Establishment of an Extracellular Acidic pH Culture System

Summary

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Abstract

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.

Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

Introduction

The tumor microenvironment plays critical roles in tumor progression and cancer cell metabolism^1,2,3. Cancer cells are often exposed to conditions such as hypoxia, nutrient deprivation, and acidic extracellular pH (pHe). However, the role of pHe in tumor progression has not been clarified as extensively as in hypoxia or nutrient starvation. The pHe in the tumor tissue can become acidic, reaching approximately pH 6.8^4,5. The acidic pH arises from aerobic and anaerobic glycolytic excretion of protons (H⁺) and lactate by proliferating cancer cells^5,6.

Recent studies revealed that acidic pHe-induced histone deacetylation, fatty acid oxidation, and exocytosis of acidic lysosomes for adaption to the severe acidic environment^7,8,9,10. However, the mechanisms through which extracellular acidification affects cancer behavior and the identity of key regulators in the acidic pH tumor microenvironment have not been fully determined. Furthermore, several reports described various acidic pH media using unclear concentrations of bicarbonate, Tris, PIPES, and HEPES buffer or lactate and HCl, but there are few reports to demonstrate the stability of the adjusted medium nor comprehensive comparison of several distinct acidic culture media^7,8,9,10

To elucidate the key regulators and metabolic changes in cancer cells in the context of extracellular acidification, we established a simple in vitro culture model to maintain an acidic pHe and examined the role of pHe in cancer cells¹¹. Using this method, we maintained an acidic culture medium with a pHe of 6.8 at 37 °C under 5% CO₂, using reduced bicarbonate concentrations in the culture medium. pH 7.4 was used as normal and control medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h during culture of PANC-1 and AsPC-1 pancreatic cancer cells. Because enhanced glycolysis accelerates excretion of not only protons but also lactate^7,8,9, we also established a culture method mimicking lactate-induced acidosis by adding lactate rather than reducing the bicarbonate concentration. Additionally, HCl-induced acidic pHe in the medium allows us to exclude the possibility that cellular responses to acidic pH culture medium are not due to a reduced concentration of bicarbonate. Moreover, using various media with a pH of 6.4 to 7.4 with different bicarbonate concentration, we can assess the extent of pHe effects on cellular responses.

Protocol

1. Preparation of Acidic Culture Medium

1. Preparation of control (pH 7.4) and low-pH (pH 6.8) culture medium
   1. Dissolve 4.75 g of DMEM powder without L-glutamine in 500 mL of water.
   2. Prepare 0.33 M NaHCO₃ solution in a pressure-tight bottle.
      Caution: It is important to use a pressure tight bottle since NaHCO₃ emits CO₂ gas when heated and thermally decomposed.
   3. Autoclave the medium and solution. Allow these buffers to cool to 25 °C.
   4. Add 5 mL of 200 mM L-glutamine, 5 mL of penicillin-streptomycin, and 50 mL of fetal bovine serum to 500 mL of DMEM without bicarbonate.
   5. For control (pH 7.4) medium, add 2.4 mL of 0.33 M NaHCO₃ solution per 100 mL of DMEM without bicarbonate. For low-pH (pH 6.8) medium, add 0.6 mL of 0.33 M NaHCO₃ solution per 100 mL of DMEM without bicarbonate.
   6. Check the medium pH after 24 h of incubation at 37 °C under 5% CO₂.
      NOTE: The amount of NaHCO₃ solution needs to be adjusted depending on the medium pH, which can differ depending on the CO₂ content of the incubator and air pressure. Ideally, for the first time, it is better to measure the medium pH with various bicarbonate concentrations to determine the appropriate amount of NaHCO₃ solution to add to DMEM.
      1. Collect culture medium immediately after removing the culture dish from the CO₂ incubator and measure the pH using a pH meter. Keep the medium temperature at 37 °C using a water bath if necessary. Measure the medium pH three times independently.
         NOTE: NaHCO₃ solution should be stored at 4 °C and each media should be freshly prepared. Commercially available products which can be used in this study are listed in Table of Materials.
2. Preparing lactate-induced or HCl-induced acidic culture medium
   1. Add 74 µL of lactate solution or 125 µL of 1 M HCl to 100 mL of control medium (prepared in step 1.1.5).
   2. Check the medium pH after 24 h of incubation at 37 °C under 5% CO₂.
      NOTE: The amount of lactate or HCl solution also needs to be adjusted according to the medium pH, which can differ depending on the CO₂ content in the incubator and air pressure. Ideally, it is better to measure the medium pH with serial lactate or HCl concentrations to determine the appropriate amount for adjusting to the desired pH of the medium.

2. Harvesting and Treating Cells

1. Start cultivating cells at 37 °C under 5% CO₂ at least 1 week before performing experiments.
   NOTE: The cell lines used in this study are listed in Table of Materials.
2. Passage cells when they reach 70 - 90% confluency. Do not allow the cells to become confluent. Change the medium every 2 - 3 days during daily cultivation.
3. Wash the cells by adding 5 mL PBS. Aspirate the PBS and add detaching enzyme such as 1 mL Trypsin.
4. Incubate at 37 °C until the cells are rounded and start to detach.
5. Add 5 mL of culture medium to the detached cells and deactivate enzyme. Transfer the cell suspension to a 15 mL tube and centrifuge for 3 min at 300 x g. Aspirate the supernatant and re-suspend the cells with culture medium.
6. Count the viable cells using a hemocytometer and Trypan-Blue.
7. Seed 5.0 x 10⁵ cells/well of a 10-cm dish in 10 mL of medium. Incubate the cells for 24 h at 37 °C under 5% CO₂.
8. Aspirate the culture medium, wash the cells with 5 mL PBS, and add 10 mL of the acidic culture medium prepared in Section 1. Incubate the cells for 24 h at 37 °C under 5% CO₂.

3. RNA Isolation

1. Aspirate culture medium and wash cells with 5 mL PBS. Disrupt the cells by adding RNA extraction reagent (see Tables of Materials).
2. Scrape the dish briefly, then remove the RNA extraction reagent with a pipette and deposit the RNA extraction reagent/cell lysate into a 1.5 mL tube. Leave at room temperature for 5 min.
3. Add 250 µL chloroform and shake the tube vigorously for about 15 s. Leave at room temperature for 5 min and centrifuge at ≥8,000 x g for 5 min.
4. Carefully remove the aqueous phase using a pipette. Leave behind some of the aqueous phase. Place in another 1.5 mL tube.
5. Add 550 µL isopropanol to the aqueous phase and mix gently. Leave at room temperature for 5 min. Centrifuge at maximal speed (≥20,000 x g) for 20 min at 4 °C.
6. Pour off the isopropanol and add 1 mL 75% ethanol in DEPC treated water. Mix gently. Centrifuge at 8,000 x g for 5 min.
7. Pipette off the ethanol and let the pellets air-dry.
8. Add approximately 15 - 25 µL (depending on the yield) of DEPC treated water to the RNA pellet. Mix thoroughly.
9. Measure the concentration of isolated RNA by measuring the OD at 260 nm using a spectrophotometer.

4. cDNA Synthesis

1. Combine the following components in a PCR reaction tube: up to 5 µg total RNA, 1 µL Oligo dT (50 µM) or Random Primer Mix (60 µM), 1 µL 10 mM dNTP, and nuclease-free water to a total volume of 14 µL.
2. Mix and briefly centrifuge the components using a small benchtop centrifuge (~5 s). Denature the sample RNA/primer for 5 min at 65 °C. Spin briefly (~5 s) using a small benchtop centrifuge and put the sample promptly on ice for at least 1 min.
3. Add the following components: 4 µL 5x Reverse Transcription buffer, 1 µL Reverse Transcriptase (200 U/µL), 1 µL RNase Inhibitor (40 U/µL).
   NOTE: Examples of commercially available kits are shown in Table of Materials
4. Cap the tube, mix, and then briefly centrifuge the contents using a small benchtop centrifuge (~5 s). Incubate the combined reaction mixture at 50 - 55 °C for 10 min.
5. Inactivate the reaction by incubating at 80 °C for 10 min.

5. Measurement of Acid-responsive Gene Expression Using Real-time PCR

1. Combine the following components in a 384-well reaction plate: 1 µL Template cDNA, 0.75 µL 10X N',N'-dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine, 0.3 µL 50 µM forward primer, 0.3 µL 50 µM reverse primer, 1 µL Taq polymerase, 2.5 µL 10x Taq polymerase buffer, 2.5 µL 20 mM dNTP, 16.65 µL water.
   NOTE: Example of commercially available kits are shown in Table of Materials. A list of primers is shown in Table 1. A 96-well reaction plate can be also used in this step. Other probes (e.g., Taqman probe) can also be used.
2. Set up the experiment and the following PCR program on a Real-time PCR System: 50 °C for 2 min, 1 cycle; 95 °C for 10 min, 1 cycle; 95 °C for 15 s and 60 °C for 1 min, 40 cycles; 95 °C for 15 s, 1 cycle; 60 °C for 15 s, 1 cycle; 95 °C for 15 s, 1 cycle.
3. Place the plate in the qPCR instrument. Start the program.
   NOTE: Upregulation of the pH responsive genes MSMO1, INSIG1, and IDI1 can be measured by protein levels.

Results

To determine the appropriate bicarbonate concentration, we prepared DMEM at range of 0 - 8 mM NaHCO₃ (final concentration in the culture medium) and succeeded in preparing culture media with pH ranging from 6.4 - 7.4 (Figure 1). We prepared DMEM with 8 mM NaHCO₃ (pH 7.4) as a control medium, and 2 mM NaHCO₃ (pH 6.8) as a medium with acidic pH according to a previous report stating that the extracellular pH reaches pH 6.8 for s...

Discussion

Here, we described a simple acidic pH culture system and its evaluation process. The combination of three methods of medium acidification, i.e., reduced bicarbonate concentration, lactate addition, and HCl addition, enabled us to investigate the pH-response mechanism thoroughly and to compare the cellular response to other tumor microenvironmental conditions such as hypoxia or nutrient starvation.

The key of this method is to determine the appropriate concentrations of bicarbonate, la...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
Dulbecco's Modified Eagle Medium“Nissui” 2	Nissui	05919
Fetal Bovine Serum	Thermo Fisher	10438
NaHCO3	Wako	191-01305
L-glutamine solution	Thermo Fisher	25030-081
Penicillin-Streptomycin Mixed Solution(Stabilized)	Nacalai	09367-34
0.5g/l-Tripsin/0.53mmol/l-EDTA Solution, with Phenol Red	Nacalai	32778-05
0.5%-Trypan Blue Stain Solution	Nacalai	29853-34
Lactic acid solution	Sigma-Aldrich	252476
Hydrochloric acid solution	Sigma-Aldrich	H9892
RNeasy Plus Mini Kit	QIAGEN	74134
SuperScript IV First-Strand Synthesis System	Thermo Fisher	18091
Power SYBR Green PCR Master Mix	Applied Biosystems	4368702
Name	Company	Catalog Number	Comments
Cell lines
PANC-1	ATCC	CRL-1469
AsPC-1	ATCC	CRL-1682
KMS-6	JCRB Cell Bank	JCRB0432
TIG	JCRB Cell Bank
MRC9	ATCC	CCL-212
HUVEC	ATCC	CRL-1730
Name	Company	Catalog Number	Comments
Equipment
NanoDrop One Microvolume UV-Vis Spectrophotometer with Wi-Fi	Thermo Fisher	ND-ONE-W
QuantStudio 5 Real-Time PCR System	Thermo Fisher	CRL-1682