Abstract 抽象的
Lysosomal inhibition elicited by palmitoyl-protein thioesterase 1 (PPT1) inhibitors such as DC661 can produce cell death, but the mechanism for this is not completely understood. Programmed cell death pathways (autophagy, apoptosis, necroptosis, ferroptosis, and pyroptosis) were not required to achieve the cytotoxic effect of DC661. Inhibition of cathepsins, or iron or calcium chelation, did not rescue DC661-induced cytotoxicity. PPT1 inhibition induced lysosomal lipid peroxidation (LLP), which led to lysosomal membrane permeabilization and cell death that could be reversed by the antioxidant N-acetylcysteine (NAC) but not by other lipid peroxidation antioxidants. The lysosomal cysteine transporter MFSD12 was required for intralysosomal transport of NAC and rescue of LLP. PPT1 inhibition produced cell-intrinsic immunogenicity with surface expression of calreticulin that could only be reversed with NAC. DC661-treated cells primed naive T cells and enhanced T cell–mediated toxicity. Mice vaccinated with DC661-treated cells engendered adaptive immunity and tumor rejection in “immune hot” tumors but not in “immune cold” tumors. These findings demonstrate that LLP drives lysosomal cell death, a unique immunogenic form of cell death, pointing the way to rational combinations of immunotherapy and lysosomal inhibition that can be tested in clinical trials.
DC661 等棕榈酰蛋白硫酯酶 1 (PPT1) 抑制剂引起的溶酶体抑制可导致细胞死亡，但其机制尚不完全清楚。 DC661 的细胞毒作用不需要程序性细胞死亡途径（自噬、细胞凋亡、坏死性凋亡、铁死亡和焦亡）。组织蛋白酶的抑制，或者铁或钙螯合，并不能挽救 DC661 诱导的细胞毒性。 PPT1 抑制诱导溶酶体脂质过氧化 (LLP)，从而导致溶酶体膜透化和细胞死亡，这种作用可以被抗氧化剂 N-乙酰半胱氨酸 (NAC) 逆转，但不能被其他脂质过氧化抗氧化剂逆转。溶酶体内运输 NAC 和拯救 LLP 需要溶酶体半胱氨酸转运蛋白 MFSD12。 PPT1 抑制产生细胞内在免疫原性，表面表达钙网蛋白，而这种免疫原性只能用 NAC 逆转。 DC661 处理的细胞引发初始 T 细胞并增强 T 细胞介导的毒性。接种 DC661 处理细胞的小鼠在“免疫热”肿瘤中产生了适应性免疫和肿瘤排斥，但在“免疫冷”肿瘤中则没有。这些发现表明，LLP 驱动溶酶体细胞死亡，这是一种独特的免疫原性细胞死亡形式，为免疫治疗和溶酶体抑制的合理组合指明了道路，并可以在临床试验中进行测试。
Keywords: Oncology
关键词：肿瘤学
Keywords: Cancer, Cellular immune response, Lysosomes
关键词：癌症，细胞免疫反应，溶酶体
Introduction 介绍
With encouraging activity in clinical trials that involve the lysosomal inhibitor hydroxychloroquine (HCQ) (1), the identification of palmitoyl-protein thioesterase 1 (PPT1) as the molecular target of chloroquine derivatives (2), and the launch of novel PPT1 inhibitors into clinical trials (3, 4), there is a need to understand the mechanism by which lysosomal inhibitors induce cell death and the effect of lysosomal inhibition on tumor immunity. The canonical mechanism of lysosomal cell death described for HCQ involves lysosomal membrane permeabilization (LMP), leakage of cathepsins, and activation of caspase-mediated apoptosis (5, 6). We have previously shown that the lysosomal inhibitor DC661 penetrates cells in the acidic tumor microenvironment and localizes to the lysosome more efficiently than HCQ (2, 7). DC661 produces potent cell death across many cancer cell lines (2). DC661 binds and inhibits PPT1, deacidifies the lysosome, and inhibits autophagy. DC661 also induces LMP and increases levels of cleaved caspase 3 (2, 7). In recent years, novel mechanisms of cell death that have immunogenic consequences have been defined and include necroptosis, ferroptosis, and pyroptosis (8). Lysosome-dependent autophagy has been shown to degrade MHC class I (9), as well as components of the immunoproteasome (10), suggesting that autophagy inhibition can enhance antigen processing. However, the immunogenic effects of lysosomal inhibition and ensuing cell death have not been fully characterized. To address this knowledge gap, we investigated the effects of DC661 on canonical cell death mechanisms to determine if lysosomal cell death is its own form of cell death or simply a precursor to one of the other established mechanisms. We found that HCQ and DC661 induced a significant increase in only a small number of proteins in the melanoma proteome and most of these were autophagy- and apoptosis-related proteins. Inhibitors of programmed cell death (apoptosis, necroptosis, ferroptosis, and pyroptosis) did not mitigate cytotoxic effects after lysosomal impairment by DC661. Lysosomal lipid peroxidation (LLP) is a major driver of LMP-induced cell death associated with calreticulin (CALR) expression on the cell the cell surface. This form of cell death can be reversed only by using the antioxidant N-acetylcysteine (NAC). NAC activity was impaired by inhibition of lysosomal cysteine importer MFSD12. LLP elicited immunogenic phenotypes promoting T cell–mediated killing. These findings demonstrate that lysosomal cell death is a potentially unique form of immunogenic cell death.
涉及溶酶体抑制剂羟氯喹 (HCQ) 的临床试验取得了令人鼓舞的进展（ 1 ), 鉴定棕榈酰蛋白硫酯酶 1 (PPT1) 作为氯喹衍生物的分子靶点 ( 2 ），以及新型 PPT1 抑制剂进入临床试验（ 3 , 4 ），需要了解溶酶体抑制剂诱导细胞死亡的机制以及溶酶体抑制对肿瘤免疫的影响。 HCQ 描述的溶酶体细胞死亡的典型机制涉及溶酶体膜透化 (LMP)、组织蛋白酶的渗漏和半胱天冬酶介导的细胞凋亡的激活。 5 , 6 ）。我们之前已经证明，溶酶体抑制剂 DC661 比 HCQ 更有效地穿透酸性肿瘤微环境中的细胞并定位到溶酶体（ 2 , 7 ）。 DC661 在许多癌细胞系中产生有效的细胞死亡（ 2 ）。 DC661 结合并抑制 PPT1，使溶酶体脱酸，并抑制自噬。 DC661 还诱导 LMP 并增加裂解的 caspase 3 的水平（ 2 , 7 ）。近年来，已经确定了具有免疫原性后果的新细胞死亡机制，包括坏死性凋亡、铁死亡和焦亡。 8 ）。溶酶体依赖性自噬已被证明可以降解 I 类 MHC（ 9 ），以及免疫蛋白酶体的成分（ 10 ），表明自噬抑制可以增强抗原加工。然而，溶酶体抑制和随后的细胞死亡的免疫原性作用尚未得到充分表征。 为了解决这一知识差距，我们研究了 DC661 对典型细胞死亡机制的影响，以确定溶酶体细胞死亡是否是其自身形式的细胞死亡，或者仅仅是其他已建立机制之一的前兆。我们发现 HCQ 和 DC661 仅诱导黑色素瘤蛋白质组中少数蛋白质显着增加，其中大部分是自噬和凋亡相关蛋白质。程序性细胞死亡（细胞凋亡、坏死性凋亡、铁死亡和细胞焦亡）抑制剂不能减轻 DC661 溶酶体损伤后的细胞毒性作用。溶酶体脂质过氧化 (LLP) 是 LMP 诱导的细胞死亡的主要驱动因素，与细胞表面的钙网蛋白 (CALR) 表达相关。这种形式的细胞死亡只能通过使用抗氧化剂 N-乙酰半胱氨酸 (NAC) 来逆转。 NAC 活性因溶酶体半胱氨酸输入蛋白 MFSD12 的抑制而受损。 LLP 引发免疫原性表型，促进 T 细胞介导的杀伤。这些发现表明，溶酶体细胞死亡是免疫原性细胞死亡的一种潜在独特形式。
Results 结果
Lysosomal inhibition induces significant changes in the proteome associated with autophagy and apoptosis.
溶酶体抑制会引起与自噬和细胞凋亡相关的蛋白质组的显着变化。
To establish the cytotoxic effects of lysosomal inhibitors, we assessed the viability of A375P melanoma, RKO colon carcinoma, and MIA PaCa-2 pancreatic cancer cell lines treated with the vacuolar H+-ATPase inhibitor bafilomycin-A1 (100 nM); PPT1 inhibitors DC661 (3 μM) or HCQ (10 μM or 30 μM); palmitate mimetic hexadecylsulfonyl fluoride (HDSF; 60 μM); cathepsin inhibitors pepstatin A (PepA; 10 μg/mL), E64 (PepA; 10 μg/mL), or PepA+E64; and lysosomal membrane disruptor Leu-Leu methyl ester hydrobromide (20 μM) for 48 hours. Only bafilomycin-A1 and DC661 caused a significant decrease in cell viability across the cancer cell lines (Figure 1A and Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI164596DS1), with DC661 producing a more profound reduction in cell viability than bafilomycin-A1. Based on these data and the pharmacological drug–like properties of chloroquine derivatives, we focused our study on chloroquine derivatives as tools to understand lysosomal cell death. An unbiased global proteome analysis was applied to A375P melanoma cells treated with DC661 (3 μM) or the less potent HCQ (10 μM or 30 μM) for 24 hours. Of the 4,264 high-confidence quantified proteins, only 87 and 55 proteins were significantly increased with DC661 (3 μM) and HCQ (30 μM), respectively; additionally, 14 and 15 proteins were significantly decreased with DC661 (3 μM) and HCQ (30 μM), respectively (absolute fold change of greater than 2; q < 0.05) with DC661 (3 μM) or HCQ (30 μM), respectively, when compared with vehicle control. The lower dose of HCQ (10 μM) showed no significant protein changes compared with vehicle control. The top 50 proteins that were significantly increased following DC661 treatment were mainly associated with autophagy and apoptosis, and similar changes were observed for the higher dosage of HCQ (Figure 1B). For these reasons, we chose to focus further studies on the more-potent DC661. Examples of some of the largest protein changes associated with autophagy and apoptosis were tax1-binding protein 1 (TAX1BP1; increased 15-fold); BCL2-interacting protein 3 (BNIP3; increased 6-fold); neighbor of BRCA1 gene 1 protein (NBR1; increased 12-fold); sequestosome-1 (SQSTM1/p62; increased 5-fold); nuclear receptor coactivator 4 (NCOA4; increased 3-fold); and LC3B (MAP1LC3B; increased 3-fold). Apolipoprotein B-100 (APOB; increased 66-fold) was derived from fetal calf serum and was not studied further. Immunoblotting confirmed that treatment with DC661 or higher concentrations of HCQ produced marked increases in the expression of autophagy cargo receptors NBR1, TAX1BP1, SQSTM1/p62, and NCOA4 in a dose- and time-dependent manner (Supplemental Figure 1, B and C). CRISPR/Cas9 KO of PPT1 in A375P cells phenocopied the effects of DC661 on these proteins when compared with their WT counterparts (Supplemental Figure 1D). However, the expression of autophagy cargo receptors and the relative increases induced by DC661 treatment was variable across colon cancer, pancreatic cancer, and other melanoma cell lines in which DC661 demonstrates cytotoxicity (Supplemental Figure 1E). This suggested that autophagy cargo receptors themselves, although increased at the protein level, are unlikely to be responsible for cell death following DC661 treatment. To confirm this, we focused on autophagy cargo receptors TAX1BP1 and BNIP3 because they have known proapoptotic effects in cancer cells (Figure 1C). TAX1BP1 is an autophagy cargo adaptor (11) and also regulates apoptosis induced by protein synthesis inhibitors or DNA-damaging agents in cancer cells (12). Knockdown of TAX1BP1 by siRNA (Figure 1D) had no effect on DC661-induced cytotoxicity in short-term (Figure 1E) and long-term viability assays (Figure 1F). These results demonstrated that TAX1BP1 does not play an essential role in DC661-mediated cytotoxicity. BNIP3 is a proapoptotic protein that impairs mitochondrial bioenergetics and regulates mitophagy (13). Effective knockdown of BNIP3 (Figure 1G) had no effect on DC661-induced cytotoxicity (Figures 1, H and I). These results showed that the cytotoxic effects of DC661 are independent of BNIP3 expression. Next, we studied the effects of depleting cells of canonical autophagy genes required for autophagosome production on DC661 efficacy by knocking down unc-51 like autophagy activating kinase 1 (ULK1) and autophagy-related gene 7 (ATG7). Effective knockdown of ULK1 or ATG7 (Supplemental Figure 2, A and B) inhibited autophagic flux (Supplemental Figure 2C) but had no effect on DC661-induced cytotoxicity (Figure 1, J–M). These results showed that the absence of the essential core autophagy machinery does not abrogate the cytotoxic effects of DC661.
Figure 1. Lysosomal autophagy inhibition induces significant changes in apoptosis and autophagy proteins.
图 1. 溶酶体自噬抑制会诱导细胞凋亡和自噬蛋白发生显着变化。
(A) Trypan blue viability assay of A375P melanoma cells treated with Bafilomycin-A1 (100 nM), DC661 (3 μM), HCQ (10 or 30 μM), hexadecylsulfonyl fluoride (HDSF, 60 μM), pepstatin A (10 μg/mL), E64 (10 μg/mL), or Leu-Leu methyl ester hydrobromide (LLoMe, 20 μM) for 48 hours. (B) LC-MS/MS–based proteome analysis of A375P cells treated with DMSO, DC661 (3 μM), or HCQ (10 or 30 μM) for 24 hours. Heatmap of the top 50 elevated proteins in DC661 verses control. Autophagy, apoptosis (names shown in bold), or other signaling pathway proteins significantly elevated (FDR, <5% and fold change, ≥2) in cells treated with 3 μM DC661, 10 μM HCQ, or 30 μM HCQ compared with those treated with vehicle control. (C) The autophagy cargo receptor proteins that have proapoptotic effects in cancer cells are shown in a Venn diagram. (D–M) A375P cells were treated with nontarget siRNA (siNT) or siRNA against TAX1BP1, BNIP3, ULK1, or ATG7 for 48 hours, followed by treatment with either DMSO or DC661 (3 μM) for 24 hours. (D and G) Immunoblotting of TAX1BP1 or BNIP3 and β-actin in the whole-cell lysates of A375P cells. (E and H) Seventy-two-hour MTT assay with 3 μM DC661 or (F and I) 7-day colony formation assay with 0.3 μM DC661 in A375P cells treated with the indicated siRNA. (J and L) Seventy-two-hour MTT assay with 3 μM DC661 and (K and M) 7-day colony formation assay with 0.3 μM DC661 in A375P cells treated with the indicated siRNA. All viability assays were performed in triplicate. ****P ≤ 0.0001. ANOVA test was used when more than 2 groups were compared. See also Supplemental Figure 1 and Supplemental Figure 2, A–C.
( A ) 用巴弗洛霉素-A1 (100 nM)、DC661 (3 μM)、HCQ (10 或 30 μM)、十六烷基磺酰氟 (HDSF, 60 μM)、胃酶抑素 A (10 μg/ mL)、E64 (10 μg/mL) 或 Leu-Leu 甲酯氢溴酸盐（LLoMe，20 μM）48 小时。 ( B ) 对用 DMSO、DC661 (3 μM) 或 HCQ（10 或 30 μM）处理 24 小时的 A375P 细胞进行基于 LC-MS/MS 的蛋白质组分析。 DC661 与对照中前 50 个升高的蛋白质的热图。与处理的细胞相比，用 3 μM DC661、10 μM HCQ 或 30 μM HCQ 处理的细胞中自噬、凋亡（名称以粗体显示）或其他信号通路蛋白显着升高（FDR，<5% 且倍数变化，≥2）与车辆控制。 ( C ) 在癌细胞中具有促凋亡作用的自噬货物受体蛋白如维恩图所示。 ( D – M ) A375P 细胞用非靶标 siRNA (siNT) 或针对TAX1BP1 、 BNIP3 、 ULK1或ATG7 的siRNA 处理 48 小时，然后用 DMSO 或 DC661 (3 μM) 处理 24 小时。 ( D和G ) A375P 细胞全细胞裂解物中 TAX1BP1 或 BNIP3 和 β-肌动蛋白的免疫印迹。 ( E和H ) 使用 3 μM DC661 进行 72 小时 MTT 测定，或 ( F和I ) 在用所示 siRNA 处理的 A375P 细胞中使用 0.3 μM DC661 进行 7 天集落形成测定。 ( J和L ) 使用 3 μM DC661 进行 72 小时 MTT 测定，( K和M ) 使用 0.0 进行 7 天集落形成测定。用指定的 siRNA 处理的 A375P 细胞中的 3 μM DC661。所有活力测定均一式三份进行。 **** P ≤ 0.0001。当比较 2 个以上的组时，使用 ANOVA 检验。参见 Supplemental Figure 1 和 Supplemental Figure 2 ，A-C。
Lysosomal inhibition by DC661 induces multiple programmed cell death pathways.
DC661 的溶酶体抑制诱导多种程序性细胞死亡途径。
The canonical viewpoint is that lysosomal cell death is due to apoptosis (5). Immunoblotting revealed that DC661 treatment resulted in activation of caspase-3, -7, and -9 and cleavage of PARP-1, confirming that DC661 activated apoptosis (Figure 2A). The pan-caspase inhibitor Z-VAD-FMK prevented caspase activation by DC661 but did not inhibit the accumulation of LC3B and p62, demonstrating that apoptosis activation and autophagy blockade are separable following lysosomal inhibition (Figure 2B). Blocking apoptosis with ZVAD-FMK did not enhance or limit DC661 cytotoxicity, suggesting that apoptosis is dispensable for lysosomal cell death (Figure 2, C and D). The cytotoxic effects of DC661 were similar in Bax/Bak double-KO primary bone marrow cells incapable of undergoing apoptosis and WT cells (Figure 2E). Blockade of apoptosis had no effect on DC661-induced cytotoxicity in colon cancer and pancreatic cancer cells as well (Supplemental Figure 2, D–F).
典型的观点是溶酶体细胞死亡是由于细胞凋亡（ 5 ）。免疫印迹显示 DC661 处理导致 caspase-3、-7 和 -9 激活以及 PARP-1 裂解，证实 DC661 激活细胞凋亡（ Figure 2A ）。泛半胱天冬酶抑制剂 Z-VAD-FMK 阻止 DC661 激活半胱天冬酶，但不抑制 LC3B 和 p62 的积累，表明溶酶体抑制后细胞凋亡激活和自噬阻断是可分离的。 Figure 2B ）。用 ZVAD-FMK 阻断细胞凋亡并不会增强或限制 DC661 细胞毒性，这表明细胞凋亡对于溶酶体细胞死亡来说是可有可无的。 Figure 2, C and D ）。 DC661 在无法凋亡的Bax/Bak双 KO 原代骨髓细胞和 WT 细胞中的细胞毒性作用相似（ Figure 2E ）。细胞凋亡的阻断对 DC661 诱导的结肠癌和胰腺癌细胞的细胞毒性也没有影响（ Supplemental Figure 2 ，D-F）。
Figure 2. DC661-induced apoptosis and necroptosis.
图 2. DC661 诱导的细胞凋亡和坏死性凋亡。
(A) Immunoblots of cleaved caspase-3 (Cl. C-3), caspase-7 (Cl. C-7), caspase-9 (Cl. C-9), PARP and β-actin from A375P cell lysates treated with indicated concentrations of DC661 for 24 hours. Staurosporine (ST; 20 ng/mL) was used as a positive control for apoptosis. (B) Immunoblots of lysates from A375P cells treated with 3 μM DC661, 80 μM pan-caspase inhibitor Z-VAD-FMK, or both for 24 hours. (C) Seventy-two-hour MTT assay plot with increasing concentrations of DC661 (0.01 to 10 μM), with and without Z-VAD-FMK 80 μM. (D) Seven-day colony formation assay in A375P cells treated with 0.3 μM DC661, 8 μM Z-VAD-FMK, or their combinations. (E) Trypan blue viability assay with and without 3 μM DC661 for 24 hours in FL5.12 and IL-3–dependent Bax−/−Bak−/− (BB-DKO) primary bone marrow cells. (F) Immunoblots of RIP1, MLKL, their phosphorylated forms, and β-actin in the lysates of A375P cells treated with DC661 for 24 hours. Necroptosis conventional TSZ (TNF-α, Smac mimetic [SM-164], and Z-VAD-FMK) treatment conditions used included the following: C, pretreatment with Z-VAD-FMK (25 μM, 1 hour), followed by SM-164 (2 μM, 1 hour) and TNF-α (20 ng/mL, 22 hours); D, pretreatment with Z-VAD-FMK (80 μM, 1 hour), followed by SM-164 (100 nM, 1 hour) and TNF-α (20 ng/mL, 22 hours). (G) Immunoblots of necroptosis proteins in lysates of A375P cells treated with necroptosis inhibitors necrostatin-1s (Nec-1s, 50 μM) and necrosulfonamide (NS, 2.5 μM) with DC661 1 μM for 24 hours. (H) Seventy-two-hour MTT assay plot with DC661 (0.01 to 10 μM), with and without necrostatin-1 (Nec-1, 50 μM), 50 μM Nec-1s, and 2.5 μM NS in A375P cells. All viability assays were performed in triplicate. ****P ≤ 0.0001; ns, nonsignificant. Two-tailed unpaired t test between 2 groups (C). ANOVA test was used when more than 2 groups were compared (E and H). See also Supplemental Figure 2, D–G.
( A ) 经 A375P 细胞裂解物处理后的裂解 caspase-3 (Cl. C-3)、caspase-7 (Cl. C-7)、caspase-9 (Cl. C-9)、PARP 和 β-actin 的免疫印迹指示 24 小时 DC661 的浓度。星形孢菌素（ST；20 ng/mL）用作细胞凋亡的阳性对照。 ( B ) 用 3 μM DC661、80 μM 泛半胱天冬酶抑制剂 Z-VAD-FMK 或两者处理 24 小时的 A375P 细胞裂解物的免疫印迹。 ( C ) 72 小时 MTT 测定图，其中 DC661 浓度不断增加（0.01 至 10 μM），有或没有 Z-VAD-FMK 80 μM。 ( D ) 用 0.3 μM DC661、8 μM Z-VAD-FMK 或其组合处理的 A375P 细胞进行 7 天集落形成测定。 ( E ) 在 FL5.12 和 IL-3 依赖的Bax −/− Bak −/− (BB-DKO) 原代骨髓细胞中，使用和不使用 3 μM DC661 进行台盼蓝活力测定 24 小时。 ( F ) DC661 处理 24 小时的 A375P 细胞裂解物中 RIP1、MLKL、其磷酸化形式和 β-肌动蛋白的免疫印迹。坏死性凋亡常规 TSZ（TNF-α、Smac 模拟物 [SM-164] 和 Z-VAD-FMK）使用的治疗条件包括： C，用 Z-VAD-FMK（25 μM，1 小时）预处理，然后用 SM -164（2 μM，1 小时）和 TNF-α（20 ng/mL，22 小时）； D，用 Z-VAD-FMK（80 μM，1 小时）预处理，然后用 SM-164（100 nM，1 小时）和 TNF-α（20 ng/mL，22 小时）预处理。 ( G ) 用坏死性凋亡抑制剂 necrostatin-1s (Nec-1s, 50 μM) 和 necrosulfonamide (NS, 2.5 μM) 与 DC661 1 μM 处理 24 小时，对 A375P 细胞裂解液中的坏死性凋亡蛋白进行免疫印迹。 ( H ) 使用 DC661 (0.A375P 细胞中含有或不含 necrostatin-1（Nec-1，50 μM）、50 μM Nec-1s 和 2.5 μM NS。所有活力测定均一式三份进行。 **** P≤0.0001 ； ns，不显着。两组之间的双尾未配对t检验 ( C )。当比较 2 个以上组（ E和H ）时，使用方差分析检验。参见 Supplemental Figure 2 ，D-G。
Because we found apoptosis to be dispensable for DC661-induced cell death, we investigated whether DC661 induces necroptosis, another form of programmed cell death regulated by receptor-interacting protein kinase (RIPK) and mixed lineage kinase domain-like protein (MLKL). Levels of the phosphorylated and activated forms of RIPK and MLKL were increased following DC661 treatment from 0.1–1 μM (Figure 2F). At 3 μM DC661 there was an absence of phosphorylated RIPK1 but persistent phosphorylated MLKL, suggesting that, at this higher concentration, additional forms of cell death could be engaged. Pretreatment with the RIPK1 inhibitors necrostatin-1 or necrostatin-1s or the MLKL inhibitor necrosulfonamide prevented phosphorylation of RIPK1 after DC661 (1 μM) treatment in melanoma cells (Figure 2G) but failed to rescue DC661-induced cytotoxicity in melanoma cells (Figure 2H) or colon cancer or pancreatic cancer cells (Supplemental Figure 2G). These findings suggest that necroptosis is activated but dispensable for DC661-mediated cell death.
因为我们发现细胞凋亡对于 DC661 诱导的细胞死亡来说是可有可无的，所以我们研究了 DC661 是否会诱导坏死性凋亡，这是另一种受受体相互作用蛋白激酶 (RIPK) 和混合谱系激酶域样蛋白 (MLKL) 调节的程序性细胞死亡形式。 DC661 处理后，磷酸化和活化形式的 RIPK 和 MLKL 水平从 0.1-1 μM 增加（ Figure 2F ）。在 3 μM DC661 下，磷酸化 RIPK1 不存在，但磷酸化 MLKL 持续存在，这表明在这个较高浓度下，可能会参与其他形式的细胞死亡。在黑色素瘤细胞中，用 RIPK1 抑制剂 necrostatin-1 或 necrostatin-1s 或 MLKL 抑制剂 necrosulfonamide 进行预处理，可以防止 DC661 (1 μM) 处理后 RIPK1 的磷酸化。 Figure 2G ）但未能挽救 DC661 诱导的黑色素瘤细胞细胞毒性（ Figure 2H ）或结肠癌或胰腺癌细胞（ Supplemental Figure 2G ）。这些发现表明坏死性凋亡被激活，但对于 DC661 介导的细胞死亡来说是可有可无的。
Lysosomes are one of the main storage sites for iron. Dysregulated intracellular iron metabolism coupled with decreased reductive capacity can trigger a nonapoptotic cell death known as ferroptosis. A hallmark of ferroptosis is upregulation of prostaglandin-endoperoxide synthase 2 (PTGS2), cation transport regulator homolog 1 (CHAC1), and cysteinyl-tRNA synthetase (CARS) (14). All 3 of these ferroptosis markers were transcriptionally upregulated by DC661 treatment (Figure 3A), suggesting that lysosomal inhibition induces ferroptosis. DC661 induced a fluorescence shift in C11-BODIPY–treated A375P cells, indicating lipid peroxidation, characteristic of ferroptosis. This DC661-induced shift was significantly reversed in the presence of ferroptosis inhibitors ferrostatin-1 or liproxstatin-1 administered at an effective concentration (Figure 3B and Supplemental Figure 3A), further indicating that DC661 induced ferroptosis. However, ferroptosis inhibition did not rescue the cytotoxicity associated with DC661 (Figure 3, C and D). Cotreatment of cancer cells with the iron chelator deferoxamine (DFO) and DC661 did not rescue the cytotoxicity of DC661 (Figure 3E). Treatment with ferrostatin-1, liproxstatin-1, or DFO did not rescue DC661 inhibition of short-term viability or long-term clonogenic growth in melanoma, colon, and pancreatic cancer cells (Supplemental Figure 3, B–E, and Supplemental Figure 4, A–D). These data demonstrate that ferroptosis is activated but dispensable for DC661-mediated cell death.
溶酶体是铁的主要储存场所之一。细胞内铁代谢失调加上还原能力下降可引发非凋亡性细胞死亡，称为铁死亡。铁死亡的一个标志是前列腺素内过氧化物合酶 2 ( PTGS2 )、阳离子转运调节剂同源物 1 ( CHAC1 ) 和半胱氨酰-tRNA 合成酶 ( CARS ) 的上调。 14 ）。所有 3 个铁死亡标记物均通过 DC661 处理进行转录上调（ Figure 3A ），表明溶酶体抑制会诱导铁死亡。 DC661 诱导 C11-BODIPY 处理的 A375P 细胞中的荧光变化，表明脂质过氧化，这是铁死亡的特征。在以有效浓度施用铁死亡抑制剂 ferrostatin-1 或 liproxstatin-1 的情况下，这种 DC661 诱导的转变显着逆转（ Figure 3B 和 Supplemental Figure 3A ），进一步表明 DC661 诱导铁死亡。然而，铁死亡抑制并不能挽救与 DC661 相关的细胞毒性（ Figure 3, C and D ）。用铁螯合剂去铁胺 (DFO) 和 DC661 共同处理癌细胞并不能挽救 DC661 的细胞毒性。 Figure 3E ）。使用ferrostatin-1、liproxstatin-1或DFO治疗并不能挽救DC661对黑色素瘤、结肠癌和胰腺癌细胞的短期活力或长期克隆生长的抑制作用。 Supplemental Figure 3 、B–E 和 Supplemental Figure 4 ，A-D）。这些数据表明铁死亡被激活，但对于 DC661 介导的细胞死亡来说是可有可无的。
Figure 3. DC661-induced ferroptosis and pyroptosis.
图 3. DC661 诱导的铁死亡和细胞焦亡。
(A) qRT-PCR showed the fold change increase in the transcriptional expression of PTGS2, CHAC, and CARS in A375P cells treated with 3 μM DC661 for 24 hours. (B) A375P cells treated for 24 hours with DC661 (3 μM), liproxstatin-1 (Liprox-1, 2 μM), or ferrostatin-1 (Ferro-1, 10 μM). Lipid peroxidation measured by C-11 BODIPY using flow cytometry. Erastin was used as positive control (see Supplemental Figure 3A). (C–E) Trypan blue cell viability assay in A375P cells treated with 3 μM DC661, with and without ferroptosis inhibitors (C) ferrostatin-1 (Ferro-1, 10 μM), (D) liproxstatin-1 (Liprox-1, 2 μM), and (E) iron chelator deferoxamine (DFO, 5 μM). (F) Western blots were probed for pyroptosis and autophagy proteins in the whole-cell lysates and HMGB1 release in cell supernatant of human WM35 empty vector (EV) and gasdermin-E–KO (KO1 and KO2) cells treated with DC661 1 μM for 48 hours. (G) Bar graph showing average DC661 IC50 values ± SEM of MTT assays in both 10% and 1% FBS conditions in mouse YUMM1.7 WT, EV, and gasdermin-E–KO (Gsdme-KO) cells from 3 independent experiments. Statistical analysis for I was applied on ΔCT values. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Two-tailed unpaired t test between 2 groups (A). ANOVA test was used when more than 2 groups were compared (B–E and G). See also Supplemental Figures 3–5. All viability assays were performed in triplicate.
( A ) qRT-PCR 显示用 3 μM DC661 处理 24 小时的 A375P 细胞中PTGS2 、 CHAC和CARS的转录表达增加倍数变化。 ( B ) 用 DC661 (3 μM)、liproxstatin-1 (Liprox-1, 2 μM) 或 ferostatin-1 (Ferro-1, 10 μM) 处理 A375P 细胞 24 小时。使用流式细胞术通过 C-11 BODIPY 测量脂质过氧化。伊拉斯汀用作阳性对照（参见 Supplemental Figure 3A ）。 ( C – E ) 用 3 μM DC661 处理的 A375P 细胞进行台盼蓝细胞活力测定，添加和不添加铁死亡抑制剂 ( C ) 铁他汀-1 (Ferro-1, 10 μM)，( D ) liproxstatin-1 (Liprox-1, 2 μM) 和 ( E ) 铁螯合剂去铁胺 (DFO, 5 μM)。 ( F ) 检测用 DC661 1 μM 处理的人 WM35 空载体 (EV) 和 Gasdermin-E–KO（KO1 和 KO2）细胞的全细胞裂解物中的焦亡和自噬蛋白以及细胞上清液中 HMGB1 的释放。 48小时。 ( G ) 条形图显示来自 3 个独立实验的小鼠 YUMM1.7 WT、EV 和 Gasdermin-E–KO ( Gsdme -KO) 细胞在 10% 和 1% FBS 条件下的 MTT 测定的平均 DC661 IC 50值 ± SEM 。 I的统计分析应用于 ΔCT 值。 * P≤0.05 ； ** P≤0.01 ； *** P≤0.001 ； **** P ≤ 0.0001。两组之间的双尾未配对t检验 ( A )。当比较 2 个以上组（ B – E和G ）时，使用方差分析检验。参见 Supplemental Figures 3–5 。 所有活力测定均一式三份进行。
Pyroptosis is a form of programmed cell death associated with an inflammatory response that involves the activation of caspases that process gasdermin (GSDM), allowing pore formation on the plasma membrane and the subsequent release of damage-associated molecular patterns, such as high-mobility group box 1 (HMGB1). DC661 treatment in multiple melanoma cell lines (A375P, A375, WM35, and WM793) resulted in the activation of initiator caspase-8 and -9 and executioner caspase-7, typical for pyroptosis, and produced cleavage of full-length GSDME, similar in extent to the known pyroptosis inducer PLX4720 and PD0325901 (Supplemental Figure 5A). DC661 produced a stronger extracellular release of HMGB1 than the known pyroptosis inducer, BRAF, and MEK inhibition in 1% FBS (15), reflecting the functional consequence of activated pyroptosis. Next, we tested whether inhibition of pyroptosis by GSDME KO would ameliorate the cytotoxic effects of DC661. DC661 treatment induced LC3II and SQSTM1/p62 accumulation and caspase activation, but HMGB1 release was nearly completely abrogated in GSDME-KO WM35 human cells, demonstrating that the functional consequence of inhibiting pyroptosis was achieved (Figure 3F). However, DC661 treatment produced equal cytotoxicity in YUMM1.7 WT, empty vector (EV), and Gsdme KO1 and KO2 cells in both 10% and 1% FBS conditions (Figure 3G and Supplemental Figure 5B). One common outcome of multiple forms of cell death is the release of LDH. DC661 produced a significant increase in LDH release, and this could not be reversed by apoptosis, necroptosis, or ferroptosis inhibition (Supplemental Figure 5C). These results showed that DC661 induces multiple modes of cell death, including apoptosis and pyroptosis as well as necroptosis and ferroptosis, but none of these modes of cell death are required for DC661-induced cell death.
细胞焦亡是一种与炎症反应相关的程序性细胞死亡形式，涉及激活处理 Gasdermin (GSDM) 的半胱天冬酶 (GSDM)，从而在质膜上形成孔，并随后释放与损伤相关的分子模式，例如高迁移率基团框 1（HMGB1）。 DC661 处理多种黑色素瘤细胞系（A375P、A375、WM35 和 WM793）会导致启动子 caspase-8 和 -9 以及刽子手 caspase-7 的激活（典型的细胞焦亡），并产生全长 GSDME 的裂解，与程度达到已知的细胞焦亡诱导剂 PLX4720 和 PD0325901（ Supplemental Figure 5A ）。 DC661 在 1% FBS 中比已知的细胞焦亡诱导剂 BRAF 和 MEK 抑制产生更强的 HMGB1 细胞外释放（ 15 ），反映了激活的细胞焦亡的功能后果。接下来，我们测试了GSDME KO 对细胞焦亡的抑制是否会改善 DC661 的细胞毒性作用。 DC661 处理诱导 LC3II 和 SQSTM1/p62 积累以及 caspase 激活，但在GSDME -KO WM35 人类细胞中 HMGB1 释放几乎完全消除，表明实现了抑制细胞焦亡的功能结果（ Figure 3F ）。然而，DC661 处理在 10% 和 1% FBS 条件下对 YUMM1.7 WT、空载体 (EV) 以及Gsdme KO1 和 KO2 细胞产生相同的细胞毒性（ Figure 3G 和 Supplemental Figure 5B ）。多种形式的细胞死亡的一个常见结果是 LDH 的释放。 DC661 使 LDH 释放显着增加，并且这不能通过细胞凋亡、坏死性凋亡或铁死亡抑制来逆转。 Supplemental Figure 5C ）。 这些结果表明，DC661诱导多种细胞死亡模式，包括细胞凋亡和细胞焦亡以及坏死性凋亡和铁死亡，但这些细胞死亡模式都不是DC661诱导的细胞死亡所必需的。
Cathepsin inhibition or calcium chelation does not prevent cell death from lysosomal membrane permeabilization.
组织蛋白酶抑制或钙螯合不能防止溶酶体膜透化导致的细胞死亡。
Having demonstrated that caspase activation (which is required for apoptosis and pyroptosis) is dispensable for DC661-induced cell death, we hypothesized that cathepsin release from lysosomes could cause caspase-dependent cell death. Chemical (DC661) or genetic (PPT1 siRNA [siPPT1]) inhibition of PPT1 produced lysosomal membrane permeabilization (LMP), while HDSF, a less-potent irreversible inhibitor of PPT1 that gets depleted rapidly in cell culture, could not induce LMP, as measured by galectin-3–positive puncta (Figure 4A). LMP results in the release of cathepsins and other lysosomal contents into the cytoplasm and is thought to be a key proximal feature of lysosome-based cell death. LMP induced by DC661 was associated with a significant increase in cytoplasmic cathepsin-L activity, which was significantly blocked with a cysteine protease inhibitor E64 (Figure 4B). Previous reports have suggested that cathepsin release from fractured lysosomes promotes caspase activation, leading to apoptotic cell death (16–18). Complete cathepsin inhibition did not prevent caspase cleavage (Figure 4C) and did not rescue DC661-induced cytotoxicity in short-term and long-term viability assays in melanoma (Figure 4, D and E), colon cancer, and pancreatic cancer cells (Supplemental Figure 6, A–C). These results counter the canonical view of lysosomal cell death as driven by cathepsin-mediated cell death. Besides cathepsin release from leaky lysosomes, calcium release from lysosomes has been implicated in cellular dysfunction when PPT1 is impaired (19). DC661 treatment resulted in extensive calcium release, which was abrogated by pretreatment of cell-permanent calcium (Ca2+) chelator, BAPTA-AM. (Figure 4F). Notably, BAPTA-AM did not prevent DC661-induced LMP (Figure 4G) or caspase cleavage (Supplemental Figure 6, D and E) and, most importantly, did not rescue DC661-induced cytotoxicity (Figure 4H). These findings were also observed in both RKO and MIA PaCa-2 cell lines, in which no significant differences in IC50 values were observed with BAPTA-AM and DC661 (Supplemental Figure 6F), which suggested that LMP-associated cell death is not dependent on calcium or cathepsins.
在证明 caspase 激活（细胞凋亡和焦亡所需）对于 DC661 诱导的细胞死亡是可有可无的，我们假设溶酶体释放的组织蛋白酶可能导致 caspase 依赖性细胞死亡。化学 (DC661) 或遗传 ( PPT1 siRNA [ siPPT1 ]) 抑制 PPT1 会产生溶酶体膜透化 (LMP)，而 HDSF（一种效力较低的 PPT1 不可逆抑制剂，在细胞培养物中会迅速耗尽）无法诱导 LMP（如测量）通过半乳糖凝集素-3-阳性点（ Figure 4A ）。 LMP 导致组织蛋白酶和其他溶酶体内容物释放到细胞质中，并且被认为是基于溶酶体的细胞死亡的关键近端特征。 DC661 诱导的 LMP 与细胞质组织蛋白酶-L 活性显着增加相关，而半胱氨酸蛋白酶抑制剂 E64 可以显着阻断该活性。 Figure 4B ）。先前的报告表明，断裂的溶酶体释放组织蛋白酶可促进半胱天冬酶激活，导致细胞凋亡。 16 – 18 ）。完全抑制组织蛋白酶并不能阻止 caspase 裂解（ Figure 4C ）并且在黑色素瘤的短期和长期活力测定中没有挽救 DC661 诱导的细胞毒性（ Figure 4, D and E ）、结肠癌和胰腺癌细胞（ Supplemental Figure 6 ，A-C）。这些结果反驳了溶酶体细胞死亡由组织蛋白酶介导的细胞死亡驱动的经典观点。除了渗漏溶酶体释放组织蛋白酶外，当 PPT1 受损时，溶酶体释放钙也与细胞功能障碍有关。 19 ）。 DC661 处理导致大量钙释放，但细胞永久钙 (Ca 2+ ) 螯合剂 BAPTA-AM 预处理可消除这种情况。 （ Figure 4F ）。 值得注意的是，BAPTA-AM 并不能阻止 DC661 诱导的 LMP（ Figure 4G ）或半胱天冬酶裂解（ Supplemental Figure 6 、D 和 E），最重要的是，没有挽救 DC661 诱导的细胞毒性（ Figure 4H ）。这些结果也在 RKO 和 MIA PaCa-2 细胞系中观察到，其中 BAPTA-AM 和 DC661 的 IC 50值没有观察到显着差异（ Supplemental Figure 6F ），这表明 LMP 相关的细胞死亡不依赖于钙或组织蛋白酶。
Figure 4. Cathepsin inhibition or calcium chelation does not prevent DC661-induced cell death.
图 4. 组织蛋白酶抑制或钙螯合不能阻止 DC661 诱导的细胞死亡。
(A) A375P-galectin-3-EGFP cells were given nontarget siRNA (siNT) or PPT1 siRNA (siPPT1) for 48 hours, followed by treatment with DMSO, 60 μM HDSF, or 3 μM DC661 for 6 hours. (B) Cathepsin-L enzyme activity (red) and quantification in A375P cells treated with 3 μM DC661, 10 μg/mL cysteine protease inhibitor E64, or the combination of both for 6 hours. (C) Immunoblots for indicated proteins in A375P cell lysates treated with pepstatin A (PepA, 10 μg/mL), 10 μg/mL E64, and PepA+E64 with or without 3 μM DC661 for 24 hours. (D) Seventy-two-hour MTT assay plot with increasing concentrations of DC661 (0.01 to 10 μM), with and without PepA, E64, and PepA+E64 in A375P cells. (E) Seven-day colony formation assay in A375P cells treated with 10 μg/mL PepA, 10 μg/mL E64, and PepA+E64 with or without 0.3 μM DC661. (F and G) A375P or A375P-galectin-3-EGFP cells were treated with 3 μM DC661, 1 μM calcium chelator BAPTA-AM, or both for 24 hours. (F) Fluorescence images of A375P cells stained with Fluo-4, AM, to detect calcium release. (G) A375P-galectin-3-EGFP cells showing galectin-3 puncta (white arrows) and quantification after treatment with DC661, BAPTA-AM, or both. (H) Seventy-two-hour MTT assay plot with increasing concentrations of DC661 (0.01 to 10 μM), with and without indicated concentrations of BAPTA-AM in A375P cells. Scale bar: 20 μm. *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001. ANOVA test was used when more than 2 groups were compared. All viability experiments were done in triplicate.
( A ) A375P-galectin-3-EGFP 细胞给予非靶标 siRNA (siNT) 或PPT1 siRNA (si PPT1 ) 48 小时，然后用 DMSO、60 μM HDSF 或 3 μM DC661 处理 6 小时。 ( B ) 用 3 μM DC661、10 μg/mL 半胱氨酸蛋白酶抑制剂 E64 或两者组合处理 6 小时的 A375P 细胞中的组织蛋白酶-L 酶活性（红色）和定量。 ( C ) 用胃酶抑素 A (PepA, 10 μg/mL)、10 μg/mL E64 和 PepA+E64（含或不含 3 μM DC661）处理 24 小时的 A375P 细胞裂解物中所示蛋白质的免疫印迹。 ( D ) A375P 细胞中 DC661 浓度不断增加（0.01 至 10 μM）、有或没有 PepA、E64 和 PepA+E64 的 72 小时 MTT 测定图。 ( E ) 用 10 μg/mL PepA、10 μg/mL E64 和 PepA+E64（含或不含 0.3 μM DC661）处理的 A375P 细胞进行 7 天集落形成测定。 ( F和G ) A375P 或 A375P-galectin-3-EGFP 细胞用 3 μM DC661、1 μM 钙螯合剂 BAPTA-AM 或两者处理 24 小时。 ( F ) A375P 细胞用 Fluo-4、AM 染色的荧光图像，用于检测钙释放。 ( G ) A375P-galectin-3-EGFP 细胞显示 galectin-3 点（白色箭头），并在用 DC661、BAPTA-AM 或两者处理后进行定量。 ( H ) A375P 细胞中 DC661 浓度不断增加（0.01 至 10 μM）、有或没有指定浓度的 BAPTA-AM 的 72 小时 MTT 测定图。比例尺：20 μm。 * P≤0.05 ； *** P≤0.001 ； **** P ≤ 0.0001。当比较 2 个以上的组时，使用 ANOVA 检验。所有活力实验均一式三份进行。
LLP drives LMP. LLP 驱动 LMP。
Having established that inhibitors of major cell death pathways (apoptosis, necroptosis, ferroptosis, and pyroptosis), cathepsins (PepA and E64), and ion chelators (BAPTA-AM [Ca2+] and DFO [Fe2+]) did not prevent cell death by DC661 and finding evidence of lipid peroxidation by C-11-BODIPY, we performed a global lipidome analysis at 2 and 4 hours after DC661 treatment. DC661 induced early and sustained 3- to 10-fold increases in every class of lysophospholipid (Figure 5A). In contrast, minimal or no changes were seen in phospholipid classes, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid (Supplemental Figure 7A). Lysophospholipid species can be generated by ROS, which oxidize the saturated fatty acid chain that is then cleaved off by phospholipases (20). To determine if antioxidants could prevent ROS-mediated lipid damage, we investigated DC661-induced cytotoxicity in the presence of the pan-ROS scavenger N-acetylcysteine (NAC) (21, 22) and putative lipid peroxidation inhibitors Trolox (23) and vitamin C (ascorbic acid) (24, 25). Unlike any of the other agents tested thus far, cotreatment with NAC rescued DC661-associated cytotoxicity across multiple cell lines (Figure 5B and Supplemental Figure 7B). Long-term CFU assays showed that NAC, unlike all the cell death inhibitors, ion chelators, and cathepsin inhibitors tested herein, prevented the cytotoxic effects of DC661 in A375P, RKO, MIA PaCa-2, B16F10, and MC38 cells (Figure 5C and Supplemental Figure 7C). Interestingly, Trolox and vitamin C did not rescue DC661 cytotoxicity in human melanoma, colorectal cancer, and pancreatic cancer cells and murine melanoma and colorectal cancer cells (Figure 5D and Supplemental Figure 7, D–H). This disparity prompted us to test whether NAC, Trolox, and vitamin C affected LMP. Our results showed that NAC was the only antioxidant that significantly reduced DC661-induced LMP (Figure 5E). We hypothesized that LLP drives LMP production by DC661, which may be reverted by NAC but not by Trolox and vitamin C. To study the process of LLP we used the fluorescent probe FOAM-LPO (26), which specifically localizes to the lysosome and produces a spectral shift from 586 to 512 nm (red to green) when exposed to lipid peroxides. We found that DC661 induced LLP compared with control (Figure 5F). NAC reduced lipid peroxidation in the lysosomes of the DC661-treated melanoma cells, while Trolox and vitamin C failed to reduce LLP (Figure 5F). NAC, Trolox, and vitamin C on their own had no significant effect on lipid peroxidation. Knockdown of PPT1 (Supplemental Figure 8A), or treatment with high concentrations of HCQ (Supplemental Figure 8B) also induced LMP that could be reversed by NAC, whereas chemical inhibition of ULK1 did not induce LMP (Supplemental Figure 8C). Importantly knockdown of siPPT1 also included LLP that could be reversed with NAC (Supplemental Figure 8D) These results showed that PPT1 inhibition induces LLP that can be rescued by NAC and is likely the cause of PPT1-inhibition-induced LMP. Further, these findings suggested that LLP is critical for lysosomal cell death.
已确定主要细胞死亡途径（细胞凋亡、坏死性凋亡、铁死亡和焦亡）、组织蛋白酶（PepA 和 E64）和离子螯合剂（BAPTA-AM [Ca 2+ ] 和 DFO [Fe 2+ ]）的抑制剂不能预防DC661 导致细胞死亡并发现 C-11-BODIPY 导致脂质过氧化的证据，我们在 2 小时和 4 小时进行了全局脂质组分析DC661治疗后。 DC661 诱导每一类溶血磷脂早期并持续增加 3 至 10 倍（ Figure 5A ）。相比之下，磷脂类别的变化很小或没有变化，包括磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰肌醇、磷脂酰甘油和磷脂酸。 Supplemental Figure 7A ）。溶血磷脂种类可以通过 ROS 产生，ROS 氧化饱和脂肪酸链，然后被磷脂酶裂解。 20 ）。为了确定抗氧化剂是否可以预防 ROS 介导的脂质损伤，我们研究了在泛 ROS 清除剂 N-乙酰半胱氨酸 (NAC) 存在的情况下 DC661 诱导的细胞毒性。 21 , 22 ）和假定的脂质过氧化抑制剂 Trolox（ 23 ）和维生素 C（抗坏血酸）（ 24 , 25 ）。与迄今为止测试的任何其他药物不同，与 NAC 共同治疗可以挽救多个细胞系中与 DC661 相关的细胞毒性（ Figure 5B 和 Supplemental Figure 7B ）。长期 CFU 测定表明，与本文测试的所有细胞死亡抑制剂、离子螯合剂和组织蛋白酶抑制剂不同，NAC 可防止 DC661 在 A375P、RKO、MIA PaCa-2、B16F10 和 MC38 细胞中的细胞毒性作用。 Figure 5C 和 Supplemental Figure 7C ）。 有趣的是，Trolox 和维生素 C 并不能挽救 DC661 对人类黑色素瘤、结直肠癌和胰腺癌细胞以及小鼠黑色素瘤和结直肠癌细胞的细胞毒性。 Figure 5D 和 Supplemental Figure 7 ，D-H）。这种差异促使我们测试 NAC、Trolox 和维生素 C 是否影响末次月经。我们的结果表明 NAC 是唯一能显着降低 DC661 诱导的 LMP 的抗氧化剂（ Figure 5E ）。我们假设 LLP 通过 DC661 驱动 LMP 产生，这可能会被 NAC 逆转，但不会被 Trolox 和维生素 C 逆转。为了研究 LLP 的过程，我们使用了荧光探针 FOAM-LPO（ 26 ），它专门定位于溶酶体，当暴露于脂质过氧化物时，会产生从 586 nm 到 512 nm（红到绿）的光谱偏移。我们发现与对照相比，DC661 诱导了 LLP（ Figure 5F ）。 NAC 减少了 DC661 处理的黑色素瘤细胞溶酶体中的脂质过氧化，而 Trolox 和维生素 C 未能减少 LLP（ Figure 5F ）。 NAC、Trolox 和维生素 C 本身对脂质过氧化没有显着影响。 PPT1的击倒（ Supplemental Figure 8A ），或用高浓度的HCQ处理（ Supplemental Figure 8B ）也能诱导 LMP，而 LMP 可以被 NAC 逆转，而 ULK1 的化学抑制不会诱导 LMP（ Supplemental Figure 8C ）。重要的是siPPT1的敲低还包括可以用 NAC 逆转的 LLP（ Supplemental Figure 8D ）这些结果表明，PPT1 抑制诱导 LLP，可由 NAC 挽救，并且可能是 PPT1 抑制诱导 LMP 的原因。此外，这些发现表明 LLP 对于溶酶体细胞死亡至关重要。
Figure 5. N-acetyl cysteine prevents DC661-induced cell death.
图 5.N-乙酰半胱氨酸可防止 DC661 诱导的细胞死亡。
(A) LC-MS/MS lipidome analysis of A375P cells treated with DMSO (white) or 3 μM DC661 (red) for 2 or 4 hours. Mean ± SD of significantly elevated lysophospholipid classes by unpaired t test. LPC, lysophosphatidyl choline; LPE, lysophosphatidyl ethanolamine; LPS, lysophosphatidyl serine; LPI, lysophosphatidyl inositol; LPG, lysophosphatidyl glycerol; LPA, lysophosphatidyl acid. (B and C) A375P, B16F10, and MC38 cells were treated with 10 mM N-acetyl cysteine (NAC), 3 μM DC661, or both for 72 hours. (B) Trypan blue viability assay after 72 hours of treatment. (C and D) Seven-day colony formation assay in A375P cells treated with 0.1 μM DC661, 1 mM NAC, 100 μM Trolox, and 100 μM vitamin C. (E and F) A375P-galectin-3-GFP cells or A375P cells were treated with 3 μM DC661, 10 mM NAC, 100 μM Trolox, and 100 μM vitamin C for 24 hours or 6 hours. (E) A375P-galectin-3-GFP cells showing galectin-3 (Gal3) puncta (white arrows) and quantification. (F) Fluorescence images of A375P cells stained with FOAM-LPO (1 μM, 5 min) to detect LLP. Scale bar: 20 μm. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. ANOVA test was used when more than 2 groups were compared. All viability experiments were done in triplicate.
( A ) 对用 DMSO（白色）或 3 μM DC661（红色）处理 2 或 4 小时的 A375P 细胞进行 LC-MS/MS 脂质组分析。通过未配对t检验显着升高的溶血磷脂类别的平均值±SD。 LPC，溶血磷脂酰胆碱； LPE，溶血磷脂酰乙醇胺； LPS，溶血磷脂酰丝氨酸； LPI，溶血磷脂酰肌醇； LPG，溶血磷脂酰甘油； LPA，溶血磷脂酸。 （ B和C ）A375P、B16F10 和 MC38 细胞用 10 mM N-乙酰半胱氨酸 (NAC)、3 μM DC661 或两者处理 72 小时。 ( B ) 处理 72 小时后台盼蓝活力测定。 ( C和D ) 用 0.1 μM DC661、1 mM NAC、100 μM Trolox 和 100 μM 维生素 C 处理的 A375P 细胞进行 7 天集落形成测定。 ( E和F ) A375P-galectin-3-GFP 细胞或 A375P 细胞用 3 μM DC661、10 mM NAC、100 μM Trolox 处理，和 100 μM 维生素 C 24 小时或 6 小时。 ( E ) A375P-galectin-3-GFP 细胞显示 galectin-3 (Gal3) 斑点（白色箭头）和定量。 ( F ) A375P 细胞用 FOAM-LPO（1 μM，5 分钟）染色以检测 LLP 的荧光图像。比例尺：20 μm。 * P≤0.05 ； ** P≤0.01 ； *** P≤0.001 ； **** P ≤ 0.0001。当比较 2 个以上的组时，使用 ANOVA 检验。所有活力实验均一式三份进行。
Import of cysteine into lysosomes by MFSD12 attenuates lysosomal cell death.
MFSD12 将半胱氨酸导入溶酶体可减轻溶酶体细胞死亡。
Of the 3 lipid peroxidation inhibitors, only NAC prevented LLP. A recent report showed that major facilitator superfamily domain containing 12 (MFSD12) is an essential component of the cysteine importer for lysosomes and melanosomes (27). We reasoned that NAC, or its metabolite cysteine, is transported into the lysosome through MFSD12, where cysteine gets oxidized to its disulfide, cystine. To test this hypothesis, we compared the ability of NAC to rescue DC661 cytotoxicity in siNT and siMFSD12 A375P-hGal3-EGFP cells. Our results showed that NAC rescued DC661-induced LMP in siNT cells but did not rescue LMP in siMFSD12 cells (Figure 6A). NAC reduced LLP of DC661-treated siNT-A375P cells but not siMFSD12 cells. siMFSD12 cells treated with DMSO or NAC had increased basal LLP (Figure 6B) compared with siNT cells. While NAC rescued DC661 cytotoxicity in siNT cells, the ability of NAC to rescue DC661 cytotoxicity was completely abrogated in siMSFD12 cells (Figure 6C). This showed that MFSD12 knockdown blocks the cytoprotective effects of NAC against DC661. NAC is deacetylated by acylase in the cytosol (28). To determine whether NAC treatment produces an accumulation of cysteine or cystine within lysosomes, we employed the lysosomal immunoprecipitation (Lyso-IP) technique (29) to purify lysosomes from DMSO and NAC-treated A375P cells (Figure 6D and Supplemental Figure 9A) and performed targeted metabolomics to quantify NAC and related metabolites. As expected, NAC was detected in NAC-treated whole-cell lysate and associated unbound fractions. L-Cysteine levels were substantially increased within NAC-treated lysosomes. L-Cystine was only detectable within NAC-treated lysosomes. Strikingly, we did not find a significant increase in glutathione (reduced) in the NAC-treated groups (Figure 6E); this was surprising because it is commonly believed that NAC treatment induces increased production of glutathione, correcting redox balance. These results suggest that cysteine imported into lysosomes through the MFSD12 importer is critical for rescuing LLP, LMP, and lysosomal cell death.
在 3 种脂质过氧化抑制剂中，只有 NAC 可以预防 LLP。最近的一份报告表明，含有 12 (MFSD12) 的主要促进子超家族结构域是溶酶体和黑素体半胱氨酸输入蛋白的重要组成部分。 27 ）。我们推测 NAC 或其代谢物半胱氨酸通过 MFSD12 转运至溶酶体，其中半胱氨酸被氧化为其二硫键胱氨酸。为了检验这一假设，我们比较了 NAC 在 siNT 和 si MFSD12 A375P-hGal3-EGFP 细胞中拯救 DC661 细胞毒性的能力。我们的结果表明，NAC 拯救了 siNT 细胞中 DC661 诱导的 LMP，但没有拯救 si MFSD12细胞中的 LMP（ Figure 6A ）。 NAC 降低 DC661 处理的 siNT-A375P 细胞的 LLP，但不降低 si MFSD12细胞。用 DMSO 或 NAC 处理的 si MFSD12细胞的基础 LLP 增加（ Figure 6B ）与siNT细胞相比。虽然 NAC 在 siNT 细胞中挽救了 DC661 细胞毒性，但在siMSFD12细胞中 NAC 挽救 DC661 细胞毒性的能力被完全消除（ Figure 6C ）。这表明MFSD12敲低阻断了 NAC 对 DC661 的细胞保护作用。 NAC 被细胞质中的酰基酶脱乙酰化（ 28 ）。为了确定 NAC 处理是否会在溶酶体内产生半胱氨酸或胱氨酸的积累，我们采用了溶酶体免疫沉淀 (Lyso-IP) 技术（ 29 ）从 DMSO 和 NAC 处理的 A375P 细胞中纯化溶酶体（ Figure 6D 和 Supplemental Figure 9A ）并进行靶向代谢组学来量化 NAC 和相关代谢物。正如预期的那样，在 NAC 处理的全细胞裂解物和相关的未结合部分中检测到了 NAC。 NAC 处理的溶酶体中 L-半胱氨酸水平显着增加。 L-胱氨酸只能在 NAC 处理的溶酶体中检测到。 引人注目的是，我们没有发现 NAC 治疗组的谷胱甘肽（减少）显着增加（ Figure 6E ）；这是令人惊讶的，因为人们普遍认为 NAC 治疗会诱导谷胱甘肽的产生增加，从而纠正氧化还原平衡。这些结果表明，半胱氨酸通过 MFSD12 输入蛋白输入溶酶体对于挽救 LLP、LMP 和溶酶体细胞死亡至关重要。
Figure 6. NAC reverses LLP in an MFSD12-dependent manner.
图 6. NAC 以依赖 MFSD12 的方式反转 LLP。
(A–C) A375P-galectin-3-GFP cells or A375P cells were treated with MFSD12 siRNA or nontarget siRNA (siNT) for 48 hours, followed by treatment with DMSO, 3 μM DC661, or 10 mM NAC for 24 hours, 6 hours, or 72 hours. (A) Quantification of galectin-3 puncta in A375P-galectin-3-GFP cells after 24 hours. Galectin-3–positive puncta are shown with white arrows. (B) Fluorescence images of A375P cells stained with FOAM-LPO (1 μM, 5 min) to detect LLP after 6 hours. (C) Trypan blue cell viability in A375P cells after 72 hours of treatment with DC661, NAC, or both. (D) Schematic of lysosomal pull down using Lyso-IP. (E) Relative quantification of metabolites in whole-cell lysates (WCL), lysosomal IP unbound fractions (UB), and lysosomal IP bound samples (Lyso IP) with NAC or vehicle treatment after 24 hours. Total peak area accounts for metabolite abundance in the entire sample. Quantifications are depicted as mean ± SD from 3 biological replicates per condition. Scale bar: 20 μm. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001. ANOVA test was used when more than 2 groups were compared.
( A – C ) A375P-galectin-3-GFP 细胞或 A375P 细胞用MFSD12 siRNA 或非靶标 siRNA (siNT) 处理 48 小时，然后用 DMSO、3 μM DC661 或 10 mM NAC 处理 24 小时，6小时，或 72 小时。 ( A ) 24 小时后 A375P-galectin-3-GFP 细胞中 galectin-3 斑点的定量。 Galectin-3 阳性斑点用白色箭头显示。 ( B ) A375P 细胞用 FOAM-LPO（1 μM，5 分钟）染色 6 小时后检测 LLP 的荧光图像。 ( C ) 用 DC661、NAC 或两者处理 72 小时后 A375P 细胞中的台盼蓝细胞活力。 ( D ) 使用 Lyso-IP 进行溶酶体下拉的示意图。 ( E ) 24 小时后，用 NAC 或载体处理全细胞裂解物 (WCL)、溶酶体 IP 未结合部分 (UB) 和溶酶体 IP 结合样品 (Lyso IP) 中代谢物的相对定量。总峰面积代表整个样品中代谢物的丰度。定量描述为每个条件 3 次生物学重复的平均值±SD。比例尺：20 μm。 * P≤0.05 ； ** P≤0.01 ； **** P ≤ 0.0001。当比较 2 个以上的组时，使用 ANOVA 检验。
LLP is immunogenic. LLP 具有免疫原性。
Some forms of regulated cell death induced by DC661 (pyroptosis, necroptosis, ferroptosis) have been identified as immunogenic. Previously, immunogenic cell death has been described for chemotherapy drugs based on characteristic features, which include display of damage-associated molecular patterns, including cell surface expression of CALR and release of HMGB1 protein and adenosine triphosphate (ATP) (30). CALR acts as an “eat-me” signal when exposed on cell surfaces during cell stress. Extracellular release of HMGB1 and ATP acts as a “find-me” signal recognized by phagocytic cells. We compared two models: B16F10 cells, which in syngeneic tumor models are almost completely devoid of tumor-infiltrating lymphocytes, and MC38 cells, which in syngeneic tumor models do have tumor-infiltrating lymphocytes. First, we demonstrated that DC661 or siPpt1 treatment significantly induces surface CALR expression that can be completely abrogated with NAC cotreatment in MC38 cells (Figures 7, A and B). Similar findings were observed in B16F10 cells (Figure 7C). Inhibitors of major cell death pathways (apoptosis, necroptosis, ferroptosis, and pyroptosis) failed to prevent the surface expression of CALR (Figure 7D). To determine if immunogenic cell death induced by DC661 is due to MHC class I upregulation, B16F10 and MC38 cells were treated with DC661 (3 μM) or DMSO for 24 hours. We found that DC661 treatment did not increase the expression of MHC class I and immunoproteasome upregulation (Figure 7E and Supplemental Figure 9, B and C).
DC661 诱导的某些形式的调节性细胞死亡（细胞焦亡、坏死性凋亡、铁死亡）已被确定为具有免疫原性。此前，化疗药物的免疫原性细胞死亡是根据特征描述的，其中包括损伤相关分子模式的显示，包括细胞表面 CALR 的表达以及 HMGB1 蛋白和三磷酸腺苷 (ATP) 的释放。 30 ）。当细胞应激期间暴露在细胞表面时，CALR 充当“吃我”信号。 HMGB1 和 ATP 的细胞外释放充当吞噬细胞识别的“找到我”信号。我们比较了两种模型：B16F10 细胞（在同基因肿瘤模型中几乎完全没有肿瘤浸润淋巴细胞）和 MC38 细胞（在同基因肿瘤模型中确实具有肿瘤浸润淋巴细胞）。首先，我们证明 DC661 或 si Ppt1处理显着诱导表面 CALR 表达，而这种表达可以通过 NAC 共处理在 MC38 细胞中完全消除（ Figures 7, A and B ）。在 B16F10 细胞中也观察到类似的结果（ Figure 7C ）。主要细胞死亡途径（细胞凋亡、坏死性凋亡、铁死亡和细胞焦亡）的抑制剂未能阻止 CALR 的表面表达。 Figure 7D ）。为了确定 DC661 诱导的免疫原性细胞死亡是否是由于 MHC I 类上调所致，用 DC661 (3 μM) 或 DMSO 处理 B16F10 和 MC38 细胞 24 小时。我们发现 DC661 治疗并没有增加 MHC I 类表达和免疫蛋白酶体上调（ Figure 7E 和 Supplemental Figure 9 、B 和 C)。
Figure 7. N-acetyl cysteine prevents DC661-induced calreticulin surface expression.
图 7.N-乙酰半胱氨酸阻止 DC661 诱导的钙网蛋白表面表达。
(A–D) Flow cytometry for calreticulin (CALR) and propidium iodide (PI), with quantification of 2–3 independent experiments. (A) Murine MC38 cells were treated with 3 μM DC661, 10 mM N-acetyl cysteine (NAC), or both for 24 hours. (B) MC38 cells were treated with siPpt11 or siNT for 48 hours in the presence or absence of 10 mM NAC for 24 hours. (C) Murine B16F10 cells were treated with 3 μM DC661, 10 mM NAC, or both for 24 hours. (D) MC38 cells were treated with cell death inhibitors (40 μM Z-VAD-FMK, 50 μM Nec-1, 2 μM Liprox-1) with and without 3 μM DC661 for 24 hours. (E) Immunoblots of MHC class I, PSMB9, PSMB8, and β-actin in the lysates of murine B16F10 and MC38 cells treated with indicated concentrations of DC661 for 6 and 24 hours. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. ANOVA test was used when more than 2 groups were compared.
( A – D ) 钙网蛋白 (CALR) 和碘化丙啶 (PI) 的流式细胞术，对 2-3 个独立实验进行定量。 ( A ) 用 3 μM DC661、10 mM N-乙酰半胱氨酸 (NAC) 或两者处理小鼠 MC38 细胞 24 小时。 ( B ) 在存在或不存在 10 mM NAC 的情况下，用 si Ppt11或 siNT 处理 MC38 细胞 48 小时，处理 24 小时。 ( C ) 用 3 μM DC661、10 mM NAC 或两者处理鼠 B16F10 细胞 24 小时。 ( D ) 用细胞死亡抑制剂（40 μM Z-VAD-FMK、50 μM Nec-1、2 μM Liprox-1）在有或没有 3 μM DC661 的情况下处理 MC38 细胞 24 小时。 ( E ) 用指定浓度的 DC661 处理 6 小时和 24 小时的小鼠 B16F10 和 MC38 细胞裂解物中 MHC I 类、PSMB9、PSMB8 和 β-肌动蛋白的免疫印迹。 * P≤0.05 ； ** P≤0.01 ； *** P≤0.001 ； **** P ≤ 0.0001。当比较 2 个以上的组时，使用 ANOVA 检验。
To understand the effects of autophagy inhibition on T cell priming, we performed an in vitro priming and coculture experiment using C57BL6/J splenocytes as previously described (31). For priming, we exposed splenocytes to DC661- or DMSO-treated B16F10 or MC38 cells. Next, these primed splenocytes were cultured with live B16F10 or MC38 cells, and cytotoxicity was measured (Figure 8A). Splenocytes primed with DC661-treated B16F10 cells produced a significant increase in IFN-γ compared with splenocytes exposed to DMSO-treated B16F10 cells. Primed T cell–mediated killing of proliferating B16F10 cells was significantly increased in DC661-primed splenocytes when compared with DMSO-primed splenocytes (Figure 8, B and C). MC38 cells treated with DC661 produced even more IFN-γ and primed T cell cytotoxicity when compared with B16F10 cells (Figure 8, D and E). NAC cotreatment with DC661 was able to completely abrogate IFN-γ release from splenocytes and blunt primed T cell cytotoxicity (Figure 8, F and G). Knockdown of calreticulin by siCalr in MC38 cells (Supplemental Figure 9D) abrogated the priming efficacy of DC661 treatment and significantly reduced primed T cell cytotoxicity (Figure 8, H and I). Taken together, these data support a mechanistic role of LLP-associated CALR upregulation in promoting antitumor cell T cell immunity.
为了了解自噬抑制对 T 细胞启动的影响，我们使用 C57BL6/J 脾细胞进行了体外启动和共培养实验，如前所述（ 31 ）。为了启动，我们将脾细胞暴露于 DC661 或 DMSO 处理的 B16F10 或 MC38 细胞。接下来，将这些引发的脾细胞与活 B16F10 或 MC38 细胞一起培养，并测量细胞毒性（ Figure 8A ）。与暴露于DMSO处理的B16F10细胞的脾细胞相比，用DC661处理的B16F10细胞引发的脾细胞产生的IFN-γ显着增加。与 DMSO 引发的脾细胞相比，DC661 引发的脾细胞中引发的 T 细胞介导的对增殖 B16F10 细胞的杀伤显着增加（ Figure 8, B and C ）。与 B16F10 细胞相比，用 DC661 处理的 MC38 细胞产生更多的 IFN-γ 和引发的 T 细胞细胞毒性（ Figure 8, D and E ）。 NAC 与 DC661 共治疗能够完全消除脾细胞的 IFN-γ 释放并减弱引发的 T 细胞的细胞毒性（ Figure 8, F and G ）。 siCalr在 MC38 细胞中敲低钙网蛋白（ Supplemental Figure 9D ）消除了 DC661 治疗的引发功效并显着降低了引发的 T 细胞的细胞毒性（ Figure 8, H and I ）。总而言之，这些数据支持 LLP 相关的 CALR 上调在促进抗肿瘤细胞 T 细胞免疫中的机制作用。
Figure 8. DC661-induced calreticulin surface expression primes T cells against tumor cells.
图 8. DC661 诱导的钙网蛋白表面表达引发 T 细胞对抗肿瘤细胞。
(A) Schema of experimental setup of splenocyte priming and coculture experiments to measure cytotoxicity in vitro by DMSO or DC661 treatment for 24 hours. (B and D) Measurement of splenocyte-secreted IFN-γ upon coculturing syngeneic splenocytes with B16F10 or MC38 cells treated with DMSO or DC661. (C and E) Measurement of percentage cytotoxicity (LDH release assay) of B16F10 or MC38 cells by DMSO- or DC661-primed splenocytes. (F and G) MC38 cells were treated with 3 μM DC661, 10 mM NAC, or both for 24 hours and then used to prime splenocytes that were used for the cytotoxicity assay. IFN-γ and percentage cytotoxicity are shown. (H and I) For calreticulin (Calr) genetic inhibition, MC38 cells were treated with calreticulin/calregulin (Calr) siRNA or nontarget siRNA (siNT) for 48 hours, followed by treatment with either DMSO or 3 μM DC661 for 24 hours. These cells were then applied to the T cell priming and cytotoxicity assay. Measurement of IFN-γ and percentage cytotoxicity are shown. All experiments were done in triplicate. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. ANOVA test was used when more than 2 groups were compared.
( A ) 脾细胞引发和共培养实验的实验装置示意图，通过 DMSO 或 DC661 处理 24 小时来测量体外细胞毒性。 ( B和D ) 将同源脾细胞与用 DMSO 或 DC661 处理的 B16F10 或 MC38 细胞共培养后测量脾细胞分泌的 IFN-γ。 （ C和E ）通过 DMSO 或 DC661 引发的脾细胞测量 B16F10 或 MC38 细胞的细胞毒性百分比（LDH 释放测定）。 ( F和G ) MC38 细胞用 3 μM DC661、10 mM NAC 或两者处理 24 小时，然后用于灌注用于细胞毒性测定的脾细胞。显示了 IFN-γ 和细胞毒性百分比。 ( H和I ) 对于钙网蛋白 ( Calr ) 基因抑制，用钙网蛋白/钙调节蛋白( Calr ) siRNA 或非靶标 siRNA (siNT) 处理 MC38 细胞 48 小时，然后用 DMSO 或 3 μM DC661 处理 24 小时。然后将这些细胞应用于 T 细胞引发和细胞毒性测定。显示了 IFN-γ 的测量结果和细胞毒性百分比。所有实验一式三份进行。 * P≤0.05 ； ** P≤0.01 ； *** P≤0.001 ； **** P ≤ 0.0001。当比较 2 个以上的组时，使用 ANOVA 检验。
Next, we expanded these in vitro findings to an in vivo antitumor vaccination study in immunocompetent and immunodeficient mice models. For this assay, in vitro freeze-thawed or DC661-treated B16F10 or MC38 cells were injected s.c. on the left flank to protect immunocompetent C57BL/6 or NOD/SCID mice against rechallenge with live tumor cells of the same kind injected 7 days later into the right flank (Figure 9A). DC661-treated B16F10 cells failed to prevent tumor rejection despite in vitro assays, suggesting that DC661 induced immunogenic cell death (Figure 9B and Supplemental Figure 9E). In contrast, inoculation of one flank with DC661-treated MC38 cells promoted complete rejection of live MC38 cells implanted on the opposite flank (Figure 9C and Supplemental Figure 9F). To determine if adaptive immunity was critical for the vaccine effect DC661 seemed to have with MC38 tumors, we repeated the experiment in NOD/SCID mice. Strikingly, we found that DC661-treated MC38 cells did not induce rejection of rechallenge tumors in immunodeficient NOD/SCID mice (Figure 9D and Supplemental Figure 9G), indicating that adaptive immunity was required for the observed vaccine effect. To test the robustness of this finding, we selected another well-known immunogenic mouse cancer cell line, CT26, and performed the vaccination experiment as above. As expected, implantation of DC661-treated CT26 cells in one flank of the mouse produced a vaccine-like effect and prevented the outgrowth of live CT26 cells implanted on the other flank (Figure 9E and Supplemental Figure 9H). Our findings suggested that DC661-induced LLP is critical for LMP-mediated immunogenic cell death, which is reversed by the import of cysteine into lysosome by lysosomal transporter MFSD12 (Figure 9F).
接下来，我们将这些体外研究结果扩展到免疫功能正常和免疫缺陷小鼠模型的体内抗肿瘤疫苗接种研究。对于该测定，将体外冻融或 DC661 处理的 B16F10 或 MC38 细胞皮下注射到左侧，以保护免疫活性 C57BL/6 或 NOD/SCID 小鼠免受 7 天后注射的同类活肿瘤细胞的再次攻击。右翼（ Figure 9A ）。尽管进行了体外试验，但 DC661 处理的 B16F10 细胞未能阻止肿瘤排斥，这表明 DC661 诱导免疫原性细胞死亡。 Figure 9B 和 Supplemental Figure 9E ）。相比之下，用 DC661 处理的 MC38 细胞接种一侧，促进了植入另一侧的活 MC38 细胞的完全排斥。 Figure 9C 和 Supplemental Figure 9F ）。为了确定适应性免疫是否对 DC661 对 MC38 肿瘤的疫苗效果至关重要，我们在 NOD/SCID 小鼠中重复了该实验。引人注目的是，我们发现 DC661 处理的 MC38 细胞不会诱导免疫缺陷 NOD/SCID 小鼠对再攻击肿瘤的排斥反应（ Figure 9D 和 Supplemental Figure 9G ），表明观察到的疫苗效果需要适应性免疫。为了测试这一发现的稳健性，我们选择了另一种众所周知的免疫原性小鼠癌细胞系CT26，并进行了如上所述的疫苗接种实验。正如预期的那样，将 DC661 处理的 CT26 细胞植入小鼠的一侧，产生了类似疫苗的效果，并阻止了植入另一侧的活 CT26 细胞的生长。 Figure 9E 和 Supplemental Figure 9H ）。我们的研究结果表明，DC661 诱导的 LLP 对于 LMP 介导的免疫原性细胞死亡至关重要，而溶酶体转运蛋白 MFSD12 将半胱氨酸导入溶酶体可逆转这种死亡。 Figure 9F ）。
Figure 9. Inoculation of DC661-treated cells produces tumor rejection in specific contexts.
图 9. 接种 DC661 处理的细胞在特定情况下会产生肿瘤排斥。
(A) Schema of tumor vaccination model. (B–E) Cells were treated with DMSO or DC661 for 36 hours and then s.c. injected (1.8 × 105 B16F10 cells, 1.5 × 106 MC38 cells, or 3.0 × 106 CT26 cells per mouse) into the left flank of immunocompetent syngeneic C57BL/6J, BALB/c mice, or immunodeficient NOD/SCID mice. Freeze-thawed (F/T) DMSO-treated cells were used as control. One week later, all mice were rechallenged and s.c. injected with live untreated cells (3 × 104 B16F10 cells, 2 × 105 MC38 cells, or 5 × 105 CT26 cells per mouse) into the right flank of corresponding vaccinated mice. Dot plot of final tumor volumes for each individual mouse in each treatment group shown. n = 4–10 per group. (F) Illustration of DC661-induced lysosomal lipid peroxidation and immunogenic cell death.
( A )肿瘤疫苗接种模型的示意图。 ( B – E ) 将细胞用 DMSO 或 DC661 处理 36 小时，然后皮下注射（每只小鼠 1.8 × 10 5 B16F10 细胞、1.5 × 10 6 MC38 细胞或 3.0 × 10 6 CT26 细胞）至免疫活性细胞的左侧同基因 C57BL/6J、BALB/c 小鼠或免疫缺陷小鼠NOD/SCID 小鼠。冻融 (F/T) DMSO 处理的细胞用作对照。一周后，对所有小鼠进行重新攻击，并将未处理的活细胞（每只小鼠 3 × 10 4 B16F10 细胞、2 × 10 5 MC38 细胞或 5 × 10 5 CT26 细胞）皮下注射到相应疫苗接种小鼠的右侧。显示每个治疗组中每只小鼠的最终肿瘤体积的点图。每组n = 4–10。 ( F ) DC661 诱导的溶酶体脂质过氧化和免疫原性细胞死亡的图示。
Discussion 讨论
Lysosomal inhibition appears to be a promising therapeutic approach in preclinical studies, and HCQ clinical trials have produced encouraging but mixed results (1, 32). PPT1 is the molecular target of HCQ, and more-potent PPT1 inhibitors, such as DC661 (2) and GNS561 (3), induce LMP-mediated cell death in vitro. The precise mechanism of lysosomal cell death and its role in tumor immunogenicity have not been fully elucidated.
溶酶体抑制似乎是临床前研究中一种有前途的治疗方法，HCQ 临床试验已经产生了令人鼓舞但好坏参半的结果（ 1 , 32 ）。 PPT1 是 HCQ 的分子靶点，还有更有效的 PPT1 抑制剂，例如 DC661（ 2 ）和GNS561（ 3 ），在体外诱导 LMP 介导的细胞死亡。溶酶体细胞死亡的确切机制及其在肿瘤免疫原性中的作用尚未完全阐明。
Antitumor immunity is enhanced when autophagy inhibition is combined with immunotherapy (9, 10, 31). Proposed mechanisms for cell-intrinsic immunogenicity following autophagy inhibition include MHC class I and immunoproteasome upregulation, which both support improved antigen processing and presentation. In addition, loss of autophagy proteins or autophagy inhibition by chloroquine augmented CD8+ T cell response by increasing surface levels of MHC class I in dendritic cells (33). We previously showed that systemic PPT1 inhibition can repolarize macrophages from an M2 to M1 phenotype. PPT1 inhibitors can enhance STING levels, leading to IFN release and augmentation of T cell–mediated killing in melanoma models (31). Here, we demonstrated that LLP itself produces a tumor cell–intrinsic immunogenic form of cell death.
当自噬抑制与免疫治疗相结合时，抗肿瘤免疫力会增强（ 9 , 10 , 31 ）。所提出的自噬抑制后细胞内在免疫原性的机制包括 I 类 MHC 和免疫蛋白酶体上调，它们都支持改善抗原加工和呈递。此外，自噬蛋白的丧失或氯喹对自噬的抑制可通过增加树突状细胞中 MHC I 类的表面水平来增强 CD8 + T 细胞反应。 33 ）。我们之前表明，系统性 PPT1 抑制可以使巨噬细胞从 M2 表型重新极化为 M1 表型。 PPT1 抑制剂可以增强 STING 水平，导致黑色素瘤模型中 IFN 释放和 T 细胞介导的杀伤作用增强（ 31 ）。在这里，我们证明了 LLP 本身会产生肿瘤细胞——细胞死亡的内在免疫原性形式。
We found that lysosomal inhibition induced very few protein changes in cancer cells, and the most significantly elevated proteins included autophagy cargo receptors, apoptosis regulators. Our approach targeting some of these genes across functions demonstrated that drug-induced proteins were not likely the main regulators of cell death. Genetic inhibition of ULK1 or ATG7 also did not rescue DC661 cytotoxicity. We demonstrated that lysosomal inhibition activates multiple forms of programmed cell death, including apoptosis, necroptosis, ferroptosis, and pyroptosis, but each of these was dispensable for drug-induced cytotoxicity. Of note, programmed cell death mechanisms can overlap and cotargeting of multiple cell death mechanisms could provide cytoprotection against cell death following lysosomal membrane permeabilization. Our work has ruled out cathepsin- and calcium-dependent mechanisms for lysosomal cell death and highlighted the importance of LLP as the critical determinant of cell death. We found evidence of LLP that was reversible by an antioxidant that was transported into the lysosome, NAC, and was critical for lysosomal membrane permeabilization and cytotoxicity. NAC was the only agent able to mitigate or reverse DC661-induced cell death, and this capacity was dependent on the presence of the lysosomal cysteine transporter MFSD12. A lack of lysosomal penetration is likely why other putative lipid peroxidation inhibitors, Trolox and vitamin C, were unable to prevent DC661 cytotoxicity. NAC is converted to cysteine, which is imported into the lysosomes and oxidizes to its disulfide form, cystine. Cystine is exported to the cytosol by another lysosomal transporter, cystinosin, where it is reduced to cysteine that remobilizes internal nutrient sources, reactivates target of rapamycin complex 1, and promotes autophagy (34). This oxidation-reduction cycling of cysteine to cystine (lysosomes) and back to cysteine (cytosol) could be a possible rescue mechanism of NAC against DC661 cytotoxicity or more general lysosomal injury across other disease contexts where NAC has proven to be useful therapeutically (35). NAC not only reversed DC661-induced LLP and LMP, but also surface expression of the immunogenic cell death marker calreticulin. Cell surface expression of CALR protein was required for the enhanced T cell–mediated cytotoxicity induced by DC661-primed splenocytes, demonstrating that lysosomal inhibition produces a specific form of cell-intrinsic immunogenicity.
我们发现溶酶体抑制在癌细胞中引起的蛋白质变化非常少，最显着升高的蛋白质包括自噬货物受体、凋亡调节因子。我们针对其中一些跨功能基因的方法表明，药物诱导的蛋白质不太可能是细胞死亡的主要调节因子。 ULK1或ATG7的基因抑制也不能挽救 DC661 的细胞毒性。我们证明，溶酶体抑制可激活多种形式的程序性细胞死亡，包括细胞凋亡、坏死性凋亡、铁死亡和细胞焦亡，但其中每一种对于药物诱导的细胞毒性都是可有可无的。值得注意的是，程序性细胞死亡机制可以重叠，并且多种细胞死亡机制的共同靶向可以提供细胞保护，防止溶酶体膜透化后的细胞死亡。我们的工作排除了溶酶体细胞死亡的组织蛋白酶和钙依赖性机制，并强调了 LLP 作为细胞死亡关键决定因素的重要性。我们发现了 LLP 的证据，该 LLP 可以被转运到溶酶体 NAC 的抗氧化剂逆转，并且对溶酶体膜通透性和细胞毒性至关重要。 NAC 是唯一能够减轻或逆转 DC661 诱导的细胞死亡的药物，这种能力取决于溶酶体半胱氨酸转运蛋白 MFSD12 的存在。缺乏溶酶体渗透可能是其他假定的脂质过氧化抑制剂（Trolox 和维生素 C）无法防止 DC661 细胞毒性的原因。 NAC 转化为半胱氨酸，半胱氨酸被输入溶酶体并氧化为其二硫键形式胱氨酸。 胱氨酸通过另一种溶酶体转运蛋白胱氨酸转运至胞质溶胶，在那里被还原为半胱氨酸，重新调动内部营养源，重新激活雷帕霉素复合物 1 的靶标，并促进自噬。 34 ）。这种半胱氨酸到胱氨酸（溶酶体）再回到半胱氨酸（胞质溶胶）的氧化还原循环可能是 NAC 对抗 DC661 细胞毒性或更普遍的溶酶体损伤的一种可能的救援机制，在其他疾病中，NAC 已被证明在治疗上有用。 35 ）。 NAC不仅逆转DC661诱导的LLP和LMP，而且还逆转免疫原性细胞死亡标记物钙网蛋白的表面表达。 CALR 蛋白的细胞表面表达是 DC661 引发的脾细胞诱导的增强 T 细胞介导的细胞毒性所必需的，这表明溶酶体抑制产生特定形式的细胞内在免疫原性。
While previous studies have demonstrated that lysosomal inhibition can enhance the antitumor activity of immune checkpoint inhibition in established flank tumors (9, 31), our study is the first to our knowledge to demonstrate a vaccine-like effect for MC38 tumors but not B16 tumors in tumor cells pretreated with DC661 prior to implantation. This demonstrates that lysosomal cell death can induce cell-intrinsic immunogenicity, but these changes by themselves are not likely enough to reverse an “immune cold” tumor microenvironment into an “immune hot” tumor microenvironment. Our previous studies in “immune cold” tumor microenvironment models B16 and the BRafCA PtenloxP Tyr:CreERT2 genetically engineered mouse models (31) demonstrated that systemic lysosomal inhibition produced effects on tumor-associated macrophages and myeloid cell–derived suppressor cells that were sufficient to enhance the efficacy of immunotherapy. It may be the case that cell-intrinsic immunogenicity also plays a role in these immune cold tumors that become more responsive to ICD following lysosomal inhibition. The vaccine-like effect of lysosomal inhibition observed in a controlled laboratory environment may or may not translate into the clinic, but it could explain why some patients treated with dabrafenib, trametinib, and HCQ in previously treated BRAF mutant melanoma had such a deep and durable response this regimen (32).
虽然之前的研究表明，溶酶体抑制可以增强免疫检查点抑制在已形成的侧翼肿瘤中的抗肿瘤活性（ 9 , 31 ），据我们所知，我们的研究是第一个证明在植入前用 DC661 预处理的肿瘤细胞对 MC38 肿瘤有类似疫苗的作用，但对 B16 肿瘤没有作用。这表明溶酶体细胞死亡可以诱导细胞固有的免疫原性，但这些变化本身不足以将“免疫冷”肿瘤微环境逆转为“免疫热”肿瘤微环境。我们之前对“免疫冷”肿瘤微环境模型 B16 和BRaf CA Pten loxP Tyr:CreER T2基因工程小鼠模型的研究（ 31 ）证明系统性溶酶体抑制对肿瘤相关巨噬细胞和骨髓细胞衍生的抑制细胞产生影响，足以增强免疫疗法的功效。细胞固有的免疫原性可能也在这些免疫冷肿瘤中发挥作用，这些冷肿瘤在溶酶体抑制后对 ICD 变得更加敏感。在受控实验室环境中观察到的溶酶体抑制的疫苗样作用可能会也可能不会转化为临床，但它可以解释为什么一些接受达拉非尼、曲美替尼和 HCQ 治疗的先前治疗过的BRAF突变黑色素瘤患者具有如此深刻和持久的效果响应该方案（ 32 ）。
Further study is needed to understand how PPT1 inhibition produces lysosomal ROS and lipid peroxidation. PPT1-dependent regulation of the V-ATPase and lysosomal acidification is not a sufficient explanation because bafilomycin inhibits lysosomal acidification but does not produce LMP (data not shown). The implications of these findings suggest that lysosomal inhibitors that enhance tumor cell–intrinsic immunogenicity can be rationally combined with therapies that enhance T cell activation, or infiltration into the tumor microenvironment, potentially yielding synergistic effects.
需要进一步研究来了解 PPT1 抑制如何产生溶酶体 ROS 和脂质过氧化。 V-ATP酶和溶酶体酸化的PPT1依赖性调节并不能充分解释，因为巴弗洛霉素抑制溶酶体酸化但不产生LMP（数据未显示）。这些发现的意义表明，增强肿瘤细胞内在免疫原性的溶酶体抑制剂可以与增强 T 细胞活化或浸润到肿瘤微环境的疗法合理结合，从而可能产生协同效应。
Methods 方法
Cell culture. 细胞培养。
Human A375P (CRL-3224), RKO (CRL-2577), DLD-1 (CCL-221), MIA PaCa-2 (CRL-1420), A549 (CRM-CCL-185), and mouse B16F10 (CRL-6475) cell lines were purchased from ATCC. Human A375 (CRL-1619, ATCC), WM35, WM793, and mouse YUMM1.7 (WT, Gsdme EV, and Gsdme KO1 and KO2) lines were obtained in-house. Mouse MC38 and CT26 cells were provided by Andy Minn, University of Pennsylvania. FL5.12 and IL-3–dependent Bax−/−Bak−/− (Bax/Bak DKO) primary bone marrow cells were obtained from Kathryn E. Wellen, University of Pennsylvania. The human Panc-1 (CRL-1469, ATCC) cell line was provided by Ben Z. Stanger, University of Pennsylvania. The mouse YUMMER 1.7 cell line was obtained from Xiaowei (George) Xu, University of Pennsylvania. All cell lines were tested for Mycoplasma biannually by University of Pennsylvania Core facilities and authenticated using short-tandem repeat fingerprinting by Wistar Institute Genomics core. A375P, RKO, DLD-1, A549, CT26, FL5.12, and Bax/Bak DKO cell lines were cultured in RPMI 1640 (Invitrogen, 11875); A375, MIA PaCa-2, Panc-1, B16F10, and MC38 cell lines were cultured in DMEM (Invitrogen, 11995); and YUMMER1.7, YUMM1.7 WT, Gsdme EV, and Gsdme KO1 and KO2) cells (15) were cultured in DMEM/F12 50/50 (Corning, 10-092-CV). Culture media were supplemented with 10% fetal bovine serum (12306C, MilliporeSigma) and 1× antibiotic antimycotic solution (Gibco, 15140-122). FL5.12 and Bax/Bak DKO cell lines were maintained in complete RPMI media supplemented with 50 μM β-mercaptoethanol (Life Technologies, 21985-023), 10 mM HEPES (H3537, MilliporeSigma), and 0.35 ng/mL and 3.5 ng/mL IL-3, respectively. YUMMER1.7 and YUMM1.7 (WT, Gsdme EV, and Gsdme KO) cell lines were maintained in complete media supplemented with 1× MEM NEAA (Gibco, 11140-050). WM35 and WM793 cells were cultured in MCDB media 153 (MilliporeSigma, M7403), containing 10% FBS in 1× Leibovitz L-15 medium (Corning, 10-045-CV), 7.5% w/v sodium bicarbonate (Corning, 25-035-CI), 1× antibiotic antimycotic solution (Gibco, 15140-122), and 5 μg/mL insulin (MilliporeSigma, I0516). Cells were grown at 37°C in the presence of 5% CO2.
人 A375P (CRL-3224)、RKO (CRL-2577)、DLD-1 (CCL-221)、MIA PaCa-2 (CRL-1420)、A549 (CRM-CCL-185) 和小鼠 B16F10 (CRL-6475) )细胞系购自ATCC。人 A375（CRL-1619、ATCC）、WM35、WM793 和小鼠 YUMM1.7（WT、 Gsdme EV、 Gsdme KO1 和 KO2）系是内部获得的。小鼠 MC38 和 CT26 细胞由宾夕法尼亚大学 Andy Minn 提供。 FL5.12 和 IL-3 依赖性Bax −/− Bak −/− ( Bax/Bak DKO) 原代骨髓细胞获自宾夕法尼亚大学 Kathryn E. Wellen。人类 Panc-1（CRL-1469，ATCC）细胞系由宾夕法尼亚大学 Ben Z. Stanger 提供。小鼠 YUMMER 1.7 细胞系获自宾夕法尼亚大学的Xiaowei (George) Xu。所有细胞系每年两次由宾夕法尼亚大学核心设施进行支原体检测，并由 Wistar 研究所基因组学核心使用短串联重复指纹进行验证。 A375P、RKO、DLD-1、A549、CT26、FL5.12 和Bax/Bak DKO 细胞系在 RPMI 1640 (Invitrogen, 11875) 中培养； A375、MIA PaCa-2、Panc-1、B16F10 和 MC38 细胞系在 DMEM (Invitrogen, 11995) 中培养；和 YUMMER1.7、YUMM1.7 WT、 Gsdme EV 以及Gsdme KO1 和 KO2）细胞（ 15 ）在 DMEM/F12 50/50（康宁，10-092-CV）中培养。培养基中补充有10%胎牛血清（12306C，MilliporeSigma）和1×抗生素抗真菌溶液（Gibco，15140-122）。 FL5.12 和Bax/Bak DKO 细胞系维持在补充有 50 μM β-巯基乙醇（Life Technologies, 21985-023）、10 mM HEPES（H3537，MilliporeSigma）以及 0.35 ng/mL 和 3.5 ng/的完全 RPMI 培养基中。分别为mL IL-3。 YUMMER1.7 和 YUMM1。7（WT、 Gsdme EV 和Gsdme KO）细胞系维持在补充有 1× MEM NEAA（Gibco，11140-050）的完全培养基中。 WM35 和 WM793 细胞在 MCDB 培养基 153（MilliporeSigma，M7403）中培养，该培养基在 1×Leibovitz L-15 培养基（Corning，10-045-CV）中含有 10% FBS，7.5% w/v 碳酸氢钠（Corning，25- 035-CI），1×抗生素抗真菌溶液（Gibco， 15140-122）和 5 μg/mL 胰岛素（MilliporeSigma，I0516）。细胞在37°C、5% CO 2存在下生长。
Chemicals and reagents. 化学品和试剂。
Chemicals purchased included HCQ Sulfate (Spectrum Chemicals, 747-36-4) and DC661 (Selleckchem, S8808). Fluo-4, AM (Thermo Fisher Scientific, F14201) was used to stain the cells for calcium as per the manufacturer’s instructions. A list of antibodies and inhibitors is provided in Supplemental Table 1 and Supplemental Table 2.
购买的化学品包括 HCQ 硫酸盐（Spectrum Chemicals，747-36-4）和 DC661（Selleckchem，S8808）。 Fluo-4，AM（赛默飞世尔科技， F14201 ）用于按照制造商的说明对细胞进行钙染色。抗体和抑制剂的列表提供于 Supplemental Table 1 和 Supplemental Table 2 。
Protein extraction and digestion for liquid chromatography–tandem mass spectrometry analysis for proteomics.
用于蛋白质组学的液相色谱-串联质谱分析的蛋白质提取和消化。
0.7 × 106 A375P melanoma cells were cultured in 60 mm culture dishes. Cells were treated with DMSO (control), DC661 (3 μM), HCQ (10 μM), or HCQ (30 μM) for 24 hours at approximately 50% confluence. Frozen cell pellets were lysed with 50 mM Tris pH 7.4, 1% SDS, 150 mM NaCl, 1 mM EDTA, 0.15 mM PMSF, 1 μg/mL pepstatin, and 1 μg/mL leupeptin. Clarified lysates (10 μg each) were electrophoresed 0.5 cm into a bis-tris gel followed by fixing and staining with colloidal Coomassie. Each 0.5 cm gel lane was excised, destained, reduced with tris (2-carboxyethyl) phosphine, alkylated with iodoacetamide, and digested with trypsin as described previously (36). Digests (1 μg) were analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) on a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) in-line with a nanoAQUITY UPLC (Waters). Analytical separation was performed on a 1.7 μm × 250 mm Peptide BEH C18 column (Waters, 186003546) using a 245-minute gradient with 0.1% formic acid in water (mobile phase A) and acetonitrile (mobile phase B) as follows: 5%–30% B over 225 minutes, 30%–80% B over 5 minutes, 15-minute hold at 80% B, and return to initial conditions. Full MS spectra were acquired at 70,000 resolution, with a scan range of 400–2,000 m/z, automatic gain control target of 3 × 106 ions, and maximum injection time of 50 milliseconds. Data-dependent MS2 spectra were acquired for the top 20 most abundant ions at 17,500 resolution, with an isolation width of 1.5 m/z, automatic gain control target of 5 × 104 ions, and maximum injection time of 50 milliseconds. Peptide match was set to preferred, and unassigned and singly charged ions were rejected (37).
0.7×10 6 A375P黑色素瘤细胞在60mm培养皿中培养。将细胞用 DMSO（对照）、DC661 (3 μM)、HCQ (10 μM) 或 HCQ (30 μM) 处理 24 小时，达到约 50% 汇合。使用 50 mM Tris pH 7.4、1% SDS、150 mM NaCl、1 mM EDTA、0.15 mM PMSF、1 μg/mL 胃酶抑素和 1 μg/mL 亮抑肽酶裂解冷冻细胞沉淀。将澄清的裂解物（每种 10 μg）电泳至 bis-tris 凝胶中 0.5 cm，然后用胶体考马斯胶固定和染色。将每个 0.5 cm 的凝胶泳道切除、脱色、用三（2-羧乙基）膦还原、用碘乙酰胺烷基化，并用胰蛋白酶消化，如前所述（ 36 ）。在与 nanoAQUITY UPLC (Waters) 联线的 Q-Exactive Plus 质谱仪 (Thermo Fisher Scientific) 上通过液相色谱-串联质谱 (LC-MS/MS) 分析消化物 (1 μg)。使用 0.1% 甲酸水溶液（流动相 A）和乙腈（流动相 B）在 1.7 μm × 250 mm 肽 BEH C18 柱（Waters，186003546）上进行 245 分钟梯度分析分离，如下：5% –30% B 持续 225 分钟，30%–80% B 持续 5 分钟，在 80% B 保持 15 分钟，然后返回初始条件。以 70,000 分辨率、400–2,000 m/z的扫描范围、3 × 10 6 个离子的自动增益控制目标和 50 毫秒的最大注射时间获取完整 MS 谱图。以 17,500 分辨率、隔离宽度为 1.5 m/z 、自动增益控制目标为 5 × 10 4离子、最大进样时间为 50 毫秒，获取前 20 个最丰富离子的数据相关 MS2 谱。肽匹配设置为首选，未分配的离子和单电荷离子被拒绝（ 37 ）。
Raw MS data were analyzed using MaxQuant 1.6.5.0 (https://maxquant.net/perseus/) with a Uniprot human sequence database (accessed on October 10, 2019) and a common contaminants database, including trypsin, keratins, bovine proteins, and mycoplasma (38). Tryptic peptide specificity with a maximum of 2 missed cleavages, fixed modification on cysteine (carbamidomethylation), and variable methionine oxidation or N-terminal acetylation were used in the search (39). A cutoff of 1% FDR was used for peptides and proteins. Match between runs was enabled; proteins were quantified using label-free quantitation (40).
使用 MaxQuant 1.6.5.0 分析原始 MS 数据（ https://maxquant.net/perseus/ ）以及 Uniprot 人类序列数据库（于 2019 年 10 月 10 日访问）和常见污染物数据库，包括胰蛋白酶、角蛋白、牛蛋白和支原体（ 38 ）。搜索中使用了最多 2 个缺失裂解的胰蛋白酶肽特异性、半胱氨酸的固定修饰（脲甲基化）以及可变的蛋氨酸氧化或 N 末端乙酰化（ 39 ）。对于肽和蛋白质，使用 1% FDR 的截止值。运行之间的匹配已启用；使用无标记定量法对蛋白质进行定量（ 40 ）。
Statistical analysis was performed using Perseus 1.6.2.3 (https://maxquant.net/perseus/) (41, 42). Proteins were required to be identified by at least 3 unique peptides and have 3 valid values (non-zero quantitation) within a sample group, and contaminants and reverse proteins were filtered from the data set. Missing values were imputed from a normal distribution. Pairwise comparisons between conditions were performed at the protein level using 2-tailed Student’s t test with permutation-based FDR with s0 = 0.1 and 250 randomizations. Significant changes were defined as FDR of less than 5% and an absolute fold change greater than 1.5 or 2.0, as specified.
使用Perseus 1.6.2.3进行统计分析（ https://maxquant.net/perseus/ ) ( 41 , 42 ）。要求蛋白质被至少3个独特的肽所识别，并且在样品组内具有3个有效值（非零定量），并且从数据集中过滤掉污染物和反向蛋白质。缺失值是根据正态分布估算的。使用 2 尾学生t检验和基于排列的 FDR（s0 = 0.1 和 250 次随机化）在蛋白质水平上进行条件之间的成对比较。根据规定，显着变化定义为 FDR 小于 5% 且绝对倍数变化大于 1.5 或 2.0。
Lyso-IP. 溶酶IP。
A375P cells were infected with pLJC5-Tmem192-3xHA lentivirus and selected using 1 mg/mL puromycin (MilliporeSigma, P4512). Approximately 3 × 106 HA tagged A375P cells were treated with either 10 mM NAC or water vehicle control for 24 hours in culture conditions previously described. After treatment, cells were washed in PBS, harvested in 0.5 mL cold KPBS, and gently homogenized using 20 strokes in a 2 mL Dounce homogenizer. About 2.5% of the homogenate was reserved for whole-cell lysate analysis, and the remainder was centrifuged at 3,000g for 2 minutes at 4°C to remove cell membrane debris. Homogenate supernatant was then transferred to a clean 1.5 mL tube and incubated with 50 μL anti-HA beads (Pierce, 88836) or anti-DDK/Flag beads (OriGene, TA150042) for 15 minutes at 4°C. Samples were then precipitated by placing tubes on DynaMag (Thermo Fisher Scientific, 12321D) and rocked gently for 2 minutes at room temperature. Supernatant was reserved for unbound fraction analysis. IP was washed 3 times with KPBS containing 8 mM CaCl2. The Lyso-IP was extracted in 80% MeOH for metabolomics analysis.
A375P 细胞用 pLJC5-Tmem192-3xHA 慢病毒感染，并使用 1 mg/mL 嘌呤霉素（MilliporeSigma，P4512）进行选择。将大约 3 × 10 6 HA 标记的 A375P 细胞在先前描述的培养条件下用 10 mM NAC 或水载体对照处理 24 小时。处理后，用 PBS 洗涤细胞，在 0.5 mL 冷 KPBS 中收获细胞，并在 2 mL Dounce 匀浆器中使用 20 次冲程轻轻匀浆。约 2.5% 的匀浆保留用于全细胞裂解液分析，其余部分在 4°C 下以 3,000 g离心 2 分钟以去除细胞膜碎片。然后将匀浆上清液转移至干净的 1.5 mL 管中，并与 50 μL 抗 HA 珠子（Pierce，88836）或抗 DDK/Flag 珠子（OriGene，TA150042）在 4°C 下孵育 15 分钟。然后将试管置于 DynaMag（Thermo Fisher Scientific，12321D）上并在室温下轻轻摇动 2 分钟来沉淀样品。保留上清液用于未结合级分分析。 IP用含有8mM CaCl 2的KPBS洗涤3次。用 80% MeOH 提取 Lyso-IP 用于代谢组学分析。
Lipid extraction and analysis using LC-MS/MS for global lipidome.
使用 LC-MS/MS 对整体脂质组进行脂质提取和分析。
Melanoma A375P cells were seeded in 60 mm dishes at 0.7 × 106 cells per dish. When cells were at approximately 50% confluence, they were treated with DMSO (control), DC661 (3 μM), or HCQ (30 μM) for 24 hours. Cells were washed 2 times with HBSS, scraped into ice-cold methanol, and transferred to glass tubes for lipid extraction with chloroform/methanol/0.88% NaCl (2:1:1) containing an EquiSPLASH internal lipid standard (Avanti Polar Lipids). Samples were vortexed and then water bath sonicated for 5 minutes on ice. After centrifugation at 500g for 15 minutes at 40°C, the lower phase was transferred to another glass tube. The upper phase was reextracted using a synthetic lower phase. After sonication and centrifugation, the lower phase was combined with the first lower phase collection. The samples were dried under nitrogen, resuspended in 10% chloroform/90% methanol, and transferred to glass LTQ vials.
将黑色素瘤 A375P 细胞以每皿 0.7 × 10 6 个细胞接种在 60 mm 培养皿中。当细胞达到约 50% 汇合时，用 DMSO（对照）、DC661 (3 μM) 或 HCQ (30 μM) 处理 24 小时。用 HBSS 洗涤细胞 2 次，刮入冰冷的甲醇中，然后转移至玻璃管中，用含有 EquiSPLASH 内部脂质标准品 (Avanti Polar Lipids) 的氯仿/甲醇/0.88% NaCl (2:1:1) 进行脂质提取。将样品涡旋，然后在冰上水浴超声处理 5 分钟。 40℃、 500g离心15分钟后，将下层相转移至另一玻璃管中。使用合成的下层相再萃取上层相。超声处理和离心后，将下层相与第一个下层相收集物合并。将样品在氮气下干燥，重新悬浮于 10% 氯仿/90% 甲醇中，然后转移至玻璃 LTQ 小瓶中。
Lipid samples were analyzed on a Thermo Fisher Scientific Q-Exactive HF-X mass spectrometer and Vanquish Horizon UHPLC system. The analytical separation used an Accucore C30 column (2.1 mm × 150 mm, Thermo Fisher Scientific) with 50:50 acetonitrile/water and 88:10:2 isopropanol/acetonitrile/water, each containing 5 mM ammonium formate and 0.1% formic acid. LC-MS/MS data were acquired separately in positive and negat