Increasing Tumor Extracellular pH by an Oral Alkalinizing Agent Improves Antitumor Responses of Anti-PD-1 Antibody: Implication of Relationships between Serum Bicarbonate Concentrations, Urinary pH, and Therapeutic Outcomes

Abstract

Acidic extracellular pH (pHe) is characteristic of the tumor microenvironment. Several reports suggest that increasing pHe improves the response of immune checkpoint inhibitors in murine models. To increase pHe, either sodium bicarbonate (NaHCO₃) or citric acid/potassium-sodium citrate (KNa-cit) was chronically administered to mice. It is hypothesized that bicarbonate ions (HCO₃⁻), produced from these alkalinizing agents in vivo, increased pHe in the tumor, and excess HCO₃⁻ eliminated into urine increased urinary pH values. However, there is little published information on the effect of changing serum HCO₃⁻ concentrations, urinary HCO₃⁻ concentrations and urinary pH values on the therapeutic outcomes of immunotherapy. In this study, we report that oral administration of either NaHCO₃ or KNa-cit increased responses to anti-programmed cell death-1 (PD-1) antibody, an immune checkpoint inhibitor, in a murine B16 melanoma model. In addition, we report that daily oral administration of an alkalinizing agent increased blood HCO₃⁻ concentrations, corresponding to increasing the tumor pHe. Serum HCO₃⁻ concentrations also correlated with urinary HCO₃⁻ concentrations and urinary pH values. There was a clear relationship between urinary pH values and the antitumor effects of immunotherapy with anti-PD-1 antibody. Our results imply that blood HCO₃⁻ concentrations, corresponding to tumor pHe and urinary pH values, may be important factors that predict the clinical outcomes of an immunotherapeutic agent, when combined with alkalinizing agents such as NaHCO₃ and KNa-cit.

INTRODUCTION

Several reports have demonstrated that the interstitial extracellular pH (pHe) of tumors is acidic (pH 6.2–6.9) compared to normal tissues (pH 7.3–7.4) and the intracellular pH of tumor cells (pHi) can be more alkaline (pH 7.1–7.7) than normal cells (pH 7.0–7.2),^1–3) which creates a reversed extracellular to intracellular pH gradient in tumors relative to normal tissues. This reversed pH gradient is produced partly by the overstimulation of several ion transporters such as the Na⁺/H⁺ exchanger (NHE-1), the Na⁺-independent and Na⁺-dependent HCO₃⁻/Cl⁻ exchangers, and the H⁺/lactate cotransporter (the monocarboxylate transporter; MCT), which secret H⁺ ions into the extracellular space.⁴⁾ In addition, tumor-associated anabolic glycolysis, called the Warburg effect, is one of the principal factors leading to the acidic tumor extracellular environment.^5–7) In tumors, the glucose transporter GLUT1, which generates ATP by glycolysis, is overexpressed and several metabolic intermediates such as lactate and pyruvate are generated. Lactate, together with H⁺ ions, via the action of MCT, is transported to the extracellular fluid, leading to its acidification.⁸⁾ Many studies reported that the reversed pH gradient across the cellular membrane is strongly related to uncontrolled progression, angiogenesis, and metastasis of tumors.^9–13)

It has been reported that tumor acidity can influence responses to immunotherapy.^14–16) Acidification of the tumor interstitial space may promote escape of solid tumors from immune surveillance and potentially limit the efficacy of immunotherapies such as an immune checkpoint inhibitor.^17,18) Pilon-Thomas et al. reported that increasing tumor pHe with sodium bicarbonate (NaHCO₃), an alkalinizing agent, increased responses to immune checkpoint inhibitors in a B16 melanoma murine model, where it was associated with increased T-cell infiltration.¹⁷⁾ These proof-of-concept studies suggest that the acidic pHe within the tumor microenvironment is a potential target to improve the response to immunotherapies.

Oral NaHCO₃ administration was used in several studies to modulate the acidic pHe in solid tumors.^18–20) Raghunand et al. have shown that NaHCO₃ effectively reversed pH gradients in tumors, but not in normal tissues.²¹⁾ The in vivo bicarbonate buffer system involves the balance of carbonic acid (H₂CO₃), HCO₃⁻ and carbon dioxide (CO₂). It plays an important role in maintaining the homeostatic control of blood pH, and as a consequence, contributes to tumor pHe.²²⁾ Oral administration of NaHCO₃ would, in theory but never elucidated, increase serum HCO₃⁻ concentrations and deliver excess HCO₃⁻ into solid tumors. The HCO₃⁻ traps a H⁺ ion in the tumor interstitial space and forms H₂CO₃, resulting in the increased tumor pHe. The hydration rate of CO₂ is much higher than the dehydration rate of H₂CO₃,²³⁾ suggesting that H₂CO₃ quickly dissociates into CO₂ and H₂O. Excess CO₂ is lost through the lungs. Renal glomerular filtration regulates blood levels of HCO₃⁻ and acid secretion.²⁴⁾ We can hypothesize that marked increases of urinary HCO₃⁻ concentration and urinary pH values resulting from increases in serum HCO₃⁻ will lead to increases in the tumor pHe. To date, however, few reports have examined the effects of an alkalinizing agent on increasing serum HCO₃⁻ concentrations, urinary HCO₃⁻ concentrations, and urinary pH values, and showed the relationships of these on the therapeutic outcomes of immunotherapies.

In this study, we show that the change in serum HCO₃⁻ concentrations and subsequent changes in urinary HCO₃⁻ concentrations, urinary pH values, and corresponding tumor pHe, by oral administration of NaHCO₃ or citric acid/potassium-sodium citrate (KNa-cit). Oral KNa-cit is a drug used to treat metabolic acidosis,²⁵⁾ and may also be a good means of generating HCO₃⁻ and alkalinizing the acidic pHe in solid tumors, because KNa-cit is effectively converted to HCO₃⁻ in a body after intestinal absorption.²⁶⁾ In addition, we show the neutralization of tumor pHe by oral administration of alkalinizing agents increases responses to anti-programmed cell death-1 (PD-1) antibodies, an immune checkpoint inhibitor, and the relationship between the change in urinary pH values and the anticancer effects.

MATERIALS AND METHODS

Materials

NaHCO₃ and magnesium oxide (MgO) were purchased from KENEI Pharmaceutical (Osaka, Japan). Anti-PD-1 antibody (InVivomAb anti-mouse PD-1 (CD279), clone: RMP1-14) was purchased from Bio X Cell (NH, U.S.A.). All other reagents were of analytical grade.

Cells and Animals

The B16 murine melanoma cells (RCB2638) and Colon-26 murine colorectal carcinoma cells (RCB2657) were purchased from the RIKEN BioResource Center (Ibaraki, Japan). The cells were cultured in RPMI-1640 medium (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) supplemented with 10% heat-inactivated fetal bovine serum (Corning, NY, U.S.A.), 100 units/mL penicillin and 100 µg/mL streptomycin (ICN Biomedicals, CA, U.S.A.) in a 5% CO₂/air incubator at 37 °C.

BALB/c mice (female, 5 weeks old) and C57BL/6 mice (female, 5 weeks old) were purchased from Japan SLC (Shizuoka, Japan). The experimental animals were allowed free access to water and mouse chow, and were housed under controlled environmental conditions (constant temperature and humidity, and a 12-h dark–light cycle). All animal experiments were evaluated and approved by the Animal and Ethics Review Committee of Tokushima University.

Tumor-Bearing Mouse Model

B16 tumor-bearing mouse model was established by subcutaneous inoculation of B16 cells (2 × 10⁶ cells/mouse) in the flank of C57BL/6 mice. Colon-26 tumor-bearing mouse model was established by subcutaneous inoculation of Colon-26 cells (2 × 10⁶ cells/mouse) at a flank region of BALB/c mice. All animal experiments were initiated when the tumors reached about 100 mm³ in size.

Measurement of Serum HCO₃⁻, Urinary HCO₃⁻ and Urinary pH Values

BALB/c mice were orally administered with a dose of NaHCO₃ (500 mg/kg) plus MgO (165 mg/kg) or KNa-cit (500 mg/kg), a citrate mixture composed of 2 mol potassium citrate, 2 mol sodium citrate and 1 mol citric acid. Each reagent was dissolved in water for oral administration. At selected time points post administration (0, 0.5, 1, 2, 4, 6, 8, and 24 h), blood was collected from the postcaval vein of the mice. Serum samples were obtained by centrifugation of the blood (3000 rpm, 4 °C, 15 min) following to incubation for 30 min at room temperature. The serum HCO₃⁻ concentrations were measured using the HCO₃⁻ measurement kit (Diacolor^® CO₂ clinical diagnostic reagent, TOYOBO, Osaka, Japan). At the same time points, urine was collected by pushing on the lower abdominal region of the mice. The urinary HCO₃⁻ concentrations were measured using the HCO₃⁻ measurement kit (Diacolor^® CO₂). Urinary pH values were measured with the pH test paper (pH 5.5–9.0, AS ONE, Osaka, Japan).

Measurement of Intratumor pH Using a Microelectrode

Colon-26 tumor-bearing mice were allowed to drink water containing 200 mM NaHCO₃ ad lib. After 14-d ingestion of NaHCO₃ solution, the interstitial pHe in the tumor was measured using microelectrode with pH meter as referred the previous publications.^20,27) The mice were anesthetized by isoflurane inhalation and then stabbed with a reference electrode (MI-401F, Microelectrodes, NH, U.S.A.) into the subcutaneous region and with a pH electrode (MI-408B TIP, Microelectrodes) into the center of the tumor. Electrodes were calibrated before the measurements using standard pH 4.01 and 7.00 buffers. One measurement was taken at each mouse and plotted.

Determination of in Vitro Expression on Phospho-S6 Ribosomal Protein (PS6RP)

B16 cells were seeded onto 6-well plates (1.0 × 10⁵ cells/well) and were pre-incubated for 24 h. The medium was changed to NaHCO₃-free fresh media at different pH (pH 6.0, 6.2, 6.4, or 7.4) adjusted by adding HCl or NaOH. After 24-h incubation, the cells were lysed with a lysis solution composed of 0.1% Triton X-100, 8 mg/mL NaCl, 8% glycerol, 0.28 mg/mL ethylenediaminetetraacetic acid (EDTA) and 2.42 mg/mL Tris–HCl (pH 7.4) including protease inhibitors (ProteoGuard™, TaKaRa BIO, Shiga, Japan). Total protein contents were measured using DC™ Protein Assay (Bio-Rad Laboratories, CA, U.S.A.). The cell extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10 µg total protein/well) using polyacrylamide gels (ATTO, Tokyo, Japan). The expression level of PS6RP in the cells was determined by Western blotting under the following conditions: blocking with 5% bovine serum albumin (BSA) in Tris-buffered saline for 1 h at 37 °C; incubation with a primary antibody of rabbit anti-mouse PS6RP antibody (Cell Signaling Technology, MA, U.S.A.) or rabbit polyclonal antibody to β-Actin (ab16039, Abcam, Cambridge, U.K.) overnight at 4 °C; incubation with a secondary antibody of horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) antibody (Abcam) for 1 h at 37 °C. The expression of PS6RP was visualized with a chemiluminescent substrate (ECL prime, GE Healthcare Bioscience, Buckinghamshire, U.K.) and analyzed with a LAS-4000 system (GE Healthcare Bioscience).

Determination of in Vivo Expression on PS6RP

B16 tumor-bearing mice were orally administered with 7 daily doses of NaHCO₃ (500 mg/kg/d) plus MgO (165 mg/kg/d). One day after the last administration, tumors were harvested and snap-frozen in optimal cutting-temperature compound (O.C.T compound, Sakura Finetek Japan, Tokyo, Japan). The frozen tumor sections (7-µm thick) were prepared using a cryostat and mounted on a MAS-coated glass slide (Matsunami Glass, Osaka, Japan). The sections were fixed by incubation in 4% paraformaldehyde for 10 min at room temperature. The tumor sections were stained with a primary antibody of rabbit anti-mouse PS6RP and then with a secondary antibody of Alexa Fluor 488-labeled anti-rabbit antibody (Thermo Fisher Scientific, MA, U.S.A.). The expression of PS6RP was imaged using a BZ-9000 fluorescence microscope (Keyence, Osaka, Japan) with a filter set at Ex. 490 nm and Em. 525 nm. Fluorescent intensity in the region of interest (ROI) was quantified using ImageJ, an open source Java-written program provided by NIH.

Therapeutic Effects of Anti-PD-1 Antibody Combined with Alkalinizing Agents in a Murine Melanoma Model

B16 tumor-bearing mice were orally administered with either NaHCO₃ (500 mg/kg/d) plus MgO (165 mg/kg/d) or KNa-cit (500 mg/kg/d) every day. The daily oral administration was performed using a feeding needle and was continued for over 14 d until the mice were terminated at 20% body weight loss. On 3 d after the first administration of alkalinizing agents, in the combination group, the mice were intraperitoneally injected with a dose of anti-PD-1 (5.0 mg/kg/d). The day when the anti-PD-1 was injected was set at Day 0 in the Figures. Volume of tumor and body weight of the treated mice were recorded twice weekly, and survival of the mice was monitored daily. Urinary pH was measured twice weekly using the pH test paper (pH 5.5–9.0, AS ONE). The tumor growth inhibition (%TGI) was calculated using the following formula (TV: Tumor volume).²⁸⁾

  

The mean survival time (MST) was recorded the mortality on a daily basis, and the increased life span (%ILS) for the treatment groups was calculated using the following formula.^29,30)

  

Effects of Combined Anti-PD-1 and KNa-Cit Treatment on the Infiltration of Immune Cells into Tumors

B16 tumor-bearing mice were orally administered, using a feeding needle, with 7 doses of KNa-cit (500 mg/kg/d) daily from Day 0 to Day 6 and were intraperitoneally injected with a single dose of anti-PD-1 antibody (5.0 mg/kg/d) on Day 4. On Day 7, the tumor was harvested, and the tumor cell suspensions were prepared using gentle dissociation with a MACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) as described previously.³¹⁾ The tumors were incubated with a mixture of Collagenase Type I (FUJIFILM Wako Pure Chemical Corporation) and Dispase II (Roche Diagnostic, Mannheim, Germany) for 40 min at 37 °C. The suspensions were further dissociated in the presence of deoxyribonuclease (DNase) I (Roche Diagnostic) and were then filtered through a cell strainer (100 µm, Corning). The prepared cells were incubated with the following 4 combinations of antibody. (1) CD8⁺ T cells: eFluor 660-labeled anti-mouse CD8a antibody (eBioscience, CA, U.S.A.), (2) natural killer (NK) cells: fluorescein isothiocyanate (FITC)-labeled anti-mouse CD49b (Integrin alpha 2) antibody (eBioscience), (3) tumor-associated macrophages (TAM): eFluor 660-labeled anti-mouse F4/80 antibody (eBioscience) and Alexa Fluor 488-labeled anti-mouse CD206 antibody (BioLegend, CA, U.S.A.), (4) regulatory T cells (T_reg): eFluor 660-labeled anti-mouse CD4 antibody (eBioscience) and Alexa Fluor 488-labeled anti-mouse CD25 antibody (eBioscience). Flow cytometry analysis was performed using a Gallios flow cytometer (Beckman Coulter, CA, U.S.A.).

Effects of the Treatment with Anti-PD-1 and pHe Condition on Secretion of Interferon-Gamma (IFN-γ) from Spleen Cells

B16 tumor-bearing mice were intraperitoneally injected with a single dose of anti-PD-1 antibody (5.0 mg/kg/d). On 3 d post injection, the spleen was harvested, and the spleen cell suspensions were prepared as described previously.³²⁾ Spleen cells were obtained by pressing spleens through a cell strainer (100 µm, Corning) and suspended in RPMI-1640 medium. Red blood cells in the cell suspension were lysed via incubation with ammonium chloride lysis buffer (0.83% NH₄Cl) for 3 min. The spleen cells were cultured in 24-well plates (5 × 10⁶ cells/well) in the presence or absence of B16 cells (spleen cells : B16 cells = 10 : 1) using NaHCO₃-free RPMI-1640 media at pH 6.2 or 7.4, adjusted by adding HCl or NaOH. After 48-h incubation, the concentration of IFN-γ in the supernatant was determined using a mouse IFN-γ enzyme-linked immunosorbent assay (ELISA) Kit (Proteintech Group, IL, U.S.A.).

Statistical Analysis

Statistical differences between the groups were evaluated by ANOVA with the Tukey post-hoc test using the Prism 8 software (GraphPad Software, San Diego, CA, U.S.A.). All values are reported as the mean ± standard deviation (S.D.). The levels of significance were set at * p < 0.05, ** p < 0.01, *** p < 0.001.

RESULTS

HCO₃⁻ Concentrations in Serum and Urine, and Urinary pH, after Oral Administration of an Alkalinizing Agent

Serum HCO₃⁻ concentrations, urinary HCO₃⁻ concentrations and urinary pH values were investigated in naive mice after a single oral administration with NaHCO₃ plus MgO (Fig. 1A). Orally administered MgO proceeds the following reactions in stomach: 2HCl + MgO→MgCl₂ + H₂O,³³⁾ and the resultant MgCl₂ absorbs undesired CO₂ gas produced by orally administered NaHCO₃. Serum HCO₃⁻ concentrations increased two-fold up until 6 h post administration and decreased to basal level by 24 h. Urinary HCO₃⁻ concentrations increased rapidly within 1 h after administration and then decreased to basal levels by 24 h. Urinary pH increased to over 8.0 within 1 h after administration and then decreased to 6.0–7.0 by 24 h. The urinary pH values were correlated with changes in serum HCO₃⁻ concentrations and urinary HCO₃⁻ concentrations. The effect of another alkalinizing agent, KNa-cit, was investigated on the same parameters (Fig. 1B). Citrate, a main component of KNa-cit, is converted to HCO₃⁻ in a body and helps to correct the acid buildup in the blood.²⁶⁾ A single administration of KNa-cit, given to naive mice, tended to increase serum HCO₃⁻ concentrations over 24 h. Urinary HCO₃⁻ concentrations increased rapidly by 1 h and then decreased to the base level by 4 h. Urinary pH increased to approx. 7.4 within 1 h after administration and then fell back to 6.0–7.0 by 4 h. These results totally demonstrates that an oral administration of an alkalinizing agent, including NaHCO₃ and KNa-cit, expands blood HCO₃⁻ concentrations, corresponding to urinary HCO₃⁻ concentrations and urinary pH values, and may subsequently increase the tumor pHe.

Fig. 1. Serum HCO₃⁻ Concentrations, Urinary HCO₃⁻ Concentrations and Urinary pH Values after an Oral Administration of Alkalinizing Agent

BALB/c mice were orally administered with a dose of either (A) NaHCO₃ (500 mg/kg) plus MgO (165 mg/kg) or (B) KNa-cit (500 mg/kg). At selected time points post administration (0, 0.5, 1, 2, 4, 6, 8, and 24 h), blood and urine were collected from the treated mice. Serum HCO₃⁻ concentrations and urinary HCO₃⁻ concentration were measured using the HCO₃⁻ measurement kit. Urinary pH values were measured using pH test papers. The data are means ± standard deviation (S.D.) (n = 5–6).

Change in Tumor pHe by Oral Administration of an Alkalinizing Agent

To assess the change of interstitial tumor pHe after treatment with NaHCO₃, several approaches were applied. pH electrode is one of adequate tools to directly measure the intratumor pH.^20,27) As shown in Fig. 2A, subcutaneous pH in normal mice, as a reference, was neutral level (pH 7.15). The intratumor pH in non-treated tumor-bearing mice was acidified at pH 6.67, which is consistent with the other observations.³⁴⁾ Meanwhile, the oral treatment with NaHCO₃ clearly increased the intratumor pH to neutral level at pH 6.90.

Fig. 2. Observation of Tumor pHe after an Oral Administration of Alkalinizing Agent

(A) Colon-26 tumor-bearing mice were allowed to drink water containing 200 mM NaHCO₃ ad lib. After 14-d ingestion of NaHCO₃ solution, the interstitial pHe in the tumor was measured using microelectrodes with pH meter. A reference electrode or a pH electrode was stabbed into the subcutaneous region or the center of tumor, respectively. One measurement was taken at each mouse and plotted with means (n = 7–9, *** p < 0.001). (B) B16 cells were cultured in 6-well plates (1.0 × 10⁵ cells/well) in media of different pH (pH 6.0, 6.2, 6.4 or 7.4). After 24 h incubation, the expression level of PS6RP and β-actin in the cells was determined by Western blotting. (C) B16 tumor-bearing mice were orally administered with 7 daily doses of NaHCO₃ (500 mg/kg/d) plus MgO (165 mg/kg/d). On one day after the last administration, the frozen-tumor sections were prepared. PS6RP in the sections was stained by immunohistochemistry. The localization of PS6RP in both peripheral area and central area in the tumor sections was imaged using a fluorescence microscope. (D) The ROI in the images was quantified using ImageJ. The data are means ± S.D. (n = 5, RFU: relative fluorescence unit, * p < 0.05 vs. control). (Color figure can be accessed in the online version.)

It has been reported that the expression of PS6RP, a downstream target of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway,³⁵⁾ was higher at physiological pHe than at acidic pHe.¹⁹⁾ Thus, the expression of PS6RP in tumor cells was studied as a function of tumor interstitial pHe. In vitro PS6RP expression in B16 melanoma cells was increased when cultured in media at physiological pH (pH 7.4) compared to acidic pHs (pH 6.0–6.6) (Fig. 2B). In vivo PS6RP expression in the tumor was then evaluated in a B16 tumor-bearing mouse model. In the peripheral area of untreated tumors, a small somewhat focal increase in PS6RP expression was detected with little activity in the interior (Fig. 2C). In tumors treated with NaHCO₃, PS6RP expression was increased both at the tumor periphery and the tumor interior compared to untreated tumors (Figs. 2C, D).

Regarding the treatment with KNa-cit, the effect on neutralizing the B16 tumor was weaker compared to NaHCO₃ (data not shown), because KNa-cit could induce the tumor neutralization via producing NaHCO₃ that is one metabolite after oral administration of KNa-cit. These results strongly suggest that the treatment with an alkalinizing agent, such as NaHCO₃ and KNa-cit, can neutralize the tumor acidified interstitial pHe, presumably owing to the increasing intratumor HCO₃⁻ concentrations, corresponding to blood HCO₃⁻ concentrations.

Combined Effects of Oral NaHCO₃ with Anti-PD-1 on B16 Tumor-Bearing Mouse Model

The effects of increasing tumor pHe by oral NaHCO₃ on tumor response of anti-PD-1 were studied in a B16 tumor-bearing mouse model. As shown in Fig. 3A, the treatment with NaHCO₃ plus MgO scarcely decrease the growth rate of tumors compared to control (%TGI by 22.3%), and the treatment with anti-PD-1 alone delayed tumor growth (%TGI by 42.1%). The combination of NaHCO₃ plus MgO with anti-PD-1 was even more effective in suppressing tumor growth (%TGI by 80.1% > 22.3 + 42.1%), which means that the combination treatment could synergistically enhance the tumor-suppressive effects of anti-PD-1. Survival of the treated mice was also monitored (Fig. 3B) and the %ILS was calculated. The treatment with NaHCO₃ plus MgO did not increase the %ILS compared to the untreated control mice. Anti-PD-1 alone, and in combination with NaHCO₃ plus MgO, increased the %ILS by 33.7 and 52.6%, respectively. Regarding the body weight changes of the mice (Fig. 3C), there are no remarkable changes in body weight among the all groups, indicating a lack of severe adverse effects. A negative correlation was observed between urinary pH values and tumor volumes at Day 14 in the groups of NaHCO₃ plus MgO and combination with anti PD-1 (Fig. 3D); the higher the urinary pH values, the lower the tumor volumes. Survival periods of time were correlated with decreasing urinary pH values in the groups of NaHCO₃ plus MgO and the combination treatment (Fig. 3E). As shown in Fig. 1A, oral administration of NaHCO₃ plus MgO increased the urinary pH value up to 8.5 to 9.0. Consistently, sequential treatment with NaHCO₃ plus MgO or the combination with anti-PD-1 increased the urinary pH up to 8.5 to 9.0 in tumor-bearing mice until Day 11 (Fig. 3E). However, on Day 14, the urinary pH in mice treated with NaHCO₃ plus MgO was clearly acidified compared to that in the combination. This would be due to the reason that the tumor in mice treated with NaHCO₃ plus MgO substantially grew up to 5000 mm³ in size (Fig. 3A), produced excess H⁺ and acidified the body. In contrast, the combination treatment of NaHCO₃ plus MgO with anti-PD-1 effectively suppressed the growth of tumor at 1300 mm³ in size on Day 14 (Fig. 3A). Under such situation, small tumor produced less H⁺. and, in turn, the combined treatment still remained the urinary pH high. These results suggest that the treatment with an alkalinizing agent can improve responses to an immune checkpoint inhibitor.

Fig. 3. Combined Treatment with NaHCO₃ Plus MgO and Anti-PD-1 in Murine B16 Melanoma

B16 tumor-bearing mice were orally administered with NaHCO₃ (500 mg/kg/d) plus MgO (165 mg/kg/d) every day. On Day 3, in the combination group, the mice were intraperitoneally injected with a dose of anti-PD-1 (5.0 mg/kg/d). The day when the anti-PD-1 was administered was set at Day 0 in the figures. (A) Tumor volumes, (B) survival and (C) body weights of the treated mice were monitored. (D, E) Urinary pH was measured using pH test papers. In the treatment groups receiving NaHCO₃ plus MgO and combination with anti-PD-1, correlations were plotted (D) between the urinary pH change and tumor volumes on Day 14 and (E) between urinary pH change and survival of the mice. The data in A, C, and pH changes in E are means ± S.D. (n = 8–10, * p < 0.05, *** p < 0.001 vs. control, ^## p < 0.01 vs. NaHCO₃ plus MgO). (Color figure can be accessed in the online version.)

Combined Effects of Oral KNa-Cit with Anti-PD-1 on B16 Tumor-Bearing Mouse Model

Effects of KNa-cit, another alkalinizing agent, on the therapeutic response of anti-PD-1 was investigated. Treatment with KNa-cit alone did not affect tumor growth rate compared to that in control group (Fig. 4A). The combined treatment with KNa-cit and anti-PD-1 showed the tendency to inhibit the tumor growth (%TGI by 41.7%). In addition, the combined treatment prolonged the survival period of time in treated model mice with good ILS (24.2%) (Fig. 4B), which was consistent with the results of the combined treatment with NaHCO₃ plus MgO and anti-PD-1 (Fig. 3B). All the treatment groups showed no severe side effects during the treatment term (Fig. 4C).

Fig. 4. Combined Treatments with KNa-Cit and Anti-PD-1 in Murine B16 Melanoma

B16 tumor-bearing mice were orally administered with KNa-cit (500 mg/kg/d) every day. On Day 3, in the combination group, the mice were intraperitoneally injected with a dose of anti-PD-1 antibody (5.0 mg/kg/d). The day when the anti-PD-1 antibodies was given was set at Day 0 in the figures. (A) Tumor volumes, (B) survival times, and (C) body weights of the treated mice were monitored. The data in A and C are means ± S.D. (n = 7–8, ** p < 0.01 vs. control). (Color figure can be accessed in the online version.)

Infiltration of Immune Cells into B16 Tumors after the Combined Treatment with an Alkalinizing Agent and Anti-PD-1

Pilon-Thomas et al. reported that increasing of tumor pHe enhances the infiltration of immune cells into tumor tissues.¹⁷⁾ In the current study, we experimentally confirmed this observation using KNa-cit, because KNa-cit could produce much comprehensive alkalinizing effects via producing a metabolite of NaHCO₃ and shifting electrical equilibrium in a body. As shown in Fig. 5A, treatment with anti-PD-1 alone increased the tumor-infiltration of CD8⁺ T cell, an important cytotoxic effector cell type in anti-tumor immunity.³⁴⁾ Combined treatment with KNa-cit and anti-PD-1 further increased the infiltration of CD8⁺ T cell into tumors to a small degree. Treatment with anti-PD-1 alone tended to increase the population of TAM, M2-phenotype macrophages that promote the proliferation and survival of tumor cells,³⁶⁾ but the combined treatment with KNa-cit and anti-PD-1 had little effect. Change in the infiltrations of NK cells and T_reg was not observed in this study. Treatment with KNa-cit alone did not affect the tumor infiltration of immune cells including CD8⁺ T cell, TAM, NK cell, and T_reg (Fig. 5B).

Fig. 5. Infiltration of Immune Cells into the B16 Tumors in Combination Treatment with an Alkalinizing Agent and Anti-PD-1

B16 tumor-bearing mice were orally administered with KNa-cit (500 mg/kg/d) daily from Day 0 to Day 6 and were intraperitoneally injected with a dose of anti-PD-1 (5.0 mg/kg/d) on Day 4. On Day 7, tumors were harvested. (A) Tumor-infiltrating immune cells (CD8⁺ T cell, TAM, NK cell, and T_reg) after the treatment with anti-PD-1 or the combination of anti-PD-1 with KNa-cit were determined by flow cytometry and are shown as the percentage of total tumor cells. (B) Tumor-infiltrating immune cells after the treatment with KNa-cit alone were determined by flow cytometry. (C) The spleen was harvested from the same mice treated with/without anti-PD-1, and the spleen cells were cultured in 24-well plates (5 × 10⁶ cells/well) in the presence or absence of B16 cells under different pH conditions (pH 6.2 or 7.4). After 48 h incubation, secreted IFN-γ in the media was determined using a mouse IFN-γ ELISA Kit. The data are means ± S.D. (n = 4, ** p < 0.01, *** p < 0.001, n.s.: not significant). (Color figure can be accessed in the online version.)

We also elucidated the activation of immune cells under our treatment conditions by studying the ex vivo secretion of the IFN-γ cytokine from spleen cells collected from tumor-bearing mice treated with/without anti-PD-1 (Fig. 5C). When co-cultured with B16 cells, the spleen cells secreted higher levels of IFN-γ at physiological pH condition (pH 7.4) compared to acidic pH condition (pH 6.2) in both treatment groups. In addition, at pH 7.4, spleen cells from control tumor-bearing mice produce higher levels of IFN-γ compared to those from the anti-PD-1-treated mice, which might depend on the exhaustion of splenic T cells by anti-PD-1 immunotherapy.³⁷⁾ Spleen cells, in the absence of B16 cells, produced little or no IFN-γ (Fig. 5C). The ex vivo results indicate that the combined treatment with an alkalinizing agent and anti-PD-1 somewhat enhances the infiltration of cytotoxic CD8⁺ T cells into the tumor and would activate the infiltrated immune cells via increasing tumor interstitial pHe by oral alkalinizing agent.

DISCUSSION

Recent clinical reports suggest that acid-base balances in vivo are important for patient health status and may be involved in the clinical outcomes. Chan et al. reported that, in colorectal cancer patients undergoing resection of their primary tumors, the group with low serum HCO₃⁻ concentrations had a significantly lower 30-d overall survival than the group with normal HCO₃⁻ levels.³⁸⁾ In addition, Hamaguchi et al. reported that, in advanced pancreas cancer patients treated with an alkalinizing therapy (an alkaline diet with supplementary oral NaHCO₃), the median overall survival from the start of alkalinizing therapy of the patients with high urinary pH (>7.0) was significantly longer than those with low urinary pH (≤7.0) (16.1 vs. 4.7 months; p < 0.05).³⁹⁾ In this study, we confirmed that the treatment with either NaHCO₃ or KNa-cit increases serum HCO₃⁻ concentrations, as predicted by increase of urinary pH values (Fig. 1), and consequently neutralize the tumor interstitial pHe at both peripheral and interior regions (Fig. 2). These suggest that the alkalinizing treatment can improve the malignant tumor microenvironment, such as acidic nature, via increasing serum HCO₃⁻ concentrations.

It is reported that tumor acidity can influence responses to immunotherapy and ionizable anticancer drugs.^14–16) Acidification of the pHe may promote escape of solid tumors from immune surveillance and potentially limit the efficacy of immunotherapies such as an immune checkpoint inhibitor.^17,40) One can hypothesize that therapeutic interventions designed to increase the pHe in patients may be able to improve therapeutic outcomes. We tested this hypothesis in vitro and in vivo in murine tumor models and confirmed that increasing tumor pHe with an oral alkalinizing agent, NaHCO₃ plus MgO or KNa-cit, increased the response to anti-PD-1 in a B16 tumor-bearing mouse model (Figs. 3, 4), and there was a correlation between urinary pH values and the antitumor effects of anti-PD-1 immunotherapy (Fig. 3). To the best of our knowledge, this is the first report to suggest that the treatment with an oral alkalinizing agent can improve the tumor-growth suppressive effects of an immune checkpoint inhibitor via elevating serum HCO₃⁻ concentrations and subsequent neutralizing the acidic tumor interstitial pHe.

In the current study, we showed that the treatments with an alkalinizing agent, either NaHCO₃ plus MgO or KNa-cit, enhanced the therapeutic effect of anti-PD-1 immunotherapy, which is consistent with the previous results.¹⁷⁾ The increased responses were hypothesized to be a result of an alkalinizing agent having the effect of increasing tumor pHe and enhancing T-cell infiltration into tumors. Acidic pHe is reported to suppress not only growth of tumor cells,⁴¹⁾ but also several immune functions relating to immune checkpoint therapy.⁴⁰⁾ In vitro studies have shown that T-cell functions, including the interleukin (IL)-2 secretion and T-cell receptor activation, were suppressed in acidic pHe conditions.⁴²⁾ Our current study demonstrated that the combined treatment with KNa-cit and anti-PD-1 increased the infiltration of CD8⁺ T cells into the tumor (Fig. 5A) and could activate the cytotoxic immune cells (Fig. 5C). It has been reported that acidic pHe resulted in an increase in the numbers of immune-suppressor cells such as myeloid-derived suppressor cells (MDSC), T_reg, and TAM, switching the immune cytokine profile from a cytotoxic T-helper 1 (Th1) phenotype to an immunosuppressed Th2 phenotype.⁴³⁾ Therefore, tumor interstitial acidification could suppress tumor-specific immune responses and increase tumor growth. We observed that combined treatment with KNa-cit and anti-PD-1 decreased the immune suppressive TAM population in the tumor (Fig. 5A), corresponding to serum HCO₃⁻ concentrations (Fig. 1) and neutralization in tumor pHe (Fig. 2), which would be expected to enhance the therapeutic effect of anti-PD-1. It would appear that increasing tumor pHe by an oral alkalinizing agent can restore T-cell functions including IFN-γ abrogation and tumor necrosis factor (TNF)-α secretion.¹⁷⁾ This could be one explanation for the improved responses that we observed for immunotherapy with anti-PD-1 in combination with an alkalinizing agent.

It has been assumed that, after oral administration, NaHCO₃ plus MgO and KNa-cit produce HCO₃⁻ via the following mechanism: the gastrointestinal pH is increased by the buffering effect of NaHCO₃, enhancing secretion of H⁺ ions to maintain homeostasis in the gastrointestinal pH and with the production of HCO₃⁻. HCO₃⁻, produced from NaHCO₃, is absorbed from the intestinal tract, and serum HCO₃⁻ concentration gradually increases (Fig. 1A). KNa-cit is absorbed from the intestinal tract into blood and dissociated into its constituent ions. The citrate anion is excreted through the urine, causing a shift in the electrical equilibrium.^44,45) In order to recover this homeostasis, serum HCO₃⁻ concentrations increase accompanied with a decrease of serum H⁺ ions,²⁶⁾ corresponding to decreasing H⁺ ions in tumor tissue. Although the mechanisms producing HCO₃⁻ are different, the rates of HCO₃⁻ appearing in, or disappearing from, the blood were similar for both alkalinizing agents (Fig. 1). It is well known that excess HCO₃⁻ in the blood is eliminated through glomerular filtration into urine; renal filtration regulates blood levels of HCO₃⁻ and acid secretion.²⁴⁾ Accordingly, urinary HCO₃⁻ concentrations and pH values were increased in conjunction with increases in serum HCO₃⁻ concentrations. Interestingly, in KNa-cit, urinary pH corresponding to serum HCO₃⁻ concentrations rapidly dropped to base levels compared to NaHCO₃ plus MgO (Fig. 1B). This might be due to the presence of excess citrate ions secreted in urine that would tend to acidify the urine. Nevertheless, both of the alkalinizing agents that we used in this study would elevate serum HCO₃⁻ concentrations, which may have been sufficient to elevate tumor pHe.

In conclusion, we demonstrated that the treatment with an alkalinizing agent increases the acidic tumor interstitial pHe via elevating serum HCO₃⁻ concentrations, corresponding to urinary HCO₃⁻ concentrations and urinary pH values. Our results imply that serum HCO₃⁻ concentrations, corresponding to tumor pHe, and urinary pH values may be important factors to predict clinical outcomes of some types of anticancer treatments combined with an alkalinizing agent.

Acknowledgments

The authors are grateful to Professor Emerita Theresa M. Allen, University of Alberta for her helpful advice in developing the English manuscript.

Conflict of Interest

K.E. is President of Delta-Fly Pharma, Inc. The other authors declare no conflict of interest.

REFERENCES

• 1) Griffiths JR. Are cancer cells acidic? Br. J. Cancer, 64, 425–427 (1991).
• 2) Wike-Hooley JL, Haveman J, Reinhold HS. The relevance of tumour pH to the treatment of malignant disease. Radiother. Oncol., 2, 343–366 (1984).
• 3) Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res., 49, 6449–6465 (1989).
• 4) Madshus IH. Regulation of intracellular pH in eukaryotic cells. Biochem. J., 250, 1–8 (1988).
• 5) Warburg O. On the origin of cancer cells. Science, 123, 309–314 (1956).
• 6) Gatenby RA, Gawlinski ET. The glycolytic phenotype in carcinogenesis and tumor invasion: insights through mathematical models. Cancer Res., 63, 3847–3854 (2003).
• 7) Gatenby RA, Gillies RJ. Why do cancers have high aerobic glycolysis? Nat. Rev. Cancer, 4, 891–899 (2004).
• 8) Neri D, Supuran CT. Interfering with pH regulation in tumours as a therapeutic strategy. Nat. Rev. Drug Discov., 10, 767–777 (2011).
• 9) Cardone RA, Casavola V, Reshkin SJ. The role of disturbed pH dynamics and the Na⁺/H⁺ exchanger in metastasis. Nat. Rev. Cancer, 5, 786–795 (2005).
• 10) Harguindey S, Orive G, Luis Pedraz J, Paradiso A, Reshkin SJ. The role of pH dynamics and the Na⁺/H⁺ antiporter in the etiopathogenesis and treatment of cancer. Two faces of the same coin—one single nature. Biochim. Biophys. Acta, 1756, 1–24 (2005).
• 11) Peppicelli S, Bianchini F, Torre E, Calorini L. Contribution of acidic melanoma cells undergoing epithelial-to-mesenchymal transition to aggressiveness of non-acidic melanoma cells. Clin. Exp. Metastasis, 31, 423–433 (2014).
• 12) Onishi I, Lin PJ, Numata Y, Austin P, Cipollone J, Roberge M, Roskelley CD, Numata M. Organellar (Na⁺,K⁺)/H⁺ exchanger NHE7 regulates cell adhesion, invasion and anchorage-independent growth of breast cancer MDA-MB-231 cells. Oncol. Rep., 27, 311–317 (2012).
• 13) Xie R, Wang H, Jin H, Wen G, Tuo B, Xu J. NHE1 is upregulated in gastric cancer and regulates gastric cancer cell proliferation, migration and invasion. Oncol. Rep., 37, 1451–1460 (2017).
• 14) Barar J, Omidi Y. Dysregulated pH in tumor microenvironment checkmates cancer therapy. Bioimpacts, 3, 149–162 (2013).
• 15) Ibrahim-Hashim A, Estrella V. Acidosis and cancer: from mechanism to neutralization. Cancer Metastasis Rev., 38, 149–155 (2019).
• 16) Wojtkowiak JW, Verduzco D, Schramm KJ, Gillies RJ. Drug resistance and cellular adaptation to tumor acidic pH microenvironment. Mol. Pharm., 8, 2032–2038 (2011).
• 17) Pilon-Thomas S, Kodumudi KN, El-Kenawi AE, Russell S, Weber AM, Luddy K, Damaghi M, Wojtkowiak JW, Mule JJ, Ibrahim-Hashim A, Gillies RJ. Neutralization of tumor acidity improves antitumor responses to immunotherapy. Cancer Res., 76, 1381–1390 (2016).
• 18) Raghunand N, Mahoney BP, Gillies RJ. Tumor acidity, ion trapping and chemotherapeutics: II. pH-dependent partition coefficients predict importance of ion trapping on pharmacokinetics of weakly basic chemotherapeutic agents. Biochem. Pharmacol., 66, 1219–1229 (2003).
• 19) Faes S, Duval AP, Planche A, Uldry E, Santoro T, Pythoud C, Stehle JC, Horlbeck J, Letovanec I, Riggi N, Demartines N, Dormond O. Acidic tumor microenvironment abrogates the efficacy of mTORC1 inhibitors. Mol. Cancer, 15, 78 (2016).
• 20) Abumanhal-Masarweh H, Koren L, Zinger A, Yaari Z, Krinsky N, Kaneti G, Dahan N, Lupu-Haber Y, Suss-Toby E, Weiss-Messer E, Schlesinger-Laufer M, Shainsky-Roitman J, Schroeder A. Sodium bicarbonate nanoparticles modulate the tumor pH and enhance the cellular uptake of doxorubicin. J. Control. Release, 296, 1–13 (2019).
• 21) Raghunand N, He X, van Sluis R, Mahoney B, Baggett B, Taylor CW, Paine-Murrieta G, Roe D, Bhujwalla ZM, Gillies RJ. Enhancement of chemotherapy by manipulation of tumour pH. Br. J. Cancer, 80, 1005–1011 (1999).
• 22) Robey IF, Baggett BK, Kirkpatrick ND, Roe DJ, Dosescu J, Sloane BF, Hashim AI, Morse DL, Raghunand N, Gatenby RA, Gillies RJ. Bicarbonate increases tumor pH and inhibits spontaneous metastases. Cancer Res., 69, 2260–2268 (2009).
• 23) Gibbons BH, Edsall JT. Rate of hydration of carbon dioxide and dehydration of carbonic acid at 25 degrees. J. Biol. Chem., 238, 3502–3507 (1963).
• 24) Wesson LG Jr, Anslow WP Jr. Relationship of changes in glomerular filtration, plasma chloride and bicarbonate concentrations and urinary osmotic load to renal excretion of chloride. Am. J. Physiol., 180, 237–248 (1955).
• 25) Starke A, Corsenca A, Kohler T, Knubben J, Kraenzlin M, Uebelhart D, Wuthrich RP, von Rechenberg B, Muller R, Ambuhl PM. Correction of metabolic acidosis with potassium citrate in renal transplant patients and its effect on bone quality. Clin. J. Am. Soc. Nephrol., 7, 1461–1472 (2012).
• 26) McNaughton L, Cedaro R. Sodium citrate ingestion and its effects on maximal anaerobic exercise of different durations. Eur. J. Appl. Physiol. Occup. Physiol., 64, 36–41 (1992).
• 27) Estrella V, Chen T, Lloyd M, Wojtkowiak J, Cornnell HH, Ibrahim-Hashim A, Bailey K, Balagurunathan Y, Rothberg JM, Sloane BF, Johnson J, Gatenby RA, Gillies RJ. Acidity generated by the tumor microenvironment drives local invasion. Cancer Res., 73, 1524–1535 (2013).
• 28) Williams JI, Weitman S, Gonzalez CM, Jundt CH, Marty J, Stringer SD, Holroyd KJ, McLane MP, Chen Q, Zasloff M, Von Hoff DD. Squalamine treatment of human tumors in nu/nu mice enhances platinum-based chemotherapies. Clin. Cancer Res., 7, 724–733 (2001).
• 29) Higgins B, Kolinsky K, Linn M, Adames V, Zhang YE, Moisa C, Dugan U, Heimbrook D, Packman K. Antitumor activity of capecitabine and bevacizumab combination in a human estrogen receptor-negative breast adenocarcinoma xenograft model. Anticancer Res., 27 (4B), 2279–2287 (2007).
• 30) Garofalo A, Naumova E, Manenti L, Ghilardi C, Ghisleni G, Caniatti M, Colombo T, Cherrington JM, Scanziani E, Nicoletti MI, Giavazzi R. The combination of the tyrosine kinase receptor inhibitor SU6668 with paclitaxel affects ascites formation and tumor spread in ovarian carcinoma xenografts growing orthotopically. Clin. Cancer Res., 9, 3476–3485 (2003).
• 31) Shimizu T, Abu Lila AS, Nishio M, Doi Y, Ando H, Ukawa M, Ishima Y, Ishida T. Modulation of antitumor immunity contributes to the enhanced therapeutic efficacy of liposomal oxaliplatin in mouse model. Cancer Sci., 108, 1864–1869 (2017).
• 32) Shimizu T, Abu Lila AS, Kawaguchi Y, Shimazaki Y, Watanabe Y, Mima Y, Hashimoto Y, Okuhira K, Storm G, Ishima Y, Ishida T. A novel platform for cancer vaccines: antigen-selective delivery to splenic marginal zone B cells via repeated injections of PEGylated liposomes. J. Immunol., 201, 2969–2976 (2018).
• 33) Yamasaki M, Funakoshi S, Matsuda S, Imazu T, Takeda Y, Murakami T, Maeda Y. Interaction of magnesium oxide with gastric acid secretion inhibitors in clinical pharmacotherapy. Eur. J. Clin. Pharmacol., 70, 921–924 (2014).
• 34) Titu LV, Monson JR, Greenman J. The role of CD8(+) T cells in immune responses to colorectal cancer. Cancer Immunol. Immunother., 51, 235–247 (2002).
• 35) Lepin EJ, Zhang Q, Zhang X, Jindra PT, Hong LS, Ayele P, Peralta MV, Gjertson DW, Kobashigawa JA, Wallace WD, Fishbein MC, Reed EF. Phosphorylated S6 ribosomal protein: a novel biomarker of antibody-mediated rejection in heart allografts. Am. J. Transplant., 6, 1560–1571 (2006).
• 36) Solinas G, Germano G, Mantovani A, Allavena P. Tumor-associated macrophages (TAM) as major players of the cancer-related inflammation. J. Leukoc. Biol., 86, 1065–1073 (2009).
• 37) Davidson TB, Lee A, Hsu M, et al. Expression of PD-1 by T cells in malignant glioma patients reflects exhaustion and activation. Clin. Cancer Res., 25, 1913–1922 (2019).
• 38) Chan JCY, Diakos CI, Engel A, Chan DLH, Pavlakis N, Gill A, Clarke SJ. Serum bicarbonate is a marker of peri-operative mortality but is not associated with long term survival in colorectal cancer. PLOS ONE, 15, e0228466 (2020).
• 39) Hamaguchi R, Narui R, Wada H. Effects of alkalization therapy on chemotherapy outcomes in metastatic or recurrent pancreatic cancer. Anticancer Res., 40, 873–880 (2020).
• 40) Lardner A. The effects of extracellular pH on immune function. J. Leukoc. Biol., 69, 522–530 (2001).
• 41) McIntyre A, Hulikova A, Ledaki I, Snell C, Singleton D, Steers G, Seden P, Jones D, Bridges E, Wigfield S, Li JL, Russell A, Swietach P, Harris AL. Disrupting hypoxia-induced bicarbonate transport acidifies tumor cells and suppresses tumor growth. Cancer Res., 76, 3744–3755 (2016).
• 42) Calcinotto A, Filipazzi P, Grioni M, Iero M, De Milito A, Ricupito A, Cova A, Canese R, Jachetti E, Rossetti M, Huber V, Parmiani G, Generoso L, Santinami M, Borghi M, Fais S, Bellone M, Rivoltini L. Modulation of microenvironment acidity reverses anergy in human and murine tumor-infiltrating T lymphocytes. Cancer Res., 72, 2746–2756 (2012).
• 43) Kolosenko I, Avnet S, Baldini N, Viklund J, De Milito A. Therapeutic implications of tumor interstitial acidification. Semin. Cancer Biol., 43, 119–133 (2017).
• 44) Kowalchuk JM, Maltais SA, Yamaji K, Hughson RL. The effect of citrate loading on exercise performance, acid-base balance and metabolism. Eur. J. Appl. Physiol. Occup. Physiol., 58, 858–864 (1989).
• 45) Parry-Billings M, MacLaren DP. The effect of sodium bicarbonate and sodium citrate ingestion on anaerobic power during intermittent exercise. Eur. J. Appl. Physiol. Occup. Physiol., 55, 524–529 (1986).