- Letter to the Editor
- Open Access
Cancer-associated fibroblast heterogeneity is associated with organ-specific metastasis in pancreatic ductal adenocarcinoma
Journal of Hematology & Oncology volume 14, Article number: 184 (2021)
Metastasis occurs in the majority of pancreatic ductal adenocarcinoma (PDAC) patients at diagnosis or following resection. Patients with liver metastasis and those with lung metastasis have significantly different prognosis. Here, we sought to understand how cancer-associated fibroblasts (CAFs) play roles in the development of organ-specific metastasis.
PDAC tumor cell lines established from the primary tumors with liver and lung metastasis potentials, respectively, in Kras/p53 mutation conditional knock-in (KPC) mice were co-cultured with matched CAFs or mouse mesenchymal stem cells. CAFs were isolated from metastases and subjected to DNA methylation and whole transcriptomic RNA sequencing analysis.
The ability of mouse PDAC tumor cell lines in developing liver or lung-specific metastases was demonstrated in orthotopic models. Tumor cells associated with liver metastasis potential, but not those associated with lung metastasis potential, induced the methylation of metabolism genes including NQO1 and ALDH1a3 and subsequent downregulated mRNA expression of a broader group of metabolism genes in CAFs. DNA methylation and downregulation of metabolism genes in CAFs in liver metastasis, but not those in lung metastasis, appeared to be regulated by DNA methyltransferase. Tumor cells associated with liver metastasis potential, but not those associated with lung metastasis potential, induce inflammatory CAF (iCAF) signatures. CAFs from liver metastasis demonstrated a more homogenous iCAF phenotype, whereas CAFs from lung metastasis maintained the heterogeneity.
PDAC with organ-specific metastatic potentials has different capacities in inducing methylation of metabolism genes in CAFs, modulating CAF phenotypes, and resulting in different levels of heterogeneity of CAFs in different metastatic niches.
To the editor
Our clinical observations revealed disease heterogeneity among pancreatic ductal adenocarcinoma (PDAC) patients with different distant metastatic sites, resulting in distinct clinical outcomes . We sought to investigate the mechanism of organ-specific metastasis in the transgenic KPC mouse model  and established two cell lines from primary tumors of KPC mouse with liver metastasis only and KPC mouse with lung metastasis only (Fig. 1a; Additional file 1: Fig. S1A). Previously, we demonstrated that PDAC tumor cells induce DNA methylation globally in CAFs [3, 4] and in macrophages  including a wide range of metabolism genes resulting in their downregulation. We chose to examine methylation of ALDH1a3 in the glucose metabolism pathway and NQO-1 in the oxidative phosphorylation (OXIPHOS) pathway (Additional file 1: Fig. S1B) and mRNA expression of their related genes (Additional file 1: Table S1). CAFs from liver metastases and lung metastases demonstrated different levels of methylation and mRNA expression in these genes (Fig. 1b–d).
We hypothesized that different methylation patterns in CAFs from different metastatic sites were induced by PDAC tumor cells with different metastasis potentials. We thus studied tumor-induced DNA methylation in the mouse mesenchymal stem cell line (moMSC), the precursor of CAFs, by co-culturing with three different primary PDAC tumor cell lines derived from a KPC mouse without metastasis (PancPrimaryTumorCell), a KPC mouse with liver metastasis only (LiverMetTumorCell), and a KPC mouse with lung metastasis only (LungMetTumorCell). Remarkably, different tumor cell lines showed differential capacities in inducing DNA methylation on NQO-1 and ALDH1a3 in moMSCs (Additional file 1: Fig. S2A and B). mRNA expression of NQO-1 as well as other metabolism genes were also downregulated in moMSC co-cultured with LiverMetTumorCells (Additional file 1: Fig. S2C–E). These results suggested that different PDAC tumor cells show different capacities in possibly shaping metabolic states of CAFs in different metastatic sites. We also developed CAF lines from primary KPC tumor with liver metastasis only (4545PancCAF) or that with lung metastasis only (3403PancCAF) and co-cultured them with matched tumor cells. Induced DNA methylation changes in NQO1 and ALDH1a3 were observed at higher degrees in 4545PancCAF from tumor co-culture compared to its mono-culture than those in 3403PancCAF (Additional file 1: Fig. S2F). Consistently, NQO-1 expression was downregulated in 4545PancCAF from tumor co-culture (Additional file 1: Fig. S2G). ALDH1a3 expression was not downregulated in 4545PancCAF from tumor co-culture, suggesting that ALDH1a3 expression is regulated by a more complex mechanism (Additional file 1: Fig. S2H).
We next sought to understand if tumor-induced gene methylation occurred in the metastatic niches where tumor cells metastasized to. We orthotopically implanted KPC cells with both liver and lung metastasis potentials (Fig. 2a). The transplanted mice developed liver and/or lung metastases. NQO-1 methylation was elevated in CAFs isolated from liver metastases compared to normal liver fibroblasts (Fig. 2b), but remained at baseline levels in CAFs from lung metastases (Fig. 2c). Supporting this notion, tumor-induced methylation of NQO-1 and ALDH1a3 and downregulation of their mRNA expression or expression of their related metabolism genes in CAFs from liver metastasis could be reversed by treatment of DNA demethylating agent, decitabine, whereas methylation in CAFs from lung metastasis was not affected (Additional file 1: Fig. S3). Thus, although the primary tumors harbor an organ-specific metastatic potential, metastatic organs possibly harbor contributive factors for an organ-specific tumor-induced metabolism gene methylation.
Nevertheless, it was unknown whether CAFs from different metastatic niches are functionally different. The functions of the subpopulations of CAFs were previously characterized and showed different transcriptomic signatures [8,9,10]. In our RNA sequencing analysis, mono-cultured moMSCs showed low expression of the majority of inflammatory CAF (iCAF) signature genes (Additional file 1: Table S2) and high expression of some of myofibroblastic CAF (myCAF) signature genes (Fig. 2d). However, upon co-cultured with LiverMetTumorCells, moMSC was programmed to express essentially all iCAF signature genes and the majority of myCAF signature genes. However, expression of the majority of iCAF and myCAF genes in moMSC co-cultured with LungMetTumorCell remained low, suggesting that LungMetTumorCell was not able to program moMSC. As expected, CAFs from lung metastasis expresses both iCAF genes and myCAF genes (Fig. 2e). By contrast, CAFs from liver metastasis did not demonstrate a myCAF signature, but expressed some iCAF genes that were not upregulated in CAFs from lung metastasis. Taken together, CAFs in liver metastasis are more homogeneous likely as a result of reprogramming of myCAF into iCAF by tumor cells that metastasize to liver, whereas CAFs in lung metastasis remain to be heterogenous (Fig. 2f). A change in the heterogeneity of CAFs to a more homogeneous iCAF phenotype may be responsible for the aggressive feature of liver metastasis and is potentially targetable by anti-IL-1β antibody .
Availability of data and materials
RNA sequencing data are provided as supplement materials.
Pancreatic ductal adenocarcinoma
Kras/p53 mutation conditional knock-in
Mouse mesenchymal stem cells
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This work was supported by an NIH grant R01 CA169702 (LZ), an NIH Grant R01 CA197296 (LZ), a National Cancer Institute Specialized Programs of Research Excellence in Gastrointestinal Cancers Grant P50 CA062924 (LZ), and a Sidney Kimmel Comprehensive Cancer Center Grant P30 CA006973.
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LZ receives grant support from Bristol-Meyer Squibb, Merck, iTeos, Amgen, NovaRock, Inxmed, and Halozyme, and received the royalty for licensing GVAX to Aduro Biotech. LZ is a paid consultant/Advisory Board Member at Biosion, Alphamab, NovaRock, Akrevia/Xilio, Ambrx, Novagenesis, Datarevive, Snow Lake Capitals, and Mingruzhiyao. LZ holds shares at Alphamab and Mingruzhiyao.
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. Representative images of lung and liver metastases from KPC mice implanted with KPC tumor cells with lung and liver metatasis potentials and schema of how represntative metabolism genes were selected for study.
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Pan, X., Zhou, J., Xiao, Q. et al. Cancer-associated fibroblast heterogeneity is associated with organ-specific metastasis in pancreatic ductal adenocarcinoma. J Hematol Oncol 14, 184 (2021). https://doi.org/10.1186/s13045-021-01203-1
- DNA methylation
- Cancer-associated fibroblast
- Organ-specific metastasis
- Pancreatic cancer