Arachidonate 12-lipoxygenase and 12-hydroxyeicosatetraenoic acid contribute to stromal aging-induced progression of pancreatic cancer

The incidence of pancreatic cancer increases with age, suggesting that chronological aging is a significant risk factor for this disease. Fibroblasts are the major nonmalignant cell type in the stroma of human pancreatic ductal adenocarcinoma (PDAC). In this study, we investigated whether the chronological aging of normal human fibroblasts (NHFs), a previously underappreciated area in pancreatic cancer research, influences the progression and therapeutic outcomes of PDAC. Results from experiments with murine xenografts and 2D and 3D co-cultures of NHFs and PDAC cells revealed that older NHFs stimulate proliferation of and confer resistance to radiation therapy of PDAC. MS-based metabolite analysis indicated that older NHFs have significantly increased arachidonic acid 12-lipoxygenase (ALOX12) expression and elevated levels of its mitogenic metabolite, 12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-(S)-HETE) compared with their younger counterparts. In co-cultures with older rather than with younger NHFs, PDAC cells exhibited increases in mitogen-activated protein kinase signaling and cellular metabolism, as well as a lower oxidation state that correlated with their enhanced proliferation and resistance to radiation therapy. Expression of ALOX12 was found to be significantly lower in PDAC cell lines and tumor biopsies, suggesting that PDAC cells rely on a stromal supply of mitogens for their proliferative needs. Pharmacological (hydroxytyrosol) and molecular (siRNA) interventions of ALOX12 in older NHFs suppressed their ability to stimulate proliferation of PDAC cells. We conclude that chronological aging of NHFs contributes to PDAC progression and that ALOX12 and 12-(S)-HETE may be potential stromal targets for interventions that seek to halt progression and improve therapy outcomes.

The incidence of pancreatic cancer increases with age; median age at diagnosis is 71 years old and the median age at death is 73 years old, suggesting that chronological aging is a significant risk factor for pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) 2 ranks fourth among cancer-related deaths in the United States: it has a very high incidence of recurrence and a dismal 5-year survival rate of less than 8% (1, 2) (RRID: SCR_006902). Recent evidence suggests that the tumor microenvironment significantly influences progression and therapy of cancer (4 -10). PDAC tissue is stroma-rich, dense, and fibrous (11,12). The stroma of PDAC includes fibroblasts, myofibroblasts, stellate cells, immune cells, and endothelial cells (8). Fibroblasts are the major noncancerous cell type contributing to the cellularity of the stroma that include synthesis and remodeling of the extracellular matrix as well as the production of secretory and nonsecretory factors that influence cancer cell proliferation and progression to end stage disease. Because age is a significant risk factor for PDAC, it is important to know whether the age of the stroma influences progression of PDAC and the mechanisms involved in this process.
Chronologically aged normal human fibroblasts (NHF) (quiescent fibroblasts aged in vitro) are known to stimulate proliferation of human breast cancer cells (13). C-C motif chemokine ligand 5, which is overexpressed in chronologically aged NHFs, has been shown to activate the extracellular signal-regulated kinase (ERK)1/2-cyclin D1 pathway in breast cancer cells resulting in the stimulation of their proliferation (13). Although quiescent cells are not actively proliferating, they are metabolically active. Significant changes in metabolism have been observed during chronological aging of NHFs (14). Metabolism of quiescent cultures of NHFs from younger (newborn to 1 year old) donors were found to be more glycolytic compared with NHFs from older (29 -70 years old) donors that exhibited significant increases in mitochondrial respiration. These previously published results led us to the novel hypothesis that an age-related change in metabolism of the stroma contributes to the progression of epithelial cancer.
An increase in mitochondrial respiration and decrease in glycolysis in older NHFs suggest that changes in lipid metabolism during aging can influence stroma-directed progression of PDAC. Lipid-signaling pathways are known to regulate proliferation and therapy of cancer cells (15)(16)(17)(18)(19). Eicosanoids are a family of lipid mediators derived from the metabolism of arachidonic acid (AA). AA is converted to eicosanoids by cyclooxygenase and arachidonic acid lipoxygenase (ALOX) enzymes. Based on their regiospecificity, human ALOXs are grouped into three major groups: ALOX5, ALOX12, and ALOX15. The enzymatic products of ALOXs are hydroperoxyeicosatetraenoic acids, which are converted to hydroxyeicosatetraenoic acids (HETEs) (20,21). 12-(S)-HETE is a mitogenic metabolite of ALOX12. The signaling and mitogenic properties of 12-(S)-HETE can be mediated by its binding to the membrane-bound G protein-coupled receptors; GPR31 is a high-affinity receptor for 12-(S)-HETE (22). Eicosanoids, which include prostaglandins and leukotrienes, are well-known for their inflammation and immune properties (23). These lipid mediators are also known to have mitogenic properties (23,24).
Results from this study show that the chronological aging of the stroma regulates progression and radiation therapy resistance of PDAC. ALOX12 and 12-(S)-HETE are potential targets of the aging stroma that can be considered to halt progression and improve therapy of PDAC.

NHFs from older healthy donors stimulate proliferation of PDAC cells
To determine whether chronological aging of NHFs regulates progression of PDAC, we used a novel murine xenograft approach where co-cultures of CellTracker Green-labeled NHFs and luciferase expressing MIA PaCa-2 cells (10:1) embedded in injectable tissue-like 3D gel matrix were injected into the flank of 30-day-old athymic female nude mice (Foxn1 nu ). Bioluminescence imaging system (Xenogen IVIS-200) was used to measure proliferation. Results clearly show the presence of CellTracker Green-labeled NHFs and luciferase expressing MIA PaCa-2 cells in the xenograft (Fig. 1A). The presence of NHFs in tumors was also evident from the confocal microscopy analysis of excised tumor tissues (Fig. 1B) and flow cytometry analysis of cell suspensions prepared from resected tumors (Fig. 1C). Measurements of tumor volume showed 2ϫ tumor volume in xenografts carrying co-cultures of MIA PaCa-2 cells and NHFs from a 61-year-old healthy donor compared with tumor volume in co-cultures of MIA PaCa-2 cells and NHFs from a 3-day-old healthy donor. Increases in tumor volume correlated with a shorter median survival of a 34-dayold compared with a median survival of 44 days of mice carrying xenograft of MIA PaCa-2 cells and NHFs from a 3-day-old donor ( Fig. 1, D and E). An age-related stimulation of proliferation of MIA PaCa-2 cells in co-cultures of older compared with young NHFs was also observed in an orthotopic xenograft approach (Fig. S1). Overall, these results support our novel hypothesis that chronological aging of the stroma regulates progression of PDAC.
Results from these in vivo experiments were also recapitulated in 3D and 2D co-cultures in vitro. A mixture of luciferase expressing MIA PaCa-2-Luc cells and quiescent cultures of NHFs (10:1) were embedded into the Cell Mate 3D matrix and co-cultured for different days. Photon counts showed approximately a 4-fold increase of MIA PaCa-2-Luc cells co-cultured with quiescent cultures of NHFs from a 61-year-old healthy donor (Fig. 2, A and B). The doubling time of MIA PaCa-2-Luc cells co-cultured with NHFs from a 61-year-old donor was ϳ20 h compared with a doubling time of 60 h for MIA PaCa-2-Luc cells co-cultured with NHFs from a 3-day-old donor (Fig. 2B). Similar to the co-existence of MIA PaCa-2-Luc cells and NHFs in tumor xenograft in vivo (Fig. 1, A-C), results from H&E staining and fluorescence microscopy also showed the presence of NHFs (black arrow) and MIA PaCa-2 cells (white arrow) in the 3D co-cultures (Fig. 2B, inset), suggesting that the 3D coculture method can partially mimic the tumor microenvironment in vitro.
To determine whether the age of the stroma can also influence therapy of PDAC, 3D co-cultures of quiescent NHFs and PDAC-Luc cells were irradiated with a shortened clinical protocol, 2 Gy ϫ 5 days. Results showed that the rate of proliferation of MIA PaCa-2-Luc cells is consistently higher in irradi-

Stromal aging and pancreatic cancer progression
ated co-cultures of NHFs from a 61-year-old donor at 4 -12 days post-irradiation compared with co-cultures with NHFs from a 3-day-old healthy donor (Fig. 2C). Comparable results were also obtained from co-cultures using a separate PDAC cell line, PANC-1-Luc (Fig. 2D). These results support the hypothesis that chronological aging of the stroma significantly affects proliferation and radiation therapy of PDAC cells.
The age difference between the two NHFs (61 years old versus 3 days old) and our earlier observation of chronological aging of NHFs stimulating proliferation of breast cancer cells (13,(25)(26)(27) suggest that the chronological aging of the stroma can significantly influence progression of PDAC. To further investigate this hypothesis, quiescent cultures of NHFs from a 3-day-old donor were aged in vitro for 7 (early) and 60 (late) days. CellTracker Green-labeled MIA PaCa-2 cells were then added on top of the unlabeled quiescent lawns of NHFs. 2D co-cultures of MIA PaCa-2 cells and NHFs were continued in vitro for an additional 2-6 days. Microscopy imaging and flow cytometry methods (13) were used to measure proliferation of MIA PaCa-2 cells in co-cultures of NHFs. Microscopy evaluation of the density of MIA PaCa-2 cells showed a significant increase in proliferation of MIA PaCa-2 cells co-cultured with late-NHFs (3-fold increase at 6 days of co-culture) (Fig. 3A). The doubling time for MIA PaCa-2 cells co-cultured with quiescent cultures of late-NHFs was calculated to be 23 h compared with a doubling time of 33 h for MIA PaCa-2 cells co-cultured with early-NHFs (Fig. 3B). These results were further confirmed by flow cytometry analysis of the percentage of MIA PaCa-2 cells in co-cultures of early-and late-NHFs; there was more than 60% increase in the number of MIA PaCa-2 cells co-cultured with late-NHFs compared with early-NHFs (Fig.  3C). These results clearly show that quiescent cultures of NHFs that are chronologically aged in vitro also promote proliferation of PDAC cells. To rigorously test this novel observation of chronological aging affecting progression of PDAC, additional 2D co-culture experiments were performed using NHFs from donors of different ages. Results showed a significant increase in the percentage of MIA PaCa-2 cells from 7% in co-cultures of quiescent cultures of NHFs from a 3-day-old donor to 18 and 30% in co-cultures with NHFs from 12-and 61-year-old healthy donors, respectively (Fig. 3, D and E). Comparable results were also observed in co-cultures of NHFs and PANC-1 and BxPC-3 PDAC cell lines (Fig. 3E). Additionally, a small but statistically significant increase in proliferation of nonmalignant H6c7 pancreatic ductal epithelial cells was also observed in co-cultures of NHFs from a 61-year-old donor (Fig. 3E). These results showed that the chronological aging of NHFs promotes proliferation of both malignant and nonmalignant pancreatic ductal epithelial cells, but the growth stimulatory effects are significantly higher in malignant compared with nonmalignant cells.
Results from the 3D co-culture experiments demonstrating radiation resistance of PDAC cells in co-cultures of old compared with young NHFs (Fig. 2D) were also recapitulated in experiments using 2D co-cultures. CellTracker Green-labeled MIA PaCa-2 cells were layered upon confluent cultures of NHFs from 3-day-old and 12-and 61-year-old healthy donors. Co-cultures at 5 days post-plating were irradiated with 0, 2, and 4 Gy ionizing radiation and continued in culture. The percentage of CellTracker Green-labeled MIA PaCa-2 cells in control and irradiated co-cultures was analyzed by flow cytometry. Results showed a significant increase in the percentage of MIA PaCa-2 cells in un-irradiated control co-cultures of 12-and 61-year-old compared with 3-day-old NHFs; approximately, 15 and 30% MIA PaCa-2 cells in co-cultures of 12-and 61-year-old NHFs compared with 5% MIA PaCa-2 cells in co-cultures of 3-day-old NHFs ( Fig. 4A). Co-cultures irradiated with 4 Gy radiation showed a significant decrease in the percentage of MIA PaCa-2 cells in all co-cultures compared with their respective un-irradiated controls (Fig. 4A). However, the decrease in the percentage of MIA PaCa-2 cells in irradiated co-cultures of 61-year-old NHFs was significantly less than co-cultures with 12-year-old and 3-day-old NHFs ( Fig. 4A), further suggesting that aging NHFs confer radiation resistance of MIA PaCa-2 cells. Indeed, flow cytometry analysis of radiation-induced toxicity of MIA PaCa-2 cells in co-cultures of NHFs showed maximal cell death in co-cultures of 3-day compared with 12-and 61-year-old NHFs (Fig. 4B). Overall, these results showed that quiescent cultures of NHFs from donors of older compared with younger healthy individuals stimulate proliferation and induce radiation therapy resistance of PDAC cells (Figs. 1-4).

ALOX12 and 12-(S)-HETE mediate fibroblasts' chronological aging-induced stimulation of proliferation of PDAC cells
We previously reported significant increases in mitochondrial respiration rates and decreases in glycolytic flux in NHFs from 29 -70 -year-old healthy donors (14), demonstrating that changes in cellular metabolism occur during chronological aging of NHFs. To determine whether specific metabolic processes are altered during chronological aging, metabolomics profiling was performed (University of Iowa Metabolomics

Stromal aging and pancreatic cancer progression
Core Facility). A semi-targeted high-resolution GC-MS protocol was used to quantitatively measure metabolites of the TCA cycle, glycolytic intermediates, amino acids, sugars, and fatty acids. Results showed significant increases in fatty acids levels of NHFs from older healthy donors, mostly in the 18 -22 carbon fatty acids (Fig. 5A). Among this group of fatty acids, levels of AA (C20:4) showed an increase of ϳ2-fold. AA is converted to eicosanoids by cyclooxygenase and ALOX enzymes. Initially, a RT-qPCR assay was performed to determine whether an agerelated increase in AA metabolism could be due to an increase in the expression of prostaglandin-endoperoxide synthase (PTGS-1 and -2) and arachidonic acid lipoxygenase (ALOX5, -12, and -15) enzymes. Results showed no significant difference in the expression of PTGS-1 and -2, and ALOX5 and ALOX15 between the 61-year-and 3-day-old NHFs. However, a significant difference in the expression of ALOX12 was observed in the 61-year-old NHFs: a ϳ6-fold increase in mRNA levels ( Fig.  5B) and 2-fold increase in protein levels ( Fig. 5B, inset). This intriguing observation of an age-related increase in ALOX12 expression was further evident from a corresponding increase in its secreted metabolite, 12-(S)-HETE. Levels of 12-(S)-HETE were significantly lower (Ͻ10 fg cell Ϫ1 ) in NHFs from a 3-dayand 5-month-old healthy donor compared with 10 -35 fg cell Ϫ1 in NHFs from the 58 -70 -year-old group (Fig. 5C). To further verify that the increases in 12-(S)-HETE levels are true representation of the chronological aging process, NHFs from a 3-day healthy donor were aged in quiescence for 7 (early) and 60 (late) days. Consistent with the results of 12-(S)-HETE levels in NHFs from donors of different ages (Fig. 5C), early-NHFs showed significantly lower levels of 12-(S)-HETE (Ͻ10 fg cell Ϫ1 ) compared with more than 20 fg cell Ϫ1 in late-NHFs (Fig.  5D). Overall, these results showed significant increases in the expression of ALOX12 and its metabolite, 12-(S)-HETE during the chronological aging of NHFs.
Considering the mitogenic property of 12-(S)-HETE (28, 29), we hypothesize that 12-(S)-HETE secreted from the aging stroma stimulates proliferation of PDAC cells via a paracrine signaling pathway. To test this novel hypothesis, we first measured mRNA levels of ALOX12 in established PDAC cell lines (MIA PaCa-2 and PANC-1), recently generated patient-derived PDAC cell lines (339 and 403), and a transformed but nonmalignant (H6c7) human pancreatic epithelial cell line. Expression of ALOX12 in all four PDAC cell lines was significantly lower compared with its expression in the nonmalignant cell line, H6c7 (Fig. 5, E and F). Next, we examined whether the receptor

Stromal aging and pancreatic cancer progression
for 12-(S)-HETE, GPR31, is differentially expressed in normal compared with PDAC cells. Expression of GPR31 was found to be significantly lower in NHFs and H6c7 cells compared with a 6 -14 -fold increase in the PDAC cell lines (Fig. 5G). These results suggest that whereas 12-(S)-HETE secreted from NHFs may not affect their own proliferation because of significantly lower expression of GPR31 and quiescent growth state, they can have a paracrine effect on neighboring PDAC cells that have significantly higher expression of GPR31. This premise is supported from results presented in Fig. 5H. A dose-dependent increase in the number of MIA PaCa-2 cells was observed when different concentrations (1-10 fg cell Ϫ1 ) of 12-(S)-HETE were added to the medium and cells were cultured for 24 -48 h (Fig.  5H). Overall, these results suggest that 12-(S)-HETE secreted from the aging stroma initiates a paracrine mitogenic effect that results in enhanced proliferation of PDAC cells.

Increases in MAPK signaling pathways and metabolism combined with a lower oxidation status of PDAC cells in co-cultures of old NHFs
We have previously shown that quiescent cultures of 3-day NHFs aged in vitro induced ERK1/2-cyclin D1 proliferative pathway in human breast cancer cells resulting in their enhanced proliferation (13). To determine whether older NHFs-induced proliferation of PDAC cells (Figs. 1-4) is associated with the induction of MAPK pathways, MIA PaCa-2 cells were first separated from co-cultures of NHFs by flow cytometry-based cell sorting. RayBiotech MAPK pathway phosphorylation arrays were used to measure the phosphorylation status of pro-proliferative signaling proteins. Although phosphorylation (active form) of most of the proliferative-signaling proteins increased in MIA PaCa-2 cells co-cultured with 61-year-old compared with 3-day-young NHFs, increases in the ERK1/2, and mTOR signaling pathways were more pronounced (Fig. 6). These results suggest that increases in the ERK1/2 and mTOR signaling pathways can mediate enhanced proliferation of PDAC cells in co-cultures of old compared with young NHFs.
Increases in glucose and mitochondrial metabolism during progression from G 0 -G 1 to S-G 2 -M phases suggest that cellular metabolism is intimately linked to cellular proliferation (30). To determine whether chronological aging of NHFs alters the metabolism of PDAC cells that results in their enhanced proliferation, flow cytometry-based cell sorting was used first to separate MIA PaCa-2 cells from 2D co-cultures of quiescent NHFs and then metabolites of MIA PaCa-2 cells were analyzed using a Thermo Q Exactive GC-MS Orbitrap instrument (UIOWA

Stromal aging and pancreatic cancer progression
HCCC Metabolomics Core). Results showed significant increases in the metabolites of the glycolytic (glucose, fructose 6-phosphate, lactate, and pyruvate) and pentose phosphate pathway (PPP: ribose 5-phosphate and sedoheptulose 7-phosphate) in MIA PaCa-2 cells co-cultured with NHFs from a 61-year-old compared with a 3-day-old healthy donor (Fig. 7A). These results suggest that increases in cellular metabolism of PDAC cells can mediate their enhanced proliferation in co-cultures of old compared with young NHFs.
To determine whether older NHFs-induced increases in glycolysis and PPP of MIA PaCa-2 cells also impact their redox status, co-cultures were incubated with dihydroethidium (DHE). DHE oxidation was analyzed by flow cytometry (30). Consistent with results presented in Fig. 3, the percentage of the MIA PaCa-2 cells was higher in co-cultures of quiescent NHFs from the 61-year-old compared with 3-day-old healthy donor (Fig. 7B). It is interesting to note that the DHE oxidation was significantly lower in MIA PaCa-2 cells co-cultured with NHFs from the 61-year-old compared with 3-day-old healthy donor (Fig. 7C). These results suggest that older NHFs facilitate a reducing environment of MIA PaCa-2 cells by rewiring their metabolism to glycolysis and PPP. Overall, these results (Figs. 6 and 7) suggest that older NHFs promote proliferation of PDAC cells by enhancing the MAPK signaling pathways and cellular metabolism that favor a reducing environment supporting proliferation of PDAC cells.

Pharmacological and molecular interventions of ALOX12 and 12-(S)-HETE in older NHFs suppress their abilities to stimulate proliferation of PDAC cells
There is growing interest in the use of polyphenols to minimize age-related health issues (31-33). Hydroxytyrosol, a major polyphenol of olives, has been shown to inhibit activity of ALOX12 (34 -36). To determine whether hydroxytyrosol treatment affects 12-(S)-HETE levels in NHFs, quiescent cultures of NHFs from a 61-year-old donor were cultured in medium containing 100 M hydroxytyrosol. An ELISA-based assay was used to measure 12-(S)-HETE in conditioned medium. Results showed more than 50% decrease in 12-(S)-HETE levels in hydroxytyrosol-treated NHFs compared with untreated controls (Fig. 8A). Consistent with earlier results (Figs. 1-4), quiescent cultures from a 61-year-old healthy donor stimulated proliferation of MIA PaCa-2 cells as evident from the significant increase in the percentage of MIA PaCa-2 cells in co-cultures of NHFs (Fig. 8B, upper panels). However, such an increase in proliferation of MIA PaCa-2 cells is significantly suppressed in co-cultures of hydroxytyrosol-treated quiescent cultures of NHFs (Fig. 8B, lower panels). Comparable results were also obtained when experiments were repeated using 3-day-old NHFs that were chronologically aged in vitro. Plating efficiency of MIA PaCa-2 cells cultured in conditioned medium collected from control treatment increased to 40% in conditioned medium collected from late-NHFs compared with 20% in medium collected from early-NHFs (Fig. 8C). However, plating efficiency of MIA PaCa-2 cells cultured in conditioned medium collected from hydroxytyrosol-treated early-and late-NHFs is significantly suppressed (Fig. 8C). These results suggest a role of 12-(S)-HETE regulating proliferation of MIA PaCa-2 cells in co-cultures of NHFs.
The causality of ALOX12 regulating old NHF-induced stimulation of proliferation of PDAC cells was also investigated using a siRNA molecular approach. Treatment with siALOX12 decreased its expression by ϳ50% in NHFs from a 61-year-old donor (Fig. 8D, upper panel). Flow cytometry analysis of 2D co-cultures that were performed using CellTracker Green-labeled NHFs and MIA PaCa-2 cells showed a significant decrease in the percentage of MIA PaCa-2 cells (ϳ35%) co-cultured with siALOX12-treated NHFs compared with 50% in cocultures with control and scramble-siRNA treated NHFs (Fig.  8D, lower panel). Overall, results from the pharmacological and molecular approaches clearly showed a mechanistic role of ALOX12 and its metabolite 12-(S)-HETE regulating old NHFs induced stimulation of proliferation of PDAC cells.

Clinical correlation of ALOX12 and 12-(S)-HETE with PDAC progression and therapy
The clinical significance of our basic science research was determined by measuring ALOX12 and 12-(S)-HETE in PDAC patients' samples. Initially, the Oncomine database was used to analyze microarray transcriptome profiling data that were collected by Buchholz et al. (37) from pancreatic intraepithelial neoplastic lesions, pancreas intraepithelial neoplasia, pancreas ductal adenocarcinoma, and normal pancreas duct. Bioinformatics evaluation of these data set revealed a significant downregulation of the expression of ALOX12 in pancreas carcinoma compared with pancreas cancer precursor and normal pancreas duct (Fig. 9A). Using de-identified surgical samples of PDAC and plasma samples (provided by Dr. J. Cullen; samples were collected from a Phase I trial, NCT number 01852890 for PDAC therapy) (38), we examined protein expression of

Stromal aging and pancreatic cancer progression
ALOX12 in biopsies and 12-(S)-HETE levels in plasma samples. Results showed that the protein levels of ALOX12 were significantly lower in PDAC cells compared with nearby nonmalignant cells (Fig. 9B), which is also consistent with the Oncomine data (Fig. 9A). A higher level of 12-(S)-HETE was observed in all patients' plasma samples post-therapy compared with pretherapy (Fig. 9C). Within the post-therapy samples, a correlation was observed between 12-(S)-HETE levels and incidence of recurrence and metastasis: higher levels of 12-(S)-HETE correlating with higher incidence of recurrence and metastasis in PDAC patients (Fig. 9, D and E). These results attest to the clinical significance of our basic science research. In summary, results from this study showed the significance of the chronological aging of normal human fibroblasts in pancreatic cancer and identified ALOX12 and 12-(S)-HETE as novel molecular regulators of stromal aging-induced progression and therapy outcomes of PDAC (Fig. 9F).

Discussion
Like all cancers, the incidence of PDAC increases exponentially with age (3). Although a majority of PDAC research focuses on understanding the cellular and molecular control of the epithelial cancer cells, we investigated whether the chronological aging of normal cells regulates progression and therapy outcomes of PDAC, and the mechanisms involved in this process. Our results showed fibroblasts from older healthy donors stimulate proliferation and confer resistance to radiation therapy of human PDAC cells. Results from pharmacological and molecular approaches identified ALOX12 and its metabolite 12-(S)-HETE contributing to older fibroblasts' induced stimulation of proliferation and radiation resistance of PDAC cells.
Replicative and chronological lifespan are two of the most widely studied modes of cellular aging (39 -41). Chronological lifespan represents the duration of quiescence during which normal cells retain their capacity to re-enter the proliferative cycle (25,26). We previously reported that the chronological aging of fibroblasts is independent of mitotic division and telomerase activity (25). Our results also showed quiescent cultures of NHFs from a 3-day-old healthy donor that were chronologically aged in culture to stimulate proliferation of human breast cancer cells (13). The present study was designed to investigate whether naturally aged NHFs influence progression and therapy of PDAC cells. Results from in vivo murine xenograft of co-cultures of NHFs and human MIA PaCa-2 cells

Stromal aging and pancreatic cancer progression
showed a higher tumor volume in co-cultures of older NHFs and MIA PaCa-2 cells, which was also associated with a lower median survival (Fig. 1, D and E). Comparable results were also obtained from 3D and 2D co-cultures of MIA PaCa-2 cells and quiescent cultures of NHFs from donors of different ages (Figs.  2, A and B, and 3). Similar results were also obtained from 2D co-culture experiments that were performed using PANC-1 and BxPC-3 PDAC cells (Fig. 3E). A small but statistically significant increase in the proliferation of H6c7 (transformed but nonmalignant) human pancreatic epithelial cells was also observed (Fig. 3E). NHFs' age-induced proliferation of PDAC cells appear to be independent of the k-ras genotype status of PDAC cells. Older NHFs stimulated proliferation of both WT (BxPC-3) and mutant (MIA PaCa-2 and PANC-1) k-ras cells ( Fig. 2A). Additional results showed that the older NHFs confer radiation resistance of MIA PaCa-2 and PANC-1 cells (Figs. 2,  C and D, and 4). Overall, our earlier results of chronologically aged in vitro cultures of NHFs stimulating proliferation of breast cancer cells (13) and results (Figs. 1-4) from this study clearly show that the chronological aging of NHFs is a critical factor in progression and therapy outcomes of PDAC.
In 1966 Stoker et al. (42) first reported quiescent cultures of rodent fibroblasts inhibiting proliferation of polyoma-transformed rodent fibroblasts. Flaberg et al. (43) used a highthroughput live-cell imaging method to study the effects of 107 human fibroblasts on proliferation of human prostate cancer cells in vitro. Although all primary cultures of fibroblasts inhibited growth of the prostate cancer cells, pediatric fibroblasts showed a higher inhibitory effect compared with adult fibroblasts. Although the authors did not address the age effect of these fibroblasts, their results did invoke the hypothesis that the age of the stroma (fibroblasts) is a significant factor in epithelial cancer progression. Indeed, laser capture microdissection and gene expression analysis of human breast cancer tissues did  (37). B, immunohistochemistry analysis of ALOX12 in paraffin-embedded and de-identified resected PDAC tissues: black arrows, PDAC cells; yellow arrows, nonmalignant stromal cells. C, 12-(S)-HETE levels in de-identified plasma samples from 14 PDAC patients. Results were evaluated for incidence of (D) recurrence and (E) metastasis. Asterisks represent significance, p Ͻ 0.05, measured by paired t test. F, an illustration showing increases in ALOX12 expression and 12-(S)-HETE levels during chronological aging of NHFs promoting PDAC progression by enhancing MAPK signaling pathways and metabolism. It is hypothesized that increases in metabolism combined with a reducing status confer resistance of PDAC cells to therapy in an aging-stromal environment.

Stromal aging and pancreatic cancer progression
show a distinct stromal-aging footprint in older compared with younger patients (44). Consistent with these literature reports, our results also showed an age-effect of NHFs stimulating proliferation of PDAC cells (Figs. 1-4). Based on single-cell transcriptomic analysis, recent studies report the presence of at least three different types of fibroblasts in the PDAC stroma: fibroblasts exhibiting myofibroblastic features (␣SMA high ) that surround the malignant PDAC and fibroblasts exhibiting inflammatory (cytokine and chemokine positive) and antigenpresenting properties that reside distally from the PDAC cells (45)(46)(47). Based on our observation of higher ALOX12 expression and higher levels of its metabolite 12-(S)-HETE (inflammatory signature) in older NHFs (Fig. 5), we speculate that the distally localized fibroblasts with the inflammatory phenotype represent the aging stroma and they contribute to the stromal age-related progression of PDAC.
We previously reported a shift in cellular metabolism from glycolysis in young to mitochondrial respiration in older NHFs (14). Decreases in extracellular acidification rates and increases in oxygen consumption rates during chronological lifespan of NHFs (14) suggest that metabolic pathways other than glycolysis are involved with the age-related increases in respiration of NHFs. Mitochondrial respiration also receives substrates from lipid metabolism, suggesting that the age-related increases in respiration of NHFs could be due to a change in their lipid metabolism. Indeed, results from MS analysis of metabolites showed significant increases in free fatty acid levels of older compared with young NHFs (Fig. 5A). An age-related increase in AA metabolism of NHFs was also evident from the corresponding increases in the expression of ALOX12 and its mitogenic metabolite, 12-(S)-HETE (Fig. 5, B-D). It is interesting to note that the expression of ALOX12 was significantly lower in established (Fig. 5E) and newly generated (Fig. 5F) patient-derived PDAC cell lines, whereas the expression of GPR31 (receptor for 12-(S)-HETE) was significantly higher in PDAC cells compared with NHFs and H6c7 nonmalignant pancreatic epithelial cells (Fig. 5, E-G). These results support the hypothesis that a paracrine signaling from older NHFs provides a mitogenic environment for PDAC cells. Although 12-(S)-HETE secreted from older NHFs may not stimulate their own proliferation due to a lower level of expression of GPR31, it is anticipated to provide mitogenic stimuli to PDAC cells due to their higher level of expression of GPR31. Indeed, exogenously added 12-(S)-HETE dose-dependently enhanced proliferation of MIA PaCa-2 cells (Fig. 5H).
Consistent with our earlier results of chronologically aged 3-day NHFs enhancing the ERK1/2 pathway that resulted in significant increases in proliferation of breast cancer cells (13), results from the MAPK arrays also showed significant increases in the ERK1/2 and mTOR pathways in MIA PaCa-2 cells that were associated with their enhanced proliferation in co-cultures of old compared with young NHFs (Fig. 6). As anticipated, increases in the ERK1/2 and mTOR pathways were also associated with increases in cellular metabolism (glycolysis and PPP) of MIA PaCa-2 cells in co-cultures of old compared with young NHFs (Fig. 7A). Whereas the nonoxidative pathway of the PPP is involved in nucleotide biosynthesis (ribose 5-phosphate), the oxidative pathway of the PPP promotes a reducing environ-ment (NADPH, GSH). Indeed, old NHFs-induced increases in the PPP of MIA PaCa-2 cells were associated with a lower oxidation status compared with the oxidation status of MIA PaCa-2 cells co-cultured with young NHFs (Fig. 7, B and C). A lower oxidation status (reducing environment) is anticipated to favor proliferation and therapy resistance of MIA PaCa-2 cells in co-cultures of old compared with young NHFs. This premise is consistent with results presented in Figs. 1-7.
Results from pharmacological and molecular approaches clearly showed a role of ALOX12 and 12-(S)-HETE contributing to old NHF-induced proliferation of PDAC cells (Fig. 8). Because expression of ALOXs is down-regulated in human bladder, breast, lung, colon, and prostate cancer cells (3,48,49), our results also suggest that ALOXs and their metabolites HEP-TEs and HETEs provide a stromal aging-derived mitogenic axis that promotes cancer progression and confer resistance to therapy. The clinical significance of our basic science research is evident from our observation of a significant decrease in ALOX12 expression in PDAC cells compared with their neighboring nonmalignant cells as well as plasma levels of 12-(S)-HETE correlating with therapy outcomes, recurrence, and metastasis ( Fig. 9, A-E).
In summary, our results showed that the chronological aging of NHFs is a critical regulator of progression and therapy outcomes of PDAC. Intervention of the aging stroma by targeting ALOX12 expression (Fig. 9F) can be a novel approach to prevent progression and improve therapy of PDAC.

Experimental procedures
Additional details of the experimental procedures are included under the "Supporting Materials".

Cell culture and reagents
All cell cultures (Table S1) were performed using Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum and antibiotics in humidified incubators with ambient oxygen concentration. Measurements in NHFs were performed using individual and primary cultures from donors of different ages; samples were not pooled. Microscopy and flow cytometry assays were used to visualize and quantitate the percentage of NHFs and PDAC cells in co-cultures. Luciferase expressing PDAC cells and Cell Mate 3D Gel matrix (BRTI Life Sciences) were used for the 3D co-culture experiments. Photon counts (Xenogen IVIS 200 system) were used to measure proliferation of luciferase expressing PDAC cells in 3D co-cultures. Hydroxytyrosol was purchased from the Cayman Chemical. A shortened clinical radiation therapy protocol (2 Gy ϫ 5 days; dose rate 0.6 Gy min Ϫ1 ) was used to irradiate co-cultures.

Murine tumor xenograft
All animal experiments were reviewed and approved by the Animal Care and Use Committee of The University of Iowa; approval number 1311211-001. Cell Mate 3D Gel matrix-embedded co-cultures of CellTracker Green-labeled NHFs and luciferase expressing MIA PaCa-2 cells (10:1) were injected into the flank of 5-week-old female athymic mice, and imaged when palpable tumors were detected. Microscopy and flow cytometry methods were used to identify NHFs and MIA PaCa-2 cells

Stromal aging and pancreatic cancer progression
in the excised xenograft. In a separate set of experiments, tumor volume of xenograft carrying co-cultures of NHFs and MIA PaCa-2 cells was measured using a digital caliper.

Analysis of metabolites
A ThermoQ Exactive Mass Spectrometer (The UIOWA Metabolomics Core Facility) was used to analyze metabolites in cells and conditioned media. Xcailbur Tracefinder 4.1 software was used for data analysis.

RT-qPCR assay
Total cellular RNA was extracted using TRIzol reagent (Invitrogen) and quantified using a NanoDrop-1000 Spectrophotometer (Thermo Fisher Scientific). One microgram of total RNA was reverse-transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). cDNAs were used for Real-Time PCR amplification using the Power SYBR Green Master Mix (Applied Biosystems) and StepOne Plus TM System (Applied Biosystems) following our published method (30).

siRNA knockdown
Quiescent cultures of NHFs were transfected with scrambled (SR30004) and siALOX12 (SR300166; OriGene) using Lipofectamine 2000 (Invitrogen) following the manufacturers protocol. Immunoblotting assay was used to measure the efficiency of siRNA knockdown of ALOX12 expression at 48 -96 h post-transfection.

Immunoblotting assay
Cell pellets were pulse-sonicated (Vibra-Cell Cup Horn; Sonics and Materials Inc.) and equal amounts of protein lysates were separated on SDS-PAGE followed by semi-dry electroblotting to nitrocellulose membrane (Bio-Rad Labs). Membranes were incubated with antibodies to human ALOX12 (Milipore-Sigma) and ␤-actin (Abcam). Pierce TM ECL 2 Western blotting Substrate (Thermo Scientific) and Typhoon FLA 7000 (GE Healthcare) were used to visualize immune-reactive polypeptides.

Immunohistochemistry assay
De-identified PDAC tissues and plasma samples were obtained from Dr. Cullen's recently completed phase I trial (NCT number 01852890) designed to examine the safety of pharmacological ascorbate with gemcitabine for the treatment of pancreatic cancer. Paraffin-embedded and de-identified surgical tissue sections were dewaxed and processed for analysis of ALOX12 expression using primary ALOX12 antibody (Millipore-Sigma), horseradish peroxidase-linked secondary antibody, and 3,3Ј-diaminobenzidine as the chromogen. Slides were counterstained with Harris hematoxylin and images were recorded using microscopy.

Statistical methods
SPSS software was used for all statistical analysis. For experiments with three or more continuous variables, statistical analysis was performed using the one-or two-way analysis of variance with Tukey's honestly significant difference. Homogeneity of variance is assumed with 95% confidence interval level. Results from at least n Ն 3 with p Ͻ 0.05 are considered significant. Group survival was summarized with Kaplan-Meier plots and compared with log-rank and Gehan-Breslow-Wilcoxon tests.

Data availability
All the data described are contained within the manuscript and associated supporting information.