentially serve to enhance diffusion-limited O2 availability. In addition, means are described for improving and increasing the uniformity of microcirculation thus possibly enhancing perfusion-limited O2 delivery. Reducing cellular respiration rate should be of benefit in both pathophysiological conditions. Because both types of O2 limitation coexist in solid tumors, appropriate combinations should be aimed at eradicating tumor hypoxia which is present in at least one third of cancers in the clinical setting.

Tumor oxygenation in anemic rats : effects of erythropoietin treatment versus red blood cell transfusion

Anemia was induced in rats by the development of a hemorrhagic ascites. These animals also bore solid tumors (DS-sarcomas) on the hind foot dorsum. The effects of two methods for anemia correction on oxygenation in the solid tumors were compared in this study. Anemia was corrected either chronically by erythropoietin administration (1000 IU/kg) over 14 days (EPO) or acutely by transfusion with red blood cells (TR). Non-anemic and untreated anemic animals served as controls. Tumor oxygenation was determined in anesthetized animals using polarographic needle electrodes and pO2 histography. The reduction in hematocrit and hemoglobin content found in anemic animals could successfully be corrected either by EPO or by TR. Anemia resulted in a worsening of tumor oxygenation which could partially be reversed by EPO or TR in small tumors (< 1.4 ml). In larger tumors (> or = 1.4 ml), neither method of anemia correction resulted in significant changes in tumor oxygenation.