Fig. 3

PRO and INV cooperate in metastasis via co-extravasation. a. In the first 24 hours after intravenous transplant of ZMEL1-PRO and -INV, a PRO-INV mixed cluster (left, arrowhead) extravasated from the caudal hematopoietic tissue (CHT) into the tail fin mesenchyme (TF)—with ZMEL1-INV leading (middle, arrow) and ZMEL1-PRO following (right, arrow). Composite images shown. b. Segmented representative image of adult zebrafish with orthotopic transplant of 1:1 mixture of ZMEL1-PRO and -INV. Arrowheads indicate polyclonal metastases, including to the kidney and caudal regions (left and right boxes, respectively). Original image shown in Figure S3b. c. Number of fish co-transplanted with a 1:1 mixture of ZMEL1-PRO and -INV that have no caudal metastases (None), caudal metastases comprised of exclusively PRO or INV, or caudal metastases formed by PRO-INV co-metastasis (Co-Met) (N=5 independent experiments with 17, 15, 16, 15, and 17 fish each; 80 fish total; p<0.001 by Mantel-Haenszel’s test for null hypothesis of no interaction). d. Percentage of fish with ZMEL1-PRO caudal metastasis 3 dpt following orthotopic transplant of ZMEL1-PRO or a 1:1 mixture of ZMEL1 PRO:INV (OR [95% CI]: 3.31 [1.10, 9.96]; p=0.033 by logistic regression). e. Proportion of fish with ZMEL1-INV caudal metastasis 3 dpt following orthotopic transplant of ZMEL1-INV or a 1:1 mixture of ZMEL1 PRO:INV (OR [95% CI]: 1.32 [0.61, 2.88]; p=0.49 by logistic regression). For c-e: N=5 independent experiments with PRO/MIX/INV 18/17/18, 13/15/14, 15/16/15, 12/15/15, and 15/17/16 fish per group, respectively; 231 fish total; plots show mean ± SD. f. Number of ZMEL1-PRO cells in Boyden Chamber assay migrating per 20X field alone or mixed with ZMEL1-INV (p=0.042 by linear regression). g. Number of ZMEL1-INV cells in Boyden Chamber assay migrating per 20X field alone or mixed with ZMEL1-PRO (p=0.91 by linear regression). For f-g: N=3 independent experiments for each EGFP and tdTomato labeling; plots show mean ± SD. See also Figure S3 and Video S4.