Extracellular Acidosis Research Articles

Cell-Type Dependent Regulation of the Electrogenic Na+/HCO3- Cotransporter 1 (NBCe1) by Hypoxia and Acidosis in Glioblastoma.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumour. It is characterised by transcriptionally distinct cell populations. In tumour cells, physiological pH gradients between the intracellular and extracellular compartments are reversed, compared to non-cancer cells. Intracellular pH in tumour cells is alkaline, whereas extracellular pH is acidic. Consequently, the function and/or expression of pH regulating transporters might be altered. Here, we investigated protein expression and regulation of the electrogenic sodium/bicarbonate cotransporter 1 (NBCe1) in mesenchymal (MES)-like hypoxia-dependent and -independent cells, as well as in astrocyte-like glioblastoma cells following chemical hypoxia, acidosis and elucidated putative underlying molecular pathways. Immunoblotting, immunocytochemistry, and intracellular pH recording with the H+-sensitive dye 2′,7′-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein were applied. The results show NBCe1 protein abundance and active NBCe1 transport. Hypoxia upregulated NBCe1 protein and activity in MES-like hypoxia-dependent GBM cells. This effect was positively correlated with HIF-1α protein levels, was mediated by TGF-β signalling, and was prevented by extracellular acidosis. In MES-like hypoxia-independent GBM cells, acidosis (but not hypoxia) regulated NBCe1 activity in an HIF-1α-independent manner. These results demonstrate a cell-specific adaptation of NBCe1 expression and activity to the microenvironment challenge of hypoxia and acidosis that depends on their transcriptional signature in GBM.

The acidic tumor microenvironment enhances PD-L1 expression via activation of STAT3 in MDA-MB-231 breast cancer cells

Tumor acidosis, a common phenomenon in solid cancers such as breast cancer, is caused by the abnormal metabolism of cancer cells. The low pH affects cells surrounding the cancer, and tumor acidosis has been shown to inhibit the activity of immune cells. Despite many previous studies, the immune surveillance mechanisms are not fully understood. We found that the expression of PD-L1 was significantly increased under conditions of extracellular acidosis in MDA-MB-231 cells. We also confirmed that the increased expression of PD-L1 mediated by extracellular acidosis was decreased when the pH was raised to the normal range. Gene set enrichment analysis (GSEA) of public breast cancer patient databases showed that PD-L1 expression was also highly correlated with IL-6/JAK/STAT3 signaling. Surprisingly, the expression of both phospho-tyrosine STAT3 and PD-L1 was significantly increased under conditions of extracellular acidosis, and inhibition of STAT3 did not increase the expression of PD-L1 even under acidic conditions in MDA-MB-231 cells. Based on these results, we suggest that the expression of PD-L1 is increased by tumor acidosis via activation of STAT3 in MDA-MB-231 cells.

A pH-sensitive field-effect transistor for monitoring of cancer cell external acid environment

The external acid environment of cancer cells is different from that of normal cells, making a profound impact on cancer progression. Here we report a simple poly-l-lysine-modified graphene field-effect transistor (PLL@G-FET) for in situ monitoring of extracellular acidosis around cancer cells. PLL is a well-known material with good biocompatibility and is rich in amino groups that are sensitive to hydrogen ions. After a simple drop-casting of PLL on the reduced graphene oxide (RGO) FET surface, the PLL@G-FET was able to realize the real-time monitoring of the localized pH change of cancer cells after the cancer cells were grown on the device. The PLL@G-FET sensor achieved a Nernstian value of 52.9 mV/pH in phosphate buffer saline from pH 4.0 to 8.0. In addition, the sensor exhibited excellent biocompatibility as well as good anti-interference ability in the cell culture medium. Furthermore, the device was used to real-time monitor the extracellular pH changes of MCF-7 cells under the intervention of different concentrations of drugs. This developed pH-sensitive FET provides a new method to study the extracellular acid environment in situ and helps us to enhance our understanding of cancer cell metabolism.

Cancer Therapeutic Targeting of Hypoxia Induced Carbonic Anhydrase IX: From Bench to Bedside.

Simple SummaryTumor hypoxia remains a significant problem in the effective treatment of most cancers. Tumor cells within hypoxic niches tend to be largely resistant to most therapeutic modalities, and adaptation of the cells within the hypoxic microenvironment imparts the cells with aggressive, invasive behavior. Thus, a major goal of successful cancer therapy should be the eradication of hypoxic tumor cells. Carbonic Anhydrase IX (CAIX) is an exquisitely hypoxia induced protein, selectively expressed on hypoxic tumor cells, and thus has garnered significant attention as a therapeutic target. In this Commentary, we discuss the current status of targeting CAIX, and future strategies for effective, durable cancer treatment.Carbonic Anhydrase IX (CAIX) is a major metabolic effector of tumor hypoxia and regulates intra- and extracellular pH and acidosis. Significant advances have been made recently in the development of therapeutic targeting of CAIX. These approaches include antibody-based immunotherapy, as well as use of antibodies to deliver toxic and radioactive payloads. In addition, a large number of small molecule inhibitors which inhibit the enzymatic activity of CAIX have been described. In this commentary, we highlight the current status of strategies targeting CAIX in both the pre-clinical and clinical space, and discuss future perspectives that leverage inhibition of CAIX in combination with additional targeted therapies to enable effective, durable approaches for cancer therapy.

Ca2+ Signalling and Hypoxia/Acidic Tumour Microenvironment Interplay in Tumour Progression.

Solid tumours are characterised by an altered microenvironment (TME) from the physicochemical point of view, displaying a highly hypoxic and acidic interstitial fluid. Hypoxia results from uncontrolled proliferation, aberrant vascularization and altered cancer cell metabolism. Tumour cellular apparatus adapts to hypoxia by altering its metabolism and behaviour, increasing its migratory and metastatic abilities by the acquisition of a mesenchymal phenotype and selection of aggressive tumour cell clones. Extracellular acidosis is considered a cancer hallmark, acting as a driver of cancer aggressiveness by promoting tumour metastasis and chemoresistance via the selection of more aggressive cell phenotypes, although the underlying mechanism is still not clear. In this context, Ca2+ channels represent good target candidates due to their ability to integrate signals from the TME. Ca2+ channels are pH and hypoxia sensors and alterations in Ca2+ homeostasis in cancer progression and vascularization have been extensively reported. In the present review, we present an up-to-date and critical view on Ca2+ permeable ion channels, with a major focus on TRPs, SOCs and PIEZO channels, which are modulated by tumour hypoxia and acidosis, as well as the consequent role of the altered Ca2+ signals on cancer progression hallmarks. We believe that a deeper comprehension of the Ca2+ signalling and acidic pH/hypoxia interplay will break new ground for the discovery of alternative and attractive therapeutic targets.

Cytoprotection of Human Progenitor and Stem Cells through Encapsulation in Alginate Templated, Dual Crosslinked Silk and Silk-Gelatin Composite Hydrogel Microbeads.

Susceptibility of mammalian cells against harsh processing conditions limit their use in cell transplantation and tissue engineering applications. Besides modulation of the cell microenvironment, encapsulation of mammalian cells within hydrogel microbeads attract attention for cytoprotection through physical isolation of the encapsulated cells. The hydrogel formulations used for cell microencapsulation are largely dominated by ionically crosslinked alginate (Alg), which suffer from low structural stability under physiological culture conditions and poor cell-matrix interactions. Here the fabrication of Alg templated silk and silk/gelatin composite hydrogel microspheres with permanent or on-demand cleavable enzymatic crosslinks using simple and cost-effective centrifugation-based droplet processing are demonstrated. The composite microbeads display structural stability under ion exchange conditions with improved mechanical properties compared to ionically crosslinked Alg microspheres. Human mesenchymal stem and neural progenitor cells are successfully encapsulated in the composite beads and protected against environmental factors, including exposure to polycations, extracellular acidosis, apoptotic cytokines, ultraviolet (UV) irradiation, anoikis, immune recognition, and particularly mechanical stress. The microbeads preserve viability, growth, and differentiation of encapsulated stem and progenitor cells after extrusion in viscous polyethylene oxide solution through a 27-gauge fine needle, suggesting potential applications in injection-based delivery and three-dimensional bioprinting of mammalian cells with higher success rates.

Abstract 3039: Decreased pH-sensitive potassium channel gene expressions in glioblastomas compared to oligodendrogliomas detected with house keeping genes may shift glioblastoma membrane potentials towards proton efflux to the microenvironment

Abstract Glial buffering of K+ prevents seizures, hence gliomas in patients on anticonvulsants may harbor potassium related anomalies. Cell membrane potentials are very sensitive to K+ and may link to tumor acidosis when they become more positive than the equilibrium potential for H+ ions (Vaupel, P, et al., Cancer Res, 1989). Kir5.1(KCNJ16) heterodimerizes with Kir4.1(KCNJ10), a major glial potassium channel, to stop K+ conductance in acidosis. Trek1&2(KCNK2&10) are also pH sensitive K+ channels. Glioblastomas (more malignant and invasive) versus oligodendrogliomas showed decreased KCNJ16 in patients on anticonvulsants (Beckner, ME, Proc AACR, A2319, 2021). However, loss of genomic integrity in tumors and potential variation between assays prompted house keeping gene (HKG) normalization to lessen confounding factors. REMBRANDT (12/31/2020, Georgetown Database of Cancer) had 220 glioblastomas and 67 oligodendrogliomas with microarray gene expression via medians of each reporter (1-8 per gene) in Adobe Flash readouts (ended 1/1/21). A subgroup, 75 glioblastomas and 24 oligodendroglioma patients, took anticonvulsants. Comparison of genes of interest (GOI) initially had t tests between types of gliomas in the subgroup. Here, differences between glioma types for each GOI reporter were compared to differences for HKGs (PPIA, RPLP0, YWHAZ, and B2M). In gliomas with anticonvulsants, paired 2-tailed t tests for a null difference of median reporters between tumor types were less sensitive than new comparisons of oligodendroglioma minus glioblastoma (OMG) differences for GOIs versus OMG differences in HKGs. For KCNJ16, the t test, p = 0.015, for a decrease in glioblastomas (versus oligodendrogliomas) in initial studies improved to p ≤ 0.0001 by using HKGs in comparisons. The KCNK2&10 decrease in glioblastomas became significant, p = 0.047. Among other glycolytic and acidosis related genes, CA12, GLO1&2, PFKFB1&3, showed stronger trends, with p values from 0.08 to 0.13 (decreases and increase) in comparisons using HKGs. Conclusion: HKG normalization presumably corrects for loss of genomic integrity, assay variations, etc. in comparisons using microarrays of different tumors. The pH-sensitive K+ channel genes studied here were expressed less in glioblastomas. The resulting pH-insensitive K+ distributions in glioblastomas putatively shift their cell membrane potentials to less negative values which are sufficient to allow passive H+ efflux that causes extracellular acidosis with accompanying activation of invasion. Acknowledgement: The data utilized in this study were provided by the Georgetown Database of Cancer (G-DOC), a project of the Georgetown Lombardi Comprehensive Cancer Center designed to provide translational research tools to the scientific community. Citation Format: Marie E. Beckner. Decreased pH-sensitive potassium channel gene expressions in glioblastomas compared to oligodendrogliomas detected with house keeping genes may shift glioblastoma membrane potentials towards proton efflux to the microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3039.

Contact-dependent, polarized acidification response during neutrophil-epithelial interactions.

Neutrophil (PMN) infiltration during active inflammation imprints changes in the local tissue environment. Such responses are often accompanied by significant extracellular acidosis that result in predictable transcriptional responses. In this study, we explore the mechanisms involved in inflammatory acidification as a result of PMN-intestinal epithelial cell (IEC) interactions. Using recently developed tools, we revealed that PMN transepithelial migration (TEM)-associated inflammatory acidosis is dependent on the total number of PMNs present during TEM and is polarized toward the apical surface. Extending these studies, we demonstrate that physical separation of the PMNs and IECs prevented acidification, whereas inhibition of PMN TEM using neutralizing antibodies enhanced extracellular acidification. Utilizing pharmaceutical inhibitors, we demonstrate that the acidification response is independent of myeloperoxidase and dependent on reactive oxygen species generated during PMN TEM. In conclusion, inflammatory acidosis represents a polarized PMN-IEC-dependent response by an as yet to be fully determined mechanism.

Extracellular acidosis stimulates breast cancer cell motility through aryl hydrocarbon receptor and c-src kinase activation.

A reduction in extracellular pH (pHe) is a characteristic of most malignant tumors. The aryl hydrocarbon receptor (AhR) is a transcription factor localized in a cytosolic complex with c-Src, which allows it to trigger nongenomic effects through c-Src. Considering that the slightly acidic tumor microenvironment promotes breast cancer progression in a similar way to the AhR/c-Src axis, our aim was to evaluate whether this pathway could be activated by low pHe. We examined the effect of pHe 6.5 on AhR/c-Src axis using two breast cancer cell lines (MDA-MB-231 and LM3) and mammary epithelial cells (NMuMG) and found that acidosis increased c-Src phosphorylation only in tumor cells. Moreover, the presence of AhR inhibitors prevented c-Src activation. Low pHe reduced intracellular pH (pHi), while amiloride treatment, which is known to reduce pHi, induced c-Src phosphorylation through AhR. Analyses were conducted on cell migration and metalloproteases (MMP)-2 and -9 activities, with results showing an acidosis-induced increase in MDA-MB-231 and LM3 cell migration and MMP-9 activity, but no changes in NMuMG cells. Moreover, all these effects were blocked by AhR and c-Src inhibitors. In conclusion, acidosis stimulates the AhR/c-Src axis only in breast cancer cells, increasing cell migration and MMP-9 activity. Although the AhR activation mechanism still remains elusive, a reduction in pHi may be thought to be involved. These findings suggest a critical role for the AhR/c-Src axis in breast tumor progression stimulated by an acidic microenvironment.

Abstract B041: Mimicking tumor acidic and hypoxic microenvironment in vitro towards generation of more predictive screening platform for solid tumors

Abstract The drug discovery process in oncology relies mainly on screening and preclinical validation platforms that use human cancer cells that are grown in standard monolayer (2D) cultures. However, 2D monolayer cell culture methods poorly reflect the tumor microenvironment (TME) and the transcriptome and epigenome attributes of the cancer cells in vivo, leading to inaccurate estimate of drug efficacy in clinic. The TME of solid tumors is complex yet it consist of generic hallmarks such as hypoxia and extracellular acidosis that are common across tumor types and directly modulate gene expression and epigenome. Here we hypothesized that in vitro culture systems that recapitulate the acidic and hypoxic features of the solid TME, more accurately reflect the gene expression profile of cells in in vivo tumors, and thus serve as improved drug screening platform that reliably determine drug efficacies. Here we surveyed systematically the impact on transcriptome and epigenome hallmarks of exposure to hypoxia and acidosis on 2D and 3D cultures of A549 lung adenocarcinoma cell line as a model for solid tumor. To recapitulate naturally occurring hypoxia in vitro, A549 cells were adapted over several passages to growth in 3D. Transcriptome data derived from cells adapted to 3D, when compared to 2D and patient tumor datasets suggested that time spent in 3D increased the concordance of transcriptome profile with tumors from patients. Furthermore adaptation to 3D revealed emergence of hypoxia hallmarks when compared to 2D. To model the combined effect of hypoxia and acidosis, the 3D adapted A549 spheroids were additionally exposed to low pH conditions (6.4) for a duration of 5 days. The additional exposure to low pH led to enhanced tumor progressive phenotypes as evidenced by enrichment in genes associated with hallmarks of inflammation, epithelial mesenchymal transition (EMT), cell invasion and drug resistance. To determine the impact of hypoxic and acidic TME on cytotoxic response to anti-cancer drugs, the 2D and 3D adapted spheroids with or without additional exposure to low pH were screened against a library consisting of 184 anti-cancer drugs. Additional exposure to low pH led to an overall increase in drug resistance. However the combined hypoxic and acidic conditions in 3D revealed novel susceptibilities for compounds that belong to the family of Aurora kinase inhibitors. These results provide indications that accurate modeling of hypoxic and acidic TME in vitro could reveal expected resistance mechanisms and specific drug susceptibilities. This study introduces a 3D screening platform that recapitulates the acidosis and hypoxia of solid TME and captures the transcriptomic profile in vitro that is highly concordant with tumors in patients. This model allows the study of resistance mechanisms and unravel novel susceptibilities that is otherwise undiscovered in standard 2D conditions and could result in the identification of anticancer drugs that are more clinically relevant. Citation Format: Nazanin Rohani Larijani, Marielle Huot, Anne Lenferink, Noël R. Raynal. Mimicking tumor acidic and hypoxic microenvironment in vitro towards generation of more predictive screening platform for solid tumors [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B041.

Pulmonary Arterial Smooth Muscle Cell Monocarboxylate Transporter 1/4 Contributes to Extracellular Acidosis following Chronic Hypoxia‐induced Pulmonary Hypertension

Pulmonary hypertension (pHTN) is defined by mean pulmonary arterial pressure at rest exceeding 20 mmHg with mortality often resulting from right ventricular failure that is secondary to increased afterload. During pHTN, increased reliance on aerobic glycolysis and inhibition of mitochondrial oxidative respiration is observed in pulmonary arterial smooth muscle cells (PASMCs). This metabolic shift is associated with hyperproliferation and resistance to apoptosis contributing to the pathogenesis of pHTN. Along with these phenotypic changes, cells undergoing aerobic respiration exhibit increased glucose uptake and subsequent lactate and H+ efflux, contributing to extracellular acidosis. Acid‐sensing ion channel 1 (ASIC1) is a voltage‐independent, proton‐gated cation channel that contributes to the development of chronic hypoxia (CH)‐induced pHTN. While the function of ASIC1 is well characterized in pHTN, it has not been determined whether the metabolic shift observed during pHTN contributes to the activation of ASIC1. Therefore, we hypothesized that enhanced glucose uptake and subsequent acidification of the extracellular microenvironment in PASMCs lead to the activation of ASIC1 in CH‐induced pHTN. As an initial step in testing this hypothesis, rats were housed in a hypobaric chamber (barometric pressure: ~380 mmHg) to induce pHTN and compared to age‐matched normoxic controls. After 4 weeks, PASMCs were collected and transiently cultured under normoxia for 3‐4 days. As expected, PASMCs from CH rats showed a reduced oxygen consumption rate (p<0.0001) and greater extracellular acidification (p<0.0001) compared to those from control animals, indicating increased glycolytic activity. Utilizing the fluorescent pH indicators SNARF‐5/AM, we found that PASMCs from CH rats exhibit intracellular alkalization (p<0.0001 vs. control) and extracellular acidosis (p<0.001 vs. control). We further examined the H+ transporter(s) involved in mediating extracellular acidosis by inhibiting carbonic anhydrase IX (CA‐IX), the Na+/H+ exchanger (NHE1), the vacuolar‐type H+‐type ATPase (V‐ATPase), or monocarboxylate transporters 1 and ‐4 (MCT1 and MCT4). The dual MCT1/4 inhibitor, syrosingopine, prevented intracellular alkalization and extracellular acidosis following CH (Figure 1A), which corresponded with an increase in MCT4, but not MCT1, expression (Figure 1B). These data suggest MCT plays an important role in regulating PASMC pH homeostasis. Future studies will assess the role of MCT to activate ASIC1 and contribute to pHTN.

Regulation of Vacuolar H+ ATPase Subunit Expression in the Mouse Brain by Developmental Stage and pH Changes

The vacuolar‐type H+ ATPase (V‐ATPase) is a ubiquitously expressed proton pump, essential for multiple cellular processes such as acidification of intracellular organelles. The V‐ATPase consists of a transmembrane domain (V0) responsible for proton translocation and a cytosolic domain (V1) which catalyzes ATP hydrolysis. The functional domains contain different subunits with tissue‐specific expression patterns. In some organs, such as the kidney and salivary glands, V‐ATPase is located in the plasma membrane and previous studies have shown that its subunits are regulated by acid‐base disturbances. It has also been proposed that V‐ATPase subunits may be regulated by growth factors. In the present study, QRT‐PCR was used to examine the expression of different V‐ATPase subunits in the mouse brain and kidney in different developmental stages. Additionally, we have examined the impact of Transforming Growth Factor β2 (TGF‐β2) on different subunit expression, as well as their regulation in mouse cortical astrocytes following extracellular acidosis. The results show that the subunits ATP6V1A1, ATP6V1C1 and ATP6V1B2 are more abundant in the mouse brain, compared to kidney. In contrast, expression of subunits ATP6V1C2 and ATP6V1G3 is higher in the kidney, compared to brain. Our data also reveal developmental changes of subunit transcriptional levels in the mouse brain. Subunit ATP6V1G1 expression is decreasing during brain development, while the expression of ATP6V1C1 is developmentally increasing in the mouse brain. Moreover, our data indicate differential regulation of distinct V‐ATPase subunits in mouse embryonic brain and kidney by TGF‐β2. Extracellular acidosis has no effect on ATP6V1A1, ATP6V1C1 and ATP6V1E1 transcript levels in cortical astrocytes. Our data provide first evidence on developmental regulation of V‐ATPase subunits in the brain.

Acidosis regulation of epithelial barrier function and immune signaling

A common but often underappreciated aspect of the inflammatory microenvironment is extracellular acidification. As with other chronic inflammatory diseases, tissue acidification has been observed in inflammatory bowel disease (IBD) patients. Colonic pH as low as 4 have been reported in IBD patients. In contrast, the pH of a healthy colon is between a pH of 7.0‐7.4. Recently, we identified an adaptive tissue response in intestinal epithelial cells (IEC) which buffered extracellular acidification during neutrophil transepithelial migration, suggesting pH homeostasis is an important aspect of acute inflammation. Extensions of these studies identified a previously undescribed CREB‐dependent pathway for sensing extracellular pH and a unique acidosis gene signature in IEC. The full impact of acidosis on IEC function remains unclear. To better understand the impact of chronic extracellular acidification on IEC function, we investigated changes in IEC immune signaling and barrier function in the setting of acute and chronic exposure to acidosis. We initially examined barrier function in T84 IEC cultured under acidic conditions and observed a significant decrease in barrier function, as determined by transepithelial electrical resistance (TER) and paracellular flux, in cells exposed to acidosis in a pH range of 6.0 – 7.4. Interestingly, following acute acidification, treatment with low pH for less than 8hr, barrier function recovered within 16hr. However, following chronic acidification, treatment with low pH beyond 16hr resulted in an appreciable barrier dysfunction for several days. We extended these studies to examine expression and localization of tight‐junction proteins in IEC exposed to acidosis and observed a time and dose‐dependent decrease in ZO‐1 and E‐cadherin expression, with almost complete loss of expression after 72hr at a pH of 6.0. Likewise, expression and localization of claudins 1, 2, and 4 decreased over time. In parallel to a loss of barrier function, IEC exposed to chronic acidosis revealed a prominent increase in the expression of inflammatory markers including several chemokines (IL‐8, CCL20, TNF) as well as anti‐microbial peptides (mucins and beta‐defensins).Thus, chronic extracellular acidosis induces the loss of IEC tight junction function, while inducing the expression of several pro‐inflammatory chemokines and the production of various anti‐microbial peptides. These results identify extracellular acidification as significant signaling mechanism which induces a loss of epithelial features and the acquisition of a more immune‐like phenotype. Furthering our understandings of the mechanisms involved in acidosis induced phenotype switching in IEC has the potential to uncover novel therapeutic drug targets.

Identification of distinct slow mode of reversible adaptation of pancreatic ductal adenocarcinoma to the prolonged acidic pH microenvironment

BackgroundPancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic neoplasm with high metastatic potential and poor clinical outcome. Like other solid tumors, PDAC in the early stages is often asymptomatic, and grows very slowly under a distinct acidic pHe (extracellular pH) microenvironment. However, most previous studies have only reported the fate of cancerous cells upon cursory exposure to acidic pHe conditions. Little is known about how solid tumors—such as the lethal PDAC originating within the pancreatic duct-acinar system that secretes alkaline fluids—evolve to withstand and adapt to the prolonged acidotic microenvironmental stress.MethodsRepresentative PDAC cells were exposed to various biologically relevant periods of extracellular acidity. The time effects of acidic pHe stress were determined with respect to tumor cell proliferation, phenotypic regulation, autophagic control, metabolic plasticity, mitochondrial network dynamics, and metastatic potentials.ResultsUnlike previous short-term analyses, we found that the acidosis-mediated autophagy occurred mainly as an early stress response but not for later adaptation to microenvironmental acidification. Rather, PDAC cells use a distinct and lengthy process of reversible adaptive plasticity centered on the early fast and later slow mitochondrial network dynamics and metabolic adjustment. This regulates their acute responses and chronic adaptations to the acidic pHe microenvironment. A more malignant state with increased migratory and invasive potentials in long-term acidosis-adapted PDAC cells was obtained with key regulatory molecules being closely related to overall patient survival. Finally, the identification of 34 acidic pHe-related genes could be potential targets for the development of diagnosis and treatment against PDAC.ConclusionsOur study offers a novel mechanism of early rapid response and late reversible adaptation of PDAC cells to the stress of extracellular acidosis. The presence of this distinctive yet slow mode of machinery fills an important knowledge gap in how solid tumor cells sense, respond, reprogram, and ultimately adapt to the persistent microenvironmental acidification.

Epithelial-Fibroblast Crosstalk Protects against Acidosis-Induced Inflammatory and Fibrotic Alterations.

Pathogenesis of chronic kidney disease (CKD) is accompanied by extracellular acidosis inflammation, fibrosis and epithelial-to-mesenchymal transition (EMT). The aim of this study was to assess the influence of acidosis on tubule epithelial cells (NRK-52E) and fibroblasts (NRK-49F) in dependence of cellular crosstalk. NRK-52E and NRK-49F were used in mono- and co-cultures, and were treated with acidic media (pH 6.0) for 48 h. The intracellular proteins were measured by Western blot. Secreted proteins were measured by ELISA. Distribution of E-cadherin was assessed by immunofluorescence and epithelial barrier function by FITC-dextran diffusion. Inflammation: Acidosis led to an increase in COX-2 in NRK-52E and TNF in NRK-49F in monoculture. In co-culture, this effect was reversed. EMT: Acidosis led to an increase in vimentin protein in both cell lines, whereas in co-culture, the effect was abolished. In NRK-52E, the E-cadherin expression was unchanged, but subcellular E-cadherin showed a disturbed distribution, and cellular barrier function was decreased. Fibrosis: Monoculture acidosis led to an increased secretion of collagen I and fibronectin in NRK-52E and collagen I in NRK-49F. In co-culture, the total collagen I secretion was unchanged, and fibronectin secretion was decreased. Intercellular crosstalk between epithelial cells and fibroblasts has a protective function regarding the development of acidosis-induced damage.

Anagliptin promotes apoptosis in mouse colon carcinoma cells via MCT-4/lactate-mediated intracellular acidosis.

Cancer cells frequently exhibit an acidic extracellular microenvironment, where inversion of the transmembrane pH gradient is associated with tumor proliferation and metastasis. To elucidate a new therapeutic target against cancer, the current study aimed to determine the mechanism by which the dipeptidyl peptidase-4 inhibitor anagliptin regulates the cellular pH gradient and concomitant extracellular acidosis during cancer progression. A total of 5x105 CT-26 cells (resuspended in phosphate buffer saline) were injected subcutaneously in the right flank of male BALB/c mice (weighing 25-28 g). The tumor samples were harvested, and lactate was detected using a lactate assay kit. Immunohistochemistry was used to detect the Ki67 and PCNA. MTT assay and flow cytometric were used to detect cell viability. Intracellular pH was detected by fluorescence pH indicator. The results revealed that anagliptin effectively reduced tumor growth, but did not affect the body weight of treated mice. Anagliptin reduced the accumulation of lactate in tumor sample. Treatment with anagliptin stimulated the apoptosis of CT-26 cells. And lactate excretion inhibition is accompanied by an increase in extracellular pH (pHe) after treatment with anagliptin. Furthermore, anagliptin induced intracellular acidification and reversed the low pHe gradient via monocarboxylate transporter-4 (MCT-4)-mediated lactate excretion. Additionally, anagliptin reversed the aberrant transmembrane extracellular/intracellular pH gradient by suppressing MCT-4-mediated lactate excretion, while also reducing mitochondrial membrane potential and inducing apoptosis. These data revealed a novel function of anagliptin in regulating lactate excretion from cancer cells, suggesting that anagliptin may be used as a potential treatment for cancer.

Differential hERG 1 subunit transcription mediates IKr inhibition by protons in human stem cell-derived cardiomyocytes

hERG 1 variants are linked with intrauterine fetal death (IUD) and sudden infant death syndrome (SIDS), yet little is known about the mechanistic contribution of hERG 1 in developing and immature cardiomyocytes. Extracellular acidosis inhibits cardiac hERG 1 current (IKr), and disrupts cardiac function. Acidosis is also believed to be a contributing factor in cases of SIDS. We hypothesized that acidosis preferentially inhibits IKr of the immature myocardium, which could promote neonatal arrhythmia and sudden infant death.

Intelligent SERS Navigation System Guiding Brain Tumor Surgery by Intraoperatively Delineating the Metabolic Acidosis

Surgeons face challenges in intraoperatively defining margin of brain tumors due to its infiltrative nature. Extracellular acidosis caused by metabolic reprogramming of cancer cells is a reliable marker for tumor infiltrative regions. Although the acidic margin‐guided surgery shows promise in improving surgical prognosis, its clinical transition is delayed by having the exogenous probes approved by the drug supervision authority. Here, an intelligent surface‐enhanced Raman scattering (SERS) navigation system delineating glioma acidic margins without administration of exogenous probes is reported. With assistance of this system, the metabolites at the tumor cutting edges can be nondestructively transferred within a water droplet to a SERS chip with pH sensitivity. Homemade deep learning model automatically processes the Raman spectra collected from the SERS chip and delineates the pH map of tumor resection bed with increased speed. Acidity correlated cancer cell density and proliferation level are demonstrated in tumor cutting edges of animal models and excised tissues from glioma patients. The overall survival of animal models post the SERS system guided surgery is significantly increased in comparison to the conventional strategy used in clinical practice. This SERS system holds the promise in accelerating clinical transition of acidic margin‐guided surgery for solid tumors with infiltrative nature.

Effect of Acidosis-Induced Signalling Pathways on Mitochondrial O2 Consumption of Tumour Cells.

Signalling pathways such as ERK1/2, p38 or PI3K are activated in tumour cells by extracellular acidosis, which is a common phenomenon in human tumours. These signalling pathways can modulate the mitochondrial function and activity. The aim of the study was to evaluate the impact of extracellular acidosis on the mitochondrial O2 consumption and, in consequence, the potential role of ERK1/2, p38 and PI3K cascades on modulating the respiratory function. The O2 consumption rate (OCR) was measured at pH7.4 and extracellular acidosis (pH6.6) in combination with inhibition of the respective signalling pathway. The activity of the pathways was determined by phosphorylation-specific western blot using the cytosolic and mitochondrial fraction of cell lysates. The experiments were performed on a rat tumour cell line (subline AT1 of the rat R-3327 prostate carcinoma) and normal cells (NRK-49F fibroblasts). Acidosis increased the OCR of AT1 cells, especially the basal OCR and the O2 consumption, which is related to ATP production. In normal NRKF cells OCR was unaffected by low pH. Inhibition of ERK1/2 and PI3K, but not p38, reduced the acidosis-induced increase of the OCR significantly in AT1 tumour cells. In this cell line acidosis also led to an ERK1/2 and PI3K activation, predominantly in the mitochondrial fraction. These results indicate that extracellular acidosis activates cellular respiration in tumour cells, presumably by activating the ERK1/2 and/or the PI3K signalling cascade. This activation of ERK1/2 and PI3K is located primarily in the mitochondrial compartment of the cells.

Role of the mTOR Signalling Pathway During Extracellular Acidosis in Tumour Cells.

The metabolic microenvironment of solid tumours is often dominated by extracellular acidosis which results from glycolytic metabolism. Acidosis can modulate gene expression and foster the malignant progression. The aim of the study was to analyse the effects of extracellular acidosis on the mTOR signalling pathway, an important regulator of anabolic and catabolic processes like cell proliferation and autophagy. The study was performed in two tumour cell lines, AT-1 prostate and Walker-256 mammary carcinoma cells. Cells were incubated at pH7.4 or 6.6 for 3h and 24h. Then RNA and protein were extracted and analysed by qPCR and western blot. mTOR and P70-S6 kinase (P70-S6K), an important downstream target of mTOR, as well as the autophagic flux were studied. The effect of acidosis on P70S6K phosphorylation was compared to pharmacological mTOR inhibition with LY294002 and rapamycin. In both cell lines the total mTOR expression was not altered by acidosis, however, the mTOR phosphorylation was reduced after 3h but not after 24h. The P70S6K phosphorylation was reduced at both time points comparable to changes by pharmacological mTOR inhibitors. The autophagic flux, also a target of mTOR and measured by LC3-II expression, was increased in both cell lines after 24h of acidosis. The results of this study indicate that mTOR signalling is inhibited by extracellular acidosis which then lead to a reduced activity of the P70-S6 kinase (modulating gene expression) and increased autophagy possibly mediated by ULK1/2 activity. These finding may offer new perspectives for therapeutic interventions in acidic tumours.