Extracellular Acidity Research Articles

Abstract PO-049: Exploiting tumor acidic microenvironment for improved therapeutics

Abstract The tumor acidic microenvironment is a fundamental characteristic of solid tumors which distinguishes the tumor from adjacent normal tissues. This unique tumor microenvironment (TME) feature directly contributes to various aspect of tumor progression including gene expression, immune suppression, drug resistance, invasion and metastasis. Acidic TME poses great challenges for the efficacy of therapeutics; extracellular acidity of cancer cells provides less permissive conditions for optimal target engagement and drug efficacy. In addition targeting the effectors of acid-base balance enriched in hypoxia and acidic regions of the tumor provide valuable tools for normalizing the extracellular acidity and delivery of therapeutics agents to areas of the tumor less malleable to conventional therapies. Thus in the case of therapeutic antibodies, strategies that specifically integrates this TME specific feature in the design could improve binding to the target under low pH conditions and serve as delivery moiety that improve tumor specificity and functional efficacy. Here we describe two examples of these therapeutic strategies that leverage the acidic and hypoxic microenvironment to overcome the challenges posed by this hostile environment with the aim of generating improved therapeutic antibodies. A. Characterization of a set of function blocking therapeutic antibodies against key target expressed in response to hypoxia which contributes to tumor acidification, we demonstrate functional efficacy of this antibody to block the activity of the target in vitro and consequently reduce tumor spheroid growth. This and similar antibodies provide tools for specific targeting of the tumor areas that are often inaccessible to most therapeutics. B. Identification of targets enriched in acidic tumor microenvironment and design of pH selective therapeutic antibodies that preferentially bind to the target and exert their function under tumor acidic conditions. We demonstrate the functional selectivity of a variant of antibody targeting HER 2, with optimized binding under low pH conditions, on the growth of BT474 spheroids. The pH selective antibody variant blocked the tumor spheroid growth when spheroids were grown in TME-relevant- low pH conditions but it remained ineffective under normal physiological pH conditions. Suggesting increased selectivity of this antibody variant towards the acidic TME that in turn lower the on-target -off-tumor toxicity. In conclusion here we provide two examples of the strategies which exploit the targeting of hypoxia induced genes and pH selective design of the therapeutic antibodies in order to improve tumor targeting capacity and reduce systemic toxicity of conventional therapeutics. Citation Format: Nazanin Rohani Larijani, Traian Sulea, Mehdi Arbabi Ghahroudi, Beatrice Paul Roc, Mylene Gosselin, Joey Sheff, John C. Zwaagstra, Anne E.G. Lenferink. Exploiting tumor acidic microenvironment for improved therapeutics [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-049.

Lapatinib-loaded acidity-triggered charge switchable polycarbonate-doxorubicin conjugate micelles for synergistic breast cancer chemotherapy

Stimulus-responsive nanosystem is a powerful method to improve the bioavailability and reduce the side effects of anticancer agents. In the present study, a customized dual pH-responsive micellar nanoplatform (DOX+LAP-M) based on polycarbonate-doxorubicin conjugate micelles was prepared to co-deliver the chemotherapeutic agent lapatinib for inhibiting tumor growth and metastasis. DOX+LAP-M micelles with spherical morphology had a size of ~112 nm and had an initial negative surface charge, which are favorable characteristics for long-term circulation in the blood. Once the micelles accumulated in tumor tissues, the intrinsic tumor extracellular acidity triggered the charge switch of DOX+LAP-M micelles from -1 to 9 mV, thereby facilitating cell internalization and tumor penetration. Subsequently, the pH-sensitive micellar core accelerated the release of doxorubicin and lapatinib in the acidic intracellular environment. DOX+LAP-M micelles effectively inhibited the proliferation, migration, and invasion of 4T1 cells in vitro; furthermore, the administration of DOX+LAP-M micelles in 4T1 xenograft-bearing mice suppressed solid tumor growth with an inhibitory rate of 90.2% and significantly decreased pulmonary metastatic nodules, without significant systemic toxicity. This multifunctional micellar system has high potential for clinical cancer therapy.

A Sequentially Responsive Nanosystem Breaches Cascaded Bio-barriers and Suppresses P-Glycoprotein Function for Reversing Cancer Drug Resistance.

Cancer chemotherapy is challenged by multidrug resistance (MDR) mainly attributed to overexpressed transmembrane efflux pump P-glycoprotein (P-gp) in cancer cells. Improving drug delivery efficacy while co-delivering P-gp inhibitors to suppress drug efflux is an often-used nanostrategy for combating MDR, which is however challenged by cascaded bio-barriers en route to cancer cells and P-gp inhibitors' adverse effects. To effectively breach the cascaded bio-barriers while avoiding P-gp inhibitors' adverse effects, a stealthy, sequentially responsive doxorubicin (DOX) delivery nanosystem (RCMSNs) is fabricated, composed of an extracellular-tumor-acidity-responsive polymer shell (PEG-b-PLLDA), pH/redox dual-responsive mesoporous silica nanoparticle-based carriers (MSNs-SS-Py), and cationic β-cyclodextrin-PEI (CD-PEI) gatekeepers. The PEG-b-PLLDA corona makes RCMSNs stealthy with prolonged blood circulation time. Once tumors are reached, extracellular acidity degrades PEG-b-PLLDA, reversing nanosystem's surface charges to be positive, which drastically improves RCMSNs' tumor accumulation, penetration, and cellular internalization. Within cancer cells, CD-PEI gatekeepers detach to allow DOX unloading in response to intracellular acidity and glutathione and functionally act as a P-gp inhibitor, dampening P-gp's efflux activity by impairing ATP production. Thus, the resultant high-efficacy drug delivery along with reduced P-gp function cooperatively reverses MDR in vitro. Importantly, in preclinical tumor models, DOX@RCMSNs potently suppress MDR tumor growth without eliciting systemic toxicity, demonstrating their potential of clinical translation.

The control of acidity in tumor cells: a biophysical model

Acidosis of the tumor microenvironment leads to cancer invasion, progression and resistance to therapies. We present a biophysical model that describes how tumor cells regulate intracellular and extracellular acidity while they grow in a microenvironment characterized by increasing acidity and hypoxia. The model takes into account the dynamic interplay between glucose and hbox {O}_2 consumption with lactate and hbox {CO}_2 production and connects these processes to hbox {H}^+ and hbox {HCO}_3^- fluxes inside and outside cells. We have validated the model with independent experimental data and used it to investigate how and to which extent tumor cells can survive in adverse micro-environments characterized by acidity and hypoxia. The simulations show a dominance of the hbox {H}^+ exchanges in well-oxygenated regions, and of hbox {HCO}_3^- exchanges in the inner hypoxic regions where tumor cells are known to acquire malignant phenotypes. The model also includes the activity of the enzyme Carbonic Anhydrase 9 (CA9), a known marker of tumor aggressiveness, and the simulations demonstrate that CA9 acts as a nonlinear hbox {pH}_i equalizer at any hbox {O}_2 level in cells that grow in acidic extracellular environments.

PH/Cathepsin B Hierarchical‐Responsive Nanoconjugates for Enhanced Tumor Penetration and Chemo‐Immunotherapy

AbstractAn ideal cancer nanomedicine should precisely deliver therapeutics to its intracellular target within tumor cells. However, the multiple biological barriers seriously hinder their delivery efficiency, leading to unsatisfactory therapeutic outcome. Herein, pH/cathepsin B hierarchical‐responsive nanoconjugates (HRNs) are reported to overcome these barriers by sequentially responding to extra‐ and intracellular stimuli in solid tumors for programmed delivery of docetaxel (DTX). The HRNs have stable nanostructures (≈40 nm) in blood circulation for efficient tumor accumulation, while the tumor extracellular acidity induces the rapid dissociation of HRNs into polymer conjugates (≈5 nm), facilitating the deep tumor penetration and cellular internalization. After being trapped into the lysosomes, the conjugates are cleaved by cathepsin B to release bioactive DTX into cytoplasm and inhibit cell proliferation. In addition to the direct inhibition effect, HRNs can trigger the in vivo antitumor immune responses via the immunogenic modulation of tumor cells, activation of dendritic cells (DCs), and generation of cytotoxic T‐cell responses. By employing a combination with α‐PD‐1 (programmed cell death 1) therapy, synergistic antitumor efficacy is achieved in B16 expressing ovalbumin (B16OVA) tumor model. Hence, this strategy demonstrates high efficiency for precise intracellular delivery of chemotherapeutics and provides a potential clinical candidate for cancer chemo‐immunotherapy.

Human IDH mutant 1p/19q co-deleted gliomas have low tumor acidity as evidenced by molecular MRI and PET: a retrospective study

Co-deletion of 1p/19q is a hallmark of oligodendroglioma and predicts better survival. However, little is understood about its metabolic characteristics. In this study, we aimed to explore the extracellular acidity of WHO grade II and III gliomas associated with 1p/19q co-deletion. We included 76 glioma patients who received amine chemical exchange saturation transfer (CEST) imaging at 3 T. Magnetic transfer ratio asymmetry (MTRasym) at 3.0 ppm was used as the pH-sensitive CEST biomarker, with higher MTRasym indicating lower pH. To control for the confounder factors, T2 relaxometry and l-6-18F-fluoro-3,4-dihydroxyphenylalnine (18F-FDOPA) PET data were collected in a subset of patients. We found a significantly lower MTRasym in 1p/19q co-deleted gliomas (co-deleted, 1.17% ± 0.32%; non-co-deleted, 1.72% ± 0.41%, P = 1.13 × 10−7), while FDOPA (P = 0.92) and T2 (P = 0.61) were not significantly affected. Receiver operating characteristic analysis confirmed that MTRasym could discriminate co-deletion status with an area under the curve of 0.85. In analysis of covariance, 1p/19q co-deletion status was the only significant contributor to the variability in MTRasym when controlling for age and FDOPA (P = 2.91 × 10−3) or T2 (P = 8.03 × 10−6). In conclusion, 1p/19q co-deleted gliomas were less acidic, which may be related to better prognosis. Amine CEST-MRI may serve as a non-invasive biomarker for identifying 1p/19q co-deletion status.

Reduced Nhe1 (Na+-H+ Exchanger-1) Function Protects ApoE-Deficient Mice From Ang II (Angiotensin II)-Induced Abdominal Aortic Aneurysms.

IgE-mediated activation of Nhe1 (Na+-H+ exchanger-1) induces aortic cell extracellular acidification and promotes cell apoptosis. A pH-sensitive probe pHrodo identified acidic regions at positions of macrophage accumulation, IgE expression, and cell apoptosis in human and mouse abdominal aortic aneurysm (AAA) lesions. Ang II (angiotensin II)-induced AAA in Nhe1-insufficient Apoe-/-Nhe1+/- mice and Apoe-/-Nhe1+/+ littermates tested Nhe1 activity in experimental AAA, because Nhe1-/- mice develop ataxia and epileptic-like seizures and die early. Nhe1 insufficiency reduced AAA incidence and size, lesion macrophage and T-cell accumulation, collagen deposition, elastin fragmentation, cell apoptosis, smooth muscle cell loss, and MMP (matrix metalloproteinase) activity. Nhe1 insufficiency also reduced blood pressure and the plasma apoptosis marker TCTP (translationally controlled tumor protein) but did not affect plasma IgE. While pHrodo localized the acidic regions to macrophage clusters, IgE expression, and cell apoptosis in AAA lesions from Apoe-/-Nhe1+/+ mice, such acidic areas were much smaller in lesions from Apoe-/-Nhe1+/- mice. Nhe1-FcεR1 colocalization in macrophages from AAA lesions support a role of IgE-mediated Nhe1 activation. Gelatin zymography, immunoblot, and real-time polymerase chain reaction analyses demonstrated that Nhe1 insufficiency reduced the MMP activity, cysteinyl cathepsin expression, IgE-induced apoptosis, and NF-κB activation in macrophages and blocked IgE-induced adhesion molecule expression in endothelial cells. A near-infrared fluorescent probe (LS662) together with fluorescence reflectance imaging of intact aortas showed reduced acidity in AAA lesions from Nhe-1-insufficient mice. This study revealed extracellular acidity at regions rich in macrophages, IgE expression, and cell apoptosis in human and mouse AAA lesions and established a direct role of Nhe1 in AAA pathogenesis.

Curcumin circumvent lactate-induced chemoresistance in hepatic cancer cells through modulation of hydroxycarboxylic acid receptor-1.

Curcumin has been demonstrated to affect the chemoresistance in cancer cells of various origins. However, its ability to modulate lactate-induced chemoresistance remains unclear. The Present investigation demonstrates that curcumin inhibits the survival of HepG2 and HuT78 cells and can modulate chemo-susceptibility of HepG2 cells. Experimental simulation of simultaneous and pre-treatment suggest cooperatively between curcumin and anticancer drugs as well as the modulation of molecular regulators. Inhibition of glucose consumption, lactate production, extracellular acidity and augmented level of Nitric oxide were observed. DAPI staining revealed hyper condensation of chromatin in curcumin-treated HepG2 cells. Curcumin also diminished the lactate-induced chemoresistance against doxorubicin in hepatic cancer cells along with down regulation of lactate receptor (hydroxycarboxylic acid receptor-1; HCAR-1/GPR81). Alteration of the extracellular milieu along with inhibited expression of genes (hif-1α, ldh-a, mct-1, mdr-1 and stat-3) and proteins (HIF-1α and HCAR-1) are indicated to be involved in curcumin-induced reversal of chemoresistance in HepG2 cells. Findings of present investigation contribute to knowledge of curcumin mediated chemosensitization and its mechanism.

Upregulation of G protein‐couple receptor 68 (GPCR68) in pulmonary artery smooth muscle cells from animals and humans with pulmonary hypertension

IntroductionPulmonary arterial hypertension (PAH) is a progressive and fatal disease with no cure. It is characterized by elevated pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP) in part due to pulmonary vascular remodeling and sustained pulmonary vasoconstriction. The increased pulmonary arterial smooth muscle cell (PASMC) proliferation contributes to excessive pulmonary vascular remodeling while a rise in cytosolic free Ca2+concentration ([Ca2+]cyt) in PASMC is a major trigger for pulmonary vasoconstriction and a key stimulus for PASMC proliferation. G‐protein coupled receptors (GPCR), which are attractive pharmacological targets, are important regulators of pulmonary vascular tone and PASMC phenotype. Stimulation of GPCR leads to Ca2+ influx and increased [Ca2+]cytdue to store‐operated Ca2+ entry (SOCE) via inositol 1,4,5‐trisphosphate (IP3) production and receptor‐operated Ca2+entry (ROCE) via diacylglycerol (DAG) production. GPCR68 (or OGR1) is a proton‐sensing GPCR that responds to extracellular acidity and regulates a variety of cellular functions. As a Gq/11‐coupled receptor, GPR68 activation is known to lead to Ca2+ release from the intracellular stores in IP3‐dependent manner but the role of GPCR68 in the development of PAH is still unknown.MethodsLung, heart, brain, liver, and kidney tissues were isolated from control rats. Pulmonary hypertension model was induced by a single dose of monocrotaline (MCT) in rats. PASMC isolated from control or MCT rats and healthy subjects or patients with idiopathic pulmonary arterial hypertension (IPAH) were cultured in 5% fetal bovine serum (FBS) smooth muscle growth media and incubated in a humidified 5% CO2 atmosphere at 37°C. Total RNA was isolated from tissue or cells using TRIZOL method. mRNA expression of GPCR68 was quantified by RT‐PCR and qRT‐PCR. The data was analyzed using ImageJ and statistical analysis was performed using paired or unpaired Student’s t‐test.ResultsRT‐PCR analysis revealed that GPCR68 is expressed in brain, lung, heart, liver, and kidney in normal rat tissues (n=5). GPCR68 mRNA level was significantly higher in lung tissue and PASMC isolated from MCT‐treated rats compared with controls. Moreover, our data shows that GPCR68 is expressed in both human PASMC and pulmonary arterial endothelial cells. qRT‐PCR showed that GPCR68 was upregulated in PASMC isolated from IPAH patients compared with cells isolated from healthy subjects.ConclusionOur finding suggest that GPR68 is involved in the development and progression of PAH and may play an important role in Ca2+ signaling and enhanced [Ca2+]cyt in PASMC from animals and humans with PAH.Support or Funding InformationThis work was supported, in part, by grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (HL125208, HL135807, and HL126609).

Extracellular acidity‑induced expression of Kallikrein‑related peptidases 7 and 8 is involved in increased invasiveness of gastric cancer cells.

In several cancers, the acidic microenvironment of cancer cells has been implicated in enhanced malignancy and metastasis. In the present study, it was observed that gastric cancer cell lines, SNU601 and AGS, exposed to an acidic medium had increased invasiveness, as assessed using Matrigel‑coated Transwell analysis. The factors regulating such acidity‑mediated enhancement of invasiveness were investigated and it was revealed that a low‑pH environment markedly increased kallikrein‑related peptidase7 (KLK7) and kallikrein‑related peptidase8 (KLK8) expression. Gene silencing assays confirmed that these peptidases were involved in acidity‑promoted invasion. Acidic conditions also increased the expression of cyclooxygenases (COX), key regulatory enzymes in the catalytic pathway of prostaglandin production. Notably, these enzymes appeared to be involved in the acidity‑mediated expression of KLK7 and KLK8, as revealed using COX inhibitors. Therefore, it was indicated that tumor invasion enhancement by extracellular acidity is regulated at least in part through the induction of the COX/KLK7 and KLK8 axis in gastric cancer cells.

Genetic Encoding of Targeted Magnetic Resonance Imaging Contrast Agents for Tumor Imaging.

Tumor-selective contrast agents have the potential to aid in the diagnosis and treatment of cancer using noninvasive imaging modalities such as magnetic resonance imaging (MRI). Such contrast agents can consist of magnetic nanoparticles incorporating functionalities that respond to cues specific to tumor environments. Genetically engineering magnetotactic bacteria to display peptides has been investigated as a means to produce contrast agents that combine the robust image contrast effects of magnetosomes with the transgenic-targeting peptides displayed on their surface. This work reports the first use of magnetic nanoparticles that display genetically encoded pH low insertion peptide (pHLIP), a long peptide intended to enhance MRI contrast by targeting the extracellular acidity associated with the tumors. To demonstrate the modularity of this versatile platform to incorporate diverse targeting ligands by genetic engineering, we also incorporated the cyclic αv integrin-binding peptide iRGD into separate magnetosomes. Specifically, we investigate their potential for enhanced binding and tumor imaging both in vitro and in vivo. Our experiments indicate that these tailored magnetosomes retain their magnetic properties, making them well suited as T2 contrast agents, while exhibiting an increased binding compared to the binding in wild-type magnetosomes.

Immunocapture-based ELISA to characterize and quantify exosomes in both cell culture supernatants and body fluids.

The immunocapture-based ELISA for extracellular vesicles (EVs)/exosomes, originally described in 2009 by Logozzi and colleagues, allows to capture, detect, characterize and quantify extracellular vesicles in both human body fluids and cell culture supernatants. It is based on the use of two antibodies directed one against a typical exosomal housekeeping protein and the second against either another exosomal housekeeping protein or a potential disease marker: the first antibody is used for the capture of exosomes, the second for the quantification and characterization of the captured vesicles. In fact, with this method it is possible both to characterize and count exosomes and to detect the presence of disease, including tumor, biomarkers. This needs of course to preliminary obtain an EVs purification from the clinical sample; the most agreed method to get to an EVs purification is the repeated rounds of ultracentrifugation, that, while far to be perfect, is the methodological approach allowing to not exclude EVs subpopulation from the separation procedure and to analyze a full range of EVs from both qualitative and quantitative point of view. The immunocapture-based approach has proven to be highly useful in screening, diagnosis and prognosis of tumors, in plasma samples. One amazing information provided by this method is that cancer patients have always significantly higher levels of EVs, in particular of exosomes, independently from the histological nature of the tumor. One microenvironmental factor that is fully involved in the increased exosome release by tumors is the extracellular acidity. However, few pre-clinical data suggest that plasmatic levels of exosomes may correlate with the tumor mass. Some recent clinical reports suggest also that circulating exosomes represent the real delivery system for some known tumor markers that are presently on trial (e.g., PSA). Here we review the pros and cons of the immunocapture-based technique in quantitative and qualitative evaluation of EVs in both health and disease.

A reciprocal feedback of Myc and lncRNA MTSS1-AS contributes to extracellular acidity-promoted metastasis of pancreatic cancer.

Rationale: Previous studies have reported on the role of extracellular acidity in the metastasis of numerous cancers. However, the involvement of long noncoding RNA (lncRNA) in the extracellular acidity-induced cancer metastasis of pancreatic cancer (PC) remains unclear.Methods: Different expression levels of lncRNAs in PC cells under normal and acidic conditions were compared by RNA sequencing (RNA-seq). The effects of antisense lncRNA of metastasis suppressor 1 (MTSS1-AS) on acidic PC cells were assessed by gain- and loss-of-function experiments. Fluorescence in situ hybridization, RNA immunoprecipitation, RNA pull-down, Western blot, luciferase reporter, and Chromatin immunoprecipitation assays were employed to determine the regulatory mechanisms of MTSS1-AS in the acidity-induced metastasis of PC cells. The expression of MTSS1-AS and associated pathways were compared in PC samples and peritumoral normal tissues.Results: RNA-seq demonstrated that MTSS1-AS was significantly downregulated in pancreatic cells cultured with the acidic medium. The overexpression of MTSS1-AS remarkably inhibited the acidity-promoted metastasis of PC cells by upregulating the expression of its sense gene metastasis suppressor 1 (MTSS1). Mechanistically, MTSS1-AS scaffolded the interaction between E3 ubiquitin-protein ligase STIP1 homology and U-box containing protein 1 (STUB1) and transcription regulator myeloid zinc finger 1 (MZF1), leading to ubiquitination-mediated degradation of MZF1. Further, MZF1 inhibited the expression of MTSS1 by binding to the MTSS1 promoter. Thus, the acidity-reduced MTSS1-AS facilitated the stability of MZF1 and its inhibitory effect on MTSS1 transcription, thereby promoting the metastasis of PC cells under acidic conditions. Moreover, MTSS1-AS was transcriptionally repressed by the binding of MYC proto-oncogene (Myc) with initiator (Inr) elements of the MTSS1-AS promoter. Meanwhile, MTSS1-AS mutually repressed the expression of Myc by impairing the MZF1-mediated transcription activation of Myc, thereby forming a negative feedback loop between MTSS1-AS and Myc in acidic PC cells. In accordance with the experimental results, MTSS1-AS and MTSS1 were downregulated in PC and correlated with poor overall survival.Conclusions: The results implicated that a reciprocal feedback loop between Myc and MTSS1-AS contributed to the extracellular acidity-promoted metastasis of PC, and indicated that MTSS1-AS was a valuable biomarker and therapeutic target for PC.

A pH-Responsive Charge-Reversal Drug Delivery System with Tumor-Specific Drug Release and ROS Generation for Cancer Therapy.

IntroductionPoor cell uptake and incomplete intracellular drug release are the two major challenges for polymeric prodrug-based drug delivery systems (PPDDSs) in cancer treatment.MethodsHerein, a PPDDS with pH-induced surface charge-reversal and reactive oxygen species (ROS) amplification for ROS-triggered self-accelerating drug release was developed, which was formed by encapsulating a ROS generation agent (vitamin K3 (VK3)) in pH/ROS dual-sensitive polymetric prodrug (PEG-b-P(LL-g-TK-PTX)-(LL-g-DMA)) based micelle nanoparticles (denoted as PVD-NPs).ResultsThe surface charge of the PVD-NPs can change from negative to positive for enhanced cell uptake in response to tumor extracellular acidity pH. After internalization by cancer cells, PVD-NPs demonstrate dual drug release in response to intracellular ROS-rich conditions. In addition, the released VK3 can produce ROS under the catalysis by NAD(P)H:quinone oxidoreductase-1, which facilitates tumor-specific ROS amplification and drug release selectively in cancer cells to enhance chemotherapy.ConclusionBoth in vitro and in vivo experiments demonstrated that the PVD-NPs showed significant antitumor activity in human prostate cancer.

Ellagic Acid Inhibits Extracellular Acidity-Induced Invasiveness and Expression of COX1, COX2, Snail, Twist 1, and c-myc in Gastric Carcinoma Cells.

Extracellular acidity has been implicated in enhanced malignancy and metastatic features in various cancer cells. Gastric cancer cell lines (AGS and SNU601) maintained in an acidic medium have increased motility and invasiveness. In this study, we investigated the effect of ellagic acid, a plant-derived phenolic compound, on the acidity-promoted migration and invasion of gastric cancer cells. Treating cells maintained in acidic medium with ellagic acid inhibited acidity-mediated migration and invasion, and reduced the expression of MMP7 and MMP9. Examining regulatory factors contributing to the acidity-mediated invasiveness, we found that an acidic pH increased the expression of COX1 and COX2; importantly, expression decreased under the ellagic acid treatment. The general COX inhibitor, sulindac, also decreased acidity-mediated invasion and expression of MMP7 and MMP9. In addition, acidity increased the mRNA protein expression of transcription factors snail, twist1, and c-myc; these were also reduced by ellagic acid. Together, these results suggest that ellagic acid suppresses acidity-enhanced migration and invasion of gastric cancer cells via inhibition of the expression of multiple factors (COX1, COX2, snail, twist1, and c-myc); for this reason, it may be an effective agent for cancer treatment under acidosis.

Amorphous Fe‐Based Nanoagents for Self‐Enhanced Chemodynamic Therapy by Re‐Establishing Tumor Acidosis

AbstractChemodynamic therapy (CDT) by introducing the Fenton‐/Fenton‐like reaction in an acidic and H2O2 environment for toxic hydroxyl radical (•OH) generation, is a newly developed tumor‐selective therapeutic. However, tumor acidosis, characterized by extracellular acidity (pHe ≈ 6.5) and intracellular alkalinity (pHi ≈ 7.2), undoubtedly confers a large chemical barrier for effective implementation of intracellular CDT and thus limits its functional activity and therapeutic efficacy. Here, the unique amorphous iron nanoparticles (AFeNPs) loaded with carbonic anhydrase IX inhibitor (CAI) are constructed to re‐establish tumor acidosis with decreased pHi and increased pHe via inhibiting the over‐expressed CA IX in cancer cells by CAI for self‐enhanced CDT. The suppression of CA IX leads to H+ accumulation in cells that could accelerate the AFeNPs‐based Fenton reaction to drastically exacerbate oxidative stress in cells and subsequently induce cell death; meanwhile, the inhibition of H+ formation outside cells efficiently represses the potential of tumor invasion and metastasis owing to the insufficient acidic ions for degradation of tumor extracellular matrix. Re‐established tumor acidosis not only assists in the optimization of CDT, but also presents an opportunity for the development of new antitumor methods that are more tumor‐acidity specific.

The Acidic Tumor Microenvironment as a Driver of Cancer.

Acidic metabolic waste products accumulate in the tumor microenvironment because of high metabolic activity and insufficient perfusion. In tumors, the acidity of the interstitial space and the relatively well-maintained intracellular pH influence cancer and stromal cell function, their mutual interplay, and their interactions with the extracellular matrix. Tumor pH is spatially and temporally heterogeneous, and the fitness advantage of cancer cells adapted to extracellular acidity is likely particularly evident when they encounter less acidic tumor regions, for instance, during invasion. Through complex effects on genetic stability, epigenetics, cellular metabolism, proliferation, and survival, the compartmentalized pH microenvironment favors cancer development. Cellular selection exacerbates the malignant phenotype, which is further enhanced by acid-induced cell motility, extracellular matrix degradation, attenuated immune responses, and modified cellular and intercellular signaling. In this review, we discuss how the acidity of the tumor microenvironment influences each stage in cancer development, from dysplasia to full-blown metastatic disease.

Extracellular pH modulating injectable gel for enhancing immune checkpoint inhibitor therapy

Clinical data from diverse cancer types shows that the increased T cell infiltration in tumors correlates with improved patient prognosis. Acidic extracellular pH is a major attribute of the tumor microenvironment (TME) that promotes immune evasion and tumor progression. Therefore, antagonizing tumor acidity can be a powerful approach in cancer immunotherapy. Here, Pluronic F-127 is used as a NaHCO3 releasing carrier to focally alleviate extracellular tumor acidity. In a mouse tumor model, intratumoral treatment with pH modulating injectable gel (pHe-MIG) generates immune-favorable TME, as evidenced by the decrease of immune-suppressive cells and increase of tumor infiltrating CD8+T cells. The combination of pHe-MIG with immune checkpoint inhibitors, anti-PD-1 and anti-TIGIT antibodies, increases intratumoral T cell function, which leads to tumor clearance. Mechanistically, extracellular acidity was shown to upregulate co-inhibitory immune checkpoint receptors and inhibit mTOR signaling pathways in memory CD8+T cells, which impaired effector functions. Furthermore, an acidic pH environment increased the expression and engagement of TIGIT and its ligand CD155, which suggested that the extracellular pH can regulate the suppressive function of TIGIT pathway. Collectively, these findings suggest that pHe-MIG holds potential as a new TME modulator for effective immune checkpoint inhibitor therapies.

Carbonic anhydrase 9 confers resistance to ferroptosis/apoptosis in malignant mesothelioma under hypoxia

Hypoxia and acidity provide microenvironment for selection under evolutionary pressure and proliferation in cancer cells. Carbonic anhydrases (CAs) are a superfamily of metalloenzymes present in all life kingdoms, equilibrating the reactions among CO2, bicarbonate and H+. CA9, a membrane-associated α-CA, has been a drug target for various cancers. Whereas iron is essential not only for cancer cells but also for all the lives on earth, little is known on the association among hypoxia, iron metabolism, extracellular acidity and redox regulation. Malignant mesothelioma (MM), an aggressive tumor with poor prognosis, is an intriguing model in that asbestos-associated pathogenesis includes excess iron environment during carcinogenesis. Re-analysis of rat asbestos-induced MM model revealed an inverse association between high CA9 expression and survival. Here we used human MMs to identify the molecular events surrounding CA9 from the viewpoint of iron metabolism. CA9 expression was significantly higher in MM cells than in MeT-5A mesothelial cells, which was further amplified under hypoxia (1%O2) with increased catalytic Fe(II). CA9 suppression by inhibitors (S4 and U104) decreased viability and migration of MM cells, accompanied by overexpression of TFRC, IREB1/2 and FPN1(SLC40A1) and by downregulation of FTH/FTL. This expressional pattern was similar to that of erastin-induced ferroptosis in the same cells. Furthermore, we observed mitochondrial fission and enhanced autophagy with increased catalytic Fe(II) in both mitochondria and lysosomes after CA9 inhibition, accompanied by increased peroxides, mitochondrial O2− and lipid peroxidation. The eventual cell death was significantly inhibited by deferoxamine, ferrostatin-1 and Z-VAD-FMK, suggesting a mixed cell death of ferroptosis and apoptosis. Therefore, CA9 plays a role in equilibrating among hypoxia, iron metabolism and redox regulation in MM cells.

The Role of Sodium Hydrogen Exchanger 1 in Dysregulation of Proton Dynamics and Reprogramming of Cancer Metabolism as a Sequela

Cancer cells have an unusual regulation of hydrogen ion dynamics that are driven by poor vascularity perfusion, regional hypoxia, and increased glycolysis. All these forces synergize/orchestrate together to create extracellular acidity and intracellular alkalinity. Precisely, they lead to extracellular pH (pHe) values as low as 6.2 and intracellular pH values as high as 8. This unique pH gradient (∆pHi to ∆pHe) across the cell membrane increases as the tumor progresses, and is markedly displaced from the electrochemical equilibrium of protons. These unusual pH dynamics influence cancer cell biology, including proliferation, metastasis, and metabolic adaptation. Warburg metabolism with increased glycolysis, even in the presence of Oxygen with the subsequent reduction in Krebs’ cycle, is a common feature of most cancers. This metabolic reprogramming confers evolutionary advantages to cancer cells by enhancing their resistance to hypoxia, to chemotherapy or radiotherapy, allowing rapid production of biological building blocks that support cellular proliferation, and shielding against damaging mitochondrial free radicals. In this article, we highlight the interconnected roles of dysregulated pH dynamics in cancer initiation, progression, adaptation, and in determining the programming and re-programming of tumor cell metabolism.