臺灣博碩士論文加值系統

節序性壞死（Necroptosis）一直以來被認為是藉由細胞所釋放損傷組織的相關分子圖譜（danger associated molecular patterns, DAMP）而引發高度免疫原性。在過去的各種研究中，節序性壞死的細胞中又經常發現它們伴隨著具高度自噬之作用，然而，此一增強的自噬作用對於節序性壞死中所產生之高度免疫原性作用中的可能作用在很大程度上卻仍然是我們尚未詳知的。在我的此一研究中，我們研究了植化物紫草素所誘導之自噬作用和紫草素誘導之節序性壞死所造成的腫瘤免疫原性之間可能的互動機制及其相關性。我們的結果顯示紫草素可以引發receptor-interacting protein kinase 1 （RIPK1）和receptor-interacting protein kinase 3 （RIPK3）之活性，進而造成節序性壞死，並且紫草素在相同濃度及處理時間下也增強了細胞內自噬作用。而我們結果亦顯示紫草素誘導的自噬作用可以促進節序性壞死細胞表面上之損傷相關分子圖譜的轉位（translocation）。有趣的是，在我們也發現中，只有紫草素誘導的節序性壞死細胞表面上之損傷相關分子圖譜能夠激活共同培養的樹突細胞（dendritic cells, DCs），而此活性並非由已釋放的損傷相關分子圖譜所造成。而當我們使用氯喹（chloroquine）來中斷了紫草素誘導的自噬作用之降解步驟時，紫草素誘導的表面上之DAMP可更進一步被用以增強以及更可激活樹突細胞活性的活化。對於潛在的臨床應用，我們初步結果發現，使用已經過氯喹和紫草素一起預先處理之腫瘤細胞製備的樹突細胞癌症疫苗製劑可以更有效地減少4T1小鼠乳癌腫瘤的轉移，並可更進一步降低阿黴素（doxorubicin）化療的有效治療劑量，因此透過紫草素和氯喹之共同處理的腫瘤細胞可望獲得更為增強的免疫原性以及抗癌疫苗效力，此舉也可提供未來使用組合藥物治療開發癌症疫苗的可能更有效治療之策略。

Programmed necrosis, necroptosis, is considered to be a highly immunogenic activity, often mediated via the release of damage-associated molecular patterns (DAMPs). Interestingly, enhanced autophagic activity is often found to be accompanied by necroptosis. However, the possible role of autophagy in the immunogenicity of necroptotic death remains largely obscure. In this study, we investigated the possible mechanistic correlation between phytochemical shikonin-induced autophagy and the shikonin-induced necroptosis for tumor immunogenicity. We show that shikonin could instigate receptor-interacting protein (RIPK)1- and RIPK3-dependent necroptosis that is accompanied by enhanced autophagy. Shikonin-induced autophagy could directly contribute to DAMP upregulation. Counterintuitively, among the released and ecto-DAMPs, only the latter were shown to be able to activate the co-cultured dendritic cells (DCs). Interruption of autophagic flux via chloroquine further upregulated ecto-DAMP activity and resultant DC activation. For potential clinical application, DC vaccine preparations treated with tumor cells that were already pre-treated with chloroquine and shikonin further enhanced the anti-metastatic activity of 4T1 tumors and reduced the effective dosage of doxorubicin. The enhanced immunogenicity and vaccine efficacy obtained via shikonin and chloroquine co-treatment of tumor cells may thus constitute a compelling strategy for developing cancer vaccines via the use of a combinational drug treatment.

Contents
Chapter 1. Introduction………………………….……………………………..1
1.1 Multiple functions of shikonin in pathological conditions…………..…1
1.2 Shikonin-induced cell death…………………………………………....3
1.3 Immunogenic cell death……………………...……………..…….……4
1.4 Necroptosis……………………………………...………………...……5
1.5 Immunogenicity in necroptosis…………………………………….....6
1.6 Functionality of autophagy………………….………………….…….7
1.7 Autophagy and cell death……………………………...……………….8
1.8 Dendritic cell based cancer vaccine………………….……………….10
Chapter 2. Specific aims and significance of this study……....….………..…12
Chapter 3. Materials & Methods………………………...…..…………….13
3.1 Cell lines……………………………………….…………………....13
3.2 Mice………………………………………………………………....13
3.3 Cell viability assay………………………………………………….13
3.4 Cell lysate preparation …………………...………………….…….….14
3.5 Western blotting……………………………...………………..…..….14
3.6 Knockdown experiment ………………………………..………….…15
3.7 Immunoprecipitation…………………….……………….………..….16
3.8 Detection of DAMP ecto-localization ……………………...……..….16
3.9 BM-DC preparation …………………………………………...…..….16
3.10 Assay for DC activation…………………………………..…….…….17
3.11 ELISA……………………………………………………………….18
3.12 Proteasome activity assay……………………...……………..……….18
3.13 Intracellular ROS level and measurement of mitochondria membrane potential……………………………………………….……………....19
3.14 Flow cytometry…………………………..…………………….……..19
3.15 Confocal microscopy………………….…………...………………….19
3.16 Construction of mpRFP-EGFP-LC3…………………...……………20
3.17 Transfection and stable pool generation…………..…………………..20
3.18 Construction of RIPK1 knockout cell line……………………………21
3.19 Animal model……………………………………....………….……21
3.20 Statistical analysis………………………..…….……………………..22
Chapter 4. Result……………………………………..…..……….…………23
4.1 Shikonin induced necroptosis in 4T1-luc2 tumor cells…………....…23
4.2 SK-treated 4T1-luc2 cells effectively immunized mice against primary tumors…………………………………………………..………..……25
4.3 SK-induced DAMP release and DAMP ecto-localization by 4T1-luc2 cells………………………………………………………...……….…27
4.4 Immunogenicity of SK-induced 4T1-luc2 ICD is in a cell-to-cell interaction dependent manner…………………………………….....28
4.5 SK-DC vaccine is effective in preventing metastasis………………30
4.6 SK Induced Autophagy in 4T1-luc2 cells………………..………...…32
4.7 SK-induced DAMP ecto-localization is closely associated with the enhanced autophagic activity……………………………..………..…33
4.8 SK + CQ benefited DC vaccine…………………………………......36
Chapter 5. Discussion…………………………...…...……………………39
Chapter 6. Conclusions……………………………….………………………48
Chapter 7. References………………………………………………………...49
Chapter 8. Table……………………………………………….…………….69
Chapter 9. Figures………………………………………..………………….72


Table of contents
Table 1. Abbreviation…………………………..………………………………56

Figure of contents
Figure 1. Chemical structure of shikonin……………….………………..…….73
Figure 2. Schematic of molecule mechanism for necroptosis………...…...... 74
Figure 3. Schematic of autophagy pathways……………………………....….75
Figure 4. The effect of SK on cell viability………………………………..…76
Figure 5. Effect of SK on enzyme markers for apoptosis.…..…………...…77
Figure 6. Effect of SK on activation of caspase 8 and PARP-1 expression…78
Figure 7. Effect of NEC-1, GSK’872, zVAD-fmk, and 3-MA on SK-induced cell death and TCZ (TNF+cycloheximide+zVAD-fmk)-induced necroptosis…..… 79
Figure 8. Efficacy of specific siRNAs for ATG5, BCN1, RIPK1, and RIPK3 in 4T1-luc2 tumor cells……………………………………………………………80
Figure 9. Effect of knocking down siATG5, siBECN1, siRIPK1, and siRIPK3 expression on SK-mediated cytotoxicity and TCZ-induced necroptosis………….…………………………………………………………81
Figure 10. Effect of SK on phosphorylation of MLKL in 4T1 tumor cells..…82
Figure 1 1. Subcellular morphology of SK-treated cells……......…………83
Figure 1 2. SK-treated 4T1-luc2 cells effectively immunized mice against primary tumors……..................................................................…………..……84
Figure 1 3. Immunization with SK-treated 4T1-luc2 cells did not prevent tumor metastasis……................................................................…………85
Figure 1 4. Effect of SK on the release of test DAMPs and LDHA from tumor cells…….........................................................................…………86
Figure 1 5. Imaging of DAMP ecto-localization in SK-treated cells................87
Figure 1 6. Time course and dosage effect of SK on 4T1 cells...………………88
Figure 1 7. Effect of SK on the release of test DAMPs from test tumor cells.…89
Figure 1 8. The dosage-dependent effect of SK on the ecto-localization of HSP70/90 and CRT………………………………………………….…………90
Figure 1 9. The effect of ecto-DAMPs versus the release of DAMPs on DC activation…………………………………………………………..…………91
Figure 20. Antibody blocking of ecto-DAMP expressions in SK-treated tumor cells suppressed DC activation……………...…………………………………93
Figure 21. The effect of SK-treated cells on expression of specific pro-inflammatory cytokines in DCs……………...…………….………………94
Figure 22. Effect of blocking ecto-DAMP expression via neutralizing antibodies on reducing SK-DC vaccine efficacy on tumor metastasis………………….…95
Figure 23. SK-DC vaccine is effective in preventing metastasis………….…97
Figure 24. Combination of SK-DC vaccine and doxorubicin is effective in preventing metastasis……………………………………………………....…98
Figure 25. Effect of SK on the expression of autophagy-related proteins in test cells………………………………………………...................................…99
Figure 26. Effect of SK on LC3 expression at the subcellular level………...100
Figure 27. Effect of SK on autophagic flux as visualized through the expression of mRFP-LC3-GFP……………………………………………...………….101
Figure 28. Subcellular morphology of SK-induced autophagy in test cells………………………………………………………..………….………102
Figure 29. Effect of knocking down ATG5, BECN1, RIPK1 and RIPK3 expression on SK-mediated DAMPs ecto-localization……………...….…….103
Figure 30. Effect of DAMP expression in immunoprecipitation of autophagosome through mRFP-GFP-LC3………………………..………..…104
Figure 31. The effect of CQ on SK-mediated DAMP ecto-localizations……..105
Figure 32. Deficient activities of autophagy and necroptosis hindered the efficacy of SK-treated tumor cells on DC activation………...……………….106
Figure 33. Effect of combinational treatment of SK and CQ in 4T1 tumor cells on the expression of CD40 and TLR4 on DCs………...…………………….107
Figure 34. Effect of SK on ROS generation in treated cells………………108
Figure 35. Effect of SK on expression of ubiquitinated proteins and 20S proteasome activities in test cells……………………………..............………109
Figure 36. The effect of ROS inhibition by NAC on SK-mediated cell death..110
Figure 37. Effect of SK on subcellular localization of ubiquinated proteins and p62 proteins, visualized by confocal microscopy…………………....………..111
Figure 38. Effect of NAC on SK-mediated ecto-DAMP expression, as determined by flow cytometric analysis……………………………...……….112
Figure 39. Effect of SK+CQ-treated tumor cells on phagocytosis function of DCs……………………………………………………………………………113
Figure 40. The effect of autophagy- and necroptosis-deficiency in SK-treated tumor cells on activation of DCs for vaccine efficacy……………………...114
Figure 41. SK + CQ treatment benefited DC vaccine………………………..116
Figure 42. Effect of SK + CQ-DC vaccine on body weight of test mice…….117
Figure 43. Expression of specific inflammatory cytokines in SK-treated 4T1-luc2 cells………………………………………………………………..118
Figure 44. Effect of SK on the expression of phosphorylated NF-kB………..119
Figure 45. Hypothetical model depicting key molecular mechanisms of autophagy-related ectoDAMP expression in SK mediated necroptosis in our test tumor cell system……………………………………………………………...120

1.Vanden Berghe T, Linkermann A, Jouan-Lanhouet S, Walczak H, Vandenabeele P: Regulated necrosis: the expanding network of non-apoptotic cell death pathways. Nat Rev Mol Cell Biol. 15:135-47, 2014.
2.Casares N, Pequignot MO, Tesniere A, Ghiringhelli F, Roux S, Chaput N, et al.: Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death. J Exp Med. 202:1691-701, 2005.
3.Galluzzi L, Vitale I, Abrams JM, Alnemri ES, Baehrecke EH, Blagosklonny MV, et al.: Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differ. 19:107-20, 2012.
4.Matzinger P: Tolerance, danger, and the extended family. Annu Rev Immunol. 12:991-1045, 1994.
5.Hartl FU: Molecular chaperones in cellular protein folding. Nature. 381:571-9, 1996.
6.Radons J: The human HSP70 family of chaperones: where do we stand? Cell Stress Chaperones. 21:379-404, 2016.
7.Garg AD, Krysko DV, Vandenabeele P, Agostinis P: DAMPs and PDT-mediated photo-oxidative stress: exploring the unknown. Photochem Photobiol Sci. 10:670-80, 2011.
8.Garg AD, Krysko DV, Vandenabeele P, Agostinis P: Hypericin-based photodynamic therapy induces surface exposure of damage-associated molecular patterns like HSP70 and calreticulin. Cancer Immunol Immunother. 61:215-21, 2012.
9.Galluzzi L, Vacchelli E, Bravo-San Pedro JM, Buque A, Senovilla L, Baracco EE, et al.: Classification of current anticancer immunotherapies. Oncotarget. 5:12472-508, 2014.
10.Michalak M, Groenendyk J, Szabo E, Gold LI, Opas M: Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum. Biochem J. 417:651-66, 2009.
11.Gardai SJ, McPhillips KA, Frasch SC, Janssen WJ, Starefeldt A, Murphy-Ullrich JE, et al.: Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte. Cell. 123:321-34, 2005.
12.Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL, et al.: Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med. 13:54-61, 2007.
13.Wiersma VR, Michalak M, Abdullah TM, Bremer E, Eggleton P: Mechanisms of Translocation of ER Chaperones to the Cell Surface and Immunomodulatory Roles in Cancer and Autoimmunity. Front Oncol. 5:7, 2015.
14.Ulloa L, Messmer D: High-mobility group box 1 (HMGB1) protein: friend and foe. Cytokine Growth Factor Rev. 17:189-201, 2006.
15.Klune JR, Dhupar R, Cardinal J, Billiar TR, Tsung A: HMGB1: endogenous danger signaling. Mol Med. 14:476-84, 2008.
16.Scaffidi P, Misteli T, Bianchi ME: Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature. 418:191-5, 2002.
17.Dumitriu IE, Baruah P, Valentinis B, Voll RE, Herrmann M, Nawroth PP, et al.: Release of high mobility group box 1 by dendritic cells controls T cell activation via the receptor for advanced glycation end products. J Immunol. 174:7506-15, 2005.
18.Kazama H, Ricci JE, Herndon JM, Hoppe G, Green DR, Ferguson TA: Induction of immunological tolerance by apoptotic cells requires caspase-dependent oxidation of high-mobility group box-1 protein. Immunity. 29:21-32, 2008.
19.Hou W, Zhang Q, Yan Z, Chen R, Zeh Iii HJ, Kang R, et al.: Strange attractors: DAMPs and autophagy link tumor cell death and immunity. Cell Death Dis. 4:e966, 2013.
20.Vercammen D, Beyaert R, Denecker G, Goossens V, Van Loo G, Declercq W, et al.: Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor. J Exp Med. 187:1477-85, 1998.
21.Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, et al.: Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol. 1:112-9, 2005.
22.Degterev A, Hitomi J, Germscheid M, Ch'en IL, Korkina O, Teng X, et al.: Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol. 4:313-21, 2008.
23.Dondelinger Y, Darding M, Bertrand MJ, Walczak H: Poly-ubiquitination in TNFR1-mediated necroptosis. Cell Mol Life Sci. 73:2165-76, 2016.
24.Wilson NS, Dixit V, Ashkenazi A: Death receptor signal transducers: nodes of coordination in immune signaling networks. Nat Immunol. 10:348-55, 2009.
25.Wang H, Sun L, Su L, Rizo J, Liu L, Wang LF, et al.: Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3. Mol Cell. 54:133-46, 2014.
26.Festjens N, Vanden Berghe T, Cornelis S, Vandenabeele P: RIP1, a kinase on the crossroads of a cell's decision to live or die. Cell Death Differ. 14:400-10, 2007.
27.Vanlangenakker N, Vanden Berghe T, Vandenabeele P: Many stimuli pull the necrotic trigger, an overview. Cell Death Differ. 19:75-86, 2012.
28.Linkermann A, Green DR: Necroptosis. N Engl J Med. 370:455-65, 2014.
29.Kaiser WJ, Sridharan H, Huang C, Mandal P, Upton JW, Gough PJ, et al.: Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL. J Biol Chem. 288:31268-79, 2013.
30.Aaes TL, Kaczmarek A, Delvaeye T, De Craene B, De Koker S, Heyndrickx L, et al.: Vaccination with Necroptotic Cancer Cells Induces Efficient Anti-tumor Immunity. Cell Rep. 15:274-87, 2016.
31.Yang H, Ma Y, Chen G, Zhou H, Yamazaki T, Klein C, et al.: Contribution of RIP3 and MLKL to immunogenic cell death signaling in cancer chemotherapy. Oncoimmunology. 5:e1149673, 2016.
32.Osborn SL, Diehl G, Han SJ, Xue L, Kurd N, Hsieh K, et al.: Fas-associated death domain (FADD) is a negative regulator of T-cell receptor-mediated necroptosis. Proc Natl Acad Sci U S A. 107:13034-9, 2010.
33.Li Y, Wang LX, Yang G, Hao F, Urba WJ, Hu HM: Efficient cross-presentation depends on autophagy in tumor cells. Cancer Res. 68:6889-95, 2008.
34.Thorburn J, Horita H, Redzic J, Hansen K, Frankel AE, Thorburn A: Autophagy regulates selective HMGB1 release in tumor cells that are destined to die. Cell Death Differ. 16:175-83, 2009.
35.Michaud M, Martins I, Sukkurwala AQ, Adjemian S, Ma Y, Pellegatti P, et al.: Autophagy-dependent anticancer immune responses induced by chemotherapeutic agents in mice. Science. 334:1573-7, 2011.
36.Li Y, Wang LX, Pang P, Cui Z, Aung S, Haley D, et al.: Tumor-derived autophagosome vaccine: mechanism of cross-presentation and therapeutic efficacy. Clin Cancer Res. 17:7047-57, 2011.
37.Menger L, Vacchelli E, Adjemian S, Martins I, Ma Y, Shen S, et al.: Cardiac glycosides exert anticancer effects by inducing immunogenic cell death. Sci Transl Med. 4:143ra99, 2012.
38.Dudek AM, Garg AD, Krysko DV, De Ruysscher D, Agostinis P: Inducers of immunogenic cancer cell death. Cytokine Growth Factor Rev. 24:319-33, 2013.
39.Duan D, Zhang B, Yao J, Liu Y, Fang J: Shikonin targets cytosolic thioredoxin reductase to induce ROS-mediated apoptosis in human promyelocytic leukemia HL-60 cells. Free Radic Biol Med. 70:182-93, 2014.
40.Wada N, Kawano Y, Fujiwara S, Kikukawa Y, Okuno Y, Tasaki M, et al.: Shikonin, dually functions as a proteasome inhibitor and a necroptosis inducer in multiple myeloma cells. Int J Oncol. 46:963-72, 2015.
41.Piao JL, Cui ZG, Furusawa Y, Ahmed K, Rehman MU, Tabuchi Y, et al.: The molecular mechanisms and gene expression profiling for shikonin-induced apoptotic and necroptotic cell death in U937 cells. Chem Biol Interact. 205:119-27, 2013.
42.Wiench B, Eichhorn T, Paulsen M, Efferth T: Shikonin directly targets mitochondria and causes mitochondrial dysfunction in cancer cells. Evid Based Complement Alternat Med. 2012:726025, 2012.
43.Chen HM, Wang PH, Chen SS, Wen CC, Chen YH, Yang WC, et al.: Shikonin induces immunogenic cell death in tumor cells and enhances dendritic cell-based cancer vaccine. Cancer Immunol Immunother, 2012.
44.Huang C, Luo Y, Zhao J, Yang F, Zhao H, Fan W, et al.: Shikonin kills glioma cells through necroptosis mediated by RIP-1. PLoS One. 8:e66326, 2013.
45.Yang H, Zhou P, Huang H, Chen D, Ma N, Cui QC, et al.: Shikonin exerts antitumor activity via proteasome inhibition and cell death induction in vitro and in vivo. Int J Cancer. 124:2450-9, 2009.
46.Shi S, Cao H: Shikonin promotes autophagy in BXPC-3 human pancreatic cancer cells through the PI3K/Akt signaling pathway. Oncol Lett. 8:1087-9, 2014.
47.Han W, Li L, Qiu S, Lu Q, Pan Q, Gu Y, et al.: Shikonin circumvents cancer drug resistance by induction of a necroptotic death. Mol Cancer Ther. 6:1641-9, 2007.
48.Gong K, Zhang Z, Chen Y, Shu HB, Li W: Extracellular signal-regulated kinase, receptor interacting protein, and reactive oxygen species regulate shikonin-induced autophagy in human hepatocellular carcinoma. Eur J Pharmacol. 738:142-52, 2014.
49.Kaur J, Debnath J: Autophagy at the crossroads of catabolism and anabolism. Nat Rev Mol Cell Biol. 16:461-72, 2015.
50.Mizushima N, Komatsu M: Autophagy: renovation of cells and tissues. Cell. 147:728-41, 2011.
51.Li WW, Li J, Bao JK: Microautophagy: lesser-known self-eating. Cell Mol Life Sci. 69:1125-36, 2012.
52.Kaushik S, Cuervo AM: Chaperone-mediated autophagy: a unique way to enter the lysosome world. Trends Cell Biol. 22:407-17, 2012.
53.Lamb CA, Yoshimori T, Tooze SA: The autophagosome: origins unknown, biogenesis complex. Nat Rev Mol Cell Biol. 14:759-74, 2013.
54.Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A, et al.: Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J Cell Biol. 182:685-701, 2008.
55.Hailey DW, Rambold AS, Satpute-Krishnan P, Mitra K, Sougrat R, Kim PK, et al.: Mitochondria supply membranes for autophagosome biogenesis during starvation. Cell. 141:656-67, 2010.
56.Ravikumar B, Moreau K, Jahreiss L, Puri C, Rubinsztein DC: Plasma membrane contributes to the formation of pre-autophagosomal structures. Nat Cell Biol. 12:747-57, 2010.
57.Mizushima N, Yoshimori T, Ohsumi Y: The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol. 27:107-32, 2011.
58.Shen HM, Mizushima N: At the end of the autophagic road: an emerging understanding of lysosomal functions in autophagy. Trends Biochem Sci. 39:61-71, 2014.
59.Fader CM, Sanchez DG, Mestre MB, Colombo MI: TI-VAMP/VAMP7 and VAMP3/cellubrevin: two v-SNARE proteins involved in specific steps of the autophagy/multivesicular body pathways. Biochim Biophys Acta. 1793:1901-16, 2009.
60.Furuta N, Fujita N, Noda T, Yoshimori T, Amano A: Combinational soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins VAMP8 and Vti1b mediate fusion of antimicrobial and canonical autophagosomes with lysosomes. Mol Biol Cell. 21:1001-10, 2010.
61.Chang TK, Shravage BV, Hayes SD, Powers CM, Simin RT, Wade Harper J, et al.: Uba1 functions in Atg7- and Atg3-independent autophagy. Nat Cell Biol. 15:1067-78, 2013.
62.Galluzzi L, Bravo-San Pedro JM, Vitale I, Aaronson SA, Abrams JM, Adam D, et al.: Essential versus accessory aspects of cell death: recommendations of the NCCD 2015. Cell Death Differ. 22:58-73, 2015.
63.Liu Y, Levine B: Autosis and autophagic cell death: the dark side of autophagy. Cell Death Differ. 22:367-76, 2015.
64.Berry DL, Baehrecke EH: Growth arrest and autophagy are required for salivary gland cell degradation in Drosophila. Cell. 131:1137-48, 2007.
65.Yu L, Wan F, Dutta S, Welsh S, Liu Z, Freundt E, et al.: Autophagic programmed cell death by selective catalase degradation. Proc Natl Acad Sci U S A. 103:4952-7, 2006.
66.Liang XH, Kleeman LK, Jiang HH, Gordon G, Goldman JE, Berry G, et al.: Protection against fatal Sindbis virus encephalitis by beclin, a novel Bcl-2-interacting protein. J Virol. 72:8586-96, 1998.
67.Maiuri MC, Le Toumelin G, Criollo A, Rain JC, Gautier F, Juin P, et al.: Functional and physical interaction between Bcl-X(L) and a BH3-like domain in Beclin-1. EMBO J. 26:2527-39, 2007.
68.Morselli E, Shen S, Ruckenstuhl C, Bauer MA, Marino G, Galluzzi L, et al.: p53 inhibits autophagy by interacting with the human ortholog of yeast Atg17, RB1CC1/FIP200. Cell Cycle. 10:2763-9, 2011.
69.Budanov AV, Karin M: p53 target genes sestrin1 and sestrin2 connect genotoxic stress and mTOR signaling. Cell. 134:451-60, 2008.
70.Kenzelmann Broz D, Spano Mello S, Bieging KT, Jiang D, Dusek RL, Brady CA, et al.: Global genomic profiling reveals an extensive p53-regulated autophagy program contributing to key p53 responses. Genes Dev. 27:1016-31, 2013.
71.Marino G, Niso-Santano M, Baehrecke EH, Kroemer G: Self-consumption: the interplay of autophagy and apoptosis. Nat Rev Mol Cell Biol. 15:81-94, 2014.
72.Marino G, Salvador-Montoliu N, Fueyo A, Knecht E, Mizushima N, Lopez-Otin C: Tissue-specific autophagy alterations and increased tumorigenesis in mice deficient in Atg4C/autophagin-3. J Biol Chem. 282:18573-83, 2007.
73.Takahashi Y, Coppola D, Matsushita N, Cualing HD, Sun M, Sato Y, et al.: Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis. Nat Cell Biol. 9:1142-51, 2007.
74.Yang S, Wang X, Contino G, Liesa M, Sahin E, Ying H, et al.: Pancreatic cancers require autophagy for tumor growth. Genes Dev. 25:717-29, 2011.
75.Guo JY, Chen HY, Mathew R, Fan J, Strohecker AM, Karsli-Uzunbas G, et al.: Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. Genes Dev. 25:460-70, 2011.
76.Bray K, Mathew R, Lau A, Kamphorst JJ, Fan J, Chen J, et al.: Autophagy suppresses RIP kinase-dependent necrosis enabling survival to mTOR inhibition. PLoS One. 7:e41831, 2012.
77.He MX, He YW: A role for c-FLIP(L) in the regulation of apoptosis, autophagy, and necroptosis in T lymphocytes. Cell Death Differ. 20:188-97, 2013.
78.Bonapace L, Bornhauser BC, Schmitz M, Cario G, Ziegler U, Niggli FK, et al.: Induction of autophagy-dependent necroptosis is required for childhood acute lymphoblastic leukemia cells to overcome glucocorticoid resistance. J Clin Invest. 120:1310-23, 2010.
79.Basit F, Cristofanon S, Fulda S: Obatoclax (GX15-070) triggers necroptosis by promoting the assembly of the necrosome on autophagosomal membranes. Cell Death Differ. 20:1161-73, 2013.
80.Steinman RM, Banchereau J: Taking dendritic cells into medicine. Nature. 449:419-26, 2007.
81.Trombetta ES, Mellman I: Cell biology of antigen processing in vitro and in vivo. Annu Rev Immunol. 23:975-1028, 2005.
82.Bennett SR, Carbone FR, Karamalis F, Flavell RA, Miller JF, Heath WR: Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature. 393:478-80, 1998.
83.Guermonprez P, Valladeau J, Zitvogel L, Thery C, Amigorena S: Antigen presentation and T cell stimulation by dendritic cells. Annu Rev Immunol. 20:621-67, 2002.
84.Diamond MS, Kinder M, Matsushita H, Mashayekhi M, Dunn GP, Archambault JM, et al.: Type I interferon is selectively required by dendritic cells for immune rejection of tumors. J Exp Med. 208:1989-2003, 2011.
85.Fuertes MB, Kacha AK, Kline J, Woo SR, Kranz DM, Murphy KM, et al.: Host type I IFN signals are required for antitumor CD8+ T cell responses through CD8{alpha}+ dendritic cells. J Exp Med. 208:2005-16, 2011.
86.Ueno H, Schmitt N, Klechevsky E, Pedroza-Gonzalez A, Matsui T, Zurawski G, et al.: Harnessing human dendritic cell subsets for medicine. Immunol Rev. 234:199-212, 2010.
87.Nabhan C: Sipuleucel-T immunotherapy for castration-resistant prostate cancer. N Engl J Med. 363:1966-7; author reply 8, 2010.
88.Anguille S, Smits EL, Lion E, van Tendeloo VF, Berneman ZN: Clinical use of dendritic cells for cancer therapy. Lancet Oncol. 15:e257-67, 2014.
89.Bol KF, Schreibelt G, Gerritsen WR, de Vries IJ, Figdor CG: Dendritic Cell-Based Immunotherapy: State of the Art and Beyond. Clin Cancer Res. 22:1897-906, 2016.
90.Vandenberk L, Belmans J, Van Woensel M, Riva M, Van Gool SW: Exploiting the Immunogenic Potential of Cancer Cells for Improved Dendritic Cell Vaccines. Front Immunol. 6:663, 2015.
91.Goldszmid RS, Idoyaga J, Bravo AI, Steinman R, Mordoh J, Wainstok R: Dendritic cells charged with apoptotic tumor cells induce long-lived protective CD4+ and CD8+ T cell immunity against B16 melanoma. J Immunol. 171:5940-7, 2003.
92.Kim HS, Choo YS, Koo T, Bang S, Oh TY, Wen J, et al.: Enhancement of antitumor immunity of dendritic cells pulsed with heat-treated tumor lysate in murine pancreatic cancer. Immunol Lett. 103:142-8, 2006.
93.Vandenberk L, Garg AD, Verschuere T, Koks C, Belmans J, Beullens M, et al.: Irradiation of necrotic cancer cells, employed for pulsing dendritic cells (DCs), potentiates DC vaccine-induced antitumor immunity against high-grade glioma. Oncoimmunology. 5:e1083669, 2016.
94.Kotera Y, Shimizu K, Mule JJ: Comparative analysis of necrotic and apoptotic tumor cells as a source of antigen(s) in dendritic cell-based immunization. Cancer Res. 61:8105-9, 2001.
95.Garg AD, Martin S, Golab J, Agostinis P: Danger signalling during cancer cell death: origins, plasticity and regulation. Cell Death Differ. 21:26-38, 2014.
96.Kepp O, Senovilla L, Vitale I, Vacchelli E, Adjemian S, Agostinis P, et al.: Consensus guidelines for the detection of immunogenic cell death. Oncoimmunology. 3:e955691, 2014.
97.Kroemer G, Galluzzi L, Kepp O, Zitvogel L: Immunogenic cell death in cancer therapy. Annu Rev Immunol. 31:51-72, 2013.
98.Yin SY, Wang CY, Yang NS: Interleukin-4 enhances trafficking and functional activities of GM-CSF-stimulated mouse myeloid-derived dendritic cells at late differentiation stage. Exp Cell Res. 317:2210-21, 2011.
99.Chow KP, Qiu JT, Lee JM, Hsu SL, Yang SC, Wu NN, et al.: Selective reduction of post-selection CD8 thymocyte proliferation in IL-15Ralpha deficient mice. PLoS One. 7:e33152, 2012.
100.Lin TJ, Lin HT, Chang WT, Mitapalli SP, Hsiao PW, Yin SY, et al.: Shikonin-enhanced cell immunogenicity of tumor vaccine is mediated by the differential effects of DAMP components. Mol Cancer. 14:174, 2015.
101.Weng D, Marty-Roix R, Ganesan S, Proulx MK, Vladimer GI, Kaiser WJ, et al.: Caspase-8 and RIP kinases regulate bacteria-induced innate immune responses and cell death. Proc Natl Acad Sci U S A. 111:7391-6, 2014.
102.Green DR, Ferguson T, Zitvogel L, Kroemer G: Immunogenic and tolerogenic cell death. Nat Rev Immunol. 9:353-63, 2009.
103.Udono H, Srivastava PK: Comparison of tumor-specific immunogenicities of stress-induced proteins gp96, hsp90, and hsp70. J Immunol. 152:5398-403, 1994.
104.Mizushima N, Yoshimori T, Levine B: Methods in mammalian autophagy research. Cell. 140:313-26, 2010.
105.Rider P, Carmi Y, Guttman O, Braiman A, Cohen I, Voronov E, et al.: IL-1alpha and IL-1beta recruit different myeloid cells and promote different stages of sterile inflammation. J Immunol. 187:4835-43, 2011.
106.Ayna G, Krysko DV, Kaczmarek A, Petrovski G, Vandenabeele P, Fesus L: ATP release from dying autophagic cells and their phagocytosis are crucial for inflammasome activation in macrophages. PLoS One. 7:e40069, 2012.
107.Kaczmarek A, Vandenabeele P, Krysko DV: Necroptosis: the release of damage-associated molecular patterns and its physiological relevance. Immunity. 38:209-23, 2013.
108.Zhang Q, Kang R, Zeh HJ, 3rd, Lotze MT, Tang D: DAMPs and autophagy: cellular adaptation to injury and unscheduled cell death. Autophagy. 9:451-8, 2013.
109.Rodriguez-Gonzalez A, Lin T, Ikeda AK, Simms-Waldrip T, Fu C, Sakamoto KM: Role of the aggresome pathway in cancer: targeting histone deacetylase 6-dependent protein degradation. Cancer Res. 68:2557-60, 2008.
110.Bruning A, Juckstock J: Misfolded proteins: from little villains to little helpers in the fight against cancer. Front Oncol. 5:47, 2015.
111.Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H, et al.: p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem. 282:24131-45, 2007.
112.Li Y, Wang LX, Pang P, Twitty C, Fox BA, Aung S, et al.: Cross-presentation of tumor associated antigens through tumor-derived autophagosomes. Autophagy. 5:576-7, 2009.
113.Gamrekelashvili J, Kapanadze T, Han M, Wissing J, Ma C, Jaensch L, et al.: Peptidases released by necrotic cells control CD8+ T cell cross-priming. J Clin Invest. 123:4755-68, 2013.
114.Maeda A, Fadeel B: Mitochondria released by cells undergoing TNF-alpha-induced necroptosis act as danger signals. Cell Death Dis. 5:e1312, 2014.
115.Spisek R, Charalambous A, Mazumder A, Vesole DH, Jagannath S, Dhodapkar MV: Bortezomib enhances dendritic cell (DC)-mediated induction of immunity to human myeloma via exposure of cell surface heat shock protein 90 on dying tumor cells: therapeutic implications. Blood. 109:4839-45, 2007.
116.Chang CL, Hsu YT, Wu CC, Yang YC, Wang C, Wu TC, et al.: Immune mechanism of the antitumor effects generated by bortezomib. J Immunol. 189:3209-20, 2012.
117.Korbelik M, Sun J, Cecic I: Photodynamic therapy-induced cell surface expression and release of heat shock proteins: relevance for tumor response. Cancer Res. 65:1018-26, 2005.
118.Srivastava PK, Menoret A, Basu S, Binder RJ, McQuade KL: Heat shock proteins come of age: primitive functions acquire new roles in an adaptive world. Immunity. 8:657-65, 1998.
119.Yang JT, Li ZL, Wu JY, Lu FJ, Chen CH: An oxidative stress mechanism of shikonin in human glioma cells. PLoS One. 9:e94180, 2014.
120.Lee MJ, Kao SH, Hunag JE, Sheu GT, Yeh CW, Hseu YC, et al.: Shikonin time-dependently induced necrosis or apoptosis in gastric cancer cells via generation of reactive oxygen species. Chem Biol Interact. 211:44-53, 2014.
121.Amos SM, Duong CP, Westwood JA, Ritchie DS, Junghans RP, Darcy PK, et al.: Autoimmunity associated with immunotherapy of cancer. Blood. 118:499-509, 2011.
122.Accapezzato D, Visco V, Francavilla V, Molette C, Donato T, Paroli M, et al.: Chloroquine enhances human CD8+ T cell responses against soluble antigens in vivo. J Exp Med. 202:817-28, 2005.
123.Joubert PE, Albert ML: Antigen Cross-Priming of Cell-Associated Proteins is Enhanced by Macroautophagy within the Antigen Donor Cell. Front Immunol. 3:61, 2012.
124.He S, Wang L, Miao L, Wang T, Du F, Zhao L, et al.: Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell. 137:1100-11, 2009.
125.Zhang DW, Shao J, Lin J, Zhang N, Lu BJ, Lin SC, et al.: RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science. 325:332-6, 2009.
126.Vince JE, Wong WW, Gentle I, Lawlor KE, Allam R, O'Reilly L, et al.: Inhibitor of apoptosis proteins limit RIP3 kinase-dependent interleukin-1 activation. Immunity. 36:215-27, 2012.
127.Lawlor KE, Khan N, Mildenhall A, Gerlic M, Croker BA, D'Cruz AA, et al.: RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL. Nat Commun. 6:6282, 2015.
128.Yatim N, Jusforgues-Saklani H, Orozco S, Schulz O, Barreira da Silva R, Reis e Sousa C, et al.: RIPK1 and NF-kappaB signaling in dying cells determines cross-priming of CD8(+) T cells. Science. 350:328-34, 2015.
129.Festjens N, Vanden Berghe T, Vandenabeele P: Necrosis, a well-orchestrated form of cell demise: signalling cascades, important mediators and concomitant immune response. Biochim Biophys Acta. 1757:1371-87, 2006.
130.Vandenabeele P, Galluzzi L, Vanden Berghe T, Kroemer G: Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat Rev Mol Cell Biol. 11:700-14, 2010.
131.Goodall ML, Fitzwalter BE, Zahedi S, Wu M, Rodriguez D, Mulcahy-Levy JM, et al.: The Autophagy Machinery Controls Cell Death Switching between Apoptosis and Necroptosis. Dev Cell. 37:337-49, 2016.
132.Gallucci S, Lolkema M, Matzinger P: Natural adjuvants: endogenous activators of dendritic cells. Nat Med. 5:1249-55, 1999.
133.Nouri-Shirazi M, Banchereau J, Bell D, Burkeholder S, Kraus ET, Davoust J, et al.: Dendritic cells capture killed tumor cells and present their antigens to elicit tumor-specific immune responses. J Immunol. 165:3797-803, 2000.
134.Sauter B, Albert ML, Francisco L, Larsson M, Somersan S, Bhardwaj N: Consequences of cell death: exposure to necrotic tumor cells, but not primary tissue cells or apoptotic cells, induces the maturation of immunostimulatory dendritic cells. J Exp Med. 191:423-34, 2000.
135.Farkas T, Daugaard M, Jaattela M: Identification of small molecule inhibitors of phosphatidylinositol 3-kinase and autophagy. J Biol Chem. 286:38904-12, 2011.
136.Wu YT, Tan HL, Huang Q, Ong CN, Shen HM: Activation of the PI3K-Akt-mTOR signaling pathway promotes necrotic cell death via suppression of autophagy. Autophagy. 5:824-34, 2009.
137.Alexander A, Cai SL, Kim J, Nanez A, Sahin M, MacLean KH, et al.: ATM signals to TSC2 in the cytoplasm to regulate mTORC1 in response to ROS. Proc Natl Acad Sci U S A. 107:4153-8, 2010.
138.Munoz-Gamez JA, Rodriguez-Vargas JM, Quiles-Perez R, Aguilar-Quesada R, Martin-Oliva D, de Murcia G, et al.: PARP-1 is involved in autophagy induced by DNA damage. Autophagy. 5:61-74, 2009.
139.Yonekawa T, Gamez G, Kim J, Tan AC, Thorburn J, Gump J, et al.: RIP1 negatively regulates basal autophagic flux through TFEB to control sensitivity to apoptosis. EMBO Rep. 16:700-8, 2015.
140.Green DR: Another face of RIPK1. EMBO Rep. 16:674-5, 2015.
141.Bao L, Haque A, Jackson K, Hazari S, Moroz K, Jetly R, et al.: Increased expression of P-glycoprotein is associated with doxorubicin chemoresistance in the metastatic 4T1 breast cancer model. Am J Pathol. 178:838-52, 2011.
142.Zhuang X, Zhang W, Chen Y, Han X, Li J, Zhang Y, et al.: Doxorubicin-enriched, ALDH(br) mouse breast cancer stem cells are treatable to oncolytic herpes simplex virus type 1. BMC Cancer. 12:549, 2012.
143.Steinhart L, Belz K, Fulda S: Smac mimetic and demethylating agents synergistically trigger cell death in acute myeloid leukemia cells and overcome apoptosis resistance by inducing necroptosis. Cell Death Dis. 4:e802, 2013.
144.Laukens B, Jennewein C, Schenk B, Vanlangenakker N, Schier A, Cristofanon S, et al.: Smac mimetic bypasses apoptosis resistance in FADD- or caspase-8-deficient cells by priming for tumor necrosis factor alpha-induced necroptosis. Neoplasia. 13:971-9, 2011.
145.Thakur R, Trivedi R, Rastogi N, Singh M, Mishra DP: Inhibition of STAT3, FAK and Src mediated signaling reduces cancer stem cell load, tumorigenic potential and metastasis in breast cancer. Sci Rep. 5:10194, 2015.
146.Ghochikyan A, Davtyan A, Hovakimyan A, Davtyan H, Poghosyan A, Bagaev A, et al.: Primary 4T1 tumor resection provides critical "window of opportunity" for immunotherapy. Clin Exp Metastasis. 31:185-98, 2014.
147.Leonhartsberger N, Ramoner R, Falkensammer C, Rahm A, Gander H, Holtl L, et al.: Quality of life during dendritic cell vaccination against metastatic renal cell carcinoma. Cancer Immunol Immunother. 61:1407-13, 2012.
148.Cheever MA, Higano CS: PROVENGE (Sipuleucel-T) in prostate cancer: the first FDA-approved therapeutic cancer vaccine. Clin Cancer Res. 17:3520-6, 2011.
149.Andersen BM, Ohlfest JR: Increasing the efficacy of tumor cell vaccines by enhancing cross priming. Cancer Lett. 325:155-64, 2012.
150.Rosenberg SA, Yang JC, Schwartzentruber DJ, Hwu P, Marincola FM, Topalian SL, et al.: Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nat Med. 4:321-7, 1998.
151.Speiser DE, Miranda R, Zakarian A, Bachmann MF, McKall-Faienza K, Odermatt B, et al.: Self antigens expressed by solid tumors Do not efficiently stimulate naive or activated T cells: implications for immunotherapy. J Exp Med. 186:645-53, 1997.
152.Overwijk WW, Theoret MR, Finkelstein SE, Surman DR, de Jong LA, Vyth-Dreese FA, et al.: Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8+ T cells. J Exp Med. 198:569-80, 2003.