Mechanisms, consequences and regulation of pH non-uniformity in 3-D tissue structures: implications on cancer therapy

Lead Research Organisation: University of Oxford
Department Name: Physiology Anatomy and Genetics

Abstract

Our aim is to study the mechanisms that regulate the acid/base balance (pH) in multi-cellular tissue, with particular emphasis on tumours.
Living cells produce acid, an end-product of metabolism. Acid must be vented out of cells, to the nearest blood capillary, in order to maintain an intracellular pH (pHi) that is permissive for healthy cell function. Efficient venting of acid is also necessary to maintain uniform pHi across multi-cellular tissue. This is particularly critical for under-perfused and metabolically-active tissues, such as developing tumours, where variable cell-to-capillary diffusion distances predispose to pHi-gradients and core-acidity.
Although significant progress has been made in the field of pHi-regulation in isolated cells, little is known about the integrated mechanisms that operate at tissue-level. Molecules likely to contribute to the latter include pH buffers, carbonic anhydrase enzymes, gap junction channels and acid/base membrane transporters. We will investigate these mechanisms using molecular biology, pH imaging and computational modelling. The research will use large, three-dimensional clusters (‘spheroids’) of cancer-derived cells as an in vitro model of developing tumours.
The good survival prospects of cancer may suggest that efficient mechanisms for venting acid are implemented in tumours. Inhibiting these mechanisms may offer novel therapeutic strategies for treating cancer. Manipulation of pH within and outside cells may also affect the cellular uptake of certain weakly basic drugs that are currently used in chemotherapy.

Technical Summary

All respiring cells produce acid (H+) which must ultimately be vented into the blood stream. Metabolic rate and cell-capillary distances can vary within a tissue. In addition, H+ ions diffuse very slowly in the presence of buffers, particularly high-molecular-weight proteins. Consequently, gradients of intra- and extracellular pH (pHi and pHe) can form, with acid accumulating at the tissue-core. Since H+ ions are key modulators of virtually all biochemical reactions, acid-displacements of pHi can have a major impact of cell function and growth, and may even trigger cell death. In adequately-perfused tissue, diffusional distances are sufficiently small to maintain pH uniformity, albeit shallow pH gradients. If capillary perfusion fails (e.g. in ischaemia) or is inadequate in relation to the metabolic rate (e.g. in cancer), pH gradients may become large and may be accompanied by pockets of hypoxia. This can lead to regional loss of function. Tumours can grow and respire at a rate that would predict considerable core-acidosis. Nonetheless, tumours can maintain integrity and thrive. Mechanisms of spatial pHi regulation across 3-D structures are not known. Pilot data suggests that three components may play a role in coordinating pHi across large cancer growths. These are (i) transmembrane flux of HCO3-, a mobile buffer, on secondary-active transporters (ii) facilitation of CO2 removal by extracellular-facing carbonic anhydrase (e.g. CA9), an enzyme and (iii) attainment of a pHi-syncitium by dissipating pHi non-uniformity through gap junctions, i.e. inter-cellular channels. The importance of these mechanisms in regulating pHi spatially will be studied in unvascularised, 3-D clusters of cells ( spheroids ) grown from tumour-derived cells that have been genetically-modified to express different combinations of proteins. The beneficial pH-unifying mechanisms operating in cancer may be targets for anti-cancer therapy. Conversely, an understanding of these adaptations can be helpful in identifying strategies to salvage other tissue such as the myocardium from ischaemic damage.

Publications

10 25 50

publication icon
Hulikova A (2013) Regulation of intracellular pH in cancer cell lines under normoxia and hypoxia. in Journal of cellular physiology

publication icon
McIntyre A (2012) Carbonic anhydrase IX promotes tumor growth and necrosis in vivo and inhibition enhances anti-VEGF therapy. in Clinical cancer research : an official journal of the American Association for Cancer Research

publication icon
Murri-Plesko MT (2011) Antibody inhibiting enzymatic activity of tumour-associated carbonic anhydrase isoform IX. in European journal of pharmacology

 
Description CRUK Development Fund
Amount £7,500 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2011 
End 07/2012
 
Description John Fell OUP Equipment fund
Amount £50,000 (GBP)
Organisation University of Oxford 
Department John Fell Fund
Sector Academic/University
Country United Kingdom
Start 10/2010 
End 09/2011
 
Description Marie Curie Initial Training Network
Amount £234,000 (GBP)
Organisation Marie Sklodowska-Curie Actions 
Sector Academic/University
Country Global
Start 01/2012 
End 12/2015
 
Description Project grant
Amount £191,000 (GBP)
Organisation Association for International Cancer Research 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2012 
End 12/2014
 
Description Royal Society Research Grant (equipment)
Amount £50,000 (GBP)
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 04/2010 
End 03/2012
 
Title Analysing tissue metabolism by imaging extracellular pH in multicellular tissue-models 
Description In many cellular physiology studies, extracellular pH (pHe) has been clamped and constant due to the design of experiments (superfusion, well-stirred chambers, large extracellular volume). We have implemented a novel, membrane-impermeant fluorescent dye (fluorescein sulphonic acid; PMID: 19458084) to measure pHe in the restricted extracellular space of 3-D multicellular tumour models (spheroids). The pHe gradients measured under different conditions (enzyme/transporter activity, diffusion distance, oxygen levels) can be analysed in terms of cellular metabolic output, to complement intracellular pH measurements. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Most studies of pH regulation have focused on intracellular pH control in single, isolated cells. At tissue-level, regulation of extracellular pH is also of great importance. In tumours, pHe is a major regulator of invasiveness and a marker of aggressiveness. In addition, low pHe has been proposed to exert selection-pressure against non-cancer cells. Many enzymes (e.g. CAIX) and many transporters are sensitive to pHe. Our imaging technology will allow researchers to extend pH studied to the extracellular compartment of tissue. 
URL http://europepmc.org/abstract/MED/19458084
 
Title Chemical assay for histidine-containing dipeptides 
Description H+ ions (commonly quantified in pH units) are major modulators of function. However, in the highly-buffered intracellular environment, H+ ions diffuse very slowly. To facilitate H+ ion mobility (and ensure good diffusive coupling and hence H+ ion uniformity), H+ ion diffusion must be facilitated by small buffer molecules. Histidine-containing dipeptides (HCDP), such as carnosine and anserine, are a family of intracellular small molecules that can facilitate H+ ion diffusion. We have developed a quantitative assay for these 'H+ ion shuttles'. Previously assays have been inaccurate due to contamination from substances such as taurine that do not shuttle H+ ions. See publication 19828673. 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact We have shown that in the heart, a tissue that relies critically on good pH control, there is a developmental upregulation of HCDP levels (neonatal to adult hearts). This may relate to greater metabolic demands placed by adult hearts and the larger dimensions (diffusion distances) in adult myocytes. 
 
Description Acid-sensing in cancer cells using G-protein coupled receptors 
Organisation University of Oxford
Department Department of Physiology, Anatomy and Genetics
Country United Kingdom 
Sector Academic/University 
PI Contribution (1) Measurements of pH and calcium during activation of acid-sensor. (2) Providing expertise in designing experiments.
Collaborator Contribution (1) Collaborator has provided biological material (neuroblastoma cell lines) to investigate the acute responses to extracellular acid. (2) Collaborator has also provided RT-PCR probes to investigate expression levels of acid-sensor in different cancer cell lines. (3) Collaborator has provided expertise in molecular biology to help train Oxford lab in studying pathways involved in cancer proliferation.
Impact Publication PMID 19249096
Start Year 2009
 
Description Characterising membrane transporters for pH regulation in cancer cell lines 
Organisation Slovak Academy of Sciences
Country Slovakia 
Sector Public 
PI Contribution Expertise and consultation in the field of cellular physiology through visits by MRC-funded postdoc from my Oxford lab.
Collaborator Contribution Collaborator and her team have provided molecular biology expertise and biological material for studies in Oxford lab.
Impact Publications 20890298, 18482982 and 19458084 have been possible thanks to cell-lines and cDNA obtained from collaborator.
Start Year 2009
 
Description Determining efficacy of antibodies with biological activity against pH-regulatory proteins 
Organisation Helmholtz Association of German Research Centres
Department Helmholtz Zentrum Munchen
Country Germany 
Sector Public 
PI Contribution To screen antibodies for biological activity in vitro, and identify candidates for further in vivo animal work
Collaborator Contribution Testing efficacy of identified antibodies in terms of tumour growth
Impact Publication: "Antitumor efficacy of a monoclonal antibody that inhibits the activity of cancer-associated carbonic anhydrase XII." Cancer Res. 2013; PMID: 24030978
Start Year 2012
 
Description Testing small molecule inhibitors for carbonic anhydrase isoforms 
Organisation University of Florence
Country Italy 
Sector Academic/University 
PI Contribution Provision of experimental data for the inhibitory potency of synthesised drugs.
Collaborator Contribution Collaborator and his team have been synthesising selective/targeted inhibitors against carbonic anhydrase isoforms. These are being tested and used as experimental tools in the Oxford lab. Future projects to design drugs targeting other cell proteins (e.g. acid/base transporters, acid-sensors) are being considered.
Impact Publications 19458084.
Start Year 2009
 
Description Using magnetic resonance imaging to study cellular physiology in whole-tissue 
Organisation University of Oxford
Department Department of Physiology, Anatomy and Genetics
Country United Kingdom 
Sector Academic/University 
PI Contribution (1) Providing mathematical diffusion-reaction models as a framework to analyse imaging data in terms of solute flux/turnover. (2) Supplementing whole-tissue data with single-cell experiments.
Collaborator Contribution Developing new methods to study metabolic flux and pH in perfused tissues/organs, using magnetic resonance imaging with hyperpolarised probes.
Impact First results of collaboration published as PMID: 20008827
Start Year 2009