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. Author manuscript; available in PMC: 2020 Sep 11.
Published in final edited form as: Cancer Discov. 2020 Jun 4;10(9):1282–1295. doi: 10.1158/2159-8290.CD-20-0329

Changes in Aged Fibroblast Lipid Metabolism Induce Age-dependent Melanoma Cell Resistance to Targeted Therapy Via the Fatty Acid Transporter FATP2

Gretchen M Alicea 1,2,3,4, Vito W Rebecca 1, Aaron R Goldman 1, Mitchell E Fane 1,3,4, Stephen M Douglass 1,3,4, Reeti Behera 1, Marie R Webster 1,5, Curtis H Kugel III 1, Brett L Ecker 1,6, M Cecilia Caino 7, Andrew V Kossenkov 1, Hsin-Yao Tang 1, Dennie T Frederick 8, Keith T Flaherty 8, Xiaowei Xu 6, Qin Liu 1, Dmitry I Gabrilovich 1, Meenhard Herlyn 1, Ian A Blair 6, Zachary T Schug 1, David W Speicher 1, Ashani T Weeraratna 1,3,4,*
PMCID: PMC7483379  NIHMSID: NIHMS1601573  PMID: 32499221

Abstract

Older melanoma patients (>50 years old) have poorer prognoses and response rates to targeted therapy compared to young patients (<50 years old), which can be driven, in part, by the aged microenvironment. Here, we show that aged dermal fibroblasts increase the secretion of neutral lipids, especially ceramides. When melanoma cells are exposed to the aged fibroblast lipid secretome, or co-cultured with aged fibroblasts, they increase the uptake of lipids, via the fatty acid transporter, fatty acid transport protein (FATP) 2, which is upregulated in melanoma cells in the aged microenvironment and known to play roles in lipid synthesis and accumulation. We show that blocking FATP2 in melanoma cells in an aged microenvironment inhibits their accumulation of lipids, and disrupts their mitochondrial metabolism. Inhibiting FATP2 overcomes age-related resistance to BRAF/MEK inhibition in animal models, ablates tumor relapse, and significantly extends survival time in older animals.

Introduction

Melanoma, like many other cancers, is a disease of aging, with incidence rising rapidly with age, and survival worsening, even when controlling for tumor grade and stage1. Melanoma is the rarest, yet deadliest form of skin cancer with an estimated 6,850 deaths in the United States for the year 2020 alone2. Contrary to other cancers such as breast and lung where incidence has been steadily decreasing, melanoma incidence has been on the rise for the past 40 years, and increased by 3% from 2006–2015 in men and woman older than 50 with a median age of diagnosis of 623. Additionally, older patients have more metastases, worse overall survival and worse response to targeted therapy relative to their younger counterparts4–6.

Targeted therapy in melanoma centers upon targeting the MAPK kinase signaling pathway, as mutations in the BRAF oncogene drive melanoma in a majority of patients. While melanoma patients initially respond to the standard of care of targeted therapy (BRAF and MEK inhibitors), resistance soon develops in most patients. One of these well established mechanisms of resistance is metabolic reprogramming, characterized by lower glycolytic and bioenergetic metabolism7. Specifically, in melanoma it has been shown that cells utilize glutamine or fats to escape therapy. In a recent study, BRAF mutant melanoma were shown to rely on oxidative phosphorylation (OXPHOS) for therapy escape, forcing the cancer cells to rely on glycolysis instead of OXPHOS via mitochondrial DNA depletion sensitized the melanoma cells to BRAF inhibition8. Additionally, these cells have different metabolic dependencies which involve inflammatory lipid metabolism through PGE2 or mitochondrial PC activity 7.

To determine the underlying mechanisms of age-related tumor progression and response to therapy, we have engineered artificial skin reconstructs built from dermal fibroblasts taken from individuals in their 20’s (young) or 60’s (aged). We have recently discovered that aged dermal fibroblasts play a significant role in driving melanoma metastasis and poorer response to targeted therapy4 in cell culture experiments, syngeneic mouse models of melanoma, and in melanoma patient samples4. In this study, we show that that melanoma cells require fatty acids secreted by aged fibroblasts to escape targeted therapy.

Fatty acid uptake, and subsequent fatty acid oxidation (FAO) play important roles in tumor cell survival and metastasis9. In tumors that are not heavily dependent upon glycolysis, FAO is thought to be the most critical bioenergetic pathway. Since therapy-resistant melanomas have been shown to switch to a less glycolytic pathway, we hypothesize that fatty acid uptake may play a role in the bioenergetics of these cells as well, and contribute to the observed age-dependent resistance of tumor cells to targeted therapy. The uptake of fatty acids in melanoma cells occurs through fatty acid transporters, in particular a family that consists of Fatty Acid Transporters1–6 (FATP1–6). FATP1 has previously been implicated in melanoma progression, where it was found that adipocytes transfer lipids to the melanoma cells through FATP1, driving invasion and metastasis10. Here we find that FATP2 expression is consistently upregulated in tumor cells in an aged microenvironment and represents the only member of the FATP family to significantly correlate with patient age. FATP2 is critical for esterification of long chain fatty acids into triglycerides (TGs), and acts as both a synthetase and transporter of fatty acids. Our data identify that targeting FATP2 ablates the uptake of lipids, and renders melanoma cells in an aged microenvironment sensitive to targeted therapy. Overall, these data support the critical importance of understanding the role of the aged microenvironment in the efficacy of treatment for patients with melanoma.

Results

In the current study, we examined the metabolic changes in the aged microenvironment, and how they impact tumor cells. We found that aged fibroblasts have increased levels of neutral lipids as defined by BODIPY 505/515 staining and higher fatty acid synthase (FASN) than young fibroblasts (Supplementary Figure 1A). We quantified and confirmed this increase in BODIPY by flow cytometry (Supplementary Figure 1B). To examine this further, we performed lipidomics analysis of young (<35) and aged fibroblasts(55<), as well as the lipid secretome of these fibroblasts. We show here the simplified lipidomes, and complete lipidomes are available upon request. In analyzing the fibroblasts themselves, we found that while the overall levels of lipid classes did not differ significantly among young and aged fibroblasts, individual lipid species differed extensively (Figure 1A). We found 257 out of 853 identified lipid species differed significantly (>1.5-fold change with a false discovery rate (FDR)<10%) with 129 elevated and 128 decreased lipid species in aged fibroblasts (Figure 1B). Most of the greatest increases were in phosphatidylglycerols (PGs) that contained at least one polyunsaturated fatty acid (PUFA) and, overall, the vast majority of PGs and all lysophosphatidylglycerols (LPGs) that significantly increased had a PUFA. Further, the vast majority of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species that significantly decreased had ether-linked fatty acids. This is particularly intriguing as peroxisomes are required for the biosynthesis of ether-linked phospholipids, and we have previously published that reactive oxygen species are increased in the aged tumor microenvironment. Furthermore, molecules that regulate ROS such as SOD3 and peroxiredoxin are decreased4. These results suggest that peroxisome function may be impaired in aged fibroblasts, consistent with previous work on age-related diseases11. Other noteworthy changes included ceramides, where most ceramides (11/14) and all glycosylceramides (6/6) that changed were increased in the aged fibroblasts, and we explore this further below.

Figure 1. Melanoma cells display elevated lipid levels when exposed to aged fibroblasts.

Figure 1.

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A. Lipidomics analysis of different lipid class profiles between aged and young fibroblasts. B. Volcano plot analysis of young and aged fibroblasts. C. Secretome analysis of differentially secreted lipid class between aged and young fibroblasts. D. Analysis of young and aged fibroblasts conditioned media after 24hrs.E. Melanoma cells were cultured with young or aged conditioned media for 48hrs. Cells were subsequently stained with dapi and BODIPY 505/515 for lipid visualization and imaged by IF microscopy. F. Melanoma cells treated with 4nM ND646 in the presence of young and aged fibroblast conditioned media for 48hrs followed by Trypan blue exclusion analysis, (two-tailed unpaired-test,p=0.0001 p=0.0024 in order) G. Melanoma cells were cultured with aged fibroblast conditioned media where FASN has been knocked down and cultured for 48 hrs H. Young and aged fibroblasts stained with BODIPY-C12, washed and co-cultured with GFP-tagged melanoma cells. Image shown is a 24h timepoint of movies supplied as supplementary movies. I. Overlap of lipidomics analysis of fibroblast secretomes, and intracellular lipid composition of melanoma cells cultured with young or aged conditioned media for 48 hours. For lipid species, statistical analysis was performed using Perseus 1.6.7.0, as described in the methods.

Because our interests are focused on the microenvironment conferred by the lipid secretome of fibroblasts and its subsequent effects on tumor cells, we next profiled the lipids in young and aged fibroblast conditioned media (CM). Here, we identified134 lipids and found that 15 changed significantly between CM from young and aged fibroblasts (|>1.5 fold change and FDR<10%). The most significant enrichment was for lipids from the ceramide class (including glycosylceramides (Figure 1C,D). This is curious given that previous studies have shown that during aging, keratinocytes decrease their ability to make sphingolipids12,13, including ceramides which are critical for maintaining the water impermeability of the skin. These findings suggest that dermal fibroblasts may attempt to compensate for the decline in keratinocyte-produced ceramides by increasing ceramide production. Whether or not fibroblast-derived ceramides are as effective as those produced by keratinocytes remains to be investigated.

To determine whether melanoma cell lipid levels were affected by lipids in their immediate microenvironment, melanoma cells were treated in vitro with aged fibroblast CM and examined for lipid accumulation. Melanoma cells increased their levels of lipids as compared to the same cells treated with young fibroblast CM (Figure 1E, quantification in Supplementary Figure 1C, and additional cell lines, including BRAF mutant lines, Supplementary Figure 1D,E, and NRAS mutant lines, Supplementary Figure 1F,G). Tumors in aged mice in vivo also had elevated lipid levels as demonstrated by perilipin staining (Supplementary Figure 1H). To determine whether the source of lipids in the melanoma cells was due to increased fatty acid synthesis in the melanoma cells, or whether the lipids in the melanoma cells were directly transported from the aged fibroblasts we took multiple approaches. We first treated melanoma cells with an acetyl coenzyme A (CoA) carboxylase inhibitor, ND646 (to inhibit fatty acid synthesis), in the presence of either young or aged fibroblast CM. We found that melanoma cells treated with ND646 in either media alone, or with young fibroblast CM underwent significant cell death (Figure 1F). In contrast, when we repeated this experiment in the presence of aged fibroblast CM, cell death was significantly decreased, suggesting that the melanoma cells in the aged media did not depend on fatty acid synthesis as the only source of endogenous lipids (Figure 1F). We then knocked down FASN in the aged fibroblasts (Supplementary Figure 1I), and showed that they no longer synthesized lipids as effectivley (Supplementary Figure 1J). Melanoma cells incubated in the CM of FASN-deficient fibroblasts no longer stained positive for lipids (Figure 1G, additional lines in Supplementary Figure 1K, quantified in Supplementary Figure 1L), suggesting that at least some of the lipids accumulating in melanoma cells might be fibroblast-derived. Additionally, we treated young and aged fibroblasts with the orange-red fluorescent C12-fatty acid BODIPY 558/568 (Thermo Scientific), a synthetic precursor of fluorescent phospholipids14. We trypsinized and washed the fibroblasts several times to ensure that any unincorporated fluorescent BODIPY was eliminated, and then incubated the BODIPY-labeled fibroblasts with GFP-tagged melanoma cells and visualized them over time. We observed that melanoma cells took up more BODIPY 558/568 from the aged fibroblasts, than from the young (Figure 1H, final time point, Supplementary Figure 1M, Supplementary movies SM1, 2). We also observed that when using CM from the young and aged fibroblasts, a similar uptake could be seen, suggesting that the lipids are being secreted by aged fibroblasts and taken up by the melanoma cells, and do not require direct cell-to-cell contact (Supplementary Figure 1N).

Since melanoma cells were thriving despite accumulating excess lipid, this suggested that the fatty acids were being packaged into neutral lipids such as triglycerides. To determine whether this was the case, we first stained melanoma cells exposed to young and aged fibroblast CM with LipoTox ® dyes that can distinguish between phospholipids (green) and neutral lipids (red). We found that both phospholipids and neutral lipids are increased in melanoma cells exposed to aged but not young fibroblast CM (Supplementary Figure 2A), and TG synthesis is also increased (Supplementary Figure 2B). This was further confirmed by our lipidomics data, where we treated the melanoma cells with young and aged fibroblast CM, and then analyzed the lipidome of the melanoma cells. As with the fibroblasts alone, melanoma cell size is significantly enlarged when exposed to aged conditioned media (Supplementary Figure 2C) and normalizing to total protein minimized any observed changes (Supplementary Figure 2D). However, when normalized to cell volume, the melanoma cell intracellular lipidome largely reflects the changes observed in the fibroblast lipid secretome and highlights an accumulation of neutral lipids such as ceramides, and TGs (Fig 1I).

To determine how lipids were being transported into the melanoma cells from the fibroblast media, we examined the expression of fatty acid transporters and identified FATP2 as the fatty acid transporter most affected by the aged microenvironment. FATP2 is primarily responsible for the transport of very long chain FA15,16 and has the dual function of facilitating the import of exogenous FA17 and the activation of FA by its intrinsic acyl-CoA synthase activity, which is a key step in the production and utilization of lipids18,19. Melanoma cells exposed to aged, but not young fibroblast CM, upregulated their levels of FATP2 (Figure 2A). FATP1 has previously been implicated in melanoma metastasis10, but showed no age-dependency in its expression in our studies. Using the TCGA database, we analyzed FATP family members 1–6 in primary melanomas and found FATP2 to be very significantly increased in primary melanomas in aged individuals (Figure 2B), whereas FATP1, 3, 4 and 5 were not significantly different between young and aged (Figure 2C). FATP6, while reaching significance (higher in aged), is not highly expressed in melanoma, and is specific to cardiac tissue. Since melanoma metastasizes to the heart, it is interesting that FATP6 is elevated in a small subset of primary melanomas. Other fatty acid transporters such as CD36 were not significantly upregulated in an age-specific manner. To further examine the specificity of FATP2 in age-dependent accumulation of lipids, we knocked down FATP2 in melanoma cells (Supplementary Figure 3A) and decreased the accumulation of lipids in melanoma cells exposed to aged fibroblast CM (Figure 2D, upper panels, additional cell lines in Supplementary Figure 3B). Conversely, knockdown of FATP1 in melanoma cells (Supplementary Figure 3C) did not affect lipid accumulation in an aged microenvironment (Figure 2D, lower panels, additional cell lines in Supplementary Figure 3D, quantifed in Supplementary Figure 3E).

Figure 2. FATP2 is upregulated in melanoma on the aged microenvironment.

Figure 2.

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A. Melanoma cells were cultured with DMEM, young or aged fibroblast conditioned media for 48 hrs. Cells were probed for FATP2 and FATP1 by immunoblotting. B. TCGA of FATP2 expression in primary melanoma. C. TCGA data analysis of transporters FATP1,3–6 and CD36 in primary melanoma D. Immunofluorescence microscopy of melanoma cells cultured with young and aged fibroblasts CM stained with BODIPY 505/515. E. FATP2 staining in melanoma skin reconstructs made with young or aged fibroblasts. F. FATP2 staining in tumor tissue from young and aged mice. G. Melanoma cells were treated with vehicle control or lipofermata (5uM) for 48hrs. Cells were subsequently stained with dapi and bodipy 505/515 for lipid visualization and imaged by IF microscopy. H. Yumm 1.7 melanoma cells were subcutaneously injected in young (8weeks) and aged (52weeks) mice. A separate cohort of aged mice bearing Yumm 1.7 tumors was treated with lipofermata (2mg/kg, twice a day for two weeks). Tumors were stained with Oil Red O to determine lipid accumulation.

We built artificial skin reconstructs as previously described4 using either young or aged fibroblasts to create a young or aged microenvironment for the same melanoma cell lines. We found that FATP2 was increased in the melanoma cells grown in aged skin reconstructs (Figure 2E). We also examined FATP2 expression in tumors in aged mice, and found increased FATP2 as compared to tumors in young mice (Figure 2F). This observed effect could also be recapitulated using Lipofermata19, a drug that specifically targets FATP2. Upon treatment with Lipofermata, lipid accumulation was decreased both in melanoma cells in an aged microenvironment (Figure 2G, quantified in Supplementary Figure 3F) and in Yumm1.7 tumors grown intradermally in aged mice (Figure 2H). These data point to a key mechanistic role for FATP2 in the age-dependent accumulation of lipids in melanoma cells.

Clinically, there also has been much interest in targeting FASN in tumors, and FASN inhibition has been shown to inhibit tumor regrowth after anti-angiogenic therapy20. There are data suggesting that melanoma cells alter metabolism to resist therapy21, and our previous data showed that aged fibroblasts can promote therapy resistance of melanoma cells in the absence of mutational changes4. Further, recent data suggest that BRAF inhibition itself can affect lipogenesis22. We therefore asked whether lipid accumulation in melanoma cells in the aged microenvironment contributed to therapy resistance. We grew BRAFV600E mutant melanoma cells in 3D spheroids in young and aged fibroblast CM, and treated them with 3μM PLX4720 (BRAF inhibitor) together with 500nM PD0325901 (MEK inhibitor), the standard treatment for malignant melanoma. We found that melanoma cells in the aged microenvironment did not respond as effectively to the treatment as those in a young microenvironment (Figure 3A). However, when we pre-treated with 5 μM Lipofermata, we could completely sensitize melanoma cells in an aged microenvironment to the PLX/PD combination (Figure 3A, bottom panels, additional melanoma lines, Supplementary Figure 4A). Genetic knockdown of FATP2 phenocopied these results (Figure 3B). Lipofermata did not affect the PO4-Erk signaling pathway (Supplementary Figure 4B), suggesting that lipids were not affecting resistance by preventing the drug from reaching its target. Next, we examined the metabolic activity of the cells, since oxidative metabolism plays a critical role in governing the resistance of melanoma cells to BRAF/MEK inhibition21,23. Here we found dramatic differences between the oxygen consumption rate of melanoma cells incubated in young vs. aged media. Melanoma cells incubated in aged media upregulate levels of CPT1, (Figure 3C), an enzyme which is responsible for the transfer of the long-chain acyl group of the Acyl-coA ester to carnitine, generating acyl-carnitine. This allows for the transport of fatty acids into the mitochondrial matrix for β-oxidation, which in turn is critical for resistance of melanoma cells to BRAF/ MEK inhibition. Etomoxir is a drug which specifically targets CPT1, and using a Seahorse assay to measure oxygen consumption rates (OCR), we treated melanoma cells with etomoxir, in the presence or absence of young or aged fibroblast CM. We found that melanoma cells treated with aged CM increased their OCR significantly as compared to melanoma cells treated with young CM, which increased their OCR very slightly (Figure 3D). Treatment of the latter cells with etomoxir did little to affect the OCR rates, suggesting that CPT1 mediated fatty acid oxidation likely did not play a role, but treatment of melanoma cells in aged CM with etomoxir dramatically reduced their OCR rate suggesting a strong reliance on the activity of CPT1 (Figure 3D).

Figure 3. In vitro inhibition of FATP2 increases response to BRAFi/MEKi therapy.

Figure 3.

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A. Melanoma cells grown in 3D spheroids were treated with PLX4720 (3μM), PD0325901 (500nM) and/or Lipofermata (5μM) in the presence of young and aged conditioned media for 48 hrs. Spheroids were subsequently stained with a viability stained and imaged with IF microscopy. Calcein-AM staining (green) signifies viable cells and TOPRO3 staining (red) signifies dead cells. B. Melanoma cells with FATP2 knockdown or empty vector grown in 3D spheroids were treated with PLX4720 (3μM), PD0325901 (500nM). Spheroids were subsequently stained with a viability stained and imaged with IF microscopy. Calcein AM staining (green) signifies viable cells and TOPRO3 staining (red) signifies dead cells C. Western analysis of CPT1 in melanoma cells exposed to young and aged fibroblast conditioned media. D. Melanoma cells exposed to non-CM, young or aged CM for 5 days with or without the CPT1 inhibitor Etomixir. Oxygen consumption rate (OCR) was measured after the treatment of oligomycin (1μM), FCCP (1.5μM) and rotenone/antimycin A (0.5μM). E. Melanoma cells exposed to non-CM, young or aged CM for 3 days prior to treatment with BRAFi/MEKi, Lipofermata or BRAFi/MEKi/Lipofermata treatment for 2 days. Oxygen consumption rate (OCR) was measured after the treatment of oligomycin (1uM), FCCP (1.5uM) and rotenone/antimycin A (0.5μM). F. Human melanoma samples stained with FATP2 and H-Score of patient samples stained with FATP2 plotted against survival in days (Mann-Whitey test, *Signifies p < 0.05). G. Pre and post treatment PDX tissue was stained with FATP2. H. Yumm1.7 melanoma cells were made resistant to the PLX4720/ PD0325901 combination therapy and grown in spheroids. Lipofermata was added to the combination of PLX/PD, and spheroids were assessed for live/ dead staining as described in A. I. Trypan blue viability assay after treatment of melanoma cells with control lipoprotein-depleted media, conditioned media from young fibroblasts, with and without PLX4720 (3μM), PD0325901 (500nM), and with PLX4720 (3μM), PD0325901 (500nM) in the presence of 14.8 ng/mL of ceramides. J. Spheroid assays of 1205LU cells after treatment with conditioned lipoprotein-depleted media from young fibroblasts, with and without PLX4720 (3μM), PD0325901 (500nM), and with PLX4720 (3μM), PD0325901 (500nM) in the presence of 14.8 ng/mL of ceramides. Spheroids were subsequently stained with a viability stained and imaged with IF microscopy. Calcein-AM staining (green) signifies viable cells and TOPRO3 staining (red) signifies dead cells. For all panels, unless otherwise specified, two-tailed unpaired t-test, *=p<0.05; **=p<0.01; ***=p<0.001.

To next determine if blocking FATP2 could also affect the OCR rate in melanoma cells cultured in aged CM, we treated melanoma cells with Lipofermata in the aged fibroblast CM, and in the presence or absence of BRAF/MEK inhibitors. This revealed that BRAF/MEK inhibition reduced the OCR of melanoma cells in aged CM, but not to the extent of either Lipofermata alone, or Lipofermata in combination with BRAF/MEK inhibitors (Figure 3E), suggesting that inhibiting FATP2 mediated transport of lipids into the melanoma cells can affect their mitochondrial activity, making them less able to resist targeted therapy. We analyzed a small cohort of patients (n=8) after BRAF/MEK inhibitor treatment for the expression of FATP2, and compared it to their overall survival time, post-treatment. FATP2 expression was markedly elevated in patients who succumbed to disease in a shorter time frame (Figure 3F). Even in this tiny cohort the relationship between FATP2 and survival was significant (p=0.03), but the number of samples was too small to get a sense of whether age was a factor as well. We also examined patient derived xenografts that were initially sensitive to BRAF/ MEK inhibition, then relapsed, for FATP2 expression before and after treatment. We found that in cases where tumors acquired resistance to the BRAF/ MEK combination, FATP2 expression increased dramatically, even if they were negative for FATP2 prior to treatment (Figure 3G). Therefore, given the ability of Lipofermata to maintain sensitivity to BRAF/MEK inhibition, we asked whether treating resistant melanoma cells with Lipofermata would re-sensitize them to BRAF/ MEK inhibitors. We created Yumm1.7 melanoma cells that were resistant to the BRAF/ MEK inhibitor combination (Yumm1.7CR cells), and then grew them in spheroid assays. The cells in the vehicle control conditions remained resistant to the PLX/PD combination, while adding Lipofermata sensitized the cells to the combination therapy (Figure 3H).

Since our LC-MS data identified ceramides as one of the most differentially expressed group of fatty acids, we wanted to analyze whether ceramides specifically contributed to therapy resistance. To calculate ceramide concentration, we used an internal standard on the LC-MS that contains a heavy labeled ceramide as well as 4 additional heavy ceramides, all of which gave similar MS signals per pmole. Lipoprotein-depleted media (1.4ng/mL of ceramides) was used to grow fibroblasts and cells grown in young media (usually 8.1ng/mL of ceramides) were reconstituted to a final concentration of 14.8 ng/mL of ceramides (concentration in aged media). Treatment of cells grown in ceramide-supplemented media showed an increased resistance to treatment with BRAF/MEK inhibitors both in a 2D trypan blue viability assay (Figure 3I), and in a 3D spheroid assay (Figure 3J). Together these data suggest that inhibiting FATP2 mediated transport of lipids could overcome resistance to targeted therapy that we observe in melanoma cells in an aged microenvironment by disrupting mitochondrially mediated mechanisms of therapy resistance.

To examine the role of FATP in vivo, we took both a pharmacological and genetic approach. First, we injected Yumm1.7 BRAFV600E mutant melanoma cells intradermally into C57/BL6 mice of either 8 weeks or 52 weeks of age, and then treated half of each group with 200 mg/kg PLX4720 (BRAF inhibitor) plus 7 mg/kg PD0325901 (MEK inhibitor). We saw that in young mice, tumors responded to the PLX/PD combination, recurring only after a long lag time of 55 days (Figure 4A). Aged mice on the other hand, initially responded, but quickly relapsed after 10–15 days (Figure 4B, teal line). Lipofermata treatment on its own did not affect tumor growth at all in young mice, but in aged mice, tumors initially responded to Lipofermata, then quickly relapsed, after 10–15 days (Figure 4B, gold line). However, where the combination of all three drugs did not further suppress tumor growth in young mice, (Figure 4A, red line), in aged mice, the triple combination ablated tumor growth (Figure 4B, red line), This is reflected in the survival curves (Figure 4C,D). The combination was well tolerated in both young and aged mice as reflected in the mouse body weights (Supplementary Figure 5A). Post-relapse analysis revealed that in tumors that recurred on BRAFi/MEKi, FATP2 staining was elevated compared to control treated mice, but not in the Lipofermata alone condition, which maintained loss of FATP2 expression (Supplementary Figure 5B).

Figure 4. FATP2 inhibition enhances BRAF and MEK combinatorial therapy “in vivo”.

Figure 4.

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A. Yumm 1.7 melanoma cells were grown in young (8weeks). Mouse tumor growth in young mice after treatment with indicated drugs (n=8 per arm). B. Yumm 1.7 melanoma cells were injected in aged mice (52 weeks). Mouse tumor growth in aged and young mice after treatment with indicated drugs (n=8 per arm). C. Survival curve for treatment in figure 4a displaying the time it took for mice to reach death (defined as time when tumor volume exceeded 750mm3). D. Survival curve for treatment on figure 4b displaying the time it took for mice to reach death (defined as time when tumor volume exceeded 750mm3). E. Yumm1.7 FATP2 Tet- inducible cell line was subdermally injected in young mice (8 weeks). Mouse tumor growth in aged and young mice after treatment with indicated drugs in the presence of absence of doxycycline (n=8 per arm). F. Yumm1.7 FATP2 Tet- inducible cell line was subcutaneously injected in aged mice (8=52 weeks). Mouse tumor growth in aged and young mice after treatment with indicated drugs in the presence of absence of doxycycline (n=8 per arm). G. Survival curve for treatment in figure 4E displaying the time it took for mice to reach death (defined as time when tumor volume exceeded 750mm3). H. Survival curve for treatment in figure 4F displaying the time it took for mice to reach death (defined as time when tumor volume exceeded 750mm3). Statistics on all curves were obtained using a linear mixed effects model. N.S., not significant, **=p<0.01, ***p=<0.001. All error bars are standard error of the mean (SEM).

We hypothesized that the difference in response to Lipofermata alone in young vs. aged mice, might be due to effects on other cell populations, such as myeloid derived suppressor cells (MDSC) in the tumor microenvironment. In support of this concept, Veglia et al. showed that Lipofermata can inhibit the activity of myeloid derived suppressor cells, rendering the tumors more susceptible to immune-mediated clearing24. Indeed, tumors in aged mice have more polymorphonuclear (PMN)-MDSCs, providing in part an explanation for these data (Supplementary Figure 5C). To test this hypothesis and to determine the effects of FATP2 inhibition on the tumor cells alone, we created melanoma cells in which we could knock down FATP2 expression in an inducible manner. We implanted these cells in young and aged mice, and then treated with the PLX/PD combination in the presence or absence of doxycycline to silence FATP2 expression (Supplementary Figure 5D). We found that downregulation of FATP2 expression specifically in melanoma cells did not affect the growth of tumors in either young or aged mice (Figure 4E,F), confirming that the observed effect of Lipofermata alone in the aged mice is likely microenvironmental. As with the Lipofermata experiment, young mice responded well to the PLX/PD combination (Figure 4E teal line), and depleting FATP2 had no further effect (Figure 4E, red line). However, in the aged mice, depletion of FATP2 in melanoma cells in combination with BRAFi/MEKi was still able to ablate tumors and sustain tumor regression (Figure 4F, red line). Survival curves for these experiments are shown in Figure 4G,H. Together these data confirm that FATP2 ablation may have the ability to overcome resistance to targeted therapy in aged patients.

Discussion

Overall, our data suggest that inhibiting FATP2 dependent accumulation of lipids can overcome therapy resistance. While our data indicate that these lipids are coming from aged fibroblasts, melanoma cells can also take up lipids from other sources such as adipocytes10. Regardless of the source, the melanoma cells are able to take up lipids, and use them to promote aggressive behavior such as metastasis10 or therapy resistance, as we show here. We show that of the lipids identified, ceramides may play a specific role. This is intriguing, because FATP2 plays a dual role as both a fatty acid transporter, and an Acyl-coA synthetase. Acyl-coA synthetase catalyzes the conversion of long chain fatty acids to fatty Acyl-coAs. Since it has been shown that cancer cells escape treatment by the conversion of ceramides into acylceramides, we used LC-MS to infer the presence of acylceramides by comparing ceramide levels in lipid extract before and after saponification. Cer(d18:1_16:0), Cer(d18:2_16:0), and Cer(d18:1_18:0) are >1.5x increased after mild saponification, suggesting presence of acylceramides with these ceramide backbones (Supplementary Figure 6A). This reaction, as well as the packaging of fatty acids into neutral TGs, which we show are increased in melanoma cells exposed to the aged TME (Supplementary Figure 2D) requires diacyl glycerol transferase (DGAT) 2. We show that DGAT2 is upregulated in melanoma cells in the aged TME (Supplementary Figure 6B). These results would need further exploration, and are merely correlative at this time.

The data in Figures 1A through 1D showed that there were no differences in the intracellular amounts of TG in fibroblasts nor were there any differences in the secreted amounts of TG in conditioned media (CM) from aged and young fibroblasts. However, there is an accumulation of neutral lipids in melanoma cells only in aged CM. Since total TG levels were the same regardless of the CM, it is likely that the fatty acids are mobilized from multiple sources including neutral lipids, phospholipids, and free fatty acids, and that it is the specific ability of FATP2 to capture and metabolize these mobilized fatty acids which directs them into de novo synthesized TG that then accumulate as lipid droplets.Together these data are highly suggestive of a dual role for FATP2 in melanoma, in its capacity both as a transporter, and as an AcylCoA synthetase, as has been shown17.

It is a point of interest that the lipids we see in the melanoma cells are packaged into TGs. Large-scale epidemiological studies have linked increased TG levels with increased risk of multiple different cancers, including prostate, ovarian and lung cancer25,26. For melanoma specifically, data from the metabolic cancer study (Me-Can) showed that patients with increased serum TGs had an increased risk of melanoma (relative risk adjusted to BMI is 1.24)25. Our study shows that TG accumulation in melanoma cells also increases the resistance to targeted therapy. These data suggest that targeting TG levels27,28, which increase in older patients, could enhance response to targeted therapy in this population of patients with increased resistance to MAPK inhibition. Macrophages may be particularly susceptible to TGs, as it has been shown that during aging, the ability of macrophages to hydrolyze and utilize TGs is decreased, due to the loss of catecholamine-induced lipolysis. Depletion of NACT, LRR, and PYD domains-containing protein (NLRP)3, an important mediator of the inflammasome, can restore catecholamine-induced lipolysis, decreasing TG levels within macrophages29. Together, these data hint that not only targeted therapies, but potentially immunotherapies may be affected by lipid accumulation in melanoma patients, and point once again to the critical role of the aged microenvironment in melanoma pathogenesis. Lipofermata in this context is an exciting drug, because it not only inhibits FATP2 in the tumor in aged individuals, but also in the immunosuppressive microenvironment, potentially increasing the susceptibility of these tumors not only to targeted therapy but also to immunotherapy.

Materials and Methods

1205 Lu, 1205-GFP, WM793, WM793-GFP, WM983B, WM164, melanoma cell lines were maintained in TU2% (MCDB153 (Sigma)/Liebovitz L-15 (Cellgro; 4:1 ratio) supplemented with 2% FCS and 1.6 mMol/L CaCl2) and transferred to DMEM (Invitrogen, Carlsbad, CA), supplemented with 5%FBS prior to conditioned media experiments. WM1361A and WM1366 were maintained in RPMI with 5% FCS. Dermal fibroblast cell lines were obtained from Biobank at Coriell Institute for Medical Research. The fibroblasts were cultured in DMEM (Invitrogen) supplemented with 10% FCS. Yumm1.7 murine melanoma cells were cultured in DMEM supplemented with 10% FCS. Keratinocytes were maintained in keratinocyte SFM supplemented with human recombinant Epidermal Growth Factor 1–53 (EGF 1–53) and Bovine Pituitary Extract (BPE) (Invitrogen). All cell lines were cultured at 37°C in 5% CO2 and medium was replaced as required. Cell stocks were fingerprinted using AmpFLSTR® Identifiler® PCR Amplification Kit from Life Technologies TM at The Wistar Institute Genomics Facility. Although it is desirable to compare the profile to the tissue or patient of origin, our cell lines were established over the course of 40 years, long before acquisition of normal control DNA was routinely performed. However, each STR profile is compared to our internal database of over 200 melanoma cell lines, as well as control lines, such as HeLa and 293T. STR profiles are available upon request. Cell culture supernatants were mycoplasma tested using Lonza MycoAlert assay at the University of Pennsylvania Cell Center Services.

Western Blot

Cell lines were plated and collected with RIPA buffer. Protein was measured with Qubit assay kit (#Q33212, Thermo Scientific) and 30ug protein was boiled and loaded into 4– 12% gel at 160V and transferred onto PVDF membranes. Blots were blocked with 5% milk for an hour and primary antibody was incubated overnight at 4C. The next day, membranes were washed with TBS with 0.1% Tween and incubated with secondary antibody for an hour at room temperature. Membranes were then washed and developed using chemiluminescence based detection reagents (#PI80196, Thermo Scientific). Chemiluminescence was detected using ImageQuant™ LAS 4000 (GE Healthcare Life Sciences, Pittsburgh, PA).

Antibodies

Antibodies were purchased from the following commercial vendors and used in the following dilutions: FATP2(1:1000, Invitrogen), FASN (1:1000, Cell Signaling), GAPDH Cell Signaling (1: 10,000), Beta Actin (1:10,000, Novus Biologicals; NB600–501), CD36 (1:1000, Novus Biologicals), FATP1 (1:1000, ABNOVA), p-ERK (1:3000, Cell Signaling), Total-ERK (1:3000, Cell Signaling), HSP90 (1:10,000, Cell Signaling), CPT1 (1:1000, Cell Signaling).

Lipidomics

Dermal fibroblasts were cultured for 24 hours in DMEM media with 2.5% lipoprotein depleted fetal bovine serum (Kalen Biomedical). Melanoma cells were incubated for 48 hours in control media or conditioned media from young/aged fibroblasts cultured for 48 hours in DMEM media with 10% fetal bovine serum. Three biological replicates were analyzed per experiment. Fibroblasts and melanoma cells were washed with cold PBS and scraped into cold methanol. Cold methanol was added to fibroblast conditioned media at five times the volume. All samples were spiked with the Splash Lipidomix of deuterium-labled lipids (Avanti Polar Lipids, Alabaster, AL), and lipids were initially extracted with chloroform/methanol/0.88% sodium chloride 2:1:1 final and re-extracted with synthetic organic phase, as described previously30. UHPLC-electrospray-tandem mass spectrometry (ESI-MS/MS) was performed in positive and negative ion modes on a Thermo Scientific Q Exactive HF-X mass spectrometer and Vanquish Horizon UHPLC system. Gradient LC separation used an Accucore C30 column (2.1 mm × 150 mm, Thermo Scientific) with 50:50 acetonitrile/water and 88:10:2 isopropanol/acetonitrile/water solvents, both containing 5 mM ammonium formate and 0.1% formic acid. MS1 scans were acquired at 120k resolution, and data dependent MS2 scans were acquired for the top 20 ions at 15k resolution with 0.4 m/z isolation width and 20/30/40 stepped NCE. Lipid species were identified and quantified using LipidSearch 4.2 (Thermo Scientific). For all data sets, lipid species were filtered by adduct and grade, and MS peak areas were normalized to the Splash Lipidomix deuterated lipid standard of the same class where available. No further normalization was performed for conditioned media as equal volumes were processed. Cellular lipidomes were further normalized by total lipid MS signal for each sample to correct for variations in total biomass content. Finally, for some comparisons, melanoma cell lipids were normalized to cell volume. Cell volume was calculated using quantitative microscopy and Huygens software. Representative data is shown in Supplementary Figure 2D. For lipid species, statistical analysis was performed using Perseus 1.6.7.0 31,32. Data were log2-transformed, and comparisons were performed using permutation-based FDR with s0=0.1 and 250 randomizations. For lipid classes, normalized peak areas were summed for each lipid species in a given class, and comparisons were performed using the Student’s t-test.

Lipidomic heatmaps

For the melanoma cells, MS1 signal was normalized to area and cell volume were used for analysis. Zero intensity values were floored to minimum detected intensity across all samples and protein groups and log2-transformed. Two sample t-test was used to estimate significance of difference between groups and correction for multiple testing was done using Benjamini-Hochberg method. Results passing FDR<5% cutoff were considered significant. Lipid molecule name with main ion was used as a unique identifier and the most significant isobars were considered to resolve duplicates. Enrichment of lipid classes was done for lipids significantly changed at least 5-fold (fibroblasts) or 2-fold (melanoma cells) and significance was estimated using Fisher Exact Test.

shRNA, lentiviral production and infection

FASN and FATP2 shRNA was obtained from the TRC shRNA library available at The Wistar Institute (TRCN0000150501, TRCN0000153400). For FASN (sh1 and 2), we used shFASN_0312 and shFASN_03125. For FATP2 (sh1 and 5), we used shFATP2_42973 and shFATP2_42977. For FATP1 (sh1 and sh2), we used shFATP1_038184 and shFATP1_038185. Lentiviral production was performed according to the protocol suggested by the Broad Institute. Briefly, 293T cells are plated at 70% confluency and co-transfected with shRNA plasmid and the lentiviral packaging plasmids (pCMV-dR8.74psPAX2, pMD2.G). pLKO.1 empty vector was used as a control. For transduction, cells were treated with lentivirus overnight and allowed to recover for 24 hours before selection using puromycin (1μg/ml).

Organotypic 3D Skin Reconstructs

Organotypic 3D skin reconstructs were generated by plating, 6.4 × 104 fibroblasts in each insert on top of the acellular layer (BD #355467 and Falcon #353092) and incubated for 45 minutes at 37°C in a 5% CO2 tissue culture incubator. DMEM containing 10% FBS was added to each well of the tissue culture trays and incubated for 4 days. Reconstructs were then incubated for 1 h at 37°C in HBSS containing 1% dialyzed FBS (wash media). Washing media was removed and replaced with reconstruct media I. Keratinocytes (4.17 × 105) and melanoma cells (8.3 × 104) were added to the inside of each insert. Media was changed every other day until day 18 when reconstructs were harvested, fixed in 10% formalin, paraffin embedded, sectioned and stained.

Drug Treatment

PLX4720 was obtained from Selleck Chem and dissolved in DMSO (stock 10mM). Cells were treated for 48 hours and analyzed. After optimization, 3μM was the final concentration utilized for the experiments. PD0325091 was obtained from Selleck Chem and dissolved in DMSO (stock 100mM). Cells were treated for 48 hours and analyzed. After optimization, 500nM was the final concentration utilized for the experiments. Lipofermata was obtained from Chembridgecorps and dissolved in 30% Kolliphor (C5135, Sigma Aldrich). Cells were treated with 5 μM for 48 hours and analyzed. ND646 from MedChemExpress was solubilized in DMSO (stock 10uM) for a final concentration of 4nM.

3D Spheroid Assays

5,000 melanoma cells were plated in 1.5% agar and spheroids were allowed to form for 3 days. Spheroids were then embedded in rat-tail collagen (Life Technologies). The next day cell lines were treated with unconditioned media, young conditioned media and aged conditioned media in presence or absence of PLX4720, PD0325901 or Lipofermata. Cell viability was measured using the LIVE/DEAD® Viability/Cytotoxicity Kit (L3224, Invitrogen). Briefly, spheroids were washed with PBS and stained with calcein-AM and Ethidium homodimer-1. The dyes were diluted in PBS and 300μl of the solution was added on the spheroid wells for 1 hour at 37°C. The spheroids were washed in PBS and imaged using a Nikon TE2000 inverted microscope.

In vivo allograft assays

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) (IACUC #1122mg/mL503X_0) and were performed in an Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) accredited facility. For Lipofermata experiments, YUMM1.7 (5 × 105 cells) overexpressing mCherry were injected subcutaneously into aged (>300 days old) and young (6 weeks old) C57/BL6 mice (Taconic). Mice were treated as follows with Lipofermata (2mg/kg, twice a day for the first two weeks and then 1 daily) or 30% Kolliphor as control. For inducible FATP2 deletion, Yumm 1.7 mCherry (5 × 105 cells) were infected with a TRIPZ Inducible Lentiviral FATP2 shRNA. This FATP2 shRNA lentiviral vector is designed with a reversible Tet-inducible vector that controls gene silencing. These cells were injected subcutaneously into aged (>300 days old) and young (6 weeks old) C57/BL6 mice (Taconic) and FATP2 expression was controlled by the administration of tetracycline (2mg/kg).

Tumor sizes were measured every 2 days using digital calipers, and tumor volumes were calculated using the following formula: volume = 0.5 × (length × width2). Time-to-event (survival) was determined by a 5-fold increase in baseline volume (~750 mm3) or was limited by the development of skin necrosis. Mice were euthanized, an tumor tissue was preserved. Half of the tissue was embedded in paraffin and other half in optimal cutting temperature compound (O.C.T, Sakura, Japan City) and flash frozen for sectioning. All reagents injected in live mice were tested for endotoxin levels at University of Pennsylvania Cell Center Services using The Associates of Cape Cod LAL test.

Seahorse Assay

Melanoma cells were incubated with young or aged conditioned media for 72 hours. After, cells were counted with trypan blue and plated at an optimal density (30,000) cells/well in XF96 cell culture plate (Agilent Technologies). Next day, cells were washed with seahorse buffer and seahorse media was added at ph7.4. Cartridge was loaded with appropriate treatments 1μM for oligomycin and 1.5 μM for FCCP and 0.5 μM for rotenone and antimycin A. Etomixir was also loaded at a final concentration of 40 μM when utilized. For the analysis of how BRAFi/MEKi, Lipofermata and/or triple combination affected the melanoma cells in the aged microenvironment, we cultured melanoma cells with aged CM for 48hours after cells were treated with either BRAFi/MEKi, Lipofermata and or the combination of all. After 48 hours, cells were trypsinized and counted with trypan blue. Viable cells were plated in the XF96well plate and the treated with 1) 1μM oligomycin 2) 1.5 μM FCCP and 3) 0.5 μM rotenone and antimycin A. For each assay, individual measurements were measured followed by injections of treatment. Cells were standardized by protein concentration at the end of the experiment.

Immunohistochemistry (IHC)

Skin reconstructs and mouse tumor samples were paraffin embedded and sectioned. Paraffin embedded sections were rehydrated through a series of xylene and different concentrations of alcohol, which was followed with a rinsed in water and washed in PBS. Slides were put with an antigen retrieval buffer (#3300, Vector Labs, Burlingame, CA) and steamed for 20 minutes. Slides were then blocked in a peroxide blocking buffer (#TA060H2O2Q, Thermo Scientific) for 15 minutes, followed by protein block (#TA-060-UB, Thermo Scientific) for 5 minutes and incubated with the primary antibody of interest which was prepared in antibody diluent (S0809, Dako). Slides were put in a humidified chamber at 4°C overnight. Next day, samples were washed with PBS and incubated in biotinylated anti-rabbit (Abcam) followed by streptavidin-HRP solution at room temperature for 20 minutes. Samples were then washed with PBS and incubated with AEC (3-amino-9-ethylcarboazole) chromogen for 10 minutes (#TA060SA, Thermo Scientific). Slides were then washed with water and incubated in Mayer’s hematoxylin (MHS1, Sigma) for 1 minute, rinsed with water, and mounted in Aquamount (#143905, Thermo Scientific).

Immunofluorescence and Quantification

Samples were fixed with 4% paraformaldehyde, for 15 minutes at room temperature. After samples were washed with PBS, they were incubated with BODIPY 493/503 or BODIPY 505/515 (1:3000, Thermofisher Scientific), LipidTOX™ Red Neutral Lipid Stain (1:125) or LipidTOX™ Green Phospholipidosis (1x) for 15 minutes at room temperature or 30 minutes for LipidTOX reagents. Samples were then washed with PBS and stain with Dapi (Invitrogen, 1:5000) for 5 minutes. After samples were washed with PBS they were mounted in Prolong Gold anti-fade reagent. For tumor, tissue was OCT embedded and sectioned. Frozen slides were fix in 4% formalin for 15 minutes. Slides were then rinse with 60% isopropanol, followed by Oil Red O in 0.5% isopropanol (#01319, Sigma Aldrich) staining for 15 minutes and rinse with 60% isopropanol. Next samples were stained with hematoxylin, rinse with water and mount in Aquamount. Lipid droplet (bodipy stain) intensity was quantified with Adobe Photoshop software. Channels were separated and melanoma cells intensity was quantified acrossed different culture conditions. Values were then compared between conditions using unpaired t-test.

Cell death assay

Melanoma cells were cultured with control media, young or aged conditioned media in the presence or absence of ND646. After 24 hours, cell viability was quantified with trypan blue.

Triglyceride Kit

Melanoma cells (1.5×10^5) were plated in 6 well plate. Next day, cells were treated with young cm, aged cm or DMEM. After 48 hours, cells were trypsinized, wash with PBS and resuspended in NP40. Cells were then plated in 96 well plate, triglycerides were measured using Triglycerides Quantification Assay Kit (#ab65336, Abcam) according to the manufacturer’s protocol. For fibroblasts, cells were plated in 6 well plates and conditioned media was collected after 48 hours and added in a 96 well plate for analysis. The plates were measured using the ELX 808 Ultra microplate reader at 570 nm.

Lipid Transfer experiment

Aged and young fibroblasts cells were seeded onto 4-well EZ MilliCell slides (EMD Millipore PEGZ0416). Lipids in aged or young fibroblasts were labeled with 5μM BODIPY 558/568 C-12 for 4 hours. After 4 hours, extracellular BODIPY was removed from the cells by washing three times with 1x Hank’s balanced salt solution (HBSS) with 0.2% fatty-acid free BSA. After washing, GFP+ human melanoma cells were seeded on top of labeled aged or young fibroblasts for 24 hours, and imaged for the entire time to generate a video. For the lipid transfer with CM, we again labeled fibroblasts with 5μM BODIPY 558/568 C-12 for 4 hours. Next, we took the CM of young and aged fibroblasts and cultured the GFP+ human melanoma cells with this media for an additional 4 hours. Cells were then fixed and imaged using confocal microscopy, where BODIPY-laden GFP+ melanoma cells could be observed. Images were captured on a Leica TCS SP5 II scanning laser confocal system.

Quantitative RT-PCR

Melanoma cells were treated with young CM or aged CM and RNA was extracted using Trizol (Invitrogen) and RNeasy Mini kit (Qiagen) as protocol instructions. 1μg RNA was used to prepare cDNA using iscript DNA synthesis kit (#1708891, Bio-Rad, CA). cDNA was diluted 1:5 before use for further reactions. Each 20μl well reaction comprised of 10μl Power SYBR Green Master mix (4367659, Invitrogen), 1μl cDNA and 1μl primer

FATP1

F’-TGACAGTCGTCCTCCGCAAGAA

R′-CTTCAGCAGGTAGCGGCAGATC

Final concentration used was 0.5μM. Standard curves were generated for all primers and each set of primers were normalized to an 18S Primer pair, acquired from Invitrogen, (AM1718).

TCGA database analysis

The RNAseq and Clinical dataset for skin cutaneous melanoma was downloaded from The Cancer Genome Atlas (TCGA; http://cancergenome.nih.gov/). Normalized mRNA expression was analyzed by quartiles. Patient ages were grouped into categories (≤50, and ≥50 years).

Acylceramides

Acylceramides were quantified indirectly by comparing ceramide levels in lipid extracts before and after mild saponification. Briefly, lipid extracts were saponified using 0.2 N KOH in methanol for 60 min at 37 °C. KOH was neutralized with HCl. Lipids were extracted with 2:1:1 chlorofom:methanol:0.88% NaCl and analyzed as described for global lipid profiling. Identified ceramides were quantitatively compared to the same ceramides identified in global lipid profiling, and putative acylceramides were defined as having a saponified:global lipid ratio greater than 1. Ratios were compared between conditions using the Student’s t-test.

Supplementary Material

1
Movie S1
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Movie S2
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Statement of Significance.

These data show that melanoma cells take up lipids from aged fibroblasts, via FATP2, and use them to resist targeted therapy. The response to targeted therapy is altered in aged individuals due to the influences of the aged microenvironment, and these data suggest FATP2 as a target to overcome resistance.

Acknowledgements

We thank the outstanding Core Facilities of the Wistar Institute, supported by P30CA010815 and Johns Hopkins Kimmel Cancer Center, P30CA00697356; A.T. Weeraratna and Q Liu are supported by R01CA174746 and R01CA207935, R01CA223256. X Xu, Q Liu, M Herlyn, and A.T. Weeraratna are also supported by P01 CA114046. I.A. Blair is supported by P30ES013508. M.R. Webster is supported by K99CA208012. AT Weeraratna is also supported by U01CA227550, the Wistar Science Discovery Fund, the Melanoma Research Foundation, a Melanoma Research Alliance/L’Oréal Paris-USA Women in Science Team Science Award, a Bloomberg Distinguished Professorship and the EV McCollum Endowment. M. Herlyn is supported by a gift from the Adelson Medical Research Foundation. DW Speicher is supported by P50CA174523. Lipidomic analysis was performed on a Q-Exactive HF hybrid quadrupole-orbitrap mass spectrometer and Vanquish Horizon ultra-high performance liquid chromatography (UHPLC) system (Thermo Scientific, San Jose, CA) purchased with NIH grant S10 OD023586. HY Tang is supported by R50CA221838. We thank Dr. Chi Van Dang, Dr. Dario Altieri and Dr. Brian Keith (Wistar Institute) and Dr. Celeste Simon (University of Pennsylvania) for critical reading of the manuscript and Dr. Fred Keeney of the Wistar Institute Imaging Core for help with image analysis.

Footnotes

Disclosures: The following conflicts are reported: Ashani Weeraratna, Phoremost Technologies, Advisory Board (unpaid); Keith Flaherty (* indicates equity holdings) Board of Directors: Clovis Oncology*, Strata Oncology*, Vivid Biosciences*, Checkmate Pharmaceuticals*, Corporate Advisory Board: X4 Pharmaceuticals*, Scientific Advisory Board: Sanofi, Amgen, Asana, Adaptimmune, Fount*, Aeglea, Shattuck Labs*, Tolero, Apricity*, Oncoceutics*, Fog Pharma*, Neon, Tvardi*, xCures*, Monopteros*, PIC Therapeutics*, Vibliome*, Consultant: Novartis, Genentech, BMS, Merck, Takeda, Verastem, Boston Biomedical, Pierre Fabre, Debiopharm.

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Supplementary Materials

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