NT5C1A expression and function in the tumor stroma

With the objective to obtain a more comprehensive picture of the clinical situation regarding NT5C1A, we analyzed both TMAs for stromal NT5C1A expression. The same scoring system was employed with score 0 indicating no stromal expression of NT5C1A and score 3 indicating strong expression of NT5C1A. The proportion of PDAC patients without stromal NT5C1A expression was between 87.7 % in TMA-1 (n = 77) and 47.3 % in TMA-2 (n = 330). Low expression was detected in 11.0 % (TMA-1) and 33.3 % (TMA-2) of these patients. Interestingly, high scores (2 and 3) of stromal NT5C1A was only given to the samples of 1.4 % (TMA-1) or 19.4 % (TMA-2) of all patients (Figure 26A-C).

Figure 26: NT5C1A expression and function in PDAC stroma in vivo. A) and B) TMA analysis for NT5C1A expression revealed very low expression in the tumor stroma of resected PDAC patients.

A semi-quantitative scoring system indicated no stromal expression with score 0 and strong stromal expression with score 3. TMA-1 from Göttingen (A) with n = 77 patients (none scored with 3) and TMA-2 from Erlangen (B) with n = 330 patient samples. C) Representative IHC of NT5C1A expression showing no NT5C1A expression (score 0), low expression (score 1), and robust stromal expression (score 2) of TMA-1. Scale bars 20 µm.

C

A B

Stroma score 0 Stroma score 1 Stroma score 2

Results – Chapter II

Page | 74 Consistent with the tumor data, stromal NT5C1A expression was not a prognostic marker.

The median post-surgical survival time was 15.2 months for patients that did not express NT5C1A (n = 156) in the tumor stroma and 17.3 months for all other patients (n = 173;

p = 0.3, TMA-2) (Figure 27). Due to the lack of treatment details, it remains to be answered whether stromal NT5C1A might be a predictive marker for gemcitabine response in PDAC patients.

Figure 27: Stromal NT5C1A expression does not correlate with overall survival in resected pancreatic cancer patients. Survival analysis of the Erlangen TMA-2 cohort comparing no NT5C1A expression with any intensity of NT5C1A expression. Median survival was 15.2 months (n = 156) for no NT5C1A expression and 17.3 months (n = 173) with stromal expression of NT5C1A (p = 0.3, log-rank test).

As shown by our group previously, NT5C1A is expressed at very low levels in CAFs and PSCs (Hessmann & Patzak et al., 2018). Furthermore, PSCs do not differ significantly regarding intracellular dFdCTP accumulation compared to CAFs (Hessmann & Patzak et al., 2018). Therefore, two PSC cell lines were stably transfected and +NT5C1A cells and vector control cells were analyzed by Western blot as previously shown (Hessmann &

Patzak et al., 2018), and also by ICC and qPCR. HA-tag expression and robust expression of NT5C1A was confirmed in the +NT5C1A-cells (Figure 28A-D). Crystal violet staining showed increased resistance of NT5C1A expressing PSCs to gemcitabine treatment (Figure 29A and 29B, Figure 30).

0 50 100 150 200 250

0 20 40 60 80 100

Time elapsed (months)

Percent survival

no NT5C1A NT5C1A

Page | 75 Figure 28: NT5C1A overexpression in stably transfected PSC cell lines. For legend, see top of next page.

C D

mMuscle PSC1 +Vector

PSC1 +NT5C1A PSC2 +Vector

PSC2 +NT5C1A 0.000

0.005 0.010 0.015 0.020

Relative RNA expression / normalized to-actin **

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A

B

PSC1 +NT5C1APSC1 +Vector

DAPI NT5C1A Merge

DAPI HA-tag Merge

PSC1 +NT5C1APSC1 +Vector

Results – Chapter II

Page | 76 Legend to Figure 28:

NT5C1A overexpression in stably transfected PSC cell lines. A) and B) Representative ICC pictures for NT5C1A and HA-tag expression in stably transfected PSCs (red; DAPI nuclear staining:

blue). HA-tag expression was exclusively shown for +NT5C1A cells (A, lower panel), control cells were devoid of immunoreactivity (A, upper panel). Very low endogenous levels of NT5C1A were demonstrated in vector control cells (B, upper panel) compared to NT5C1A expression in the NT5C1A-overexpressing cells (B, lower panel). Two technical replicates were analyzed per cell line and antibody. Scale bars 50 µm. C) Strong NT5C1A mRNA expression was shown in both +NT5C1A murine pancreatic stellate cell lines, as analyzed by quantitative RT-PCR (PSC1: p = 0.003 and PSC2: p = 0.0001). NT5C1A expression was hardly detectable in vector control cells. Diagram indicates mean ± SEM of three biological replicates. Murine muscle sample served as positive control. Values were normalized to β-actin as housekeeping gene. D) Western blot analysis of transfected PSCs showing robust NT5C1A and HA-tag protein expression in cells expressing NT5C1A and no detectable expression in vector control cells. Representative image of three independent experiments is shown with murine muscle lysate as positive control for NT5C1A expression.

Figure 29: NT5C1A function in PDAC stroma. A) Murine PSCs were treated with increasing concentrations of gemcitabine for six days and crystal violet assays were performed. The staining was more pronounced in NT5C1A expressing cell lines. Representative images of two independent experiments, with each two technical replicates, are shown. B) Crystal violet stain intensity was

Page | 77 Figure 30: Stromal NT5C1A expression enhances gemcitabine resistance in vitro.

Quantification of crystal violet assay for stably transfected PSC2 cells with and without NT5C1A expression. Two-way ANOVA with Sidak’s multiple comparisons test was performed (7 nM:

p = 0.002, 12 nM: p < 0.0001).

Considering our previously published findings that +NT5C1A-PSCs accumulate significant lower amounts of intracellular gemcitabine triphosphate (Hessmann & Patzak et al., 2018), we hypothesized that gemcitabine availability would be significantly increased in the supernatant of NT5C1A-PSCs. To test this, MTT assays with conditioned media (CM) of transfected PSCs were performed. PSCs were incubated with 25 nM of gemcitabine-hydrochloride for 24 h and CM was then transferred to two KPC tumor cell lines (Figure 31A). Cell viability was determined 72 h later and demonstrated significantly decreased tumor cell viability with CM of +NT5C1A-PSCs, compared to vector control cells with only endogenous levels of NT5C1A. In the two KPC cell lines the viability decreased to 73 % and 75 %, respectively (KPCa: p = 0.003 and KPCb: p = 0.047) (Figure 31B).

Figure 31: Stromal cells expressing NT5C1A increase available amounts of gemcitabine for tumor cells in vitro. For legend, see top of next page.

A B

Results – Chapter II

Page | 78 Legend to Figure 31:

Stromal cells expressing NT5C1A increase available amounts of gemcitabine for tumor cells in vitro. A) and B) MTT cell viability assay for KPC cell lines treated with CM of NT5C1A expressing PSCs and control CM. A) Schematic experimental overview. CM from PSCs (+NT5C1A) and control PSCs (+vector) was obtained by preincubation with 25 nM gemcitabine-hydrochloride for 24 h.

Subsequently, tumor cells were treated for 72 h with CM of PSCs and viability was assessed using MTT cell viability assay. B) Tumor cell viability of two different murine KPC cell lines was significantly decreased following treatment with CM of +NT5C1A-expressing PSCs (KPCa: 73 %; p = 0.003 and KPCb: 75 %; p = 0.047). Graphs indicate mean ± SEM of four biological replicates.

3.2.6 Reduced accumulation of the cytotoxic gemcitabine metabolite dFdCTP in NT5C1A expressing stromal cells

The transfected PSCs were incubated with gemcitabine for 2 h and subjected to LC-MS/MS-analysis to investigate the hypothesis that NT5C1A expression in stromal cells would decrease intracellular gemcitabine accumulation, as it was already shown for tumor cell lines. Indeed, the concentration of intracellular dFdCTP was significantly decreased in both NT5C1A re-expressing PSC lines (Figure 32) (Hessmann & Patzak et al., 2018). As a result, it could be assumed that the transfected cells would consequently increase the amount of available gemcitabine for tumor cells.

Figure 32: Pharmacokinetic analysis of the gemcitabine metabolite dFdCTP in murine PSCs.

Cells were treated with 1 µM of gemcitabine-hydrochloride for 2 h and cell pellets were harvested.

Concentrations of the cytotoxic gemcitabine metabolite dFdCTP were determined using LC-MS/MS.

Significantly lower amounts of dFdCTP were found in both transfected fibroblast cell lines (+NT5C1A) (compared to control cells (+vector) (PSC1: 166 vs. 71 ng/ 1x10⁶ cells; p = 0.006 and PSC2: 228 vs.

100 ng/ 1x10⁶ cells; p = 0.0002). Graph shows mean ± SEM of three technical replicates. Adapted from (Hessmann & Patzak et al., 2018).

PSC1 PSC2

0 50 100 150 200 250

+Vector +NT5C1A

**

LC-MS/MS analysis in cell lines Concentration dFdCTP (ng / 1*106 cells)

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