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. 2026 Mar 16;11(12):18590–18600. doi: 10.1021/acsomega.5c05706

Tumor Lipidomic Changes after Treatment with Polymeric Hydroxychloroquine in a Syngeneic Murine Model of Pancreatic Cancer

Sai Sundeep Kollala †,‡,§, Ling Ding ‡,§, Sudipta Panja ‡, Amar B Singh ∥,⊥, David Oupicky ‡,#, Daryl J Murry §,#,*
PMCID: PMC13044646  PMID: 41939310

Abstract

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most lethal malignancies, with limited treatment options. Altered lipid metabolism, including dysregulated eicosanoid pathways, plays a role in PDAC progression and therapy resistance. In this study, we investigated the impact of a treatment with polymeric hydroxychloroquine (PCQ), a novel macromolecular derivative of hydroxychloroquine (HCQ), on tumor lipid metabolism in a syngeneic mouse model of PDAC. Using a validated LC-MS/MS method, we quantified 72 eicosanoids in tumors to assess the metabolic changes induced by PCQ and HCQ treatment. Our findings revealed significant PCQ-mediated downregulation of multiple protumorigenic eicosanoids, including PGD2, PGJ2, LTE4, LTB4, and HETEs, and upregulation of antitumor resolvins. This was further confirmed by Partial Least Squares Discriminant Analysis (PLS-DA) and heatmap analysis. The parent compound HCQ showed minimal impact on eicosanoid concentrations in the tumors, with no significant alterations at 24-h and modulation of only six eicosanoids downregulated at 72-h post treatment. Overall, our data highlight a novel role of PCQ in modulating tumor lipid metabolism, suggesting a unique mechanism of action and its potential as an adjuvant agent in the therapy of PDAC.

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Introduction

Pancreatic ductal adenocarcinoma (PDAC) is currently the third leading cause of cancer-related deaths in the US and is projected to become the second leading cause of death by 2030. Several key factors contribute to the poor prognosis of PDAC, including late-stage detection, early onset of metastasis, and resistance to conventional chemotherapy. The current 5-year survival rate of PDAC is 13%. Surgical removal of the primary tumor, combined with chemotherapy offer limited treatment option and there is a lack of effective treatment strategies for managing advanced metastatic disease. Despite recent advancements in radiation therapy, immunotherapy, and targeted therapy, response rates remain low.

Disrupted cellular metabolism is considered one of the hallmarks of cancers. To meet their high energy requirements, cancer cells enhance and maintain an elevated metabolic rate. − In addition to the basic metabolic pathways, such as aerobic glycolysis and glutamine metabolism, that are highly upregulated in pancreatic cancer, there has been a significant focus on understanding the abnormalities in lipid pathways and risk for metastatic disease as lipids play a crucial role in cancer development and progression. Lipids have been shown to be involved in modulating the tumor microenvironment, , promoting metastasis, , inducing resistance to therapy, and immune cells activation. ,

Arachidonic acid, an important constituent of the cell membrane, plays a crucial role in intra cellular signaling pathways through its derivatives prostaglandins (PG), thromboxanes (TX), and leukotrienes (LT). Arachidonic acid is released from the cell membrane by enzyme phospholipase A2 (cPLA2α) by hydrolyzing membrane phospholipids for the synthesis of eicosanoids. Cyclooxygenases (COXs) are responsible for generating PG, prostacyclin, and TX, while lipoxygenases (LOXs) produce LT and hydroxy eicosatetraenoic acids (HETE). Eicosanoids comprising PG, prostacyclin, TX, and LT play a pivotal role in various functions including inflammation, blood clotting, immune modulation, allergic reactions, cancer growth, and progression. − It is well established that the eicosanoids are highly upregulated across different cancers. − Multiple studies have highlighted the potential of genetically ablating select eicosanoids in achieving antitumor effects. − Therefore, efforts have been made to develop inhibitors that target eicosanoids and lipid pathways. However, these inhibitors have been associated with adverse side effects, particularly increasing the risk for cardiovascular diseases such as hypertension, and ischemic stroke. Thus, developing novel agents that selectively target individual eicosanoid synthesis pathways may offer a more beneficial approach for patients limiting the existing side effects.

Chloroquine (CQ) is a derivative of the naturally occurring heterocyclic aromatic alkaloid compound quinine. CQ has been extensively used to treat and prevent malaria caused by the Plasmodium species for more than 80 years. However, CQ induces cardiotoxicity, causes gastrointestinal disturbances, and results in irregular blood pressure. , Hydroxychloroquine (HCQ) is a derivate of quinone that has gained importance due to its anti-inflammatory effects in the treatment of autoimmune disorders such as rheumatoid arthritis, lupus, and primary sjögren’s syndrome. Numerous studies have demonstrated HCQs anti-inflammatory role in disorders such as Inflammatory Bowel Disease (IBD) and various cancers. , HCQ was approved in the treatment of type II diabetes where a significant improvement of lipid profile and insulin levels along with lower hemoglobin A1c was observed. Though HCQ proved to be a potent drug to treat a variety of disorders, the safety limitations are still a concern as blurred vision, retinopathy is still prevalent among patients taking HCQ. HCQ in combination with ERK inhibition served as an excellent treatment option for pancreatic cancer. Additionally, HCQ has been shown to inhibit autophagy in cancer cells, sensitizing to chemotherapy. Furthermore, recent studies have indicated that HCQ attenuates tumor growth in a mouse- TNBC, ovarian and hepatocellular xenograft models when combined with chemotherapy drugs.

To minimize the systemic exposure and overcome systemic side effects, such as retinopathy, we synthesized a polymeric form of HCQ, called polymeric hydroxychloroquine (PCQ). We have previously demonstrated the anti-inflammatory role of oral PCQ in the treatment of IBD with minimal systemic absorption. Additionally, our recent studies have shown that the administration of PCQ resulted in significant tumor accumulation and penetration of the drug in an orthotopic pancreatic cancer model and was accompanied by a robust antimetastatic activity. Studies have shown the role of HCQ in altering lipid metabolism, cholesterol synthesis, and absorption. , Our current study aims to elucidate the lipidomic alterations that are induced by PCQ in a syngeneic mouse model of pancreatic cancer through a previously developed and validated LC-MS/MS method for the simultaneous quantification of 72 eicosanoids representing the major eicosanoid pathways.

Results

Synthesis of PCQ

RAFT polymerization was employed to synthesize PCQ using two comonomers, CQMA and DHPMA. CQMA was synthesized through an EDC coupling reaction, while DHPMA was obtained by the epoxy ring opening reaction of glycidyl methacrylate. The polymer had a molecular weight of 25 kDa and was a random copolymer composed of 14 “CQMA” and 124 “DHPMA” repeating units. The chemical structures of HCQ and PCQ are shown in Figure .

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Chemical structure of HCQ and the novel PCQ.

Tumor Monitoring through Ultrasound Imaging

Ultrasound imaging was performed 12 days post intrapancreatic cell injection to monitor tumor growth and volume. All ultrasound imaging studies were performed in the UNMCs ultrasound imaging core facility. Representative ultrasound 3D images for two randomly chosen mice are shown in Figure which illustrates the tumor size and suggests the presence of tumor.

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Representative ultrasound 3D tumor images from two randomly selected animals.

Lipidomic Changes Associated with PCQ at 24-h and 72-h

To analyze overall lipidomic changes associated with PCQ treatment compared to the control at 24-h, we plotted multivariate Partial Least Squares Discriminate Analysis (PLS-DA). As shown in Figure a, the tumor samples from the control group were largely separated from the tumor samples that were treated with PCQ. To understand significant changes in eicosanoid concentrations between control and PCQ treated groups, we created a heatmap, Figure b. We observed that a total of 23 eicosanoids were downregulated upon treatment with PCQ. As shown in the heatmap, we have observed decreased concentrations of eicosanoids such as LTE4, PGD2, PGJ2, PGF3, LTD4, LTB4, PGB2, 11-HETE, 12-HETE, 8-HETE, and 15-HETE. To visualize the eicosanoids that showed at least a 2-fold change, we further created a volcano plot, Figure c. Overall, there was a downregulation of eicosanoids within the PCQ-treated group compared with the untreated tumor-bearing controls.

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Changes in eicosanoids with PCQ at 24-h. (a) PLS-DA plot showing the distinct metabolic alterations upon treatment with PCQ. Red circles indicate untreated controls, and green dots indicate PCQ treated tumor samples (n = 4). (b) Heatmap representing the differentially altered metabolites upon treatment with PCQ (n = 4) p ≤ 0.05 considered significant using student’s t test. (c) Volcano plot representing the downregulated metabolites on left and upregulated metabolites on the right.

To analyze the lipidomic changes associated with PCQ treatment at 72-h, we repeated the same process and plotted multivariate PLS-DA as in Figure a. Tumor samples from the control group were clearly separated from the tumor samples that were treated with PCQ, as shown in Figure a. Further, to understand the statistically significant eicosanoids between control and PCQ treated groups, we created a heatmap, Figure b. A total of 13 eicosanoids had lower concentrations upon treatment with PCQ, including 14,15 LTC4, 13­(S)-HOTrE, 30_PGF1, LTE4, LTD4, LTB4, and 39-PGD3PGB2 following PCQ administration. To visualize eicosanoids that exhibited at least a 2-fold change, we created a volcano plot, Figure c. Overall, we observed a downregulation of eicosanoid concentrations within the PCQ treated group at 72-h when compared with untreated tumor-bearing controls. These findings collectively validate the role of PCQ in downregulating important eicosanoid lipid mediators 24-h and 72-h post PCQ treatment.

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Changes in eicosanoids with PCQ at 72-h. (a) PLS-DA plot showing the distinct metabolic alterations upon treatment with PCQ. Red circles indicate untreated controls, and green dots indicate PCQ treated tumor samples (n = 4). (b) Heatmap representing the differentially altered metabolites upon treatment with PCQ (n = 4) p ≤ 0.05 considered significant using student’s t test. (c) Volcano plot representing the downregulated metabolites on left and upregulated metabolites on the right.

Lipidomic Changes Associated with HCQ at 24-h and 72-h

To analyze overall lipidomic changes associated with HCQ treatment compared to controls at 24-h, we plotted multivariate PLS-DA shown in Figure a. The tumor samples from the control group were not largely separated from the tumor samples that were treated with HCQ. To understand significant changes in eicosanoid concentrations between control and HCQ treated groups, we further created a heatmap, as shown in Figure b. However, in our analysis of the eicosanoids with statistically significant difference between control and HCQ treated groups, we found that none. The heatmap analysis with top 20 eicosanoids as shown in Figure b further validates the above conclusion. Taken together, unlike PCQ treated samples, parent compound HCQ did not significantly affect the eicosanoid levels at 24-h post treatment.

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Changes in eicosanoids with HCQ at 24-h. (a) PLS-DA plot showing the distinct metabolic alterations upon treatment with HCQ. Red circles indicate untreated controls, and green dots indicate HCQ treated tumor samples (n = 4). (b) Heatmap representing the top 20 metabolites. However, none of the metabolites are statistically significant (n = 4) p ≤ 0.05 considered significant using student’s t test.

To analyze the lipidomic changes associated with HCQ treatment at 72-h, we repeated the same process and plotted multivariate Partial Least Squares Discriminate Analysis (PLS-DA) as shown in Figure a. Tumor samples from the control group were separated from the tumor samples that were treated with HCQ, as shown in Figure a. Further, to understand the statistically significant eicosanoids between control and HCQ treated groups, we created a heatmap, Figure b. A total of 6 eicosanoids had lower concentrations upon treatment with HCQ. As indicated in the heatmap, we observed a decrease in the concentration of eicosanoids such as PGDM, LTB4, LTD4, LTE4, and Tetranor-PGFM following HCQ administration. Overall, the above data suggested that the PCQ was statistically more effective in downregulating eicosanoid concentrations in tumors at both 24-h and 72-h compared to HCQ treated tumors.

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Changes in eicosanoids with HCQ at 72-h. (a) PLS-DA plot showing the distinct metabolic alterations upon treatment with HCQ. Red circles indicate untreated controls, and green dots indicate HCQ treated tumor samples (n = 4). (b) Heatmap representing the differentially altered metabolites upon treatment with HCQ (n = 4) p ≤ 0.05 considered significant using student’s t test.

Discussion

This study is the first to demonstrate that treatment with PCQ leads to broad suppression of multiple arachidonic acid-derived eicosanoids in a PDAC tumor model. These findings provide a mechanistic explanation for our previously reported antitumor and antimetastatic effects of PCQ where PCQ showed a higher accumulation, thus highlighting enhanced drug delivery to tumors. While individual eicosanoids such as PGE2, LTB4, and LTD4 are known to promote tumor growth, migration, chemoresistance, and immunosuppression, our results show that PCQ suppresses not just one mediator but an entire cluster of pro-metastatic lipid signals. This multitarget inhibition represents a unique therapeutic advantage compared to single-pathway agents. Our study demonstrated a strong downregulation of the arachidonic acid pathway members, both cyclooxygenase and lipoxygenase pathway. We have previously shown that PCQ can reduce PDAC cancer cell growth and migration in vitro. Additionally, PCQ has been shown to reduce the number of macroscopic metastatic nodules in kidney, abdominal wall, liver, and spleen. Further, a 25% decrease in the tumor weight was reported in the mice that were treated with PCQ compared to HCQ and untreated tumor-bearing controls. This supports the evidence that PCQ has antitumor effects in inhibiting cancer cell migration and metastasis.

Arachidonic acid pathway members are one of the key players in cancer initiation and progression. It is, thus, important to understand the role of PLA2α and other eicosanoids in cancer. Multiple studies have highlighted the increased expression of the cytosolic PLA2α across different cancers. ,− Inhibition of PLA2α has been implicated in reducing the onset of metastasis in breast cancer. , Further, pro-metastatic roles of cPLA2α and its downstream products such as LTB4 and PGE2 have been reported in several cancers such as lung, colorectal, hepatocellular carcinoma, and triple-negative breast cancer. ,, PGE2, a crucial class of PGs, has been shown to enhance the ability to form lung and liver metastasis in preclinical models. Inhibiting PGE synthase has been demonstrated to reduce lung metastasis, thereby restoring CD8+ T cells and NK cells and promoting anticancer effects. Inhibition of PGE2 has been shown to decrease chemoresistance, increase apoptosis, and decrease tumoral proliferation and vascularization. , Combinatorial immunotherapies that target CTLA4 or PD-1 along with eicosanoid mediators such as PGE2 have shown promising results in sensitizing cancers. ,, These studies collectively demonstrate the importance of various eicosanoids and the beneficial effects of inhibiting them across cancers and cancer metastasis.

We have demonstrated that treatment with PCQ led to distinct downregulation of various eicosanoid concentrations at 24-h and 72-h compared to untreated controls. Moreover, the parent compound, HCQ, exhibited no statistically significant alterations in eicosanoids at 24-h and only six eicosanoids at 72-h compared to untreated controls. This finding underscores the potent effect of PCQ in inhibiting eicosanoids compared to HCQ. Moreover, inhibiting a single eicosanoid such as PGE2 or LTD4 has been shown to significantly reduce tumor aggressiveness, chemoresistance, decreased metastasis, and restore immune cell mediated cytotoxicity. Our analysis revealed that PCQ treatment led to substantial downregulation of not just one but multiple eicosanoids in the PDAC model. This further highlights the pronounced effect on eicosanoid markers was due to the higher PCQ delivery and accumulation as highlighted in our previous study. This marks the first time that such multiple eicosanoid inhibition has been demonstrated following drug administration. Moreover, we previously demonstrated the impact of PCQ on PDAC metastasis and tumor weight and thus highlight a mechanism by which PCQ inhibits metastasis incidence through significantly downregulating or inhibiting the eicosanoid mediators of the arachidonic acid pathway. We have previously shown that PCQ inhibits CXCR4/SDF1 mediated cancer cell migration which in turn supports the role of PCQ in being antitumorigenic.

Another characteristic of cancer cells is the heightened inflammatory response. A recent study has demonstrated the inhibitory effect of PCQ on pro-inflammatory cytokines such as IL1β and IL6 in IBD. Eicosanoids have been reported to play crucial roles in cellular infection and inflammation. We posit that PCQ, through its action in reducing eicosanoid concentrations, leads to an overall decrease in the level of cellular inflammation. Given the multifaceted anticancerous benefits of targeting single or combination of eicosanoids, numerous studies have attempted to develop inhibitors of eicosanoid members. However, these inhibitors have exhibited severe side effects such as cardiotoxicity, blood clotting, platelet aggregation, and edema. In contrast, PCQ mitigates the safety concerns associated with CQ, leading us to anticipate that PCQ will not manifest extreme side effects.

The prominent eicosanoids that were found to be common with PCQ treatment at 24-h and 72-h were LTB4, LTD4, LTE4, 14,15 LTC4, 39_PGD3, Tetranor-PGFM, 6 Keto PGF1, PGJ2, and Resolvin D1. All of these eicosanoids other than resolvins have been shown to have increased expression in cancers, including pancreatic cancer. Ablating the genes that encode for these eicosanoids at a genetic level has been associated with antitumorigenic properties. For instance, increased expression of LTD4 has been shown to promote proliferation of PDAC cancer cells and targeting LTD4 with a small molecule inhibitor has led to cell cycle arrest at G0/G1 phase. Similar antiproliferative effects were observed with inhibiting LTB4 using an LTB4 antagonist. Increased LTD4 has been shown to activate MAPK/ERK kinase (MEK) 1/2 kinases in PDAC cells. A recent study identified LTB4 as a diagnostic biomarker for PDAC. Further, LTB4 has been linked to activate the transcription factor NF-κB and thereby induce tumor promotion. LTC4 has been shown to reduce breast cancer cell migration. An increased expression of 6 Keto PGF1 was observed in cancer patients which corelates with poor survival. , PGJ2 has been known to reduce PDAC invasiveness by reducing the activity of matrix metalloproteinases (MMPS) and thereby inhibiting the process of epithelial to mesenchymal transition (EMT) and in turn metastasis. PGJ2 and PGE2 has been shown to downregulate CXCR4 on cancer cells through NF-κB and PPARγ signaling. Moreover, our group has already shown the inhibitory effect of CXCR4/SDF1 mediated cell migration.

Previous published studies from our lab have shown IP injections of nanoparticles exhibited more tumor accumulation than IV in PDAC models, not only in the primary tumor, but also sites of metastasis. , To investigate the reasons behind the high tumor accumulation of the nanoparticles after IP administration, our lab examined H&E-stained tissue sections of normal organs, primary pancreatic tumors, and metastatic foci. We found that both the primary pancreatic tumors and the organs containing metastatic lesions lacked the visceral peritoneum, which is normally composed of flat mesothelial cells. This characteristic absence of mesothelium on the tumor surface likely contributed to the enhanced localization of nanoparticles to tumor.

Our previous studies also compared the IP and IV administration of the nanoparticles in terms of the plasma circulation. Following IV administration, the drug was rapidly removed from the plasma and nearly undetectable by 24-h due to fast clearance of the nanoparticles from the blood. In contrast, we observed widespread local retention of the IP injected nanoparticles at 4 and 24-h post injection. The results suggested that a significant portion of the IP injected nanoparticles was retained in the peritoneum and tissues with limited systemic absorption. Moreover, we previously compared PCQ tumor accumulation following IV and IP administration. The results indicate that IV-administered PCQ primarily accumulated in the kidneys, whereas IP-administered PCQ predominantly accumulated in the tumors. These studies highlight our rationale for using an IP mode of injection compared with an IV method. Therefore, we selected IP administration for PDAC treatment due to improved tumor accumulation and reduced blood clearance observed in prior studies.

From our previous study, we have developed PCQ as a macromolecular agent, taking advantage of the multivalency effect to increase biological activity and its macromolecular nature to favorably modify pharmacokinetic and disposition profile. Our subsequent study showed that replacing the ester linker between polymer and HCQ with a nondegradable bond has no effect on the antimigration and other biological activity, validating our hypothesis that it is the multivalent macromolecular version of PCQ that is responsible for the observed activity. We have previously reported that the ester linker in PCQ is remarkably stable and resistant to esterase action within the time frame of the studies in this manuscript as shown by negligible release of free HCQ from PCQ in vivo over 24 h. Overall, all our prior studies have demonstrated that all developed PCQs had superior inhibitory effect on cancer cell migration in vitro and metastasis inhibition in vivo when compared with HCQ control. ,, These prior studies provide the framework for the present evaluation of the effects of PCQ on tumor eicosanoids because of their crucial role in the process of cancer migration and metastasis. We have also demonstrated that PCQ accumulates significantly more in the tumor sites compared to HCQ, thus highlighting PCQs delivery efficiency to tumors in comparison to the parent compound HCQ. Also, our IP route of delivery showed better tumor accumulation highlighting significantly increased delivery, and in this current study we show that PCQ treatment led to a more pronounced downregulation of pro-tumorigenic eicosanoids and upregulation of anti-inflammatory resolvins. Furthermore, since we used equivalent molar concentrations for both PCQ and HCQ (HCQ 30 mg/kg, and PCQ 30 mg/kg equivalent of HCQ) we believe, in combination with our prior findings, that the lipidomic differences observed are due to an increased drug delivery efficiency. This highlights the significance of PCQ as a promising therapeutic strategy for the PDAC.

Another interesting finding that we observed in tumors treated with PCQ was the upregulation of Resolvins D1 and Resolvin D2. Resolvins are important anti-inflammatory, proresolving autacoids involved in inhibiting the tumor cell debris formed after chemo/targeted therapy. These resolvins have been shown to suppress tumor growth and enhance cancer therapy by activating macrophage clearance of tumor cell debris. With the upregulation of Resolvin D1 and Resolvin D2 in response to PCQ, we propose PCQ as a novel therapeutic agent that may augment cytotoxic cancer therapy by fortifying the clearance of tumor cell debris.

Conclusions

Our study demonstrates that PCQ exerts a distinct and more pronounced effect on tumor lipid metabolism in pancreatic ductal adenocarcinoma compared to HCQ. PCQ significantly downregulates several protumorigenic eicosanoids such as PGD2, PGJ2, and LTE4 and promotes the upregulation of antitumor lipid mediators such as resolvins. The metabolic alterations suggest that PCQ acts through a novel mechanism distinct from HCQ positioning it as a promising candidate for combination therapy in PDAC. Targeting dysregulated eicosanoid pathways with PCQ may offer a new avenue to overcome therapy resistance and improve treatment outcomes in this highly lethal malignancy. These studies unequivocally underscore the significance of targeting eicosanoids in PDAC, and we strongly advocate for the utilization of PCQ in PDAC sensitization. In summary, we propose that PCQ may be used as an adjuvant in the therapy of PDAC to improve the therapeutic response. Further investigations into the impact of PCQs on immunometabolism signaling and therapeutic synergy are warranted.

Methods

Chemicals and Reagents

HCQ sulfate (98%) was obtained from Acros Organics (Fisher Scientific, Pittsburgh, PA). Dichloromethane (DCM), methanol, acetonitrile, and acetic acid (UPLC grade) were purchased from Fisher Scientific. 2,3-dihydroxypropyl methacrylate (DHPMA) and N-(3-aminopropyl) methacrylamide hydrochloride (APMA HCl) were purchased from Polysciences (Warrington, PA). Phosphate buffered saline (PBS), Dulbecco’s Modified Eagle Medium (DMEM), sodium pyruvate, glucose, essential and nonessential amino acids, penicillin-streptomycin (10,000 U/ML), TrypLE were purchased from Gibco (Life technologies, Grand Island, NY). Fetal Bovine Serum (FBS) was purchased from Atlanta Biologicals (Flowery Branch, GA). BD Matrigel for intrapancreatic surgery was purchased from Thermo Fisher Scientific. All eicosanoid standards and deuterated internal standards (IS) were purchased from Cayman Chemicals (Ann Arbor, MI, USA).

Synthesis of PCQ

The synthesis of PCQ was accomplished by copolymerizing CQMA with 2,3-dihydroxypropyl methacrylate (DHPMA) using the reversible addition–fragmentation chain-transfer (RAFT) copolymerization reaction as previously described. The mixture containing the monomers (CQMA and DHPMA), a chain-transfer agent (CTA), and AIBN (22′-azobis­(isobutyronitrile)) in a molar ratio of 20:1:0.25 was dissolved in 1:1 v-v 1,4-dioxane and DMSO within a Schlenk tube. The solution was then purged with nitrogen for 30 min to remove dissolved oxygen. Polymerization was initiated by placing the Schlenk tube into an oil bath at 70 °C, where it was stirred continuously for 24-h. Afterward, the reaction was terminated by immersing the Schlenk tube in liquid nitrogen. The reaction mixture was then acidified to pH ∼ 4 by adding a 0.1 N acetic acid solution. The resulting polymer was purified by dialyzing it against deionized water for 3 days using a 3.5 kDa dialysis tube. Finally, the purified polymer was dried using a lyophilizer and its chemical structure was confirmed through 1H NMR spectroscopy and molecular weight by size exclusion chromatography.

Pancreatic Cancer Cell Line

Mouse KPC 1245 cell, a well-established model of pancreatic ductal adenocarcinoma, was a generous gift from Dr. Michael. A. Hollingsworth (Eppley Institute, University of Nebraska Medical Center, Omaha, NE). These cells carry KRAS G12D and TP53R172H point mutations. The KPC 1245 cell line was cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin (0.5 mg/mL), streptomycin (0.5 mg/mL) and incubated in a humidified chamber with 5% CO2 at 37 °C.

In Vivo Mouse Studies

A total of 25 male C57BL/6 mice (6 weeks old) were purchased from Charles River Laboratories (Wilmington, MA, USA) and housed in the animal facility at the University of Nebraska Medical Center, Omaha, USA. Male mice were preferred for our study due to factors such as hormonal influence on tumor progression. Estrogen in female mice can slow tumor growth and affect metabolic responses, potentially introducing variability. Additionally, hormonal fluctuations across the estrous cycle may impact the tumor metabolism. To minimize these variables and ensure more consistent and reproducible results, male mice were selected. Moreover, PDAC’s incidence and mortality rates are slightly higher in males than females. All animal procedures were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee in accordance with NIH guidelines. KPC 1245 cells (5 × 103) were orthotopically implanted in the pancreas tail of the C57BL/6 mice. Following implantation, mice were maintained for 12 days, and then ultrasound imaging was performed to determine tumor formation. Following tumor presence identification (50 mm3 tumor), mice were randomly distributed using simple randomization into 3 groups. Group 1: untreated/control where 5 mice were intraperitonially injected with saline; Group 2: HCQ treated group: 10 mice were intraperitonially injected with HCQ (30 mg/kg); and Group 3: PCQ treated group: 10 mice were intraperitonially injected with 30 mg/kg equivalent of HCQ, which is approximately 163 mg/kg of PCQ. All of the administrations were performed through intraperitoneal (IP) injection. Further, the treated mice were subdivided into two groups where the first group (n = 5) was identified to collect tumors at 24-h and the other group (n = 5) was identified to collect tumors at 72-h after treatment. At 24-h and 72-h time point, tumors were collected after euthanizing the mice using CO2 inhalation followed by cervical dislocation. Tumors were snap frozen using dry ice and stored in −80 °C.

Metabolite Isolation

In order to isolate the lipid metabolites from tumor tissues collected from control, HCQ, and PCQ groups, we performed tissue homogenization on equally weighed tissues across conditions using a TissueLyserII (Qiagen Science Germantown, MD, USA). Tumor tissues were weighed and triple-distilled deionized water (Thermo Fisher Scientific) added at a 6-fold dilution (5 parts water in μL added to 1 part tumor weighed in mg). Tumor tissue was homogenized, and tumor homogenate (25 μL) was spiked with a mix of deuterated 15 HETE, PGE2, and TXB2 internal standards (IS; 20 μL) and 5% acetic acid (800 μL). Simultaneously, a set of standards with 9 serial dilutions and 4 quality controls (QCs) were prepared with IS (20 μL) and 5% acetic acid (800 μL) for standard curve calibration. The processed samples, standards, and QCs were loaded onto the solid phase extraction (SPE) cartridge (Oasis HLB 3 cc/60 mg; Waters Corporation, Milford, MA, USA) following conditioning the cartridges with methanol. Following sample loading, the cartridge was washed twice with 5% methanol (0.5 mL). The sample was eluted with methanol (1 mL) and then dried in a nitrogen manifold (Zymark TurboVap LV Evaporator) at room temperature. The dried eluent was reconstituted in 50% acetonitrile and water (100 μL) as previously described in .

Liquid Chromatography and Mass Spectrometry

Extracted samples for eicosanoid determination along with standards and QCs were analyzed using an LC-MS/MS 8060 NX system (Shimadzu Scientific Instruments, Columbia, MD, USA) with a dual ion source in negative electrospray ionization mode. LC and MS parameters were optimized accordingly to the previously developed method. Chromatographic separations were performed on a Shim-pack XR-ODSIII, 2.0 × 150 mm analytical column (Shimadzu Scientific Instruments, Columbia, MD, USA) as previously described. A complete list of eicosanoid metabolites analyzed is shown in Table.

1. List of the Eicosanoid Metabolites Analyzed.

s. no analyte name s. no analyte name s. no analyte name
1 PGJ2 26 13,14-DiOH-15-Keto-PGE1 51 8-HEPE
2 20-OH-PGE2 27 PGD1 52 11-HEPE
3 PGB2 28 13,14-DiOH-PGE1 53 12-HEPE
4 PGD2 29 TXB3 54 15-HEPE
5 PGE2 30 15-deoxy-delta 12,14 PGJ2 55 9(S)-HOTrE
6 AA 31 PGE1 56 13(S)-HOTrE
7 15-HETE 32 PGE3 57 5-OxoETE
8 12-HETE 33 PGD3 58 12-OxoETE
9 11-HETE 34 PGF3 59 15-OxoETE or 15-KETE
10 8-HETE 35 14,15 LTC4 60 9-OxoODE or 9-KEDE
11 5-HETE 36 Tetranor-PGEM 61 15-OxoEDE or 15-KEDE
12 LTE4 37 Tetranor-PGFM 62 Tetranor-PGDM
13 LTD4 38 11-De TXB3 63 Tetranor-PGJM
14 LTC4 39 2,3 Dinor 8-iso PGF2 64 resolvin d1
15 LTB4 40 docosahexaenoic acid 65 resolvin d2
16 13,14-DiOH-15-Keto-PGE2 41 9(10)-DiHOME 66 resolvin d3
17 11-B-PGF2 42 12(13)-DiHOME 67 TXB2-d4
18 8-iso-PGF2 43 4-HDHA 68 PGE2-d4
19 PGF2 44 7-HDHA 69 AA-d8
20 15-Keto-PGE2 45 8-HDHA 70 15 -HETE-d8
21 6 Keto-PGF1 46 10-HDHA 71 LTB4-d4
22 TXB2 47 11-HDHA 72 resolvin d1–d5
23 13,14-DiOH-PGF2 48 11-HEDE    
24 PGF1 49 15-HEDE    
25 13,14-DiOH-15-Keto-PGF2 50 5-HEPE    
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Metabolomics Data Processing

Raw peaks for all of the lipid metabolites was corrected according to the specific retention time values and fragmentation pattern using Lab Solutions software v5.18 (Shimadzu Scientific, Inc., Columbia, MD, USA). Further, these data were converted to concentration data (ng/g). The obtained concentration values were normalized to the initial tumor weights. Metaboanalyst 6.0 (https://www.metaboanalyst.ca) was used for data analysis.

Statistical Analysis

Data analysis included multivariate analysis consisting of Principal Component Analysis (PCA) and Partial Least squares-Discriminate Analysis (PLS-DA). Volcano plots and heatmaps were employed to visualize fold change differences and statistically significant metabolites, respectively. A student’s t test was used to determine the statistically significant metabolites between groups with a p-value cutoff < = 0.05. Fold change thresholds of 2.0 or greater were set for identifying significant fold changes. All results presented are based on n = 4 samples for each group, excluding one replicate that exhibited more than 2 standard deviations difference, thereby eliminating it from all analyses.

Supplementary Material

ao5c05706_si_001.pdf (167.1KB, pdf)

Acknowledgments

This research was funded by the University of Nebraska Medical Center and supported by grants R01 DK124095, P30 GM127200, R01 CA235863, R01 DK120533 and University of Nebraska Medical Center’s Presidential Graduate Fellowship and graduate studies fellowship under program of excellence awarded to S.S.K. The authors thank M.A Hollingsworth (University of Nebraska Medical Center UNMC) for providing the KPC cell line. The authors thank B.T Hackford of Ultrasound Imaging Facility (UNMC) for helping longitudinal imaging of tumors with 3D volumetric analysis. Major instrumentation has been provided by the Office of Vice Chancellor for Research, The University of Nebraska Foundation, the Nebraska Banker’s Fund, and by the NIH-NCRR shared Instrument Program. The Table of Contents graphic was created using Biorender.com. The content and interpretations presented in this publication are solely the responsibility of the authors.

The data underlying this study are available in the published article and its Supporting Information.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c05706.

  • Additional figures for the differences between HCQ and PCQ treated tumors at 24-h and 72-h, PL-SDA plot showing distinct metabolic alterations between HCQ treated group and PCQ treated group at 24-h and 72-h, along with a heatmap showing the statistically significant lipid metabolites (PDF)

S.S.K.: Conceptualization, writing the original draft, validation, methodology, generation of the image for table of contents, and investigation. Dr. L.D.: Data curation and methodology development. Dr. S.P.: Data curation and methodology development. Dr. A.B.S.: Supervision, review, and editing. Dr. D.O.: Conceptualization, supervision, review, and editing. Dr. D.J.M.: Conceptualization, supervision, review, and editing. All of the authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

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Supplementary Materials

ao5c05706_si_001.pdf (167.1KB, pdf)

Data Availability Statement

The data underlying this study are available in the published article and its Supporting Information.

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