Lidia Moyano-Galceran

Supplementary Figure S3. IFN measurement and organ BLI of the short-term in vivo study. A. IFN quantification by ELISA in post-mortem mouse blood plasma. B. Representative BLI images of isolated organs from mice treated with PBS, NT-T, and CAR-T cells at the indicated T cell doses after 32 days. C. Organ BLI quantification. N=3 for control. N=5 for all groups, except N=2 for NT-T 1M, N=3 for CAR-T 1M, and N=4 NT-T 2M. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison tests. (A) ***p=0.0005, ****p=0.0001. (C) *p=0.04, **p≤0.001 to PBS group. Not significant, n.s., differences were attributed to p≥0.05. (M=1x106).

Supplementary Figure S2. Short-term in vivo study, dose-response, and weight monitoring. A. Treatment groups and used T cell doses. B. Weight monitoring in the indicated mice groups. CAR-T 3M treated mice showed a trend of mild weight loss. (M=1x106).

Supplementary Figure S6. Expression of SSEA-4 in normal mouse tissues. Immunofluorescence detection of SSEA-4 (green) in the indicated tissues of healthy 10 weeks old (C57BL/6) mouse using anti-SSEA-4 antibody.

SSEA-4 expression in an ovarian cancer cohort.

Supplementary Figure S5. Weight monitoring during the long-term in vivo study. Mice treated with 2M CAR-T dose showed pronounced weight loss and health deterioration at earlier time points than mice treated with 1M CAR-T dose. PBS, tumor-free CAR/NT-T 1M, and NT-T groups did not show weight loss and remained healthy during the duration of the experiment. Unless otherwise indicated, mice groups are tumor-bearing. (M=1x106).

Supplementary Figure S7. T cell detection in mouse tissues after the long-term survival study. Indicated tissues were stained with HOECHST 33342 for nuclei (blue), anti-EGFR to detect CAR-T cells (middle panel; green) and anti-CD3 for T cells (right panel, white). Left panel shows a merged image of all three channels. Arrows highlight CAR-T and CD3 positive cells, while arrowheads highlight erythrocytes with an intense background staining.

Supplementary Figure S1. SSEA-4 staining specificity and LDH cytotoxicity assay. A. SSEA-4 immunohistochemistry in paraffin-embedded gastric cancer tumor microarrays. B. Upper panel sections were treated with standard xylene/ethanol series used in paraffin embedded section treatment, whereas lower panel samples were untreated. C. Consecutive sections of fresh frozen pancreatic cancer patient samples were stained with anti-SSEA-4 antibody. The staining was blocked with SSEA-4 hexaose (middle panel) or SSEA-3 (right panel). D. Representative immunofluorescence micrographs of SSEA-4 (red) in OVCAR-4 cells (high expression) and Caov-3 cells (no expression). Nucleus (blue; Hoechst 33342). N=3. E. LDH assay to measure toxicity of anti-SSEA-4 CAR-T cells on SSEA-4 positive OVCAR-4 cells and SSEA-4 negative Caov-3 cells as a control (N=2). LDH assay was performed after 72 hours of CAR/NT-T cell treatment. ****p=0.0001, ***p=0.0002 and **p=0.0025.

Supplementary Data S1. Pathology Report of the long-term survival study. Post-mortem lung, intestine and kidney tissue samples collected and analyzed by Histowiz.