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. 2018 Mar;67(3):497-507.
doi: 10.1136/gutjnl-2016-311954. Epub 2017 Jan 10.

Fibroblast drug scavenging increases intratumoural gemcitabine accumulation in murine pancreas cancer

E Hessmann  1 , M S Patzak  1 , L Klein  1 , N Chen  1 , V Kari  2 , I Ramu  1 , T E Bapiro  3   4 , K K Frese  5 , A Gopinathan  3 , F M Richards  3 , D I Jodrell  3   6 , C Verbeke  7   8 , X Li  9 , R Heuchel  9 , J M Löhr  9 , S A Johnsen  2 , T M Gress  10 , V Ellenrieder  1 , A Neesse  1
Affiliations

Fibroblast drug scavenging increases intratumoural gemcitabine accumulation in murine pancreas cancer

E Hessmann et al. Gut. 2018 Mar.

Abstract

Objective: Desmoplasia and hypovascularity are thought to impede drug delivery in pancreatic ductal adenocarcinoma (PDAC). However, stromal depletion approaches have failed to show clinical responses in patients. Here, we aimed to revisit the role of the tumour microenvironment as a physical barrier for gemcitabine delivery.

Design: Gemcitabine metabolites were analysed in LSL-KrasG12D/+ ; LSL-Trp53R172H/+ ; Pdx-1-Cre (KPC) murine tumours and matched liver metastases, primary tumour cell lines, cancer-associated fibroblasts (CAFs) and pancreatic stellate cells (PSCs) by liquid chromatography-mass spectrometry/mass spectrometry. Functional and preclinical experiments, as well as expression analysis of stromal markers and gemcitabine metabolism pathways were performed in murine and human specimen to investigate the preclinical implications and the mechanism of gemcitabine accumulation.

Results: Gemcitabine accumulation was significantly enhanced in fibroblast-rich tumours compared with liver metastases and normal liver. In vitro, significantly increased concentrations of activated 2',2'-difluorodeoxycytidine-5'-triphosphate (dFdCTP) and greatly reduced amounts of the inactive gemcitabine metabolite 2',2'-difluorodeoxyuridine were detected in PSCs and CAFs. Mechanistically, key metabolic enzymes involved in gemcitabine inactivation such as hydrolytic cytosolic 5'-nucleotidases (Nt5c1A, Nt5c3) were expressed at low levels in CAFs in vitro and in vivo, and recombinant expression of Nt5c1A resulted in decreased intracellular dFdCTP concentrations in vitro. Moreover, gemcitabine treatment in KPC mice reduced the number of liver metastases by >50%.

Conclusions: Our findings suggest that fibroblast drug scavenging may contribute to the clinical failure of gemcitabine in desmoplastic PDAC. Metabolic targeting of CAFs may thus be a promising strategy to enhance the antiproliferative effects of gemcitabine.

Keywords: CHEMOTHERAPY; DRUG METABOLISM; PANCREATIC CANCER; PANCREATIC FIBROSIS.

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Conflict of interest statement

Competing interests: None declared.

Figures

Figure 1
Figure 1
Pharmacokinetic profile of gemcitabine metabolites in primary pancreatic tumours, liver metastases and normal liver tissue in corresponding KPC mice. (A) Necropsy view of a KPC mouse with a large pancreatic tumour (dotted blue circle) and several liver metastases (white arrows). (B) H&E staining of a representative KPC tumour with matched liver metastasis. NL, normal liver, LM, liver metastasis. Scale bars, 50 µm. (C) Tumour-bearing KPC mice were treated with one dose of gemcitabine at 100 mg/kg intraperitoneally. Tumour tissues, liver metastases and normal liver tissue were excised and assessed for gemcitabine metabolites 2 hours later by liquid chromatography-mass spectrometry/mass spectrometry (n=15). Gemcitabine is significantly elevated in primary tumours compared with liver metastases (p<0.05) and normal liver tissue (p<0.02). (D) The deaminated and inactive metabolite 2′,2′-difluorodeoxyuridine (dFdU) shows no significant differences among the three groups. (E) The triple phosphorylated active gemcitabine metabolite 2′,2′-difluorodeoxycytidine-5′-triphosphate (dFdCTP) is significantly increased in primary pancreatic tumours as compared with normal liver tissue (p<0.01).
Figure 2
Figure 2
Primary tumours display higher stromal content than liver metastases. (A) Representative pictures of collagen, secreted protein acidic and rich in cysteine (SPARC) and α-smooth muscle actin (α-SMA) stains from primary murine pancreatic tumours and matched liver metastases (LM) with adjacent normal liver (NL) reveal increased cellular and acellular desmoplasia in primary tumours. Scale bars, 50 µm. (B and C) Automated quantification of n=8 primary tumours and n=8 liver metastases reveal significant increase in collagen and α-SMA area in primary tumours (p<0.01; Wilcoxon matched-pairs signed-rank test). (D) Western blot analysis of whole tissue lysates from KPC primary tumours (n=5), liver metastases (n=5) and normal liver tissue (n=1) confirm higher α-SMA, SPARC and fibronectin levels compared with liver metastases and normal liver. HSP90, heat shock protein 90. (E) Immunohistochemical CD31 analysis reveals comparable mean vessel density (MVD) in primary tumours (n=8) and liver metastases (n=8), whereas normal liver tissue (n=8) featured significantly higher MVD (p<0.01; Wilcoxon matched-pairs signed-rank test). Scale bars, 25 µm. PDAC, pancreatic ductal adenocarcinoma.
Figure 3
Figure 3
Human primary tumours reveal higher α-smooth muscle actin (α-SMA) content compared with matched liver metastases. (A) α-SMA immunohistochemistry of human primary pancreatic ductal adenocarcinoma (PDAC) with matched liver metastases of n=11 patients. Scale bars, 50 µm. (B) The α-SMA score was significantly higher in primary tumours compared with matched liver metastases (p<0.004; Wilcoxon matched-pairs signed-rank test).
Figure 4
Figure 4
Fibroblasts accumulate activated gemcitabine while inactivation is decreased. (A) Typical morphology and α-smooth muscle actin immunoreactivity of cancer associated fibroblasts (CAFs) using immunocytochemistry. (B) Murine CAFs (n=2) and pancreatic stellate cells (PSCs) (n=2) as well as primary cell lines from KPC pancreatic tumours (n=4) and metastatic foci (n=4) were cultured and treated with 1 µM gemcitabine for 2 hours. Cell pellets and cell supernatants were subjected to liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) for analysis. 2′,2′-Difluorodeoxycytidine-5′-triphosphate (dFdCTP) was significantly increased in cell pellets from fibroblasts (PSCs and CAFs), compared with tumour cells (p<0.03). (C) The inactivated 2′,2′-difluorodeoxyuridine (dFdU) was significantly decreased in fibroblasts compared with tumour cells (p<0.03) indicating a greatly reduced gemcitabine inactivation in those cells. (D) Equal amounts of native gemcitabine (2′,2′-difluorodeoxycytidine (dFdC)) was detectable 2 hours after administration in cell culture supernatant. (E and F) 72 hours MTT assay with conditioned media (CM) of CAF1 and CAF2 preincubated for 24 hours with 30 nM gemcitabine in KPC1 (GI50 32 nM) and KPC2 (GI50 25 nM) cells shows significant increase in cell viability compared with CAF1 and CAF2 control media with fresh 30 nM gemcitabine prior to 72 hours treatment (KPC1—CAF1: p<0.002; KPC1—CAF2: p<0.04 and KPC2—CAF1: p<0.01, KPC2—CAF2: p<0.03; two-tailed, unpaired t-test).
Figure 5
Figure 5
Gemcitabine inactivating genes are expressed at low levels in stromal cells in vitro and in vivo. RNA isolated from murine cancer associated fibroblasts (CAFs) (n=2) and pancreatic stellate cells (PSCs) (n=2) as well as primary cell lines from KPC pancreatic tumours (n=4) and metastatic foci (n=4) were subjected to quantitative reverse transcription-PCR. Gemcitabine metabolising enzymes were significantly downregulated in fibroblasts compared with tumour cells for (A) 5′-nucleotidase, cytosolic IA (Nt5c1A, p<0.03 and <0.03, respectively), (B) 5′-nucleotidase, cytosolic III (Nt5c3, p<0.03). (C) Deoxycytidine kinase (dCK) was not significantly different in fibroblasts compared with tumour cells (p=0.4); ns, not significant. (D) Gemcitabine treatment with 1 µM for 2 hours in PSC1 and PSC2 stably overexpressing Nt5c1A shows significant reduction of 2′,2′-difluorodeoxycytidine-5′-triphosphate (dFdCTP) (p<0.01 and <0.001, respectively, two-tailed, unpaired t-test). (E) Representative immunohistochemical pictures of murine and human tumour tissue showing cytidine deaminase (Cda), deoxycytidylate deaminase (Dctd) and Nt5c1A expression in tumour cells, whereas stromal cells (arrows) are almost completely devoid of immunoreactivity. dCK is robustly expressed in stromal cells (quantification see online supplementary figure S6). Scale bars, 50 µm.
Figure 6
Figure 6
Gemcitabine treatment does not induce apoptosis in fibroblasts in vivo but reduces metastatic burden in KPC mice. (A and B) Archived tissue from primary pancreatic KPC tumours was evaluated retrospectively. Gemcitabine treatment had been administered every 3–4 days for 9 days. The last dose was given 2 hours prior sacrifice. Co-immunofluorescence (Ki67, red; α-smooth muscle actin (α-SMA), green) shows no significant difference in proliferation rate in α-SMA-positive cells after 9 days gemcitabine treatment (two-tailed, unpaired t-test), Scale bar, 50 µm. (C and D) Co-immunochemistry for α-SMA and CC3 does not show significant differences on gemcitabine treatment in KPC mice (two-tailed, unpaired t-test; arrow indicates apoptotic fibroblast). Scale bar, 50 µm. (E and F) Total number of all liver metastases and small liver metastases in 10 serial liver sections in a historical cohort of KPC mice treated with gemcitabine (n=8) or vehicle (n=8) for 9 days shows reduction of liver metastases by >50%. (G) Representative H&E of a small liver metastasis from KPC mouse (dotted circle) and normal liver tissue (NL). (H) Ultrasound volume measurements of corresponding mice reveals marginal response (−14%) on gemcitabine treatment for 9 days.
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