Rheumatoid Synovial Fluid and Acidic Extracellular pH Modulate the Immunomodulatory Activity of Urine-Derived Stem Cells.

Urine-derived stem cells (UdSCs) possess a remarkable anti-inflammatory and immune-modulating activity. However, the clinical significance of UdSCs in autoimmune inflammatory diseases such as rheumatoid arthritis (RA) is yet to be explored. Hence, we tested the UdSCs response to an articular RA microenvironment. To simulate the inflamed RA joint more authentically in vitro, we treated cells with rheumatoid synovial fluids (RASFs) collected from RA patients, serum deprivation, acidosis (pH 7.0 and 6.5), and their combinations. Firstly, the RASFs pro-inflammatory status was assessed by cytokine quantification. Then, UdSCs were exposed to the RA environmental factors for 48 h and cell proliferation, gene expression and secretion of immunomodulatory factors were evaluated. The immunosuppressive potential of pre-conditioned UdSCs was also assessed via co-cultivation with activated peripheral blood mononuclear cells (PBMCs). In all experimental conditions, UdSCs' proliferation was not affected. Conversely, extracellular acidosis considerably impaired the viability/proliferation of adipose tissue-derived stem cells (ATSCs). In the majority of cases, exposure to RA components led to the upregulated expression of IL-6, TSG6, ICAM-1, VCAM-1, and PD-L1, all involved in immunomodulation. Upon RASFs and acidic stimulation, UdSCs secreted higher levels of immunomodulatory cytokines: IL-6, IL-8, MCP-1, RANTES, GM-CSF, and IL-4. Furthermore, RASFs and combined pretreatment with RASFs and acidosis promoted the UdSCs-mediated immunosuppression and the proliferation of activated PBMCs was significantly inhibited. Altogether, our data indicate that the RA microenvironment certainly has the capacity to enhance UdSCs' immunomodulatory function. For potential preclinical/clinical applications, the intra-articular injection might be a reasonable approach to maximize UdSCs' therapeutic efficiency in the RA treatment.

Acidic extracellular pH drives accumulation of N1-acetylspermidine and recruitment of protumor neutrophils.

An acidic tumor microenvironment plays a critical role in tumor progression. However, understanding of metabolic reprogramming of tumors in response to acidic extracellular pH has remained elusive. Using comprehensive metabolomic analyses, we demonstrated that acidic extracellular pH (pH 6.8) leads to the accumulation of N1-acetylspermidine, a protumor metabolite, through up-regulation of the expression of spermidine/spermine acetyltransferase 1 (SAT1). Inhibition of SAT1 expression suppressed the accumulation of intra- and extracellular N1-acetylspermidine at acidic pH. Conversely, overexpression of SAT1 increased intra- and extracellular N1-acetylspermidine levels, supporting the proposal that SAT1 is responsible for accumulation of N1-acetylspermidine. While inhibition of SAT1 expression only had a minor effect on cancer cell growth in vitro, SAT1 knockdown significantly decreased tumor growth in vivo, supporting a contribution of the SAT1-N1-acetylspermidine axis to protumor immunity. Immune cell profiling revealed that inhibition of SAT1 expression decreased neutrophil recruitment to the tumor, resulting in impaired angiogenesis and tumor growth. We showed that antineutrophil-neutralizing antibodies suppressed growth in control tumors to a similar extent to that seen in SAT1 knockdown tumors in vivo. Further, a SAT1 signature was found to be correlated with poor patient prognosis. Our findings demonstrate that extracellular acidity stimulates recruitment of protumor neutrophils via the SAT1-N1-acetylspermidine axis, which may represent a metabolic target for antitumor immune therapy.

Norcantharidin Induces Immunogenic Cell Death of Bladder Cancer Cells through Promoting Autophagy in Acidic Culture.

The acidic tumor microenvironment stands as a major obstacle to the efficient elimination of tumor cells. Norcantharidin (NCTD) is a powerful antitumor agent with multiple bioactivities. However, the effect of NCTD under acidic conditions is still unclear. Here, we report that NCTD can efficiently kill bladder cancer (BC) cells in acidic culture, and more intriguingly, NCTD can induce immunogenic cell death (ICD), thereby promoting antitumor immunity. In NCTD-treated BC cells, the surface-exposed calreticulin (ecto-CALR) was significantly increased. Consistently, co-culture with these cells promoted dendritic cell (DC) maturation. The NCTD-induced ICD is autophagy dependent, as autophagy inhibition completely blocked the NCTD-induced ecto-CALR and DC maturation. In addition, the DC showed a distinct maturation phenotype (CD80high CD86low) in acidic culture, as compared to that in physiological pH (CD80 high CD86high). Finally, the NCTD-induced ICD was validated in a mouse model. NCTD treatment significantly increased the tumor-infiltrating T lymphocytes in MB49 bladder cancer mice. Immunizing mice with NCTD-treated MB49 cells significantly increased tumor-free survival as compared to control. These findings demonstrate that NCTD could induce ICD in an acidic environment and suggest the feasibility to combine NCTD with anticancer immunotherapy to treat BC.

Neutralization of Acidic Tumor Microenvironment (TME) with Daily Oral Dosing of Sodium Potassium Citrate (K/Na Citrate) Increases Therapeutic Effect of Anti-cancer Agent in Pancreatic Cancer Xenograft Mice Model.

Extracellular pH (pHe) of tumor cells is characteristic of tumor microenvironment (TME). Acidic TME impairs the responses of tumors to some anti-cancer chemotherapies. In this study, we showed that daily oral dosing of sodium potassium citrate (K/Na citrate) increased blood HCO3- concentrations, corresponding to increase of HCO3- concentrations and pHs in urine, and neutralized the tumor pHe. Neutralization of acidic TME by alkaline substance like HCO3-, an active metabolite of K/Na citrate, well potentiated the therapeutic effect of anticancer agent TS-1®, an orally active 5-fuluoro-uracil derivative, in Panc-1 pancreatic cancer-xenograft murine model. Neutralization of acidic TME by using an alkaline K/Na citrate is a smart approach for enhancement of the therapeutic effects of anticancer agents for pancreatic cancer in the end stage.

Adaptation to chronic acidic extracellular pH elicits a sustained increase in lung cancer cell invasion and metastasis.

Acidic extracellular pH (pHe) is an important microenvironment for cancer cells. This study assessed whether adaptation to acidic pHe enhances the metastatic phenotype of tumor cells. The low metastatic variant of Lewis lung carcinoma (LLCm1) cells were subjected to stepwise acidification, establishing acidic pHe-adapted (LLCm1A) cells growing exponentially at pH 6.2. These LLCm1A cells showed increased production of matrix metalloproteinases (MMPs), including MMP-2, -3, -9, and -13, and pulmonary metastasis following injection into mouse tail veins. Although LLCm1A cells exhibited a fibroblastic shape, keratin-5 expression was increased and α-smooth muscle actin expression was reduced. Despite serial passage of these cells at pH 7.4, high invasive activity through Matrigel® was sustained for at least 28 generations. Thus, adaptation to acidic pHe resulted in a more invasive phenotype, which was sustained during passage at pH 7.4, suggesting that an acidic microenvironment at the primary tumor site is important in the acquisition of a metastatic phenotype.

Imatinib Uptake into Cells is Not Mediated by Organic Cation Transporters OCT1, OCT2, or OCT3, But is Influenced by Extracellular pH.

BACKGROUND: Cancer cells undergo genetic and environmental changes that can alter cellular disposition of drugs, notably by alterations of transmembrane drug transporters expression. Whether the influx organic cation transporter 1 (OCT1) encoded by the gene SLC221A1 is implicated in the cellular uptake of imatinib is still controversial. Besides, imatinib ionization state may be modulated by the hypoxic acidic surrounding extracellular microenvironment. OBJECTIVE: To determine the functional contribution of OCTs and extracellular pH on imatinib cellular disposition. METHODS: We measured imatinib uptake in two different models of selective OCTs drug transporter expression (transfected Xenopus laevis oocytes and OCT-expressing HEK293 human cells), incubated at pH 7.4 and 6, using specific mass spectrometry analysis. RESULTS: Imatinib cellular uptake occurred independently of OCT1- OCT2- or OCT3-mediated drug transport at pH 7.4. Uptake of the OCTs substrate tetraethylammonium in oocytes remained intact at pH 6, while the accumulation of imatinib in oocytes was 10-fold lower than at pH 7.4, irrespectively of OCTs expressions. In OCT1- and OCT2-HEK cells at pH 6, imatinib accumulation was reduced by 2- 3-fold regardless of OCTs expressions. Since 99.5% of imatinib at pH6 is under the cationic form, the reduced cellular accumulation of imatinib at such pH may be explained by the lower amount of uncharged imatinib remaining for passive diffusion across cellular membrane. CONCLUSION: Imatinib is not a substrate of OCTs 1-3 while the environmental pH modulates cellular disposition of imatinib. The observation that a slightly acidic extracellular pH influences imatinib cellular accumulation is important, considering the low extracellular pH reported in the hematopoietic leukemia/ cancer cell microenvironment.

Cancer metabolism: New insights into classic characteristics.

Initial studies of cancer metabolism in the early 1920s found that cancer cells were phenotypically characterized by aerobic glycolysis, in that these cells favor glucose uptake and lactate production, even in the presence of oxygen. This property, called the Warburg effect, is considered a hallmark of cancer. The mechanism by which these cells acquire aerobic glycolysis has been uncovered. Acidic extracellular fluid, secreted by cancer cells, induces a malignant phenotype, including invasion and metastasis. Cancer cells survival depends on a critical balance of redox status, which is regulated by amino acid metabolism. Glutamine is extremely important for oxidative phosphorylation and redox regulation. Cells highly dependent on glutamine and that cannot survive with glutamine are called glutamine-addicted cells. Metabolic reprogramming has been observed in cancer stem cells, which have the property of self-renewal and are resistant to chemotherapy and radiotherapy. These findings suggest that studies of cancer metabolism can reveal methods of preventing cancer recurrence and metastasis.

Metadherin contributes to epithelial-mesenchymal transition and paclitaxel resistance induced by acidic extracellular pH in nasopharyngeal carcinoma.

Paclitaxel resistance is a challenge to the treatment of nasopharyngeal carcinoma (NPC). An acidic extracellular pH (pHe), a hallmark of solid tumors, is demonstrated to decrease the efficacy of chemotherapy. However, the precise function of acidic pHe in mediating chemotherapy in NPC remains unknown. In the present study, acidic pHe significantly decreased the cytotoxicity of paclitaxel in NPC cells. In addition, epithelial-mesenchymal transition (EMT)-like changes were observed in NPC cells cultured at acidic pHe. Metadherin (MTDH), a novel oncogene, is expressed in multiple types of solid tumor, and is associated with several malignant cell characteristics, including malignant cell transformation, proliferation, angiogenesis, chemoresistance, invasion and metastasis. In the present study, MTDH expression was increased in NPC cells that had been cultured at an acidic pHe. Furthermore, the silencing of MTDH expression reversed EMT molecular marker expression and sensitized NPC cells to paclitaxel. Taken together, the results of the present study provide evidence to support an association between acidic pHe-induced paclitaxel resistance and MTDH-mediated EMT in NPC cells. Thus, targeting MTDH may provide a novel strategy for overcoming chemoresistance in NPC therapy.

TRPM5 mediates acidic extracellular pH signaling and TRPM5 inhibition reduces spontaneous metastasis in mouse B16-BL6 melanoma cells.

Extracellular acidity is a hallmark of solid tumors and is associated with metastasis in the tumor microenvironment. Acidic extracellular pH (pH e ) has been found to increase intracellular Ca2+ and matrix metalloproteinase-9 (MMP-9) expression by activating NF-κB in the mouse B16 melanoma model. The present study assessed whether TRPM5, an intracellular Ca2+-dependent monovalent cation channel, is associated with acidic pH e signaling and induction of MMP-9 expression in this mouse melanoma model. Treatment of B16 cells with Trpm5 siRNA reduced acidic pH e -induced MMP-9 expression. Enforced expression of Trpm5 increased the rate of acidic pH e -induced MMP-9 expression, as well as increasing experimental lung metastasis. This genetic manipulation did not alter the pH e critical for MMP-9 induction but simply amplified the percentage of inducible MMP-9 at each pH e . Treatment of tumor bearing mice with triphenylphosphine oxide (TPPO), an inhibitor of TRPM5, significantly reduced spontaneous lung metastasis. In silico analysis of clinical samples showed that high TRPM5 mRNA expression correlated with poor overall survival rate in patients with melanoma and gastric cancer but not in patients with cancers of the ovary, lung, breast, and rectum. These results showed that TRPM5 amplifies acidic pH e signaling and may be a promising target for preventing metastasis of some types of tumor.

Acidic extracellular pH promotes epithelial mesenchymal transition in Lewis lung carcinoma model.

BACKGROUND: Epithelial mesenchymal transition (EMT) is thought to be an essential feature of malignant tumor cells when they spread into the stroma. Despite the extracellular acidity of tumor tissues, the effect of acidic extracellular pH (pH e ) on EMT in carcinoma models, including the Lewis lung carcinoma (LLC) model, remains unclear. METHODS: High and low metastatic LLC variants were generated by repeated tail vein injection of metastatic cells. DMEM/F12 medium, which has been supplemented with 15 mM HEPES, 4 mM phosphoric acid, and 1 g/L NaHCO3 and adjusted to the desire pH with HCl or NaOH, was used for cell culture. EMT marker gene expression was determined by quantitative reverse transcription-polymerase chain reaction. Migration and invasion activities were analyzed by wound healing assay and the Boyden chamber assay through Matrigel®, respectively. RESULTS: Low metastatic variant LLCm1 cells showed a cobble-stone like morphology at pH e 7.4. At pH e 6.8, however, their morphology became fibroblastic, similar in shape to high metastatic variant LLCm4 cells. Steady state levels of matrix metalloproteinase-9 (Mmp9) mRNA were induced by acidic pH e , maximizing at pH 6.8, with the levels of Mmp9 mRNA higher in LLCm4 than in LLCm1 cells. Both variants showed decreased levels of E-cadherin and increased levels of vimentin at pH e 6.8. Acidic pH e also induced expression of mRNAs encoding the E-cadherin repressors, Zeb2, Twist1 and Twist2, as well as enhancing cell motility and in vitro invasion through Matrigel®. CONCLUSIONS: Acidic pH e can induce EMT in some types of carcinoma.