Exosome antagonized lipid peroxidation and desensitized ferroptosis in LUAD
Excessive lipid peroxidation is a hallmark of ferroptosis, and HNF4a and HIC1 play opposite roles in ferroptosis . MDA, a byproduct of lipid peroxidation, was anti-correlated with anti-ferroptotic HNF4a, while was correlated with pro-ferroptotic HIC1 in LUAD (Supplementary Fig. S1A-B), confirming that MDA reflects ferroptosis-associated lipid peroxidation.
We were curious to know whether lipid peroxidation is suppressed in LUAD, and indeed, MDA levels were lower in LUAD tissues in comparison to the matched adjacent tissues (Fig. 1A). In addition, 39.9% LUADs (75/188) also showed downregulation of 4-HNE, another byproduct of lipid peroxidation in LUAD (Fig. 1B). As known, LUADs are commonly driven by mutations [2, 3]; however, after screening common mutations within EGFR (Del19, L858R and T790M), kRAS (G12C, G12S, G12A and G13C) and p53 (R196P, H179Y, P250L, R249S and R248G), we found that MDA at least might not be determined by these driver mutations (Fig. 1C). Collectively, lipid peroxidation is suppressed possibly via a driver mutation-independent manner in a large proportion of LUAD.
Then, we tested whether exosome suppresses lipid peroxidation and desensitizes LUAD cells to ferroptosis. Ex vivo primary LUADs from patients for curative therapy were treated with or without GW4869 and DMA, two exosome biogenesis inhibitors, and they both elevated 4-HNE (Fig. 1D), hinting that prevention of exosome generation stimulates lipid peroxidation. Does exosome from LUAD plasma desensitize ferroptosis? To this end, plasma exosome from healthy individuals and LUAD patients were extracted and verified by TEM (Fig. 1E). The exosome diameters were mainly between 30 and 150 nm (Supplementary Fig. S1C), which were similar to that reported in prior studies [23, 24]. Except endoplasmic reticulum (ER) biomarker Calnexin, exosome biomarkers including CD63, TSG101, ALIX and CD9 were all clearly detected in our samples (Supplementary Fig. S1D), further demonstrating the quality of the exosome. A549 and H1975 LUAD cell lines were picked up for further study because they exhibited the least and the most sensitivity to erastin and RSL3, two ferroptosis agonists, among the LUAD cell lines tested in 3D spheroid culture (Supplementary Fig. S1E-F). By pre-co-incubating H1975 cells with exosome, we found that plasma exosome from a large proportion of LUAD patients could significantly prevent erastin- and RSL3-reduced cell viability and -induced cell death and lipid ROS generation. By comparison, plasma exosome from healthy individuals failed to do so (Fig. 1F). The results from 3D spheroid culture, which is a better way to reflect cell-cell interaction than monolayer culture, also supported that only the exosome from LUAD plasma had the capacity to prevent erastin- and RSL3-induced cell death in H1975 spheroids (Fig. 1G). Reasoning that the sensitivity to ferroptosis was varied between A549 and H1975 cells, we wondered whether the exosome from A549 cells was required for H1975 cells to obtain a less sensitivity to ferroptosis. As expected, pre-co-incubation with exosome that extracted from A549-cultured DMEM or directly the DMEM itself desensitized H1975 cells to the induction of erastin and RSL3 (Fig. 1H). However, fresh DMEM, DMEM from cultured H1975 cells and A549 cells following GW4869 treatment did not have such effects (Fig. 1H). Together, a role of exosome to antagonize lipid peroxidation and desensitize LUAD cells to ferroptosis was established.
A link between exosomal- and intracellular-cir93 in LUAD
Then, important contents in the exosome were investigated. Emerging studies have demonstrated the critical roles of exosomal-circRNAs in tumorigenesis [25, 26]; we thereby tried to identify such circRNAs in LUAD exosome. Firstly, the available circRNA-chip data (Zhu, et al., Ref. 27 and Yang, et al., GSE101586) evaluating differential circRNA expression between LUAD and normal lung were mined. Two circRNAs, i.e. cir93 and cir34 were identified as the common upregulated intracellular-circRNAs in LUAD between the two datasets (Fig. 2A). Further verifications indicated that only cir93, but not cir34 was elevated in our LUAD cohort (n=250, Fig. 2B-C and Supplementary Fig. S2A-B), and this led us to focus on cir93 hereafter. Dislike NUP107 mRNA, the transcript of cir93 parental gene, cir93 could only be PCR amplified from cDNA but not gDNA template of A549 and H1299 cells using circRNA-specific primer sets (Supplementary Fig. S2C), suggesting that cir93 is a genuine circRNA. Of note, an elevation of plasma exosomal-cir93 was also observed in LUAD (n=200) as compared to healthy individuals (n=200, Fig. 2D). Reasoning that circRNA can be transmitted via exosome between host and target cells , we suggested that elevation of intracellular-cir93 is closely linked with exosomal-cir93 in LUAD.
Exosome secreted by tumor cells themselves was essential for elevating intracellular-cir93 in LUAD
Because tumor and tumor stroma are both important for tumorigenesis [29, 30], we thereby wanted to ascertain whether they are involved separately or in combined for the link between exosomal- and intracellular-cir93. After measuring cir93 in LUAD tissue specimens, we found 68.0% of them (34/50) exclusively expressing cir93 in the tumor part. By contrast, only 4.0% (2/50) exhibited an exclusive cir93 expression in the stroma part (Fig. 2E). Those specimens with cir93 exclusively expressed in tumor part were further evaluated by IHC using antibodies against αSMA; a biomarker of fibroblast and to some extent capable of distinguishing stroma from tumor parts. Indeed, all of them (34/34) showed mutual exclusion of cir93 and αSMA within the same areas (Fig. 2F). EpCAM is a surface pan-carcinoma antigen. By sorting patient-derived EpCAM (+) and EpCAM (-) cells in primary tumors from 3 distinct LUAD patients, we found that intracellular-cir93 was much higher in EpCAM (+) cells than that in EpCAM (-) cells (Fig. 2G). Taken together, tumor part is the more likely site to establish the link between exosomal- and intracellular-cir93 in LUAD.
Subsequently, we tried to investigate whether exosome secreted by tumor cells themselves elevates intracellular-cir93 in the tumor part of LUAD. To this end, primary EpCAM (+) and EpCAM (-) cells from case #1 and #2 in Fig. 2G were used for further analysis. Before co-incubating cultured-DMEM from the first cells with the secondary cells, the exosome concentrations in cultured DMEM were ensured to be similar between EpCAM (+) and EpCAM (-) cells (Fig. 2H, lanes, 1-4); however, intracellular-cir93 in case #2 EpCAM (+) cells could only be sustained after co-incubating with the cultured-DMEM from case #1 EpCAM (+) cells, and vice versa (Fig. 2H, lanes, 1-2). By contrast, in both case #1 and #2, co-incubation of cultured-DMEM from EpCAM (-) cells failed to sustain intracellular-cir93 in EpCAM (+) cells (Fig. 2H, lanes, 3-4). The critical roles of exosome in EpCAM (+) cells were further verified because reduction of exosome in the cultured-DMEM of the first EpCAM (+) cells following GW4869 treatment failed to sustain intracellular-cir93 in the secondary EpCAM (+) cells (Fig. 2H, lanes, 5-6). In addition to the DMEM, the results from extracted exosome also demonstrated that only that exosome from EpCAM (+) but not EpCAM (-) cells had the capacity to elevate intracellular-cir93 in EpCAM (+) cells (Fig. 2I-J). The valid exosome-mediated cir93 transmission between A549 and H1299 cells was further confirmed by incubating one cell line with extracted PKH67-marked exosome from another cell line expressing mCherry-labeled cir93 followed by tracing the exosome (green) and cir93 (red) under microscope (Fig. 2K). Together, exosome secreted by tumor cells themselves is essential for elevating intracellular-cir93 in the tumor part of LUAD.
Elevated intracellular-cir93 desensitized LUAD cells to ferroptosis via regulating AA
Despite elevation of intracellular-cir93 is sustained by exosome in LUAD (Fig. 2), whether exosome antagonizes ferroptosis via elevating intracellular-cir93 remains unclear. Ferroptosis is characterized by the presence of smaller than normal mitochondria with condensed densities of mitochondrial membrane . Expectedly, treating H1975 cells with erastin and RSL3 resulted in such effects; however, they were prevented by overexpressing cir93 (Fig. 3A). By pre-infecting H1975 cells with dual-cir93 and GFP-expressing lenti-virus and staining the cells with PI, a small fluorescent molecule that binds to DNA but cannot penetrate into live cells, elevating cir93 was also revealed to prevent erastin- and RSL3-induced cell death (Fig. 3B). In addition, like ferroptosis inhibitors Fer-1 and DFO, overexpressing cir93 was capable of holding back erastin- and RSL3-induced reduction of cell viability and induction of lipid ROS generation in H1975 cells (Fig. 3C and Supplementary Fig. 3A). Interestingly, cir93 itself could not be affected by erastin and RSL3 (Supplementary Fig. S3B), suggesting that cir93 is an independent factor to influence ferroptosis. If cir93 truly desensitizes LUAD cells to ferroptosis, anti-cir93 could exert opposite function. Expectedly, administrating anti-cir93 downregulated intracellular-cir93 in A549 cells (Supplementary Fig. S3C), and aggravated erastin- and RSL3-induced reduction of cell viability and induction of cell death and lipid ROS generation (Supplementary Fig. S3D-F). Thus, the above data demonstrated a role of elevating cir93 to mitigate ferroptosis in vitro.
To investigate whether elevating intracellular-cir93 also desensitizes LUAD cells to ferroptosis in vivo, H1975 cell-implanted intrapulmonary LUAD-bearing mice were intranasal infected with adeno-associated virus 5 (AAV5) dual-expressing cir93 and GFP before further administrating with PKE, an in vivo stable erastin derivative. Compared to the control, cir93 was indeed overexpressed, and PKE-induced elevation of 4-HNE and MDA were largely prevented in intrapulmonary LUAD (Fig. 3D), suggesting that elevating cir93 suppresses ferroptosis-associated lipid peroxidation in vivo. To further investigate whether anti-cir93 oppositely stimulates lipid peroxidation within plasma membrane fraction in mouse LUAD, a fluorescent probe C11-BODIPY581/591 was used to directly measure oxidized lipid and conA-AlexaF was employed to visualize membrane. Images were captured at emission at 580/600 nm (the non-oxidized form, red) and 490/510 nm (the oxidized form, green) and then merged to demonstrate the fraction of the oxidized C11-BODIPY581/591. As shown in Fig. 3E, the fraction of oxidized C11-BODIPY581/591 (green) within the plasma membrane was remarkably increased following administrating with anti-cir93. Combined with the data showing that overexpressing cir93 desensitizes cells to ferroptosis that induced by both erastin and RSL3 (Fig. 3A-C), and reasoning that treating two small molecules both lead to lipid peroxidation , we supposed that elevating intracellular-cir93 might desensitize LUAD cells to ferroptosis via suppressing lipid peroxidation.
Next, we investigated how elevating intracellular-cir93 suppresses lipid peroxidation in LUAD cells. Excessive peroxidation of PUFA, such as AA and AdA are prerequisite for the trigger of ferroptosis . Plasma membrane incorporation of AA is also essential for such a process . By a click chemistry-based method using alkyne-labeled AA or AdA and Fluro-488-labeled Azide, we found that elevating cir93 in H1975 cells was capable of reducing AA but not AdA incorporation into the plasma membrane (Fig. 3F-G and Supplementary Fig. S3G-H). Moreover, global-AA could be simultaneously reduced by overexpressing cir93 in H1975 cells (Fig. 5E and 5G). These results suggested that suppression of lipid peroxidation by elevating cir93 might be conducted by modulation of AA.
Finally, we examined whether exosome suppresses lipid peroxidation in a cir93-dependent way. Reasoning that MRC-5 and WI-38, two established lung fibroblasts, demonstrated remarkable lower levels of intracellular-cir93 and exosomal-cir93 in cultured DMEM than LUAD cell lines (Supplementary Fig. S3I), we compared the effects of extracted exosome from MRC-5, WI-38 and LUAD A549 cells on lipid ROS generation following erastin treatment in H1975 cells. We noticed that only exosome from A549 cells significantly reduced lipid ROS generation that induced by erastin (Fig. 3H), hinting that the role of exosome to suppress lipid peroxidation in LUAD cells is via a cir93-dependent way.
FABP3 worked as a downstream of cir93, interacting and being upregulated by cir93
Then, the downstream effectors of cir93 were further investigated. CircRNAs interact with proteins [34, 35]; we hence performed proteomics to seek potential proteins that cir93 might influence. Via pull-down experiments with cir93, anti-sense cir93, or without RNA (blank) followed by proteomics, 143 proteins were identified specifically interacted with cir93 (Fig. 4A). To narrow candidates, we wanted to know which proteins are also cir93-regulated. Because intracellular-cir93 was higher in A549 cells than H1975 cells (Supplementary Fig. S3I), we inhibited cir93 in A549 cells while overexpressed cir93 in H1975 cells before subjecting samples into proteomics again. Only FABP3 was identified upregulated by cir93. Unfortunately, no proteins were identified downregulated by cir93. Interestingly, FABP3 was also one of those 143 proteins interacting with cir93 (Fig. 4A). The upregulation of FABP3 by cir93 was further verified by IB (Fig. 4B). Like cir93, FABP3 could not be regulated by erastin and RSL3 (Fig. 4C and Supplementary Fig. S3B), indicating that FABP3 is also an independent factor to influence ferroptosis. Moreover, elevating cir93-desensitized LUAD cells to erastin- and RSL3-induced ferroptosis and ferroptosis-associated lipid ROS generation were all indispensable of FABP3, because compared to WT H1975 cells, cir93 was ineffective in FABP3-/- cells (Fig. 4C and Supplementary Fig. S4A-C). Together, FABP3 interacts and being upregulated by cir93, through which facilitates FABP3 working as a downstream of cir93 to desensitize LUAD cells to ferroptosis.
Molecular basis behind cir93-FABP3 interaction and its essential role in modulating FABP3, AA, lipid peroxidation and sensitivity of LUAD cells to ferroptosis
Subsequently, we investigated the molecular basis for cir93 to interact and upregulate FABP3. To further verify that FABP3 interacts with cir93, PAR-CLIP was performed, and we found that FABP3 did not interact with cir34, but rather, interacted with cir93 (Supplementary Fig. S4D). Molecular basis between FABP3 and cir93 was then predicated by catRAPID online software. Three regions, named as P#1 (26th~77th a.a), P#2 (44th~95th a.a) and P#3 (51st~102rd a.a) located within FABP3, and two regions, named as R#1 (57th~108th nt) and R#2 (151st ~202rd nt) located within cir93 were predicted with interaction potentials (Fig. 4D and Supplementary Fig. S4E). Cir93 pull-down experiments demonstrated that deletion of the R#2 region of cir93 exclusively abolished cir93-FABP3 interactions in H1975 cells (Fig. 4E). Reconstitution of FABP3-/- H1975 cells by Myc-tagged WT or FABP3 without P#3 region followed by RIP experiments using anti-Myc antibodies indicated that the P#3 region of FABP3 contains critical domain responsible for the cir93-FABP3 interaction (Fig. 4F). The P#1 and P#2 regions of FABP3 were excluded because FABP3 still interacted with cir93 even when they were deleted (Supplementary Fig. S4F). Because P#1, P#2 and P#3 have overlapped regions and only the region compassing 96th~102rd a.a in P#3 is unique as compared to P#1 and P#2 (Fig. 4D), we speculated participation of this region (hereafter named as P#4) in the cir93-FABP3 interaction, and expectedly, deletion of P#4 disrupted cir93-FABP3 interaction in H1975 cells (Fig. 4F). Furthermore, besides P#3, P#4 was also prerequisite for cir93 to upregulate FABP3 (Supplementary Fig. S4G). Together, the molecular basis for cir93-FABP3 interaction and its essential roles to upregulate FABP3 in LUAD cells was elucidated.
Does cir93-FABP3 interaction also influence AA, lipid peroxidation and sensitivity of LUAD cells to ferroptosis? Like elevating cir93, overexpressing FABP3 reduced AA but not AdA incorporation into the plasma membrane of H1975 cells; however, the effects no longer exist once upon the P#4 site was deleted. Deletion of the R#2 region also prevented the roles of cir93 to suppress AA membrane incorporation (Fig. 3F, 4G and Supplementary Fig. S4H-I). Data from Fig. 4H further demonstrated that the cir93-FABP3 interaction was required for the reduction of global-AA (Fig. 4H). To get clinical support, intracellular-cir93 and the open reading frame (ORF) of FABP3 in 30 randomly chosen LUAD tissue specimens were sequenced. Only one specimen (3.3%, 1/30) each contained a mutation in R#2 of cir93 and P#4 of FABP3, respectively (Fig. 4I). The disruption of cir93-FABP3 interaction resulted by the two mutations were subsequently verified (Supplementary Fig. S4J-K). Of note, mutations in cir93 and FABP3 increased 4-HNE concentrations in LUAD (Fig. 4J), further confirming the importance of cir93-FABP3 interaction in reducing lipid peroxidation. Moreover, disruption of cir93-FABP3 interaction abolished the effects of cir93 and FABP3 to desensitize H1975 cells to erastin- and RSL3-induced cell death (Fig. 4K). Collectively, cir93-FABP3 interaction and its role to upregulate FABP3 are essential for modulating AA and reducing lipid peroxidation and sensitivity of LUAD cells to ferroptosis.
Exosome functioned to upregulate FABP3 via the cir93-FABP3 interaction in LUAD
Because the cir93-FABP3 interaction is required for cir93 to upregulate FABP3 (Fig. 4B and Supplementary Fig. S4G), and exosome is essential for elevating intracellular-cir93 in LUAD (Fig. 2); we wondered whether plasma exosome from LUAD lifts intracellular FABP3 expression via the cir93-FABP3 interaction. To address this, plasma exosome from LUAD (n=200) was co-incubated with engineered H1975 cells expressing WT or FABP3 without P#4 region. More than two-fold upregulation of FABP3 in FABP3WT-expressing H1975 cells was observed following co-incubation of 68.5% (137/200) exosome, while only 3.5% (7/200) exosome exhibited similar functions in those cells expressing FABP3△P#4 (Fig. 4L-M), suggesting the cir93-FABP3 interaction is also the basis for exosome to upregulate FABP3 in LUAD.
Taurine was essential for cir93-mediated upregulation of FABP3 to modulate AA and desensitize LUAD cells to ferroptosis
Next, we investigated the outcome following upregulation of FABP3 in LUAD. Because FABP3 acts as a transporter responsible for transporting AA to the location where it can react with other metabolites [36, 37], the metabolites both regulated by FABP3 and associated with AA could determine sensitivity of LUAD cells to ferroptosis. To figure out such metabolites, metabolomics were performed to compare metabolites in LUAD tissues expressing high and low levels of FABP3. Among 362 metabolites, 63 were upregulated while 26 were downregulated in FABP3 high-expressing LUADs (n=30) in comparison to the low-expressing ones (n=30, Fig. 5A and Supplementary Fig. S5A). To the best of our knowledge, among all the identified metabolites, taurine was the only one that can also react with AA to generate NAT (Fig. 5A-B and Ref. 38). Because taurine and AA are substrates while NAT are products of this reaction (illustrated in Fig. 5B), a reduction of taurine in FABP3 high-expressing LUAD (Fig. 5A) might be explained by FABP3-mediated acceleration of the reaction. Unfortunately, a decrease of global-AA and an increase of NAT were not detected by metabolomics, which may due to the methodology limitation . However, after evaluated by ELISA and targeted MS, we found that global-AA and NAT were indeed reduced and induced following overexpressing FABP3 in H1975 cells (Supplementary Fig. S5B-C). FABP3 transports AA via its phenylalanine (F)16 residue , we hence replaced this F by a serine (S) to test whether reduction of taurine is mediated by the transport function of FABP3, and expectedly, overexpressing FABP3F16S failed to reduce taurine as compared to FABP3WT in H1975 cells (Fig. 5C). These results suggested that upregulation of FABP3 stimulates transport of AA to react with taurine and thus reduces global-AA in LUAD cells.
Reasoning that FABP3 works as a downstream of cir93 (Fig. 4C and Supplementary Fig. S4B-C), we wondered whether cir93 is also linked with FABP3-mediated manipulation of taurine and global-AA. Overexpressing cir93 in H1975 cells led to simultaneous reductions of taurine and global-AA, and they were entirely abolished upon knocking out FABP3 (Fig. 5D-E), demonstrating that cir93 regulates taurine and global-AA via FABP3. By contrast, inhibiting cir93 via anti-cir93 in A549 cells induced taurine and global-AA at the same time (Fig. 5F-G). Thus, manipulating taurine and global-AA is the downstream effect of cir93 in LUAD cells. In addition, co-incubation H1975 cells with plasma exosome from LUAD also resulted in lower levels of taurine and global-AA as compared to the exosome from healthy individuals (Fig. 5H-I). Due to the importance of AA in ferroptosis, exosome-desensitized LUAD cells to ferroptosis might partially via reduction of global-AA in a cir93-FABP3-taurine-dependent way.
To further verify the essential roles of taurine to reduce global-AA and desensitize LUAD cells to ferroptosis, we tried to eliminate endogenous taurine in LUAD cells. CDO1 and CSAD are critical for the synthesis of taurine (illustrated in Fig. 5J). By simultaneously knocking CDO1 and CSAD down in H1975 cells, taurine was significantly reduced and could not be further declined anymore by overexpressing cir93 or FABP3 (Fig. 5K), suggesting that sufficient taurine are prerequisites for cir93 and FABP3 to regulate taurine itself. Of note, taurine was indeed essential for cir93 and FABP3 to reduce global-AA (Fig. 5L). We then investigated whether taurine is indispensable for cir93 and FABP3 to desensitize LUAD cells to ferroptosis and reduce lipid ROS generation. Once upon taurine was depleted, erastin- and RSL3-induced ferroptosis and lipid ROS generation were significantly aggravated at basal levels; however, the restore effects by cir93 and FABP3 were all blocked (Fig. 5M and Supplementary Fig. S5D), suggesting that taurine is basically anti-ferroptotic and essential for cir93 and FABP3 to desensitize LUAD cells to ferroptosis. Via evaluating the fraction of the oxidized C11-BODIPY581/591 and MDA concentration in the implanted LUAD in mice, the essential role of taurine for cir93 and FABP3 to reduce lipid peroxidation following administrating PKE in vivo was further verified (Fig. 5N-O and Supplementary Fig. S5E).
NAT prevented AA incorporation into the plasma membrane in LUAD cells
As described above, we used a click chemistry-based method to evaluate AA incorporation into the plasma membrane (Fig. 3F and 4G); however, click chemistry was performed when excessive exogenous alkyne-labeled AA was added. In such conditions, overexpressing cir93 and FABP3 still prevented AA incorporation into the plasma membrane although endogenous global-AA could also be reduced by cir93 and FABP3 (Fig. 5E, 5G, 5L, 6A-B and Supplementary Fig. S6A), hinting that other mechanism might be simultaneously involved. Reasoning that AA consume is accompanied with NAT generation (illustrated in Fig. 5B); we speculated that the nascent NAT might be essential for the function of cir93 and FABP3. As expected, the relationships among cir93, FABP3 and NAT were established in H1975 cells (Fig. 6C-D). Also, taurine was essential for cir93 and FABP3 to elevate NAT in H1975 cells and implanted tumors (Fig. 6E and Supplementary Fig. S6B). Interestingly, compared to plasma exosome from healthy individuals, co-incubation of the one from LUAD led to a higher level of intracellular-NAT in H1975 cells (Fig. 6F), further indicating that elevating NAT is a critical event for exosome to desensitize LUAD cells to ferroptosis. To provide direct evidences supporting the role of NAT to prevent AA incorporation into the plasma membrane, NAT was directly incubated with H1975 cells, and such function of NAT was confirmed (Fig. 6G-H). Then, we investigated whether NAT desensitizes LUAD cells to ferroptosis, and found that erastin- and RSL3-induced induction of cell death and lipid peroxidation as well as reduction of cell viability could be mitigated in the presence of NAT (Fig. 6I-J and Supplementary Fig. S6C-E). Thus, cir93- and FABP3-desensitized LUAD cells to ferroptosis might also via stimulated generation of NAT to prevent AA incorporation into the plasma membrane.
Next, we investigated the related mechanisms. As known, ACSL4, LPCAT3 and PLTP are enzymes involved in incorporation of AA into the plasma membrane (illustrated in Fig. 6K, and Ref. 40, 41, we therefore tested whether NAT affects these enzymes, and noticed that they were all reduced by NAT dose-dependently in H1975 cells (Fig. 6L). By comparing fold changes of ACSL4, LPCAT3 and PTLP with or without co-incubation of plasma exosome, we found that 78% (78/100), 67% (67/100) and 70% (70/100) of the one from LUAD patients could reduce ACSL4, LPCAT3 and PTLP to a remaining level of less than 50%; however, similar effects were found for only 12% (12/100), 12% (12/100) and 15% (15/100) of the one from healthy individuals (Fig. 6M-N). Together, NAT-mediated downregulation of enzymes that associated with AA incorporation into the plasma membrane is equally important for exosome, cir93 and FABP3 to desensitize LUAD cells to ferroptosis.
The correlations among exosomal- and intracellular-cir93, FABP3, taurine and NAT in LUAD
To further verify the conclusions draw from above cell- and mice-based experiments (Fig. 1-6), we evaluated correlations among exosomal- and intracellular-cir93, FABP3, taurine and NAT in human LUAD specimens. Similar to intracellular-cir93 (Fig. 2B-C), FABP3 was also elevated in our LUAD cohort (n=250, Supplementary Fig. S7A). A significant correlation between FABP3 and intracellular-cir93 was revealed in LUAD (n=250, Fig. 7A). Moreover, taurine was negatively correlated with intracellular-cir93 and FABP3 (Fig. 7B-C). By contrast, positive correlations among NAT, intracellular-cir93 and FABP3 were identified in LUAD (Fig. 7D-E). To further evaluate the close relationship between exosomal- and intracellular-cir93, matched plasma and tissue specimens were obtained from same LUAD patients (n=15), and we found that exosomal-cir93 correlated well with intracellular-cir93 and FABP3 (Supplementary Fig. S7B-C). Therefore, the close relationships among exosomal- and intracellular-cir93, FABP3, taurine and NAT were established in LUAD.
Ferroptosis resistance and poor survival outcome predicted by high levels of cir93 and FABP3 in LUAD
PDX mouse models are promising tools to evaluate drug efficacy in vivo. We compared efficacy of PKE in two PDX mice models expressing distinct levels of cir93 and FABP3 (Fig. 7F), and noticed that higher levels of cir93 and FABP3 in PDX#2 resulted in less suppressions of tumor growth and lower 4-HNE levels following PKE administration as compared to PDX#1, which had relative lower levels of cir93 and FABP3 (Fig. 7F-G and Supplementary Fig. S7D), further demonstrating that cir93 and FABP3 play roles to stimulate tumor growth and desensitize cells to ferroptosis in LUAD. Via evaluating tissue slices of PDX ex vivo, we also found that PDX#2 was more resistant to erastin-induced ferroptosis in comparison to PDX#1 (Fig. 7H). Overall survival (OS) was subsequently evaluated for PDX mouse models, and PDX#2 demonstrated a shorter OS than PDX#1 (Fig. 7I). Shorter OS was also observed in those LUAD patients with higher levels of cir93 and FABP3 (n=41) compared to those with lower levels (n=41, Fig. 7J). These data demonstrated that ferroptosis resistance and poor survival in LUAD can be predicted by high levels of cir93 and FABP3.
Blocking exosome improved ferroptosis-based treatment
Reasoning that exosome from LUAD desensitizes cells to ferroptosis (Fig. 1-6); we wondered whether blocking biosynthesis of exosome could improve ferroptosis-based treatment. To this end, we further co-administrated PKE-treated A549-based CDX mice with GW4869. We found that co-treating mice with GW4869 resulted in more significant impairments of tumor growth and longer OS as compared to those treated with PKE alone (Fig. 7K-M). The aggravated elevation of MDA further confirmed that the effects were through a lipid peroxidation-dependent manner (Fig. 7N). Together, co-treating with agents that can block biosynthesis and function of exosome might be helpful to improve ferroptosis-based therapy against LUAD.