Fig S1 Figure S1: 5T4 expression in B cell development. Adapted from microarray data published online by van Zelm et al

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Fig S1 Figure S1: 5T4 expression in B cell development. Adapted from microarray data published online by van Zelm et al [11]

(i)(ii) C Fig S2 Figure S2 Expression of 5T4 by B-ALL relapse patients blasts Immunofluorescence 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-1REHSup-B15NALM-6TOM-1 5T4 CXCR4 Sup-B15 TOM-1 AB 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. 5T4CXCR4compositeZ stackbrightfield 5T4CXCR4 composite Z stackbrightfield 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 marrowOvary Sup5T4 5T4 Sup Figure S5: 5T4 phenotypes are stable after iv challenge and only Sup5T4 is found in the ovary Flow cytometric analysis representative of bone marrow and ovary cells isolated from mice (n = 4) injected with the Sup-5T4 sublines (5x10 6 ; 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 Control PBMC PBMC + ANYARA 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.

IdentifierReferenceSup-B15Sup5T4Sup AMEL_1 XXXX AMEL_2 YYYY CSF1PO_1 11 CSF1PO_2 12 D13S317_ D13S317_2 14 D16S539_1 11 D16S539_2 12 D5S818_1 12 D5S818_2 13 D7S820_1 10 D7S820_ TH01_ TH01_2 9.3 TPOX_ TPOX_ vWA_1 15 vWA_2 17 Table S1: Authentication of Sup-B15, Sup & Sup5T4 cells [13]