Compartmental pH regulation in cancer and antitumor immunity: therapeutic opportunities and challenges

Abstract

Dysregulated pH homeostasis is a defining feature of tumor biology and a potential therapeutic vulnerability. Cancer cells maintain an alkaline intracellular pH, whereas the tumor microenvironment remains acidic and intracellular organelles preserve compartment-specific luminal acidity, together supporting tumor growth, metabolic adaptation, immune evasion, and therapeutic resistance. Recent studies show that modulation of pH regulation across the tumor microenvironment, cytoplasm, and intracellular organelles disrupts ionic balance, impairs organelle function, and triggers regulated cell death, including alkaliptosis and organelle stress–associated lethality. Beyond direct tumor killing, pH modulation also reshapes antitumor immunity by relieving extracellular acidosis, restoring immune-cell function, and inflammatory signaling. In this review, we summarize recent advances in compartmental pH regulation in cancer, with particular emphasis on the distinction between experimentally demonstrated mechanisms and emerging conceptual models. We discuss how alkalization-oriented interventions may suppress tumor growth in selected contexts, but may also produce neutral, adaptive, or even protumorigenic consequences depending on tumor type, buffering capacity, transporter activity, metabolic state, and immune composition. We further evaluate pH-dependent immune regulation, organelle alkalization, alkaliptosis, quantitative pH measurement, and monitoring technologies. Finally, we highlight major translational barriers. A more quantitative, compartment-resolved, and context-aware understanding of pH biology will be required before pH modulation can be reliably translated into clinically useful therapeutic strategies.

Introduction

Cancer progression is increasingly understood as a process driven not only by genetic alterations but also by dynamic remodeling of the tumor microenvironment and intracellular homeostatic systems [1]. Among these adaptive networks, dysregulated pH homeostasis has emerged as a defining feature of tumor biology and a potential therapeutic vulnerability [2, 3]. Although conventional treatments such as radiotherapy and chemotherapy have improved patient outcomes, their efficacy remains limited by dose-related toxicity, acquired resistance, and insufficient tumor selectivity [4, 5]. This has prompted growing interest in strategies that target the microenvironmental and intracellular systems that support tumor growth and adaptation [6].

Intracellular pH homeostasis is essential for normal cellular function [7, 8]. Enzyme activity, membrane dynamics, vesicular trafficking, and signal transduction all depend on tightly controlled proton balance [9]. Under physiological conditions, cytosolic pH is maintained within a narrow range, whereas intracellular organelles such as lysosomes and the Golgi apparatus preserve distinct acidic luminal environments required for protein degradation, trafficking, and sorting [10]. These compartment-specific pH gradients are established primarily by proton pumps and transport systems, including vacuolar-type H⁺-adenosine triphosphatase (V-ATPase) and solute carrier (SLC) family transporters [11, 12].

In tumor cells, however, this pH organization is frequently disrupted. Cancer cells often maintain a relatively alkaline intracellular pH that favors proliferation, metabolic adaptation, and resistance to stress, whereas the extracellular space becomes acidic, largely owing to enhanced glycolysis and proton export associated with the Warburg effect [13, 14]. This reversed pH gradient is now recognized as an important feature of malignant progression and contributes to invasion, immune evasion, and therapeutic resistance [15].

Compared with the extensively studied role of extracellular acidosis in tumor progression, the biological and therapeutic significance of alkalinizing shifts has only recently begun to emerge. Increasing evidence suggests that pH modulation across different tumor compartments, including the extracellular tumor microenvironment, cytoplasm, and intracellular organelles, can directly influence tumor cell fate. Such perturbations may disrupt ionic homeostasis, impair organelle function, and activate multiple forms of regulated cell death, including alkaliptosis [16], ammonia-induced cell death [17], and organelle stress–associated lethality [18, 19]. Similarly, extracellular buffering strategies, such as sodium bicarbonate administration, can alleviate tumor-associated acidosis, reverse local immunosuppression, and improve sensitivity to conventional therapies [20]. These findings suggest that pH modulation is not merely a biochemical consequence of tumor metabolism but may represent a therapeutically exploitable vulnerability in cancer.

In this review, we summarize recent advances in how compartmental pH regulation influences tumor cell fate and antitumor immunity across the tumor microenvironment, cytoplasm, and intracellular organelles. Rather than providing an exhaustive catalog of pH regulators, we focus on representative mechanisms that illustrate the interplay between pH dynamics, metabolic stress, organelle dysfunction, and regulated cell death, while highlighting their implications for biomarker development, dynamic monitoring, and therapeutic translation. A deeper understanding of these spatially organized pH alterations may facilitate the development of more precise strategies based on targeted modulation of pH across tumor compartments.

Evidence hierarchy for compartmental pH coupling: established mechanisms versus emerging concepts

A major challenge in interpreting pH-targeted cancer studies is the difference between compartment-specific observations and systems-level inference. Several mechanisms are well supported experimentally, including extracellular acidosis in many solid tumors, alkaline-shifted intracellular pH in cancer cells, acidification of lysosomes and endosomes, and the dependence of vesicular trafficking and antigen processing on organelle pH. In contrast, the idea that extracellular, cytoplasmic, and organellar pH compartments operate as a coordinated tumor-wide regulatory network remains an emerging conceptual model.

For clarity, we classify the current evidence into three levels. First, established compartment-specific mechanisms include transporter-mediated proton extrusion, carbonic anhydrase-dependent extracellular acidification, lysosomal V-ATPase-dependent acidification, and pH-sensitive immune-cell dysfunction. Second, partially supported cross-compartmental interactions include extracellular pH effects on intracellular pH regulation, lysosome–mitochondria crosstalk during stress, and cytoplasmic pH shifts affecting metabolic enzymes or cell death regulators. Third, broader systems-level claims, such as coordinated propagation of pH perturbations across the whole tumor ecosystem, remain largely inferential and require direct longitudinal, quantitative, and spatially resolved validation.

This evidence hierarchy is important therapeutically because an intervention that alkalinizes one compartment may not necessarily produce predictable effects in another. Tumor cells can compensate through proton transporters, bicarbonate transporters, carbonic anhydrases, metabolic rewiring, altered perfusion, and stromal buffering. Therefore, compartmental selectivity and adaptive feedback should be considered central determinants of therapeutic efficacy.

Against this background, the following sections examine how pH alterations in distinct compartments regulate specific cell-death programs, and how these mechanistic insights inform the design and limitations of pH-targeted cancer therapies.

Therapeutic alkalization–driven regulation of tumor cell death across compartments

Compartment-specific pH gradients are essential for tumor cell survival and adaptation, supporting metabolic flexibility, organelle function, stress tolerance, and therapeutic resistance. Disruption of this balance, particularly through alkalinizing shifts, can alter tumor cell fate by triggering stress responses and engaging multiple regulated cell death pathways. In this section, we summarize the molecular mechanisms by which therapeutic alkalization drives tumor cell death across extracellular, cytoplasmic, and organellar compartments (Fig. 1).

Fig. 1.

pH alkalization and tumor cell death. A Tumor microenvironment (TME): Strategies such as oral buffering (e.g., NaHCO₃), antibody–urease conjugates, bicarbonate-based embolization (TILA–TACE), and metal-based agents locally increase extracellular pH, thereby modulating tumor acidity and sensitizing tumors to therapy. B Cytoplasm: Cytoplasmic alkalization, induced by osmotic stress, NaHCO₃, optogenetic tools (AR3), or small molecules (e.g., JTC801, PF-429242 combined with chloroquine), elevates intracellular pH (pHi) and activates multiple regulated cell death pathways, including alkaliptosis, apoptosis, necroptosis, and autophagy-associated cell death. Key regulatory mechanisms involve SLC9A1-mediated proton flux, metabolic buffering pathways, and lysosome–cytosol pH disequilibrium. C Organelles: Alkalization of intracellular organelles, particularly lysosomes and the Golgi apparatus, disrupts vesicular trafficking, protein degradation, and signaling pathways. Lysosomal alkalization, mediated by V-ATPase inhibition or ion channel modulation (e.g., TMEM175, SLC7A11, CLCN family), can trigger multiple forms of cell death, including ferroptosis, pyroptosis, apoptosis, and necroptosis. Golgi alkalization, regulated by SLC9A7 and STING1, impairs trafficking and secretion while contributing to organelle crosstalk and stress signaling

Extracellular alkalization and tumor microenvironment–dependent cell death

Rapid tumor growth and hypoxia-driven metabolic reprogramming frequently generate an acidic extracellular niche that promotes invasion, metastasis, immune evasion, and therapeutic resistance [21–24]. Accordingly, local neutralization of extracellular acidity emerges as a promising anticancer strategy (Fig. 1A). The underlying principle is to selectively increase extracellular pH through buffering systems or by inhibiting proton production and export.

Clinically, oral buffering strategies such as sodium bicarbonate (NaHCO₃) are being explored in combination with gemcitabine for unresectable pancreatic cancer (NCT01350583, NCT01846429, NCT01198821), although their application remains limited by gastrointestinal toxicity and poor patient compliance [25]. To improve tumor specificity, antibody-conjugated urease systems are developed to locally generate ammonium (NH₄⁺) and bicarbonate (HCO₃⁻) within tumors. Preclinical studies show encouraging tolerability and antitumor activity in non-small cell lung cancer models [26]. In hepatocellular carcinoma, bicarbonate-integrated transarterial chemoembolization demonstrates effective local control with manageable safety profiles [27]. Additional approaches, including metal-based thermochemical agents capable of inducing local heating and alkalization, also show efficacy in breast cancer models [28].

However, extracellular alkalization is not uniformly beneficial. Systemic administration of NaHCO₃ accelerates malignant progression in primary breast cancer by enhancing acid extrusion capacity [29]. These findings indicate that extracellular pH correction should not be interpreted simply as reversal of tumor acidosis. Instead, it may impose a selection pressure that favors tumor subclones with stronger buffering capacity, higher transporter activity, or greater metabolic plasticity. The outcome of alkalization is therefore likely to depend on tumor type, baseline extracellular pH, perfusion, hypoxia, stromal buffering, carbonic anhydrase expression, monocarboxylate transporter activity, sodium–hydrogen exchanger activity, and immune-cell composition. This context dependence may partly explain why pH-buffering strategies that are effective in controlled animal models have not yet achieved broad clinical success. Another important issue is whether extracellular alkalization directly induces tumor cell death or primarily modifies the conditions under which other therapies operate. Many studies showing enhanced tumor control after buffering involve combination with chemotherapy, radiotherapy, immunotherapy, hyperthermia, or nanomedicine-based delivery systems. In these settings, alkalization may improve drug distribution, relieve immune suppression, or alter stress signaling rather than independently triggering a defined cell-death program.

In conclusion, extracellular alkalization should be viewed as a double-edged sword intervention. It may alleviate acid-mediated invasion and immunosuppression in certain tumors, but it may also trigger adaptive buffering mechanisms that support tumor survival or progression. In addition, a key unresolved question is how extracellular alkalization influences intracellular pH homeostasis and whether sustained perturbation of extracellular proton balance can be translated into durable intracellular stress sufficient to trigger tumor-selective cell death.

Cytoplasmic alkalization and regulated cell death pathways

Direct induction of cytoplasmic alkalization exploits the distinct buffering capacity of cancer cells, pushing them beyond their homeostatic threshold and triggering regulated cell death.

Alkaliptosis

Alkaliptosis is a pH-dependent regulated cell death program induced by JTC801 and characterized by lethal cytosolic alkalization associated with lysosome–cytosol pH disequilibrium [30, 31]. Rather than being mediated by a single linear signaling pathway, alkaliptosis arises from coordinated perturbations in multiple layers of the pH regulatory network, including transcriptional buffering programs, lysosomal proton handling, nutrient scavenging pathways, and nuclear stress regulators, thereby providing several potential therapeutic targets (Fig. 1B).

In pancreatic ductal adenocarcinoma (PDAC), JTC801 induces nuclear factor kappa B subunit 1 (NFKB1)- dependent suppression of CA9, thereby promoting alkaliptosis [16]. In parallel, acyl-CoA synthetase short chain family member 2 (ACSS2)-driven acetyl-CoA accumulation further enhances NFKB1 signaling through acetylation, thereby amplifying alkaliptosis and linking this death program to central carbon and lipid metabolism [32, 33]. Macropinocytosis of fatty acids (e.g., oleic acid) suppresses alkaliptosis by sustaining lysosomal function, representing a metabolic buffering mechanism [34], likely because macropinocytosis sustains lysosomal degradation capacity and helps preserve organellar pH stability [35].

JTC801 also can directly stabilize ATPase H⁺ transporting V0 subunit D1 (ATP6V0D1). which complexes with signal transducer and activator of transcription 3 (STAT3) to disrupt lysosomal pH homeostasis, causing cytosolic alkalinization and alkaliptosis [31]. In paclitaxel-resistant ovarian cancer, elevated ATP6V0D1 expression suppresses ATP binding cassette subfamily B member 1 (ABCB1) and promotes alkaliptosis, indicating that modulation of this pathway may simultaneously disrupt intracellular proton balance and inhibit drug efflux–mediated resistance [36].

Conversely, sterol regulatory element binding transcription factor 2 (SREBF2)-driven upregulation of cytochrome P450 family 51 subfamily A member 1 (CYP51A1) prevents lysosomal cholesterol accumulation by reducing ubiquitin-mediated degradation of transmembrane protein 175 (TMEM175), thereby suppressing alkaliptosis in pancreatic cancer [30].

Collectively, these findings support the view that alkaliptosis is a network-level consequence of cytoplasmic alkalization rather than an isolated signaling event. Further clarification of how cytoplasmic alkalization intersects with other regulated cell death programs will be essential for understanding its therapeutic potential and for designing effective combination strategies. Despite these advances, alkaliptosis remains an emerging and incompletely validated form of regulated cell death. Most current evidence derives from defined experimental systems using pharmacological or genetic perturbations. Therefore, alkaliptosis should currently be considered a promising conceptual and experimental framework rather than an established dominant mechanism of pH-targeted therapy. Future studies will need to define its molecular execution machinery, quantitative pH thresholds, temporal sequence relative to organelle dysfunction, and diagnostic markers in patient-derived models and clinical samples.

Other cytoplasmic alkalization–associated death programs

Cytoplasmic alkalization is not restricted to alkaliptosis alone but instead can engage branching death responses within an interconnected pH regulatory network (Fig. 1B). For example, under osmotic stress, solute carrier family 9 member A1 (SLC9A1)-mediated intracellular pH elevation activates receptor interacting serine/threonine kinase 3 (RIPK3)-dependent necroptosis in L929 sarcoma and colon cancer cells, linking membrane pH regulation to inflammatory cell death pathways [37]. In hepatocellular carcinoma cells, bicarbonate-induced cytoplasmic alkalization suppresses mitochondrial oxidative phosphorylation and activates AMP-activated protein kinase (AMPK)-dependent autophagy. When lysosomes and autolysosomes subsequently become alkalinized, autophagic flux is blocked, resulting in cell death [38]. This illustrates how cytoplasmic and lysosomal pH dysregulation can cooperate to amplify cellular stress.

Temporally controlled alkalization models further support the causal role of cytoplasmic pH shifts in cell death. Optogenetic expression of archaerhodopsin 3 (AR3) enables light-triggered intracellular alkalization and induces apoptosis both in vitro, including MC38 and B16F10 cancer cells, and in vivo, demonstrating the feasibility of precisely manipulating intracellular pH as an experimental and potentially therapeutic approach [39].

In addition, the combination of the site-1 protease inhibitor PF-429242 with chloroquine induces pH-dependent cell death in hepatocellular carcinoma cells [40]. This phenotype can be reversed by acidic culture conditions or N-acetylcysteine (NAC), suggesting that intracellular alkalization and oxidative stress act cooperatively in this setting. This combination also alters ATP6V0D1 and STAT3 expression, indicating partial mechanistic convergence with lysosome-centered alkaliptosis pathways.

Mechanistically, these observations indicate that cytoplasmic alkalization may acts as an upstream stress signal capable of converging on multiple death programs, including necroptosis, apoptosis, and autophagy-associated cell death. The therapeutic response to pH-directed interventions will therefore likely depend on the dominant death-routing and compensatory buffering programs present within a given tumor type.

Organelle-specific alkalization as an execution hub of tumor cell death

Intracellular organelles rely on tightly controlled luminal pH to sustain function. Even modest pH perturbations disrupt trafficking, proteostasis, and nutrient recycling, thereby compromising tumor cell survival. Lysosomes and the Golgi apparatus are especially vulnerable to alkalinization, establishing organelle-specific pH disruption as a central driver of tumor cell death (Fig. 1C).

Lysosomes

Lysosomal alkalization can induce tumor cell death in selected experimental settings through interconnected mechanisms, including hydrolase inactivation, disruption of mechanistic target of rapamycin complex 1 (mTORC1) signaling, calcium (Ca²⁺) imbalance, and membrane destabilization with release of death-promoting contents. As a key hub for nutrient sensing, autophagic flux, and stress signaling, the lysosome functions as a central node in pH-dependent cell death pathways [41].

Macrolide inhibitors such as bafilomycin A1, concanamycin A, and verucopeptin directly bind specific subunits of V-ATPase, thereby suppressing proton pumping and elevating lysosomal pH. For example, verucopeptin inhibits the growth of multidrug-resistant tumor cells, including A375, U2OS, HL-60, and K562 cells, as well as mouse xenograft models [42]. Similarly, the natural product toosendanin inhibits V-ATPase activity, disrupts lysosomal acidification, and impairs cathepsin maturation, thereby blocking protective autophagy and sensitizing A549 and HeLa cancer cells to chemotherapy [43].

Beyond luminal pH regulation, disruption of V-ATPase activity also affects broader lysosomal signaling networks. For example, EN6 covalently binds cysteine 277 within ATPase H⁺ transporting V1 subunit A (ATP6V1A), disrupting lysosomal localization of mTORC1 and thereby activating autophagy in various cells [44]. These findings suggest that targeting lysosomal proton handling may simultaneously impair organelle function and suppress compensatory growth signaling pathways.

Lysosomal pH is also shaped by proton leak and ion-coupled conductance pathways. TMEM175 and solute carrier family 7 member 11 (SLC7A11) are described as fast and slow proton leak pathways, respectively, in multiple cell models (HAP1 and HeLa cells and HT1080 cells) [45, 46]. In addition, TMEM165 mediates Ca²⁺-driven proton efflux from lysosomes in HeLa and MCF7 cells, indicating that excessive Ca²⁺ loading may also promote lysosomal alkalization [47]. Members of the chloride voltage-gated channel family, including CLCN3 and CLCN7, further influence lysosomal acidity through Cl⁻/H⁺ exchange. Functionally, CLCN7 deficiency is associated with lysosomal alkalization, impaired degradative capacity, and α-synuclein accumulation in patient-derived neuronal cells [48]. These adaptive “leak and counter-leak” processes may buffer therapeutic pH stress and should be considered when predicting responses to lysosome-targeted therapies.

Severe lysosomal dysfunction can propagate into multiple downstream death programs, including ferroptosis [49], pyroptosis [50], apoptosis [51], necroptosis [52], ammonia-associated cell death [17], and alkaliptosis [31, 53]. At the same time, lysosomal membrane repair pathways mediated by endosomal sorting complexes required for transport (ESCRT) [54], the endoplasmic reticulum [55, 56], sphingomyelin [57], annexins [58], and conjugation of ATG8 to single membranes (CASM) may partially restore lysosomal integrity [59]. When damage exceeds the repair threshold, severely compromised lysosomes can themselves be eliminated through autophagy [60, 61]. Thus, these findings identify lysosomal alkalization as a key determinant of tumor cell death and treatment response.

Golgi apparatus

In addition to lysosomes, the Golgi apparatus is highly sensitive to pH perturbation. Disruption of Golgi luminal acidity impairs tumor cell fitness and triggers stress responses associated with cell death. For example, inhibition of solute carrier family 9 member A7 (SLC9A7) induces Golgi alkalization, promotes cytoplasmic acidification, and compromises pancreatic cancer cell adaptation through dysregulated actin remodeling [62]. Conversely, intracellular delivery of human lactoferrin activates SLC9A7-dependent proton transport, acidifies organelles, and suppresses proliferation in lung cancer cells [63]. These findings indicate that both alkalinization and re-acidification of the Golgi apparatus influence tumor growth, depending on the metabolic and secretory state of the tumor.

Emerging evidence further indicates that Golgi pH regulation is closely linked to innate immune signaling and organelle crosstalk. Stimulator of interferon response cGAMP interactor 1 (STING1), beyond its canonical role in immune signaling [64], functions as a proton channel within the Golgi apparatus [65]. STING1-mediated proton efflux promotes Golgi deacidification and contributes to non-canonical autophagy, stress signaling, and cell death–associated responses in U2OS, HT1080, and BJ cells [66]. STING1-dependent pH elevation within the Golgi and Golgi-derived vesicles also serves as an early step in lysosome biogenesis [67], highlighting a direct functional link between Golgi pH homeostasis and lysosomal dynamics.

Additional perturbations, including viral oncoproteins and ionophores such as monensin, likewise induce Golgi alkalization and remodel exocytosis, adhesion, and glycosylation pathways in multiple cancer cell types (such as NIH-3T3, NSCLC, breast and colorectal cancer cells) [68–70]. These findings support the view that the Golgi apparatus is not merely a passive trafficking organelle but an active pH-sensitive regulatory hub that coordinates secretion-dependent tumor adaptation and inter-organelle communication.

Thus, Golgi pH homeostasis represents an additional layer of vulnerability in tumor cells. Its close coupling to vesicular trafficking, secretory stress, and lysosome biogenesis suggests that Golgi-targeted pH modulation may complement lysosome-directed strategies in organelle-based cancer therapy.

pH alkalization in antitumor immune regulation

Abnormal pH homeostasis within tumors not only affects tumor cell survival but also actively shapes the immune microenvironment. Extracellular acidosis, cytoplasmic buffering, and organelle-specific proton dynamics collectively regulate immune-cell metabolism, antigen processing, inflammatory signaling, and responsiveness to immunotherapy. Accordingly, therapeutic modulation of pH across distinct tumor compartments emerges as an important strategy for restoring antitumor immunity. Beyond directly inducing tumor cell death, pH modulation can relieve local immunosuppression, enhance immune-cell function, and improve sensitivity to immune-based therapies. In this section, we summarize how compartment-specific pH regulation shapes tumor–immune interactions across multiple cellular levels (Fig. 2).

Fig. 2.

pH regulation and antitumor immunity. A Tumor microenvironment (TME): Extracellular pH modulation through oral sodium bicarbonate (NaHCO₃), inhibition of carbonic anhydrase 9 (CA9), solute carrier family 4 member 4 (SLC4A4), or lactate dehydrogenase A (LDHA), and alkaline culture conditions promotes local alkalization, enhances antigen exposure (e.g., mesothelin, MSLN), and improves immunotherapy responsiveness. In contrast, acidic TME signaling suppresses T-cell receptor (TCR) signaling through UBASH3B–CBLB complex formation, while lactate promotes M2 macrophage polarization and immune suppression. B Cytoplasm: Cytoplasmic pH regulation influences immune escape and immunogenic cell death. CARNS2-dependent carnosine buffering promotes galectin-9 release and immune evasion. Therapeutic cytoplasmic hyperalkalization induced by hydrogen sulfide (H₂S) releasers combined with carbonic anhydrase inhibitors or manganese carbonate (MnCO₃) nanoparticles enhances immunotherapy and promotes immunogenic pyroptosis. In contrast, SLC16A4-dependent cytosolic alkalization supports immune escape. Alkaliptosis induces damage-associated molecular pattern (DAMP) release and activates advanced glycosylation end-product specific receptor (AGER)–stimulator of interferon response cGAMP interactor 1 (STING1) signaling. C Organelles: Organelle-specific pH modulation regulates innate and adaptive immune responses. Golgi pH homeostasis influences STING1-dependent proton efflux and cytokine secretion, while compound C53 selectively modulates STING1 proton channel activity. Lysosomal alkalization induced by agents such as naphplatin and lithium carbonate enhances macrophage polarization and CD8⁺ T-cell metabolic fitness. In contrast, excessive lysosomal alkalization in senescent cells or chronically activated CD8⁺ T cells may promote ferroptosis resistance or impair immune effector function through ammonia-mediated neutralization

Tumor microenvironment remodeling and immune activation

An acidic tumor microenvironment imposes major metabolic and signaling constraints on antitumor immunity [71]. Rather than being merely a byproduct of tumor metabolism, extracellular acidosis actively reshapes immune-cell function by altering nutrient competition, signaling thresholds, stromal–immune interactions, and cytokine responsiveness. Accordingly, local alkalization of the tumor microenvironment represents an important strategy for restoring immune activity and improving responses to immunotherapy (Fig. 2A).

Several studies have shown that extracellular buffering can improve antitumor immune responses in vivo. Oral administration of sodium bicarbonate (NaHCO₃) or tris(hydroxymethyl)aminomethane (Tris) elevates extracellular pH in mouse models of breast, pancreatic, and prostate cancer, suppresses metastasis, and enhances the efficacy of immunotherapy [72, 73]. Beyond direct buffering, targeting the acidification axis itself has also shown promise. Inhibition of CA9 and solute carrier family 4 member 4 (SLC4A4) reduces proton and bicarbonate transport, thereby lowering the acid burden within tumors such as pancreatic cancer [74, 75].

Microenvironmental alkalization can also promote the transition from immunologically “cold” tumors to “hot” tumors by enhancing antigen exposure and immune-cell engagement. For example, alkaline culture conditions or sodium bicarbonate treatment increase mesothelin (MSLN) surface abundance and enhance MSLN–chimeric antigen receptor (CAR) T cells killing in vitro and in vivo across MSLN-high tumors, including triple-negative breast cancer, pancreatic cancer, and ovarian cancer [76]. Mechanistically, extracellular alkalization–induced autophagy and lysosomal dysfunction impair MSLN degradation, thereby increasing its surface expression and improving CAR-T recognition [76].

Metabolic rewiring within the acidic microenvironment further influences immune-cell function. Inhibition of lactate dehydrogenase A (LDHA) or restriction of lactate efflux enhances immune-mediated killing in melanoma models, and LDHA expression negatively correlates with T-cell activation markers in human melanoma [77]. In addition, extracellular acidity can directly suppress immune signaling pathways. For example, acidic conditions promote formation of a pH-sensitive ubiquitin associated and SH3 domain containing B (UBASH3B)–Cbl proto-oncogene B (CBLB) complex downstream of the T-cell receptor, thereby inhibiting T-cell activation in prostate and melanoma tumor models [78]. Similarly, lactate acts as a paracrine signal that drives macrophage M2 polarization and promotes angiogenesis in multiple tumor models [79].

From an immunometabolic perspective, local alkalization of the tumor microenvironment sustains immunomodulatory effects in preclinical models, especially when combined with inhibition of acid production or proton export.

Cytoplasmic pH regulation in tumor–immune interactions

Cytosolic pH serves as a hub that integrates plasma membrane proton flux, intracellular buffering metabolites, and organelle pH stability. As a result, perturbations in cytoplasmic pH can reshape tumor–immune interactions, either by disrupting adaptive buffering mechanisms or by triggering immunogenic forms of cell death (Fig. 2B).

One representative example is carnosine, a mobile buffering metabolite that accumulates under hypoxic conditions and binds cytosolic protons, thereby facilitating net proton efflux. Under hypoxia, carnosine synthase 2 (CARNS2)-driven carnosine synthesis sustains lysosomal activity and accelerates degradation of nuclear transcription factor X-box binding protein 1 (NFX1), promoting galectin-9 release and T-cell–mediated immune evasion [80]. In contrast, CARNS2 knockdown reduces cytosolic pH, increases CD8⁺ T-cell infiltration, and prolongs survival in hepatocellular carcinoma models [80], highlighting the role of intracellular buffering in immune escape.

Therapeutic induction of cytoplasmic alkalization shows immune-enhancing effects. A hybrid platform combining hydrogen sulfide (H₂S) releasers with tumor-associated carbonic anhydrase inhibitors induces lethal cytosolic hyperalkalization while modulating extracellular pH, thereby restoring sensitivity of various cancer cells to immune-mediated killing as well as to chemotherapy and radiotherapy [81]. Similarly, in vivo, manganese carbonate (MnCO₃)-based nanotherapy alleviates immunosuppressive acidity in 4T1 tumors and induces endoplasmic reticulum stress–dependent immunogenic pyroptosis while elevating cytosolic pH through proton consumption and lactate regulation [82]. This cytoplasmic alkalization–associated enhancement of antitumor immunity has also been validated in melanoma models [83].

In some tumor contexts, cytoplasmic alkalization itself is directly linked to malignant growth. A representative example is the epigenetic enhancement of SLC16A4 activity, which promotes intracellular alkalization required for acute myeloid leukemia progenitor proliferation [84]. Targeting SLC16A4 eliminates malignant cells without relying on conventional cytotoxic chemotherapy, suggesting that intracellular pH buffering may represent an immune-relevant metabolic vulnerability.

Certain forms of cytoplasmic alkalization–associated cell death are intrinsically immunogenic. Mechanistically, alkaliptotic stimulation induces release of high mobility group box 1 (HMGB1), which activates advanced glycosylation end-product specific receptor (AGER, also known as RAGE)–STING1 signaling in macrophages and promotes pro-inflammatory cytokine production [85]. This directly links cytoplasmic pH disruption to tumor cell death and immune activation.

These observations suggest that cytoplasmic pH targeting not only promotes direct tumor cell killing but also propagates downstream immune activation through lysosomal and Golgi-associated signaling pathways.

Organelle pH homeostasis and immune signaling

In tumor cells, lysosomal pH is often highly adapted to support metabolic recycling, proteostasis, and immune evasion [86, 87]. Accordingly, lysosomal alkalization can suppress tumor-protective autophagy while simultaneously enhancing antitumor immunity through overlapping mechanisms (Fig. 2C).

Lysosomal alkalization can drive immune metabolic reprogramming. Naphplatin modulates lysosomal pH in bone marrow-derived macrophages through its basic chemical structure, triggering mucolipin TRP cation channel 1 (MCOLN1)-Ca²⁺-dependent activation of the mitogen-activated protein kinase 14 (MAPK14)/nuclear factor kappa B subunit 1 (NFKB1) pathway and promoting M1 polarization of tumor-associated macrophages [88]. Similarly, lithium carbonate disrupts lysosomal acidification by interfering with V-ATPase, restoring lysosomal diacylglycerol–protein kinase C theta signaling and promoting SLC16A1 (also known as MCT1) localization to mitochondria [89]. This facilitates lactate utilization as an energy source for CD8⁺ T cells and improves the efficacy of immunotherapy in melanoma, colon cancer, and breast cancer models.

Lysosomal alkalization–associated lysosomal membrane permeability can also promote immune activation. Increased membrane permeability leads to release of cathepsins and damage-associated molecular patterns, including HMGB1 and adenosine triphosphate (ATP), thereby activating antigen-presenting cells and supporting T-cell expansion [90]. However, the immunological effects of lysosomal alkalization are context dependent. In ferroptosis-related settings, alkalization may promote tumor resistance through lysosomal ferrous iron retention and reduced lipid peroxidation [91]. In chronically activated effector CD8⁺ T cells, excessive ammonia generated from mitochondrial glutamine catabolism neutralizes lysosomal acidity through Rh family C glycoprotein (RHCG), thereby impairing cytotoxic function [17]. These observations suggest that the same organelle pH shift may be beneficial in tumor cells but detrimental in effector immune cells.

In addition to lysosomes, Golgi and mitochondrial pH regulation also contribute to immune signaling. In stimulator of interferon genes–associated vasculopathy with onset in infancy (SAVI) mouse models, ADP-ribosylation factor GTPase-activating protein 2 (ARFGAP2) deficiency suppresses STING1-mediated Golgi proton efflux and reduces inflammatory pathology [92]. Likewise, compound C53 selectively blocks STING1 proton channel activity without abolishing interferon signaling [65], suggesting that STING1 immune signaling and pH-regulatory functions can be pharmacologically separated. Mitochondrial pH gradients are similarly important for energy metabolism and are functionally coupled with lysosomal and cytoplasmic pH homeostasis, together contributing to intracellular metabolic and immune balance [93–95].

Overall, the immunological consequences of organelle-specific pH modulation remain highly context dependent. Their compartment selectivity may prove valuable for predicting therapeutic efficacy, immune response, and treatment-related toxicity.

Immune-cell trade-offs and selective pH targeting

The opposing immune effects of pH modulation can be reconciled by considering spatial, cellular, and temporal selectivity. Extracellular buffering is most likely to be beneficial when it preferentially acts in acidic tumor regions and is timed to relieve acid-mediated immune suppression during immune activation or checkpoint blockade. However, alkalization of intracellular organelles, particularly lysosomes and endosomes, can have divergent consequences depending on the cell type. In antigen-presenting cells, endolysosomal pH controls antigen degradation, cross-presentation, and inflammatory signaling. Excessive or mistimed alkalization may impair antigen processing, whereas moderate modulation may enhance cross-presentation in selected settings. In tumor cells, lysosomal alkalization may promote lysosomal stress and cell death, but in immune cells it may disrupt antigen presentation or autophagic homeostasis.

Several strategies may reduce these risks. First, extracellular buffering agents should be designed to minimize systemic alkalosis and preferentially modify acidic tumor regions. Second, organelle-directed alkalizing agents should ideally be delivered selectively to tumor cells through antibody conjugation, ligand-directed carriers, tumor-enzyme-activated systems, or pH-responsive nanoparticles. Third, treatment scheduling should consider immune-cell biology, transient extracellular buffering may be compatible with immunotherapy, whereas prolonged lysosomal alkalization during antigen priming may suppress dendritic-cell and macrophage function. Fourth, immune monitoring should accompany pH-targeted therapy, including assessment of antigen presentation, T-cell activation, cytokine production, macrophage polarization, and cytotoxic effector function.

Therefore, targeted pH buffering can address immune-related toxicity only if it achieves adequate spatial, cellular, and temporal selectivity. Otherwise, the same perturbation that stresses tumor cells may also compromise immune-mediated tumor elimination.

Therapeutic modulators of pH alkalization

Therapeutic modulation of pH can suppress tumor growth in selected contexts through multiple mechanisms, including extracellular buffering, inhibition of acid extrusion, cytoplasmic pH disruption, and organelle-specific pH perturbation. These approaches can be broadly classified according to the compartment or regulatory node they primarily target, including tumor microenvironment buffering, carbonic anhydrase and proton transporter inhibition, cytoplasmic pH modulation, lysosomal or endosomal alkalization, and pH-responsive drug delivery (Table 1).

Table 1.

Representative pH modulators and their mechanisms of action

Modulators	Function	Primary targets / mechanisms	Models	Refs
Sodium bicarbonate	Enhance tumor sensitivity to chemotherapeutic agents such as mitomycin and doxorubicin	Neutralizing the acidity of TME	Breast cancer, liver cancer and PDAC, etc	[25, 27, 120]
LDH NPs	Slowly releases OH⁻ to inhibit tumor cell autophagy, reprograms macrophages into an M1 antitumor phenotype, and increases T cell infiltration	Alkalinization of the TME	Melanoma, colon cancer	[97]
CHHD-Cu NPs	Inhibit CA9, elevate extracellular pH, improve the immunosuppressive microenvironment, and enhance effector T cell function	CA9	H22 mouse tumor model	[98]
JTC801	Selectively inducing cytoplasmic alkalization triggers alkaliptosis	Induction of cytoplasmic alkalization (involving multiple layers of regulatory networks)	PDAC, ovarian cancer	[16, 30, 31, 36]
Bafilomycin A1	Inhibits lysosomal acidification, disrupts its function, and induces cancer cell death	V-ATPase	Various cancer cell lines	[121–125]
EN6	Activate V-ATPase, but under specific conditions may also affect cancer cells by disrupting pH homeostasis	V-ATPase	Various cancer cell lines	[44, 91]
Monensin	Disrupting the pH homeostasis of the Golgi apparatus induces cancer cell death	Golgi apparatus	Fibrosarcoma, NSCLC	[69]

However, these strategies should be interpreted cautiously. Although many pH-modulating agents are designed to act within a defined compartment, downstream responses may propagate through metabolism, ion transport, vesicular trafficking, mitochondrial stress, and immune signaling. Conversely, pH modulation in one compartment may be buffered or uncoupled from other compartments by compensatory transporters and metabolic adaptation. Therefore, the therapeutic effect of a pH-modulating intervention cannot be inferred solely from its nominal target. It requires direct measurement of target-compartment pH, assessment of compensatory responses, and validation of the relevant cell-death pathway.

At the level of the tumor microenvironment, sodium bicarbonate increases the sensitivity of breast cancer models to mitomycin and doxorubicin without elevating extracellular pH in normal tissues [96]. Similar effects are observed in liver cancer models [27]. However, conventional buffering systems are often limited by short duration of action and poor tumor selectivity, which has prompted the development of nanotechnology-based approaches. For example, layered double hydroxide nanoparticles slowly hydrolyze within tumors to release hydroxide ions (OH⁻), inhibit autophagy, promote macrophage polarization toward an M1-like phenotype, and increase T-cell infiltration in melanoma and colon cancer models [97]. Similarly, coupled hydroxyethyl starch-based copper nanoparticles elevate tumor extracellular pH by inhibiting CA9, thereby improving the immunosuppressive microenvironment and enhancing effector T-cell infiltration and function [98].

Within tumor cells, several agents directly modulate intracellular pH. Latrunculin A elevates intracellular pH through SLC9A1 following cytoskeletal disruption and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) activation, inducing both cytoplasmic and organellar alkalization in multiple cell models. Similar effects have been reported for blebbistatin and Y-27632 [99]. In contrast, the small molecule JTC801 more selectively induces cytoplasmic alkalization and triggers alkaliptosis, showing potent anticancer activity in pancreatic cancer and drug-resistant ovarian cancer models. This process is frequently accompanied by lysosomal pH disruption, further supporting lysosomes as a therapeutically actionable target.

Among organelle-directed strategies, targeting lysosomal pH homeostasis, particularly V-ATPase, is especially promising. Multiple V-ATPase inhibitors, including salicylihalamide A [100], lobatamide [101], diphyllin [102], bafilomycin A1, and concanamycin A [102, 103], as well as the activator EN6 [44], demonstrate significant anticancer efficacy across multiple tumor types. Clinically used gastric acid inhibitors such as omeprazole, which also affect V-ATPase activity, may provide an additional translational strategy [104]. The small molecule 249C shows selective activity in RAS-mutant lung, colon, and pancreatic cancers through V-ATPase inhibition, providing preclinical proof of concept for mutation-guided pH-targeted therapy [105].

Other organelle-targeted alkalization strategies also show therapeutic potential. Golgi-directed alkalinizing agents show favorable antitumor effects in fibrosarcoma and non-small cell lung cancer models [68, 69]. Despite differences in compartment selectivity, many of these interventions ultimately converge on disruption of the global intracellular pH regulatory network and activation of downstream cell death pathways.

From a translational perspective, continued investigation of the signaling connections within this spatially organized pH regulatory network will be essential for developing safer and more effective pH-targeted therapies.

Quantitative pH biology and attribution of biological effects

A critical issue in pH-targeted cancer therapy is the magnitude, duration, and localization of pH changes required to produce biological effects. Tumor cells tightly regulate intracellular pH, and many reported differences between malignant and nonmalignant cells, or between treated and untreated conditions, are within a narrow range of tenths of a pH unit. Such small changes may be biologically meaningful for pH-sensitive enzymes and signaling proteins, but they also raise important interpretive challenges. In many studies, cell death is attributed to alkalization or acid neutralization without direct demonstration that the relevant pH compartment has changed sufficiently to explain the observed phenotype.

Future studies should therefore report quantitative pH values rather than qualitative descriptions such as alkalization or acidification. Ideally, pH measurements should be compartment-specific, calibrated, time-resolved, and linked to functional endpoints. For example, extracellular pH measurements should distinguish bulk medium pH from pericellular pH in poorly perfused tumor regions. Cytoplasmic pH should be distinguished from lysosomal, endosomal, Golgi, and mitochondrial pH. Organelle pH measurements should consider probe localization, calibration range, photostability, and perturbation caused by the probe itself.

It is also essential to determine whether observed biological effects are directly attributable to pH changes or to secondary pharmacological consequences. Many pH-modulating agents affect ion gradients, membrane potential, metabolism, vesicular trafficking, autophagy, mitochondrial function, and drug accumulation independently of pH. Therefore, mechanistic studies should combine direct pH measurement with genetic perturbation, orthogonal pharmacological tools, rescue experiments, and pathway-specific cell-death assays. Such quantitative validation will be necessary to distinguish true pH-dependent regulated cell death from nonspecific toxicity or indirect stress responses.

Multiscale monitoring of pH dynamics in cancer

In this section, we focus on the representative techniques and methods for monitoring different cell compartments, and further point out the limitations of these methods.

Monitoring measures

Because the efficacy and toxicity of pH-targeted interventions depend on the compartment engaged and the extent of compensatory buffering, dynamic pH monitoring is increasingly important for both mechanistic studies and clinical translation. Approaches that capture compartment-specific pH dynamics help define treatment response, predict toxicity, and guide patient stratification (Fig. 3; Table 2). Beyond providing mechanistic insight, these technologies also serve as potential biomarkers for real-time assessment of therapeutic efficacy and resistance.

Fig. 3.

Monitoring pH dynamics in cancer. Monitoring technologies enable dynamic and compartment-specific assessment of pH changes across extracellular, cytoplasmic, and organelle compartments, thereby supporting mechanistic studies, biomarker discovery, and therapeutic response evaluation. 1) Extracellular microenvironment: Ultra-pH-sensitive (UPS) nanoprobes detect severely polarized extracellular acidic regions (SPEARs; pH < 5.3) generated by lactate–proton co-export. Near-infrared II (NIR-II) ratiometric probes enable high-sensitivity imaging of dynamic tumor microenvironment pH changes in vivo. pH-sensitive photoacoustic chemical imaging (PACI) nanoparticles provide non-invasive three-dimensional pH mapping and correlate tumor microenvironment alkalization with improved chemotherapy response. LET-R photosensitizers further reveal immunosuppressive tumor regions. 2) Cytoplasm: Cytoplasmic pH is monitored using chemical probes such as BCECF-AM, seminaphthorhodafluor-1 acetoxymethyl ester (SNARF-1-AM), and pHrodo, as well as genetically encoded sensors including pHluorin. These approaches enable dynamic quantification of intracellular pH within the physiological range of approximately 7.2–7.4. 3) Intracellular organelles: Organelle-targeted probes, including LysoSensor Yellow/Blue, lysosome-targeted pHluorin, Golgi-targeted pHluorin, and B26 probes, allow high-fidelity mapping of subcellular pH dynamics, particularly within lysosomes and the Golgi apparatus

Table 2.

Technologies for monitoring tumor pH dynamics: advantages and limitations

Technology/probes	Application context	Advantages	Disadvantages	Models	Ref
Relative pH magnetic resonance imaging	Extracellular microenvironment	Non-invasive, high spatial resolution, capable of quantitatively mapping pH heterogeneity within tumors, and utilizing iron-based contrast agents with high safety	Equipment is expensive and technical operations are complex	4T1 breast cancer mouse model	[126]
Photoacoustic imaging	Extracellular microenvironment	Non-invasive, quantitative, with three-dimensional spatial resolution, and superior penetration depth compared to purely optical imaging	Relies on exogenous contrast agents (nanoparticles), with technology currently in the preclinical research phase	Patient-derived breast cancer xenograft model	[108]
NIR-II FRET ratio fluorometric probes	Extracellular microenvironment	Near-infrared region II offers superior penetration depth, high ratio measurement accuracy, and enables dynamic visual monitoring	It remains necessary to overcome the scattering and absorption of light by biological tissues	Living tumor model	[107]
BCECF-AM, SNARF-1-AM, pHrodo	Cytoplasm	High sensitivity, easy to use, minimal cell damage, and some models feature ratio measurement capabilities	Prone to leaking from cells, susceptible to photo-bleaching	Various cell lines	[127, 128]
pHluorin	Cytoplasm; organelles	Heritable, low cytotoxicity, suitable for long-term dynamic observation, capable of targeted fusion for specific organelle localization	Sensitivity range is typically limited to pH 5–8 and may be affected by interference from the complex intracellular environment	Various cell lines after transfection	[129–132]
LysoSensor	Acidic organelles such as lysosomes	Specific targeting of acidic compartments, pKa matching, low fluorescence background in neutral environments	Detection may be affected by factors such as phototoxicity, probe concentration, and cell type	Various cell lines	(45, 46, 133)

At the extracellular level, ultra-pH-sensitive (UPS) nanoprobes identify severely polarized extracellular acidic regions (SPEARs) formed by lactate–proton co-export through monocarboxylate transporters, with pH values below 5.3 across multiple cancer cell lines as well as three-dimensional tumor models. Notably, SPEARs correlate with cytotoxic T-cell exclusion [106]. Similarly, the ratiometric near-infrared region II fluorescence resonance energy transfer probe pTAS enables high-accuracy imaging of dynamic tumor microenvironment pH changes in vivo [107]. pH-sensitive photoacoustic chemical imaging (PACI) further provides non-invasive, quantitative, three-dimensional pH mapping and links tumor microenvironment alkalization to improved paclitaxel efficacy in breast cancer models [108]. Enzyme-activated anthocyanin photosensitizers, such as LET-R, generate both fluorescent and photoacoustic signals upon activation by carboxylesterase in hepatocellular carcinoma cells, thereby enabling enzyme activity–based stratification [109].

In contrast to the acidic extracellular microenvironment, the cytosolic pH of most cancer cells remains within a relatively narrow alkaline range, typically around 7.2–7.4. High-precision, dynamic, and minimally invasive monitoring of this core physiological parameter remains an important area for understanding tumor biology and developing novel therapies. Most currently available cytosolic pH probes are fluorescence-based. These reagents generally enter cells as membrane-permeable dyes and generate pH-dependent fluorescent signals after esterase activation. Common examples include BCECF-AM [110], pHrodo [111], and seminaphthorhodafluor-1 acetoxymethyl ester (SNARF-1-AM) [112]. In addition, theranostic probes based on CA9 inhibitors linked to weakly basic moieties such as morpholine specifically target tumors, disrupt lysosomal pH homeostasis, induce alkaliptosis [113], and simultaneously enable visualization of the therapeutic process. However, these probes are primarily suited for in vitro applications and remain limited for long-term dynamic monitoring.

Compared with conventional chemical dyes, pH-sensitive green fluorescent protein variants, known as pHluorins, exhibit lower cytotoxicity and are better suited for long-term live-cell imaging under physiological conditions. Fusion of pHluorin with cytoplasmic localization sequences or intracellular proteins enables compartment-selective monitoring of cytoplasmic pH. Examples include fusion constructs with cytoskeletal proteins or metabolic enzymes [114–116]. Nevertheless, restricted sensitivity windows and susceptibility to local ionic and protein-context effects remain important limitations.

At the organelle level, commercial probes such as LysoSensor Yellow/Blue are widely used for lysosomal pH monitoring [117]. However, signal accuracy is often influenced by phototoxicity, probe concentration, cell type, and cross-reactivity with other organelles. Fusion of pHluorin with lysosome-specific proteins enables long-term dynamic monitoring of lysosomal pH [115], and the same strategy is readily applicable to the Golgi apparatus. In addition, newer probes such as B26 provide improved sensitivity for detecting lysosomal alkalization in live cells, particularly within the near-neutral pH range, and this performance is also validated in zebrafish models [118].

From a translational perspective, multiscale pH monitoring should be regarded as an essential platform for biomarker development, response prediction, and clinical implementation of pH-targeted therapies.

Limitations of pH monitoring for clinical translation

Although pH-sensitive probes and imaging platforms have substantially advanced the study of tumor pH, several limitations restrict their immediate clinical translation. First, calibration remains challenging because fluorescence intensity, probe distribution, tissue penetration, local concentration, oxygenation, and optical scattering can influence apparent pH values. Second, tumor pH is spatially heterogeneous and temporally dynamic, meaning that single time-point measurements may not capture clinically relevant pH states. Third, interpatient variability in perfusion, metabolism, stromal composition, and prior treatment may complicate standardization of pH thresholds for patient selection. Fourth, different technologies measure different pH compartments, including extracellular pH, intracellular pH, or organelle pH, and these values should not be treated as interchangeable.

Clinical implementation will also require reproducibility, regulatory validation, safety assessment, and integration with therapeutic decision-making. A useful monitoring strategy should not only detect tumor acidity or alkalization but also determine whether a pH-targeted intervention reaches the intended compartment, achieves sufficient target engagement, and correlates with biological outcomes such as cell death, immune activation, or therapeutic response. Therefore, pH imaging should be developed alongside biomarker frameworks and clinical trial designs that explicitly test whether measured pH changes predict treatment benefit.

Conclusions and future perspectives

Recent advances support compartmental pH regulation as an important and potentially targetable feature of cancer biology, but its therapeutic exploitation remains highly context dependent. Rather than representing a single uniform vulnerability, tumor pH regulation consists of multiple spatially organized and dynamically adaptive processes involving extracellular acidity, cytoplasmic buffering, transporter activity, and organelle-specific proton homeostasis. Current evidence strongly supports many compartment-specific pH functions, whereas the broader concept of an integrated tumor-wide pH regulatory network should be regarded as a useful working model that requires further experimental validation [119]. Therapeutic alkalization can suppress tumor growth, promote cell death, and improve antitumor immunity in selected preclinical settings. However, alkalization can also trigger compensatory buffering, transporter adaptation, metabolic rewiring, impaired antigen processing, immune-cell dysfunction, or even protumorigenic effects depending on biological context. Thus, the key translational challenge is not simply to neutralize acidity or induce alkalization, but to achieve quantitative, durable, cell-type-specific, and compartment-selective pH modulation.

A central concept emerging from current studies is that pH modulation should not be viewed simply as acid neutralization, but as a strategy to target specific regulatory nodes within a dynamic proton-control network. Depending on the site engaged, alkalization induces distinct biological outcomes, including extracellular immune reprogramming, cytoplasmic stress signaling, and organelle-centered execution pathways. This spatial selectivity is likely to be a major determinant of therapeutic efficacy and toxicity.

Despite substantial progress, several challenges remain. The mechanisms coordinating pH coupling across biological scales remain incompletely understood, particularly how local perturbations propagate through the pH regulatory network and how tumors activate compensatory buffering pathways. In addition, current pH modulators are limited by insufficient selectivity, short duration of action, and off-target toxicity. Biomarkers and longitudinal monitoring tools suitable for clinical translation also remain underdeveloped, restricting patient stratification and response prediction. The factor contributing to this barrier to clinical translation may be the high degree of redundancy in pH regulation. Tumor cells express a variety of acid–base regulatory factors. The inhibition or buffering effect on one of these components can be compensated for by others. Furthermore, systemic pH regulation is also limited by the host’s physiological functions. The pH of blood and tissues is strictly regulated; sustained systemic alkalization may be limited by metabolic alkalosis, electrolyte imbalances, gastrointestinal tolerance issues, and patient compliance. Additionally, drug delivery poses challenges because the most relevant pH compartments are spatially heterogeneous and dynamically changing.

Another key unresolved question is how tumor heterogeneity, lineage-specific buffering capacity, and the differential sensitivity of immune versus malignant cells shape therapeutic responses in a context-dependent manner. Addressing these issues will be critical for improving selectivity and minimizing unintended toxicity.

Future studies should prioritize integration of site-selective pH modulation with precision delivery systems, dynamic imaging platforms, and combination strategies that block compensatory buffering pathways. Rational combinations with immunotherapy, targeted therapy, and nanomedicine may enhance tumor selectivity and therapeutic durability. A deeper understanding of signaling crosstalk within the spatially organized pH regulatory network will be essential for translating pH-targeted strategies into clinically actionable therapies.

In conclusion, pH-targeted therapy remains a promising but incompletely validated direction in cancer treatment. Its successful translation will require a shift from broad systemic alkalization toward mechanism-guided, quantitatively monitored, and compartment-selective intervention strategies.

Author contributions

All authors contribute to the writing, revision, and final approval of the manuscript.

Funding

National Natural Science Foundation of China, Guangzhou Health Science and Technology Young Talents Cultivation Program, China Postdoctoral Science Foundation, Postdoctoral Fellowship Program of the China Postdoctoral Science Foundation, Guangzhou Municipal Science and Technology Bureau.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors declare no competing interests.