Modulation of mitochondrial function by extracellular acidosis in tumor cells and normal fibroblasts: Role of signaling pathways.

In many tumors pronounced extracellular acidosis resulting from glycolytic metabolism is found. Since several environmental stress factors affect the mitochondrial activity the aim of the study was to analyze the impact of acidosis on cellular oxygen consumption and which signaling pathways may be involved in the regulation. In two tumor cell lines and normal fibroblasts cellular oxygen consumption rate (OCR) and mitochondrial function were measured after 3 h at pH 6.6. Besides the activation of ERK1/2, p38 and PI3K signaling in the cytosolic and mitochondrial compartment, the mitochondrial structure and proteins related to mitochondria fission were analyzed. The acidic extracellular environment increased OCR in tumor cells but not in fibroblasts. In parallel, the mitochondrial membrane potential increased at low pH. In both tumor lines (but not in fibroblasts), the phosphorylation of ERK1/2 and PI3K/Akt was significantly increased, and both cascades were involved in OCR modulation. The activation of signaling pathways was located predominantly in the mitochondrial compartment of the cells. At low pH, the mitochondrial structure in tumor cells showed structural changes related to elongation whereas mitochondria fragmentation was reduced indicating mitochondria fusion. However, these morphological changes were not related to ERK1/2 or PI3K signaling. Acidic stress seems to induce an increased oxygen consumption, which might further aggravate tumor hypoxia. Low pH also induces mitochondria fusion that is not mediated by ERK1/2 or PI3K signaling. The mechanism by which these signaling cascades modulate the respiratory activity of tumor cells needs further investigation.

Development and evaluation of a software system for medical students to teach and practice anamnestic interviews with virtual patient avatars.

BACKGROUND AND OBJECTIVES: Taking a medical history is a core competence of the diagnostic process. At the beginning of their study medical students need to learn and practice the necessary techniques, initially focusing on good structuring and completeness. For this purpose, an interactive software system (ViPATalk) was developed in which the student can train to pose questions to virtual patient avatars in free conversation. At the end, the student receives feedback on the completeness of the questioning and an explanation of the essential items. The use of this software was compared to the traditional format of student role play in a randomized trial. METHODS: The central component of ViPATalk is a chatbot based on the AI language AIML, which generates an appropriate answer based on keywords in the student's question. To enable a realistic use, the student can enter the question via microphone (speech-to-text) and the answer generated by the chatbot is presented as a short video sequence, where the avatar is generated from a real image. Here, the transition between the sequences is seamless, resulting in a continuous movement of the avatar during the conversation. RESULTS: The learning success by practicing with ViPATalk was tested in an anamnestic interview with actors as simulated patients. The completeness of the conversation was evaluated with regard to numerous aspects and also certain behaviors during the conversation. These results were compared with those after practicing using peer role play. CONCLUSIONS: It was found that practicing with ViPATalk was mostly equivalent to the students' role play. In the subsequent survey of the students, the wish was expressed that the ViPATalk software should also be used as an online tool for self-study and that there should be more cases for practicing.

The Role of microRNAs in Gene Expression and Signaling Response of Tumor Cells to an Acidic Environment.

Many tumors are characterized by marked extracellular acidosis due to increased glycolytic metabolism, which affects gene expression and thereby tumor biological behavior. At the same time, acidosis leads to altered expression of several microRNAs (Mir7, Mir183, Mir203, Mir215). The aim of this study was to analyze whether the acidosis-induced changes in cytokines and tumor-related genes are mediated via pH-sensitive microRNAs. Therefore, the expression of Il6, Nos2, Ccl2, Spp1, Tnf, Acat2, Aox1, Crem, Gls2, Per3, Pink1, Txnip, and Ypel3 was examined in acidosis upon simultaneous transfection with microRNA mimics or antagomirs in two tumor lines in vitro and in vivo. In addition, it was investigated whether microRNA expression in acidosis is affected via known pH-sensitive signaling pathways (MAPK, PKC, PI3K), via ROS, or via altered intracellular Ca2+ concentration. pH-dependent microRNAs were shown to play only a minor role in modulating gene expression. Individual genes (e.g., Ccl2, Txnip, Ypel3) appear to be affected by Mir183, Mir203, or Mir215 in acidosis, but these effects are cell line-specific. When examining whether acid-dependent signaling affects microRNA expression, it was found that Mir203 was modulated by MAPK and ROS, Mir7 was affected by PKC, and Mir215 was dependent on the intracellular Ca2+ concentration. Mir183 could be increased by ROS scavenging. These correlations could possibly result in new therapeutic approaches for acidotic tumors.

Correlation between Circulating miR-16, miR-29a, miR-144 and miR-150, and the Radiotherapy Response and Survival of Non-Small-Cell Lung Cancer Patients.

Despite the success of current therapy concepts, patients with advanced non-small-cell lung cancer (NSCLC) still have a very poor prognosis. Therefore, biological markers are urgently needed, which allow the assessment of prognosis, or prediction of the success of therapy or resistance in this disease. Circulating microRNAs (miRs) have potential as biomarkers for the prognosis and prediction of response to therapy in cancer patients. Based on recent evidence that circulating miR-16, miR-29a, miR-144 and miR-150 can be regulated by ionizing radiation, the concentration of these four miRs was assessed in the plasma of NSCLC patients at different time points of radiotherapy by digital droplet PCR (ddPCR). Furthermore, their impact on patients' prognosis was evaluated. The mean plasma levels of miR-16, miR-29a, miR-144 and miR-150 significantly differed intra- and inter-individually, and during therapy in NSCLC patients, but showed a strong positive correlation. The individual plasma levels of miR-16, miR-29a and miR-144 had prognostic value in NSCLC patients during or at the end of radiotherapy in Cox's regression models. NSCLC patients with low levels of these three miRs at the end of radiotherapy had the worst prognosis. However, miR-150 plasma levels and treatment-dependent changes were not predictive. In conclusion, circulating miR-16, miR-29a and miR-144, but not miR-150, have a prognostic value in NSCLC patients undergoing radiotherapy.

Role of acidosis-sensitive microRNAs in gene expression and functional parameters of tumors in vitro and in vivo.

BACKGROUND: The acidic extracellular environment of tumors has been shown to affect the malignant progression of tumor cells by modulating proliferation, cell death or metastatic potential. The aim of the study was to analyze whether acidosis-dependent miRNAs play a role in the signaling cascade from low pH through changes in gene expression to functional properties of tumors in vitro and in vivo. METHODS: In two experimental tumor lines the expression of 13 genes was tested under acidic conditions in combination with overexpression or downregulation of 4 pH-sensitive miRNAs (miR-7, 183, 203, 215). Additionally, the impact on proliferation, cell cycle distribution, apoptosis, necrosis, migration and cell adhesion were measured. RESULTS: Most of the genes showed a pH-dependent expression, but only a few of them were additionally regulated by miRNAs in vitro (Brip1, Clspn, Rif1) or in vivo (Fstl, Tlr5, Txnip). Especially miR-215 overexpression was able to counteract the acidosis effect in some genes. The impact on proliferation was cell line-dependent and most pronounced with overexpression of miR-183 and miR-203, whereas apoptosis and necrosis were pH-dependent but not influenced by miRNAs. The tumor growth was markedly regulated by miR-183 and miR-7. In addition, acidosis had a strong effect on cell adhesion, which could be modulated by miR-7, miR-203 and miR-215. CONCLUSIONS: The results indicate that the acidosis effect on gene expression and functional properties of tumor cells could be mediated by pH-dependent miRNAs. Many effects were cell line dependent and therefore do not reflect universal intracellular signaling cascades. However, the role of miRNAs in the adaptation to an acidic environment may open new therapeutic strategies.

Impact of the acidic environment on gene expression and functional parameters of tumors in vitro and in vivo.

BACKGROUND: The low extracellular pH (pHe) of tumors resulting from glycolytic metabolism is a stress factor for the cells independent from concomitant hypoxia. The aim of the study was to analyze the impact of acidic pHe on gene expression on mRNA and protein level in two experimental tumor lines in vitro and in vivo and were compared to hypoxic conditions as well as combined acidosis+hypoxia. METHODS: Gene expression was analyzed in AT1 prostate and Walker-256 mammary carcinoma of the rat by Next Generation Sequencing (NGS), qPCR and Western blot. In addition, the impact of acidosis on tumor cell migration, adhesion, proliferation, cell death and mitochondrial activity was analyzed. RESULTS: NGS analyses revealed that 147 genes were uniformly regulated in both cell lines (in vitro) and 79 genes in both experimental tumors after 24 h at low pH. A subset of 25 genes was re-evaluated by qPCR and Western blot. Low pH consistently upregulated Aox1, Gls2, Gstp1, Ikbke, Per3, Pink1, Tlr5, Txnip, Ypel3 or downregulated Acat2, Brip1, Clspn, Dnajc25, Ercc6l, Mmd, Rif1, Zmpste24 whereas hypoxia alone led to a downregulation of most of the genes. Direct incubation at low pH reduced tumor cell adhesion whereas acidic pre-incubation increased the adhesive potential. In both tumor lines acidosis induced a G1-arrest (in vivo) of the cell cycle and a strong increase in necrotic cell death (but not in apoptosis). The mitochondrial O2 consumption increased gradually with decreasing pH. CONCLUSIONS: These data show that acidic pHe in tumors plays an important role for gene expression independently from hypoxia. In parallel, acidosis modulates functional properties of tumors relevant for their malignant potential and which might be the result of pH-dependent gene expression.

A hydraulic model of cardiovascular physiology and pathophysiology embedded into a computer-based teaching system for student training in laboratory courses.

Functional understanding of the different parts of the cardiovascular system is essential for an insight into pathomechanisms of numerous diseases. During training cardiovascular physiology, students and early-stage medical personnel should understand the role of different functional parameters for systolic and diastolic blood pressure, as well as for blood flow. The impact of isolated parameters can only be studied in models. Here physical hydraulic models are an advantage in which the students have a direct contact to the mechanical properties of the circulatory system. But these models are often difficult to handle. The aim of the present study was to develop a comprehensive model of the cardiovascular system, including a mechanical heart with valves, an elastic aorta, a more rigid peripheral artery system, a total peripheral resistance, and a venous reservoir representing the variable cardiac preload. This model allows one to vary systematically several functional parameters and to continuously record their impact on pressure and flow. This model is embedded into a computer-based teaching system (LabTutor) in which the students are guided through the handling of the model (as well as the systematic variation of parameters), and the measured data can be analyzed. This hybrid teaching system, which is routinely integrated in physiology laboratory courses of medical students, allows students to work with a complex hydraulic model of the cardiovascular system and to analyze systematically the impact of influencing variables (e.g., increased peripheral resistance or changed cardiac preload) as well as pathophysiological dysfunctions (e.g., reduced aortic compliance).

Causes and Consequences of A Glutamine Induced Normoxic HIF1 Activity for the Tumor Metabolism.

The transcription factor hypoxia-inducible factor 1 (HIF1) is the crucial regulator of genes that are involved in metabolism under hypoxic conditions, but information regarding the transcriptional activity of HIF1 in normoxic metabolism is limited. Different tumor cells were treated under normoxic and hypoxic conditions with various drugs that affect cellular metabolism. HIF1α was silenced by siRNA in normoxic/hypoxic tumor cells, before RNA sequencing and bioinformatics analyses were performed while using the breast cancer cell line MDA-MB-231 as a model. Differentially expressed genes were further analyzed and validated by qPCR, while the activity of the metabolites was determined by enzyme assays. Under normoxic conditions, HIF1 activity was significantly increased by (i) glutamine metabolism, which was associated with the release of ammonium, and it was decreased by (ii) acetylation via acetyl CoA synthetase (ACSS2) or ATP citrate lyase (ACLY), respectively, and (iii) the presence of L-ascorbic acid, citrate, or acetyl-CoA. Interestingly, acetylsalicylic acid, ibuprofen, L-ascorbic acid, and citrate each significantly destabilized HIF1α only under normoxia. The results from the deep sequence analyses indicated that, in HIF1-siRNA silenced MDA-MB-231 cells, 231 genes under normoxia and 1384 genes under hypoxia were transcriptionally significant deregulated in a HIF1-dependent manner. Focusing on glycolysis genes, it was confirmed that HIF1 significantly regulated six normoxic and 16 hypoxic glycolysis-associated gene transcripts. However, the results from the targeted metabolome analyses revealed that HIF1 activity affected neither the consumption of glucose nor the release of ammonium or lactate; however, it significantly inhibited the release of the amino acid alanine. This study comprehensively investigated, for the first time, how normoxic HIF1 is stabilized, and it analyzed the possible function of normoxic HIF1 in the transcriptome and metabolic processes of tumor cells in a breast cancer cell model. Furthermore, these data imply that HIF1 compensates for the metabolic outcomes of glutaminolysis and, subsequently, the Warburg effect might be a direct consequence of the altered amino acid metabolism in tumor cells.

Correction to: Tumor pH and metastasis: a malignant process beyond hypoxia.

The authors have noticed a typographical error in the published article. The term "epithelial-to-mesenchymal transition" should have been used instead of the term "endothelial-to-mesenchymal transition" throughout the manuscript.

Extracellular Acidosis Modulates the Expression of Epithelial-Mesenchymal Transition (EMT) Markers and Adhesion of Epithelial and Tumor Cells.

Epithelial-to-mesenchymal transition (EMT) is an important process of tumor progression associated with increased metastatic potential. EMT can be activated by external triggers such as cytokines or metabolic parameters (e.g. hypoxia). Since extracellular acidosis is a common finding in tumors, the aim of the study is to analyze its impact on the expression of EMT markers in vitro and in vivo as well as the functional impact on cell adhesion. Therefore, three tumor and two normal epithelial cell lines were incubated for 24 h at pH 6.6 and the expression of EMT markers was studied. In addition, mRNA expression of transcription and metabolic factors related to EMT was measured as well as the functional impact on cell adhesion, either during acidic incubation or after priming cells in an acidic milieu. E-cadherin and N-cadherin were down-regulated in all tumor and normal cell lines studied, whereas vimentin expression increased in only two tumor and one normal cell line. Down-regulation of the cadherins was seen in total protein and to a lesser extent in surface protein. In vivo an increase in N-cadherin and vimentin expression was found. Acidosis up-regulated Twist1 and Acsl1 but down-regulated fumarate hydratase (Fh). Cell adhesion during acidic incubation decreased in AT1 prostate carcinoma cells whereas preceding acidic priming increased their subsequent adhesion. Low tumor pH is able to modulate the expression EMT-related proteins and by this may affect the stability of the tissue structure.

Cellular and radiobiological effects of carbonic anhydrase IX in human breast cancer cells.

Hypoxia­induced carbonic anhydrase IX (CAIX) is involved in intracellular and extracellular pH regulation, which is critical for tumor growth and metastasis. CAIX is overexpressed in breast cancer and is associated with the poor survival of patients after radiotherapy. Therefore, we evaluated the cellular and radiobiological effects of CAIX inhibition in human breast cancer cells. We used CA9 siRNA and the CA inhibitor (CAI) U104, respectively, to inhibit CAIX expression and activity in basal triple­negative MDA­MB­231 and luminal MCF­7 cells under hypoxic conditions. We investigated the effects of CAIX inhibition on CA9 mRNA and CAIX protein level, as well as on CAIX activity, intracellular pH, proliferation, apoptosis, clonogenic survival, migration, cell cycle distribution and radiosensitivity. CA9 siRNA and CAI U104 decreased CA9 mRNA and CAIX protein level in MDA­MB­231 and MCF­7 cells. Furthermore, incubation with CAI U104 significantly decreased carbonic anhydrase activity and reduced the intracellular pH. Additionally, CA9 siRNA or U104 reduced clonogenic survival, migration and the number of cells in the G0/G1 phase, induced apoptosis and demonstrated additive or synergistic effects in combination with irradiation. In conclusion, combination of CAIX inhibition and irradiation is a promising treatment strategy against breast cancer with hypoxia­induced CAIX expression.

Tumor pH and metastasis: a malignant process beyond hypoxia.

Tumors often show, compared to normal tissues, a markedly decreased extracellular pH resulting from anaerobic or aerobic glycolysis in combination with a reduced removal of acidic metabolites. Several studies indicate that acidosis induces (independently from hypoxia) hematogenous and lymphatic spread of tumor cells worsening the long-term prognosis of tumor patients. This review gives an overview on the impact of low pH on different steps of metastasis including (a) local tumor cell invasion and angiogenesis, (b) intravasation of tumor cells and detachment into the circulation, and (c) adherence of circulating tumor cells, transmigration and invasion in the new host tissue. The review describes pH-dependent cellular mechanisms fostering these steps such as epithelial-to-mesenchymal transition (EMT), activation of cell migration, degradation of the extracellular matrix, or angiogenesis. The review discusses mechanisms of tumor cells for proton sensing including acid-sensitive ion channels (ASICs, TRPs) or ion transporters (NHE1) and G protein coupled H+-sensors. Finally, the review describes several intracellular signaling cascades activated by H+ sensing mechanisms leading to transcriptional, post-transcriptional, or functional changes in the cell relevant for the metastatic spread. From these studies, different therapeutical approaches are described to overcome tumor acidosis or to interfere with the signaling cascades to reduce the metastatic potential of tumors.

Acidic extracellular environment affects miRNA expression in tumors in vitro and in vivo.

Hypoxia can control the expression of miRNAs in tumors which play an important role for the control of the malignant behavior. The aim of the study was to analyze whether extracellular acidosis, a common feature of tumors, also has an impact on the miRNA expression in isolated cells as well as in solid tumors. MiRNA expression was analyzed in two rat tumor cell lines (AT1 prostate and Walker-256 mammary carcinomas) by NGS and qPCR. In vivo the same cell lines were implanted subcutaneously and the tumor pH was modulated by inspiratory hypoxia and inhibition of the respiratory chain. In addition, the expression of five genes (Brip1, Ercc6l, Ikbke, Per3, Tlr5) which are potential targets of the miRNAs were analyzed on mRNA level. Screening showed that 38 (AT1) resp. 41 (Walker-256) miRNAs were pH-dependent. Validation by qPCR revealed that only 4 miRNAs were consistently regulated in both cell lines: miR-183, miR-203a, miR-215 and miR-7a. The expression of miR-7a was increased by low pH whereas all others were decreased. In the tumors in vivo all 4 miRNAs were down-regulated. The potential targets showed pH dependency in both cell lines. In conclusion, extracellular acidosis regulates the expression of miRNAs in vitro and in vivo. The expression of targets of these miRNAs were also pH-dependent. The pH may therefore affect the biological behavior of tumors via miRNAs.

Hyperbaric oxygenation and glucose/amino acids substitution in human severe placental insufficiency.

In the first case, the AA and glucose were infused through a perinatal port system into the umbilical vein at 30 weeks' gestation due to severe IUGR. The patient received daily hyperbaric oxygenation (HBO, 100% O2 ) with 1.4 atmospheres absolute for 50 min for 7 days. At 31+4  weeks' gestation, the patient gave birth spontaneously to a newborn weighing 1378 g, pH 7.33, APGAR score 4/6/intubation. In follow-up examinations at 5 years of age, the boy was doing well without any neurological disturbance or developmental delay. In the second case, the patient presented at 25/5  weeks' gestation suffering from severe IUGR received HBO and maternal AA infusions. The cardiotocography was monitored continuously during HBO treatment. The short-time variations improved during HBO from 2.9 to 9 msec. The patient developed pathologic CTG and uterine contractions 1 day later and gave birth to a hypotrophic newborn weighing 420 g. After initial adequate stabilization, the extremely preterm newborn unfortunately died 6 days later. Fetal nutrition combined with HBO is technically possible and may allow the prolongation of the pregnancy. Fetal-specific amino-acid composition would facilitate the treatment options of IUGR fetuses and extremely preterm newborn.

Long-term biodistribution study of HPMA-ran-LMA copolymers in vivo by means of 131I-labeling.

BACKGROUND: For the evaluation of macromolecular drug delivery systems suitable pre-clinical monitoring of potential nanocarrier systems is needed. In this regard, both short-term as well as long-term in vivo tracking is crucial to understand structure-property relationships of polymer carrier systems and their resulting pharmacokinetic profile. Based on former studies revealing favorable in vivo characteristics for 18F-labeled random (ran) copolymers consisting of N-(2-hydroxypropyl)methacrylamide (HPMA) and lauryl methacrylate (LMA) - including prolonged plasma half-life as well as enhanced tumor accumulation - the presented work focuses on their long-term investigation in the living organism. METHODS: In this respect, four different HPMA-based polymers (homopolymers as well as random copolymers with LMA as hydrophobic segment) were synthesized and subsequent radioactive labeling was accomplished via the longer-lived radioisotope 131I. In vivo results, concentrating on the pharmacokinetics of a high molecular weight HPMA-ran-LMA copolymer, were obtained by means of biodistribution and metabolism studies in the Walker 256 mammary carcinoma model over a time-span of up to three days. Besides, a direct comparison with the 18F-radiolabeled polymer was drawn. To consider physico-chemical differences between the differently labeled polymer (18F or 131I) on the critical micelle concentration (CMC) and the size of the polymeric micelles, those properties were determined using the 19F- or 127I-functionalized polymer. Special emphasis was laid on the time-dependent correlation between blood circulation properties and corresponding tumor accumulation, particularly regarding the enhanced permeability and retention (EPR) effect. RESULTS: Studies revealed, at first, differences in the short time (2h) body distribution, despite the very similar properties (molecular structure, CMC and size of the micellar aggregates) of the non-radioactive 19F- and 127I-functionalized polymers. Long-term investigations with the 131I-labeled polymer demonstrated that, despite a polymer clearance from the blood within 72h, there was still an increase in tumor uptake observed over time. Regarding the stability of the 131I-label, ex vivo biodistribution experiments, considering the uptake in the thyroid, indicated low metabolism rates. CONCLUSION: The observed in vivo characteristics strongly underline the EPR effect. The findings illustrate the need to combine information of different labeling approaches and in vivo evaluation techniques to generate an overall pharmacokinetic picture of potential nanocarriers in the pre-clinical setting. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENTS: The in vivo behavior of the investigated HPMA-ran-LMA copolymer demonstrates great potential in terms of an effective accumulation in the tumor.

Correction to: Oxygen Transport to Tissue XL.

The chapters "Changes in Cytochrome-C-Oxidase Account for Changes in Attenuation of Near-Infrared Light in the Healthy Infant Brain" and "Fibreless Multiwavelength NIRS System for Imaging Localised Changes in Cerebral Oxidised Cytochrome C Oxidase" are made as open access as per the author's request in this revised version of the book.

Assessing Glomerular Filtration in Small Animals Using [68Ga]DTPA and [68Ga]EDTA with PET Imaging.

PURPOSE: Determining the glomerular filtration rate (GFR) is essential for clinical medicine but also for pre-clinical animal studies. Functional imaging using positron emission tomography (PET) allows repetitive almost non-invasive measurements. The aim of the study was the development and evaluation of easily synthesizable PET tracers for GFR measurements in small animals. PROCEDURES: Diethylenetriaminepentaacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA) were labeled with Ga-68. The binding to blood cells and plasma proteins was tested in vitro. The distribution of the tracers in rats was analyzed by PET imaging and ex vivo measurements. From the time-activity-curve of the blood compartment (heart) and the total tracer mass excreted by the kidney, the GFR was calculated. These values were compared directly with the inulin clearance in the same animals. RESULTS: Both tracers did not bind to blood cells. [68Ga]DPTA but not [68Ga]EDTA showed strong binding to plasma proteins. For this reason, [68Ga]DPTA stayed much longer in the blood and only 30 % of the injected dose was eliminated by the kidney within 60 min whereas the excretion of [68Ga]EDTA was 89 ± 1 %. The calculated GFR using [68Ga]EDTA was comparable to the measured inulin clearance in the same animal. Using [68Ga]-DPTA, the measurements led to values which were 80 % below the normal GFR. The results also revealed that definition of the volume of interest for the blood compartment affects the calculation and may lead to a slight overestimation of the GFR. CONCLUSIONS: [68Ga]EDTA is a suitable tracer for GFR calculation from PET imaging in small animals. It is easy to be labeled, and the results are in good accordance with the inulin clearance. [68Ga]DTPA led to a marked underestimation of GFR due to its strong binding to plasma proteins and is therefore not an appropriate tracer for GFR measurements.

Inhibition of Carbonic Anhydrase IX by Ureidosulfonamide Inhibitor U104 Reduces Prostate Cancer Cell Growth, But Does Not Modulate Daunorubicin or Cisplatin Cytotoxicity.

Carbonic anhydrase (CA) IX has emerged as a promising target for cancer therapy. It is highly upregulated in hypoxic regions and mediates pH regulation critical for tumor cell survival as well as extracellular acidification of the tumor microenvironment, which promotes tumor aggressiveness via various mechanisms, such as augmenting metastatic potential. Therefore, the aim of this study was to analyze the complex interdependency between CA IX and the tumor microenvironment in prostate tumor cells with regard to potential therapeutic implications. CA IX was upregulated by hypoxia as well as acidosis in prostate cancer cells. This induction did not modulate intracellular pH but led to extracellular acidification. Pharmacological inhibition of CA IX activity by U104 (SLC-0111) resulted in a reduction in tumor cell growth and an increase in apoptotic cell death. Intracellular pH was reduced under normoxic and even more so under hypoxic conditions when CA IX level was high. However, although intracellular pH regulation was disturbed, targeting CA IX in combination with daunorubicin or cisplatin did not intensify apoptotic tumor cell death. Hence, targeting CA IX in prostate cancer cells can lead to intracellular pH dysregulation and, consequently, can reduce cellular growth and elevate apoptotic cell death. Attenuation of extracellular acidification by blocking CA IX might additionally impede tumor progression and metastasis. However, no beneficial effect was seen when targeting CA IX in combination with chemotherapeutic drugs.

Tumor Acidosis and Hypoxia Differently Modulate the Inflammatory Program: Measurements In Vitro and In Vivo.

Inflammatory mediators produced by the tumor cells are of importance for immune response but also for malignant progression. The aim of the study was to analyze the expression of monocyte chemoattractant protein-1, interleukin-6 (IL-6), tumor necrosis factor-α, inducible isoform of nitric oxide synthase (iNOS), cyclooxygenase-2, and osteopontin in vitro in two different tumor cell lines under hypoxia (pO2≈1.5 mmHg) and/or acidosis (pH=6.6) for up to 24 hours since hypoxia and acidosis are common characteristics of solid tumors. Additionally, the same tumor cell lines implanted in vivo were made hypoxic and acidotic artificially for 24 hours, after which the cytokine expression was measured. Finally, the activation of ERK1/2 and p38 by acidosis/hypoxia and their impact on cytokine expression were studied. The results indicate that acidosis and hypoxia have fundamentally different (often opposing) effects on cytokine expression. In addition, these effects were tumor cell line specific. When combining hypoxia and acidosis, the overall changes reflect an additive effect of both conditions alone, indicating that hypoxia and acidosis act by independent mechanisms. The in vivo changes corresponded well with the results obtained in the isolated tumor cells. Only iNOS expression was downregulated in vivo but increased in cell culture. For IL-6 expression, the acidosis-induced changes were dependent on ERK1/2 activation. In conclusion, it was demonstrated that the environmental pO2 and pH strongly affect the expression of inflammatory mediators in tumor cells. In vivo, most of the inflammatory mediators were downregulated, which could limit the activation of immune cells and by this foster the immune escape of tumors.

Endocytotic uptake of HPMA-based polymers by different cancer cells: impact of extracellular acidosis and hypoxia.

BACKGROUND: Polymeric nanoparticles allow to selectively transport chemotherapeutic drugs to the tumor tissue. These nanocarriers have to be taken up into the cells to release the drug. In addition, tumors often show pathological metabolic characteristics (hypoxia and acidosis) which might affect the polymer endocytosis. MATERIALS AND METHODS: Six different N-(2-hydroxypropyl)methacrylamide (HPMA)-based polymer structures (homopolymer as well as random and block copolymers with lauryl methacrylate containing hydrophobic side chains) varying in molecular weight and size were analyzed in two different tumor models. The cellular uptake of fluorescence-labeled polymers was measured under hypoxic (pO2 ≈1.5 mmHg) and acidic (pH 6.6) conditions. By using specific inhibitors, different endocytotic routes (macropinocytosis and clathrin-mediated, dynamin-dependent, cholesterol-dependent endocytosis) were analyzed separately. RESULTS: The current results revealed that the polymer uptake depends on the molecular structure, molecular weight and tumor line used. In AT1 cells, the uptake of random copolymer was five times stronger than the homopolymer, whereas in Walker-256 cells, the uptake of all polymers was much stronger, but this was independent of the molecular structure and size. Acidosis increased the uptake of random copolymer in AT1 cells but reduced the intracellular accumulation of homopolymer and block copolymer. Hypoxia reduced the uptake of all polymers in Walker-256 cells. Hydrophilic polymers (homopolymer and block copolymer) were taken up by all endocytotic routes studied, whereas the more lipophilic random copolymer seemed to be taken up preferentially by cholesterol- and dynamin-dependent endocytosis. CONCLUSION: The study indicates that numerous parameters of the polymer (structure, size) and of the tumor (perfusion, vascular permeability, pH, pO2) modulate drug delivery, which makes it difficult to select the appropriate polymer for the individual patient.