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Isomeric lipid signatures reveal compartmentalised fatty acid metabolism in cancer

Cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular fatty acids (FA) and de novo synthesis. Given that oxidation of de novo synthesised FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates - however hitherto FAs were considered part of a common pool. To probe FA metabolic partitioning, cancer cells were supplemented with stable-isotope labelled FAs. Structural analysis of the resulting glycerophospholipids revealed that labelled FAs from uptake were largely incorporated to canonical (sn-)positions on the glycerol backbone. Surprisingly, labelled FA uptake disrupted canonical isomer patterns of the unlabelled lipidome and induced repartitioning of n-3 and n-6 polyunsaturated-FAs into glycerophospholipid classes. These structural changes evidence differences in the metabolic fate of FAs derived from uptake or de novo sources and demonstrate unique signalling and remodelling behaviours usually hidden to conventional lipidomics. Highlights Lipid isomers reveal discrete metabolic compartmentalisation in cancer FAs derived from uptake and de novo synthesis have different metabolic fates Stearate uptake signals for PUFA (n-3 and n-6) repartitioning between lipid classes sn-positional isomers are a marker for aberrant lipid metabolism


Summary
Cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular fatty acids (FA) and de novo synthesis. Given that oxidation of de novo synthesised FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates -however hitherto FAs were considered part of a common pool. To probe FA metabolic partitioning, cancer cells were supplemented with stable-isotope labelled FAs. Structural analysis of the resulting glycerophospholipids revealed that labelled FAs from uptake were largely incorporated to canonical (sn-)positions on the glycerol backbone. Surprisingly, labelled FA uptake disrupted canonical isomer patterns of the unlabelled lipidome and induced repartitioning of n-3 and n-6 polyunsaturated-FAs into glycerophospholipid classes. These structural changes evidence differences in the metabolic fate of FAs derived from uptake or de novo sources and demonstrate unique signalling and remodelling behaviours usually hidden to conventional lipidomics.

Introduction
Although seemingly banal, the structure-function relationship of glycerophospholipids (GPL) is far more complex and insightful than first meets the eye. While the amphoteric nature of the molecule importantly allows for chemical partitioning, the individual functional groups serve numerous other specific purposes. The charged hydrophilic head group creates the possibility for lipids to be directed to particular subcellular compartments whereby the lipophilic FAs can impart variable physicochemical properties to the molecule and initiate signalling. 1 Arguably, two of the most important processes in dictating the modelling and remodelling of mammalian GPL structure are the Kennedy pathway and the Lands cycle.
While the Kennedy pathway describes the de novo synthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from cytidine-diphosphate diacylglycerol, 2 the Lands cycle chronicles the process by which phospholipase and acyltransferase enzymes remodel GPL-FAs. 3 While these processes logically have implications in cellular energy production, whether de novo generated FAs or extracellular FAs (from uptake) have identical or varied roles is not well understood. Given that cancer cells are known to modulate and switch between de novo and uptake FA metabolism to opportunistically source energy for growth, [4][5] understanding if subfractions of FAs serve discrete purposes is of importance when elucidating the molecular mechanisms of cancer. For example, a cancer cell actively increasing the production of FAs only to oxidise these to meet energy demands is counterproductive and thus the cell might instead be compelled to source extracellular FAs. For this to be possible however, there must be compartmentalisation and discrete metabolic treatment of de novo or uptake FAs -a sentiment that is contrary to the current idea that the cell has a common "pool" of FAs, used to meet metabolic demand. 4,6 One of the more important enzymes within the Lands cycle is phospholipase-A2 (PLA2), a lipase which is responsible for the enzymatic cleavage of the FA located at the sn-2 position within GPLs. Notably, PLA2 has been shown to exhibit a high affinity of action towards GPLs containing polyunsaturated FAs (PUFA), such as arachidonic acid (AA; 20:4n-6) and docosahexaenoic acid (DHA; 22:6n-3). 7 Amongst other functions the release of AA and DHA in turn initiate pro-or anti-inflammatory responses through the cyclooxygenase and lipoxygenase pathways. [8][9] Concurrent with the release of the sn-2 FA is the formation of a 1acyl-2-lyso-GPL, or 2-lyso-GPL, with a hydroxyl group at the sn-2 position of the glycerol-backbone. While these lyso-species are themselves signalling mediators, 10 they also readily undergo re-esterification, reverting to diacyl-GPLs via acyltransferase enzymes, such as the lyso-phosphatidic acid acyltransferase (LPAAT) or lyso-phosphatidylcholine acyltransferase (LPCAT) isoforms. 3,[11][12] The labile nature of lyso-GPLs also allows physicochemical properties such as pH and temperature to influence a behaviour known as acyl chain migration, whereby a FA is able to migrate from one sn-position to a free hydroxyl group on the glycerolbackbone. Unlike the sn-selectivity of PLA2, acyltransferase enzymes are not reported to exhibit preference for 1-lyso or 2-lyso species and as such acyl chain migration that occurs prior to esterification will influence lipid molecular structure. Because Kennedy and Lands metabolic pathways affect different structural motifs of the GPL molecule, they are able to dictate both the transport of lipids around the cell and the initiation of signalling cascades through the release of biologically active FAs. This allows for independent organelle membrane modifications as well as the initiation of signalling cascades through the release of biologically active FAs. Although the various mechanisms and outcomes of lipid remodelling have been studied extensively, little is known about the impact that sn-isomeric lipid species have on remodelling and transport, or conversely, the impact these mechanisms have on lipid isomer populations. While it is now known that the GPLs of both prokaryotic and eukaryotic cells exhibit a preference for unsaturated FAs to be at the sn-2 position, prior to 2003 there was little-to-no indication that sn-positional isomers were present in eukaryotic cells. [13][14] Ekroos et al. displayed that not only was there a quantifiable population of sn-1 unsaturated GPLs, but the relative abundance of these regioisomer populations can vary between mammalian GPL samples. 13 This was exemplified in more recent studies into mammalian GPL regioisomers, which revealed that bovine liver, 15 egg yolk and sheep kidney 16 exhibited a stronger preference for canonical sn-positions than synthetically prepared standards.
Although an independent lipase enzyme, phospholipase A1 (PLA1), is known to catalyse the cleavage of FAs in the sn-1 position of specific GPL classes, 17 the preference for unsaturated FAs to exist in the sn-2 position is thought to be strongly linked with Lands cycle lipid remodelling and PLA2 functionality. 7,9,18 This enhanced sn-positional specificity in biological systems implies that cellular intervention is required for maintenance. Studies into the peroxidation of unilamellar liposomes comprised of specific phosphatidylcholine sn-isomers (containing palmitic acid and linoleic acid) revealed oxidation rates were sn-isomer dependent. 19 Using radiolabelled FAs in mouse models, others showed that while the supplemented cis-FA (oleic acid) showed esterification specificity to the sn-2 position of GPLs, the trans-FA species (elaidic acid) was equally esterified to sn-1 and -2 positions. More recently, molecular dynamics simulations revealed that unsaturated FAs at the sn-1 position of GPLs create more ordered (and hence less fluid) membranes than their unsaturated sn-2 counterparts. 20 These functional differences may help explain variations in sn-isomer distribution that are observed to exist in recent tissue models. In a pivotal study, Paine et al. showed that lipid isomers were tightly regulated and highly organised within the grey and white matter structures of brain tissue, and were subsequently disrupted in murine cancer tumours. 21 Therefore, because lipid species containing saturated FAs at the sn-1 and unsaturated FAs at the sn-2 are the typical structures identified as the majority isomer in lipid canon, to aid with explanation throughout, these isomers will be referred to as 'canonomers'.
Conversely, lipids displaying atypical or apocryphal sn-isomeric structure (with the unsaturated FA contained at the sn-1 position) will be referred to 'apocromers'.
Using stable isotope labelled FAs and a prostate cancer cell line (LNCaP), here we trace the metabolic fate of isotopically labelled FAs in cellular lipids, specifically tracking the snposition (sn-position) at which they are incorporated into glycerophospholipids (GPL). Using a tandem mass spectrometric technique that combines collision-induced dissociation (CID) and ozoneinduce dissociation (OzID; i.e., CID/OzID), we determine that lipids incorporating FAs from supplementation have isomer profiles that are distinct from lipids incorporating de novo synthesised (or previously stored) FAs. Monitoring the distribution of sn-isomers across resected LNCaP xenograft tumours (from mice) using isomer-resolved molecular imaging reveals that apocromeric species are spatially correlated to PUFA containing GPLs and lysolipids. Treating mice with a potent and selective phospholipase A2 Group IIA (sPLA2-IIA) inhibitor (KH064), and conducting the same correlative analysis reveals that spatial correlation between apocromer, PUFA-GPLs and lyso-lipids is weakened. Given that there are multiple known changes to the physico-chemical environment of cancer tumours 22-23 and lyso-lipid acyl chain migration is known to be affected by pH and temperature, 24 we propose that lipid apocromer species are the product of lipid remodelling in a perturbed physico-chemical environment and show potential as a biomarker for aberrant lipid metabolism in cancer tumours.
Observing the sn-isomer compositions of PC 32:1 within the de novo-lipidome ( Fig. 2A) Notably, this showed that the uptake (but not incorporation) of extracellular FA increased the relative abundance of apocromer species in the de novo-lipidome. As with changes to FA snposition, variation in the distribution of DB locational isomers within each supplement experiment was observed ( Fig. 2A pie charts). Comparative to NS cells, FA supplemented cells (12PA, 13PA, 13SA) exhibited slight decreases of n-8 (≤1%), n-9 (1-3%) and n-10 (1-2%) and a concomitant increase of n-7 (3-5%). Together, this combination of increased apocromer Investigating the sn-isomer distributions of the isotope labelled GPLs that constituted the uptake-lipidome of 13PA and 13SA cells, the labelled FAs from uptake were observed to either be directly incorporated into the lipid or were incorporated in combination with cellular modification (e.g., desaturation, elongation, or β-oxidation). It has previously been shown that the ordering of these metabolic events determines the species of the FA. 25 The strength of supplementing with isotopically labelled FAs combined with mass spectrometric

The relationship between sn-and DB position
To investigate an explicit relationship between DB and sn-positional isomers, mass spectral dimensionality was increased (MS 4 ) to combine both OzID and CID/OzID experiments (i.e., ordered as OzID/CID/OzID). As such, the distribution of canonomer and apocromer for each FA DB location is quantitative, however absolute mole quantitation of each DB FA sn-isomer would still require establishing OzID reaction rates or developing calibration curves. This technique is not without limitations, however; to achieve the analysis requires: (i) the lipid to be detectable in positive polarity MS, (ii) the precursor ions ability to form sodiated adducts, and (iii) high concentrations of ozone to be generated and passivated into the high-pressure trapping regions of the MS instrument. As such, applications of this technique are currently limited to PC lipids with high cellular abundance such as PC 32:1 and PC 34:1.
Applying this methodology to PC 32:1 from NS LNCaP ( snposition occupancy that appeared dependant on fatty acyl composition (i.e., increased relative abundance of both PC 16:1n-9/16:0 and PC 14:0/18:1n-9 isomers). An equivalent full structure analysis for PC 34:1 can be found in Fig. S1E and showed that within the PC 16:0_18:1 species, n-7 and n-10 DBs were associated with sn-1 isomers, while n-9 showed strong preference for the sn-2 position.   Instead, this result implies that stearic acid from uptake induced lipid remodelling to include twice as much PUFA ≥4 to the unlabelled de novo-PS fractions, while this effect is only minor in any newly formed PS that incorporated the 13SA label in the labelled uptake-lipidome. This effect was apparent within all monitored GPLs (cf. Fig. S2).

Influence of saturated FA supplements on unsaturated membrane lipids
Considering the independent changes to the degree of unsaturation within the de novoand uptake-lipidomes after FA supplementation, it was decided that individual lipid speciation should be explored for any variation. To achieve this, a heat map was created where the mol% of FA sum-compositions across five GPLs from the de novoand uptake-lipidomes of 13PA and 13SA cells were compared against 12PA cells as a fold-change (Fig. 4C). Overall, this effect of increased PUFA for dn-13SA could be observed across all GPL classes and was exemplified by increases in PI, PS, PC and PE 36:5 and 36:6, and also PI, PS, PC, PG and PE 38:6. Although affecting fewer GPL classes, the up-13SA profiles also displayed this effect but additionally showed decreases in PE 38:4 and PE 40:4 and increases in PC, PE and PI 34:4. The change within GPLs with 5 or 6 degrees of unsaturation (i.e., EPA or DHA) was less significant in both dn-and up-13PA profiles, however the impact 13PA supplementation had on GPLs with 4 degrees of unsaturation (i.e., AA) was more widespread than 13SA.
As mentioned previously, PUFA ≥4 is broadly representative of "dietary" FAs, and its increase amongst all monitored dn-GPLs after PA or SA supplementation suggests that these PUFAs are being sourced and remodelled from another lipid class, such as other GPLs or more likely, being sequestered from triacylglycerols (TG). [27][28] Interestingly, within the dn-13SA profiles an increase within the PC 40:5, PC 40:6 and PE, PS, PI 40:6 could be observed. This is in contrast to the up-13SA profiles, where PS, and PE, 40:5 and 40:6 were seen to decrease. Together these increases in 40:5 and 40:6 from the de novo-lipidome and decreases in PUFA PS and PE from the uptake-lipidome suggest that the remodelling of PUFA from other lipid classes after FA uptake was GPL class specific. Overall, these results display that the uptake of FAs not only impacted the pre-existing (de novo) lipidome but was also influential in the remodelling of PUFAs between the GPLs (and other unmonitored classes) in a class-specific manner.    5H) and compared against the spatial distribution of PUFA-GPLs (Fig. 5G-H), the apocromer showed high positive correlation with the presence of these species. Given that PUFA-GPLs (e.g., PC 18:0_20:4 and PC 18:0_22:6) are the primary substrate for PLA2, 7 and the intermediary products of this activity are lyso-lipids, the presence of a lyso-PC (LPC) 18:0 (Fig. 5D) in this same region is suggestive of PLA2 activity. With the altered pH environments that are commonplace in tumours, 22 acyl chain migration 24 occurring in these lyso-lipids prior to lipid-remodelling and the reformation of diacyl-lipids would lead to the occurrence of snapocromer lipid species. Indeed, the overexpression of group IIA secretory-PLA2 (sPLA2-IIA) is observed in LNCaP cells and is correlated with poor clinical prognosis in prostate cancers. 32 Within murine models that were treated with an sPLA2-IIA inhibitor (KH064), correlations between PUFA GPLs and apocromeric lipids were less apparent, suggesting that this migration activity can be partially attenuated by limiting lipase activity (cf. Fig. S3).
An unexpected result (Fig. 5I) is the correlation between TG species and the region of extensive lipid remodelling in the host adipocyte cells. Recent studies investigating how external stimuli can affect cancer cell lipid homeostasis found that the degree of FA unsaturation in TG and GPLs was disrupted by nutrient deprivation and hypoxia. 27,33 Given that the physical conditions in the study attenuate cellular FA desaturation, researchers were able to show that in order to maintain appropriate ratios of membrane-bound saturated and unsaturated FAs, cells sequestered unsaturated FAs from TG fractions and transferred to membrane GPLs. Therefore, due to the colocalization of TG and PUFA-GPLs, the increased abundance of PUFA species in GPLs is assumed to come from the repartitioning of TG stores.
Similar to the TG localisation, the fatty tissue appears to house an increased abundance of PUFA ether-lipid species (PC O-32:0 and PC O-34:1) and the tentatively assigned TG ethers (cf.

Discussion
With the exception of mitochondria, FA modification is predominantly undertaken in the endoplasmic reticulum (ER) and thus trafficking of FAs and lipids is necessary to reach the plasma and organelle membranes. Although there are exceptions, lipids mainly require active transport mechanisms to shuttle around the cell. The three conventionally accepted trafficking mechanisms involve: membrane transport (e.g., budding from a donor membrane), carrier proteins (e.g., fatty acyl binding proteins [FABP]), or transfer across membrane contact sites (e.g., ER/mitochondria lipid exchange). 38 Alongside de novo lipogenesis, cells can also obtain extracellular FAs from uptake and incorporate these into lipid structures. From our work presented herein, these differential trafficking mechanisms can be inferred from the variations in the PC (and other GPL) lipid species profile(s) (vide supra Fig. 1D; for other GPLs cf. Fig. S2). For example, tracing the incorporation and modification(s) of labelled palmitic and stearic acids reveals that each FA is either incorporated into more highly unsaturated GPLs or are themselves desaturated more frequently (Fig. 1D; up 13PA/up 13SA; e.g., PC 32:2, PC 34:2, PC 34:3 and PC 36:3). In contrast, GPLs solely incorporating FAs synthesised by cellular machinery appear to be influenced by the supplemented FA such that unlabelled PC 34:0 and PC 36:6 species are discretely increased ( Fig. 1D; dn 13PA/dn 13SA).
These differences suggest that the cellular management of FAs is dependent both on FA source-origin and FA speciation. Thus we propose that there are distinct mechanisms for the trafficking of FAs originating from de novo lipogenesis and those the cell has acquired from uptake. Previous research has also revealed FA-dependent trafficking differences. One such example can be found in FABP5, a FA binding protein that delivers FAs from the cytosol to nuclear peroxisome proliferator-activated receptor beta or delta (PPARβ/δ). Researchers were able to show that while FAs from uptake all complexed with FABP5, a saturated FA (palmitic acid) inhibited FABP5 activation of the PPARβ/δ pathway, while a polyunsaturated FA (linoleic acid; LA; FA 18:2n-6) instead activated PPARβ/δ. 39 This FA-driven inhibition/activation of PPARβ/δ has downstream effects in the activation of lipometaboliccentred genes such as pyruvate dehydrogenase kinase 4 (PDK4), angiopoietin 4 (ANGPTL4), perilipin 2 (Plin2) and cluster of differentiation/FA translocase (CD36). Considering the changes that occur to FA metabolism in cancer, especially including FA uptake and energy production from FAs, the translation of these genes into proteins has widespread implications for oncometabolism. [40][41][42][43][44][45] Furthermore, this FABP5/PPARβ/δ mechanism highlights how FA trafficking mechanisms can be entangled with signalling events, while this work provides the foundation for the discovery of the proteins involved in partitioning of dn-and up-FAs.
Given the widespread impact FA trafficking can have on signalling for gene expression and protein translation, it is logical to assume that the cell has extremely fine control over FA uptake, FA trafficking and regulation of lipid remodelling. This is exemplified in our work by the DB or sn-position differences observed between labelled or unlabelled FAs, and possibly relates to the known compartmentalisation of desaturase enzymes. Within the pie charts of Fig. 2D, the percentage of n-10 (requiring FADS2 desaturation) is observed to be higher when the labelled palmitic acid is modified by desaturation or by combined desaturation and elongation, while n-7 and n-9 (requiring SCD-1 desaturation) are seen to be higher within labelled lipids where the unlabelled FA is the unsaturated chain. This would suggest that a higher proportion of FAs from uptake are transported to the mitochondria (where FADS2 desaturation occurs) 46 while de novo synthesised FAs are more readily transported to the ER (where SCD-1 desaturation occurs). 47 Further, this would indicate that FA speciation and origin (i.e., uptake or de novo) both determine subcellular destination as opposed to the conventional theory that all FAs form a common "pool". One explanation for this compartmentalisation of FA fractions would be for net-positive energy production.
Catabolising FAs that have been actively de novo synthesised, although sometimes required, is counterproductive to energy production. Instead, trafficking FAs from uptake to β-oxidation sites would provide externally produced energy (and carbon) to the cell. Interestingly, FA uptake influences PPARβ/δ activity and subsequent activation of PDK4, which regulates the conversion of pyruvate and glucose to acetyl-CoA and supresses ferroptosis. 40 These functions directly implicate compartmentalised FA metabolism as mitochondria are responsible for cellular energy production via β-oxidation and FA synthesis, while the peroxisomes mediate an alternate FA β-oxidation pathway and are largely responsible for the formation of reactive oxidative species and ferroptosis.
A related result that supports the hypothesis of specific FA species compartmentalisation can be found in the unusual lipid DB profile displaying 16:1n-12 and increased 16:1n-9 (Fig. 2D) after a labelled stearic acid has undergone partial β-oxidation and desaturation to FA 16:1*.
As stated, within the cell both the mitochondria and peroxisomes have the capacity for βoxidation, however peroxisome β-oxidation and stimulation has been shown to have a complex relationship with the genetic expression of fads1 and fads2 and hence FADS1 and FADS2 enzymes. [48][49] These enzymes catalyse Δ4, Δ5, Δ6 and Δ8 desaturation 31,46 and in conjunction with β-oxidation would lead to the formation of the unusual 16:1n-12 species observed within Fig. 2D (mid). This may suggest that the stearic acid supplement is being split between two trafficking pathways: (i) to the ER for SCD-1 desaturation and then membranecontact transport to the peroxisomes for β-oxidation to yield 16:1n-9 and stimulate the peroxisomal influence on FADS2, and (ii) to the mitochondria for FADS2 desaturation and βoxidation to yield 16:1n-12.
Another example of remodelling compartmentalisation can be observed in Fig. 3 (left), where PC 16:0_16:1n-9 and PC 16:0_16:1n-10 from non-supplemented LNCaP showed equal apocromer/canonomer distribution, while PC 16:0_16:1n-7 markedly favoured canonomeric incorporation. This is a remarkable result as both β-oxidation and FADS2 desaturation (leading to FA 16:1n-9 and n-10, respectively) are mitochondrial processes, while the SCD-1 desaturation forming FA 16:1n-7 would occur at the ER. Similarly, the PC 14:0_18:1 in Fig. 3 (right) shows that snisomer distribution of FA 18:1n-7 and n-10 is quite similar, while n-9 is distinctly different. To form PC 14:0_18:1, both FA 16:1n-7 and FA 16:1n-10 would require elongation, while FA 18:1n-9 would be directly desaturated from FA 18:0. Interestingly, subcellular organelles are observed to have discrete pH ranges with mitochondrial pH being 7.58 for the matrix and 6.88 for the intermembrane space, 50  which mediate an anti-inflammatory response through resolvins, protectins, maresins and the disruption of lipid rafts. 8,62 Further, the presence or absence of specific FA species within particular GPL classes and the degree of unsaturation in membrane lipid FAs has been shown to be influential factors in cell signalling. [63][64] As seen in Fig. 4C, notably our work shows that the uptake of stearic acid drives a sharp increase of DHA into GPLs. Given that these GPL-DHA species are prime targets for PLA2 activity, the increase in these substrates would likely lead to the release of DHA from the lipid and mediate anti-inflammatory signalling cascades. This signal by uptake FAs presents a promising target for inducing pro-or anti-inflammatory responses via non-toxic interventions. For example, by pre-exposing the cells to DHA substrates for a period of time and then supplementing with stearic acid. This should repartition DHA into the GPLs, which upon lipase activity would initiate a strong antiinflammatory effect and provide a non-toxic alternative to conventional anti-inflammatory drug therapies.
Given that FAs from uptake are shown to signal for the remodelling of PUFAs into the GPL lipid fraction (cf. Fig. 4), one explanation for this distinction between de novo and uptake FAs might lie within homeostasis. Within Fig. 2 it is observed that FA uptake causes a large variation to the abundance of lipid apocromers in the de novo-lipidome fraction comparative to the nil-supplemented control. In contrast, the supplementation and incorporation of palmitic acid (and its metabolic progeny) into the uptake-lipidome is seen to influence the apocromer abundance such that it replicates the nil-supplemented control. In the more biologically relevant instance where the cell is constantly taking up extracellular FAs that are causing the signalling for remodelling events, it is logical to assume the incorporation of these new FAs into lipids would follow a homeostatic cellular template. Accordingly, the more that FAs are taken up, the larger this uptake-lipid fraction becomes until it exists in majority and thus dilutes the lipid fraction that was perturbed by remodelling -thereby restoring homeostasis. Disruption of these mechanisms, such as changes to environmental physicochemical properties, would therefore lead to the incorrect formation of lipid snisomers and have dire implications on homeostatic metabolism. The spatial correlations observed in Fig. 5 between PUFA lipids, lipid apocromers, lyso-lipids and TGs indicate that there was dynamic lipid remodelling occurring within the fatty tissue of the xenograft model, however this same remodelling is not observed within the bulk of the tumour (round bolus).
Here, it is speculated that the hallmark switch to FA metabolism by tumour cells led to the production and secretion of palmitic and stearic acids, while simultaneously creating a more acidic environment for the surrounding tissues and an increased expression of sPLA2-IIa in attempt to decrease inflammation. Subsequently, the secreted palmitic and stearic acids were taken up by the host adipocyte cells, which caused the repartitioning of PUFAs from TGs to GPLs, especially PIs which are mainly located on the outer leaflet of the plasma membrane. These outwardly exposed PUFA PIs are a prime target for the increased presence of sPLA2-IIa, which would in turn lead to the formation of lyso-lipid and free FA species. The hypothetical change in the pH caused by the tumour would then lead to lyso-acyl chain migration and the formation of apocromeric lipid species. Consequently, lipids normally containing signalling  Interestingly, the formation of ROS species is also controlled by the peroxisomes, which along with previous implications, peroxisomal function/dysfunction can also be implied through the co-distribution of ether-lipid species (peroxisomal lipid class) to the PUFA-GPL tissue regions.
TG speciation and spatial distribution (cf. Fig. S4) also alludes to changes in lipid droplet formation in PUFA-GPL tissue regions, while Plin2 is known to interact with lipid droplets to indirectly regulate droplet structure and function. 43 Lastly, changes to FA/lipid uptake and trafficking are readily observed throughout this work, which are known functions of CD36 44 and Together the relationship between FA metabolism and these downstream effects of FA activated PPARβ/δ activity suggests that the trafficking of FAs and lipids is tightly regulated and highly influential in cell homeostasis.
Although previous research has provided a wealth of knowledge with regards to lipid remodelling, trafficking, and signalling, the recent progress made in lipid isomer research calls for an update on how these processes are impacted by or impact upon isomeric lipid species.
Within, we present an array of isomeric changes that occur after FA supplementation and how these mechanisms are manifesting in tumour tissues. Importantly, these metabolic differences are heavily related to known oncogenic enzymes that are influential in cancer pathogenesis. Together, the indication that there exists distinct subcellular fractions of FA that have different metabolic fates, functions and flow-on effects suggests that lipid isomers could be potential biomarkers for lipid remodelling and disease progression in tumour tissues.   Matrigel® (100 μL; Corning®). Bloods were collected once weekly and prostate specific antigen (PSA) was measured by ELISA to monitor PCa progression. Mice were weighed and tumour volume was measured using digital callipers thrice weekly. Tumours were allowed to grow until PSA reached 25-50 ng/mL before mice were castrated. One week following castration, mice were randomised to receive an sPLA2-IIa inhibitor (KH064; 5 mg -1 kg -1 day -1 by oral gavage) or vehicle control (0.5% Carboxymethylcellulose). Mice were culled when the ethical endpoint was reached, defined as maximum tumour volume (1000 mm 3 ) or when an Animal Ethics Committee-approved welfare score required euthanasia. Xenografts and tissues were collected and weighed. Tumours were hematoxylin and eosin (H&E) stained for morphological assessment, while tissues were halved and either: (i) placed in liquid nitrogen until moved to storage at −80°C, or (ii) stored in neutral buffered formalin fixation and paraffin-embedded (FFPE).

Tissue sectioning and mounting
Frozen resected tissues were freeze mounted to the microtome block using water and sectioned at 10 μM thickness for lipid analyses using a CM 1950 Cryostat (Leica Biosystems, Nussloch, Germany), and using a blade that was free from optimal cutting temperature (OCT) compound. Tissue sections were thaw mounted on standard glass slides (SuperFrost Plus, Menzel-Gläser, Braunschweig, Germany) before MALDI-MSI-OzID protocol (see Method 8.3.1) and the procedure used for H&E staining (see Method 8.2.3).
Hematoxylin and Entellan® were purchased from Merck (Darmstadt, Germany) and eosin Y from J.T. Baker (Center Valley, USA).

Lipid extraction
Cell lipids were extracted using methods similar to those described by Matyash et al. 65  Capped vials were vortexed for 20 s, before 1.5 h bench-top agitation. Phase separation was induced by adding 200 µL of aqueous ammonium acetate (150 mM) before samples were vortexed for 20 s and centrifuged for 5 min at 2,000 x g. The organic supernatant was pipetted off to a clean labelled 2 mL glass vial and stored at -20 °C before analyses.

Mass spectrometry imaging of lipid isomers
Samples for MALDI-MSI were placed into microcentrifuge tubes, purged with nitrogen gas and stored on dry ice for inter-laboratory shipping. Samples were stored at -80 °C upon arrival.
Immediately prior to analysis, tissues were first sectioned as per Method 8.2.2 and thinly coated with 12 passes (45 mm spray height, 30 °C, 10 psi, 2 mm track spacing) of 100 mM sodium acetate dissolved in 2:1 methanol/chloroform using an HTX TM-Sprayer (HTXImaging, Chapel Hill, NC, USA). 2,5-dihydroxyacetophenone was then sublimated (40 mg, 160 °C, 4 mins) to the sample slides using a sublimator (HTXImaging, Chapel Hill, NC, USA). Two separate MALDI-MSI experiments were undertaken on 10 µm tissue sections that were adjacent to H&E stained tissues. The first was the identification and spatial distribution of sum composition lipid species via high mass-resolution (120k at m/z 400) mass analysis (≤3 Δppm), and the second was the identification and spatial distribution of PC 34:1 sn-isomers (using MALDI-MSI-CID/OzID). All MSI experiments were performed on an Orbitrap Elite (Thermo Scientific, Bremen, Germany) mass spectrometer with MALDI-MSI pixel size of ~50 µm. The commercial ion source and stacked ring ion guide were replaced with an elevated-pressure MALDI ion source incorporating a dual-ion funnel interface (Spectroglyph LLC, Kennewick, USA) as has been described previously. 66 The MALDI laser was operated at a repetition rate of 100 Hz and pulse energy of ~1.0 µJ. The laser was focused to a spot size of ~15x12 µm as determined by the size of ablation craters in desorbed matrix. Pressure within the ion source was set to 10 mbar in the first ion funnel, and 2 mbar in the second ion funnel. The mass spectrometer was additionally modified to allow for ozonolysis, as previously described. 21 Briefly, using oxygen as a feed gas (99.999% purity, Linde Gas Benelux BV, The Netherlands

Direct infusion ESI-OzID of lipid double bonds
The DBs of intact glycerophospholipids were determined via mass spectrometry using an Orbitrap Elite high-resolution mass spectrometer (Thermo Scientific, Bremen, Germany) modified with an ozone generator (Titan-30UHC Absolute Ozone, Edmonton, Canada), as described in Method 8.3.1. In this instance, a diverter valve was placed on the nitrogen gas inlet to the higher collisional dissociation (HCD) cell, and nitrogen was replaced with generated ozone gas. 67  This experiment was then repeated for the remaining OzID-aldehyde product ions from each DB location.

Conventional lipidomics for phospholipid profiles
Lipid extracts from cell lines were run through an automated lipidomics workflow using an

MALDI-MSI OzID for lipid double bond imaging
MS spectral data files were first stitched together with MALDI imaging co-ordinate positions and collated as a MATLAB data table. TIC normalisation of the data was achieved by dividing each spectral peak by the total ion abundance in each MSI pixel and comparing this to all other pixels. Lipid abundance and distribution data were then visualised through MATLAB scripts developed by Gert. B Eijkel. 21,66 Functional distribution images (FDI) were calculated by plotting the intensity of one monitored ion divided by the combined intensity of all monitored ions (i.e., Ia / [Ia + Ib])

Conventional lipidomics for phospholipid profiles
Lipidview (Version 1.3 beta, SCIEX, Concord, ON, Canada) was used for data processing of SCIEX data files obtained from the QTRAP 6500. Lipid assignments were based on the software lipid tables and shortlisted to include even-chain lipids with 0-6 s. Odd-chain/etherlipid data was obtained but was not included in this study due to the ambiguity in assigning isobars in low resolution mass spectrometry. Isotope correction factors were applied, and MS peaks were ratioed to the isotope corrected internal standard included in each scan type. The inclusion of deuterated and odd chain fatty acids within the internal standard lipids sufficiently mass shifted internal standards away from any biological lipids, therefore allowing accurate and reliable peak intensity measurements to be discerned. Data tables were extracted from Lipidview and imported to Microsoft Excel for cell count normalization, internal standard concentration factoring, statistical analysis, and graphing.

Statistics and error analysis
The mean with 95% confidence intervals was used for error analysis on column-charts throughout and was calculated using Microsoft Excel and conventional equations. For Figure   1, the mean and variance were established using Microsoft Excel to calculate a paired t test.   KH064 treated xenograft model. An increased abundance of PUFA lipid and decreased abundance of apocromer can be observed across the tissue. Although spatial correlation is still apparent, these changes to lipid species abundance thereby reduces the correlation between either species. Both still appear to be positively correlated with host adipocyte cells. Due to a sampling malfunction, directly adjacent tissue sections could not be analysed, instead the H&E displayed is 3-4 section depths (~30-40 µm) distal to the MALDI-MSI images. were also observed to be spatially correlated to host adipocyte cells, PUFA lipids and increased apocromer abundance (H&E stain). While TG O-appears mostly homogenous across the region, PC O-appears to be more abundant around the perimeter. All species were identified by exact mass (Δppm ≤ 3), with no reasonable alternative identifications found using LIPID MAPS® or other lipid databases. Phosphatidylethanolamine ethers (PE O-) were also potentially identified but were not included due to their isobaric overlap with [PC +K] + precursor ions. All high mass-resolution MALD-MSI images are normalised to self, displaying zero abundance (dark blue) ranging through to highest abundance (orange/red).