Elevated Expression of the C-Type Lectin CD93 in the Glioblastoma Vasculature

Author Manuscript Published OnlineFirst on September 11, 2015; DOI: 10.1158/0008-5472.CAN-14-3636 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Elevated expression of the C-type lectin CD93 in the glioblastoma vasculature

regulates cytoskeletal rearrangements that enhance vessel function and reduce

host survival

*'1# ,%#,) +.SQ#'& ,%SQWQ- #02 3% ,-SQWQ3 3 ,%SQ + "-0*1'0 3*& 11 ,TQ

0' #-0% , )'SQ!#1 +" 88 0SQ$-& ,$SQ #-0%#0#,"#*#, 30%UQ %,3111 ,"SQ

(0#"0'))-,2M,SQ,( ,+'21TQVQ,, .'+ #0%S#

1. Department of Immunology, Genetics and Pathology, The Rudbeck Laboratory, Uppsala

University, Uppsala, Sweden

2. Department of Neuroscience, Neurology, University Hospital Uppsala, Uppsala, Sweden

3. Dept. of Neurology, University Medicine Goettingen, Robert-Koch Str. 40, 37075 Göttingen,

Germany

4. Department of Neurology, Danish Epilepsy Center, Dianalund, Denmark

5. These authors contributed equally to the work.

#0-00#1.-,"',% 32&-0S,, .'+ #0%Q.#. 02+#,2-$2++3,-*-%7Q #,#2'!1 ,") 2&-*-%7Q

3" #!) -0 2-07Q5..1 * 5,'4#01'27Q,VYWSZW5..1 * Q,5#"#,T)&-,#S&VXYRTSXXV[X

( 6S&VXSZWWZ[US+ '*S,, T.'+ #0%'%.T33T1#

Running title

0.[U0#%3* 2#1 ,%'-%#,#1'1',%*'- * 12-+

Key words

%*'- * 12-+ Q ,%'-%#,#1'1Q0V27.#*#!2',Q#,"-2&#*' *!#** "'-,Q4 1!3* 0$3,!2'-,

Conflicts of interest

&# 32&-01& 4#,-!-,$*'!2-$',2#0#122-"'1!*-1#T

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2015 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 11, 2015; DOI: 10.1158/0008-5472.CAN-14-3636 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

ABSTRACT

*'- * 12-+ '1 , %%0#11'4# 0 ',23+-0!& 0 !2#0'8#" 7 , ,-0+ * *--"4 1!3* 230#2& 2

'1&7.#0.#0+# *#T#0#5#0#.-02 ,-4#*0-*#$-00.[U',0#%3* 2',% ,%'-%#,#1'1',2&'1

1#22',% 7+-"3* 2',%!#**V!#** ,"!#**V+ 20'6 "'-,-$#,"-2&#*' *!#**1T'113#+'!0- 00 7

, *71'1"#+-,120 2#"2& 24 1!3* 0#6.0#11'-,-$0.[U!-00#* 2#"5'2&.--01304'4 *',

!*','! *!-&-02-$. 2'#,215'2&&'%&V%0 "# 120-!72'!%*'-+ T,'+'* 0*7Q',20 !0 ,' *%0-52&',2&#

TXS+-31#+-"#*-$%*'-+ 5 1"#* 7#"1'%,'$'! ,2*7',0.[UV V&-121Q0#13*2',%','+.0-4#"

1304'4 *!-+. 0#"2-5'*"V27.#+'!#T&'1#$$#!25 1 11-!' 2#"5'2&',!0# 1#"4 1!3* 0

.#0+# '*'27 ,""#!0# 1#"4 1!3* 0.#0$31'-,-$23+-01Q',"'! 2',%0#"3!#"4#11#*

$3,!2'-, *'27',2&# 1#,!#-$0.[UTC'V+#"' 2#" 22#,3 2'-,-$0.[U',#,"-2&#*' *!#**1

"'+','1&#"D (V',"3!#"23 #$-0+ 2'-,', U.!-** %#,%#*T0.[U5 10#/3'0#"$-0#$$'!'#,2

#,"-2&#*' *!#**+'%0 2'-, ,".0-.#0!#**.-* 0'8 2'-,',4'20-T(302�Q',#,"-2&#*' *!#**1

5�#0.[U5 1 22#,3 2#"Q"#!0# 1#"!#**1.0# "',%*#"2- 1#4#0#0#"3!2'-,',!#** "'-,Q

* !)-$.0-.#0!#**!-,2 !21Q *-11-$DV! "�', ," #00 ,2 !2',120#11$' #0$-0+ 2'-,TF30

0#13*21'"#,2'$70.[U 1 )#70#%3* 2-0-$%*'-+ ,%'-%#,#1'1 ,"4 1!3* 0$3,!2'-,Q !2',%4'

!72-1)#*#2 *0# 00 ,%#+#,210#/3'0#"$-0!#**V!#** ,"!#**V+ 20'6 "'-,T

INTRODUCTION

*'- * 12-+ 1 0#&'%&*7+ *'%, ,2 0 ',23+-012& 2 0#!& 0 !2#0'8#" 7,3!*# 0

27.' Q&'%&.0-*'$#0 2'4#',"#6Q,#!0-1'1Q#,"-2&#*' *.0-*'$#0 2'-, ,".*#-+-0.&'!Q ,-0+ *

4#11#*1SQTT&#23+-04#11#*1 0#+ *$3,!2'-,',% ,"&7.#0.#0+# *#Q %%0 4 2',%2&#

!-,"'2'-, 7%'4',%0'1#2- 0 ',#"#+ UT�#$-0#Q!-+ ',',%!-,4#,2'-, *! ,!#02� .7

5'2&1.#!'$'!2 0%#2',%-$2&# ,-0+ *23+-04#11#*10#.0#1#,21 , 220 !2'4# ..0- !&$-0

20# 2+#,2-$%*'-+ . 2'#,21T,2'V ,%'-%#,'!2� .7,#320 *'8',%4 1!3* 0#,"-2&#*' *%0-52&

$ !2-0D (-0'210#!#.2-0D (T'+.0-4#11304'4 *', ,'+ *+-"#*1-$%*'-+ ,"'1',

!*','! *31# 11#!-,"V*',#20# 2+#,2$-0%*'- * 12-+ VQWT,2'**Q2� .'#12 0%#2',%2&#

D ( D (T. 2&5 7& 4#,-20#13*2#"',.0-*-,% 2'-,-$-4#0 **1304'4 *-$. 2'#,215'2&

%*'- * 12-+ Q"#1.'2# 1'%,'$'! ,2'+.0-4#+#,2',.0-%0#11'-,V$0##1304'4 *T#1'12 ,!#

+#!& ,'1+1*# "',%2-3.V0#%3* 2'-,-$-2�.0-V ,%'-%#,'!$ !2-01-02-6'!'2'#1 0'1',%"3#2-

2&#'+.-02 ,20-*#-$D ( D (T1'%, *',%',,-0+ *.&71'-*-%7+ 7!-,20' 32#2-2&#

"'1 ..-',2',%0#13*21T, *2#0, 2'4#120 2#%7'12-#6.*-'22&##0-%#,#'27-$%#,##6.0#11'-,

',23+-0#,"-2&#*' *!#**1 1!-+. 0#"2-,-0+ *#,"-2&#*' *!#**1Q ,"2 0%#2+-*#!3*#1

1.#!'$'! **7#6.0#11#"',2&#23+-04 1!3* 230#2& 2!-,20' 32#2- ,%'-%#,#1'1 ,"4 1!3* 0

+ *$-0+ 2'-,',%*'- * 12-+ XV[T

&0-3%&* 1#0! .230#+'!0-"'11#!2'-,$-**-5#" 7+'!0- 00 7 , *71'1-$%#,#

#6.0#11'-,Q5#& 4#0#!#,2*7!& 0 !2#0'8#"2&#20 ,1!0'.2-+#-$ *--"4#11#*1',&3+ ,

%*'- * 12-+ Q*-5V%0 "#%*'-+ ,"!-,20-* 0 ',2'113#T!#'"#,2'$'#"[W%#,#12& 2 0#

1.#!'$'! **7#6.0#11#"',%*'- * 12-+ 4 1!3* 230#Q',!*3"',%2&#1',%*#V. 1120 ,1+#+ 0 ,#

%*7!-.0-2#',0.[UYT0.[U!-,1'121-$ ,#620 !#**3* 0. 025'2& 0V27.#*#!2',V*')#"-+ ',

0.Q$'4#2 ,"#+#.'"#0+ *%0-52&$ !2-0 (V*')#0#.# 21Q ,#0V&00'!&+3!',V*')#

"-+ ',Q 20 ,1+#+ 0 ,#"-+ ', ," 1&-02!72-.* 1+'!"-+ ',SRT22'1.0-+',#,2*7

#6.0#11#"',#,"-2&#*' *!#**1 ,"1-+#&#+ 2-.-'#2'!13 1#21Q ,"! , #1&#" ,"0#*# 1#"',

1-*3 *#$-0+10.[U3.-,',$* ++ 2-0712'+3*'SSQSTT0.[UV V+'!#& 4#,-0#.-02#"

4 1!3* 0"#$#!21 ," 0# -0, 2,-0+ *+#,"#*' ,0 2'-1Q 321&-5"#$#!21',!*# 0 ,!#-$

.-.2-2'!!#**1 ," 1*'%&2*7"#!0# 1#"+ ',2#, ,!#-$ ,2' -"71#!0#2'-,SUQSVT

0#!#,2123"7& 1'"#,2'$'#"0.[U 1-,#-$2&#2-.TR%#,#1-$ !-0#&3+ ,

.0'+ 0723+-0 ,%'-%#,#1'11'%, 230#&'%&*7#6.0#11#"',&# " ,",#!)1/3 +-31!#**

! 0!',-+ 1Q 0# 12! ,!#01 ,"!*# 0!#**0#, *!#**! 0!',-+ 1SWT0-,1'12#,25'2& 0-*#$-0

0.[U', ,%'-%#,#1'1Q 0#!-+ ', ,2.0-2#',!-,2 ',',%2&# (V*')#"-+ ', ,"2&#1#0',#V

2&0#-,',#0'!&+3!',V*')#"-+ ',-$0.[U& 1 ##,"#+-,120 2#"2-& 4#.0-V ,%'-%#,'!

.0-.#02'#1Q 322&##$$#!215#0#+-"#0 2# ,"*#11#4'"#,2',2-2 *10.[U5�#2&#0.'1

',!*3"#"SXT -0#-4#0Q , ,2' -"70#!-%,'8',%2&##620 !#**3* 0"-+ ',-$0.[U'1 *#2-

',&' '2.0-*'$#0 2'-,Q+'%0 2'-, ,"1.0-32',%-$&3+ ,#,"-2&#*' *!#**1SYT*2&-3%&2&#+ ',

1'2#-$0.[U#6.0#11'-,'12&#4 1!3* 0#,"-2&#*'3+Q . 02$0-+2&# -4#+#,2'-,#"123"'#1Q'21

0-*#',4 1!3* 0 '-*-%7 ,"$3,!2'-,& 1,-2 ##,',4#12'% 2#"T

-3,"#012 ,"2&#0-*#-$0.[U',%*'- * 12-+ 4#11#*1 ,"'21#$$#!2-,23+-0

%0-52&Q5#',4#12'% 2#"0.[U$3,!2'-,',23+-0 ,%'-%#,#1'1 ,"#,"-2&#*' *!#** '-*-%7T

2,2#0#12',%*7Q5#- 1#04#".0-*-,%#"1304'4 *-$0.[UV V+'!# # 0',%-02&-2-.'! TXS%*'-+ 1

1!-+. 0#"2-5'*"V27.#+'!#Q 11-!' 2#"5'2&"#!0# 1#"23+-04#11#*.#0$31'-, ,"#,& ,!#"

*# ) %#',0.[UV V+'!#T0-,1'12#,25'2& 0-*#-$0.[U',-0!x 2',% ,%'-%#,#1'1Q5#$-3,"

2& 2),-!)"-5,-$0.[U',&' '2123 #$-0+ 2'-,Q "'-, ,"+'%0 2'-,-$#,"-2&#*' *!#**1in

vitroQ"3#2-"#$#!21',!72-1)#*#2 *0#V 00 ,%#+#,2 ,"*-11-$#,"-2&#*' * "�#,!#(3,!2'-,1T

2+.-02 ,2*7Q&'%&0.[U#6.0#11'-,',23+-04#11#*11'%,'$'! ,2*7!-00#* 2#"5'2&1&-02#0

1304'4 *', !*','! *!-&-02-$. 2'#,215'2&%0 "#222V2D 120-!72'!%*'-+ 1T*2-%#2�Q-30" 2

'+.*72& 20.[U'1 )#70#%3* 2-0-$%*'-+ ,%'-%#,#1'1 ,"4 1!3* 0$3,!2'-,2&0-3%&2&#

!-,20-*-$!#**V!#** ,"!#**V+ 20'6 "'-,T

MATERIALS & METHODS

Tissue Microarray and Image analysis

D 1!3* 0#6.0#11'-,-$0.[U5 1 , *78#"',2'113#+'!0- 00 71!-,2 ',',%

"3.*'! 2#2'113#!-0#1.#01 +.*#S++"' +#2#0-$TUW '-.1'#1-$&3+ ,*-5V%0 "#!F

%0 "#22 ,"&'%&V%0 "#!F%0 "#222 ,"2D%*'-+ 1-$4 0'-31&'12-*-%'! *13 27.#1Q ,"

,-,V+ *'%, ,2 0 ',2'113#%*'-1'1 ,",-0+ *%0#7 ,"5&'2#+ 22#031#" 1!-,20-*1

,3..*#+#,2 07 *#ST3+ ,2'113#5 1- 2 ',#"', + ,,#0!-+.*' ,25'2&2&#

.#!* 0 2'-,-$#*1',)'T&#2&'!1#4'#5"- 0"',5..1 * ..0-4#"2&#31#-$&3+ ,1 +.*#1

,". 02'!'. 2'-,-$. 2'#,21-!!300#" $2#0',$-0+#"!-,1#,2T

2++3,-&'12-!&#+'! *12 ',',%5 1.#0$-0+#" 1"#1!0' #"SZ31',% 0 '2

,2' -"72-0.[U)RR[URRQ2* 1,2' -"'#1Q,2-!)&-*+Q,5#"#, ,"U_U_V

"' +',- #,8'"',# 113 120 2#T&#$0#/3#,!7-$.-1'2'4#*712 ',#"4#11#*15 11!-0#"',

*',"#"$ 1&'-,-, 1! *#$0-+R2-TR-,-4#11#*112 ',#"QS-+',-0'27-$4#11#*112 ',#"QT-

+ (-0'27-$4#11#*112 ',#"T

Tumor cell and primary endothelial cell and pericytes culture

TVS$' 0-1 0!-+ Q"SXT(SR+#* ,-+ +#0'! ,7.#03*230#0-**#!2'-,Q

, 11 1QDQ5,4' 0,2 ," 0"1Q ," TXS%*'-+ !#**1)',"%'$2$0-+.0T #8 , $0 ,7Q

C2Q"3" .#12#,%',##0#"2-#6.0#11*3!'$#0 1#',&-31#& ,%et alTQ3,.3 *'1&#"5#0#

!3*230#"-,SRV!+!3*230#"'1',.3* #!!-_1 -"'$'#" %*# #"'3+'$##!&,-*-%'#1Q

0 0*1 "Q0Q5,13..*#+#,2#"5'2&SR$$#2 *! *$1#03+(0,R,'%+ V*"0'!&Q,2T-3'1Q FQ

5, 2UY-0 ,"W$0FT [W$ '0', &3+'"'$'#"!& + #0T

3+ ,.#0+ * '!0-4 1!3* 0,"-2&#*' *0#**1. 0RU"'-+#"'! *Q5..1 * Q

,5#"#,Q ,")0-+-0#**Q#'"#* #0%Q #0+ ,75#0#!3*230#"3.2-. 11 %#SW-,%#* 2',V!- 2#"

!3*230#"'1',,"-2&#*' *0#**" 1 * #"'3+5'2&$3**13..*#+#,21" V DTQ)0-+-0#** 2

UY-0 ,"W$0FT [W$ '0', &3+'"'$'#"!& + #0T3+ ,"0 ',)#0'!72#1")RU

"'-+#"'! *Q5..1 * Q,5#"#,5#0#!3*230#"-,!3*230#"'1',)#0'!72# #"'3+) U

"'-+#"'! * 2UY-0 ,"W$0FT [W$ '0', &3+'"'$'#"!& + #0T0#***',#15#0#,-2

32&#,2'! 2#" $2#0.30!& 1#"SXQTVS-0 $2#020 ,1$#0$0-+-2�* -0 2-0'#1 TXS 32

5#0#0-32',#*72#12#",#% 2'4#$-0+7!-.* 1+ 31',%2&# 7!-.* 1+ .#2#!2'-,M'2-,8 T

Mice

0.[UV V+'!#SU5#0# 0#"',&-31#T0WY"* X5'*"V27.#+'!#5#0#.30!& 1#"

$0-+ !-,'! ""-+&-*2Q.#,+ 0)T** ,'+ *5-0)5 1.#0$-0+#" !!-0"',%2-2&#

%3'"#*',#1$-0 ,'+ *#6.#0'+#,2 2'-, ,"5#*$ 0#.0-4'"#" 75..1 * 5,'4#01'27 ,"

..0-4#" 72&#5..1 * 0-3,270#%'-, *#2&'!1!-++'22##T

Tumor studies

TXS%*'-+ !#**1#6.0#11',%*3!'$#0 1#5#0#-02&-2-.'! **7',(#!2#"',2&# 0 ',

-$0.[UV V ,"5'*"V27.#0WY"* X+'!# ,"23+-0"#4#*-.+#,25 1+-,'2-0#" 7

'-*3+',#1!#,!#'+ %',% 1"#1!0' #"',,3..*#+#,2 07 2#0' *1 ," #2&-"1T

(-02&#1304'4 *123"7Q+'!#5#0#1 !0'$'!#" 7!#04'! *"'1*-! 2'-,5&#,2

1&-5#"+-0#2& ,SR$5#'%&2*-11Q ," 0 ',15#0##6!'1#"Q1, .V$0-8#,-,'1-.#,2 ,# "07V'!#

,"12-0#" 2VZR-0$-0$302� , *71'1T

-123"74 1!3* 0.#0$31'-,Q+'!#5#0#',(#!2#"5'2&SRR=*-$S+% +*(20V

* #*#"-0 '-2',V* #*#"*7!-.#01'!-,#1!3*#,23+*#!2',D#!2-0 -0 2-0'#1Q"30*',% +#Q0Q

5,',2&#2 '*4#',-02&#-0 '2 *.*#631 2" 7TSVTVT&#*#!2',5 1 **-5#"2-!'0!3* 2#$-0SRV

UR+',32#1Q $2#05&'!&2&#+'!#5#0#.#0$31#"5'2&)",$-**-5#" 7V$)(2&0-3%&2&#&# 02

3,"#0'1-$*30 ,#-0)#2 +',# 67* 8',# ,#12' T&# 0 ',15#0##6!'1#"Q$'6#"',V$)($-0

T&-301 ,""#&7"0 2#"',UR$13!0-1# )",-4#0,'%&2Q ,"#'2�#+ #""#"',F0007-+-3,2

'12-* 1Q 2# -0%Q,5#"#, ,"1, .V$0-8#,-,'1-.#,2 ,# "07V'!#$-0!07-1#!2'-,',%Q-0

"'0#!2*731#"$-01#!2'-,',%-, 4' 0 2-+#T

0.[UV V ,"5'*"V27.#0WY"* X+'!#5#0#13 !32 ,#-31*7',(#!2#"',2&#0'%&2

$* ,)5'2&SRRQRRR"SXT(SR-0WRRQRRRTVS!#**1',SRR=*.&-1.& 2#V 3$$#0#"1 *',#3,"#0

,#12' 7',& * 2'-,-$'1-$*30 ,# FTT3+-015#0#+# 130#" 7! *'.#01 ,"2&#4-*3+#

5 1! *!3* 2#" !!-0"',%2-2&#$-0+3* S23+-04-*3+#-RQWT#*#,%2'"2&TT2" 7SW

"SXT(SR-0" 7TRTVSQ+'!#5#0#1 !0'$'!#" 7!#04'! *"'1*-! 2'-, ,"23+-015#0#

#6!'1#"T&#5#2V5#'%&25 1"#2#0+',#" ,"23+-015#0#1, .V$0-8#,-,'1-.#,2 ,# "07V'!#

,"12-0#" 2VZR-0T

siRNA transfections

. 05#0#',!3 2#"5'2&1!0 + *#"!-,20-*1'C-01'C2-0.[U

10.[US ,"10SQSWQ(*#6'3 #QQ' %#,Q'*"#,Q #0+ ,7 2 !-,!#,20 2'-,-$T, ',

+'6230#-$TR$F.2' #+'$##!&,-*-%'#1',#,"-2&#*' *!#**+#"'3+13..*#+#,2#"5'2&

UR=* +*C' R*'.-$#!2 +',#'$##!&,-*-%'#1$-0VVX&-301Q $2#05&'!&2&#+#"'3+5 1

0#.* !#"5'2&$0#1&+#"'3+T6.#0'+#,215#0#.#0$-0+#" 2" 7TVU $2#01'C20 ,1$#!2'-,T

Lentivirus construction, production and HDMEC infection

!.C1#/3#,!#1$-0&3+ ,0.[U!V0.[U ,"0.[U5'2& W +',- !'"1

XRXVXSR"#*#2'-,0.[UV072-W Q0#.-02#"+-#1', ',"',%1'2#5#0#"#1'%,#" ,"+32 ,2

. 05#0#- 2 ',#" 1"#1!0' #"',,3..*#+#,2 07 2#0' * ," #2&-"1T

Tube formation assay

1'CV20 ,1$#!2#". 05#0#1##"#"-,2- U.!-** %#,VS%#* ,"12'+3* 2#"

5'2&WR,% +*D ($-0TV&-301 1"#1!0' #"S[T&#0#13*2',%23 #15#0#$'6#"',',!('6Q

12 ',#"5'2&#6 1#"V* #*#".& **-'"','$##!&,-*-%'#1 ," , *78#" 7!-,$-! *

+'!0-1!-.7T&#2-2 *.& **-'"',V.-1'2'4#23 # 0# 5 1/3 ,2'$'#"31',%2+ %#$ ,",-0+ *'8#"

2-2&#,3+ #0-$-#!&12V.-1'2'4#,3!*#'T

"1#"V20 ,1$#!2#". 05#0#20 ,1$#!2#"5'2&1!0 + *#"1'C ,"+'6#"',

23 #$-0+ 2'-, 11 75'2&3,* #*#"!#**12& 25#0#20 ,1$#!2#"5'2&1'C2-0.[UQ ,"4'!#

4#01 T3 #15#0#4'13 *'8#" 712 ',',%5'2&*#6 (*3-0XVYV* #*#".& **-'"', ," , *78#"

7!-,$-! *+'!0-1!-.7T&#. 0 '*'272-$-0+*3+','8#"23 #V*')#1203!230#15 1 11#11#"

1.0#4'-31*7"#1!0' #"TRT,##,3..*#+#,2 07 2#0' *1 ," #2&-"1.

Proliferation and apoptosis

)0-*'$#0 2'-,-$1'C20 ,1$#!2#". 0!3*230#"'," ,"")!3*230#"',

!-,"'2'-,#"+#"' "#0'4#"$0-+1'CV20 ,1$#!2#". 05 1 11#11#" 7!#**!-3,2',%

"30',% 2'+#.#0'-"-$V" 71T.-.2-1'1',1'CV20 ,1$#!2#". 05 1 11#11#" 7

"#2#!2'-,-$!*# 4#"! 1. 1#VU !2'4'2731',% !-*-0'+#20'! 11 7 1"#1!0' #"',

Migration assay

0-,$*3#,21'CV20 ,1$#!2#". 05#0#1#03+12 04#"'," 5'2&S$(0,T

,!0 .#V5-3,"15#0#.0-"3!#"-,2&#!#**+-,-* 7#0 ,"5-3,"0#. '05 1 , *78#"3,"#0

SWR6+ %,'$'! 2'-,)* ,.-,STR6- (#!2'4#3.2-Z&-301Q31',% 12#0#-+'!0-1!-.#,2#F

.'1!-4#07TDSTQ#'11 22 !&#"2- "'%'2 *! +#0 6'-0 +Q#'11T*2#0, 2'4#*7Q 2V&-301

$2#05-3,"',%Q2&#!#**15#0#$'6#"$-0'++3,-* #*',% ,"!-,$-! *+'!0-1!-.7 , *71'1 1

"#1!0' #"',2&#,3..*#+#,2 07 2#0' *1 ," #2&-"1T

Adhesion assay

1'CV20 ,1$#!2#". 05#0#1##"#"-32'," 13..*#+#,2#"5'2&RTT$",

2SRQRRR!#**1.#05#**', %#* 2',V!- 2#"[XV5#**.* 2#T$2#0UR+',Q2&#5#**15#0#5 1&#"

5'2&)",2-0#+-4#,-,V "�#,2!#**1 ,"2&#,3+ #0-$ "�',%!#**15 1/3 ,2'$'#"31',%

2&#07Q5CV !#**/3 ,2'$'! 2'-,)'2'$##!&,-*-%'#1T(*3-0#1!#,!#5'2& ,#6!'2 2'-, 2

VZR,+ ,"#+'11'-, 2WTR,+5 1+# 130#"31',% ! ** !D'!2-0$*3-0#1!#,!#.* 2#0# "#0T

*2#0, 2'4#*7Q 2UR+', $2#01##"',%Q!#**15#0#$'6#"5'2&V$)($-0'++3,-* #*',% ,"

!-,$-! *+'!0-1!-.7T

Western blot and co-immunoprecipitation

)0-2#',*#4#*-$0.[U ,"+-#1',5 1 11#11#" 75#12#0, *-2 ,"!-V

'++3,-.0#!'.'2 2'-,$-00.[U5 1.#0$-0+#"5'2&2&#)'#0!#!-V'++3,-.0#!'.'2 2'-,)'2

�+-,!'#,2'$'! 1"#1!0' #"',2&#,3..*#+#,2 07 2#0' *1 ," #2&-"1T

RNA extraction and qPCR

C$0-+1'CV20# 2#"#,"-2&#*' *!#**1in vitro5 1#620 !2#"31',%2&#C# 17

)*31 ','M'2Q' %#,T-2 *C5 120 ,1!0' #"31',%,3.#01!0'.22220#4#01#20 ,1!0'.2 1#',

TRV=*2-2 *4-*3+#!-,2 ',',%TWR,%-$0 ,"-+ +#01 ,"VR3,'21-$C1#F5',&' '2-0

**$0-+'$##!&,-*-%'#1T+C#6.0#11'-,-$0.[U5 1/3 ,2'$'#"0#* 2'4#2-2&#&-31#

)##.',%%#,#) 70# *V2'+#)0',"3.*'! 2#0# !2'-,1.#01 +.*#5'2&RTTW= $-05 0" ,"

0#4#01#.0'+#0',,S" 0##,)0 12#0 '6'$##!&,-*-%'#1T

Immunofluorescent staining of HDMEC

1'CV20 ,1$#!2#". 05#0#'++3,-$*3-0#1!#,2*712 ',#"$-0 -*%',V[Y2-

4'13 *'8#2&# -*%'!-+.*#6 ,"$-0DV0 "�',2-4'13 *'8#2&#!#**V!#**(3,!2'-,1 1"#1!0' #"

',2&#,3..*#+#,2 07 2#0' *1 ," #2&-"1T

Immunofluorescent and immunohistochemical staining of tumor sections

007-1#!2'-,1 ,"4' 0 2-+#1#!2'-,1$0-+ TXS%*'-+ 1 ,"TVS$' 0-1 0!-+ 1

5#0#'++3,-$*3-0#1!#,2*712 ',#"$-00.USQ0.VWQ0.[UQ.-"-! *76',Q!*# 4#"! 1. 1#VUQ

,"'++3,-&'12-!&#+'! **712 ',#"$-00.[U 1"#1!0' #"',2&#

,3..*#+#,2 07 2#0' *1 ," #2&-"1T

Statistical analysis

,2 2'12'! *1'%,'$'! ,!#-$2&#- 1#04#""'$$#0#,!#15 1 ""0#11#" 712 2'12'! *

2#12131',% 0 .&) ")0'1+DXTRS 0 .&) ",-$25 0#Q, ,.'#%-Q0Q5,T

,304'4 *!304#15#0#.*-22#" !!-0"',%2-2&#M .* ,V #'#0+#2&-".0-"3!2V*'+'2

+#2&-" ,"2&#*-%V0 ,).0- '*'272#12 ,2#*V0-6#12'+ 2#"2&#.0-%,-12'!4 *3#-$2&#

0.[U#6.0#11'-,',2&#3,'4 0' 2# , *71'1T&#0-6.0-.-02'-, *& 8 0"+-"#*5 131#"2-

! *!3* 2#2&#'+. !2-$0.[U#6.0#11'-,',2&#+3*2'4 0' 2# , *71'1-$&'%&V%0 "#%*'-+ 1

2-%#2�5'2&#12 *'1&#".0-%,-12'!$ !2-01+ *'%, ,!7%0 "#Q. 2'#,2 %#Q#62#,2-$23+-0

0#1#!2'-, ,".-12-.#0 2'4#2� .7T&#*#4#*$-0!-,$-3,"#012- #0#+-4#"$0-+2&#+-"#*

5&#, "(312#"$-04 0' *#1 *0# "7',2&#+-"#*.V2-V0#+-4#5 11#22-1RTSTC 230 *

*-% 0'2&+*,!3+3* 2'4#& 8 0".*-215#0#+ "#2-!-,$'0+2&# 113+.2'-,-$2&#

.0-.-02'-, *& 8 0"$3,!2'-,1T&#1'%,'$'! ,2.0-%,-12'!$ !2-01',2&#12#.5'1#+-"#*5#0#

*1- , *71#" 1.0-"3!212-+','+'8#2&#.-11' '*'27-$',2#0 !2'-,T$ )Q4#01'-,SRTR,,

2,12'232#2,!TQ0 07QC-02&0 0-*', Q5,5 131#"$-012 12'! * , *71'1T

RESULTS

Vascular expression of CD93 correlates to tumor grade in human glioma

!#.0#4'-31*7"#+-,120 2#"2& 2&'%&4 1!3* 0"#,1'27Q4 1!3* 0 ,-0+ *'8 2'-,

,"$-0+ 2'-,-$%*-+#03*-'"+'!0-4 1!3* 0.0-*'$#0 2'-,1 0#!& 0 !2#0'12'!$# 230#1-$%0 "#2D

%*'-+ Q ,"2& 22# #00 ,24#11#*1.0-"3!#&'%&*#4#*1-$0.[U+C ,".0-2#',YT2,

-0"#02-#62#,"-30 , *71'1-$0.[U.0-2#',#6.0#11'-,',23+-04#11#*1', * 0%#0+ 2#0' *Q5#

1!-0#"2&#$0 !2'-,-$ *--"4#11#*112 ',',%.-1'2'4#$-00.[U',1#4#0 *23+-02'113#

+'!0- 00 712& 2!-,2 ',#" 2-2 *-$TUW '-.1'#1-$&3+ ,!-,20-* 0 ', ,"%0 "#22Q%0 "#222

,"%0 "#2D%*'-+ 2'113#1-$4 0'-31&'12-*-%'! *13 27.#1T&'1"#+-,120 2#"2& 2',YU$-$

2&#%0 "#2D%*'-+ 1 +.*#12&#+ (-0'27-$2&# *--"4#11#*112 ',#".-1'2'4#$-00.[U('%TSV

"T&#$0 !2'-,-$0.[UV.-1'2'4#4#11#*15 11'%,'$'! ,2*7*-5#0',0#1.#!2'4#*7%0 "#222 ,"

%0 "#22%*'-+ Q ,"#4#,$302�0#"3!#"',!-,20-* 0 ',1 +.*#1Q5�#UU$"'",-2"'1.* 7

,70.[U12 ',',%',2&#4 1!3* 230# 2 **T,2 ',',%-$* 0%#01#!2'-,1-$&3+ ,%*'- * 12-+

2'113#0#4# *#"2& 20.[UV.-1'2'4#4#11#*15#0#.0#1#,2 -2&5'2&',2&#!-0# ,"2&#',4 "',%

$0-,2-$2&#23+-0('%TS0T1"#+-,120 2#"',,3..*#+#,2 07 *#SQ2&#$0 !2'-,-$0.[UV

.-1'2'4#4#11#*15 11*'%&2*7&'%�',#.#,"7+-+ !-+. 0#"2--*'%- 120-!72-+ ,"

-*'%-"#,"0-%*'-+ Q5&'*#2&#* 0%#120.[UV.-1'2'4'275 1$-3,"',%*'-1 0!-+ ,"

%*'- * 12-+ T

CD93 deficiency impairs GL261 glioma and T241 fibrosarcoma growth in female mice

-',4#12'% 2#2&#0-*#-$0.[U',%*'-+ %0-52&Q5##+.*-7#"2&#+30',# TXS

%*'-+ +-"#*'+.* ,2#"-02&-2-.'! **7',2&#+-31# 0 ',T!#$'012!&#!)#"2&##6.0#11'-,

. 22#0,-$0.[U', TXS%*'-+ 7'++3,-&'12-!&#+'! * ,"'++3,-$*3-0#1!#,212 ',',%T

&'10#4# *#"2& 20.[U5 1+ ',*7#6.0#11#"',2&#4 1!3* 230#-$2&# TXS%*'-+ Q ,"2& 2

0.[U12 ',',%5 1+-0#',2#,1#',2&#23+-04#11#*12& ,',2&#4 1!3* 230#-$2&#1300-3,"',%

0 ',('%TTT -0#-4#0Q2&# TXS%*'-+ 4#11#*1"'1.* 7#" , ,-0+ *.&#,-27.#-$

+ *$-0+#"4#11#*1Q1'+'* 02-2& 2$-3,"',&3+ ,%*'- * 12-+ ('%TST2++3,-$*3-0#1!#,2

!-V12 ',',%5'2&0.US ,"0.VW"#+-,120 2#"2& 20.[U5 1&'%&*7#6.0#11#" 72&#

4 1!3* 230# ,",-2"#2#!2#"',',$'*20 2',%*#3)-!72#1-0 TXS23+-0!#**1('%TT"V0TC-0.[U

12 ',',%5 1"#2#!2#"', TXS23+-01',0.[UV V+'!#('%TT.V(T

!#'+.* ,2#" TXS!#**1#6.0#11',%*3!'$#0 1#12#0#-2 !2'! **7',2&# 0 ',1-$

$#+ *#0.[UV"#$'!'#,2 ,"5'*"V27.#+'!#Q ,".#0$-0+#" '-*3+',#1!#,!#'+ %',%2-+# 130#

23+-04-*3+# 2"'$$#0#,2" 71 $2#023+-0!#**',-!3* 2'-,T!&'*#2&#%*'-+ %0-52&!304#1

..# 0#"2-& 4# 1'+'* 01*-.#Q2&# 0 ',23+-01-$0.[UV V+'!#0# !&#" +# 130 *#1'8#

1SRRQRRR '-*3+',#1!#,!#3,'21-, 4#0 %#SS" 71* 2#02& ,2&# 0 ',23+-01-$5'*"V27.#

+'!#('%TT VT&'15 1 11-!' 2#"5'2& 1'%,'$'! ,2*7*-,%#01304'4 * $2#023+-0!#**

',(#!2'-,',0.[UV"#$'!'#,2+'!#!-+. 0#"2-5'*"V27.#+'!#('%TT2T

- , *78#5�0.[UV"#$'!'#,!7 $$#!21%0-52&-$-2�23+-027.#1 15#**Q

5##+.*-7#"2&#13 !32 ,#-31*7%0-5',%TVS$' 0-1 0!-+ ,""SXT(SR+#* ,-+ +-"#*1T

!#$-3,"2& 2TVS%0-52&5 11'%,'$'! ,2*70#"3!#"',$#+ *#0.[UV"#$'!'#,2+'!#!-+. 0#"

2-5'*"V27.#+'!#('%TT$T&'15 1!-,$'0+#" 72&#"'$$#0#,!#1',5#'%&2-$2&#23+-012 )#,

2" 7TR $2#023+-0!#**',(#!2'-,1('%TTMT2,!-,20 12Q"SX+#* ,-+ 23+-01%0#5#/3 **7

5#**',0.[UV V ,"5'*"V27.#+'!#('%T,SV"T2,2#0#12',%*7Q',+ *#0.[UV V+'!#Q5#"'",-2

"#2#!2 ,7"'$$#0#,!#',23+-0%0-52&',#'2�2&# TXS-02&#TVS23+-0+-"#*('%T,S0VT

Knockdown of CD93 inhibits formation of tube-like structures in vitro

-',4#12'% 2#2&#0-*#-$0.[U',#,"-2&#*' *!#** '-*-%7Q5#31#"C

',2#0$#0#,!#2-),-!)"-5,'21#6.0#11'-,',&3+ ,"#0+ *+'!0-4 1!3* 0#,"-2&#*' *!#**1

. 0in vitroT0 ,1$#!2'-,-$. 05'2&1'C2-0.[U0#"3!#"'21+C#6.0#11'-, 7

[WV[[$Q5&'!&0#13*2#"', ,#$$'!'#,2"-5,0#%3* 2'-,-$0.[U.0-2#',2& 2.#01'12#"$-0 2*# 12

2&0##" 71('%TUT

!#$'012',4#12'% 2#"'$0.[U& 1 0-*#',#,"-2&#*' *!#**.0-*'$#0 2'-,-01304'4 *Q

,"$-3,"2& 21'CV+#"' 2#"),-!)"-5,-$0.[U"'",-2 $$#!22&#.0-*'$#0 2'-,0 2#-0

.-.2-1'1-$. 0('%T,TV"T"#! 31#5#.0#4'-31*71&-5#"2& 20.[U+C'1

3.0#%3* 2#"5&#,. 0$-0+23 #V*')#1203!230#1-, U.V!-** %#,%#*',0#1.-,1#2-D (

YQ5#,#62',4#12'% 2#"&-51'CV+#"' 2#"),-!)"-5,-$0.[U $$#!212&#$-0+ 2'-,-$

2#23 #1T!&'*#!-,20-*V20 ,1$#!2#"!#**1$-0+#" ,#25-0)-$23 #V*')#1203!230#1',2&#

!-** %#,%#* $2#0TV&-301-$12'+3* 2'-,5'2&D (Q!#**12& 25#0#20 ,1$#!2#"5'2&1'C2-

0.[U*-122&#! . !'272-$-0+!-,2 !215'2&,#'%& -0',%!#**1 ,"#12 *'1&23 #1('%TU0V(T

Q3 ,2'$'! 2'-,-$2&#2-2 * 0# -$2&#23 3* 0,#25-0)Q,-0+ *'8#"2-!#**,3+ #0Q0#4# *#"2& 2

0.[U),-!)"-5,1'%,'$'! ,2*7'+. '0#"23 #$-0+ 2'-, 7. 0('%TU"T

,',!#2&##$$'!'#,!7-$23 3* 0,#25-0)$-0+ 2'-,'1 *1-',$*3#,!#" 72&#"#,1'27

-$2&#!#**1Q5# !!-3,2#"$-0"'$$#0#,!#1',!#**,3+ #0 7+'6',%!-,20-*V20 ,1$#!2#""1#"V

* #*#". 0-, SSS ," SST0 2'-5'2&3,* #*#". 02& 25#0#20 ,1$#!2#"5'2&1'C

2-0.[UT)& **-'"',* #*',%2-4'13 *'8#2&#3,* #*#"!#**1('%TU R"#.'!2#"',%0##,0#4# *#"

2& 2-,*72&#"1#"V* #*#"!-,20-*!#**15#0# *#2-. 02'!'. 2#',2&#,#25-0)-$23 #V$-0+',%

!#**1Q5&'*#3,* #*#"!#**1',5&'!&0.[U#6.0#11'-,5 1),-!)#""-5,"'",-22 )#. 02',

$-0+ 2'-,-$2#1203!230#1T!&#,2&#0#4#01##6.#0'+#,25 1.#0$-0+#"Q-,*72&#3,* #*#"

. 05'2&,-0+ **#4#*1-$0.[U2--). 02',2&#$-0+ 2'-,-$23 #V*')#1203!230#1('%TUT

&0-3%&"'1.#01'-,-$#,"-2&#*' *!#**15'2&', !-** %#,%#*Q5#$-3,"2& 2 *2&-3%&#,"-2&#*' *

!#**1$ '*#"2-+ )#*-,%23 3* 0,#25-0)1 $2#00.[U),-!)"-5,Q2&#1&-0223 #V*')#1203!230#1

2& 2$-0+#"!-,2 ',#"*3+#,1('%TU2V)T&'113%%#1212& 20.[U!-,20' 32#12- ,%'-%#,#1'1 7

.0-+-2',%23 3* 0+-0.&-%#,#1'1Q 322& 20.[U'1"'1.#,1 *#$-0*3+#,$-0+ 2'-,T

Knockdown of CD93 inhibits endothelial cell adhesion and migration in vitro

&#.0-!#11-$23 3* 0+-0.&-%#,#1'10#/3'0#1!#**12- "�#2-2&##620 !#**3* 0

+ 20'6Q+'%0 2# ,"2-#12 *'1&!#**V!#**!-,2 !215'2&-,# ,-2�T-',4#12'% 2#5&'!&-$2#

1.#!21 0#!-,20-**#" 70.[UQ5#$'012123"'#"5�0.[U),-!)"-5, $$#!212&#+'%0 2-07

! . !'27-$. 0', 1!0 2!& 11 7T!&'*#!-,20-*V20 ,1$#!2#". 0& "!-4#0#" 0-3,"

ZR$-$2&#5-3," 0# 2Z&-301 $2#02&#1!0 2!&Q. 020 ,1$#!2#"5'2&0.[U1'C& "

!-4#0#"-,*7 0-3,"WX$-$2&#5-3," 0# Q1&-5',%2& 20.[U),-!)"-5,',&' '21. 0

+'%0 2'-,('%TVV"T-"#2#0+',#'$2&#',&' '2'-,-$+'%0 2'-,3.-,0.[U),-!)"-5,5 1

0#* 2#"2-"#$#!21',!72-1)#*#2 *0# 00 ,%#+#,2Q5#12 ',#"2&#+'%0 2',%$0-,2',!-,20-* ,"

0.[U1'CV20# 2#"!#**15'2& , ,2' -"70#!-%,'8',%%-*%',V[Y2-4'13 *'8#2&# -*%'T&#

-0'#,2 2'-,-$2&# -*%'',0#* 2'-,2-2&#"'0#!2'-,-$+'%0 2'-,5 1 , *78#" 1 + 0)#0-$

!-00#!2!#**.-* 0'8 2'-,('%T,T0V.TST!#$-3,"2& 2',+-!)V20 ,1$#!2#" ,"!-,20-*1'CV

20 ,1$#!2#"!#**1Q2&#+ (-0'27-$#,"-2&#*' *!#**15#0#!-00#!2*7.-* 0'8#"Q5&'*#0.[U

),-!)"-5,0#13*2#"', 1'%,'$'! ,2',!0# 1#',2&#,3+ #0-$,-,V.-* 0'8#"!#**1 ,"

"'1-0% ,'8#"*# "',%#"%#-$2&#+'%0 2',%$0-,2('%TV0V T

!#2&#,2#12#"5�0.[U),-!)"-5, $$#!21#,"-2&#*' *!#**1 "'-,2-

%#* 2',V!- 2#"130$ !#T2++3,-$*3-0#1!#,212 ',',%-$!#**1UR+',32#1 $2#01##"',%0#4# *#"

2& 2),-!)"-5,-$0.[U0#13*2#"',+ 0)#"*7"'+','1&#"1.0# "',%-$#,"-2&#*' *!#**1('%T

VVMT0-,1'12#,25'2&2&'1Q0.[U),-!)"-5,1'%,'$'! ,2*70#"3!#"2&#,3+ #0-$ "�#,2

!#**1('%TVT12&#',20 !#**3* 0"-+ ',-$0.[U& 1 ##,"#+-,120 2#"2- ',"2-+-#1',Q

+#+ #0-$2&##80', 0 "'6', +-#1', $ +'*7-$.0-2#',12& 2',2#0 !25'2&!72-1)#*#2 *

!2',TTQ5#',4#12'% 2#"&-5),-!)"-5,-$0.[U $$#!212&#-0% ,'8 2'-,-$2&#!72-1)#*#2-,

7'++3,-$*3-0#1!#,2*7* #*',%!#**15'2&.& **-'"',T!&'*#2&#!-,20-*!#**1$-0+#" 0#%3* 0

+-,-* 7#0Q2&#!#**1',5&'!&0.[U5 1),-!)#""-5,"'1.* 7#"120-,%120#11$' #0$-0+ 2'-,

,"$-0+#"-,*7*'+'2#"!-,2 !215'2&-,# ,-2�('%TWVT2++3,-$*3-0#1!#,212 ',',%-$

DV! "�',!-,$'0+#"120')',%"#$#!21', "�#,1(3,!2'-,$-0+ 2'-, $2#00.[U),-!)"-5,

('%TW2V)T&#$-0+ 2'-,-$',2#0V#,"-2&#*' *(3,!2'-,15 10#12-0#" 7*#,2'4'0 *20 ,1$#!2'-,

5'2& 5'*"V27.#0.[U!-,1203!2Q 32,-2 720 ,1$#!2'-,5'2& +32 ,20.[U!-,1203!2* !)',%

2&#0#.-02#"+-#1', ',"',%1'2#072-W 0.[UT&'1'1!-,1'12#,25'2& 0-*#$-00.[U',

!-,20-**',%!72-1)#*#2 *-0% ,'8 2'-, 2*# 12. 02' **72&0-3%&',2#0 !2'-,5'2&+-#1',('%T,TV

Perfusion of glioma vasculature is reduced in CD93-deficient mice

-"#2#0+',#5& 23,"#0*'#12&#"#!0# 1#"23+-0%0-52&',0.[UV V+'!#Q5#

, *78#",#!0-1'1Q&7.-6' ," .-.2-1'1-$23+-0!#**1',TVS ," TXS23+-01T.#!0# 1#"

,#!0-1'15 1$-3,"', TXS23+-01Q 32,-2TVS23+-01Q',0.[UV V+'!#!-+. 0#"2-5'*"V

27.#+'!#Q5&'!&!-00#* 2#"2-2&#1+ **#023+-01'8#('%T,UVT!#,-2#" 20#,"2-5 0"1

',!0# 1#"&7.-6' Q 1 11#11#" 7 5VS12 ',',%Q', -2&TVS ," TXS23+-015&'*#

#,& ,!#" .-.2-1'15 1$-3,"-,*7',TVS23+-01',0.[UV V+'!#('%T,U2VT

"#! 31#-30" 2 13%%#12 0-*#$-00.[U',23 3*-+-0.&-%#,#1'1Q5#',4#12'% 2#"

5�0.[U"#$'!'#,!7 $$#!2#"23+-0 ,%'-%#,#1'1', TXS23+-01T,* 0%#"4#11#*15'2&

, ,-0+ *+-0.&-*-%75#0#$-3,"', TXS23+-01-$ -2&5'*"V27.# ,"0.[UV V+'!#Q ,"

,-"'$$#0#,!#1',4#11#*"#,1'27-04 1!3* 0+-0.&-*-%75#0#,-2#" 712#0#-*-%'! * , *71'1-$

4#11#* 0# ,""' +#2#0', TXS%*'-+ ('%TXV0Q('%T,V"V0Q,-0',TVS$' 0-1 0!-+ ('%T

,VT,'+'* 0*7QTVS ," TXS23+-04#11#*1',0.[UV V ,"5'*"V27.#+'!#5#0##/3 **7

!-4#0#" 7"#1+',V.-1'2'4#.#0'!72#1T0-,1'12#,25'2&2&'1Q#,"-2&#*' *V"#0'4#"1-*3 *#0.[U"'"

,-2 $$#!2in vitro .#0'!72#.0-*'$#0 2'-,('%T,V(V T

,',!#0.[U),-!)"-5, $$#!2#"#,"-2&#*' *.-* 0'8 2'-,5'2&0#1.#!22-2&#

"'0#!2'-,-$+'%0 2'-,('%TV0V Q5# , *78#".-* 0'8 2'-,-$23+-04#11#*1 712 ',',%$-0

.-"7! *76',*3+', *+ 0)#0',!-+ ', 2'-,5'2&!-** %#,2D 1#+#,2+#+ 0 ,#T!#$-3,"

2& 25&'*#4#11#*1',TVS23+-015#0#!-00#!2*7.-* 0'8#"', -2&0.[UV V ,"5'*"V27.#+'!#Q

TXS23+-04#11#*15#0#1203!230 **7 ,-0+ * ,"2&#.-* 0'8 2'-,5 1 2*# 12. 02' **7

"'103.2#"', -2&%#,-27.#1('%T,WVFT2,2#0#12',%*7Q2&0-3%&12 ',',%$-0#,"-%#,-312% Q5#

$-3," 120')',%',!0# 1#',4 1!3* 0.#0+# '*'27', TXS23+-01$0-+0.[UV V+'!#('%TX.V

(T)#0$31'-,-$%*'-+ V # 0',%+'!#5'2&*7!-.#01'!-,#1!3*#,23+2-+ 2-*#!2',0#4# *#"2& 2

2&#.#0!#,2 %#-$4#11#*12& 25#0#.#0$31#"5 11'%,'$'! ,2*70#"3!#"',0.[UV V+ *# ,"

$#+ *#+'!#('%TX VT2+.-02 ,22-,-2#'12& 2#4#,4#11#*12& 21&-5#"-,*71+ **20 !#1-$

*#!2',V.-1'2'4'27('%TX2VM5#0#!-,1'"#0#" 1.#0$31#"',2&#/3 ,2'$'! 2'-,T#4#11#*1

!-,12'232#"2&#+ (-0'27-$.#0$31#" *--"4#11#*1',0.[UV V+'!#Q ,"!0# 2#" 120-,%!-,20 12

5'2&2&#5#**V.#0$31#"4#11#*1',5'*"V27.#+'!#2& 21&-5#"2&#.0#1#,!#-$(20V*#!2', *-,%

2&#5&-*#4 1!3* 0*',',%('%TX(VT#" 2 "#+-,120 2#2& 20.[U.0-+-2#1$-0+ 2'-,-$

$3,!2'-, *4 1!3* 230#',+-31#%*'-+ T

Tumor vascular expression of CD93 is indicative of poor survival for patients with high-

grade astrocytic gliomas

.3#2-2&#"'$$#0#,!#',%*'-+ %0-52& ,"23+-04 1!3* 0.#0$31'-, #25##,

0.[UV V ,"5'*"V27.#+'!# ,"2&#120')',%#$$#!21-$0.[U),-!)"-5,-,2&##,"-2&#*' *

!72-1)#*#2 *0#V 00 ,%#+#,2Q5# , *78#"'$#6.0#11'-,-$0.[U',23+-04#11#*1!-00#* 2#"2-

1304'4 *T(-02&'1 , *71'1Q5#',!*3"#"2&#&'%&V%0 "# 120-!72'!23+-012& 25#0#. 02-$

.0#4'-31*7"#1!0' #"!*','! *!-&-02TUT'%&V%0 "##.#,"7+-+ 1Q%*'-1 0!-+ 1 ,"

, .* 12'!-*'%-"#,"0-%*'-+ 15#0##6!*3"#"$0-+2&'1 , *71'1T) 2'#,215#0#"'!&-2-+'8#"

',2-&'%&-0*-54 1!3* 00.[UV#6.0#11',%%0-3.1 !!-0"',%2-2&# 4#0 %#1-$2&#0.[U12 ',',%

',2&#%*'-+ 2'113#+'!0- 00 71('%30#SQ,3..* *#ST&#M .* ,V #'#01304'4 *!304#

0#4# *#"2& 2. 2'#,215'2&!*# 00.[U#6.0#11'-,',2&#+ (-0'27-$2&#'023+-04 1!3* 230#

0.[U1!-0#3STW& " 1'%,'$'! ,2*71&-02#01304'4 *2& ,. 2'#,212& 2#6.0#11#"0.[U',2&#

+',-0'27-$4#11#*10.[U1!-0#2STR('%TYQ.-RTRVSYQ-%V ,)2#12T0.[U#6.0#11'-,5 1

'"#,2'$'#" 1 ,',"#.#,"#,2.0#"'!2-0-$1304'4 *',2&#+3*2'4 0' 2# , *71'1.-RTRVY[Q0-6

.0-.-02'-, *& 8 0"Q2-%#2�5'2&#12 *'1&#"!*','! *.0-%,-12'!$ !2-01$-02#23+-01T

2,2#0#12',%*7Q5&'*#5#$-3," 1'%,'$'! ,2',!0# 1#',0.UV 0# ',23+-015'2& &'%&

#6.0#11'-,-$0.[U('%TY"Q0.UV 0# per se"'",-2!-00#* 2#2-',!0# 1#". 2'#,21304'4 *

.-RTWRRXQ0-60#%0#11'-,+-"#*T&'1',"'! 2#12& 2&'%&0.[U#6.0#11'-, ," *2#0 2'-,-$2&#

4 1!3* 0.&#,-27.#'1 11-!' 2#"5'2&.--01304'4 *',2#23+-01Q!-,1'12#,25'2&2&#

.0-.-1#" '-*-%'! *0-*#-$0.[U',2&#23+-04 1!3* 230#-$&3+ ,%*'-+ 1T

DISCUSSION

62#,1'4# ,%'-%#,#1'1 ,"+ 0)#"*7 ,-0+ * *--"4#11#*1 0# & **+ 0)-$

%0 "#2D%*'-+ ,"2&# ,-0+ *Q+ *$3,!2'-,',%4#11#*1!-,20' 32#2-2&#1#4#0'27-$2&#

"'1# 1#T)0#4'-31*7Q5#0#.-02#"2& 22#. 2&-*-%'! **7 *2#0#"4#11#*1',%0 "#2D%*'-+

#6.0#11&'%&*#4#*1-$0.[UQ 20 ,1+#+ 0 ,#.0-2#',$0-+2&#0V27.#13.#0$ +'*7-$*#!2',1

5'2& ! *!'3+V"#.#,"#,2! 0 -&7"0 2#V ',"',%"-+ ',YT0.[U5 1','2' **713%%#12#"2-

.* 7 0-*#', ,%'-%#,#1'1"3#2-'21&'%&#6.0#11'-,', 0# 1-$ *--"4#11#*0#+-"#*',%',2&#

"#4#*-.',%4 1!3* 230#-$+-31##+ 07-1TVT!#,-5"#+-,120 2#2& 20.[U& 1 $3,!2'-, *

0-*#',+-"3* 2',% ,%'-%#,#1'1 ,"4 1!3* 0$3,!2'-,',%*'-+ Q ,"2& 2&'%&*#4#*-$0.[U

.0-2#',!-00#* 2#15'2&1&-02#01304'4 *',. 2'#,215'2&&'%&V%0 "# 120-!72'!%*'-+ 1T

2,*',#5'2&-30','2' *$',"',%-,#*#4 2#"0.[U#6.0#11'-,',%*'- * 12-+

4 1!3* 230#Q 13 1#/3#,2123"7'"#,2'$'#"0.[U 1-,#-$2&#2-.%#,#1-$ !-0#&3+ ,.0'+ 07

23+-0 ,%'-%#,#1'11'%, 230#Q&'%&*7#6.0#11#"',&# " ,",#!)1/3 +-31!#**! 0!',-+ 1Q

0# 12! ,!#01 ,"!*# 0!#**0#, *!#**! 0!',-+ 1YQSWT#123"'#1 0#2&#$'012-,#12-

0#.-020.[U 1 23+-04 1!3* 0+ 0)#0T&#-2� 0-3.R2D+#+ #01-$2&#0V27.#*#!2',

13.#0$ +'*7Q13!& 12&0-+ -+-"3*', ,"#,"-1' *', *1-),-5, 123+-0#,"-2&#*' *+ 0)#0

SQ& 4# *0# "7',# 0*'#0123"'#1 ##,0#.-02#"2- #&'%&*7#6.0#11#"',+ *'%, ,223+-0

4 1!3* 230#Q',!*3"',% 0 ',23+-01ZQTWVTYT

&#&'%&*#4#*-$0.[U#6.0#11'-, ,"'21!-00#* 2'-,5'2& .--01304'4 *-32!-+#

',%*'- * 12-+ . 2'#,215 00 ,2123"'#1 '+#" 23,"#012 ,"',%2&##$$#!2-$0.[U-,%*'-+

"#4#*-.+#,2T-2&'1#,"Q5##+.*-7#"2&# TXS+-31#%*'-+ +-"#*Q5&'!&'1 ,#6!#**#,2

+-"#*$-0-30.30.-1#Q 1'214 1!3* 0.&#,-27.# ,"0.[U#6.0#11'-,. 22#0,+'+'!2& 2-$

&3+ ,%*'- * 12-+ T!#1&-5#"2& 22&# 1#,!#-$0.[U',$#+ *#+'!#*# "12-"#* 7#"

-32%0-52&-$ TXS%*'-+ ,"'+.0-4#"1304'4 *T&'15 1 11-!' 2#"5'2& 120')',%*7

',!0# 1#".#0+# '*'27 ," 0#"3!#".#0$31'-,-$2&#%*'-+ 4 1!3* 230#',0.[UV"#$'!'#,2

+'!#T-%#2�5'2&-30in vitro- 1#04 2'-,12& 20.[U'1#11#,2' *$-02&##12 *'1&+#,2-$

#,"-2&#*' *!#**V!#** ,"!#**V+ 20'6!-,2 !21Q+'%0 2'-, ,"23 3* 0+-0.&-%#,#1'1Q2#" 2

13%%#122& 20.[U.0-+-2#12&#$-0+ 2'-,-$5#**V$3,!2'-,',%23+-04#11#*1TF30- 1#04 2'-,1

$302�+-0#13%%#122& 2"#$#!21',',2#0V#,"-2&#*' *(3,!2'-,1 ,"',!0# 1#".#0+# '*'27',2&#

1#,!#-$0.[UQ0 2�2& ,* !)-$*3+#,$-0+ 2'-,Q3,"#0*'#'21#$$#!2-,4 1!3* 0.#0$31'-,T

F30" 2 0#',*',#5'2& 0#!#,2*7.3 *'1&#"123"7 7F0* ,"','et alTQ2& 20#.-02#"2& 2

+-,-!*-, * ,2' -"72-0.[U'1 *#2-',&' '22&#$-0+ 2'-,-$4#11#*V*')#1203!230#1 7. 0

-,+ 20'%#*in vitro ,"in vivoQ13%%#12',%2& 2 ,2' -"72 0%#2',%-$0.[U+ 7',&' '223+-0

,%'-%#,#1'1SYT,'+'* 02--30123"'#1Q2&# 32&-01 *1-1&-5#"2& 20.[U1'*#,!',% 7

*#,2'4'0 *V+#"' 2#"#6.0#11'-,-$ 1+ **& '0.',C'+. '01&3+ ,#,"-2&#*' *!#**+'%0 2'-,

,"1.0-32',%in vitroT-5#4#0Q5&'*#F0* ,"','et alT"#+-,120 2#"2& 21&CV+#"' 2#"

),-!)"-5,-$0.[U',&' '2#".0-*'$#0 2'-,-$5D0Q5#!-3*",-2!-,$'0+2& 2',-30. 0T

*2-%#2�Q2#" 2 13%%#122& 20.[U'1 .-2#,2' *2 0%#2$-0 ,2' ,%'-%#,'!2� .7$-0

! ,!#0T

22'1,-2),-5,5�2&##620 !#**3* 0CV2#0+', *"-+ ',-$0.[U ',"12-

-2�+-*#!3*#1T*2&-3%&0.[U5 1-,!#2&-3%&22- #2&#0#!#.2-0$-0!-+.*#+#,2

!-+.-,#,20S/SRQ+-0#0#!#,2123"'#1"#+-,120 2#"2& 20S/V+#"' 2#".& %-!72-1'1"-#1

,-2"#.#,"-,0.[USU ,"2& 20.[U"-#1,-2 ',"0S/TZQT[T&#',20 !#**3* 00V2#0+', *

"-+ ',-$0.[U',2#0 !215'2&+-#1',Q +#+ #0-$2&# #80', 0 "'6', +-#1',$ +'*7-$

.0-2#',12& 2! ,*',)0.[U2-2&#!72-1)#*#2-,T,3!&*',) %#-$20 ,1+#+ 0 ,#.0-2#',12-

!72-1)#*#2 * !2',& 1 ##,1&-5,2-!-,20' 32#2- 0#"'120' 32'-,-$2&# !2',!72-1)#*#2-,2& 2

'1#11#,2' *$-0+'%0 2'-, ," "'-,URT&'1!-3*"#6.* ',2&#0#"3!2'-,',!#**+'%0 2'-,

," "'-,Q ,"2&# ,-0+ *!72-1)#*#2 *.&#,-27.#',#,"-2&#*' *!#**13.-,0.[U

),-!)"-5,T,'+'* 0*7Q ,-2� 0-3.R2D+#+ #0-$2&#0V27.#*#!2',13.#0$ +'*7Q

2&0-+ -+-"3*',Q!-,20-*1+-0.&-*-%7 ,"+'%0 2'-,-$#.'2&#*' *!#**14' 2&# .0-2#',

#80',UST&#*',) %#-$0.[U2-2&# !2',!72-1)#*#2-,'1 *1-#11#,2' *$-0'21.0#4'-31*7

"#1!0' #"0-*#',.& %-!72-1'1TTQUTT)0#4'-31123"'#1& 4#.-',2#"2- 0-*#$-00.[U',

*#3)-!72#',$'*20 2'-,',4 0'-31',$* ++ 2-07+-"#*1UUQUVT-5#4#0Q5#"'",-2"#2#!2 ,7

"'$$#0#,!#',*#3)-!72#',$'*20 2'-,',2- TXS ,"TVS23+-01 #25##,0.[UV"#$'!'#,2 ,"

5'*"V27.#+'!#('%T,V.VT&'113%%#1212& 20.[UV#6.0#11',%*#3)-!72#1"-,-2 $$#!2 TXS

.0-%0#11'-,Q!-,1'12#,25'2&0.[U #',%#6.0#11#"#6!*31'4#*7 723+-04#11#*1',2&'1+-"#*T

,',2#0#12',%- 1#04 2'-,'12& 22&# 1#,!#-$0.[U"#* 7#"23+-0%0-52&-,*7

',$#+ *#+'!#Q -2&', TXS%*'-+ 1 ,"TVS$' 0-1 0!-+ 1T10.[U'1+ ',*74 1!3* 0V

#6.0#11#"Q2&'1.-',212- 1#6"'$$#0#,!##'2�',2&#4 1!3* 230#-0',23+-0%0-52&',2#

+-31#+-"#*1T,#6&-0+-,#1& 4# ##,"#+-,120 2#"2- $$#!2#,"-2&#*' *!#** '-*-%7Q

,%'-%#,#1'1Q *--"4#11#*$3,!2'-, ,"2&#',!'"#,!# ,". 2&-*-%7-$! 0"'-4 1!3* 0"'1# 1#1

UWVUYT -0#-4#0Q! ,!#0131!#.2' '*'27'1"'$$#0#,2 #25##,2&#25-1#6#1$-0+ ,727.#1-$

! ,!#0UZQ ,"%*'- * 12-+ 1 0#VR$+-0#!-++-,',+#,2& ,',5-+#,SQU[TF$,-2#Q

&-5#4#0Q'12&#- 1#04 2'-,2& 2#4#,2&-3%&2&# 1#,!#-$0.[U"'",-2 $$#!223+-0%0-52&

1.##"',+ *#+'!#Q'2'+. '0#".#0$31'-,-$2&#%*'-+ 4 1!3* 230#',+ *#+'!##/3 **75#** 1

',$#+ *#+'!#T&'113%%#1212& 20.[U!-,20-*14 1!3* 0$3,!2'-, -2&',+ *#1 ,"$#+ *#1T

+-,%&3+ ,%*'-+ . 2'#,21Q0.[U5 1#6.0#11#"2- ,#/3 *#62#,2',23+-04#11#*1-$+#,

,"5-+#,Q ,"2&#!-00#* 2'-,2-.--01304'4 *5 1-,*71'%,'$'! ,25&#, -2&+#, ,"

5-+#,5#0#',!*3"#"',2&#1304'4 * , *71'1('%T,XV.T

*2-%#2�Q-30" 2 '+.*72& 20.[U'1 )#70#%3* 2-0-$%*'-+ ,%'-%#,#1'1T

&0-3%&2&#!-,20-*-$!#**V!#** ,"!#**V+ 20'6 "'-,Q0.[U!-,20-*14#11#* 0!&'2#!230#

"30',%. 2&-*-%'! * ,%'-%#,#1'1 ,".0-+-2#123+-0.0-%0#11'-, 7#,& ,!',%2&#$-0+ 2'-,

-$5#**V$3,!2'-,',%23+-04#11#*1T12&#0.[U#6.0#11'-,*#4#*!-00#* 2#15'2&1304'4 *-$

. 2'#,215'2&&'%&V%0 "# 120-!72'!%*'-+ 1Q$3230#123"'#11&-3*"$-!31-,#4 *3 2',%'21

.-2#,2' * 12� .#32'!2 0%#2$-0 ,2' ,%'-%#,'!2� .7-0 1 '-+ 0)#0$-0%*'- * 12-+

.0-%0#11'-,T

Acknowledgements

2+ %',%5 1.#0$-0+#"5'2&#/3'.+#,2+ ',2 ',#" 72&#,!'#,!#$-0'$# "'-D'1

)* 2$-0+Q5..1 * T!#2& ,).0T '!& #*"#0%/4'12 ,".0T,'+-,)+ ,Q.#. 02+#,2-$

"'-*-%7QF,!-*-%7 ," "' 2'-,,!'#,!#Q5..1 * 5,'4#01'27Q$-0 !!#112-2'113#

+'!0- 00 71-$&'%&V%0 "#%*'-+ . 2'#,21T

REFERENCES

1. Wen PY, Kesari S. Malignant gliomas in adults. N Engl J Med 2008;359(5):492-507. 2. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, et al. The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol 2007;114(2):97- 109. 3. Jain RK, di Tomaso E, Duda DG, Loeffler JS, Sorensen AG, Batchelor TT. Angiogenesis in brain tumours. Nat Rev Neurosci 2007;8(8):610-22. 4. Jansen M, de Witt Hamer PC, Witmer AN, Troost D, van Noorden CJ. Current perspectives on antiangiogenesis strategies in the treatment of malignant gliomas. Brain Res Brain Res Rev 2004;45(3):143-63. 5. Wong ML, Prawira A, Kaye AH, Hovens CM. Tumour angiogenesis: its mechanism and therapeutic implications in malignant gliomas. J Clin Neurosci 2009;16(9):1119-30. 6. Langenkamp E, Molema G. Microvascular endothelial cell heterogeneity: general concepts and pharmacological consequences for anti-angiogenic therapy of cancer. Cell Tissue Res 2009;335(1):205-22. 7. Dieterich LC, Mellberg S, Langenkamp E, Zhang L, Zieba A, Salomaki H, et al. Transcriptional profiling of human glioblastoma vessels indicates a key role of VEGF-A and TGF-beta2 in vascular abnormalization. The Journal of pathology 2012. 8. St.Croix B, Rago C, Velculescu V, Traverso G, Romans KE, Montgomery E, et al. Genes expressed in human tumor endothelium. Science 2000/8/18;289(5482):1197-202.

9. Langenkamp E, Vom Hagen FM, Zwiers PJ, Moorlag HE, Schouten JP, Hammes HP, et al. Tumor Vascular Morphology Undergoes Dramatic Changes during Outgrowth of B16 Melanoma While Proangiogenic Gene Expression Remains Unchanged. ISRN Oncol 2011;2011:409308. 10. Nepomuceno RR, Henschen-Edman AH, Burgess WH, Tenner AJ. cDNA cloning and primary structure analysis of C1qR(P), the human C1q/MBL/SPA receptor that mediates enhanced phagocytosis in vitro. Immunity 1997;6(2):119-29. 11. Bohlson SS, Silva R, Fonseca MI, Tenner AJ. CD93 is rapidly shed from the surface of human myeloid cells and the soluble form is detected in human plasma. J Immunol 2005;175(2):1239-47. 12. Greenlee MC, Sullivan SA, Bohlson SS. Detection and characterization of soluble CD93 released during inflammation. Inflammation research : official journal of the European Histamine Research Society [et al] 2009;58(12):909-19. 13. Norsworthy PJ, Fossati-Jimack L, Cortes-Hernandez J, Taylor PR, Bygrave AE, Thompson RD, et al. Murine CD93 (C1qRp) contributes to the removal of apoptotic cells in vivo but is not required for C1q-mediated enhancement of phagocytosis. Journal of immunology 2004;172(6):3406-14. 14. Chevrier S, Genton C, Kallies A, Karnowski A, Otten LA, Malissen B, et al. CD93 is required for maintenance of antibody secretion and persistence of plasma cells in the bone marrow niche. Proceedings of the National Academy of Sciences of the United States of America 2009;106(10):3895-900. 15. Masiero M, Simoes FC, Han HD, Snell C, Peterkin T, Bridges E, et al. A Core Human Primary Tumor Angiogenesis Signature Identifies the Endothelial Orphan Receptor ELTD1 as a Key Regulator of Angiogenesis. Cancer cell 2013;24(2):229-41. 16. Kao YC, Jiang SJ, Pan WA, Wang KC, Chen PK, Wei HJ, et al. The epidermal growth factor-like domain of CD93 is a potent angiogenic factor. PloS one 2012;7(12):e51647. 17. Orlandini M, Galvagni F, Bardelli M, Rocchigiani M, Lentucci C, Anselmi F, et al. The characterization of a novel monoclonal antibody against CD93 unveils a new antiangiogenic target. Oncotarget 2014;5(9):2750-60. 18. Uhlen M, Oksvold P, Fagerberg L, Lundberg E, Jonasson K, Forsberg M, et al. Towards a knowledge-based Human Protein Atlas. Nature biotechnology 2010;28(12):1248-50. 19. Mellberg S, Dimberg A, Bahram F, Hayashi M, Rennel E, Ameur A, et al. Transcriptional profiling reveals a critical role for tyrosine phosphatase VE-PTP in regulation of VEGFR2 activity and endothelial cell morphogenesis. FASEB J 2009/5;23(5):1490-502. 20. Koh W, Stratman AN, Sacharidou A, Davis GE. In vitro three dimensional collagen matrix models of endothelial lumen formation during vasculogenesis and angiogenesis. Methods in enzymology 2008;443:83-101. 21. Bisel B, Calamai M, Vanzi F, Pavone FS. Decoupling polarization of the Golgi apparatus and GM1 in the plasma membrane. PLoS One 2013;8(12):e80446. 22. Zhang M, Bohlson SS, Dy M, Tenner AJ. Modulated interaction of the ERM protein, moesin, with CD93. Immunology 2005;115(1):63-73. 23. Elsir T, Edqvist PH, Carlson J, Ribom D, Bergqvist M, Ekman S, et al. A study of embryonic stem cell-related proteins in human astrocytomas: identification of Nanog as a predictor of survival. Int J Cancer 2014;134(5):1123-31. 24. Petrenko O, Beavis A, Klaine M, Kittappa R, Godin I, Lemischka IR. The molecular characterization of the fetal stem cell marker AA4. Immunity 1999;10(6):691-700. 25. Maruno M, Yoshimine T, Isaka T, Kuroda R, Ishii H, Hayakawa T. Expression of thrombomodulin in astrocytomas of various malignancy and in gliotic and normal brains. J Neurooncol 1994;19(2):155-60. 26. Carson-Walter EB, Winans BN, Whiteman MC, Liu Y, Jarvela S, Haapasalo H, et al. Characterization of TEM1/endosialin in human and murine brain tumors. BMC Cancer 2009;9:417.

27. Maia M, DeVriese A, Janssens T, Moons M, Lories RJ, Tavernier J, et al. CD248 facilitates tumor growth via its cytoplasmic domain. BMC Cancer 2011;11:162. 28. McGreal EP, Ikewaki N, Akatsu H, Morgan BP, Gasque P. Human C1qRp is identical with CD93 and the mNI-11 antigen but does not bind C1q. J Immunol 2002;168(10):5222-32. 29. Greenlee-Wacker MC, Galvan MD, Bohlson SS. CD93: recent advances and implications in disease. Current drug targets 2012;13(3):411-20. 30. Tsukita S, Yonemura S. Cortical actin organization: lessons from ERM (ezrin/radixin/moesin) proteins. J Biol Chem 1999;274(49):34507-10. 31. Hsu YY, Shi GY, Kuo CH, Liu SL, Wu CM, Ma CY, et al. Thrombomodulin is an ezrin-interacting protein that controls epithelial morphology and promotes collective cell migration. Faseb J 2012;26(8):3440-52. 32. Greenlee MC, Sullivan SA, Bohlson SS. CD93 and related family members: their role in innate immunity. Current drug targets 2008;9(2):130-8. 33. Harhausen D, Prinz V, Ziegler G, Gertz K, Endres M, Lehrach H, et al. CD93/AA4.1: a novel regulator of inflammation in murine focal cerebral ischemia. Journal of immunology 2010;184(11):6407-17. 34. Greenlee-Wacker MC, Briseno C, Galvan M, Moriel G, Velazquez P, Bohlson SS. Membrane- associated CD93 regulates leukocyte migration and C1q-hemolytic activity during murine peritonitis. Journal of immunology 2011;187(6):3353-61. 35. Sieveking DP, Lim P, Chow RW, Dunn LL, Bao S, McGrath KC, et al. A sex-specific role for androgens in angiogenesis. J Exp Med 2010;207(2):345-52. 36. Sader MA, Celermajer DS. Endothelial function, vascular reactivity and gender differences in the cardiovascular system. Cardiovasc Res 2002;53(3):597-604. 37. Pepine CJ, Nichols WW, Pauly DF. Estrogen and different aspects of vascular disease in women and men. Circ Res 2006;99(5):459-61. 38. Dorak MT, Karpuzoglu E. Gender differences in cancer susceptibility: an inadequately addressed issue. Front Genet 2012;3:268. 39. Ricard D, Idbaih A, Ducray F, Lahutte M, Hoang-Xuan K, Delattre JY. Primary brain tumours in adults. Lancet 2012;379(9830):1984-96.

FIGURE LEGENDS

Figure 1: CD93 expression was increased in high-grade human glioma vasculature.

2++3,-&'12-!&#+'! *12 ',',%-$0.[U#6.0#11'-,A ,"1#+'V/3 ,2'2 2'4#1!-0',%-$2&#

$0 !2'-,-$.-1'2'4# *--"4#11#*1B',&3+ ,%*'-+ 2'113#+'!0- 00 71T2, 2-2 *-$TUW

&3+ ,%*'-+ ,",-,V+ *'%, ,2!-,20-* 0 ', '-.1'#1Q2&#$0#/3#,!7-$.-1'2'4#*712 ',',%

4#11#*15 11!-0#"-, 1! *#$0-+R2-TR-,-4#11#*112 ',#"QS-+',-0'27-$4#11#*112 ',#"Q

T-+ (-0'27-$4#11#*112 ',#"T#13*215#0# 4#0 %#" ,".*-22#" 1.#0!#,2 %#-$1 +.*#1.#0

1!-0#',,-,V+ *'%, ,2!-,20-* 0 ',[ '-.1'#1Q%0 "#22ZT '-.1'#1Q%0 "#222WV '-.1'#1Q

,"%0 "#2D1 +.*#1[R '-.1'#1Rp0RQRRRSQM031) *V! **'12#125'2&.3,,#_1.-122#12TC

0.[U"'120' 32'-,',%0 "#2D%*'-+ 4 1!3* 230#T&#*-5#0+ %,'$'! 2'-,'+ %#1&-51,-

%0-11"'$$#0#,!#',0.[U"'120' 32'-,5'2&',2&#23+-0 0# T.-22#"*',#0#.0#1#,212&#

+'%0 2',%$0-,2-$23+-0!#**1T

Figure 2: CD93 deficiency delayed GL261 glioma outgrowth in female mice.

A-CS0.[U'1.0-+',#,2*7#6.0#11#"',2&# ,-0+ * *--"4#11#*1-$ TXS%*'-+ Q7#2*#11

',2#,1#',2&#4 1!3* 230#-$2&#1300-3,"',% 0 ',R'++3,-&'12-!&#+'! *12 ',',%-$0.[UQ

,",-2',2&#23+-0V',$'*20 2',%*#3)-!72#1" ,"0R'++3,-$*3-0#1!#,2!-V12 ',',%-$0.[U

5'2&0.US ,"0.VWTD-FSD *'" 2'-,-$0.[U ,2' -"71.#!'$'!'27', TXS%*'-+ %0-5,',

0.[UV V+'!#T2++3,-&'12-!&#+'! *12 ',',%-$0.[U.T2++3,-$*3-0#1!#,2!-V12 ',',%-$

0.[U5'2&0.US ,"0.VW',0.[UV V+'!#V(TG-I: TXS%0-52&',0.[UV V ,"5'*"V27.#

$#+ *#0WY"* X+'!#T S"'-*3+',#1!#,!#V 1#"/3 ,2'$'! 2'-,-$23+-01'8# # ,(,.Q,-Y$-0

52 ,",-W$-00.[U)-Q2&'1',!*3"#125-52 ,"-,#0.[UV V+'!#5'2&-3223+-02 )#TS

D *3#1"#.'!22&#,3+ #0-$" 71 $2#05&'!&2&#23+-00# !&#" *3+',#1!#,!#',2#,1'27

1SRRQRRR5,'21 ," #! +#4'1' *#',2&#2D2,'+ %#0Q! *!3* 2#"$0-+2&#" 7-$',(#!2'-,T !&

4 *3#!-00#1.-,"12--,#+-31##p0RTRWQ ,,V!&'2,#752#12TISM .* ,V #'#01304'4 *!304#

-$+'!#',(#!2#"5'2& TXS%*'-+ !#**1Q+'!#5#0#1 !0'$'!#"5&#,213$$#0#"$0-++-0#

2& ,SW$*-11-$ -"75#'%&2Q0#* 2'4#2-2&# -"75#'%&2 22&#12 02-$2&##6.#0'+#,2

#p0RTRWQ,-SRVSUQ #& ,V"0#1*-5V!'*!-6-,2#12TJ-K:3+-04-*3+#1$R # ,(,.Q,-SW ,"

#,"V.-',25#'%&2MR # ,-$TVS$' 0-1 0!-+ %0-5',%13 !32 ,#-31*7',2&#0'%&2$* ,)-$

$#+ *#0.[UV V ,"5'*"V27.#+'!#T3+-01'8#15#0#+# 130#" 7! *'.#01#4#07TVU" 71Q ,"

23+-05#'%&215#0#"#2#0+',#" 2" 7TR#p0RTRWQ#p0RTRRRSQ ,,V!&'2,#752#12T

Figure 3: CD93 knockdown impaired tube formation of HDMEC.

AS&##6.0#11'-,-$0.[U',. 05 1#$$'!'#,2*7',&' '2#" 7C',2#0$#0#,!#TD *3#1

"#.'!2$-*"!& ,%#',0.[U+C#6.0#11'-,0#* 2'4#2-) 1 &-31#)##.',%%#,# # ,&

,.Q ," ,"1"#.'!20.[U.0-2#', ," !2', 1 &-31#)##.',%%#,# 2S ,"U" 71 $2#0

1'C20 ,1$#!2'-,TB-FS. 020 ,1$#!2#"5'2&1'C2-0.[U ,"!-,20-*V20 ,1$#!2#"!#**1

5#0# **-5#"2-$-0+23 #V*')#1203!230#1-, U.V!-** %#,%#*3,"#0D (12'+3* 2'-,$-0TV&Q

$'6#" ,"12 ',#"5'2&.& **-'"',T-2 *23 3* 0 0# 5 1/3 ,2'$'#" ," "(312#"2-2&#,3+ #0

-$-#!&12V.-1'2'4#,3!*#'2- !!-3,2$-0"'$$#0#,!#1',!#**,3+ #0"Q# !&4 *3#"#.'!212&#

+# ,-$UVV5#**1', 1#. 0 2##6.#0'+#,2R#p0RTRWQF,#V5 7CFD5'2&"-,$#00-,'

!-00#!2'-,$-0+3*2'.*#!-+. 0'1-,1TG-HS2, 1#. 0 2##6.#0'+#,2Q"1#"V* #*#". 0

5#0#20 ,1$#!2#"5'2&!-,20-*1'CQ ,"+'6#"', 23 #$-0+ 2'-, 11 75'2&3,* #*#"!#**1

2& 25#0#20 ,1$#!2#"5'2&1'C2-0.[UT2, ""'2'-,Q2&#0#4#01##6.#0'+#,25 1.#0$-0+#"T

)& **-'"',*#6 XVY* #*',%"#.'!2#"',%0##,5 131#"2-4'13 *'8#2&#3,* #*#"!#**1 1

5#**TF,*72&#!-,20-*V20 ,1$#!2#"!#**1!-,20' 32#"2-2&#$-0+ 2'-,-$23 #V*')#1203!230#1Q

5&'*#!#**120 ,1$#!2#"5'2&1'C2-0.[U"'",-22 )#. 02',2&#23 #V$-0+',%,#25-0)T I-PS

(-0+ 2'-,-$*3+','8#"23 #V*')#1203!230#1 7. 0T. 020 ,1$#!2#"5'2&1'C2-0.[U

,"!-,20-*V20 ,1$#!2#"!#**15#0# **-5#"2-$-0+23 #V*')#1203!230#1-, U.V!-** %#,%#*

3,"#0D (12'+3* 2'-,$-0UV" 71Q$'6#" ,"12 ',#"5'2&S$2-*3'"', *3#T&#*-5#0

+ %,'$'! 2'-,.'!230#12VQSR6- (#!2'4#1&-5'+. '0#"23 #V*')#1203!230#$-0+ 2'-,3.-,

0.[U),-!)"-5,1'0.[US ,"1'0.[UW!-+. 0#"2-!-,20-*!#**1 -!) ,"1'020*T&#

&'%&+ %,'$'! 2'-,.'!230#1 V)QVR6- (#!2'4#"#+-,120 2#2& 2 -2&!-,20-* ,"0.[U

),-!)"-5,!#**1 0# *#2-$-0+*3+','8#"23 3* 01203!230#1 00-5&# "1T

Figure 4: CD93 knockdown impaired migration, polarization and cell-matrix adhesion of

HDMEC.

A-BS '%0 2'-,-$. 0', 1!0 2!& 11 7TD *3#10#.0#1#,2/3 ,2'$'! 2'-,-$5-3,"!*-130# 2

"'$$#0#,22'+#.-',21 $2#02&#1!0 2!&"R # ,&,.-$2&0##1#. 0 2##6.#0'+#,21Q*p0RTRWQ

,23"#,21tV2#12TC-G:2++3,-$*3-0#1!#,2* #*',%5'2& -*%',V[Y%0##, ,".& **-'"',0#"-$

. 0',2&#+'%0 2',%$0-,2-$ 1!0 2!& 11 7T 0%# 00-51"#.'!22&#"'0#!2'-,-$+'%0 2'-,T

&#,3+ #0-$.-* 0'8#"!#**1!*-1#" 00-5&# "1 ,",-,V.-* 0'8#"!#**1-.#, 00-5&# "1

',2&#*# "',%#"%#5#0#!-3,2#" ,"2&#.#0!#,2 %#-$,-,V.-* 0'8#"!#**15 1! *!3* 2#" Q

# ,&,.-$2&0##1#. 0 2##6.#0'+#,21Q#.0RTRWQ,23"#,21tV2#12TH-L: "'-,-$. 02-

%#* 2',V!- 2#".* 2#1 2UR+', $2#01##"',%T2++3,-$*3-0#1!#,2* #*',%5'2&.& **-'"',0#"

0#4# *#" "'$$#0#,!#',!#**1.0# "',% $2#00.[U),-!)"-5,TQ3 ,2'$'! 2'-,-$,3+ #0-$

"�',%!#**1R # ,&,.-$2&0##1#. 0 2##6.#0'+#,21QSR5#**1.#0!-,"'2'-,Q#p0RTRWQ

F,#V5 7CFD5'2&"-,$#00-,'!-00#!2'-,T

Figure 5: CD93 knockdown affected cytoskeletal organization and cell-cell junctions in

HDMEC.

A-HS2++3,-$*3-0#1!#,2* #*',%-$!-,20-*V20 ,1$#!2#". 0 ,". 020 ,1$#!2#"5'2&

1'C2-0.[U5'2&.& **-'"',2-4'13 *'8#2&# !2',!72-1)#*#2-,%0##,T,* 0%#+#,21-$2&#

',1#211&-5 ,',!0# 1#', !2',120#11$' #0$-0+ 2'-,TI-P:2++3,-$*3-0#1!#,2* #*',%5'2&DV

! "�',%0##, ,".& **-'"',0#"-$!-,20-*V20 ,1$#!2#". 0 ,". 020 ,1$#!2#"

5'2&1'C2-0.[UT&##,* 0%#".'!230#1"#+-,120 2# !*# 0', '*'27-$1'0.[UV20 ,1$#!2#"

. 02-',2#0 !25'2&,#'%& -0',%!#**1T

Figure 6: CD93 deficiency impaired vascular perfusion in GL261 glioma.

A-BS2++3,-$*3-0#1!#,212 ',',%-$ TXS%*'-+ $-00.US0#4# *#",-%0-11"'$$#0#,!#',

4#11#*+-0.&-*-%7Q'+ %#12 )#, 2SR6+ %,'$'! 2'-,TC:Q3 ,2'$'! 2'-,-$0.USV.-1'2'4#

4 1!3* 0130$ !# 0# ', TXS%*'-+ # ,Q,-SVVSXQ ,,V!&'2,#752#12 ,",23"#,2_1tV

2#120#4# *#",-12 2'12'! **71'%,'$'! ,2"'$$#0#,!#TD-E: 2++3,-$*3-0#1!#,212 ',',%-$ TXS

%*'-+ $-0#,"-%#,-312% 0#4# *#"',!0# 1#"23+-04 1!3* 0.#0+# '*'27',0.[UV V+'!#

!-+. 0#"2-52+'!#T,! *# 0SWRRV+TF:Q3 ,2'$'! 2'-,-$#,"-%#,-312% 0#* 2'4#2-2-2 *

23+-0 0# ',52 ,"0.[UV V+'!# # ,Q,-UVVQ,23"#,2_1tV2#12TG-N: TXS%*'-+ V # 0',%

+ *# ,"$#+ *#+'!#5#0#.#0$31#"5'2&0#1.#!2'4#*7(20V* #*#"-0 '-2',V* #*#"

*7!-.#01'!-,#1!3*#,23+2-+ 2-*#!2',T"0 ',23+-0!07-1#!2'-,1-04' 0 2-+#1#!2'-,15#0#

'++3,-$*3-0#1!#,2*712 ',#"$-00.US2-4'13 *'8#2&#4 1!3* 230#Q ,"2&#,3+ #0-$.#0$31#"

4#11#*15 1/3 ,2'$'#" ,"#6.0#11#" 1 .#0!#,2 %#-$2-2 *4#11#*,3+ #0 VR,-UVVQW

0#.0#1#,2 2'4#'+ %#15#0# , *78#".#0+-31#Q#p0RTRWQ##p0RTRSQ,23"#,2_1tV2#12T"*--"

4#11#*15#0#!-,1'"#0#".#0$31#"#4#,5&#,1+ **20 !#1-$*#!2',5#0#4'1' *#T&# *--"4#11#*

-$2&#0.[UV V+-31#',2&#%0 .&'12�#$-0#!-3,2#" 1.-1'2'4#',2&#/3 ,2'$'! 2'-,2VCTS#2Q

2&#.0#1#,!#-$(20V*#!2',5 1*'+'2#"2--,*7!#02 ',1.-21',2&#+ (-0'27-$.#0$31#"4#11#*1

',0.[UV V+'!#Q5&'*#(20V*#!2',.#0$31'-,!-4#0#"2&#5&-*#4 1!3* 0*',',%', *--"4#11#*1-$

5'*"V27.#+'!#T

Figure 7: High vascular CD93 expression in human glioma correlated with poor survival.

A:M .* ,V #'#0!304#1-$&'%&V%0 "#%*'-+ . 2'#,21%0 "#222 ,"2D5'2&*-54 1!3* 00.[U

#6.0#11'-, 4#0 %#0.[U1!-0#-$R-0SQ,-SU-0&'%&4 1!3* 00.[U#6.0#11'-, 4#0 %#

0.[U1!-0#-$STW-0TQ,-XZT,2 2'12'! * , *71'1-$1304'4 *0 2#15 1 11#11#" 73,'4 0' 2#

-%V ,)2#12Q.-RTRVYS ,"+3*2'4 0' 2#0-6)0-.-02'-, * 8 0"1Q.-RTRVY[+-"#*1TB:

0-00#* 2'-, #25##,4 1!3* 0'27 ,"0.[U#6.0#11'-,',&'%&V%0 "#%*'-+ %0 "#222 ,"%0 "#

2DT&#%0 .&0#.0#1#,212&#.#0!#,2 %#-$2&#0.UV.-1'2'4# 0# !-+. 0#"2-0.[U

#6.0#11'-,1!-0',%0.[U1!-0#R&S ,"SQW&TR,-T[QSRZR ,,V!&'2,#752#12 ,",23"#,2_1tV

2#12T

Elevated expression of the C-type lectin CD93 in the glioblastoma vasculature regulates cytoskeletal rearrangements that enhance vessel function and reduce host survival

Elise Langenkamp, Lei Zhang, Roberta Lugano, et al.

Cancer Res Published OnlineFirst September 11, 2015.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-14-3636

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2021/03/03/0008-5472.CAN-14-3636.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/early/2015/09/11/0008-5472.CAN-14-3636. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.