Inflammation, ischemia or the microenvironment of solid tumors is often accompanied by a reduction of extracellular pH (acidosis) that stresses the cells and acts on cellular signaling and transcription. The effect of acidosis on the expression of various inflammatory markers, on functional parameters (migration, phagocytic activity) and on signaling pathways involved was studied in monocytic cells and macrophages. In monocytic cell lines acidosis led to a reduction in expression of most of the inflammatory mediators, namely IL-1ß, IL-6, TNF-α, MCP-1, COX-2 and osteopontin. In primary human monocytes MCP-1 and TNF-α were reduced but COX-2 and IL-6 were increased. In RAW264.7 macrophage cell line IL-1ß, COX-2 and iNOS expression was increased, whereas MCP-1 was reduced similar to the effect in monocytic cells. For primary human monocyte-derived macrophages the regulation of inflammatory markers by acidosis depended on activation state, except for the acidosis-induced downregulation of MCP-1 and TNF-α. Acidosis affected functional immune cell behavior when looking at phagocytic activity which was increased in a time-dependent manner, but cellular motility was not changed. Neither ERK1/2 nor CREB signaling was stimulated by the reduction of extracellular pH. However, p38 was activated by acidosis in RAW264.7 cells and this activation was critical for the induction of IL-1ß, COX-2 and iNOS expression. In conclusion, acidosis may impede the recruitment of immune cells, but fosters inflammation when macrophages are present by increasing the level of COX-2 and iNOS and by functionally forcing up the phagocytic activity.

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.

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.

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.

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 (pO 2 ≈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, pO 2 ) modulate drug delivery, which makes it difficult to select the appropriate polymer for the individual patient.