Fig. 1.

Distinct DNA methylation patterns in metabolism genes were observed in CAFs isolated from liver metastasis versus lung metastasis. a Establishment of primary mouse PDAC tumor cell lines with organ-specific metastasis potentials. Different KPC tumor cell lines were established from primary pancreatic tumor of KPC mice that developed liver metastasis only and lung metastasis only, respectively. These KPC cell lines were tested for their capacities to generate metastasis in mice through the hemispleen injection model of liver metastasis and the inferior vena cava injection model of lung metastases as described previously [6, 7]. Five mice per group were used to test each KPC cell line using both mouse models, respectively. Numbers of mice that developed large metastatic lesions were indicated. Longer arrows indicate large metastatic lesions; shorter arrows indicate small metastatic lesions. Note that the KPC cell line established from primary tumor with liver metastases consistently gave rise to liver metastasis macroscopically but not lung metastasis (5/5), whereas the KPC cell line established from primary tumor with lung metastases consistently gave rise to lung metastasis (5/5). The lung met tumor cell line was only able to develop a small number of macroscopically visualized metastatic foci when the cell line was injected by the hemispleen technique (Additional file 1: Fig. S1A). Liver and lung were examined microscopically and confirmed that the liver met tumor cell line only developed micro-metastases in lung (Additional file 1: Fig. S1A). b and c Percentages of methylation in the ALDH1a3 and NQO-1 gene were measured in tumor cells and CAFs isolated from primary pancreatic tumor and liver or lung metastases from KPC mouse with only liver or lung metastasis, respectively. Four different CAFs including: (1) CAFs isolated from primary KPC tumors of a mouse that developed liver metastasis only; (2) CAFs isolated from primary KPC tumors of a mouse that developed lung metastasis only; (3) CAFs isolated from liver metastases of a KPC mouse that developed liver metastasis only (liver mets CAFs); (4) CAFs isolated from lung metastases of a KPC mouse that developed lung metastasis only (lung mets CAFs) were all compared to normal pancreas (indicated with black arrow), liver (indicated with red arrow) and lung (indicated with green arrow) fibroblasts. Note that, for both NQO-1 and ALDH1a3 genes, DNA methylation levels were found to be elevated in liver mets CAFs from KPC mouse that developed liver metastasis compared to normal liver fibroblasts. NQO-1 and ALDH1a3 did not show an elevated DNA methylation in lung mets CAFs from KPC mouse that developed lung metastasis compared to normal lung fibroblasts, even though they demonstrated a high-level DNA methylation in CAFs from primary tumors that developed lung metastasis only. The methylation of NQO-1 and ALDH1a3 remained the same in tumor cells from both primary tumors and liver/lung metastases although the methylation level of ALDH1a3 was higher than that of NOQ-1 in tumor cells. Methylation percentage was quantified by MethySYBR real-time PCR (MSP). Black, red, green arrows indicate different comparison groups. Triplicate experimental results are presented as mean ± SEM. *Unpaired t test, p < 0.05. Independent experiments were conducted twice. d Heatmap was generated from the RNA sequencing analysis of mouse CAFs isolated from a KPC mouse that developed lung metastasis (3404LungCAF) only and from a KPC mouse that developed liver metastasis only (4545LiverCAF) to compare expression of selected metabolic genes. Mouse homologs of genes in the ALDH1-associated metabolism pathway and NQO-1-associated oxidative phosphorylation pathway previously found to have a significantly increased methylation level and also a significantly decreased mRNA expression level in CAFs following co-culture with human PDAC tumor cells [3] were selected. Heatmap was generated using transcripts per million (TPM) scores