The glucose detection checkerboard and lactic acid checkerboard were obtained from the Jiancheng Institute of Biological Engineering (Nanjing, China). RIPA cracking liquid kits were purchased from Biyuntian Biological Co., Ltd. (Shanghai, China). Dimethyl sulfoxide was obtained from Sigma Co., Ltd. (Beijing, China). Dulbecco's modified Eagles medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone (Logan, UT, USA). Transwell chambers were purchased from Millipore (Billerica, MA, USA). Matrigel and the One-Step RT-PCR kit were purchased from BD Biosciences (Franklin Lakes, NJ, USA). LDHA, PKM2, monocarboxylate transporter 1 (MCT1), SDH, fumarate hydratase (FH), matrix metalloprotease (MMP)-2 and MMP-9 and β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The MCT1-specific blocker was from Sigma.

Human pancreatic cancer cells [BxPc-3, Panc-1; obtained from the American Tissue Type Collection (ATCC; Manassas, VA, USA)] were maintained in DMEM supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), 0.1 mM nonessential amino acids, 0.2 mM glutamine, 1 mM pyruvate, and 10% heat-inactivated FBS and incubated in 5% CO₂ humidified atmosphere at 37̊C. Cells were grown to 80% confluence. In the invasion and migration experiments, the cells were cultured in DMEM without FBS.

Normal fibroblast (NF) and CAF cells were derived from patients with pancreatic cancer and pancreatic trauma from the Second Affiliated Hospital of Xi'an Jiaotong University. All patients were newly diagnosed and had not received any relevant treatment prior to surgery. Informed consent was obtained from all patients prior to obtaining the specimens. Fibroblast isolation was conducted as previously described (13). First, the tissue was trimmed to 1×1×1 mm and washed gently with phosphate-buffered saline (PBS) three times (5 min each). Next, the tissues were washed once with the medium and placed in fresh cell cultural medium containing 15% fetal calf serum, 2 mM L-glutamine and 10% penicillin. The tissue was cut with a sterile scalpel blade and sections of cells were gently scraped with a blunt blade. The cells were cultured in an incubator for 3–5 days at 37̊C and 5% CO₂. The medium was replaced once and every three days thereafter; after 14 days, the cells fully covered the Petri dish. When the cell density reached 80–90%, the cells were digested with trypsin and regenerated at a rate of 1:3. The CAFs and NFs used in the experiment were the 3rd and 5th generations of cells cultured in vitro, respectively, and showed no obvious aging phenotype.

CAFs were added to 6-well plates at a density of 1.5×10⁵/ml and rinsed with PBS after 24 h. The medium was replaced with serum-free medium and cultured for 48 h, after which the culture broth was collected and centrifuged to remove the cells and debris; the supernatant obtained was the CAF conditioned medium. These samples were stored at 4̊C. The conditioned medium of NFs was collected in the same manner.

The pancreatic cancer cells BxPc-3 and Panc-1 were added to Petri dishes at a density of 1.5×10⁵/ml; after 24 h, CAF-CM was added and the cells were cultured for 48 h. Cells in PBS or serum-free medium were used as controls. An inverted phase contrast microscope was used to observe the morphology and growth of pancreatic cancer cells in each Petri dish. Proteins were extracted from the cells.

Cell migration capability was evaluated using a scratch test. First, 10×10⁵ BxPc-3 and Panc-1 cells were seeded into 1.5 ml media in each well of a 24-well plate. The cells were grown to a confluent layer (48 h), and then, a scratch was made in each well using a pipette tip. Subsequently, the cells were washed gently with PBS, and then culture broths of CAFs and NFs were added to the respective wells. An image was captured at time point 0. The cells were then incubated at 37̊C in 5% CO₂ and images were acquired after 24 h. The 24 h time point was chosen to decrease the potential impact of proliferation on the closing of the scratch. ImagePro Plus 5.0 from the NIH (Bethesda, MD, USA) was used to standardize the results.

Cell invasion was examined using Transwell assays. Following incubation for 48 h, 3×10⁴ cells were transferred to the top of the Matrigel-coated invasion chambers (BD Biosciences) in serum-free DMEM. DMEM containing 10% FBS was added to the lower chamber. After 24 h, the non-invading cells were removed and the invading cells were fixed using 95% ethanol, stained with 0.1% crystal violet, and photographed at a magnification of ×100 under an inverted phase contrast microscope (Olympus CKX31/41; Olympus, Tokyo, Japan). The experiments were repeated three times.

According to Fischer et al (14) the glucose uptake rate is reflected by the amount of [³H]-2DG taken up by the cells. After 24 h in serum-free culture, the medium was changed to low-sugar DMEM, 37 kBq/ml [³H]-2DG was added, and the cells were cultured for another 24 h. After digestion, the small fraction of cells remaining was counted and other cells were lysed in 0.5 M NaOH for 15 min; the same volume of 0.5 M hydrochloric acid was added for neutralization. The dpm value in the cell lysate solution was examined using a microplate reader. [³H]-2DG intake by the cells was calculated as follows: Total cellular radioactivity - non-specific binding radioactivity)/24 h.

Cells in the 12-well plate were washed once with PBS, the medium was replaced with phenol-free red medium, and the cells were cultured for 20 h. The supernatant was collected according to the instructions of the lactic acid detection kit. Lactic acid content was examined using a DRY-CHEM FDC3500 analyzer (Fuji, Tokyo, Japan); additionally, digested cells were counted. The result reflected the amount of lactic acid generated/10⁶ cells.

After culturing the NF and CAF cells for 24 h, the solution was used to culture pancreatic cancer cells for 24 h. Cells grown in PBS or serum-free medium were used as blank controls. Fresh DMEM containing mitochondrial fluorescent probes (1:200) was added at 500 µl/well and incubated for an additional 30 min. The cells were stored and produced in a darkroom and immediately observed using an inverted fluorescence microscope.

Total RNA was extracted from the cells using TRIzol reagent. In total, 2 µg RNA was reversed-transcribed into first-strand cDNA using the RevertAid First Strand cDNA Synthesis kit (Thermo Scientific, Waltham, MA, USA). PCR primer sequences were as follows: MMP-2 forward primer, 5′-TGGTCCTGGTGCTCCTGGTG-3′ and reverse primer, 5′-GCTGCCTGTCGGTGAGATTGG-3′; MMP-9 forward primer, 5′-TGGTCCTGGTGCTCCTGGTG-3′ and reverse primer, 5′-GCTGCCTGTCGGTGAGATTGG-3′; LDHA forward primer, 5′-CCAACATGGCAGCCTTTTCC-3′ and reverse primer, 5′-TCACGTTACGCTGGACCAAA-3′; PKM2 forward primer, 5′-ATTATTTGAGGAACTCCGCCGCCT-3′ and reverse primer, 5′-ATTCCGGGTCACAGCAATGATGG-3′; MCT1 forward primer, 5′-TCGGTATCTTTGGATGGAGAGG-3′ and reverse primer, 5′-TGGTAACTTCATTTGGCTTCCC-3′; SDH forward primer, 5′-CGGAAGAGTGTATGGACCA-3′ and reverse primer, 5′CGACGTAGTCCTTGTTGAC-3′; FH forward primer, 5′-GCGGATCCGGACATTGAGT-3′ and reverse primer, 5′-CGAATTCTCACTTTGGACCCA-3′; β-actin forward primer, 5′-ATCGTGCGTGACATTAAGGAGAAG-3′ and reverse primer, 5′-AGGAAGGAAGGCTGGAAGAGTG-3′. The PCR conditions were as follows: an initial reaction at 42̊C for 1 h was used for cDNA synthesis, followed by denaturation at 94̊C for 5 min and 22 cycles of the following reactions: 94̊C for 30 sec, 55̊C for 30 sec and 72̊C for 30 sec. After the final cycle, the reaction was amplified at 72̊C for 10 min. The housekeeping gene β-actin was used as an internal reference.

In total, 5×10⁵ cells in the logarithmic growth phase were added to 0.5 ml of pre-chilled cell lysis buffer and incubated on ice for 30 min. After centrifugation, the supernatant was collected and the protein contents were measured. The proteins were separated by 10% SDS-PAGE and blotted onto a nitrocellulose membrane by semi-dry transfer. Next, the membrane was immersed in Tris-buffered saline with Tween-20 containing 5% skim milk for blocking followed by overnight incubation with the primary antibody at 4̊C. On the following day, the membrane was incubated with the secondary antibody conjugated to horseradish peroxidase at 1:2,000 dilution (Santa Cruz Biotechnology) at room temperature for 2 h, and then an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Amersham, UK) was used for staining. The membrane was photographed and the results were analyzed.

Each experiment was repeated at least three times. The data are expressed as mean ±SD and analyzed using the Student's t-test. P<0.05 indicates a statistically significant difference.

After 10 days, cell morphology was observed using an inverted phase contrast microscope, and NFs and CAFs both showed a spindle shape (Fig. 1). The CAFs were spindle- or fusiform-shaped, had inconsistent sizes, showed dense growth and exhibited a disordered arrangement. For NFs, the cells showed multiple flat stellate shapes with similar cell sizes, were arranged in the same direction, and had a radially outward appearance (Fig. 1A). Transmission electron microcopy observations showed that CAFs had large cell nuclei, were evenly colored, and contained one or two nucleoli. In the cytoplasm, a large number of rough endoplasmic reticulum, mitochondria, and bundles of parallel subcapsular filaments were observed. For NFs, the cells had an irregular cell nucleus, were rich in rough endoplasmic reticulum in the cytoplasm, and had no filaments (Fig. 1B). Immunohistochemical results of CAFs showed that vimentin and α-smooth muscle actin expression was positive. In NFs, vimentin showed positive expression, whereas α-smooth muscle actin expression was negative (Fig. 1C and D).

According to previous studies, CAF cells exhibit the ‘Warburg effect’, similarly to tumor cells; lactate is produced, secreted and absorbed by tumor cells to synthesize anabolic biomass to facilitate the rapid proliferation of tumor cells. In order to determine whether glycolysis occurs in CAFs, we examined the lactate levels of glucose uptake in the culture medium. The results demonstrated that glucose uptake and lactate production in CAFs were significantly increased compared to that in NFs (Fig. 2A and B). To further verify the change in glycolysis in CAFs, RT-PCR was conducted to detect the mRNA expression of the CAF glycolytic enzymes lactate dehydrogenase (LDHA) and pyruvate kinase m2 (PKM2). The results showed that LDHA and PKM2 were highly expressed in CAFs (Fig. 2C and E). The western blotting and PCR test results were consistent: LDHA and PKM2 protein expression in CAFs was higher than that in the control group (Fig. 2D and F). These results further confirmed that in vitro, glycolysis occurs in CAFs.

In order to explore whether the capacity of aerobic oxidation is strengthened in cancer cells after co-culture, we observed the fluorescence intensity in the mitochondria of cancer cells by fluorescence microscopy. After co-culture, pancreatic cancer cell mitochondrial activity in the CAF group was higher than that in the NF group and cells cultured alone (Fig. 3A). The western blot results showed that in cancer cells, SDH, FH and MCT1 protein expression in the CAF group was higher than that in the NF group and for cells cultured alone (Fig. 3B and C). This indicates that a CAF (glycolysis)-cancer cell (aerobic oxidation) metabolic coupling mode exists.

In order to determine whether CAFs promote the migration of pancreatic cancer cells, an indirect co-culture model was developed for 48 h, and the influence of CAFs on cancer cell migration was examined in a scratch test. The results showed that compared with that in the CAF-CM group, PBS alone or NF-CM induced the migration abilities in the BxPc-3 and Panc-1 cells (Fig. 4). This indicates that CAFs enhance the migration of pancreatic cancer cells in vitro.

In order to detect the influence of CAFs on pancreatic cancer cell invasion, we first adopted an indirect co-culture model and processed the cells for 48 h, after which a Transwell assay was used to examine cell invasion capability. The results showed that the number of cells penetrating the CAF-CM group was significantly increased compared to that in the PBS or NF-CM groups (Fig. 5), indicating that CAF enhances the invasive ability of pancreatic cancer cells.

To further verify the impact of CAFs on pancreatic cancer invasion and metastasis, we used RT-PCR and western blotting to detect the expression of matrix MMP-2 and MMP-9 mRNA and protein, respectively (Fig. 6). The PCR results showed that MMP-2 and MMP-9 mRNA levels were significantly higher in the CAF-CM group than levels in the control group. Western blot results showed that the MMP-2 and MMP-9 protein expression levels were significantly increased in the CAF-CM group compared with levels in the control group (Fig. 6). These results indicate that CAF enhances cell invasion and migration abilities.

Based on the above results, CAFs are generally glycolytic and release lactic acid as nutrients through MCT4/1 transport; these nutrients can be used by pancreatic cancer cells to maintain their progression. In order to verify whether removing metabolic coupling affects the progression of pancreatic cancer, an MCT1-specific blocker was used to pre-treat pancreatic cancer cells and for co-culture with CAFs. The results showed that after removal of ‘cancer cell-CAF’ metabolic coupling, there was no significant difference in the cell invasion ability among the three groups (Fig. 7). These data indicate that the ‘CAF-cancer cell’ metabolic coupling ring is an important mechanism for enhancing the progression of pancreatic cancer cells.