Cancer-associated fibroblasts reuse cancer-derived lactate to maintain a fibrotic and immunosuppressive microenvironment in pancreatic cancer

Glycolysis is highly enhanced in pancreatic ductal adenocarcinoma (PDAC) cells; thus, glucose restrictions are imposed on nontumor cells in the PDAC tumor microenvironment (TME). However, little is known about how such glucose competition alters metabolism and confers phenotypic changes in stromal cells in the TME. Here, we report that cancer-associated fibroblasts (CAFs) with restricted glucose availability utilize lactate from glycolysis-enhanced cancer cells as a fuel and exert immunosuppressive activity in the PDAC TME. The expression of lactate dehydrogenase A (LDHA), which regulates lactate production, was a poor prognostic factor for patients with PDAC, and LDHA depletion suppressed tumor growth in a CAF-rich murine PDAC model. Coculture of CAFs with PDAC cells revealed that most of the glucose was taken up by the tumor cells and that CAFs consumed lactate via monocarboxylate transporter 1 to enhance proliferation through the TCA cycle. Moreover, lactate-stimulated CAFs upregulated IL-6 expression and suppressed cytotoxic immune cell activity synergistically with lactate. Finally, the LDHA inhibitor FX11 reduced tumor growth and improved antitumor immunity in CAF-rich PDAC tumors. Our study provides insight regarding the crosstalk among tumor cells, CAFs, and immune cells mediated by lactate and offers therapeutic strategies for targeting LDHA enzymatic activity in PDAC cells.


LDHA knockdown-rescue experiments
To rescue the expression of LDHA in the stable LDHA knockdown clones in Panc02, we generated a piggybac expression vector plasmid to overexpress the shRNA-resistant LDHA ORF sequence (Vector Builder, vector ID: VB230220-1513drq).The expression vector plasmid was transfected into Panc02 shLDHA clones with the hyPBase piggybac transposon vector (Vector Builder).Cells were then treated with blasticidin (Wako) after transfection, and LDHA expression was confirmed by western blotting.

Lactate assay performed with glucose starvation medium
RPMI 1640 (no glucose) medium with L-glutamine and phenol red (Fuji Film Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) was used as the glucose starvation medium.Lactic acid (Fuji Film Wako, Osaka, Japan) was added, and the concentration was adjusted to 5 mM or 10 mM before use.

Transwell assay
To evaluate the crosstalk between PDAC cells and CAFs, cells were cocultured using Transwell inserts (Corning, NY USA).To measure lactate-related protein expression in PDAC cells, PANC-1 or PK8 cells were seeded in the lower wells at a density of 1.5×10 4 , and CAFs were seeded at the same density in the upper wells.For the measurement of lactate-related protein expression in CAFs, human CAFs were seeded in the lower wells at a density of 1.5×10 4 , and PANC-1 or PK8 cells were seeded at the same density in the upper wells.Cells were incubated with 5% CO2 at 37°C for 72 h, and total protein was harvested from the cells in the lower wells.

Metabolomic analysis
Human CAFs were pretreated with lactate for 24 hours.Any protein was removed with filtration according to the company's protocol, and the intracellular metabolites of 2×10 6 human CAFs were stored at -80°C after extraction.Finally, the metabolites were measured by Human Metabolic Technology (HMT Yamagata Japan).For isotope tracing experiments, 4 x 10 6 human CAFs were cultured with glucose-free RPMI 1640 supplemented with 10% FBS overnight.The next day, the medium was changed to glucose-free RPMI 1640 supplemented with 10% FBS and 10 mM 13 C-labeled lactate (Cambridge Isotope Laboratories, Inc., CLM-1579-0) or nonlabeled lactate.After 24 hours, cells were harvested, and metabolomic analysis was performed at the Clinical and Translational Research Center, Keio University Hospital, Tokyo, Japan.

Lactate and IL6 stimulation assay performed with immune cells
Blood donated by healthy volunteers was processed to isolate PBMCs.PBMCs were seeded in a 96-well plate at 1.0×10 5 cells per well.Then, 10 mM lactate and 500 pg/ml recombinant human IL6 (PeproTech, London, UK) were added, and the cells were incubated with 5% CO2 for 48 h at 37°C.

Quantitative Reverse Transcription-Polymerase Chain Reaction
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to determine messenger RNA (mRNA) levels in pancreatic cancer cell lines.Total RNA was isolated from cells using TRIzol (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and the concentration of purified RNA was measured by comparing the A260 and A280 values using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).Complementary DNA (cDNA) was generated from total RNA using a ReverTra Ace qPCR RT kit (Toyobo Co. Ltd, Osaka, Japan) according to the manufacturer's instructions and subsequently used as the template for PCR.qRT-PCR was performed as described previously [1].Transcript levels were measured in a duplicate set of reactions for each gene.
Relative hypoxanthine phosphoribosyltransferase 1 levels were used for normalization.The sequences of the primers used in this study are listed in Table S2.

The Cancer Genome Atlas data analysis
RNA-sequencing data for 177 PDAC patients in The Cancer Genome Atlas (TCGA) database were obtained from the Genomic Data Commons Data Portal (https://portal.gdc.cancer.gov/) in November 2021.The PDAC patients were divided into LDHA-high (n = 89) and LDHA-low (n = 88) groups based on LDHA gene expression.The RNA-sequencing data of the LDHAhigh and LDHA-low PDAC patients were analyzed to estimate CD8+ T-cell infiltration in the tumor microenvironment using the CIBERSORT tool (https://cibersort.stanford.edu/).