Engineering Tumour Cell-Binding Synthetic Polymers with Sensing. Dense Transporters Associated with Aberrant Glutamine Metabolism

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Edward Ray
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1 Supplmntry Informtion Enginring Tumour Cll-Binding Synthti Polymrs with Snsing Dns Trnsportrs Assoitd with Arrnt Glutmin Mtolism oki Ymd, Yuto ond, iroysu Tkmoto, Tkhiro omoto, Mkoto Mtsui, Kishiro Tomod, Msmitsu Konno, idshi Ishii, Mski Mori, nd ouhiro ishiym* S1

2 Supplmntry Figurs zido-3,6,9-trioxundn-1-min CA-Lys(TFA) DMS r.t., 3 dys F F F 3 3 n Azid-PLys(TFA) F F F M / 5 q. r.t., 8 h 3 3 n Azid-PLys 3 + Supplmntry Figur S1. Synthti produr for zid-plys. 3 3 n Azid-PLys + 3 Bo Bo-Glu-Bzl DMT-MM, TEA, DMS r.t., ovrnight 3 3 n Azid-PLys(Bo-Glu-Bzl) Bo 1. M /.5 q. r.t., 18h 2. A / 3 Cl q. r.t., 18h 3 3 n Azid-PLys(Gln) Supplmntry Figur S2. Synthti produr for zid-plys(gln) S2

3 3 3 n Azid-PLys + 3 Bo-Glu(Bzl)- Bo DMT-MM, TEA, DMS r.t., ovrnight 3 3 n Azid-PLys(Bo-Glu(Bzl)) Bo 1. M /.5 q. r.t., 18h 2. A / 3 Cl q. r.t., 18h 3 3 n Azid-PLys(α-Glu) Supplmntry Figur S3. Synthti produr for zid-plys(α-glu). 3 3 n Azid-PLys(Gln) R Cy5-DBC 1 mm C 3 q. p 7.4 r.t., ovrnight R 3 n PLys(Gln) S S 3 - R = + 3 S Supplmntry Figur S4. Synthti produr for PLys(Gln). S3

4 3 3 n 3 n R Cy5-DBC Azid-PLys(α-Glu) R PLys(α-Glu) 1 mm C 3 q. p r.t., ovrnight S S 3 - R = + 3 S Supplmntry Figur S5. Synthti produr for PLys(α-Glu) solv. d d TSP δ (ppm) Supplmntry Figur S6. 1 MR sptrum of zid-plys(gln)-1 (D 2 ). S4

5 solv. d d TSP δ (ppm) Supplmntry Figur S7. 1 MR sptrum of zid-plys(gln)-5 (D 2 ) solv. d d TSP δ (ppm) Supplmntry Figur S8. 1 MR sptrum of zid-plys(gln)-3 (D 2 ). S5

6 solv. d d TSP δ (ppm) Supplmntry Figur S9. 1 MR sptrum of zid-plys(α-glu)-1 (D 2 ) solv. d d TSP δ (ppm) Supplmntry Figur S1. 1 MR sptrum of zid-plys(α-glu)-5 (D 2 ). S6

7 solv. d d TSP δ (ppm) Supplmntry Figur S11. 1 MR sptrum of zid-plys(α-glu)-3 (D 2 ). ) ) ) Elution volum (ml) Elution volum (ml) Elution volum (ml) d) ) f) Elution volum (ml) Elution volum (ml) Elution volum (ml) Supplmntry Figur S12. Siz xlusion hromtogrphy of sris of PLys(Gln) nd PLys(α-Glu). ) PLys(Gln)-1, ) PLys(Gln)-5, ) PLys(Gln)-3, d) PLys(α-Glu)-1, ) PLys(α-Glu)-5, f) PLys(α-Glu)-3. [olumn, Suprdx 2 inrs 1/3GL; lunt, 1 mm phospht uffr (p 7.4) ontining 15 mm Cl; flow rt,.6 ml/min; tmprtur, r.t.; dttor, fluorsn (x/m = 62 nm/67 nm)] S7

8 rt Lung Spln Supplmntry Figur S13. Immunohistohmil nlysis of tissus from mi ring suutnous BxPC3 tumours. Rd, nti-humn/murin ASCT2 ntiody; lu, nuli. Sl r, 1 µm. Supplmntry Figur S14. Rltiv xprssion lvl of ASCT2 quntifid y ELISA. Dt r mn ± S.D. (n=3). p* <.1 (on-wy AVA with Tuky s multipl omprison tst). S8

9 Fluorsn intnsity (.u.) * * PLys(Gln)-1 PLys(Gln)-5 PLys(Gln)-3 Supplmntry Figur S15. Cllulr uptk nlysis of polymrs in pg2 lls. Th p* <.1 (on-wy AVA with Tuky s multipl omprison tst) Fluorsn intnsity (.u.) PLys(α-Glu)-1 PLys(α-Glu)-5 PLys(α-Glu)-3 Supplmntry Figur S16. Cllulr uptk nlysis of sris of PLys(α-Glu)-n in BxPC3 lls. Th lls wr inutd with S.D. (n=3). S9

12 PLys(Gln)-5 % of without inhiitors 1 8 6 4 2 2.5 mm BzlSr 5 mm BzlSr 1 mm BzlSr 2.5 mm MAIB 5 mm MAIB 1 mm MAIB 2.5 mm Gln 5 mm Gln 1 mm Gln Without inhiitors 2.

Cllulr uptk nlysis of PLys(Gln)-5 in BxPC3 lls with inhiitors (BzlSr, ASCT2 inhiitor; MAIB, systm A inhiitor; BC, systm L inhiitor; ) 37 ºC 4 ºC ) PLys(Gln)-5 Cy5 fluorsn

10 12 PLys(Gln)-5 % of without inhiitors mm BzlSr 5 mm BzlSr 1 mm BzlSr 2.5 mm MAIB 5 mm MAIB 1 mm MAIB 2.5 mm Gln 5 mm Gln 1 mm Gln Without inhiitors 2.5 mm BC 5 mm BC 1 mm BC Supplmntry Figur S17. Cllulr uptk nlysis of PLys(Gln)-5 in BxPC3 lls with inhiitors (BzlSr, ASCT2 inhiitor; MAIB, systm A inhiitor; BC, systm L inhiitor; ) 37 ºC 4 ºC ) PLys(Gln)-5 Cy5 fluorsn intnsity (%) PLys(Gln)-5 * 37 C 4 C Supplmntry Figur S18. ) Confol lsr snning mirosopi imgs of BxPC3 lls trtd with PLys(Gln)-5. Rd, Cy5-lld polymrs; grn, lt ndosom/lysosom; lu, nulus. Sl r, 2 mm. ) Cy5 fluorsn intnsitis of BxPC3 lls ftr 3 h trtmnt with PLys(Gln)-5 t 37 C nd 4 C. Dt p* <.1 (Studnt s t-tst). S1

11 ) ) ) PLys(Gln)-1 PLys(Gln)-5 Fluorsn intnsity (% of without BzlSr) [BzlSr] (mm) Fluorsn intnsity (% of without BzlSr) [BzlSr] (mm) Supplmntry Figur S19. Cll-sd omptitiv inhiition urv of PLys(Gln)-1 (), PLys(Gln)-5 (), nd PLys(α-Glu)- Fluorsn intnsity (% of without BzlSr) 1 5 PLys(α-Glu) [BzlSr] (mm) Fluorsn intnsity (.u.) PLys(α-Glu)-1 t p 7.4 PLys(α-Glu)-1 t p 6.5 PLys(Gln)-1 t p 6.5 Supplmntry Figur S2. Cllulr uptk nlysis of PLys(Gln)-1 nd PLys(α-Glu)-1 in BxPC3 lls t p 6.5. S11

12 Supplmntry Mthods ELISA Th rltiv xprssion lvl of ASCT2 in ulturd lls ws quntifid using ELISA Strtr Assory Kit (Bthyl Lortoris, In., Montgomry, TX) ording to th mnuftur s produr. Brifly, th lls ( lls) wr trnsfrrd to 1.5 ml tus nd wshd thr tims with old PBS. Th lls wr thn lysd y sonition in 5 µl of lysis uffr (TBS ontining 1 mm EDTA,.1% P-4 (Sigm Aldrih Corportion), nd prots inhiitor oktil (Roh, Mnnhim, Grmny), p 7.5). Th rsulting solutions wr to ollt th suprntnts. Protin onntrtion of th olltd suprntnts ws quntifid using BCA ssy (Thrmo Fishr Sintifi). To prform th ELISA, mirotitr plts wr otd with 5 µg of th protin lysts y inuting ovrnight Th plts wr wshd thr tims with wshing uffr (TBS with.5% Twn 2, p 8.) nd trtd with 1% BSA in TBS t mint tmprtur for 3 min. Aftr wshing thr tims with wshing uffr, th plts wr inutd with 1 µl of rit nti-humn ASCT2 polylonl ntiody solution (1:3 dilution in TBS ontining 1% BSA nd.5% Twn 2, Snt Cruz Biothnology) t mint tmprtur for 1.5 h. Th plts wr wshd thr tims with wshing uffr nd inutd with 1 µl of horsrdish proxids (RP)-onjugtd got nti-rit IgG ntiody (1:8 dilution in TBS ontining 1% BSA nd.5% Twn 2, Thrmo Fishr Sintifi) t mint tmprtur for 1 h. Aftr wshing fiv tims with wshing uffr, 1 µl of 3,3',5,5'-ttrmthylnzidin (TMB) sustrt solution ws ddd to h wlls for th rtion with RP. Th rtion ws stoppd y th S12

13 ddition of 1 µl of.18 M 2 S 4, nd th sorn t 45 nm ws msurd. Cllulr uptk nlysis t p 6.5 To invstigt th fft of p on th llulr uptk hvior of PLys(α-Glu)-1 nd PLys(Gln)-1 in BxPC3 lls, th p of th ssy uffr (PBS ontining 1% FBS) ws djustd to 6.5 using Cl quous solution. Th produr ws sm in llulr uptk study dsrid ov xpt for th p of th ssy uffr. S13

Above. Below H&E. Above CD4. CD11b. Fluoromyelin BDA BDA BDA. Cerebral cortex. Focal lesion. Thoracic cord. Lumbar cord. Hindlimb placing score

Above. Below H&E. Above CD4. CD11b. Fluoromyelin BDA BDA BDA. Cerebral cortex. Focal lesion. Thoracic cord. Lumbar cord. Hindlimb placing score 1. 1. 1 1 3 Tim tr lsion inution () wks wks 3 1 1 1 3 Tim tr lsion inution () i Ltrl sor. Mil Aov Blow.. 1 wk On sgmnt ov g 3. Lsion Fluoromylin Fluoromylin h CD11 Hinlim pling sor CD Aov Blow Fol lsion