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Experimental Oncology 34, 79–84, 2012 (June) 79
EXTRACELLULAR ACIDITY AS FAVOURING FACTOR OF TUMOR
PROGRESSION AND METASTATIC DISSEMINATION
L. Calorini*, S. Peppicelli, F. Bianchini
Dipartimento di Patologia e Oncologia Sperimentali, Universitа degli Studi di Firenze, e Istituto Toscano
Tumori, Firenze, Italy
The bidirectional interactions between tumor cells and the so-called “host reactive stroma” play a critical role in most of the events
characterizing tumor progression and distant organ colonization. This review discusses critical components of tumor environment
involved in tumor cell dissemination. More specifically, it addresses some of the experimental evidences providing that acidity of tumor
environment facilitates local invasiveness and metastasis formation, independently from hypoxia, with which acidity may be associ-
ated. Besides, acidity renders tumor cells resistant to radiation therapy and chemotherapeutic drugs. Therefore, this review examines
the strategies for raising the low extracellular pH of tumors that might have considerable potential in cancer therapy.
Key Words: extracellular acidity, invasiveness, metastatic dissemination, proton pump inhibitors.
CANCER AS A COMPLEX SCENARIO
OF TUMOR CELLS AND AN ACTIVE STROMA
Capacity of cancer to evolve and change has been
named “tumor progression” [1]. Biological character-
istics that define tumor progression have been exten-
sively described, although the underlying mechanisms
remain unknown. Malignant tumor cells accumulate
increasingly genetic alterations, generated by random
mutational events, leading them to assume all the
characteristics of invasive cells. In concert with this
“genetic instability”, a key role in favouring changes
in tumor cells is played by local host factors [2, 3].
Among local factors, particular attention has been
devoted to the interactions that tumor cells establish
with various host cells that reside in or are attracted into
tumor environment. The bidirectional interaction be-
tween tumor cells and host cells, is recognized as cru-
cial for the decision whether tumor cells progress
toward metastatic dissemination or remain dormant
[4–8]. Indeed, tumor growth and metastasis is sig-
nificantly reduced in fibroblast-deficient mice, while
injection of wild-type fibroblasts into these mice can
reverse this phenotype, providing a clear evidence for
the involvement of fibroblasts in the emergence of me-
tastasis [9–11]. This type of activated cells, commonly
identified by the expression of -smooth muscle actin
(-SMA) and referred as “myofibroblasts” [12], was
named cancer-associated fibroblasts (CAFs) and ac-
tively participates at all stages of metastatic cascade.
In addition cells of monocyte/macrophage lineage
enter into the tumor mass via blood vessels throughout
life span of tumors, from early-stage lesions to late-
stage tumors that are invasive and metastatic, and are
indicated as tumor-associated macrophages (TAMs)
[13, 14]. TAMs are remarkable for the diverse activi-
ties in which they can engage on different occasions.
Quiescent macrophages respond to immune or bac-
terial stimuli by expressing new functional activities,
resulting in their capacity to recognize and destroy
transformed cells. On the contrary, macrophages
isolated from experimental and spontaneous tumors
show a reduced level of cytotoxic activities and was
proved to be relevant to tumor progression and mes-
tastases [15, 16]. Plasticity of both CAFs and TAMs
may be exploited by tumor cells to elicit distinct func-
tions at different stages of tumor progression. It is also
possible that changes expressed by these host cells
during tumor development might be related to their
location inside the tumor mass. Most tumors develop
an environment characterized by low oxygen tension
(hypoxia), elevated interstitial fluid pressure, low glu-
cose concentration and high lactate concentration.
These changes are largely caused by a combination
of poor tissue perfusion due to abnormal tumor vas-
culature, uncontrolled proliferation and altered energy
metabolism [17].
Cells require oxygen and nutrients for their survival
and growth. Likewise, neoplastic cells depend on near-
by capillaries for growth and once aggregates of tumor
cells reach the diffusion limit for critical nutrients and
oxygen, tumor cells become dormant. Indeed, some
human tumors can remain dormant for a number
of years at a stage where tumor cell proliferation and
death are balanced. But once new blood vessel forma-
tion is initiated, the so-called angiogenic switch, tumor
progression and metastasis follow [18]. The new vessel
formation governed by a balance of pro- and anti-
angiogenenic factors is often disturbed in tumors lea-
ding to a vasculature characterized by dilated, tortuous
and incomplete vessels. The molecular mechanisms
causing this abnormal vascular architecture are still
debated, but the uncontrolled vascular endothelial
Received: May 23, 2012.
*Correspondence: E-mail: lido.calorini @unifi.it
Abbreviations used: α-SMA — α-smooth muscle actin; CA — car-
bonic anhydrase; CAFs — cancer-associated fibroblasts; ECM —
extracellular matrix; HIF — hypoxia-inducible factor; MCTs — mono-
carboxylate transporters; MMPs — matrix metalloproteinases;
NHEs — Na+/H+ exchangers; PAI — plasminogen activator inhibitors;
TAMs — tumor-associated macrophages; uPA — urokinase-type
plasminogen activator; uPAR — uPA receptor; V-ATPase — vacuolar
H+-ATPase; VEGF-A — vascular endothelial growth factor A.
Exp Oncol 2012
34, 2, 79–84
80 Experimental Oncology 34, 79–84, 2012 (June)
growth factor A (VEGF-A) signalling may be a key
contributor. VEGF-A is a strong mitogen and survival
growth factor for vascular endothelial cells and induces
mobilization and recruitment of endothelial precursor
cells [19, 20]. Furthermore, VEGF-A contributes to the
angiogenic phenotype by increasing the permeability
of existing vessels, permitting extravasation of fibrino-
gen and clotting factors and resulting in a fibrin-rich
stroma that supports migration of endothelial cells
and formation of new vasculature. However, the
uncontrolled secretion of VEGF-A results in a lower
perfusion rates in tumors than in many normal tissues.
Blood flow in tumors is unevenly distributed and can
even reverse its direction in some vessels, therefore,
regions with poor perfusion are common. These
environmental features vary widely in different areas
of tumors, reflecting tumor cell heterogeneity. In addi-
tion, the uncontrolled growth of tumor cells compress
the intra-tumor lymphatic vessels. Consequently, there
are no functional lymphatic vessels inside solid tumors,
whereas functional lymphatic vessels are present only
in peri-tumoral tissues [21, 22]. Both, the high perme-
ability of tumor blood vessels and the lack of functional
lymphatics are keys contributors to the development
of an interstitial hypertension in neoplastic tissues
[23]. As a result, the hydrostatic and colloid osmotic
pressures become almost equal between intravascular
and extravascular spaces, compromising the delivery
of nutrients as well as therapeutic agents. Since tumor
interstitial hypertension is a reflection of global patho-
physiology of tumors, it may be used for diagnosis
and/or prognosis. The consequent metabolic hallmark
of tumor environment is hypoxia. Hypoxia character-
izes the microenvironment of many solid tumors and
it has been shown to affect many biological proper-
ties of tumor cells implicated in tumor progression,
response to therapy, including clinical outcome of pa-
tients [24–26]. The mechanism behind these effects
is related to the induction of hypoxia-inducible factor
(HIF) family of transcription factors. Under conditions
of acute or chronic hypoxia, HIF-1 is stabilized, form
a heterodimer with HIF-1, allowing this factor to bind
a core sequence and increase transcription of target
genes. This factor regulates many cellular processes
including apoptosis, cell proliferation, angiogenesis
and glucose metabolism [27, 28]. Thus, hypoxia in-
creases genetic instability, blood vessel formation and
a switch to anaerobic metabolism.
Hypoxia, elevated interstitial fluid pressure, low
glucose and high lactate concentration resulting from
a predominant anaerobic metabolism, are responsible
of low extracellular pH (pHe) in tumor tissues. As a con-
sequence, the second metabolic hallmark of tumor
environment is tumor acidosis.
In this review, we will discuss evidence that aci-
dity of tumor extracellular space represents a direct
contributor to the process of tumor progression and
that normalization of pHe could be considered a new
strategy for tumor therapy.
CONSEQUENCES OF TUMOR ACIDITY
In contrast to normal cells, which rely on mitochon-
drial oxidative phosphorylation to generate the energy
needed for cellular processes, most cancer cells,
even in the presence of sufficient oxygen to support
mitochondrial respiration, use “aerobic glycolysis”,
a phenomenon termed “the Warburg effect” [29,
30]. This phenomenon was first reported by Warburg
in the 1920s, leading to hypothesis that cancer results
from impaired mitochondrial metabolism. Although
the “Warburg hypothesis” has proven incorrect,
an increased conversion of glucose to lactic acid
in tumor cells has been continuously demonstrated
(½ (glucose) = lactate- + H+). The clinical application
of the imaging technique positron-emission tomog-
raphy (PET) using the glucose analog 2-(18F)-fluoro-
2-deoxy-D-glucose (FDG) tracer, demonstrated that
most primary and metastatic human lesions express
a high glucose uptake [31]. FDG-PET combined with
computer tomography (PET/CT) has a high sensitivity
and specificity for the detection of metastases of most
epithelial cancers. A possible explanation for the
switch to aerobic glycolysis is that proliferating tumor
cells have important metabolic requirements beyond
ATP, and some glucose must be diverted to macromo-
lecular precursors such as acetyl-CoA for fatty acids,
glycolytic intermediates for nonessential amino acids
and ribose for nucleotides. Moreover, some tumors
possess a greater capacity to pump lactic acid and
protons out to the extracellular space through specific
transporters, to maintain an appropriate neutral/slight
alkaline intracellular pH essential for cell proliferation.
The inefficient removal of protons and lactic acid from
the extracellular spaces, due to the poorly perfused
tumor tissue and absence of functional lymphatic
vessels, creates a reversed pH gradient characte-
rized by an acidic pHe and an alkaline intracellular pH
(pHi) [32].
In vitro and in vivo studies revealed that tumor cells
have pHi ranging from 7.12 to 7.56 (pHi of normal cells:
6.99-7.20), and pHe of 6.2-6.9 (pHe of normal extracel-
lular space: 7.3-7.4). Degree of acidity in tumors tends
to be associated with a poorer prognosis [33]. Indeed,
tumor acidity contributes to aggressiveness of tumor
cells, stimulating increased mutation rate [34]. Acute
and chronic acidosis, hypoxia and reoxygenation injury
all together promote DNA instability even in very small
tumors leading to the selection of cells with additional
genetic defects. Moreover, a minimum in pHe has been
observed near tumor periphery, where tumor cells are
invading normal tissues [35]. Hypoxia, also, stimulates
invasiveness in tumor cells [27]. Could be expected
that low extracellular pH and hypoxia always co-
localize within tumor regions, instead, there is often
a lack of spatial correlation among these parameters.
Potential explanations of this lack of correlation could
be due to the enhanced glucose uptake for glycolytic
ATP generation in conditions of high oxygen tension,
or to the possibility that some tumor vessels carrying
hypoxic blood, are unable to deliver adequate quan-
Experimental Oncology 34, 79–84, 2012 (June) 81
tity of oxygen to the cells, but are able to carry away
the waste products (e.g., lactic acid). Low pHe has
shown to affect several steps of metastatic cascade.
In some tumor cells, low pH promotes angiogenesis
through VEGF [36] and IL-8 [37], however in other
models of tumor cells acidosis inhibits VEGF [38].
Role of acidic pH in angiogenesis is still not completely
understood. On the other hand, influence of acidity
in invasiveness of tumor cells into host tissues is well
demonstrated. Invasiveness is a multistep process
based on extracellular matrix-degrading proteinases,
such as serine and metallo-proteinases, reorganiza-
tion of cytoskeleton and an integrin-mediated for-
mation and release of focal adhesion contacts [39].
It has been reported that an acidic pHe may enhance
invasion of tumor cells facilitating the redistribution
of active cathepsin B, a lysosomal aspartic proteinase
with acidic pH optima, to the surface of malignant cells
[40, 41]. Acid-activated catepsins L also participate
to amplify proteinase cascade through activation of
urokinase-type plasminogen activator (uPA) [42]. The
uPA system, made by uPA, two main plasminogen
activator inhibitors (PAI-1, PAI-2) and uPA receptor
(uPAR), is critical for tumor cell-driven degradation
of extracellular matrix (ECM) in many steps of meta-
static cascade [43, 44]. Activation of cathepsins D and
L in an acidic tumor environment reduce perfusion
of tumor regions, generating angiogenesis inhibitors
such as angiostatin [45] and endostatin [46] from pro-
teolysis of plasminogen and collagen, enhancing the
chaotic vascular organization of tumors. Furthermore,
acidic pH can promote the conversion of matrix me-
talloproteinases (MMPs) in their active forms. MMPs
have long been associated with invasiveness and dis-
semination of tumor cells, due to their capacity to help
tumor cells to cross structural barriers, inclu ding base-
ment membranes and structural components of the
ECM, such as collagen fibers [47–49]. Degradation
of structural components of ECM is considered es-
sential in tumor-induced angiogenesis. MMPs also
participate in the release of cell-membrane-precursor-
forms of many growth factors. The expression of MMPs
in tumors is regulated in a paracrine manner by growth
factors and inflammatory cytokines secreted by tumor
infiltrating inflammatory cells as well as tumor cells
themselves, and a continuous cross talk between tu-
mor cells and inflammatory cells during the invasion
process was demonstrated. Incubation of human and
mouse melanoma cells in a low pH medium stimulate
MMP expression and an increase in vitro invasiveness
and in vivo metastasis formation in immunodeficient
mice [50–53].
Another important component of basement mem-
brane to be degraded by tumor cells to disseminate
are the heparan sulphate chains. Toyoshima and
Nakajima report that heparanase has an optimal
pH of 4.2, but a significant heparanase activity persists
at pH 6.0–6.5, suggesting that the acidic environment
of tumors may activate the degrading properties of tu-
mor heparanases [54].
More recently, two of the most important H+ trans-
porters, the ubiquitously expressed Na+/H+ exchanger
isoform (NHE1) [55] and the plasma membrane type
of vacuolar H+-ATPases (V-ATPases) [56] were found
to be implicated in migration of tumor cells. NHE1 in-
fluences the formation of invadopodia, structures that
regulate cell motility [57]. Cell motility is driven by cy-
cles of actin polymerization, integrin-mediated cell
adhesion and acto-myosin contraction. Thus the
moving tumor cells, in the absence of proteinases,
make contact with collagen fibers and proceed along
fibers [39]. V-ATPases are a family of ATP-dependent
proton pumps particularly expressed by invasive pan-
creatic [58] and breast carcinomas [59], and inhibition
of V-ATPases expression in hepatocarcinoma using
siRNA abrogates invasion and metastatic diffusion
of these tumor cells [60]. On the whole, an acidic
condition may potentiate several proteinases critical
for tumor cells when they detach from the primary tu-
mor, migrate into the blood, extravasate and colonize
in distant host tissues.
Moreover, extracellular lactic acid can suppress
tumoricidal activity of cytotoxic lymphocytes and
natural killer cells, an effect mediated by lactate/H+
co-transporter that under neutral conditions remove
lactic acid from leukocytes [61]. Acidification of the ex-
tracellular space may also influence radiation therapy
and chemotherapy. Indeed, acidity of tumors reduce
sensitivity of tumor cells to radiation therapy [62–64].
This protective effect is considered to be due to the
decreased fraction of proliferating tumor cells [65]
and the reduced fixation of radiation-induced DNA
damage [66].
Extracellular acidity also confers a special re-
sistance against weakly basic drugs to tumor cells.
It has been reported that chronic and acute sodium
bicarbonate-induced alkalosis is able to circumvent
this drug resistance and enhance the anti-tumor ac-
tivities of two weakly basic drugs, such as doxorubicin
[67] and mitoxantrone [68]. These results suggest
that induction of metabolic alkalosis using sodium
bicarbonate can produce a net gain in the therapeutic
index of the several chemotherapeutic agents, and
open up the possibility that normalization of pHe may
have a therapeutic utility.
MANIPULATION OF TUMOR ACIDIFICATION
Since acidity of tumor environment appears
to contribute to cancer aggressiveness, chemo- and
radiation resistance and, even, evasion of immune
reactions, measures to normalize pHe of tumors may
be used in tumor therapy.
A number of researches have explored the pos-
sibility to correct the extracellular acidity of tumors.
Studies revealed that in tumors levels of CO2 are
higher and concentration of bicarbonate, the princi-
pal physiologic buffer used to control pH, are lower
than in blood or in healthy tissues [69, 70]. Therefore,
it is possible that an increased concentration of sodium
bicarbonate can reduce aggressiveness of tumor
82 Experimental Oncology 34, 79–84, 2012 (June)
cells. Indeed, the alkalization of melanoma-bearing
animals by sodium bicarbonate was found to inhibit
the development of spontaneous metastases [71]. In-
terestingly, a similar dose of bicarbonate used in these
latter experiments has been administered chronically
(>1 year) in patients with renal tubular acidosis [72]
and sickle cell anemia without adverse effects [73].
Computer simulation used to verify the ability of so-
dium bicarbonate to increase pHe of tumors in vivo
also indicates that the normalization of tumor acidity
reduces invasiveness of tumor cells without altering
the pH of blood or normal tissues [74].
As an alternative strategy for correcting low pHe,
several authors explored the inhibition in tumor cells
of key pH regulators that maintain a neutral/alkaline
intracellular pH by extruding lactate or protons [75].
pH regulators in tumor cells include extracellular
forms of carbonic anhydrase (CA), Na+/HCO3- co-
transporters, Na+/H+ exchangers (NHEs), mono-
carboxylate transporters (MCTs) and the vacuolar
H+-ATPase (V-ATPases). The raise of pHe promoted
by these inhibitors is constantly associated with a de-
crease of intracellular pH. Acidity of pHi tends to sup-
press the efficiency of glycolysis, sustaining the raise
of pHe [76], and may exert anti-proliferative and pro-
apoptotic effects on tumor cells themselves [77–79].
Consequently, pH regulators might be considered true
anticancer drugs.
Two CA isozymes, CA9 and CA12, are overex-
pressed in tumor cells and their activity is associated
with malignancy and resistance to therapy [80]. Sul-
phonamide CA inhibitors that target CA9 were found
effective to block the growth of primary tumor and
metastases in a mouse model of breast cancer [81].
Some of these compounds are in advanced preclini-
cal evaluation. V-ATPases, while originally identified
in intracellular compartments, they have increasingly
been shown to play essential roles in proton transport
across the plasma membrane of a variety of cell types,
including tumor cells [59, 76, 82]. The likely similarity
between V-ATPase and the H+/K+ ATPase, the enzyme
involved in proton secretion in gastric parietal cells,
prompted the investigation of proton pump inhibitors
(PPIs), such as omeprazole and esomeprazole, for
inhibiting V-ATPase. When activated by acidic pHe
of tumors, these drugs can inhibit V-ATPase by a co-
valent interaction. Both in vitro and in vivo experi-
ences indicate that non-toxic doses of PPIs, analo-
gous to those used for treatment of Zollinger-Ellison
syndrome, exert anti-proliferative and pro-apoptotic
effects on melanoma cells [83]. PPIs were also dem-
onstrated to inhibit growth of B-cell lymphoma cells
transplanted into severe combined immunodeficient
mice [77]. Knockdown of V-ATPase expression by siR-
NA in cells isolated from a human hepatocarcinoma
markedly reduced metastatic dissemination of these
cells [84]. Importantly, Hashioka et al report that PPIs
have anti-inflammatory effects and decrease mono-
cytic neurotoxicity [85]. Recently, Lee et al. found that
omeprazole exerts a cancer-preventive role against
colitis-induced carcinogenesis, a chemopreventive
action independent of gastric acid suppression [86].
Evidence that PPIs play a role in normalization of low
pH and abrogate inflammation, renders these drugs
suitable to target critical mechanisms involved in tumor
progression. Moreover, clinical data provide that PPIs
have a very low level of systemic toxicity as compared
with standard chemotherapeutic agents. NHE is crucial
in pH regulation and is expressed in eve ry cell type.
There are several NHE inhibitors, structu rally related
to amiloride and cariporide, however the diffused
presence of NHE in many tissues and its role in crucial
physiological processes, confers to this class of agents
potential risk of side effects. Inhibitors of V-ATPase
and NHE have been shown to have an additive im-
pact on intracellular pH and on thermosensitization
[87]. Therefore, it is crucial to develop agents that
selectively target NHE in tumor. At the same time,
potent, non-toxic selective MCT inhibitors are needed.
MCTs, are overexpressed in many tumors and the
isoform MCT1 regulates the entry and exit of lactate
from tumor cells. The inhibition of MCT1 was found
to induce a switch from lactate-fuelled respiration
to glycolisis, which was accompanied by a retardation
of tumor growth in a mouse model of lung carcinoma
and in transplanted human colorectal carcinoma [88].
CONCLUSION
Tumor stroma manifest some degree of plasti-
city, a property controlled by tumor cells themselves.
Indeed, tumor cells influence host stromal elements
to produce relevant effectors that act as tumor pro-
moters. The metabolic hallmarks of this space are
hypoxia and acidosis. We have elucidated how the
extracellular acidity per se, may promote an aggres-
sive and metastatic phenotype in tumor cells and how
these findings suggest the possibility of a novel and
effective therapeutic strategy based on the control
of tumor acidity.
ACKNOWLEDGEMENTS
This study was funded by grants from Istituto
Toscano Tumori, Ente Cassa di Risparmio di Firenze.
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