Polyunsaturated fatty acids-induced ferroptosis suppresses pancreatic cancer growth

Despite recent advances in science and medical technology, pancreatic cancer remains associated with high mortality rates due to aggressive growth and no early clinical sign as well as the unique resistance to anti-cancer chemotherapy. Current numerous investigations have suggested that ferroptosis, which is a programed cell death driven by lipid oxidation, is an attractive therapeutic in different tumor types including pancreatic cancer. Here, we first demonstrated that linoleic acid (LA) and α-linolenic acid (αLA) induced cell death with necroptotic morphological change in MIA-Paca2 and Suit 2 cell lines. LA and αLA increased lipid peroxidation and phosphorylation of RIP3 and MLKL in pancreatic cancers, which were negated by ferroptosis inhibitor, ferrostatin-1, restoring back to BSA control levels. Similarly, intraperitoneal administration of LA and αLA suppresses the growth of subcutaneously transplanted Suit-2 cells and ameliorated the decreased survival rate of tumor bearing mice, while co-administration of ferrostatin-1 with LA and αLA negated the anti-cancer effect. We also demonstrated that LA and αLA partially showed ferroptotic effects on the gemcitabine-resistant-PK cells, although its effect was exerted late compared to treatment on normal-PK cells. In addition, the trial to validate the importance of double bonds in PUFAs in ferroptosis revealed that AA and EPA had a marked effect of ferroptosis on pancreatic cancer cells, but DHA showed mild suppression of cancer proliferation. Furthermore, treatment in other tumor cell lines revealed different sensitivity of PUFA-induced ferroptosis; e.g., EPA induced a ferroptotic effect on colorectal adenocarcinoma, but LA or αLA did not. Collectively, these data suggest that PUFAs can have a potential to exert an anti-cancer effect via ferroptosis in both normal and gemcitabine-resistant pancreatic cancer.

cell growth by LA and αLA treatment compared with BSA control at day 2, while from day 3, an entire cell death was observed in MIA-Paca2 cells (Fig. 1A).On the other hand, SA treatment did not affect the cell proliferation of MIA-Paca2, but the proliferation was significantly increased with OA treatment (Fig. 1A).Similarly, in Suit-2 cells, LA and αLA treatment induced cell death at day 2, while OA and SA significantly increased cell proliferation (Fig. 1A), suggesting that PUFAs possessing two or more double bonds can have the potentials to suppress the pancreatic cancer cell growth.Moreover, when we examined earlier time points for detailed changes below 48 h of treatment, we confirmed that the lethal event with LA and αLA occurred between 12 and 24 h after treatment (Fig. 1B).Timelapse imaging demonstrated that cells continued to proliferate until around 13 h, then started swelling, ballooning, and being ruptured (Fig. 1C, supplemental movie), which could be observed in the phase of necrosis 30 as well as ferroptosis 31 .Furthermore, we did not observe any difference in cleaved caspase 3 levels between BSA-and LA/αLA-treated MIA-Paca2 or Suit2 cells (Fig. 1D), suggesting that apoptosis might not be in the cascade of LA and αLA-induced cell death.

LA and αLA induced pancreatic cancer cell death through ferroptosis
Given that necroptotic/ferroptotic phenotypes were observed in LA and αLA treated pancreatic cancer cell lines, we investigated if ferrostatin-1, a ferroptosis inhibitor, could affect the pancreatic cancer cell growth in order to confirm the involvement of LA and αLA in ferroptosis pathway.Proliferation assay revealed that ferrostatin-1 did not change the proliferation rate of either MIA-Paca2 or Suit-2 when treated with BSA, however, the effect of LA and αLA on pancreatic cancer cell death was negated by treatment with ferrostatin-1, with proliferation rate being restored back to the same levels as BSA-treated control (Fig. 2A).Furthermore, upregulated lipid peroxidation by LA and αLA treatment in MIA-Paca2 or Suit-2 were also suppressed by ferrostatin-1 treatment to the same level with BSA control (Fig. 2B), suggesting that LA and αLA might induce pancreatic cancer cell death through ferroptosis.

LA and αLA induced ferroptosis pathway leads to RIP/MLKL dependent necroptosis pathway
The molecular mechanism of necroptosis and ferroptosis has been under intensive investigation in recent years.The receptor-interacting protein (RIP) kinase, together with mixed lineage kinase domain-like pseudokinase (MLKL), are considered to be the key mediator of necroptosis 32,33 .Therefore, we assessed the phosphorylation of RIP3 and MLKL in MIA-Paca2 at 12 h and 18 h after FAs treatment by Western blot, and we observed increased phosphorylation levels of RIP3 at 12 h and 18 h after LA treatment and only at 12 h after αLA treatment (Fig. 3A).Similarly, the phosphorylation level of MLKL was significantly increased at 12 h after LA and αLA treatment (Fig. 3A).Furthermore, in consistence with our proliferation results, LA and αLA-induced upregulation of phosphorylation levels on RIP3 and MLKL were negated by treatment with ferrostatin-1 at 12 h (Fig. 3B).We also investigated if necrosis inhibitor GSK'782 could reverse the effect of LA and αLA-induced cell death.Proliferation assay revealed that GSK'782 partially reduced the rate of cell death induced by LA and αLA in a dose dependent manner, although it did not return to the levels observed in the BSA treated cells (Fig. 3C).In addition, although we could not observe a change in GPX4 expression in LA and αLA treated cells (Fig. 3A), deferasirox (DFX), an iron chelator leading to the reduction of lipid peroxidation, successfully negated the LA and αLA-induced cell death in MIA-Paca2 cells (Fig. 3D).These results suggest that the effects of LA/αLA are induced through both a ferroptosis dependent pathway upstream of RIP/MLKL, and a ferroptosis/necroptosisdependent pathway via the RIP/MLKL pathway.

Intraperitoneal administration of LA and αLA suppressed the growth of pancreatic cancer in the xenograft model
Next, the roles of LA and αLA were examined in vivo using the Suit-2 subcutaneous xenograft model.In the mouse i.p. administrated with BSA, apparent cancer growth could be observed in 11 days after transplantation (Fig. 4A-C) and their survival rate started to be decreased at 13 days after transplantation (Fig. 4D).In contrast, i.p. injection of both LA and αLA significantly suppressed the cancer growth (Fig. 4A-C) and ameliorated the decreased survival rate of transplanted mice (Fig. 4D).Consistently with the in vitro results, ferrostatin-1 negated the anti-cancer effect of LA at 19 days after transplantation and the effect of αLA at 15 days after transplantation (Fig. 4A-C), resulting in decrease of survival rate (Fig. 4D).

LA and αLA had different anti-cancer effects on Gemcitabine-resistant pancreatic cancers
To examine the anti-cancer effect on drug-resistant pancreatic cancers, Gemcitabine-resistant cells RPK-1 and RPK-9, which we previously established 34 , were treated with LA and αLA.Additionally, given the predominance of KRAS mutation in pancreatic cancer cells, we used a Bcpx3 lacking the KRAS mutation to further evaluate the ferroptosis/necroptosis effect of LA and αLA.Proliferation assay revealed that LA and αLA successfully induced cell death in normal PK1 and PK9 cells similar to other pancreatic cancer cell lines including MIA-Paca2 and Suit-2, however the effect on RPK-1 and RPK9 was not consistent.Although LA induced cell death in RPK-1 4 days after treatment, αLA did not change the proliferation rate (Fig. 5A).On the other hand, in RPK-9, αLA induced cell death 3 days after treatment, but LA showed only a suppressive effect in cell growth compared with control (Fig. 5A).Interestingly, LA-induced cell death in RPK-1, suppression of proliferation in RPK-9, and αLA-induced cell death in RPK-9 were completely negated by ferrostatin-1, resulting in being restored back to the same levels as BSA-treated control (Fig. 5B).In the case of Bxpc3 cells, which do not possess KRAS mutation generally found in pancreatic cancers, LA and αLA induced an intensive effect of cell death via ferroptosis pathway (Fig. 5C,D).These results suggest that drug-resistant pancreatic cancers may have differential ferroptosis resistance with specific PUFA treatment.Since double bonds possessed by PUFAs are prone to oxidation, it is important to examine the correlation between the number of double bonds in PUFAs and susceptibility of pancreatic cancers to ferroptosis.Among the commonly known and researched PUFAs, arachidonic acid (AA, 20:4), eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6) have been shown by previous studies to have some anti-tumor properties 35 .Thus, we assessed the role of these PUFAs on pancreatic cancer cell growth.In MIA-Paca2, AA, EPA and DHA demonstrated an anti-cancer effect more clearly compared to LA (Fig. 6A).On the other hand, in Suit-2 cells, DHA treatment suppressed cell growth compared to BSA control without causing apparent cell death even at 24 h, while AA induced cell death already at 8 h after treatment (Fig. 6B).EPA treatment induced the cell death at the similar level as with LA treatment (Fig. 6B).

Differential sensitivity to PUFA-induced ferroptosis in lung, skin, liver, colon and brain tumors
To understand the anti-tumor effect on other types of tumor cells including lung, skin, liver, colon and brain tumors, different cell lines were treated with LA, αLA and EPA.In A549 cells (human carcinoma), Skmel23 cells (human melanoma) and HepG2 cells (human hepatoma), all FAs exhibited marked effects on cell death (Fig. 7).
On the other hand, LA and αLA were ineffective in the colon cancers DLD-1 or HCT15 (human colorectal adenocarcinoma), while EPA caused cell death to both cell lines (Fig. 7).In brain tumor cells, all the FAs showed cell death in U251 cells (human glioblastoma) at day 2 after treatment but could only partially suppress the tumor growth in U87 cells (human glioma) (Fig. 7).

Discussion
Extensive studies have recently suggested that ferroptosis plays a pivotal role in tumor suppression and could be harnessed for cancer therapy 36,37 .Ferroptosis is defined as an intracellular iron-dependent form of cell death, and is distinct from other forms of cell death, including apoptosis and necrosis.However, in recent years, several studies have reported a crosstalk between necrosis and ferroptosis phenotypes.Although the activation of RIP1 and RIP3 kinase has been recognized as the key signaling in the necrosis pathway, Tong et al. demonstrated that RIP activation could be observed in RSL3 (a classic ferroptosis inducer)-induced ferroptosis 38 .In addition, GPX4 is proposed to be a master regulator in the ferroptosis process with a unique function to interrupt lipid peroxidation 11 and GPX4-independent ferroptosis pathways, including enzymatic, metabolomic and lipidomic pathways, have been recently discovered in various physiological and pathophysiological processes 12,[39][40][41] .Despite the numerous researches in recent years focusing on both ferroptosis and necrosis, the mechanism behind these cell death processes is only partially understood.However, what is importantly common between ferroptosis and necrosis is the increase in lipid peroxidation in the cell membrane, which triggers the cell swelling, ballooning, and rupture, as observed in Fig. 1C and supplemental movie.So far, numerous studies over several decades have suggested potential anti-tumor effects of PUFAs using multiple targets, including transcriptional and mitochondrial regulation, anti-inflammation, including cell apoptosis as well as necroptosis 42,43 .Based on numerous recent researches, PUFAs are considered to be ultimate The proliferation rate of PK-1, RPK-1, PK-9 and RPK-9 cells (A) and BxPC3 cells (C) treated with BSA, 60 µM LA, and 60 µM αLA (A).Live cells were counted successively for 4 days.One-way ANOVA followed by the Tukey test was used for multiple comparisons.Data shown are the means ± SEM ****P < 0.00001 BSA-control versus LA, #### P < 0.00001 BSA-control and αLA.(B and D) The proliferation rate of RPK-1 and RPK-9 cells (B) and Bxpc3 cells (D) treated with 60 µM LA, and 60 µM αLA alone or together with 10 µM ferrostatin-1.Live cells were counted successively for 4 days.One-way ANOVA followed by the Tukey test was used for multiple comparisons.Data shown are the means ± SEM ****P < 0.00001 between with and without ferrostatin-1 in LA-treated cells, #### P < 0.00001 between with and without ferrostatin-1 in αLA-treated cells.triggers of ferroptosis because increased lipid absorption can enrich the PUFA content of cell membranes 44 , driving the accumulation of lipid peroxidation and excess iron which produces ROS 10 .This ultimately leads to the porosity, destruction of the barrier function, and alteration in membranes permeability 45,46 .Here, we found that the treatment of LA and αLA among 18-carbon FAs increased lipid peroxidation (Fig. 2B) and suppressed pancreatic cancer growth with necroptotic/ferroptotic morphological change (Fig. 1A-C).Previous studies have shown that ferrostatin-1 is able to slow the accumulation of lipid hydroperoxides by acting as a radical-trapping antioxidants 47 and also able to prevent the oxidative destruction of membrane lipid PUFAs 48 .In this study, ferrostatin-1 expectedly negated the increase of lipid peroxidation and cell death by LA and αLA treatment, restoring the levels back to the normal cell proliferation rate (Fig. 2A,B).Similarly to our results, molecular biological studies on ferroptosis in pancreatic cancers revealed that ARF6-silencing increased the expression of ACSL4, which enrich cellular lipid membrane with long PUFAs 27 , resulting in RLS3-induced ferroptotic cell death of PANC-1 and MIA-Paca2 49 .In addition, MGST1 limits lipid peroxidation via interaction with ALOX5, resulting in inhibition of ferroptotic pancreatic cancer cell death 50 .These results suggest that PUFAs enriched in pancreatic cancer cell membrane might induce ferroptosis/necrosis, possibly through excess ROS production derived from lipid peroxidation.We further demonstrated that ferrostatin-1 negated the LA and αLA-induced increased phosphorylation of both RIP3 (Ser166) and MLKL (Ser358), restoring back to normal cell proliferation rate in MIA-Paca2 (Fig. 3A,B).In addition, necroptosis inhibitor GSL'782 partially suppressed the LA and αLA-cell death although it did not return to the same level with BSA control (Fig. 3C).Collectively, these results indicate that LA/αLA induced lipid peroxidation is upstream of RIP/MLKL dependent necrosis pathway.Therefore, PUFA induced cell death might be dependent on both ferroptosis and necroptosis (Fig. 8).
Given that development of resistance to cancer therapy remains a major challenge, a number of preclinical and clinical studies have focused on overcoming drug resistance.Although gemcitabine is the main chemotherapy treatment for pancreatic cancer, resistance to gemcitabine begins to develop after a few weeks of treatment 51 .Gemcitabine, a deoxycytidine analog, has its main effects on DNA synthesis and promots DNA damage.However, long usage of gemcitabine leads to the accumulation of cytidine deaminase (CDA), which enhances drug detoxification of gemcitabine itself 52 .Other studies have demonstrated that excessive activation of PI3K/AKT Figure 6.Ferroptosis effect by PUFA on pancreatic cancers is not dependent on the increasing number of carbon or double bonds in the PUFA.(A and B) The proliferation rate of MIA-Paca2 cells (A) and Suit-2 cells (B) treated with BSA, 60 µM LA, 60 µM AA, 60 µM EPA and 60 µM DHA alone or together with 10 µM ferrostatin-1.Live cells were counted successively up to 24 h.One-way ANOVA followed by the Tukey test was used for multiple comparisons.Data shown are the means ± SEM *P < 0.00001, #### P < 0.00001, ┼┼┼┼ P < 0.00001, ǂ ǂ ǂ ǂ P < 0.00001 BSA-control versus LA, DHA, AA, EPA, respectively.aaaa P < 0.00001 between with and without ferrostatin-1 treatment.and nuclear factor-κ-gene binding (NF-κB) signaling can lead to gemcitabine resistance in pancreatic cancers 53,54 .Although multiple mechanisms of gemcitabine resistance have been identified, we hypothesized that direct incorporation of PUFAs into gemcitabine-resistant pancreatic cancers can cause ferroptosis cell death similarly to other drug-sensitive pancreatic cancer cells including MIA-Paca2 and Suit-2.Unexpectedly, LA induced cell toxicity in RPK-1 and suppressed cell growth in RPK9 at 3-4 days after treatment, while αLA induced cell toxicity in RPK-9, but did not affect the RPK-1 growth (Fig. 5A).Ferrostatin-1 successfully restored these anticancer effect back to the control levels, suggesting that specific PUFAs partially induce ferroptosis cell death in gemcitabine-resistant pancreatic cancer cells.Considering the effect of gemcitabine in DNA synthesis, consequent altered lipid metabolism in RPK-9 may selectively affect the anti-cancer effect of αLA.In fact, Doll et al. have demonstrated that the ACSL4-deficient Pfa1 cells (TAM-inducible disruption of Gpx4 in mouse embryonic fibroblast cells) are refractory to ferroptosis, which can be re-sensitized by re-expression of ACSL4 27 .Other studies also demonstrated increased levels of fatty acid synthase (FASN) in gemcitabine-resistant pancreatic cancers, and FASN silencing downregulated the resistance 55 .These pieces of evidence together with our results may suggest that the difference in lipid metabolism between normal PK cells and gemcitabine-resistant PK cells exhibits different sensitization to ferroptosis induced by PUFAs including LA and αLA.Our challenge to examine the correlation between the number of double bonds in PUFAs and the susceptibility of pancreatic cancers to ferroptosis demonstrated unexpected results.Considering the pro-inflammatory pathway of ROS production from AA, it was reasonable that AA exhibited a strong ferroptotic effect on MIA-Paca2 and Suit2 cells (Fig. 6A,B).EPA also demonstrated ferroptotic effects attaining entire cell death at 24 h and at a similar level as with LA and AA in Suit-2 cells (Fig. 6B).However, DHA, which possesses a larger number of double bonds used in this study, showed a mild anti-cancer effect compared to the drastic effects of LA, AA and EPA (Fig. 6B).It should be noted that stressed cells caused by PUFA overload from the microenvironment activate protective membrane remodeling mechanisms to redistribute these PUFAs phospholipid membrane pools, and sequestered them into lipid droplets (LD) and TAGs pools, thereby preventing PUFA oxidation and cellular damage [56][57][58][59] .Moreover, it has been shown that the increase in LD formation in cells treated with FAs correlates with the number of double bonds in the FAs, and especially DHA-treated colorectal cancer cells (SiHa and HCT-116) showed the largest number of LDs formed 60 .Additionally, PUFAs are hydrolyzed by the phospholipase A2 (PLA 2 ) enzymes and released from membrane phospholipids and then imported into cells through various fatty acid transport proteins, such as fatty acid translocase (FAT/CD36), fatty acid transport protein, and fatty acid binding protein 61,62 .Particularly, AA and EPA are preferentially released by cytoplasmic PLA 2 , whereas DHA is released by Ca 2+ -independent PLA 2 63 .Together, these data corroborate our findings of DHA showing a mild effect on ferroptosis compared to other PUFAs.The mechanisms of how altered lipid metabolism differentiate the preference of FAs for ferroptosis will be further explored in future studies.
Increasing evidence has demonstrated that PUFAs play significant roles in tumor risk and progression 64 .However, despite numerous pre-clinical mechanistic studies using cell lines and mouse models to determine molecular targets of PUFAs, they have not been translated into clinical trials.Lipid metabolic alterations frequently occur in malignant tumors, and thus, understanding the mechanisms that maintain lipid homeostasis in drug-resistant tumor cells may reveal the metabolic characteristics that could be applied to the clinical setting.Having shown the effect of PUFAs in pancreatic cancer cells, we further challenged different tumor cell lines with PUFAs treatment (Fig. 7).We indeed observed a differential sensitivity to PUFA-induced ferroptosis in different cancer cell lines and these findings support the previous observation of PUFAs-induced ferroptotic cell death in tumors.In fact, docosapentaenoate (DPA, n-6 FA) and DHA treatment induced ferroptotic cell death in effect in HCT-116 cells (human colorectal carcinoma) 60 .Furthermore, in melanoma A375 and B16F10, AA synergizes with interferon (IFN)γ to induces ferroptotic cell death 65 .These results suggest that different tumors may have a specific preference in PUFAs uptake into cell membrane or may have differential effects on lipid metabolism.In this study, we demonstrated that only EPA induced ferroptosis in human colorectal carcinoma including DLD-1 and HCT-15, but not LA and αLA (Fig. 7).Moreover, previous studies have shown unique inherent susceptibility of different cancer cells to ferroptosis due to the differences in metabolic reprograming, imbalance in ferroptosis defenses and genetic mutations [66][67][68] .For example, it has been shown that in gastrointestinal cancer cells, DNA methylation-mediated silencing of very long-chain fatty acid protein 5 (ELOVL5) and fatty acid desaturase 1 (FADS1), as well as the inability of these cells to generate AA and adrenic acid (AdA) from LA to be oxidized by lipoxygenases, render them resistant to ferroptosis 66 .On the other hand, it has been shown that in isocitrate dehydrogenase 1 (IDH1) mutant cells such as glioblastomas, the oncometabolite, 2-Hydroxyglutarate, increases erastin-induced lipid ROS accumulation and sensitizes cells to ferroptosis 68 .As such, further research to elucidate www.nature.com/scientificreports/ the cancer type-specific requirement of different PUFAs for ferroptotic as well as necroptotic cell death is needed to advance therapeutic targeting of these diseases.

Conclusion
In this study, we newly demonstrated that LA and αLA induced ferroptotic cell death in MIA-Paca2 and Suit-2, which is negated by ferrostatin-1 restoring back to BSA control levels.Consistently, intraperitoneally administration of LA and αLA suppressed subcutaneously transplanted Suit-2 cell growth in xenograft model mice.In addition, LA and αLA partially induced a ferroptotic effect on the gemcitabine-resistant-PK cells, although the effect was exerted late compared to treatment of normal-PK cells.The trial to validate the importance of double bonds in PUFAs in ferroptosis revealed that AA and EPA showed marked effects of ferroptosis on pancreatic cancer cells, while DHA showed mild suppression of cancer growth.Further challenges into other tumor types revealed different sensitivity of PUFA-induced ferroptosis.Taken together, these data suggest that PUFAs can have a potential to exert anti-cancer effect via ferroptosis in both normal and gemcitabine-resistant pancreatic cancers.

Ethics statement
We confirmed that all methods were performed in accordance with the relevant guidelines and regulations.The research was carried out in compliance with the ARRIVE guidelines (https:// arriv eguid elines.org).This study also complies with all relevant ethical regulations in Japan.The animal protocols and procedures for experiments were approved by the Animal Committee of the Tohoku University School of Medicine Ethics Committee (2022MdA-012), and all experiments were conducted according to the ethical and safety guidelines of the Institute.

Antibodies and reagents
Primary antibodies used in this study were rabbit polyclonal anti-cleaved caspase-

Cell culture and treatment
Human pancreatic cell lines MIA Paca2 and Suit-2, human lung cancer cell line A549, human hepatocellular carcinoma HepG2, human colon adenocarcinoma DLD-1, HCT-15, and Colo-205, and human glioblastoma cell line U87 were obtained from Cell Resource Centre for Biomedical Research in Tohoku University.Human melanoma cell lines SKmel23 were kindly provided by Prof. Yutaka Kawakami in Keio University 69 .Human glioblastoma cell line U251 was purchased from the JCRB Cell Bank (Osaka, Japan).Human pancreatic cancer cell lines and acquired gemcitabine-resistant human pancreatic cancer cell lines were established as described 34,70,71 .MIA Paca2, Suit-2, PK-1, RPK-1, PK-9, RPK-9 and Skmel23 were maintained in RPMI1640 Medium (Sigma Aldrich, Co., St. Louise, MO, USA) and other cell lines in Dulbecco's modified Eagle's medium (Sigma Aldrich, Co.) both supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific Inc.) and 1% (v/v) penicillin/streptomycin (Thermo Fisher Scientific Inc.) at 37 °C with 5% CO 2 .All cell lines were routinely tested for mycoplasma by Polymerase chain reaction (PCR).Cells were cultured in appropriate dishes and serum-deprived for 24 h prior to fatty acids (FAs) treatment.FAs were prepared in FA-free BSA-DPBS vehicle as described previously 59 .In brief, 1.2 mM FA stock was mixed with 3 mM FA-free BSA-DPBS vehicle and rotated for 1 h at 37 °C for conjugation.The FA mixture was then diluted in 1% FBS medium to a final concentration of 60 µM FA/ 150 µM BSA.1% FBS medium containing 150 µM BSA was used as control.When required, ferrostatin-1 (10 μM), GSK'782 (10 nM, 20 nM, 50 nM) and DFX (100 μM) were co-treated with FA treatment.

Cell proliferation assay
Cells were seeded in 12-well plates at a density of 1 × 10 4 cells/well.Cell proliferation was evaluated using an automated cell counter (CellDrop FL, DeNovix, DE, USA).Cells were counted using the Trypan blue exclusion staining to discriminate between live and dead cells.Cells were also washed twice to eliminate any dead cells before counting.

Timelapse imaging
Cells were seeded in a 35-mm glass bottom dish at a density of 5 × 10 3 cells/well.Twenty-four hours after serum deprivation, the dishes were placed in the microscope (BZ-X800, Keyence, Osaka, Japan) with a time-lapse

Figure 1 .
Figure 1.LA and αLA pushed cell death into necroptotic/ferroptotic phenotypes.(A) The proliferation rate of MIA-Paca2 and Suit-2 cells treated with BSA (control) or different 60 µM FAs (SA, OA, LA, and αLA).Live cells were counted successively for 4 days.One-way ANOVA followed by the Tukey test was used for multiple comparisons.Data shown are the means ± SEM *P < 0.05, ***P < 0.001, ****P < 0.0001 versus BSA-control.(B) The proliferation rate of MIA-Paca2 and Suit-2 cells treated with LA, and αLA.Live cells were counted in early time points up to 48 h.Data shown are the means ± SEM *P < 0.05, ****P < 0.0001 versus at 12 h in LA treatment, # P < 0.05, ## P < 0.001, #### P < 0.00001 versus at 12 h in αLA treatment.(C) Representative phase contrast images showing the time course for the morphological change in Mia-Paca2 treated with LA. (D) Western blot for cleaved caspase 3 and caspase 3 in MIA-Paca2 and Suit-2 cells treated with BSA, LA, and αLA for 12 h and 18 h.Control is the cells treated with medium containing 1% FBS only.

Figure 2 .
Figure 2. LA and αLA induced pancreatic cancer cell death through ferroptosis.(A) The proliferation rate of MIA-Paca2 and Suit-2 cells treated with BSA, 60 µM LA, and 60 µM αLA alone or together with 10 µM ferrostatin-1.Live cells were counted successively up to 48 h.One-way ANOVA followed by the Tukey test was used for multiple comparisons.Data shown are the means ± SEM ****P < 0.00001 versus with LA treatment without ferrostatin-1, #### P < 0.00001 versus with αLA treatment without ferrostatin-1 (B) Bar graph showing lipid peroxidation of LA and αLA in MIA-Paca2 and SUIT-2 cells treated with BSA, 60 µM LA, and 60 µM αLA alone or together with 10 µM ferrostatin-1.Data shown are the means ± SEM ****P < 0.00001.

Figure 5 .
Figure 5. LA and αLA had different anti-cancer effects on Gemcitabine-resistant pancreatic cancers.(A and C)The proliferation rate of PK-1, RPK-1, PK-9 and RPK-9 cells (A) and BxPC3 cells (C) treated with BSA, 60 µM LA, and 60 µM αLA (A).Live cells were counted successively for 4 days.One-way ANOVA followed by the Tukey test was used for multiple comparisons.Data shown are the means ± SEM ****P < 0.00001 BSA-control versus LA, #### P < 0.00001 BSA-control and αLA.(B and D) The proliferation rate of RPK-1 and RPK-9 cells (B) and Bxpc3 cells (D) treated with 60 µM LA, and 60 µM αLA alone or together with 10 µM ferrostatin-1.Live cells were counted successively for 4 days.One-way ANOVA followed by the Tukey test was used for multiple comparisons.Data shown are the means ± SEM ****P < 0.00001 between with and without ferrostatin-1 in LA-treated cells, #### P < 0.00001 between with and without ferrostatin-1 in αLA-treated cells.

Figure 8 .
Figure 8. Schematic illustration how PUFAs induces ferroptotic cell death in the pancreatic cancers.Exogenous PUFAs including LA and αLA taken up into the cells undergo lipid peroxidation leading to ferroptotic cell death.In addition, ROS accumulation from LA and αLA peroxidation may induce phosphorylation of RIP3 and MLKL leading to membrane disruption.