Fig S1

Figure S1: 5T4 expression in B cell development.Adapted from microarray data published online by van Zelm et al http://franklin.et.tudelft.nl/#bcell [11]

(i) (ii)

C(ii)

Fig S2

Figure S2 Expression of 5T4 by B-ALL relapse patients blastsImmunofluorescence staining for 5T4 on DAPI labelled cytospins of bone marrow samples from three relapse patients with pre-B ALL (i-iii) ; magnification x 40, scale bar 5 µm.

SD-1 REH Sup-B15NALM-6 TOM-1

5T4

CXCR4

Sup-B15

TOM-1

A B

Fig S3

C

Figure S3: 5T4 expression by Pre-B ALL cell lines (A) Flow cytometric analyses show the surface expression of 5T4 on a subpopulation of Sup-B15 cells and at low levels on Tom-1 cells, but not by SD-1, REH or Nalm-6 cells. Surface CXCR4 expression is detected on all cells except SD-1. (B) Characteristic punctate 5T4 (green) labelling of cytospins of Sup-B15 and Tom-1 cells (nuclei labelled with DAPI; blue).(C) Immunofluorescence labelling for 5T4 (green), CXCR4 (red ), composite, Z stack and brightfield images for Sup5T4 and Sup cells; bar is 5µM.

5T4 CXCR4 composite Z stack brightfield

5T4 CXCR4 composite Z stack brightfield

Sup5T4

Sup

Fig S4

Sup

Sup5T4

Day 27 ventral images showing assessment of upper and lower abdomen tumour distribution which are presented in Figure 6C as % of total ventral photon count

Fig S5

Bone marrow Ovary

Sup5

T4

5T4

Sup

Figure S5: 5T4 phenotypes are stable after iv challenge and only Sup5T4 is found in the ovaryFlow cytometric analysis representative of bone marrow and ovary cells isolated from mice (n = 4) injected with the Sup-5T4 sublines (5x106 ; i.v.) at day 30 and gated on the mcherry positive leukemic cells. The 5T4 status of the sublines remained unaltered in vivo over time.

Fig S6

Co

ntr

ol

PB

MC

PB

MC

+ A

NY

AR

A

Figure S6: Immunotherapy of Sup5T4 in NSG mice. Ventral images of mice at day 27 after challenge with no treatment, PBMC or PBMC and ANYARA.

Identifier Reference Sup-B15 Sup5T4 Sup

AMEL_1 X X X X

AMEL_2 Y Y Y Y

CSF1PO_1 11 11 11 11

CSF1PO_2 12 12 12 12

D13S317_1 8 8 8 8

D13S317_2 14 14 14 14

D16S539_1 11 11 11 11

D16S539_2 12 12 12 12

D5S818_1 12 12 12 12

D5S818_2 13 13 13 13

D7S820_1 10 10 10 10

D7S820_2 11 10 10 10

TH01_1 6 6 6 6

TH01_2 9.3 9.3 9.3 9.3

TPOX_1 8 8 8 8

TPOX_2 9 9 9 9

vWA_1 15 15 15 15

vWA_2 17 17 17 17

Table S1: Authentication of Sup-B15, Sup & Sup5T4 cells [13]