ArticlePDF AvailableLiterature Review

Natural modulators of the hallmarks of immunogenic cell death

April 2019
Biochemical Pharmacology 162(26)

DOI:10.1016/j.bcp.2018.12.016

Authors:

Flavia Radogna

Laboratoire de Biologie Moléculaire et Cellulaire du Cancer

Mario Dicato

Centre Hospitalier de Luxembourg

Marc F Diederich

Seoul National University

Natural compounds act as immunoadjuvants as their therapeutic effects trigger cancer stress response and release of damage-associated molecular patterns (DAMPs). These reactions occur through an increase in the immunogenicity of cancer cells that undergo stress followed by immunogenic cell death (ICD). These processes result in a chemotherapeutic response with a potent immune-mediating reaction. Natural compounds that induce ICD may function as an interesting approach in converting cancer into its own vaccine. However, multiple parameters determine whether a compound can act as an ICD inducer, including the nature of the inducer, the premortem stress pathways, the cell death pathways, the intrinsic antigenicity of the cell, and the potency and availability of an immune cell response. Thus, the identification of hallmarks of ICD is important in determining the prognostic biomarkers for new therapeutic approaches and combination treatments.

Effects of ICD-induced cancer cells as anticancer vaccines in immunocompetent mice. ICD-induced cancer cells can act as anticancer vaccines by inducing a variety of premortem stress pathways. Each of these pathways leads to the release of several DAMPs by increasing the immunogenicity of stressed and dying cancer cells and by triggering immunological memory. Scheme was generated in part with ScienceSlides (Visiscience).

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The mechanisms of action of natural compounds on the hallmarks of immunogenic cell death.

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Premortem stress pathways involved in ICD. In response to a variety of stress stimuli such as Ca ⁠ 2+ overload and/or the ROS generation, pro-survival pathways such as ER stress, UPR and autophagy switch to ICD. Ca ⁠ 2+ leakage, ROS generation, and caspase-8 activation are essential for ICD. Scheme was generated with ScienceSlides (Visiscience).

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Key ICD events influenced by ICD inducers. ER stress, autophagy, TLR3 activation and HMGB1 release are important events in ICD. In ER stress, UPR induces the pre-apoptotic exposure of the 'eat me' signal CRT that leads to the engulfment of dying cancer cells and tumor antigen uptake for DCs. Autophagy induces the release of ATP that activates the NLRP3/ inflammasome pathway in DCs and IL1β production for the activation of the tumor specific CD8 ⁠ +

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Schematic representation of the effect of ICD inducers on ICD hallmarks. Scheme was generated in part with ScienceSlides (Visiscience).

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Figures - uploaded by Marc F Diederich

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Content uploaded by Marc F Diederich

Content may be subject to copyright.

UNCORRECTED PROOF

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1. Introduction

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UNCORRECTED PROOF

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2. Overview of ICD: Immunogenic versus tolerogenic cell death

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9FG 6M 75GD5G9G 5B8 H<9 7@ :5A=@M C: DFCH9=BG ,<=G DFC;F5AA98 57

H=J=HM <5G 699B 7@5GG=R98 5G HC@9FC;9B=7 5G H<9 D<5;C7MHCG=G H<5H F9GI@HG

:FCA H<=G HMD9 C: 79@@ 89A=G9 =G BCB=AAIBC;9B=7 /<9F95G B97FCH=7

79@@ 895H< =G 7<5F57H9F=N98 6M 79@@I@5F GK9@@=B; 5B8 GI6G9EI9BH FIDHIF9

C: H<9 D@5GA5 A9A6F5B9 H<5H =G 7CBG=89F98 =AAIBC;9B=7 5B8 9@=7=HG =B

S5AA5H=CB

&9J9FH<9@9GG 5DCDHCG=G 75B 5@GC 69 <=;<@M =AAIBC;9B=7 89D9B8

=B; CB H<9 7CADCG=H=CB C: H<9 79@@ GIF:579 5B8 H<9 DFC8I7HG G97F9H98

6M H<9 8M=B; 79@@G +9J9F5@ HMD9G C: 5GG5MG <5J9 699B IG98 HC =BJ9GH=

;5H9 B9K 5BH=75B79F 5;9BHG H<5H 75B =B8I79 5B8 8=GH=B;I=G< =AAIBC

;9B=7 5B8CF HC@9FC;9B=7 79@@ 895H< BH=75B79F 5;9BHG K9F9 RFGH =89B

H=R98 =B 7I@HIF98 <IA5B 79@@ @=B9G H<9B H9GH98 =B =AAIBC7CAD9H9BH

5B8 =AAIBC89R7=9BH GMB;9B9=7 A=79 695F=B; ;F5:H98 ;9B9H=75@@M8F=

J9B CF 7<9A=75@@M–=B8I798 GI67IH5B9CIG HIACFG 34 +CA9 7<9ACH<9F

5D9IH=7 5;9BHG GI7< 5G 7=GD@5H=B 75BBCH =B8I79 ! 5B8 5F9 ACF9 HC@9FC

;9B=7 K=H< F9GD97H HC =AAIB9 F97C;B=H=CB 'H<9F 5;9BHG GI7< 5G CL

5@=D@5H=B <CK9J9F 5F9 <=;<@M 9:R7=9BH =B HF=;;9F=B; ! 34 59G5F

9H 5@ 34 89ACBGHF5H98 H<5H 8CLCFI6=7=B=B8I798 5DCDHCG=G HF=;;9FG

5B 5BH=;9BGD97=R7 =AAIB9 F9GDCBG9 !B GIDDCFH C: H<=G C6G9FJ5H=CB

HIACFG 89F=J98 :FCA 7C@CF97H5@ , 75B79F 79@@G F9;F9GG 5:H9F 8CL

CFI6=7=B HF95HA9BH -B@=?9 H<9 F9GI@HG G99B =B H<9 =AAIBC7CAD9H9BH

A=79 =AAIBC89R7=9BH A=79 HF95H98 K=H< 8CLCFI6=7=B 8=8 BCH 9LD9F=

9B79 HIACF F9;F9GG=CB CLCFI6=7=BHF95H98 B97FCH=7 , 79@@G :5=@98

HC GH=AI@5H9 5BM =AAIB9 F9GDCBG9 =B H<9 DF9G9B79 C: H<9 D5B75GD5G9

=B<=6=HCF N.58 ,<9G9 F9GI@HG 89ACBGHF5H9 H<5H 5DCDHCG=G 75B HF=;;9F

5B 5BH=;9BGD97=R7 =AAIB9 F9GDCBG9 34 %CF9CJ9F H<9 , 79@@G

H<5H :5=@98 HC =B8I79 ! IB89F B97FCH=7 7CB8=H=CBG @57?98 H<9 F979D

HCF=BH9F57H=B; G9F=B9H<F9CB=B9DFCH9=B ?=B5G9  *!(# 5 A5>CF DFC

H9=B F9EI=F98 HC =B8I79 5 F9;I@5H98 :CFA C: B97FCG=G 75@@98 B97FCD

HCG=G ,<IG B97FCDHCG=G =G 5GGC7=5H98 K=H< =AAIBC;9B=7=HM 34

'H<9F ACIG9 75B79F 79@@G 9LDF9GG=B; DFCH9=BG F9EI=F98 :CF B97FCDHC

G=G 75B 57H=J5H9 ! 34 5B79F 79@@G H<5H 8=9 :C@@CK=B;

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UNCORRECTED PROOF

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Fig. 2. <9A=75@ GHFI7HIF9 C: ! =B8I79FG %C@97I@5F GHFI7HIF9G K9F9 8F5KB K=H< <9A8F5K (FC:9GG=CB5@ 

Table 1

,<9 A97<5B=GAG C: 57H=CB C: B5HIF5@ 7CADCIB8G CB H<9 <5@@A5F?G C: =AAIBC;9B=7 79@@ 895H<

!<5@@A5F?G

<9A=75@7@5GG

CADCIB8 'F=;=B =;IF9 %97<5B=GAC:57H=CB *9:9F9B79G

*5IHCD<5;MD5BB9L=B

7<5BB9@G

BH<F57M7@=B9 !86+5842=)+7 GD  (F95DCDHCH=7*,9LDCGIF995F@M5DCDHCH=7,(G97F9H=CB@5H9

5DCDHCH=7 %F9@95G9

34

* +<=?CB=B &/)'4  5F@M5DCDHCH=7*,95F@M5DCDHCH=7 +( 34

*5IHCD<5;MD5BB9L=B

7<5BB9@G

5F8=57;@M7CG=89G /-/8'1/7596596+'  (F95DCDHCH=7*,9LDCGIF995F@M5DCDHCH=7,(G97F9H=CB@5H9

5DCDHCH=7 %F9@95G9

34

* (57@=H5L9@ "'<97(6+:/,41/'  (F95DCDHCH=7*,9LDCGIF9@5H95DCDHCH=7 %F9@95G9 34

* C@7<=7=B9 41)./)9275  5F@M5DCDHCH=7 +( +( % 34

* (5HID=@CB9 !46'3-/92

)+11914792

 (F95DCDHCH=7*,9LDCGIF9@5H95DCDHCH=7 %F9@95G9 34

* M7@CD<CGD<5A=89 +MBH<9H=75@?M@5H=B;

5;9BH

 (F95DCDHCH=7*,9LDCGIF9@5H95DCDHCH=7 %F9@95G9 34

* /C;CB=B !)98+11'6/'

('/)'1+37/7

 5F@M5DCDHCH=7 +( +( % 34

,( 589BCG=B9 HF=D<CGD<5H9 *, 75@F9H=7I@=B * 9B8CD@5GA=7 F9H=7I@IA % <=;< AC6=@=HM ;FCID 6CL  +( <95H G<C7? DFCH9=B 

HF95HA9BH K=H< BCB=AAIBC;9B=7 H<9F5D=9G IB89F;C HC@9FC;9B=7 D<5;C

7MHCG=G ,<=G A97<5B=GA =G A98=5H98 6M H<9 =BH9F57H=CB 69HK99B ‘95H

A9’G=;B5@G CB H<9 75B79F 79@@G H<5H 5F9 8M=B; 5B8 H<9=F F9GD97H=J9

7C;B5H9 F979DHCFG CB D<5;C7MH9G H<9G9 F979DHCFG :57=@=H5H9 =AAIBCGID

DF9GG=CB 8F=J9B 6M 5BH==BS5AA5HCFM 7MHC?=B9G 'B H<9 CH<9F <5B8

75B79F 79@@G H<5H 8=9 5:H9F HF95HA9BH K=H< =AAIBCGH=AI@5BHG CF =A

AIBC58>IJ5BHG =B8I79FG C: ! IB89F;C =AAIBC;9B=7 D<5;C7MHC

G=G ,<=G A97<5B=GA =G A98=5H98 6M H<9 =BH9F57H=CB 69HK99B =A

AIBC;9B=7 ‘95H A9’G=;B5@G 5B8 H<9=F F9GD97H=J9 7C;B5H9 F979DHCFG

H<9G9 F979DHCFG =B8I79 =AAIBCGH=AI@5H=CB H<5H =G 8F=J9B 6M DFC=B

S5AA5HCFM 7MHC?=B9G 9@@ 895H< HF=;;9F98 6M 5BH=75B79F AC@97I@9G

75B =B8I79 5 56+2468+2 75B79F GHF9GG F9GDCBG9 K<=7< =G <=;<@M =A

AIBC;9B=7 ,<=G =AAIBC;9B=7=HM =G 5 F9GI@H C: H<9 9A=GG=CB C: %(G

5B8 CH<9F =AAIBCGH=AI@5HCFM AC@97I@9G H<5H 75B 9@=7=H ,79@@ A98=

5H98 5BH=HIACF =AAIB=HM ,<9F9:CF9 H<9 GHF9GG D5H<K5MG =B

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Fig. 3. * GHF9GG 5B8 -(* D5H<K5MG /<9B =BHF59LHF579@@I@5F GHF9GGCFG @958 HC A=G:C@898 DFCH9=B 577IAI@5H=CB *(=( 6=B8G HC H<9 H<F99 -(* G9BGCFG (*# !*α 5B8 , =B

H<9 * (*# D<CGD<CFM@5H9G 9!αH<5H =B<=6=HG DFCH9=B GMBH<9G=G HC 5HH9BI5H9 * DFCH9=B GMBH<9G=G K=H<CIH 5::97H=B; , 57H=J5H=CB !*α5B8 , =B7F95G9 7<5D9FCB9 9LDF9GG=CB 5B8

GMBH<9G=G C: DFCH95G9G =BJC@J98 =B DFCH9=B 89;F585H=CB +7<9A9 K5G ;9B9F5H98 K=H< +7=9B79+@=89G .=G=G7=9B79

Fig. 4. (F9ACFH9A GHF9GG D5H<K5MG =BJC@J98 =B ! !B F9GDCBG9 HC 5 J5F=9HM C: GHF9GG GH=AI@= GI7< 5G 5V CJ9F@C58 5B8CF H<9 *'+ ;9B9F5H=CB DFCGIFJ=J5@ D5H<K5MG GI7< 5G * GHF9GG

-(* 5B8 5IHCD<5;M GK=H7< HC ! 5V @95?5;9 *'+ ;9B9F5H=CB 5B8 75GD5G9 57H=J5H=CB 5F9 9GG9BH=5@ :CF ! +7<9A9 K5G ;9B9F5H98 K=H< +7=9B79+@=89G .=G=G7=9B79

JC@J98 =B 9LDCGIF9 5B8 F9@95G9 C: 5 GD97=R7 D5B9@ C: %(G @958G HC

! F5H<9F H<5B 79@@ 895H< =HG9@: 34

,<9 89H97H=CB C: <5@@A5F?G C: ! =;  =B 7I@HIF98 <IA5B 75B

79F 79@@G =G =ADCFH5BH =B 89H9FA=B=B; DFC;BCGH=7 6=CA5F?9FG HC =89B

H=:M B9K 8FI;G K=H< =AAIBC;9B=7 DFCD9FH=9G ,<=G 89H97H=CB 7CI@8 F9

GI@H =B 5 7<9ACH<9F5D9IH=7 F9GDCBG9 HF5BG:CFA=B; =BHC 5 ACF9 DCH9BH

=AAIB9A98=5H=B; F9GDCBG9 &5HIF5@ 7CADCIB8G H<5H 75B =B8I79 !

A=;<H 69 5B =BH9F9GH=B; 5DDFC57< =B 7CBJ9FH=B; 75B79F =BHC =HG CKB J57

7=B9

3. Natural compounds as ICD inducers

+9J9F5@ D5F5A9H9FG 89H9FA=B9 K<9H<9F 5 7CADCIB8 75B 57H 5G 5B

! =B8I79F CF =: H<9 7CADCIB8 75B 9J9B 9L<=6=H 5BM A95B=B;:I@ 9:

:97H +I7< D5F5A9H9FG 75B =B7@I89 H<9 B5HIF9 C: H<9 =B8I79F H<9 56+

2468+2 GHF9GG D5H<K5MG 57

H=J5H98 6M H<9 =B8I79F H<9 79@@ 895H< D5H<K5MG H<5H H<9 =B8I79F A5M

HF=;;9F =BHF=BG=7 5BH=;9B=7=HM C: H<9 79@@ 5B8 H<9 DCH9B7M 5B8 5J5=@56=@

=HM C: H<9 =AAIB9 79@@ F9GDCBG9 =G7CJ9FM C: BCJ9@ ! =B8I79FG 75D5

6@9 C: 9@=7=H=B; 8=J9FG9 =AAIBC;9B=7 ‘95H A9’G=;B5@G =G 5@GC 7FI7=5@ :CF

7<9ACH<9F5DM IG=B; H<=G 5DDFC57< ,C GIDD@9A9BH DF9G9BH 7<9ACH<9F

5D=9G B9K D@5BH89F=J98 7CADCIB8G H<5H 57H 5G =AAIBCAC8I@5HCFG

:FCA B5HIF5@ DFC8I7HG <5J9 ;5FB9F98 =BH9F9GH +CA9 7<9ACH<9F5D9IH=7

7CADCIB8G H<5H 5F9 IGI5@@M IG98 =B 7@=B=7G 5G =B8I79FG C: ! =B7@I89

B5HIF5@ 7CADCIB8G @=?9 H<9 5BH<F57M7@=B9 8CLCFI6=7=B :FCA !86+584

2=)+7 5+9)+8/97 =;  34 6IH ACGH@M GMBH<9H=7 5;9BHG @=?9 A=

HCL5BHFCB9 5B 5BH<F579B98=CB9 IG98 HC HF95H 57IH9 AM9@C=8 @9I?9A=5

%$ =;  6CFH9NCA=6 5 DFCH95GCA5@ =B<=6=HCF 5DDFCJ98 :CF H<9

HF95HA9BH C: AM9@CA5 5B8 @MAD<CA5 =;  34 5B8 CL5@=D@5H=B

=;  34 !BH9F9GH=B;@M D@5BH89F=J98 DF97@=B=75@ 9LD9F=A9BH5@

7CADCIB8G GI7< 5G G<=?CB=B =GC@5H98 :FCA 2=75C 5 HF58=H=CB5@ <=

B9G9 A98=7=B9 ,% :FCA H<9 8F=98 FCCH

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Fig. 5. #9M ! 9J9BHG =BSI9B798 6M ! =B8I79FG * GHF9GG 5IHCD<5;M ,$* 57H=J5H=CB 5B8 % F9@95G9 5F9 =ADCFH5BH 9J9BHG =B ! !B * GHF9GG -(* =B8I79G H<9 DF95DCDHCH=7

9LDCGIF9 C: H<9 ‘95H A9’G=;B5@ *, H<5H @958G HC H<9 9B;I@:A9BH C: 8M=B; 75B79F 79@@G 5B8 HIACF 5BH=;9B IDH5?9 :CF G IHCD<5;M =B8I79G H<9 F9@95G9 C: ,( H<5H 57H=J5H9G H<9 &$*(

=BS5AA5GCA9 D5H<K5M =B G 5B8 !$βDFC8I7H=CB :CF H<9 57H=J5H=CB C: H<9 HIACF GD97=R7 V , 79@@G ,$* G=;B5@=B; =B8I79G 5B 5IHC7F=B9D5F57F=B9 HMD9 ! !& DFC8I7H=CB :C@@CK98

6M H<9 DFC8I7H=CB C: 0$ ,<9 F9@95G9 C: % 8IF=B; H<9 @5H9 GH5;9G C: 5DCDHCG=G @958G HC ,$* @=;5B8 57H=J5H=CB =B G +7<9A9 K5G ;9B9F5H98 =B D5FH K=H< +7=9B79+@=89G .=G=

G7=9B79

Fig. 6. +7<9A5H=7 F9DF9G9BH5H=CB C: H<9 9::97H C: ! =B8I79FG CB ! <5@@A5F?G +7<9A9

K5G ;9B9F5H98 =B D5FH K=H< +7=9B79+@=89G .=G=G7=9B79

C: /8.475+6292 +6=8.646./>43 CF 75F8=57 ;@M7CG=89G  :CF 9L5AD@9

:FCA /-/8'1/7 596596+' =;  G99 5@GC ,56@9  G<CK98 DFCA=G

=B; DCH9BH=5@ 5G BCJ9@ =B8I79FG C: H<9 <5@@A5F?G C: ! ,<9 D<MG=7C

7<9A=75@AC@97I@5F 7<5F57H9F=GH=7G C: G9J9F5@ ! =B8I79FG D@5M 5 ACF9

7FI7=5@ FC@9 =B H<9=F 57H=CBG F5H<9F H<5B H<9 ‘CBH5F;9H’9::97HG H<9M 9L

<=6=H  DF98=7H98 ! G7CF9 :CF 7CADCIB8G DF9G9BH =B H<9 5DDFCJ98

8FI; @=6F5FM K5G 75@7I@5H98 65G98 CB H<9 7CFF9@5H=CB 69HK99B 6=C@C;

=75@ 5B8 D<MG=7C7<9A=75@ 7<5F57H9F=GH=7G IG=B; RJ9 6=C@C;=75@ G=;BG C:

! D<CGD<CFM@5H=CB C: 9I?5FMCH=7 =B=H=5H=CB :57HCF 9!α *, 9LDC

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, 79@@G DF9J9BH98 B9K HIACF ;FCKH< =B A=79 %CF9CJ9F H<9 A5H

IF5H=CB C: G D<5;C7MHCG=G 5B8 H<9 IDF9;I@5H=CB C: ,MD9 ! !& F9

GDCBG9 K=H< 7@CB5@ 9LD5BG=CB C: V 5B8 V , 79@@G <5J9 699B 8C7

IA9BH98 :H9F – K99?G C: HF95HA9BH K=H< MD(, =B F5HG H<9 9GH56

@=G<98 1 HIACFG H<5H K9F9 9L<=6=H=B; 5 GHFCB; =B=H=5@ F9GDCBG9 HC H<9

HF95HA9BH GI::9F98 5 F9@5DG9 ,<=G F9@5DG=B; =B8=75H98 H<9 F9:F57HCF=B9GG

C: H<9G9 HIACFG HC MD(, HF95HA9BH 1 75B79F 79@@G 75B F9G=GH H<9

57H=CB C: 5 6CB5 R89 ! =B8I79F ,<=G <5G A589 H<9 1 75B79F 79@@

@=B9 5B =BH9F9GH=B; 9LD9F=A9BH5@ AC89@ :CF H<9 GHI8M C: H<9 F9G=GH5B79

C: 5BH=75B79F J577=B5H=CB 89GD=H9 H<9 HMD9 C: ! =B8I79FG IG98 34

,<9 F9G=GH5B79 C6G9FJ98 =B H<=G AC89@ =G 5GGC7=5H98 K=H< 5 89:97H=J9

97HC*, <=;<@=;<H=B; H<9 =ADCFH5B79 C: H<9 ‘95H A9’85B;9F G=;B5@ =B

D<5;C7MH=7 7@95F5B79 5B8 H<9 5BH=75B79F =AAIB9 F9GDCBG9 HCK5F8 5B

H=75B79F J577=B5H=CB LC;9BCIG F97CA6=B5BH *, =ADFCJ98 H<9 D<5;C

7MH=7 F9ACJ5@ C: ! =B8I79FHF95H98 1 79@@G %CF9 =ADCFH5BH@M 5

G=;B=:=75BH =B7F95G9 =B H<9=F J577=B5H=CB 56=@=HM K5G C6G9FJ98 ,<9G9 DF9

7@=B=75@ 85H5 K9F9 GIDDCFH98 6M 5 GI6G9H C: 75B79F D5H=9BHG K=H< 5 J5F=

9HM C: 75B79FHMD9G H<5H DCGG9GG98  "V@CKHIACFG ,<9 9LDF9GG=CB @9J9@G

C: *, K9F9 DF98=7H=J9 C: H<9 D5H=9BH’G 7@=B=75@ F9GDCBG9 HC !=B8I7

=B; 5BH=75B79F H<9F5D=9G ,<9 HIACF *,V<=;<D<9BCHMD9 K5G C6G9FJ98 HC

69 5 ;CC8 DFC;BCGH=7 :57HCF :CF 5 DCG=H=J9 7@=B=75@ F9GDCBG9 HC H<9F5DM

,<=G F9GDCBG9 K5G C6G9FJ98 K=H< ! =B8I79FG GI7< 5G F58=CH<9F5DM 5B8

D57@=H5L9@ =B @IB; 5B8 CJ5F=5B 75B79F D5H=9BHG F9GD97H=J9@M 34 CK

9J9F :IFH<9F J5@=85H=CB =B 7@=B=75@ HIACF H=GGI9 G5AD@9G :/' =AAIBC<=G

HC7<9A=GHFM =G F9EI=F98

 3)41=8/) :/697

J9B H<CI;< BC B5HIF5@ 7CADCIB8G 5+6 7+ CB7C@MH=7 J=FIG9G 75B 69

7@5GG=R98 5G G9@:F9D@=75H=B; “B5HIF5@ 9BH=H=9G”H<5H 5F9 HIACF G9@97H=J9

5B8 CB7C@MG5H9 HIACF 79@@G *979BH@M H<9 56=@=HM C: CB7C@MH=7 J=FIGA9

8=5H98 HIACF 79@@ 895H< HC 9@=7=H 5B =BB5H9 =AAIB9 F9GDCBG9 K5G 89ACB

GHF5H98 ,<9 9:R757M C: 79FH5=B CB7C@MH=7 GHF5=BG K5G 5@GC G<CKB HC =A

DFCJ9 +C :5F H<F99 CB7C@MH=7 J=F5@ GHF5=BG 75B 69 7CBG=89F98 ,MD9 !! !

=B8I79FG =;   7CLG57?=9J=FIG  . CB7C@MH=7 589BCJ=FIG 5B8

&9K75GH@9 8=G95G9 J=FIG &. =;  ,<9G9 GHF5=BG =B8I79 5B * GHF9GG

H<FCI;< H<9 DFC8I7H=CB C: J=F5@ 9BJ9@CD9 DFCH9=BG :C@@CK=B; H<9 =B:97

H=CB C: HIACF 79@@G !B BCBGA5@@ 79@@ @IB; 75F7=BCA5 5B8 589BC75F7=

BCA5 79@@G . 5B8 CB7C@MH=7 589BCJ=FIG9G =B8I79 H<9 GIF:579 9LDF9G

G=CB C: *, :C@@CK98 6M H<9 F9@95G9 C: ,( 5B8 % 34 !BHF5

HIACF5@ 58A=B=GHF5H=CB C: . =B BI89 A=79 =B8I798 H<9 =BR@HF5H=CB

C: &# 79@@G A57FCD<5;9G ;F5BI@C7MH9G 5B8 A5HIF9 G =B H<9 HIACF

H=GGI9 34  8=::9F9BH A97<5B=GA =G IH=@=N98 6M &. HC =B8I79 B97FCD

HCG=G 577CAD5B=98 6M H<9 GIF:579 9LDCGIF9 C: *, 5B8 H<9 DCGHB97FCD

HCH=7 F9@95G9 C: % =B $ ;@=CA5 79@@G 34

@H<CI;< BC DF97@=B=75@ 85H5 9L=GH :CF /3 :/:4 J577=B9GH<9G9 J=FIG9G

5F9 ?BCKB HC 5::97H  A5HIF5H=CB 5B8 H<9 57H=J5H=CB C: H<9 =AAIB9

GMGH9A 65G98 J577=B5H=CB =G H<9F9:CF9 5 DFCA=G=B; GHF5H9;M :CF 75B

79F =AAIBCH<9F5DM <CK9J9F H<9 =AAIBCGIDDF9GG=J9 HIACF A=7FC9B

J=FCBA9BH ,% GIDDF9GG98 5BH=HIACF =AAIB=HM 5B8 =B<=6=H98 H<9 9:

R757M C: H<9 J577=B9 ,C CJ9F7CA9 H<9G9 C6GH57@9G 5B8 9B<5B79 H<9

9:R7=9B7M C: 65G98 J577=B5H=CB CB7C@MH=7



UNCORRECTED PROOF

 '*4-3'+8'1 /4).+2/)'1.'62')414-= <<<  <<<<<<

J=FIG9G K9F9 IG98 5G 5 GI=H56@9 H<9F5D9IH=7 58>IJ5BH K=H< HIACF8=

F97H98 GMGH9A=7  J577=B5H=CBG HC 9@=A=B5H9 =AAIB9 GIDDF9GG=CB 5B8

DFCACH9  :IB7H=CB 34 G 89F=J98 :FCA 6F95GH 75B79F D5H=9BHG

HF95H98 K=H< &.CB7C@MG5H9G G<CK98 =B7F95G98 9LDF9GG=CB C: =AAIBCG

H=AI@5HCFM AC@97I@9G 5B8 H<9 GH=AI@5H=CB C: , 79@@G K<9B 7CAD5F98 HC

G :FCA BCB=B:97H98 6F95GH 75F7=BCA5 79@@G 34 ,<IG =BHF5HIACF5@

HF95HA9BH C: &. K=H< 5 GI67IH5B9CIG J577=B5H=CB 7CBH5=B=B; G

@C5898 K=H< &.=B:97H98 6F95GH 75B79F 79@@G DFCACH98 @CB;@5GH=B; HI

ACFGD97=R7 , 79@@ F9GDCBG9G 5B8 8=G95G9 GH56=@=N5H=CB ,<9 CB7C@MH=7

A95G@9G J=FIG K5G 5@GC 89ACBGHF5H98 HC =B8I79 H<9 F9@95G9 C: %

5G K9@@ 5G H<9 9LDCGIF9 C: G HC A95G@9G J=FIGHF95H98 A9@5BCA5

79@@G /3 :/864 ,<9 9:R7=9BH@M DF=A98 A9@5BCA5GD97=R7 79@@G K9F9 ?=@@98

6M !&γDFC8I7=B; V , 79@@G %CF9CJ9F A95G@9G J=FIG=B:97H98

A9@5BCA5 79@@G DFCJ=898 ACF9 9::97H=J9 HIACF CB7C@MG5H9G :CF H<9 F9

7FI=HA9BH C: G H<5B H<9 IB=B:97H98 A9@5BCA5 79@@ @MG5H9G 34

4. Mechanisms leading to ICD

  786+77 #  '3* )'16+8/)91/3 +<54796+

,<9 * =G 5 ?9M CF;5B9@@9 :CF 5V GHCF5;9 @=D=8 6=CGMBH<9G=G :C@8

=B; AC8=R75H=CB 5B8 HF5:R7?=B; C: G97F9HCFM 5B8 HF5BGA9A6F5B9 DFC

H9=BG (<MG=C@C;=7 GHF9GG 75B =B8I79 *GHF9GG F9GI@H=B; =B 7F=H=75@ 9::97HG

CB =BHF579@@I@5F <CA9CGH5G=G 34 ,<9 D<MG=C@C;=75@ GHF9GG9G =B7@I89 =B

7F95G98 DFCH9=B CJ9F@C58=B; 5 F9GI@H C: =B7F95G98 DFCH9=B HF5BG@5H=CB CF

H<9 =B<=6=H=CB C: 89;F585H=CB C: A=G:C@898 DFCH9=BG F98CL =A65@5B79

C: H<9 * @IA9B K<=7< =AD5=FG H<9 :CFA5H=CB C: 8=GI@R89 6CB8G 8MG

F9;I@5H=CB C: H<9 6IT9F=B; 75D57=HM C: *5V <CA9CGH5G=G <MDCL=5

5B8 BIHF=9BH 89DF=J5H=CB 34 ,<9 * F9GDCB8G HC GHF9GG 6M 57H=J5H

=B; 5B 585DH=J9 A97<5B=GA 75@@98 -(* 34 K<=7< 5=AG HC F99GH56

@=G< * <CA9CGH5G=G 5B8 DFCACH9 GIFJ=J5@ 6M F9GHCF=B; H<9 *5GGC7=5H98

DFCH9=B :C@8=B; A57<=B9FM H<FCI;< H<9 F98I7H=CB C: DFCH9=B HF5BG@5H=CB

CA9CGH5G=G =G 57<=9J98 6M 5 F98I798 HF5BG@C75H=CB C: DFCH9=BG HC H<9

* =B7F95G=B; H<9 GMBH<9G=G C: 7<5D9FCB9G 5B8CF 6M =B7F95G=B; DFC

H9=B 89;F585H=CB :/' H<9 + DFCH95GCA9 34 !BCG=HC@F9EI=F=B; 9BNMA9

!*α (*# 5B8 57H=J5H=B; HF5BG7F=DH=CB :57HCF , 5F9 H<9 ?9M

* GHF9GG G9BGCFG H<5H F9;I@5H9 H<9 -(* 34 ,<9G9 DFCH9=BG 5F9 5@GC IB

89F H<9 B9;5H=J9 7CBHFC@ C: *(=( H<5H A5=BH5=BG H<9 DFCH9=BG =B 5B

=B57H=J9 GH5H9 IB89F D<MG=C@C;=75@ 7CB8=H=CBG -B89F * GHF9GG *(

=( 8=GGC7=5H9G :FCA H<9G9 * GHF9GG G9BGCFG 57H=J5H=B; H<9 (*# D5H<

K5M ,<=G 57H=J5H=CB H<9B @958G HC H<9 =B8I7H=CB C: 9!αD<CGD<CFM@5

H=CB K<=7< =B HIFB =B<=6=HG DFCH9=B GMBH<9G=G =;  7H=J5H98 (*# =G

F9EI=F98 :CF !5GGC7=5H98 97HC*, 9LDF9GG=CB =B J=HFC 5B8 ! =B J=JC

34 CK9J9F 57H=J5H=CB C: (*# 5@CB9 8C9G BCH 5@K5MG F9GI@H =B =B

7F95G98 97HC*, 34 ,<IG * GHF9GG =G F9EI=F98 6IH =G BCH GI:R7=9BH HC

=B8I79 H<9 HF5BG@C75H=CB C: *, HC H<9 79@@ GIF:579 34 ,<=G HF5BG@C

75H=CB C: *, C77IFG DF=CF HC HF5BG@C75H=CB C: D<CGD<5H=8M@G9F=B9 (+ HC

H<9 D@5GA5 A9A6F5B9 5G 5 DF95DCDHCH=7 9J9BH 34 7HC*, G9FJ9G

5G 5 DCH9BH ‘95H A9’G=;B5@ H<5H =G F97C;B=N98 6M H<9  F979DHCF CB

D<5;C7MH9G 5B8 GH=AI@5H9G H<9 9B;I@:A9BH C: 8M=B; 75B79F 79@@G :CF @C75@

G

77CF8=B;@M =H =G 9GG9BH=5@ HC =BJ9GH=;5H9 BCJ9@ 7CADCIB8G 56@9

HC HF=;;9F 79@@I@5F 5@H9F5H=CBG <=BH=B; 5H 5B !=B8I7=B; DCH9BH=5@

CIA5F=BG 5F9 B5HIF5@ 7CADCIB8G K=H< 5BH=CL=85BH 5BH==BS5AA5HCFM

5B8 5BH=75B79F DCH9BH=5@ ?BCKB HC AC8I@5H9 =BS5AA5HCFM D5H<K5MG

=G7CIA5F=B ', @98 HC 79@@ 7M7@9 5FF9GH 5B8 CBG9H C: G9B9G79B79 CB

 @IB; 75B79F 79@@G 7CB7CA=H5BH K=H< * 5B8 C@;= GHF9GG @958=B;

HC A9H56C@=7 5@H9F5H=CBG !BH9F9GH=B;@M 7CA6=B5H=CB HF95HA9BHG C: ',

K=H< GI6HCL=7 7CB79BHF5H=CBG C: 7@L$ 5B8 %7@H5F;9H=B;   DFCH9=B

=B<=6=HCFG HF=;;9F98 5DD95F5B79 C: GMB9F;=GH=7 =AAIBC;9B=7 79@@ 895H<

A5F?9FG 34 +=A=@5F@M 7MHCGH5H=7 6=G<M8FCLM7CIA5F=B 89F=J5H=J9

', K<=7< 7CAD@=98 K=H< H<9 $=D=BG?=G FI@9 C:  5B8 =B8I798 8=::9F

9BH=5@ HCL=7=HM =B J5F=CIG 7<FCB=7 AM9@C=8 @9I?9A=5 %$ 79@@ AC89@G

', HF=;;9F98 * GHF9GG @958=B; HC 75BCB=75@ 75GD5G989D9B89BH 5DCD

HCG=G 5B8 F9@95G9 C: %(G =B7@I8=B; *, % 5B8 *D 34

,<9 57H=J5H=CB C: H<9 -(* DFCGIFJ=J5@ D5H<K5M AC8I@5H9G H<9 ,%

HC GIDDCFH HIACF=;9B9G=G 6M =B7F95G=B; H<9 G97F9H=CB C: =BS5AA5HCFM

7MHC?=B9G 9; !$ !$β !$ !$ 5B8 ,&α34 ,<9 -(*

75B 5@GC AC8I

@5H9 5BH=;9B=7=HM :/' H<9 @5GG ! %  K<=7< =G @C5898 K=H< 5B 5BH=;9B=7

D9DH=89 K=H<=B H<9 * 69:CF9 69=B; HF5BGDCFH98 HC H<9 79@@ GIF:579 =A

D@=75H=CB C: * GHF9GG =G GH=@@ 8965H98 34 CK9J9F K<9B * GHF9GG =G

7CBG=89F98 HCC G9J9F9 H<9 DFCGIFJ=J5@ A97<5B=GA GK=H7<9G =BHC 5 79@@

895H< D5H<K5M 8F=J9B 6M H<9 , 5B8 ,9B<5B79F6=B8=B; DFC

H9=B ( <CAC@C;CIG DFCH9=B  '( ,<9 HF5BG=H=CB =G 5@GC 7<5F

57H9F=N98 6M H<9 F9@95G9 C: 85B;9F G=;B5@G K<=7< HF=;;9F 5B 5BH=HIACF

=AAIB9 F9GDCBG9 34 !H <5G 699B C6G9FJ98 H<5H 5V @95?5;9 A5M

69 F9EI=F98 :CF * GHF9GG 5B8 *, 9LDCGIF9 34 ,<9 8=5@?M@ F9GCF

7=BC@ GH9AD<C@ 5 D<MHCHCL=B :FCA !8+25.=1/92 -14(91/,+692 =B8I798 5

HMD9 C: 7CBHFC@@98 B97FCG=G H<5H =G 89D9B89BH CB A=HC7<CB8F=5@ D9FA9

56=@=HM HF5BG=H=CB DCF9 A(,( CD9B=B; !ADCFH5BH@M H<9 HF5BG@5H=CB5@

=AAIBC;9B=7 DCH9BH=5@ C: GH9AD<C@ K5G J5@=85H98 6M A95GIF9A9BH C:

% F9@95G9 =B GH9AD<C@HF95H98 %$ D5H=9BH 79@@G +H9AD<C@=B

8I798 BCB75BCB=75@ DFC;F5AA98 79@@ 895H< @98 HC =B7F95G98 7MHCGC@=7

5V @9J9@G 34 ! =B8I7H=CB K=H< G K5G =B<=6=H98 6M 5V 7<9@5

HCFG GI;;9GH=B; H<5H 5V =CBCD<CF9G A=A=7 H<9 *,9LDCG=B; 57H=J

=HM 34 *'+ 5F9 5@GC 7F=H=75@ :CF 5BH<F57M7@=B9=B8I798 ! 5B8 HF95H

A9BH K=H< &579HM@7MGH9=B9 & F98I798 H<9 HF5BG@C75H=CB C: *, HC

H<9 D@5GA5 A9A6F5B9 34 ,<9 CL=8=N=B;:C@8=B; 9BJ=FCBA9BH C: H<9

* 5B8 H<9 DF9G9B79 C: 56IB85BH *F9G=89BH DFCH9=BG GI7< 5G DFCH9=B

8=GI@R89 =GCA9F5G9 (! 5F9 ?BCKB HC DF98CA=B5BH@M :57=@=H5H9 8=GI@

R89 6CB8 :CFA5H=CB 8IF=B; H<9 DFC79GG C: DFCH9=B :C@8=B; ,<=G * 9B

J=FCBA9BH =G DF9J9BH=B; 5;;F9;5H=CB CF 577IAI@5H=CB C: IB:C@898 DFC

H9=BG =B H<9 * @IA9B @H9F5H=CBG =B H<9 CL=85H=J9 9BJ=FCBA9BH C: H<9

* 75IG9 H<9 DFC8I7H=CB C: * GHF9GG=B8I798 *'+ 34 MGH9=B9 5B8

;@IH5H<=CB9 +  <5J9 699B 89ACBGHF5H98 HC 6IT9F *'+ 577IAI@5H=CB

5B8 HC A5=BH5=B * F98CL <CA9CGH5G=G *'+ 7CI@8 69 ;9B9F5H98 8IF

=B; H<9 HF5BG:9F C: 9@97HFCBG :FCA DFCH9=B H<=C@G HC AC@97I@5F CLM;9B

6M * CL=8CF98I7H=B *' 5B8 (! CF 8IF=B; DFCH9=B A=G:C@8=B;

8I9 HC H<9 89D@9H=CB C: + 34 :H9F IH=@=N=B; +  H<=C@G 5F9 F9

D5=F98 9B56@=B; H<9A HC =BH9F57H K=H< *'(! 5B8 HC 69 F9CL=8=N98

,<9G9 F9D9H=H=J9 7M7@9G C: 8=GI@R89 6CB8 6F95?5;9 5B8 :CFA5H=CB ;9B

9F5H9 *'+ =;  CK9J9F *'+ 5@CB9 5F9 =BGI:R7=9BH HC =B8I79 !

5G *'+ ;9B9F5H98 =B H<9 * 5F9 =BGI:R7=9BH HC =B=H=5H9 CL=85H=J9 GHF9GG

34 5@7=IA @95?5;9 :FCA H<9 * K5G DFCDCG98 5G H<9 A=GG=B; @=B?

69HK99B * GHF9GG 5B8 *'+ =B 7<9ACH<9F5DM=B8I798 ! 5@7=IA F9

@95G98 6M H<9 * 7CI@8 =B7F95G9 A=HC7<CB8F=5@ 5V @C58=B; :/' A=HC

7<CB8F=55GGC7=5H98 A9A6F5B9G %%G 34 K<=7< DFCACH9 A=HC

7<CB8F=5@ *'+ ;9B9F5H=CB 34 %=HC7<CB8F=5@ *'+ ;9B9F5H=CB G9BG=

H=N9G * 5V 7<5BB9@G HC F9@95G9 5V 5B8 =B7F95G9G *'+ ;9B9F5H=CB :/'

5 :998657? @CCD 34 5@7=IA=B8I798 A=HC7<CB8F=5@ *'+ HC;9H<9F

K=H< *'+ ;9B9F5H98 =B H<9 * @IA9B A5M F957< H<9 7F=H=75@ H<F9G<C@8

F9EI=F98 :CF ! 5@7=IAA98=5H98 *'+ DFC8I7H=CB A5M 9L579F65H9 *

GHF9GG @958=B; HC 79@@ 895H< H<5H =G A5=B@M 9L97IH98 H<FCI;< 5DCDHCG=G

34 CF HC J5F=CIG :CFAG C: F9;I@5H98 B97FCG=G GI7< 5G B97FCDHCG=G =;

 34 G 5B 9L5AD@9 GH9AD<C@ =B8I798 BCB75BCB=75@ DFC;F5AA98

79@@ 895H< K5G HF=;;9F98 6M =BCG=HC@ HF=GD<CGD<5H9 !(89D9B89BH

7MHCGC@=7 5V 577IAI@5H=CB K<=7< @958 HC * GHF9GG 5B8 A=HC7<CB8F=5@

GK9@@=B; 5GGC7=5H98 K=H< 7C@@5DG98 7F=GH59 89D9B8=B; CB A=HC7<CB8F=5@

D9FA956=@=HM HF5BG=H=CB DCF9 A(,( CD9B=B; 34 ,<9 6=G<M8FCLM

7CIA5F=B 89F=J5H=J9 ', =BGH958 K5G 56@9 HC HF=;;9F * GHF9GG @958

=B; HC 75BCB=75@ 75GD5G989D9B89BH 5DCDHCG=G 5B8 F9@95G9 C: %(G =B

J5F=CIG 7<FCB=7 AM9@C=8 @9I?9A=5 %$ 79@@ AC89@G 34

5GD5G9A98=5H98 57H=J5H=CB C: 5DCDHCG=G =G 9GG9BH=5@ :CF ! 34

* GHF9GG DFCACH9G 5V F9@95G9 :FCA H<9 * K<=7< @958G HC H<9 57

H=J5H=CB C: H<9 5V89D9B89BH DFCH95G9 75@D5=B K<=7< 57H=J5H9G 75G

D5G9A98=5H98 79@@ 895H< 7H=J5H98 75GD5G9 7@95J9G  79@@ F979DHCF

5GGC7=5H98 DFCH9=B 5D 34 :CFA=B; 5 DFC5DCDHCH=7 D :F5;A9BH

H<5H =BH9F57HG K=H< H<9 5DCDHCG=G F9;I@5HCF  79@@ @MAD<CA5 7@ HC

F9@95G9 G9EI9GH9F98 7@5GGC7=5H98 DFCH9=B U 5L  5B8 7@ 5;CB=GH

?=@@9F  5? 34 =;  ,<9 7<9A=75@ GHFI7HIF9 C: H<9 8=::9F9BH

! =B8I79FG =G BCH @=B?98 HC H<9=F :IB7H=CB 5G G<CKB 6M H<9 8=::9F

9BH =AAIBC;9B=7 DCH9BH=5@ C: HKC GHFI7HIF5@@M F9@5H98 7CADCIB8G CL

5@=D@5H=B 5B8 7=GD@5H=B CBG=89F=B; H<9 =ADCFH5B79 C: HF=;;9F=B; 5B *

GHF9GG 6M ! =B8I79FG H<9 J5F=CIG * GHF9GG ?=B9H=7G 5B8 =BH9BG=HM A5M

<5J9 5B =ADCFH5BH =AD57H CB H<9 EI5@=H5H=J9 5B8 EI5BH=H5H=J9 9::97HG C:

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UNCORRECTED PROOF

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+9J9F5@ ! =B8I79FG CL5@=D@5H=B A=HCL5BHFCB9 8CLCFI6=7=B 75B

75IG9 ,( F9@95G9 :FCA 8M=B; HIACF 79@@G 34 IHCD<5;M F9DF9

G9BHG H<9 DF=A5FM A97<5B=GA H<5H =B8I79G <=;< 9LHF579@@I@5F ,( F9

@95G9 :FCA !HF95H98 8M=B; HIACF 79@@G ,<9F9:CF9 H<=G A97<5B=GA

=G G99B HC 57H 5G 5 DCH9BH 7<9ACH57H=7 :57HCF H<5H 5HHF57HG AM9@C=8

79@@G =BHC H<9 A=7FC9BJ=FCBA9BH 8F=J=B; H<9 57H=J5H=CB C: G :/' H<9

DIF=B9F;=7 (*0 =;  IHCD<5;M =G 5 75H56C@=7 DFC79GG H<5H 89

;F589G 7MHCD@5GA=7 DFCH9=BG 5;;F9;5H9G 5B8 85A5;98 CF;5B9@@9G ,<=G

89;F585H=CB C77IFG H<FCI;< 5B 5IHCD<5;CGCA9@MGCGCA9 7CAD@9L =B

JC@J=B; @MGCGCA5@ DFCH95G9G H<5H 9BGIF9G H<9 HF5BGA=HH5B79 C: 9B9F;M HC

H<9 79@@G H<FCI;< H<9 F9@95G9 C: ,( :FCA 8M=B; 79@@G =;  34

IHCD<5;M =G 5 DF9ACFH9A GHF9GG 585DH5H=CB A97<5B=GA K<=7< =G

5 F9GDCBG9 HC 5 J5F=9HM C: GHF9GG GH=AI@= =B7@I8=B; GH5FJ5H=CB <MDCL=5

* GHF9GG 5B8 CL=85H=J9 GHF9GG ,<9G9 GH=AI@= HF=;;9F 5 DFCGIFJ=J5@ D5H<

K5M 5B8 @958 HC 79@@ 895H< 5JC=85B79 9D9B8=B; CB H<9 H<F9G<C@8 5I

HCD<5;M D@5MG 5 FC@9 =B 95F@M 5DCDHCG=G G=B79 A=HCL5BHFCB9 5B8 CL

5@=D@5H=B 6CH< =B<=6=H ,( G97F9H=CB :FCA 8M=B; 79@@G 6M GIDDF9GG=B;

, , 5B8 697@=B 34 IHCD<5;M 5@@CKG ,( F9@95G9 6M

5 A97<5B=GA H<5H F9EI=F9G H<9 @MGCGCA5@5GGC7=5H98 A9A6F5B9 DFCH9=B

$%( K<=7< =G HF5BG@C75H98 HC H<9 D@5GA5 A9A6F5B9 =B 5 75GD5G9

5B8 D5BB9L=B  (&089D9B89BH A5BB9F &9J9FH<9@9GG 5IHCD<5;M

5B8 $%( 8C BCH =BSI9B79 H<9 CD9B=B; C: H<9 (&0 7<5BB9@ K<=7<

=G F9EI=F98 :CF H<9 !5GGC7=5H98 HF5BG@C75H=CB C: $%( HC H<9 D@5GA5

A9A6F5B9 5B8 :CF @MGCGCA5@ 9LC7MHCG=G 34 $MGCGCA9G 7CBGH=HIH9

H<9 A5=B GCIF79 C: J9G=7I@5F ,( F9@95G9 :FCA H<9 =BHF579@@I@5F 9BJ=FCB

A9BH H<FCI;< H<9 :IG=CB C: @MGCGCA5@ 5B8 D@5GA5 A9A6F5B9G 9J9BHI

5@@M 5@@CK=B; ,( 9LC7MHCG=G 3–4 ,<9 GH=AI@5H=CB C: ,$* @958G

HC H<9 @MGCGCA5@ 5@?5@=N5H=CB 5B8 F9@95G9 C: ,( 5B8 @MGCGCA5@ 7CBH9BHG

:FCA 6CH< CDH=7 B9FJ9 <958 5GHFC7MH9G 5B8 F9H=B5@ D=;A9BH98 9D=H<9@=5@

79@@G =BJC@J=B; H<9 A,'* G=;B5@=B; D5H<K5M 34 $MGCGCA5@ ,( G9

7F9H=CB K5G G<CKB HC F9;I@5H9 5V <CA9CGH5G=G =B , ( <IA5B ACBC

7MH=7 79@@G :/' 57H=J5H=CB C: DIF=B9F;=7 (1 F979DHCFG ,( G97F9H=CB 5B8

GI6G9EI9BH (1 F979DHCF 57H=J5H=CB 7CBHF=6IH9G G=;B=:=75BH@M HC 5V G=;

B5@=B; ;9B9F5H98 6M @MGCGCA9 8=GFIDH=CB HF=;;9F98 6M H<9 75H<9DG=B 

GI6GHF5H9 ;@M7M@$D<9BM@5@5B=B969H5B5DH<M@5A=89 (& 34

(F95DCDHCH=7 G97F9H=CB C: ,( A5M 89D9B8 CB H<9 (*# DFCL=A5@

G97F9HCFM D5H<K5M 5B8 D<CGD<5H=8M@=BCG=HC@6=GD<CGD<5H9 ?=B5G9

(!#89D9B89BH 9LC7MHCG=G ,( 75B 69 G97F9H98 =B H<9 @5H9 5DCDHCH=7

GH5;9 6M D5GG=J9 F9@95G9 CK=B; HC D@5GA5 A9A6F5B9 D9FA956=@=N5H=CB

/<9B H<9 -(* :5=@G HC F9GHCF9 DFCH9=B HF5:R7?=B; @958=B; HC 577I

AI@5H=CB C: @5F;9 5ACIBHG C: IB:C@898 DFCH9=BG H<5H 5F9 BC @CB;9F 89

;F5898 6M H<9 DFCH95GCA5@ D5H<K5M =B H<9 7MHCGC@ * GHF9GG 75B =B8I79

5IHCD<5;M ,<9 DFCH95GCA9 79FH5=B@M 7CBHF=6IH9G HC 89;F589 G<CFH@=J98

DFCH9=BG 34 K<9F95G 5IHCD<5;M =G F9EI=F98 HC 6F95? 8CKB @CB;@=J98

CF <95J=@M 5;;F9;5H98 DFCH9=BG 34 ,<IG H<9 CJ9FK<9@A98 DFCH95

GCA9 6M 9L79GG=J9 DFCH9=B 5;;F9;5H=CB 75B 69 GIDDCFH98 6M ! =B

8I79FG HF=;;9F=B; 5IHCD<5;M HC 5@@9J=5H9 H<=G 9L79GG C: IB:C@898 DFC

H9=BG -(* 5B8 5IHCD<5;M D5H<K5MG H<9F9:CF9 KCF? HC;9H<9F HC F9G7I9

H<9 79@@G 5H 5 79FH5=B H<F9G<C@8 5B8 H<IG 9B<5B79 ! * GHF9GG 75B

5@GC 8=F97H@M DFCACH9 5IHCD<5;M 6M D<CGD<CFM@5H=B; 7@ 34 K<=7<

@958G HC H<9 8=GGC7=5H=CB C: 697@=B :FCA 7@ K<=7< =G 5 ?9M F9;I@5

HCFM GH9D =B 5IHCD<5;M DFC;F9GG=CB *9;I@5H=B; H<9 HF5BG7F=DH=CB C: ,G

75B 5@GC @958 HC H<=G 8=GGC7=5H=CB ,<9 9!α, D5H<K5M 75B 57H=J5H9

5IHCD<5;M 6M IDF9;I@5H=B; H<9 9LDF9GG=CB C: H<9 GHF9GGF9;I@5H98 DFC

H9=B D 5B8 =HG 8CKBGHF95A H5F;9H DG9I8C?=B5G9 ,F=66@9G <CAC@C;I9 

,* 34 5B =B<=6=HCF C: H<9 #,A5AA5@=5B H5F;9H C: F5D5AM7=B

7CAD@9L A,'* =;  34 9B9H=7 =B<=6=HCFG C: 5IHCD<5;M GI7<

5G 7<@CFCEI=B9 5B8 <M8FCLM7<@CFCEI=B9 F9GHCF9 7<9ACG9BG=H=J=HM 5B8

9B<5B79 HIACF 79@@ 5DCDHCG=G =B A5BM HMD9G C: <9A5HCDC=9H=7 A5@=;

B5B7=9G 9GD=H9 =HG DFCGIFJ=J5@ FC@9 5IHCD<5;M A5M 5@GC F9DF9G9BH 5

A97<5B=GA C: 79@@ 895H< 89RB98 5G 5IHCD<5;=7 79@@ 895H< 34 F

G9B=7 HF=CL=89 =B8I79G 5IHCD<5;=7 79@@ 895H< =B @9I?9A=7 79@@ @=B9G 5B8

%$ DFC;9B=HCFG K<=7< 7CI@8 69 F9J9FG98 6M ?BC7?8CKB C: 697@=B

CF H; %CF9CJ9F H<9 CJ9F9LDF9GG=CB C: 697@=B 89@=J9F98 6M 5B CB

7C@MH=7 J=FIG =B8I798 5IHCD<5;=7 79@@ 895H< =B 5 J5F=9HM C: @9I?9A=7 79@@

@=B9G 5B8 DF=A5FM @9I?9A=7 6@5GHG 34

,( G=;B5@=B; <5G 5B 5IHC7F=B9D5F57F=B9 9::97H 'B79 F9@95G98 =BHC

H<9 9LHF579@@I@5F 9BJ=FCBA9BH ,( 75B 5HHF57H G 5B8 57H=J5H9 H<9

=BS5AA5GCA9 D5H<K5M HC 7F95H9 5 7FCGG@=B? 69HK99B H<9 =BB5H9 5B8

585DH=J9 =AAIB9 F9GDCBG9G !B8998 ,( G=;B5@=B; :/' H<9 (*0 F9

79DHCFG 57H=J5H9G H<9 & , $** 5B8 (1 8CA5=BG7CBH5=B=B; DFC

H9=B &$*( =BS5AA5GCA9 D5H<K5M =B G 5B8 A57FCD<5;9G 34

,<9 &$*( D5H<K5M 57H=J5H9G 75GD5G9 K<=7< @958G HC H<9 G97F9H=CB C:

A5HIF9 DFC=BS5AA5HCFM !$β5B8 !$ DFCH9=BG +I6G9EI9BH@M γδ ,

79@@G 75B 69 F97FI=H98 @958=B; HC !&γDFC8I7=B; HIACFGD97=R7 V ,

79@@G =;  34 CK9J9F ,( A5M 5@GC 69 =AD@=75H98 =B 7CB:9FF=B;

HC@9FC;9B=7 :95HIF9G HC H<9 =BR@HF5H=B; G :/' IDF9;I@5H=B; =B8C@95A=B9

8=CLM;9B5G9 !' 34 5B 9BNMA9 ?BCKB HC 69 =BJC@J98 =B H<9

;9B9F5H=CB C: 5B =AAIBCGIDDF9GG=J9 A=7FC9BJ=FCBA9BH =B %$ ,<9 F9

@95G9 C: 9LHF579@@I@5F ,( :FCA 7<9ACH<9F5DMHF95H98 8M=B; 79@@G K5G

G<CKB HC =B7F95G9 H<9 BIA69F C: ,V*G 9LDF9GG=B; ( 5B8 !'V

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=@M ,<=G 7MHC?=B9 5@GC <5G =AAIBCAC8I@5HCFM 57H=J=H=9G 5B8 A5M 7CB

HF=6IH9 HC <CGH 5BH=J=F5@ 89:9BG9 34 ,$* =G 5 ?9M 9B8CGCA5@ (**

:CF 8CI6@9 GHF5B898 8G*& 5B8 =G ?BCKB HC HF=;;9F HMD9 ! !& =B 5B

H=J=F5@ =AAIB9 F9GDCBG9G 34 BH<F57M7@=B9G 34 9@=7=H H<9 ,$*

D5H<K5M =B 75B79F 79@@G @958=B; HC 5IHC7F=B9D5F57F=B9 HMD9 ! !& G=;

B5@=B; 5B8 H<9 GI6G9EI9BH G97F9H=CB C: H<9 7<9AC?=B9 0 ACH=:

@=;5B8 0$ ,<9G9 DFC79GG9G GI;;9GH H<5H 5BH<F57M7@=B9A98=5H98

=AAIB9 F9GDCBG9G A=A=7 H<CG9 =B8I798 6M J=F5@ D5H<C;9BG !&G 6=B8

HC H<9=F 7C;B5H9 F979DHCFG <9H9FC8=A9FG C: =BH9F:9FCB αβF979DHCF !

&* 5B8  HF=;;9F=B; H<9 D<CGD<CFM@5H=CB C: G=;B5@ HF5BG8I79FG 5B8

57H=J5HCFG C: HF5BG7F=DH=CB +,, HF5BG7F=DH=CB :57HCFG 5B8 H<9 HF5B

G7F=DH=CB C: !& F9GDCBG9 ;9B9G ,<9G9 F957H=CBG H<9B @958 HC H<9 F9@95G9

C: 0$ 5B8 7CBHF=6IH9 HC 5B 5BH=75B79F =AAIB9 F9GDCBG9 =; 

9B9H=75@@M ?BC7?=B; CIH ,$* CF !&* :5=@98 HC F9GI@H =B 5 F9GDCBG9

HC 7<9ACH<9F5DM IB@9GG ,MD9 ! !& CF 0$ K5G 58A=B=GH9F98 F9GD97

H=J9@M =B H<9 ?BC7?CIHG ,<=G =B7@IG=CB H<9F9:CF9 ;I5F5BH99G H<9 J577=

B5H=CB C: A=79 5;5=BGH HIACF F97<5@@9B;9 34 ,<9 HMD9 ! !& G=;B5

HIF9 =G 5@GC 5 ;CC8 DFC;BCGH=7 :57HCF H<5H DF98=7HG 7@=B=75@ F9GDCBG9G HC

5BH<F57M7@=B965G98 7<9ACH<9F5DM H<5H <5G 699B 7<5F57H9F=N98 6M DCCF

DFC;BCG=G =B 6F95GH 75F7=BCA5 D5H=9BHG

5. Perspectives

9D9B8=B; CB H<9 B5HIF9 C: H<9 ! =B8I79FG 5B8 H<9 =BH9BG=HM C: H<9

GH=AI@= 75B79F 79@@G A5M 8=9 5B8 A5B=:9GH 5 J5F=9HM C: 56+2468+2 GHF9GG

F9GDCBG9G K<=@9 =B8I7=B; 8=::9F9BH 79@@ 895H< D5H<K5MG :CF 5 @C75@ 9:

:97H %(G 8=7H5H9 H<9 F9@5H=CBG<=D 69HK99B GHF9GG98 5B8 8M=B; 79@@G

5B8 H<9 A=7FC9BJ=FCBA9BH 6M 5::97H=B; H<9 F97FI=H98 AM9@C=8 5B8 @MA

D<C=8 57HCFG ,<IG %(G 75B 69 89G7F=698 5G H<9 89H9FA=B5BH C: H<9

RB5@ CIH7CA9 =B H<9 7CBH9LH C: H<9 @C75@ 9::97H ,<9 B5HIF9 C: D<5;C7MH9G

/+ HC@9FC;9B=7 :7 =AAIBC;9B=7 5B8 H<9 7<C=79 C: H<9 @9I?C7MH9G 5H

HF57H98 HC H<9 8M=B; 79@@G A5M =BSI9B79 H<9 =BS5AA5HCFM 5B8 =AAIB9

7CBG9EI9B79G C: 79@@ 895H< ,<IG H<9 5J5=@56=@=HM 5B8 G9@97H=CB C: H<9

F97FI=H98 @9I?C7MH9G H<5H F9GDCB8 HC %(G 5B8 H<9 =BS5AA5HCFM =B

H9FA98=5HCFG H<5H A5M GH=AI@5H9 CF =B<=6=H H<9 @C75@ F9GDCBG9 5F9 =ADCF

H5BH D5F5A9H9FG HC 7CBG=89F =B H<9 G9@97H=CB C: B5HIF5@ ! =B8I79FG

/9 <5J9 G99B H<5H G9J9F5@ 7CADCIB8G =B8I79 ! 5B8 A=;<H 5@GC 69

5 DFCA=G=B; 5DDFC57< =B 7CBJ9FH=B; 75B79F =BHC =HG CKB J577=B9 =; 

,<IG H<9 89H97H=CB C: <5@@A5F?G C: ! =G =ADCFH5BH =B =89BH=:M=B; B9K

8FI;G K=H< =AAIBC;9B=7 DFCD9FH=9G :CF 5 DCH9BH=5@ 7@=B=75@ CIH7CA9 !B

D5FH=7I@5F ! 5B8 %( G=;B5@=B; 7CI@8 697CA9 9GG9BH=5@ :CF H<9 G9

@97H=CB C: D5H=9BHGD97=R7 7<9ACH<9F5DM 5B8 =B HIACFGD97=R7 H<9F5D9I

H=7 GHF5H9;=9G ,<=G 5DDFC57< KCI@8 9J9BHI5@@M 5@@CK HC :IFH<9F D9FGCB5@

=N9 5BH=75B79F H<9F5D=9G 65G98 CB ! 7<5F57H9F=GH=7G

Con*ict of interest

,<9 5IH<CFG 897@5F9 BC 7CBS=7H C: =BH9F9GH

Acknowledgements

* =G GIDDCFH98 6M 5 ;F5BH :FCA ,O@OJ=9 $IL9A6IF; *9G95F7< 5H

$% =G GIDDCFH98 6M H<9 “*97<9F7<9 5B79F 9H +5B;”:CIB85H=CB

“*97<9F7<9G +7=9BH=REI9G $IL9A6CIF;”“9B 59FN RF ?F==6G?F5B? #5B

B9F” 7H=CB $!'&+ “.5=B7F9 @9 5B79F” 5B8 ,O@OJ=9 $IL9A6CIF; %

=G GIDDCFH98 6M H<9 *9G95F7< !BGH=HIH9 C: (<5FA579IH=75@ +7=9B79G C@

@9;9 C: (<5FA57M +9CI@ &5



UNCORRECTED PROOF

 '*4-3'+8'1 /4).+2/)'1.'62')414-= <<<  <<<<<<

H=CB5@ -B=J9FG=HM #CF95 H<9 ,IACF %=7FC9BJ=FCBA9BH *

 :FCA H<9 &5H=CB5@ *9G95F7< CIB85H=CB :IB898 6M H<9

%=B=GHFM C: +7=9B79 5B8 !, C: #CF95 5B8 H<9 F95H=J9(=CB99F=B; *9

G95F7<9FG (FC;F5A H<FCI;< +9CI@ &5H=CB5@ -B=J9FG=HM +&- 3IB8=B;

BIA69F 4 + 5B8 % HC F5=B #CF95 #  (@IG DFC

;F5A #CF95

References

34 ( %5HN=B;9F ,C@9F5B79 85B;9F 5B8 H<9 9LH9B898 :5A=@M BBI *9J !AAIBC@

  –

34 +$ ,CD5@=5B + C8= "* F5<A9F +& 9HH=B;9F  +A=H<  %79F

ACHH " (CK89F@M * 5FJ5>5@ " +CGA5B % H?=BG ( $9A=B; *

+D=;9@ +" BHCB=5 $ CFB  F5?9 % (5F8C@@ $ <9B /  +<5F:A5B

* B89FG "% ,5I69 ,$ %7%=@@9F  0I " #CFA5B % "IF9#IB?9@ +

;F5K5@  %7CB5@8  #C@@=5  IDH5 "% /=;;=BHCB % +NBC@ +5:9HM

57H=J=HM 5B8 =AAIB9 7CFF9@5H9G C: 5BH=( 5BH=6C8M =B 75B79F & B;@ " %98

   –

34 "* F5<A9F ++ ,M?C8= $) <CK /" KI +$ ,CD5@=5B ( KI 

F5?9 $  5A57<C " #5I< # '8IBG=  (=HCH ' 5A=8 + <5H=5 * %5F

H=BG # 5HCB + <9B ,% +5@5M + @5D5FH<M " FCGGC " #CFA5B +%

(5F?9F + ;F5K5@ +% C@869F; % (5F8C@@  IDH5 "% /=;;=BHCB +5:9HM

5B8 57H=J=HM C: 5BH=($ 5BH=6C8M =B D5H=9BHG K=H< 58J5B798 75B79F & B;@ "

%98    –

34  <9B $ %=@@9F % *969@5HHC 1 F9K5<  *CK9 $ @5F?9 % N5D=;5 #

*CG9BH<5@ , !A5A=7<= 1 <9B + <5B; (+ <CK8<IFM  &5=A5B 1

/5B;  15B;  IA6@9G * 9F6GH ( +=AG + =B8I798 =BS5AA5

HCFM F9GDCBG9G 5F9 A98=5H98 6M 8=GH=B7H 85A5;9 5GGC7=5H98 AC@97I@5F D5HH9FBG

%( F979DHCFG =B J=HFC 5B8 =B J=JC ($C+ 'B9    9

34  9FB5B89N ( I969B9F * +7<K569 5A5;95GGC7=5H98 AC@97I@5F D5HH9FBG

=B 75B79F 5 8CI6@998;98 GKCF8 'B7C;9B9    –

34 2 15B; "" 5B 'DD9B<9=A @5FA=BG 5B8 =AAIB=HM !AAIBC@ *9J  

 –

34 . #FMG?C  5F;  #57NA5F9? ' #FMG?C ( ;CGH=B=G ( .5B89B5699@9

!AAIBC;9B=7 79@@ 895H< 5B8 %(G =B 75B79F H<9F5DM &5H *9J 5B79F  

 –

34 %% I6=B 0 2<5B;  +7<IGH9F  5FCB "( /5F8 , &C;I7<= 1 !J5BCJ5 "

IB85@  FH<IF /" #F9669F  %I@89F % ,C969G % .9G9@M ++ $5A

" #CFA5B "( @@=GCB " F99A5B   +<5FD9 $ (95F79 ,& +7<I

A57<9F * 969FGC@8  *5AA9BG99 " %9@=9: * %5F8=G / =@@5B89FG

%& FHMCACJ * +7<F9=69F <97?DC=BH 6@C7?589 75B79F =AAIBCH<9F5DM H5F

;9HG HIACIFGD97=R7 AIH5BH 5BH=;9BG &5HIF9    –

34 $ CB;  B;@9A5B 9B8F=H=7 79@@G =B 75B79F =AAIBCH<9F5DM BBI *9J !A

AIBC@   –

34 ' (FCI8:CCH  (CIB=CH=G # +<9B;  $CJ9@5B8  (=9H9FGN 9B8F=H=7 79@@

J577=B5H=CB LD9FH *9J .577=B9G    –

34 % .9G9@M %  #9FG<5K * +7<F9=69F %" +AMH< &5HIF5@ =BB5H9 5B8 585D

H=J9 =AAIB=HM HC 75B79F BBI *9J !AAIBC@   –

34 * F99B , 9F;IGCB $ 2=HJC;9@  #FC9A9F !AAIBC;9B=7 5B8 HC@9FC;9B=7

79@@ 895H< &5H *9J !AAIBC@    –

34  .=J=9F   *5I@9H  %CF9HH5 % 5@=;=IF= $ 2=HJC;9@ $$ $5B=9F /%

1C?CM5A5 + -;C@=B= !BB5H9 CF 585DH=J9 =AAIB=HM ,<9 9L5AD@9 C: B5HIF5@ ?=@@9F

79@@G +7=9B79    –

34 ' #9DD $ +9BCJ=@@5 ! .=H5@9  .577<9@@= + 8>9A=5B ( ;CGH=B=G $ D9HC<

 F5B85 . 5FB565 & @CM $ F577= # F97?DCH  FCI;<  IEI9 %

5GHFC % =FCB9 %! C@CA6C ! F9A9F + 9A5F=5 $ =B=  @=CDCI@CG

 5;;=CB= + CFA9BH= " I7=?CJ5 $ 56F=9@9 -+ 5=D@ " 5@CB  5F;

 <=F=B;<9@@= & =9G9 2+ IC  9AA=B?= % 9FFA5BB "/ C8;9 +

C@89BF=989F " CB9M7<IF7< % I 0 I5B; ,% !@@=8;9 # #CBC % #CF

69@=? . #FMG?C + $C= (* $CK9BGH9=B  $I;@= 1 %5  %589C  %5B

:F98= ! %5FH=BG  %5J=@=C $ %9B;9F & %9F9B8=BC % %=7<5I8  %=;BCH

#$ %CGGA5B  %I@H<CT * '9<@9F  (5@CA6C , (5B5F9H5?=G " (C@  (FC=

9HH= " *=77=  *=;5BH= ( *CJ9F9)I9F=B=  *I65FH9@@=  +=GH=;I %" +AMH<

" +CBB9A5BB * +D=G9? " +H5;; ) +I??IFK5@5  ,5FHCIF  ,<CF6IFB + 

,<CFB9 ( .5B89B5699@9  .9@CHH= +, /CF?9B<9  15B; /0 2CB; $ 2=HJC

;9@  #FC9A9F $ 5@@INN= CBG9BGIG ;I=89@=B9G :CF H<9 89H97H=CB C: =AAIBC

;9B=7 79@@ 895H< 'B7C=AAIBC@C;M    9

34  9F9@@5 %  ,9=H9B  *58C;B5 % =75HC % =989F=7< FCA B5HIF9 HC 698

G=89 DFCGIFJ=J5@ 5B8 79@@ 895H< A97<5B=GAG 5G H<9F5D9IH=7 H5F;9HG =B 75B79F HF95H

A9BH =CH97<BC@ 8J    –

34 % =989F=7<  9F9@@5 &CB75BCB=75@ DFC;F5AA98 79@@ 895H< A97<5B=GAG HF=;

;9F98 6M B5HIF5@ 7CADCIB8G +9A=B 5B79F =C@ –  –

34  %5NIA89F  9F9@@5 % =989F=7< &5HIF5@ G75::C@8G =B 5BH=75B79F H<9F5DM

5B8 DF97=G=CB A98=7=B9 =CH97<BC@ 8J    –

34  %CF795I + <5H95IJ=9IL % 'FG=B=  ,F97I@ % =75HC % =989F=7< &5H

IF5@ 7CADCIB8G 5B8 D<5FA579IH=75@G F9DFC;F5A @9I?9A=5 79@@ 8=::9F9BH=5H=CB

D5H<K5MG =CH97<BC@ 8J   (H   –

34 % +7<B9?9B6IF;9F % =75HC % =989F=7< (@5BH89F=J98 9D=;9B9H=7 AC8I@5HCFG

:CF 75B79F HF95HA9BH 5B8 DF9J9BH=CB =CH97<BC@ 8J    –

34  ,9GB=9F9  +7<@9AA9F . C=;9 ' #9DD ! %5FH=BG  <=F=B;<9@@= $

MA9F=7 % %=7<5I8 $ D9HC< $ 5F5I@H " %9B8=6CIF9 "( (=;BCB . "CCGH9

( J5B B89FH % I7F9IL $ 2=HJC;9@  (=5F8  #FC9A9F !AAIBC;9B=7 895H<

C: 7C@CB 75B79F 79@@G HF95H98 K=H< CL5@=D@5H=B 'B7C;9B9    –

34 & 5G5F9G %' (9EI=;BCH  ,9GB=9F9  <=F=B;<9@@= + *CIL & <5DIH 

+7<A=HH  5A5= + 9FJ5G+HI66G % '69=8  CIH5BH  %9H=J=9F 

(=7<5F8 ( I7CIHIF=9F  (=9FFCB  5FF=8C $ 2=HJC;9@  #FC9A9F 5G

D5G989D9B89BH =AAIBC;9B=7=HM C: 8CLCFI6=7=B=B8I798 HIACF 79@@ 895H< " LD

%98    –

34 %$ @69FH  +5IH9F & <5F8K5> 9B8F=H=7 79@@G 57EI=F9 5BH=;9B :FCA 5DCDHCH=7

79@@G 5B8 =B8I79 7@5GG !F9GHF=7H98 ,$G &5HIF9    –

34  !BCI9 # ,5B= %I@H=AC85@ =AAIBC;9B=7 75B79F 79@@ 895H< 5G 5 7CBG9EI9B79 C:

5BH=75B79F 7MHCHCL=7 HF95HA9BHG 9@@ 95H< =::9F    –

34 2+ IC 2 $=I $ 5FH@9HH 'B7C@MH=7 =AAIBCH<9F5DM 8M=B; H<9 F=;<H K5M =G 5

?9M HC 9@=7=H=B; DCH9BH 5BH=HIACF =AAIB=HM FCBH 'B7C@   

34  15B; 1 %5  <9B  2<CI , 15A5N5?=  #@9=B  (=9HFC7C@5  .57

7<9@@= + +CIEI9F9  +5IJ5H $ 2=HJC;9@ ' #9DD  #FC9A9F CBHF=6IH=CB C:

*!( 5B8 %$#$ HC =AAIBC;9B=7 79@@ 895H< G=;B5@=B; =B 75B79F 7<9ACH<9F5DM 'B

7C=AAIBC@C;M    9

34  *58C;B5 % =989F=7< +HF9GG=B8I798 79@@I@5F F9GDCBG9G =B =AAIBC;9B=7 79@@

895H< =AD@=75H=CBG :CF 75B79F =AAIBCH<9F5DM =C7<9A (<5FA57C@  

–

34 * +D=G9?  <5F5@5A6CIG  %5NIA89F   .9GC@9 + "5;5BB5H< %. <C

85D?5F CFH9NCA=6 9B<5B79G 89B8F=H=7 79@@ A98=5H98 =B8I7H=CB C: =AAIB=HM

HC <IA5B AM9@CA5 J=5 9LDCGIF9 C: 79@@ GIF:579 <95H G<C7? DFCH9=B  CB 8M=B; HI

ACF 79@@G H<9F5D9IH=7 =AD@=75H=CBG @CC8    –

34 + %=M5ACHC  !BCI9 , &5?5AIF5 % 15A585  +5?5ACHC 1 -F5H5 ,

'?5N5?= , %5FIACHC  ,5?5<5G<= # ,5?5M5A5 1 &5?5B=G<=  +<=A=NI #

,5B= CLG57?=9J=FIG  =G 5B CB7C@MH=7 J=FIG K=H< =AAIBCGH=AI@5HCFM DFCD9FH=9G

H<5H =G 57H=J9 5;5=BGH @IB; 589BC75F7=BCA5 5B79F *9G   

–

34 % I89?  5F; . #FMG?C  9 *IMGG7<9F ( ;CGH=B=G !B8I79FG C: =A

AIBC;9B=7 75B79F 79@@ 895H< MHC?=B9 FCKH< 57HCF *9J   

–

34 & *I:C  5F; ( ;CGH=B=G ,<9 IB:C@898 DFCH9=B F9GDCBG9 =B =AAIBC;9B=7

79@@ 895H< 5B8 75B79F =AAIBCH<9F5DM ,F9B8G 5B79F    –

34  *CB ( /5@H9F +=;B5@ =BH9;F5H=CB =B H<9 9B8CD@5GA=7 F9H=7I@IA IB:C@898 DFC

H9=B F9GDCBG9 &5H *9J %C@ 9@@ =C@    –

34 ! %5FH=BG ' #9DD  +7<@9AA9F + 8>9A=5B % ,5=@@9F + +<9B % %=7<5I8

$ %9B;9F  8CIF5 & ,5>988=B9  ,9GB=9F9 $ 2=HJC;9@  #FC9A9F *9GHCF5

H=CB C: H<9 =AAIBC;9B=7=HM C: 7=GD@5H=B=B8I798 75B79F 79@@ 895H< 6M 9B8CD@5GA=7

F9H=7I@IA GHF9GG 'B7C;9B9    –

34 + = @5G=C !% /CFH9@  J5B @589@ $ 89 .F=9G , I=J9A5B 89 C9F #

/CF5< & 89 55G +! IG7<CK !" 89 .F=9G  =;8CF +. 5HC IA5B

7 G 5F9 7F=H=75@ 79@@I@5F A98=5HCFG C: =AAIB9 F9GDCBG9G =B8I798 6M =A

AIBC;9B=7 79@@ 895H< 'B7C=AAIBC@C;M    9

34 $ %9B;9F  .577<9@@= + 8>9A=5B ! %5FH=BG 1 %5 + +<9B , 15A5N5?= )

+I??IFK5@5 % %=7<5I8  %=;BCH  +7<@9AA9F  +I@D=79  $C7<9F 0

=8FC@  <=F=B;<9@@= & %C8>H5<98= $ 5@@INN=  B8F9 $ 2=HJC;9@ ' #9DD

 #FC9A9F 5F8=57 ;@M7CG=89G 9L9FH 5BH=75B79F 9::97HG 6M =B8I7=B; =AAIBC;9B=7

79@@ 895H< +7= ,F5BG@ %98    F5

34 + ?A5B "  CFCG<CK , IF?9 % =N85FC;@I & 65G9 AC8=R75H=CBG

=B8I798 =B =GC@5H98 <IA5B 7<FCA5H=B 6M & 89<M8FC;9B5G975H5@MN98 F98I7

H=CB C: 8CLCFI6=7=B =C7<9A=GHFM    –

34 % 5A5;;= * CAD5FG=  +HFC77<=  ,9GHCB=  B;9@9@@=  (5BBIH= D=FI

6=7=B 5B8 8CLCFI6=7=B 7CAD5F5H=J9 A9H56C@=GA 5B8 D<5FA57C?=B9H=7G 

7FCGGCJ9F GHI8M 5B79F <9ACH<9F (<5FA57C@    –

34 % 5A5;;=  +HFC77<= ( 5F=G=  %5FHCB=  ,CBCB= % I5F5@8= %

+HFC@=B9B989HH=  :H<MA=CDCI@CG  (5BBIH= !85FI6=7=B A9H56C@=GA 5B8 D<5F

A57C?=B9H=7G 5:H9F =BHF5J9BCIG 5B8 CF5@ 58A=B=GHF5H=CB =B 75B79F D5H=9BHG 5

7FCGGCJ9F GHI8M 5B79F <9ACH<9F (<5FA57C@    –

34 " I7=?CJ5 ( #F5@=?CJ5  =5@CJ5 , FHB=7?M $ *C6 " 5FHIB?CJ5 * +D=G9?

IA5B HIACF 79@@G ?=@@98 6M 5BH<F57M7@=B9G =B8I79 5 HIACFGD97=R7 =AAIB9 F9

GDCBG9 5B79F *9G    –

34 $ D9HC<  <=F=B;<9@@=  ,9GB=9F9 % '69=8  'FH=N  F=C@@C  %=;BCH

% %5=IF=  -@@F=7< ( +5I@B=9F  15B; + A=;CF9B5  *M::9@ " 5FF5H

( +5:H=;  $9J= * $=89F95I  &C;I9G "( %=F5  <CADF9H . "CI@=B 

@5J9@<5D9@CB " CIF<=G  B8F9 + 9@5@C;9 , ,IFGN  #FC9A9F $ 2=HJC

;9@ ,C@@@=?9 F979DHCF 89D9B89BH 7CBHF=6IH=CB C: H<9 =AAIB9 GMGH9A HC 5BH=

75B79F 7<9ACH<9F5DM 5B8 F58=CH<9F5DM &5H %98    –

34 % '69=8  ,9GB=9F9  <=F=B;<9@@= % =A=5 $ D9HC< "$ (9F:9HH=B= %

5GH98C  %=;BCH , (5B5F9H5?=G & 5G5F9G  %9H=J=9F & $5FC7<9HH9 ( J5B

B89FH  =77CG5BH= % (=579BH=B= $ 2=HJC;9@  #FC9A9F 5@F9H=7I@=B 9LDCGIF9

8=7H5H9G H<9 =AAIBC;9B=7=HM C: 75B79F 79@@ 895H< &5H %98    –

34  +=GH=;I , 15A5N5?=  .577<9@@= # <565 ( BCH " 85A ! .=H5@9 

CI65F  5F577C  *9A98=CG $ 9B8  5BB5B= $ MA9F=7 1 %5 %

&=GC+5BH5BC ' #9DD "$ +7<I@HN9 , ,IH=B;  9@5F89@@= $ F577= . $5

+CFG5  2=77<988I ( +9GH=@=  -F65B= % 9@CF9BN= % $57FC=L,F=?= .

)I=8J=@@9 * CB:CFH= "( +D5BC $ (IGNH5= . (C=F=9FC@5A9 + 9@5@C;9  (9

B5I@H$@CF75 + $58C=F9 $ FBCI@8 " MFH5 % 9GGC@=9FG  ;;9FACBH %

=5B7<= % (=HH9H  B;6@CA  (RFG7<?9 0 (F9J=@@9  -N9 * +7<F9=69F

%, <CK %" +AMH<  (FC=9HH=  B8F9  #FC9A9F $ 2=HJC;9@ 5B79F

79@@5IHCBCACIG 7CBHF=6IH=CB C: HMD9 ! =BH9F:9FCB G=;B5@=B; HC H<9 9:R757M C:

7<9ACH<9F5DM &5H %98    –

34 % %=7<5I8 ! %5FH=BG ) +I??IFK5@5 + 8>9A=5B 1 %5 ( (9@@9;5HH= +

+<9B ' #9DD % +7C5N97  %=;BCH + *9@@C.5FCB5 % ,5=@@9F $ %9B;9F 

.577<9@@= $ 5@@INN=  <=F=B;<9@@=  8= .=F;=@=C $ 2=HJC;9@  #FC9A9F I

HCD<5;M89D9B89BH 5BH=75B79F =AAIB9 F9GDCBG9G =B8I798 6M 7<9ACH<9F5D9IH=7

5;9BHG =B A=79 +7=9B79    –

34  <=F=B;<9@@= $ D9HC<  ,9GB=9F9 $ MA9F=7 1 %5  'FH=N # .9FA59@9B

, (5B5F9H5?=G  %=;BCH  -@@F=7< "$ (9F:9HH=B=  +7<@9AA9F  ,5G89A=F %

-<@ ( 9B=B  =J5G  *M::9@ " #5B9@@CDCI@CG " ,G7<CDD  B8F9 * $=8

9F95I &% %7$5I;<@=B &% 5MB9G %" +AMH<  #FC9A9F $ 2=HJC;9@ 7H=

J5H=CB C: H<9 &$*( =BS5AA5GCA9 =B 89B8F=H=7 79@@G =B8I79G !$69H589D9B89BH

585DH=J9 =AAIB=HM 5;5=BGH HIACFG &5H %98    –

34 % =989F=7<  %I@@9F  9F9@@5 5F8=57 ;@M7CG=89G :FCA AC@97I@5F H5F;9HG HC

=AAIBC;9B=7 79@@ 895H< =C7<9A (<5FA57C@   –

34 ! (F5GG5G ( =5A5B8=G &CJ9@ H<9F5D9IH=7 5DD@=75H=CBG C: 75F8=57 ;@M7CG=89G

&5H *9J FI; =G7CJ    –

34 &2 +7<B9=89F  9F9@@5 "1 $99  %5NIA89F #* #=A  89 5FJ5@<C "

%IB?9FH *% (58I5 / #F9=G #/ #=A  <F=GHCJ % =75HC " #=A /

5B  F5;5 %' +=AC9G % =989F=7< 5F8=57 ;@M7CG=89 ;@I7C9J5

HFCACBCG=89 =B8I79G 75B79F HMD9GD97=R7 79@@ 895H< FCBH (<5FA57C@  

 

34  9F9@@5  5=;B95IL  %5NIA89F "1 $99  +5@5B8  *58C;B5 , 5F;9

 .9F;9N  *97<9F " +5FFM #/ #=A 1 +<=B % =75HC % =989F=7<

7@ DFCH9=B :5A=@M 9LDF9GG=CB D5HH9FB 89H9FA=B9G GMB9F;=GH=7 DFC5DCDHCH=7 9:

:97HG C:   A=A9H=7G

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K=H< <9A=GMBH<9H=7 75F8=57 ;@M7CG=89 -&+ =B 57IH9 AM9@C=8 @9I?9A=5

$9I?9A=5    –

34  *58C;B5  9F9@@5  5=;B95IL  <F=GHCJ % =75HC % =989F=7< 9@@

HMD989D9B89BH *'+ 5B8 A=HCD<5;M F9GDCBG9 @958G HC 5DCDHCG=G CF B97FCDHCG=G =B

B9IFC6@5GHCA5 'B7C;9B9    –

34  9F9@@5  %I@@9F  5=;B95IL  *58C;B5  .=FM + <5H95IJ=9IL % =

75HC % =989F=7< 5F@M 8CKBF9;I@5H=CB C: %7@ F9;I@5H9G 5DCDHCG=G HF=;;9F98 6M

75F8=57 ;@M7CG=89 -&+ 9@@ 95H< =G   9

34 % +@=B;9F@5B8  9F9@@5 " I7<9@55F % =989F=7<  9@89F6@CA 5F8=57

;@M7CG=89G =B 75B79F H<9F5DM :FCA DF97@=B=75@ =BJ9GH=;5H=CBG HCK5F8G 7@=B=75@ HF=5@G

!BJ9GH &9K FI;G    –

34  9F9@@5 % =75HC % =989F=7< GG9A6@=B; H<9 DINN@9 C: 5BH=75B79F A97<5

B=GAG HF=;;9F98 6M 75F8=57 ;@M7CG=89G %=HC7<CB8F=CB    –

34 , "IB7?9F  9F9@@5 %  ,9=H9B  %CF795I % +7<IA57<9F " <9@R 

55G7<H % +7<B9?9B6IF;9F  9BFM % =75HC % =989F=7< -&+ 5

GH9FC=8 75F8=57 ;@M7CG=89 =B8I7=B; 5DCDHCH=7 79@@ 895H< =B <IA5B @9I?9A=5 79@@G

=C7<9A (<5FA57C@    –

34 , "IB7?9F % +7<IA57<9F % =75HC % =989F=7< -&+ :FCA 5@CHFCD=G

DFC79F5 5G 5 F9;I@5HCF C: G=;B5@=B; D5H<K5MG =BJC@J98 =B DFC@=:9F5H=CB 5B8 79@@

895H< =C7<9A (<5FA57C@    –

34 , %=>5HCJ=7 ! *C@5B8  .5B )I5EI969?9  &=@GGCB  %5H<=9I  .5B .MB7?H

 5FFC  @5B7C . 577<=B= * #=GG ,<9 5@D<5 GI6IB=H C: H<9 GC8=IA DIAD

7CI@8 F9DF9G9BH 5 BCJ9@ H5F;9H HC 7CA65H BCBGA5@@ 79@@ @IB; 75B79FG " (5H<C@

   –

34  $9:F5B7 , %=>5HCJ=7 1 #CB8C + +5IJ5;9 ! *C@5B8 ' 969=F  #FGH=7 .

.5G=7 ( 5=@@M + #CB8C  @5B7C * #=GG ,5F;9H=B; H<9 5@D<5  GI6IB=H C: H<9

GC8=IA DIAD HC 7CA65H ;@=C6@5GHCA5 79@@G &9IFCGIF;9FM    DD

– 8=G7IGG=CB –

34 &2 +7<B9=89F  9F9@@5 %' +=AC9G % =989F=7< BH=75B79F 5B8 =A

AIBC;9B=7 DFCD9FH=9G C: 75F8=57 ;@M7CG=89G %C@97I@9G   

34 ' #9DD $ %9B;9F  .577<9@@= + 8>9A=5B ! %5FH=BG 1 %5 ) +I??IFK5@5

% %=7<5I8 $ 5@@INN= $ 2=HJC;9@  #FC9A9F BH=75B79F 57H=J=HM C: 75F8=57

;@M7CG=89G 5H H<9 :FCBH=9F 69HK99B 79@@5IHCBCACIG 5B8 =AAIBC@C;=75@ 9::97HG

'B7C=AAIBC@C;M    –

34 $ %9B;9F  .577<9@@= + 8>9A=5B ! %5FH=BG 1 %5 + +<9B , 15A5N5?= )

+I??IFK5@5 % %=7<5I8  %=;BCH  +7<@9AA9F  +I@D=79  $C7<9F 0

=8FC@  <=F=B;<9@@= & %C8>H5<98= $ 5@@INN=  B8F9 $ 2=HJC;9@ ' #9DD

 #FC9A9F 5F8=57 ;@M7CG=89G 9L9FH 5BH=75B79F 9::97HG 6M =B8I7=B; =AAIBC;9B=7

79@@ 895H< +7= ,F5BG@ %98    F5

34 $ 9NI  +5IJ5H " IA95I $ CA9G85+=@J5 # !F=65FF9B + CFJ9=@@9 (

5F7=5 $ 2<5C ( $=I $ 2=HJC;9@ $ +9BCJ=@@5 ' #9DD  #FC9A9F 9!5@D<5

D<CGD<CFM@5H=CB =G D5H<C;BCACB=7 :CF =AAIBC;9B=7 79@@ 895H< 9@@ 95H< =::9F

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34 ( $=I $ 2<5C  $CCG # !F=65FF9B + $57<?5F  2<CI $ CA9G85+=@J5 

<9B $ 9NI  CB7CAD5=B  (9F9N $ 2=HJC;9@ ' #9DD  #FC9A9F !89BH=R

75H=CB C: D<5FA57C@C;=75@ 5;9BHG H<5H =B8I79 % F9@95G9 +7= *9D  

 

34  .5@9BH  (9B5I@H$@CF75  5MF9  #FC9A9F <5B;9 =B * * ;9B9

GH5HIG 5:H9F H5L5B965G98 7<9ACH<9F5DM :CF 6F95GH 75B79F DC@MD@C=8=N5H=CB 75B

@958 HC 8=5;BCGH=7 D=H:5@@G K=H< DCH9BH=5@ =AD57H :CF 7@=B=75@ A5B5;9A9BH 5B79F

9B9H  –  –

34 $ +9BCJ=@@5 ! .=H5@9 ! %5FH=BG % ,5=@@9F  (5=@@9F9H % %=7<5I8 $ 5@@INN=

+ 8>9A=5B ' #9DD % &=GC+5BH5BC + +<9B  %5F=BC  F=C@@C  C=@9J9

 "C6 + $58C=F9  <=F=B;<9@@=  +=GH=;I , 15A5N5?= + *9@@C.5FCB5 

$C7<9F . (C=F=9FC@5A9 % ,5@6CH  .5@9BH  9F5F8=B9@@=  BHC77=5  =

77CG5BH= % =A=5 % (=579BH=B=  I9MC &$ %9GG=B5 % $= " <5B .

+=;@  (CIF7<9F  *I7?9BGHI<@  5FACB5IH=9FF9N . $5N5F "% (9BB=B;9F

 %589C  $CD9N'H=B %" +AMH< $ 2=HJC;9@ % 5GH98C  #FC9A9F B =A

AIBCGIFJ9=@@5B79 A97<5B=GA 7CBHFC@G 75B79F 79@@ D@C=8M +7=9B79  

 –

34  9&5F8C " F9BB5B  *9L<9D5>  *I::9@@ +$ +<=5C + %5889B

/% 5@@5;<9F & /58<K5B= + #9=@ + "IB5=8 + *I;C + K5B; #

"=FGHFCA $ /9GH $% CIGG9BG $9I?C7MH9 7CAD@9L=HM DF98=7HG 6F95GH 75B79F

GIFJ=J5@ 5B8 :IB7H=CB5@@M F9;I@5H9G F9GDCBG9 HC 7<9ACH<9F5DM 5B79F =G7CJ 

  –

34 $/ (:5BB9BGH=9@ ++ $5A $ A9BG % "5::99 , FAGHFCB; (57@=H5L9@

9B<5B79G 95F@M 89B8F=H=7 79@@ A5HIF5H=CB 5B8 :IB7H=CB H<FCI;< ,$* G=;B5@=B; =B

A=79 9@@ !AAIBC@    –

34 $ .C@?F5D9F # 5@@  F=;;G + *5>DIH ( #C<=C  9&5F8C + *5B (57@=

H5L9@ H<9F5DM DFCACH9G 6F95GH 75B79F A9H5GH5G=G =B 5 ,$*89D9B89BH A5BB9F

5B79F *9G    –

34 % *CG9@@= . 9F985 % 8= 5F= . CFA=75  +D=@5  "C7<9AG  5FG57= *

CB5<I9 "$ I@@9M " +7<@CA  I585;B= ::97HG C: 7CBJ9BH=CB5@ H<9F5D9IH=7

=BH9FJ9BH=CBG CB H<9 BIA69F 5B8 :IB7H=CB C: F9;I@5HCFM , 79@@G 'B7C=AAIBC@C;M

   9

34 ## (957<A5B % *5C * (5@A9F % 2=85B=7 / +IB * @J=B; +/ *CH<

K9@@ IB7H=CB5@ A=7FCHI6I@9G 5F9 F9EI=F98 :CF 5BH=;9B DFC79GG=B; 6M A57FCD<5;9G

5B8 89B8F=H=7 79@@G !AAIBC@ $9HH    –

34 */ "57?A5B % *<C58G  CFBK9@@ + #5B85F=5B %=7FCHI6I@9A98=5H98

&?5DD5 57H=J5H=CB =B H<9 ,&5@D<5 G=;B5@=B; D5H<K5M LD 9@@ *9G  

 –

34 & %=NIACHC " 5C  %5HGIG<=A5 1 ';5K5  ,5B5?5  ,5?5G<=A5 =G7CJ

9FM C: BCJ9@ =AAIBCGH=AI@5BHG 6M 89B8F=H=779@@65G98 :IB7H=CB5@ G7F99B=B; @CC8

   –

34 & %=NIACHC  ,5B5?5  %5HGIG<=A5 % .=G<K5B5H<  ,5?5G<=A5

C@7<=7=B9 DFCACH9G 5BH=;9B 7FCGGDF9G9BH5H=CB 6M AIF=B9 89B8F=H=7 79@@G " !B

J9GH 9FA5HC@    –

34  /9B % <9B ++ <9B $, I5B; /, <5B; / /9= $ <CI (

FI@G9@J5B " /I + #IC &+ 15B; +D97=R7 A=7FCHI6I@989DC@MA9F=N=B;

5;9BHG 5I;A9BH 9:R757M C: 89B8F=H=7 79@@65G98 75B79F J577=B9G " =CA98 +7=

  

34 " CTA5BB ! .=H5@9  I7<A5BB $ 5@@INN= / +7<K989 $ +9BCJ=@@5 /

+?I65@@5 + .=J9H * $=7<HB9F "% .=79B7=C , (5B5F9H5?=G  +=9A9=GH9F 

$5;9 $ &5BHM + 5AA9F # %=HH9@GH598H + /=BG9@ " G7<9B6F9BB9F %

5GH98C  9A5F7<9 - #@5F  #FC9A9F !ADFCJ98 79@@I@5F D<5FA57C?=B9H=7G

5B8 D<5FA57C8MB5A=7G IB89F@=9 H<9 K=89 5BH=75B79F 57H=J=HM C: G5;CD=@CB9 5B

79F *9G    –

34  15B; ( 2<CI  I5B;  <9B & %5 ) I= + +<9B / CB; 0

2<5B; / $=5B 0 /5B; )( CI " $=I +<=?CB=B 9L9FHG 5BH=HIACF 57H=J=HM J=5

DFCH95GCA9 =B<=6=H=CB 5B8 79@@ 895H< =B8I7H=CB =B J=HFC 5B8 =B J=JC !BH " 5B79F

   –

34 0( IC 01 2<5B; + 2<5B; @=B=75@ HF=5@ CB H<9 9::97HG C: G<=?CB=B A=LHIF9

CB @5H9F GH5;9 @IB; 75B79F 2<CB; 0= 1= "=9 9 25 2<=    DD –



34 % <9B (  /5B; ++ <9B  /9B 1  <9B / 15B; &+ 15B;

+<=?CB=B =B8I79G =AAIBC;9B=7 79@@ 895H< =B HIACF 79@@G 5B8 9B<5B79G 89B8F=H=7

79@@65G98 75B79F J577=B9 5B79F !AAIBC@ !AAIBCH<9F   

–

34  +<=< $$ 15B; *9@5L5BH 9::97H =B8I798 6M KC;CB=B :FCA +7IH9@@5F=5

65=75@9BG=G CB F5H =GC@5H98 IH9F=B9 GACCH< AIG7@9 (<5FA =C@   

–

34 1 15B; 0" $= 2 <9B 00 2<I " /5B; $ 2<5B; $ )=5B; 1" %5 21 $=

)$ IC ) 1CI /C;CB=B =B8I798 75@F9H=7I@=B5BB9L=B  9LDCGIF9 8=7H5H9G

H<9 =AAIBC;9B=7=HM C: 75B79F 79@@G =B 5 (*##, 89D9B89BH A5BB9F ($C+ 'B9

   9

34  +7<=5JCB=  +=GH=;I % .5@9BH=B=  %5HH9= ( +9GH=@=  +D585FC % +5B7<9N

+ $CF9BN= %, -FGC  9@5F89@@= $ 56F=9@9  (FC=9HH= $ F577= M7@CD<CG

D<5A=89 GMB9F;=N9G K=H< HMD9 ! =BH9F:9FCBG H<FCI;< GMGH9A=7 89B8F=H=7 79@@ F957H=

J5H=CB 5B8 =B8I7H=CB C: =AAIBC;9B=7 HIACF 5DCDHCG=G 5B79F *9G   

–

34  <=F=B;<9@@=  %9B5F8 ( (I=; + $58C=F9 + *CIL  %5FH=B  +C@5FM 

$9 9GB9 $ 2=HJC;9@  <5I::9FH %9HFCBCA=7 7M7@CD<CGD<5A=89 F9;=A9B G9@97

H=J9@M 89D@9H9G  F9;I@5HCFM , 79@@G 5B8 F9GHCF9G , 5B8 &# 9::97HCF

:IB7H=CBG =B 9B8 GH5;9 75B79F D5H=9BHG 5B79F !AAIBC@ !AAIBCH<9F  

 –

34  5F; ( ;CGH=B=G * GHF9GG 5IHCD<5;M 5B8 =AAIBC;9B=7 79@@ 895H< =B D<C

HC8MB5A=7 H<9F5DM=B8I798 5BH=75B79F =AAIB9 F9GDCBG9G (<CHC7<9A (<CHC6=C@

+7=    –

34  5F;  %59G * J5B .@=9H ( ;CGH=B=G ,5F;9H=B; H<9 <5@@A5F?G C: 75B79F

K=H< H<9F5DM=B8I798 9B8CD@5GA=7 F9H=7I@IA * GHF9GG %C@ 9@@ 'B7C@  

 9

34  5F; . #FMG?C ( .5B89B5699@9 ( ;CGH=B=G MD9F=7=B65G98 D<CHC8M

B5A=7 H<9F5DM =B8I79G GIF:579 9LDCGIF9 C: 85A5;95GGC7=5H98 AC@97I@5F D5HH9FBG

@=?9 +( 5B8 75@F9H=7I@=B 5B79F !AAIBC@ !AAIBCH<9F   

–

34  5F; . #FMG?C , .9F:5=@@=9  #57NA5F9?  9FF9=F5 , %5FMG59@ &

*I6=C % =F7NI?  %5H<=9I " *C96FC9? / BB59FH " C@56 ( 89 /=HH9

( .5B89B5699@9 ( ;CGH=B=G  BCJ9@ D5H<K5M 7CA6=B=B; 75@F9H=7I@=B 9LDCGIF9

5B8 ,( G97F9H=CB =B =AAIBC;9B=7 75B79F 79@@ 895H< %' "   

–

34  5F; + @G9B . #FMG?C ( .5B89B5699@9 ( 89 /=HH9 ( ;CGH=B=G *9G=G

H5B79 HC 5BH=75B79F J577=B5H=CB 9::97H =G 7CBHFC@@98 6M 5 75B79F 79@@5IHCBCACIG

D<9BCHMD9 H<5H 8=GFIDHG =AAIBC;9B=7 D<5;C7MH=7 F9ACJ5@ 'B7CH5F;9H  

 –

34 ! =57CBI . 9FI@@C %$ =FJ=B9B + G7IH9B5=F9 % -;C@=B= +# (9GCB9B +

F5A5BH9 + (5FJ=5=B9B  #5B9FJ5 + $CG?C;  @=CDCI@CG + (9GCB9B 

9AA=B?= !AAIB9 F9GDCBG9 =G 5B =ADCFH5BH 5GD97H C: H<9 5BH=HIACF 9::97H DFC

8I798 6M 5 $9B7C8=B; CB7C@MH=7 589BCJ=FIG 5B79F *9G   

–

34  #C?G  5F; % <F<5F8H % *=J5 $ .5B89B69F? $ CCB + 9

.@99G7<CIK9F ( ;CGH=B=G & F5: +/ .5B CC@ &9K75GH@9 8=G95G9 J=FCH<9F5DM

=B8I79G @CB;H9FA GIFJ=J5@ 5B8 HIACFGD97=R7 =AAIB9 A9ACFM =B CFH<CHCD=7

;@=CA5 H<FCI;< H<9 =B8I7H=CB C: =AAIBC;9B=7 79@@ 895H< !BH " 5B79F  

 –

34 +& 2<5B; !# <C= "  I5B; "1 1CC #" <C= ' 1IB 'DH=A=N=B; 

J577=B5H=CB 6M 7CA6=B5H=CB K=H< CB7C@MH=7 589BCJ=FIG 7C9LDF9GG=B; !$ 5B8

%+ %C@ ,<9F    –

34 & /C@@9F + #BC7?9  %IB8H  IF@9J=? & +HFIJ9F  #@CCG  CCN5F= (

+7<57<9 %( %5BBG &( %5@9? , +D5FK5GG9F $ 29B89F , /=FH< + #I

6=7?5  #I<B9@ .=FIG=B8I798 HIACF =BS5AA5H=CB :57=@=H5H9G 9::97H=J9  75B79F

=AAIBCH<9F5DM =B 5 ,F9;89D9B89BH A5BB9F =B A=79 " @=B !BJ9GH  

 –

34 $ 5= " #CCDA5BB  =C@5 ( CIFB=9F . +7<=FFA57<9F 9B8F=H=7 79@@G DI@G98

K=H< J=F5@ CB7C@MG5H9G DCH9BH@M GH=AI@5H9 5IHC@C;CIG , 79@@G :FCA 75B79F D5H=9BHG

!BH " 'B7C@    –

34 ' CBB9@@M  FF=B;HCB%5=G $ +H99@9  5857 . "9BB=B;G # +7CHH 

(957< *% (<=@@=DG " CB8  (5B8<5 # 5FF=B;HCB * .=@9 + *IGG9@@ (

+9@6M  %9@7<9F %95G@9G J=FIG 75IG9G =AAIBC;9B=7 79@@ 895H< =B <IA5B

A9@5BCA5 9B9 ,<9F    –

34 + 96F9A9G?9@  "C<BGHCB CB79DHG 5B8 A97<5B=GAG IB89F@M=B; 7<9ACH<9F5DM

=B8I798 =AAIBC;9B=7 79@@ 895H< =AD57H CB 7@=B=75@ GHI8=9G 5B8 7CBG=89F5H=CBG :CF

7CA6=B98 H<9F5D=9G 'B7CH5F;9H    –

34 , (5B5F9H5?=G ' #9DD - FC7?A9=9F  ,9GB=9F9  >CF?@IB8  <5D

A5B % IF7<G7<@5; & "CN5  (=9FFCB ( J5B B89FH " 1I5B $ 2=HJC;9@ 

%589C  /=@@=5AG  #FC9A9F %97<5B=GAG C: DF95DCDHCH=7 75@F9H=7I@=B 9LDC

GIF9 =B =AAIBC;9B=7 79@@ 895H< %' "    –

34 % 89 FIMB .* /=9FGA5 / 9@:F=7< ( ;;@9HCB  F9A9F ,<9 9J9F9LD5B8

=B; =AAIBCAC8I@5HCFM FC@9 C: 75@F9H=7I@=B =B 75B79F =AAIB=HM FCBH 'B7C@

  

34 "1 $99 ' ,5@<=  "5B;  9F9@@5  5=;B95IL #/ #=A "/ $99 % =

75HC # 57<5F= / 5B %+ +=@J5  'F@=?CJ5 % =989F=7< MHCGH5H=7 <M

8FCLM7CIA5F=B ', =B8I79G *C@;= GHF9GG 5B8 +,, =B<=6=H=CB HF=;;9F=B;

BCB75BCB=75@ 79@@ 895H< 5B8 GMB9F;M K=H<   A=A9H=7G =B @IB; 75B79F 5B79F

$9HH   –

34  %5NIA89F "1 $99 ' ,5@<=  9F9@@5 + <5H95IJ=9IL  5=;B95IL *

CB; " #5B; 1 $99 #/ #=A / #=A 1 +<=B % =75HC # 57<5F=

%+ +=@J5  'F@=?CJ5CM9F % =989F=7< M8FCLM7CIA5F=B ', ?=@@G %$

79@@G 5@CB9 CF =B GMB9F;M K=H< =A5H=B=6 CF +MBF=6C =BJC@J9A9BH C: * GHF9GG 5B8

%( F9@95G9 5B79F $9HH   –

34 +! F=J9BB=?CJ * F9H9B % #5F=B !AAIB=HM =BS5AA5H=CB 5B8 75B79F 9@@

   –

34 %( *5IG7< * +9FH=@  GHF9GG:I@ A=7FC9BJ=FCBA9BH CDDCG=B; 9::97HG C: H<9 9B

8CD@5GA=7 F9H=7I@IA GHF9GG F9GDCBG9 =B H<9 GIDDF9GG=CB 5B8 9B<5B79A9BH C: 585D

H=J9 HIACF =AAIB=HM !BH *9J !AAIBC@    –

34 % %5IF9@ ( %7F5H< # %B=7< + 95@M  <9J9H  +5A5@= CBHFC@@=B; H<9

IB:C@898 DFCH9=B F9GDCBG9A98=5H98 @=:9 5B8 895H< 897=G=CBG =B 75B79F +9A=B

5B79F =C@   –

34 * ,IR , (5B5F9H5?=G # =5B7<=  F=C@@C  5N=  = +5BC  ,9GB=9F9 '

#9DD ( (5H9F@=B=F97<CH $ 2=HJC;9@ % (=579BH=B=  +N5658?5=  #FC9A9F

*98I7H=CB C: 9B8CD@5GA=7 F9H=7I@IA 5 @9J9@G :5JCFG D@5GA5 A9A6F5B9 GIF:579

9LDCGIF9 C: 75@F9H=7I@=B 9@@ 95H< =::9F    –



UNCORRECTED PROOF

 '*4-3'+8'1 /4).+2/)'1.'62')414-= <<<  <<<<<<

34 + "= "1 $99 " +7<FCF  %5NIA89F % "5B; + <5H95IJ=9IL % +7<B9?9B

6IF;9F * CB;  <F=GHCJ " #5B; 1 $99 / 5B #/ #=A 1 +<=B

% =75HC  9F9@@5 % #CB=;  'F@=?CJ5 % =989F=7< ,<9 8=5@?M@ F9GCF7=

BC@ GH9AD<C@ 8=GFIDHG 75@7=IA <CA9CGH5G=G HC HF=;;9F DFC;F5AA98 =AAIBC;9B=7

B97FCG=G =B 75B79F 5B79F $9HH   –

34 % 299G<5B   $99 * #=A " <59 B8CD@5GA=7 F9H=7I@IA GHF9GG 5B8

5GGC7=5H98 *'+ !BH " %C@ +7=    

34  =;5  <9J9H *98CL G=;B5@=B; @CCDG =B H<9 IB:C@898 DFCH9=B F9GDCBG9 9@@

+=;B5@    –

34  <5B85FM  %5F5<5HH5 * #=A " <59 B =BJC@J9A9BH C: CL=85H=J9

GHF9GG =B 9B8CD@5GA=7 F9H=7I@IA GHF9GG 5B8 =HG 5GGC7=5H98 8=G95G9G !BH " %C@ +7=

   –

34 $ =@=DD=B (" %5;5@<59G  = 9B989HHC % C@9@@5 , (CNN5B +H56@9 =BH9F57

H=CBG 69HK99B A=HC7<CB8F=5 5B8 9B8CD@5GA=7 F9H=7I@IA 5@@CK F5D=8 577IAI@5H=CB

C: 75@7=IA =B 5 GI6DCDI@5H=CB C: A=HC7<CB8F=5 " =C@ <9A   

–

34 %" 5BGGCB * %5BGGCB + %CFCH5  -7<=BC , #5@@IF , +IA= & !G<== %

+<=A5NI % #99D  "9;CFCJ  @A9F 5@7=IA=B8I798 ;9B9F5H=CB C: F957H=J9

CLM;9B GD97=9G =B 6F5=B A=HC7<CB8F=5 =G A98=5H98 6M D9FA956=@=HM HF5BG=H=CB F99

*58=7 =C@ %98    –

34 + 5BG5;<= , C@9B5F % %589G<  GCF85G + *5A57<5B8F5*5C # +<5FA5

! 1I@9 +# "CG9D<  5>BC7N?M !GC:CFA 5B8 GD97=9GGD97=R7 7CBHFC@ C: =BCG=

HC@ HF=GD<CGD<5H9 !( F979DHCFG 6M F957H=J9 CLM;9B GD97=9G " =C@ <9A

   –

34 + +5J9@>9J5 +$ %7 $5I;<@=B ( .5B89B5699@9  +5A5@= %" 9FHF5B8 B8C

D@5GA=7 F9H=7I@IA GHF9GG =B8I79G @=;5B8=B89D9B89BH ,&*A98=5H98 B97FCDHCG=G

=B $ 79@@G 9@@ 95H< =G   9

34  F97?9BF=8;9 % +HC>5BCJ=7 * %5F79@@IG  +<CF9 5GD5G9 7@95J5;9

DFC8I7H C: ( =B8I79G A=HC7<CB8F=5@ RGG=CB H<FCI;< 9B8CD@5GA=7 F9H=7I@IA

75@7=IA G=;B5@G 9B<5B7=B; 7MHC7<FCA9 7 F9@95G9 HC H<9 7MHCGC@ " 9@@ =C@ 

  –

34 , &5A65  ,=5B # <I +1 K5B; #/ 1CCB + MIB % =F5?=  %5B8=

BCJ5 +/ $99 !(( 7CAD@9L HF5BG8I79G 5DCDHCH=7 G=;B5@G :FCA 9B8C

D@5GA=7 F9H=7I@IA HC A=HC7<CB8F=5 IB89F 9B8CD@5GA=7 F9H=7I@IA GHF9GG 9@@ *9D 

  –

34  #FC9A9F $ 5@@INN= ' #9DD $ 2=HJC;9@ !AAIBC;9B=7 79@@ 895H< =B 75B79F

H<9F5DM BBI *9J !AAIBC@   –

34 % %=7<5I8 0 0=9 "% F5JC+5B (98FC $ 2=HJC;9@  /<=H9  #FC9A9F B

5IHCD<5;M89D9B89BH 5BH=75B79F =AAIB9 F9GDCBG9 89H9FA=B9G H<9 9:R757M C:

A9@5BCA5 7<9ACH<9F5DM 'B7C=AAIBC@C;M    9

34 ! %5FH=BG 1 /5B; % %=7<5I8 1 %5 ) +I??IFK5@5 + +<9B ' #9DD 

%9H=J=9F $ 5@@INN= "$ (9F:9HH=B= $ 2=HJC;9@  #FC9A9F %C@97I@5F A97<5

B=GAG C: ,( G97F9H=CB 8IF=B; =AAIBC;9B=7 79@@ 895H< 9@@ 95H< =::9F  

 –

34 2 2<5B;  <9B / 2<CI  +CB; , 0I ) $IC / /5B; 0+ I + I5B

*9;I@5H98 ,( F9@95G9 :FCA 5GHFC7MH9G H<FCI;< @MGCGCA9 9LC7MHCG=G &5H 9@@ =C@

   –

34 1 CI " /I ) $= + )=B 1 /5B; " $=  $CI 2 <9B 0% $= )%

$IC + I5B %=7FC;@=5@ A=;F5H=CB A98=5H98 6M ,(=B8I798 ,( F9@95G9 :FCA

@MGCGCA9G 9@@ *9G    –

34 . +=J5F5A5?F=G<B5B + =8I@5  5ADK5@5  #5H=?5B9B= +" CIBH5=B CB

GH=HIH=J9 @MGCGCA9 9LC7MHCG=G F9@95G9G ,( 5B8 9B;5;9G (1 F979DHCFG =B <IA5B

ACBC7MH9G " 9@@ +7=  (H   –

34 "% 97?9@ &% CA9N / $I # 5AD5;BC  &569H  @65@5K= " $=A

# C9GN95HH5;@=5   %=H7<9@@ +H=AI@5H=CB C: ,$* HF=;;9FG F9@95G9 C: @MGCGC

A5@ ,( =B 5GHFC7MH9G 5B8 9D=H<9@=5@ 79@@G H<5H F9EI=F9G ,*(%$ 7<5BB9@G +7=

*9D    

34 % <CK8<IFM  B9B?9@ !BHF579@@I@5F 8MB5A=7G C: H<9 I6=EI=H=BDFCH95

GCA9GMGH9A *9G   

34  *C69FHG . 9F9H=7 IHCD<5;=7 DFCH9C@MG=G C: @CB;@=J98 DFCH9=BG =B BCB@=J9F

79@@G %9H<C8G %C@ =C@   –

34 , .9F:5=@@=9 % +5@5N5F  .9@5G7C ( ;CGH=B=G $=B?=B; * GHF9GG HC 5IHCD<5;M

DCH9BH=5@ =AD@=75H=CBG :CF 75B79F H<9F5DM !BH " 9@@ =C@   

34 & '<C?5 + 1CG<== , 5HHCF= # 'BCN5?=  5M5G<= ,* 5 BCJ9@ *

GHF9GG=B8I7=6@9 ;9B9 =G =B8I798 J=5 , '( D5H<K5M 5B8 =G =BJC@J98 =B 79@@

895H< %' "    –

34 #& 5@6M ! ,9?989F9@=  $CD9N9F9GH9=B  'NDC@5H ,5F;9H=B; H<9 DFC895H<

5B8 DFCGIFJ=J5@ :IB7H=CBG C: 5IHCD<5;M 5G BCJ9@ H<9F5D9IH=7 GHF5H9;=9G =B 75B79F

IHCD<5;M    –

34  J5B;9@=GH=  J5B;9@=GH=  <=5F=B=  $CB9HH=  ICBH9ADC $% &9F= "

%7I6F9M % %5FH9@@= IHCD<5;M =B 57IH9 @9I?9A=5G 5 8CI6@998;98 GKCF8

K=H< =ADCFH5BH H<9F5D9IH=7 =AD@=75H=CBG =C7<=A =CD<MG 7H5 %C@ 9@@ =C@

$=D=8G    –

34 % $977=GC  '758@=?CJ5 + +5B;5@9HH= + ,F565B9@@=  9 %5F7<=  'F=C@= 

(9;CF5FC ( (CFH5F5FC  "5B8IG  CBH58=B=  *985J=8 . +5@J9GHF=B= (

*CA9FC %( C@CA6C  = .=F;=@=C % 5JC  8=BC@R  IFH= ,( F9@95G9

:FCA 7<9ACH<9F5DMHF95H98 8M=B; @9I?9A=5 79@@G 9@=7=HG 5B =AAIB9 GIDDF9GG=J9 9:

:97H 6M =B7F95G=B; F9;I@5HCFM , 79@@G 5B8 HC@9FC;9B=7 89B8F=H=7 79@@G FCBH !A

AIBC@   

34  *58C;B5 % =75HC % =989F=7< 5B79FHMD9GD97=R7 7FCGGH5@? 69HK99B 5I

HCD<5;M B97FCDHCG=G 5B8 5DCDHCG=G 5G 5 D<5FA57C@C;=75@ H5F;9H =C7<9A (<5FA5

7C@    –

34 $+ =7?9BG !* (CK@9M % I;<9G % %575F@5B9 ,<9 7CAD@9L=H=9G C: @=:9

5B8 895H< 895H< F979DHCF G=;B5@@=B; D@5H:CFAG LD 9@@ *9G   

–

34 ( .5B89B5699@9 $ 5@@INN= , .5B89B 9F;<9  #FC9A9F %C@97I@5F A97<5

B=GAG C: B97FCDHCG=G 5B CF89F98 79@@I@5F 9LD@CG=CB &5H *9J %C@ 9@@ =C@ 

  –

34 + $57CIF ' %=7<95I  5AA5BB . FCI=B95I8 " ,G7<CDD  +C@5FM %,

=A5B7<9C=HF9@ <9ACH<9F5DM 9B<5B79G ,&F9@5H98 5DCDHCG=G=B8I7=B; @=;5B8

!+ 5GG9A6@M =B , <IA5B 7C@CB 75B79F 79@@G 'B7C;9B9   

–

34 # =5B7<= ( %9=9F  H5B;@98 K96 C: I6=EI=H=B 7<5=BG 6F95?=B; B9KG =B ,&*

G=;B5@=B; %C@ 9@@    –

34  9J=B  CC? 1 $=B 1 *C8F=;I9N % #9@@=<9F 2 $=I ,<9 8=GH=B7H FC@9G C:

,* 5B8 *!( =B !## 57H=J5H=CB 6M ,&* ,* F97FI=HG !## HC ,&* K<=@9

*!( A98=5H9G !## 57H=J5H=CB !AAIB=HM    –

34 %" 9FHF5B8 + %=@IH=BCJ=7 #% =7?GCB / C  CI8F95I@H " IF?=B

"/ =@@5F8 " "5EI=H< +" %CFF=G ( 5F?9F 7!( 5B8 7!( :57=@=H5H9 75B

79F 79@@ GIFJ=J5@ 6M :IB7H=CB=B; 5G  @=;5G9G H<5H DFCACH9 *!( I6=EI=H=B5H=CB

%C@ 9@@    –

34 // *9=@9M / "=B " $99  /F=;<H 0 /I  ,9K5@H ,' $9CB5F8 

&CF6IFM $ =HND5HF=7? % 2<5B; + +IB 9I6=EI=H=B5H=B; 9BNMA9 1$ B9;5

H=J9@M F9;I@5H9G H<9 I6=EI=H=B89D9B89BH ?=B5G9 ,5? 5B8 DF9J9BHG 56BCFA5@ , 79@@

F9GDCBG9G " LD %98    –

34 ' %=7<95I " ,G7<CDD !B8I7H=CB C: ,& F979DHCF !A98=5H98 5DCDHCG=G J=5 HKC

G9EI9BH=5@ G=;B5@=B; 7CAD@9L9G 9@@    –

34  BB=65@8= ( %9=9F <97?DC=BHG =B ,&=B8I798 79@@ 895H< =AD@=75H=CBG =B =B

S5AA5H=CB 5B8 75B79F ,F9B8G %C@ %98    –

34 $ 5@@INN=  IEI9 ' #9DD $ 2=HJC;9@  #FC9A9F !AAIBC;9B=7 79@@ 895H< =B

75B79F 5B8 =B:97H=CIG 8=G95G9 &5H *9J !AAIBC@    –

34 , 15A5N5?=  5BB5B= . (C=F=9FC@5A9 + $58C=F9  $C7<9F  +=GH=;I &

(F585 + 8>9A=5B "( 5H5B= % F9I89B69F;  5@5BCG  B8F9  #FC9

A9F $ 2=HJC;9@ 9:97H=J9 =AAIBC;9B=7 79@@ 895H< C: %89R7=9BH HIACFG

7CAD9BG5HCFM H<9F5DM K=H< ,$* 5;CB=GHG 9@@ 95H< =::9F    –

34  .9B9F95I % 5G5@;F5B8= % +7<=F5@8= " BHC=B9  5HH5B9C  9

%5F7<=G " $=I  BHCB9@@=  (F9H= $ *59@= ++ +<5AG  15B; $ .5F5B= -

B89FGGCB #" ,F579M  57<= % -;I77=CB= % =5B7<= %IHI5@@M 9L7@IG=J9

F98CL :CFAG C: % DFCACH9 79@@ F97FI=HA9BH CF DFC=BS5AA5HCFM 7MHC?=B9 F9

@95G9 " LD %98    –

34 $ <5=GK=B; , '69F@9M LHF579@@I@5FA=7FC9BJ=FCBA9BH5@ F98CL GH5H9 B

H=CL=8 *98CL +=;B5@    –

34 * #5B; ) 2<5B; " 29< F8 %, $CHN9  ,5B; % =B 75B79F ;CC8

658 CF 6CH< @=B 5B79F *9G    –

34 $$ (C@=75GHFC !$ !65B9N  &CH7CJ=7<  IF5B '$ (C8<5>79F ,<9 HIACF

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Chromomycin A5 induces bona fide immunogenic cell death in melanoma

Article

Full-text available

Nov 2022

Purpose Some first-line cytotoxic chemotherapics, e.g. doxorubicin, paclitaxel and oxaliplatin, induce activation of the immune system through immunogenic cell death (ICD). Tumor cells undergoing ICD function as a vaccine, releasing damage-associated molecular patterns (DAMPs), which act as adjuvants, and neoantigens of the tumor are recognized as antigens. ICD induction is rare, however it yields better and long-lasting antitumor responses to chemotherapy. Advanced metastatic melanoma (AMM) is incurable for more than half of patients. The discovery of ICD inducers against AMM is an interesting drug discovery strategy with high translational potential. Here we evaluated ICD induction of four highly cytotoxic chromomycins A (CA 5-8 ). Methods ICD features and DAMPs were evaluated using several in vitro techniques with metastatic melanoma cell line (B16-F10) exposed to chromomcins A 5-8 such as flow cytometry, western blot, RT-PCR and luminescence. Additionally in vivo vaccination assays with CA 5 -treated cells in a syngeneic murine model (C57Bl/6) were performed to confirm ICD evaluating the immune cells activation and their antitumor activity. Results B16-F10 treated with CA 5-8 and doxorubicin exhibited ICD features such as autophagy and apoptosis, externalization of calreticulin, and releasing of HMGB1. However, CA 5 -treated cells had the best profile, also inducing ATP release, ERp57 externalization, phosphorylation of eIF2α and altering expression of transcription of genes related to autophagy, endoplasmic reticulum stress, and apoptosis. Bona fide ICD induction by CA 5 was confirmed by vaccination of C57BL/6 mice with CA 5 -treated cells which activated antigen-presenting cells and T lymphocytes and stimulated antitumor activity. Conclusion CA 5 induces bona fide immunogenic cell death on melanoma.

An Immunogenic Cell Death-Related Classification Predicts Prognosis and Response to Immunotherapy in Hepatocellular carcinoma

Preprint

Full-text available

Oct 2022

Hepatocellular carcinoma (HCC) is one of the world's malignant tumors with high mortality and a worse prognosis. Immunogenic cell death is caused by tumor cells stimulated by stressors to display or release Danger Associated Molecular Patterns (DAMPs)that attract and activate antigen-presenting cells. It would be advantageous to identify biomarkers associated with Immunogenic cell death(ICD) and stratify patients according to their benefits from ICD immunotherapy. Here, we identified two subtypes ICD-related by consensus clustering. ICD-high Subtype has an excellent clinical outcome, abundant immune cell infiltration, and highly active immune response signaling. In addition, we developed and validated an ICD-related risk model that predicts HCC patients' survival and correlates with the tumor immune microenvironment. In conclusion, we have established a new classification system for HCC based on ICD signatures. This stratification has significant clinical outcomes for estimating prognosis and immunotherapy in HCC patients.

Schweinfurthin induces ICD without ER stress and caspase activation

Article

Full-text available

Dec 2022

Our previous study showed that one of the schweinfurthin compounds, 5'-methoxyschweinfurthin G (MeSG), not only enhances the anti-tumor effect of anti-PD1 antibody in the B16F10 murine melanoma model, but also provokes durable, protective anti-tumor immunity. Here we further investigated the mechanisms by which MeSG treatment induces immunogenic cell death (ICD). MeSG induced significant cell surface calreticulin (CRT) exposure in a time and concentration dependent manner as well as increased phagocytosis of tumor cells by dendritic cells in vitro. Interestingly, this CRT exposure differs from the canonical pathway in several aspects. MeSG does not cause ER stress and does not require PERK to induce CRT exposure. Caspase inhibitors partially rescue cells from MeSG-induced apoptosis, but fail to reduce CRT exposure. MeSG does not cause ERp57 exposure and the absence of ERp57 expression does not reduce CRT exposure. Finally, an intact ER to Golgi transport system is required for this phenomenon. These results lend support to the development of the schweinfurthin family as drugs to enhance clinical response to immunotherapy and highlight the need for additional research on the mechanisms of ICD induction.

An Overview on Immunogenic Cell Death in Cancer Biology and Therapy

Article

Full-text available

Jul 2022

Immunogenic cell death (ICD) is a modality of regulated cell death that is sufficient to promote an adaptive immune response against antigens of the dying cell in an immunocompetent host. An important characteristic of ICD is the release and exposure of damage-associated molecular patterns, which are potent endogenous immune adjuvants. As the induction of ICD can be achieved with conventional cytotoxic agents, it represents a potential approach for the immunotherapy of cancer. Here, different aspects of ICD in cancer biology and treatment are reviewed.

Phase I study of PT-112, a novel pyrophosphate-platinum immunogenic cell death inducer, in advanced solid tumours

Article

Full-text available

Jul 2022

Background PT-112, the first pyrophosphate-platinum conjugate, causes immunogenic cell death in experimental models, leading to recruitment of tumour-infiltrating lymphocytes. PT-112 also associates with bone (osteotropism), likely driven by its pyrophosphate moiety. This is the first-in-human study of PT-112 monotherapy, exploring its safety and efficacy in a patient population where standard of care therapies were exhausted and novel treatment options are needed. Methods Patients with progressing, advanced solid tumours received PT-112 intravenously (1 h) on days 1, 8, 15 of a 28-day cycle in an open-label, multi-centre 3 + 3 dose-escalation trial, conducted at four US research sites. The primary objective was to assess safety and pharmacokinetics, and to identify a recommended phase 2 dose (RP2D). Eligibility criteria included: age ≥18 years, Eastern Collaborative Oncology Group (ECOG) Performance Status of 0–1, and disease evaluable by Response Evaluation Criteria in Solid Tumours (RECIST) v1·1 or by informative tumour markers. Patients receiving ≥1 dose of PT-112 were included in the safety and pharmacokinetic analyses, with the exploratory efficacy analysis including patients receiving ≥1 dose at 125 mg/m². This study is registered at ClinicalTrials.gov, number NCT02266745, with the dose-escalation portion of the study closed. Findings Between July 7th, 2014 and September 18th, 2018, 66 heavily pre-treated patients (median 4 prior lines, IQR 2–6) were enrolled and treated across 11 doses (12–420 mg/m²). Treatment-related adverse events included fatigue (23 patients, 35%), nausea (16 patients, 24%), and peripheral neuropathy (14 patients, 21%). Grade 3 events were experienced by 18 patients (27%), with no grade 4–5 events observed. The recommended phase 2 dose was determined to be 360 mg/m². Nine (17%) of the 54 efficacy evaluable patients achieved progression-free survival ≥6 months. Durable partial responses were induced in non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), and thymoma. Radiographic and serum marker reductions were observed among ten patients with metastatic castration resistant prostate cancer, four of whom survived two years or longer. Interpretation PT-112 is safe and well-tolerated in a heavily pre-treated population. Prolonged responses were noted against thymoma and lung cancer, along with radiographic and serum marker improvement in prostate cancer. Given the heterogeneous patient population, subsequent studies will be needed to characterize the risk/benefit ratio in more homogenous settings. Further development of PT-112 is ongoing, as single-agent and in combination with immune checkpoint inhibition. Funding Funding was provided by Promontory Therapeutics Inc.

Nanodelivery of scutellarin induces immunogenic cell death for treating hepatocellular carcinoma

Article

Jun 2023
INT J PHARMACEUT

Hepatocellular carcinoma (HCC) causes the immunosuppressive tumor microenvironment (TME) resistant to current immunotherapy. The immunogenic apoptosis (currently termed immunogenic cell death, ICD) of cancer cells may induce the adaptive immunity against tumors, thereby providing great potential for treating HCC. In this study, we have confirmed the potential of scutellarin (SCU, a flavonoid found in Erigeron breviscapus) for triggering ICD in HCC cells. To facilitate in vivo application of SCU for HCC immunotherapy, an aminoethyl anisamide-targeted polyethylene glycol-modified poly(lactide-co-glycolide) (PLGA-PEG-AEAA) was produced to facilitate SCU delivery in this study. The resultant nanoformulation (PLGA-PEG-AEAA.SCU) remarkably promoted blood circulation and tumor delivery in the orthotopic HCC mouse model. Consequently, PLGA-PEG-AEAA.SCU reversed the immune suppressive TME and achieved the immunotherapeutic efficacy, resulting in significantly longer survival of mice, without inducing toxicity. These findings uncover the ICD potential of SCU and provide a promising strategy for HCC immunotherapy.

Combining in situ vaccination and immunogenic apoptosis to treat cancer

Article

Feb 2023
IMMUNOTHERAPY-UK

Immunization approaches are designed to stimulate the immune system and eliminate the tumor. Studies indicate that cancer immunization combined with certain chemotherapeutics and immunostimulatory agents can improve outcomes. Chemotherapeutics-based immunogenic cell death makes the tumor more recognizable by the immune system. In situ vaccination (ISV) utilizes established tumors as antigen sources and directly applies an immune adjuvant to the tumor to reverse a cold tumor microenvironment to a hot one. Immunogenic cell death and ISV highlight for the immune system the tumor antigens that are recognizable by immune cells and support a T-cell attack of the tumor cells. This review presents the concept of immunogenic apoptosis and ISV as a powerful platform for cancer immunization.

3′-epi-12β-hydroxyfroside-mediated autophagy degradation of RIPK1/RIPK3 necrosomes leads to anergy of immunogenic cell death in triple-negative breast cancer cells

Article

Dec 2022
PHARMACOL RES

Increasing studies have suggested that some cardiac glycosides, such as conventional digoxin (DIG) and digitoxin, can induce immunogenic cell death (ICD) in various tumors. We previously found that 3′-epi-12β-hydroxyfroside (HyFS), a novel cardenolide compound isolated by our group, could induce cytoprotective autophagy through inactivation of the Akt/mTOR pathway. However, whether HyFS can induce ICD remains unknown. In this study, we extend our work to further investigate whether HyFS could induce both autophagy and ICD, and we investigated the relationship between autophagy and ICD in three TNBC cell lines. Unexpectedly, compared to DIG, we found that HyFS could induce complete autophagy flux but not ICD in three human triple-negative breast cancer (TNBC) cell lines and one murine TNBC model. Inhibition of HyFS-induced autophagy resulted in the production of ICD in TNBC MDA-MB-231, MDA-MB-436, and HCC38 cells. A further mechanism study showed that formation of RIPK1/RIPK3 necrosomes was necessary for ICD induction in DIG-treated TNBC cells, while HyFS treatment led to receptor-interacting serine-threonine kinase (RIPK)1/3 necrosome degradation via an autophagy process. Additionally, inhibition of HyFS-induced autophagy by the autophagy inhibitor chloroquine resulted in the reoccurrence of ICD and reversion of the tumor microenvironment, leading to more significant antitumor effects in immunocompetent mice than in immunodeficient mice. These findings indicate that HyFS-mediated autophagic degradation of RIPK1/RIPK3 necrosomes leads to inactivation of ICD in TNBC cells. Moreover, combined treatment with HyFS and an autophagy inhibitor may enhance the antitumor activities, suggesting an alternative therapeutic for TNBC treatment.

Nitric Oxide Induces Immunogenic Cell Death and Potentiates Cancer Immunotherapy

Article

Mar 2022

Cytotoxic triterpene and steroidal glycosides from the seeds of Digitalis purpurea and the synergistic cytotoxicity of steroidal glycosides and etoposide in SBC-3 cells

Article

Feb 2022
BIOORG CHEM

The phytochemical investigations of the seeds of Digitalis purpurea have revealed their richness in cardenolide and pregnane glycosides exhibiting potent cytotoxicity; further chemical examinations of the D. purpurea seeds have achieved the isolation of six triterpene glycosides (1–6), six spirostanol glycosides (7–12), and three furostanol glycosides (13–15), including seven previously unidentified compounds (1–3, 10–12, and 14). Here, the structures of 1–3, 10–12, and 14 were determined via extensive spectroscopic analyses, including two-dimensional (2D) NMR; hydrolysis, followed by chromatographic and spectroscopic analyses; and X-ray crystallographic analysis. The cytotoxic activities of the isolated compounds (1–15) against SBC-3 small cell lung carcinoma and TIG-3 normal human diploid fibroblast cells were evaluated. Triterpene glycoside 3 and spirostanol glycoside 9 exhibited considerable cytotoxicity with IC50 values of 1.0 and 1.7 µM, respectively; they induced apoptotic cell death, which was accompanied by the activation of caspase-3 in SBC-3 cells. Spirostanol glycoside 7 exhibited cytotoxicity toward the SBC-3 cells (IC50 1.3 μM). Additionally, 7 at 0.1 and 1.0 µM synergistically enhanced the cytotoxicity of etoposide against SBC-3 cells; compound 7 induced the release of DAMPs; the release of HMGB1, the secretion of ATP, and the exposure of CALR in the SBC-3 cells. Furthermore, the combination of 7 and etoposide resulted in increasing the extracellular release of DAMPs. These data indicated that 7, as well as its combination with etoposide, might potentially cause immunogenic cell death.

Stimulation of TLR3 Triggers Release of Lysosomal ATP in Astrocytes and Epithelial cells that Requires TRPML1 Channels

Article

Full-text available

Apr 2018

Cross-reactions between innate immunity, lysosomal function, and purinergic pathways may link signaling systems in cellular pathologies. We found activation of toll-like receptor 3 (TLR3) triggers lysosomal ATP release from both astrocytes and retinal pigmented epithelial (RPE) cells. ATP efflux was accompanied by lysosomal acid phosphatase and beta hexosaminidase release. Poly(I:C) alkalinized lysosomes, and lysosomal alkalization with bafilomycin or chloroquine triggered ATP release. Lysosomal rupture with glycyl-L-phenylalanine-2-naphthylamide (GPN) eliminated both ATP and acid phosphatase release. Secretory lysosome marker LAMP3 colocalized with VNUT, while MANT-ATP colocalized with LysoTracker. Unmodified membrane-impermeant 21-nt and “non-targeting” scrambled 21-nt siRNA triggered ATP and acid phosphatase release, while smaller 16-nt RNA was ineffective. Poly(I:C)-dependent ATP release was reduced by TBK-1 block and in TRPML1−/− cells, while TRPML activation with ML-SA1 was sufficient to release both ATP and acid phosphatase. The ability of poly(I:C) to raise cytoplasmic Ca2+ was abolished by removing extracellular ATP with apyrase, suggesting ATP release by poly(I:C) increased cellular signaling. Starvation but not rapamycin prevented lysosomal ATP release. In summary, stimulation of TLR3 triggers lysosomal alkalization and release of lysosomal ATP through activation of TRPML1; this links innate immunity to purinergic signaling via lysosomal physiology, and suggests even scrambled siRNA can influence these pathways.

Cardiac Glycoside Glucoevatromonoside Induces Cancer Type-Specific Cell Death

Article

Full-text available

Mar 2018

Cardiac glycosides (CGs) are natural compounds used traditionally to treat congestive heart diseases. Recent investigations repositioned CGs as potential anticancer agents. To discover novel cytotoxic CG scaffolds, we selected the cardenolide glucoevatromonoside (GEV) out of 46 CGs for its low nanomolar anti-lung cancer activity. GEV presented reduced toxicity toward non-cancerous cell types (lung MRC-5 and PBMC) and high-affinity binding to the Na+/K+-ATPase α subunit, assessed by computational docking. GEV-induced cell death was caspase-independent, as investigated by a multiparametric approach, and culminates in severe morphological alterations in A549 cells, monitored by transmission electron microscopy, live cell imaging and flow cytometry. This non-canonical cell death was not preceded or accompanied by exacerbation of autophagy. In the presence of GEV, markers of autophagic flux (e.g. LC3I-II conversion) were impacted, even in presence of bafilomycin A1. Cell death induction remained unaffected by calpain, cathepsin, parthanatos, or necroptosis inhibitors. Interestingly, GEV triggered caspase-dependent apoptosis in U937 acute myeloid leukemia cells, witnessing cancer-type specific cell death induction. Differential cell cycle modulation by this CG led to a G2/M arrest, cyclin B1 and p53 downregulation in A549, but not in U937 cells. We further extended the anti-cancer potential of GEV to 3D cell culture using clonogenic and spheroid formation assays and validated our findings in vivo by zebrafish xenografts. Altogether, GEV shows an interesting anticancer profile with the ability to exert cytotoxic effects via induction of different cell death modalities.

ATP Release from Chemotherapy-Treated Dying Leukemia Cells Elicits an Immune Suppressive Effect by Increasing Regulatory T Cells and Tolerogenic Dendritic Cells

Article

Full-text available

Dec 2017

Chemotherapy-induced immunogenic cell death can favor dendritic cell (DC) cross-priming of tumor-associated antigens for T cell activation thanks to the release of damage-associated molecular patterns, including ATP. Here, we tested the hypothesis that in acute myeloid leukemia (AML), ATP release, along with its well-known immune stimulatory effect, may also contribute to the generation of an immune suppressive microenvironment. In a cohort of AML patients, undergoing combined daunorubicin and cytarabine chemotherapy, a population of T regulatory cells (Tregs) with suppressive phenotype, expressing the immune checkpoint programmed cell death protein 1 (PD-1), was significantly increased. Moving from these results, initial in vitro data showed that daunorubicin was more effective than cytarabine in modulating DC function toward Tregs induction and such difference was correlated with the higher capacity of daunorubicin to induce ATP release from treated AML cells. DCs cultured with daunorubicin-treated AML cells upregulated indoleamine 2,3-dioxygenase 1 (IDO1), which induced anti-leukemia Tregs. These data were confirmed in vivo as daunorubicin-treated mice show an increase in extracellular ATP levels with increased number of Tregs, expressing PD-1 and IDO1+CD39+ DCs. Notably, daunorubicin failed to induce Tregs and tolerogenic DCs in mice lacking the ATP receptor P2X7. Our data indicate that ATP release from chemotherapy-treated dying cells contributes to create an immune suppressive microenvironment in AML.

Targeting mutant p53 for efficient cancer therapy

Article

Full-text available

Dec 2017
NAT REV CANCER

The tumour suppressor gene TP53 is the most frequently mutated gene in cancer. Wild-type p53 can suppress tumour development by multiple pathways. However, mutation of TP53 and the resultant inactivation of p53 allow evasion of tumour cell death and rapid tumour progression. The high frequency of TP53 mutation in tumours has prompted efforts to restore normal function of mutant p53 and thereby trigger tumour cell death and tumour elimination. Small molecules that can reactivate missense-mutant p53 protein have been identified by different strategies, and two compounds are being tested in clinical trials. Novel approaches for targeting TP53 nonsense mutations are also underway. This Review discusses recent progress in pharmacological reactivation of mutant p53 and highlights problems and promises with these strategies.

Hydroxycoumarin OT-55 kills CML cells alone or in synergy with Imatinib or Synribo: involvement of ER stress and DAMP release

Article

Sep 2018
CANCER LETT

We synthetized and investigated here the anti-leukemic potential of the novel cytostatic bis(4-hydroxycoumarin) derivative OT-55 which complied with the Lipinski's rule of 5 and induced differential toxicity in various chronic myeloid leukemia (CML) cell models. OT-55 triggered ER stress leading to canonical, caspase-dependent apoptosis and release of danger associated molecular patterns. Consequently, OT-55 promoted phagocytosis of OT-55-treated CML cells by both murine and human monocyte-derived macrophages. Moreover, OT-55 inhibited tumor necrosis factor α-induced activation of nuclear factor-кB and produced synergistic effects when used in combination with imatinib to inhibit colony formation in vitro and Bcr-Abl+ patient blast xenograft growth in zebrafish. Furthermore, OT-55 synergized with omacetaxine in imatinib-resistant KBM-5 R cells to inhibit the expression of Mcl-1, triggering apoptosis. In imatinib-resistant K562 R cells, OT-55 triggered necrosis and blocked tumor formation in zebrafish in combination with omacetaxine.

Natural scaffolds in anticancer therapy and precision medicine

Article

May 2018
BIOTECHNOL ADV

The diversity of natural compounds is essential for their mechanism of action. The source, structures and struc- ture activity relationship of natural compounds contributed to the development of new classes of chemotherapy agents for over 40years. The availability of combinatorial chemistry and high-throughput screening has fueled the challenge to identify novel compounds that mimic nature's chemistry and to predict their macromolecular targets. Combining conventional and targeted therapies helped to successfully overcome drug resistance and prolong disease-free survival. Here, we aim to provide an overview of preclinical investigated natural compounds alone and in combination to further improve personalization of cancer treatment.

Stress-induced cellular responses in immunogenic cell death: Implications for cancer immunotherapy

Article

Feb 2018
BIOCHEM PHARMACOL

Cancer is evading the host's defense mechanisms leading to avoidance of immune destruction. During tumor progression, immune-evading cancer cells arise due to selective pressure from the hypoxic and nutrient-deprived microenvironment. Thus, therapies aiming at re-establishing immune destruction of pathological cells constitute innovating anti-cancer strategies. Accumulating evidence suggests that selected conventional chemotherapeutic drugs increase the immunogenicity of stressed and dying cancer cells by triggering a form of cell death called immunogenic cell death (ICD), which is characterized by the release of danger-associated molecular patterns (DAMPs). In this review, we summarize the effects of ICD inducers on DAMP signaling leading to adjuvanticity and antigenicity. We will discuss the associated stress response pathways that cause the release of DAMPs leading to improved immune recognition and their relevance in cancer immunotherapy. Our aim is to highlight the contribution of adaptive immunity to the long-term clinical benefits of anticancer treatments and the properties of immune memory that can protect cancer patients against relapse.

eIF2α phosphorylation is pathognomonic for immunogenic cell death

Article

Jan 2018
CELL DEATH DIFFER

The phosphorylation of eIF2α is essential for the endoplasmic reticulum (ER) stress response, the formation of stress granules, as well as macroautophagy. Several successful anticancer chemotherapeutics have the property to induce immunogenic cell death (ICD), thereby causing anticancer immune responses. ICD is accompanied by the translocation of calreticulin (CALR) from the ER lumen to the plasma membrane, which facilitates the transfer of tumor-associated antigens to dendritic cells. Here we systematically investigated the capacity of anticancer chemotherapeutics to induce signs of ER stress. ICD inducers including anthracyclines and agents that provoke tetraploidization were highly efficient in enhancing the phosphorylation of eIF2α, yet failed to stimulate other signs of ER stress including the transcriptional activation of activating transcription factor 4 (ATF4), the alternative splicing of X-box binding protein 1 (XBP1s) mRNA and the proteolytic cleavage of activating transcription factor 6 (ATF6) both in vitro and in cancers established in mice. Systematic analyses of clinically used anticancer chemotherapeutics revealed that only eIF2α phosphorylation, but none of the other signs of ER stress, correlated with CALR exposure. eIF2α phosphorylation induced by mitoxantrone, a prototype ICD-inducing anthracyline, was mediated by eIF2α kinase-3 (EIF2AK3). Machine-learning approaches were used to determine the physicochemical properties of drugs that induce ICD, revealing that the sole ER stress response relevant to the algorithm is eIF2α phosphorylation with its downstream consequences CALR exposure, stress granule formation and autophagy induction. Importantly, this approach could reduce the complexity of compound libraries to identify ICD inducers based on their physicochemical and structural characteristics. In summary, it appears that eIF2α phosphorylation constitutes a pathognomonic characteristic of ICD.

Cytostatic hydroxycoumarin OT52 induces ER/Golgi stress and STAT3 inhibition triggering non-canonical cell death and synergy with BH3 mimetics in lung cancer

Article

Mar 2018
CANCER LETT

Coumarins are natural compounds with antioxidant, anti-inflammatory and anti-cancer potential known to modulate inflammatory pathways. Here, non-toxic biscoumarin OT52 strongly inhibited proliferation of non-small cell lung cancer cells with KRAS mutations, inhibited stem-like characteristics by reducing aldehyde dehydrogenase expression and abrogated spheroid formation capacity. This cytostatic effect was characterized by cell cycle arrest and onset of senescence concomitant with endoplasmic reticulum and Golgi stress, leading to metabolic alterations. Mechanistically, this cellular response was associated with the novel capacity of biscoumarin OT52 to inhibit STAT3 transactivation and expression of its target genes linked to proliferation. These results were validated by computational docking of OT52 to the STAT3 DNA-binding domain. Combination treatments of OT52 with subtoxic concentrations of Bcl-xL and Mcl-1-targeting BH3 protein inhibitors triggered synergistic immunogenic cell death validated in colony formation assays as well as in vivo by zebrafish xenografts.

The dialkyl resorcinol stemphol disrupts calcium homeostasis to trigger programmed immunogenic necrosis in cancer

Article

Mar 2018
CANCER LETT

Stemphol (STP) is a novel druggable phytotoxin triggering mixed apoptotic and non-apoptotic necrotic-like cell death in human acute myeloid leukemia (AML). Use of several chemical inhibitors highlighted that STP-induced non-canonical programmed cell death was Ca2+-dependent but independent of caspases, poly (ADP-ribose) polymerase-1, cathepsin, or calpains. Similar to thapsigargin, STP led to increased cytosolic Ca2+ levels and computational docking confirmed binding of STP within the thapsigargin binding pocket of the sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA). Moreover, the inositol 1,4,5-trisphosphate receptor is implicated in STP-modulated cytosolic Ca2+ accumulation leading to ER stress and mitochondrial swelling associated with collapsed cristae as observed by electron microscopy. Confocal fluorescent microscopy allowed identifying mitochondrial Ca2+ overload as initiator of STP-induced cell death insensitive to necrostatin-1 or cycloheximide. Finally, we observed that STP-induced necrosis is dependent of mitochondrial permeability transition pore (mPTP) opening. Importantly, the translational immunogenic potential of STP was validated by HMGB1 release of STP-treated AML patient cells. STP reduced colony and in vivo tumor forming potential and impaired the development of AML patient-derived xenografts in zebrafish.

Natural modulators of the hallmarks of immunogenic cell death

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