Figure 1. In vivo selection for tumor re-initiation.

(a) Schematic of the in vivo selection strategy used to derive tumorigenic-enriched (TE) populations. Breast cancer cells were injected into the mammary fat pads of immunodeficient mice at low cell numbers and the resulting tumors formed were dissociated into single-cell suspensions and re-injected over multiple rounds of serial dilution. (b) Flow chart depicting the generation of tumorigenic-enriched (TE) derivatives from the MDA-MB-231 (MDA-231) and CN34 parental cell lines (left). Table depicting the number of injected cells and the numbers of tumors formed per mammary fat pad injection into immunodeficient NOD scid mice during the process of generating the in vivo selected derivatives (right). (c) MDA-TE3 cells exhibited enrichment for tumor re-initiation capacity as compared to MDA-parental cells following orthotopic injection of 1×10² cancer cells into NOD scid mice. MDA-TE3 cells yielded tumors in 12/20 sites compared to 1/20 sites for the MDA-parental cells after 10 weeks (left). Gross tumor explants (right). n = 20 independent mammary fat pad injections (pooled from 5 mice with 4 injections each per condition and represented as open squares, right). (d) CN34-TE2 cells exhibited enrichment for tumor re-initiation capacity as compared to CN34-parental cells following orthotopic injection of 2×10³ cancer cells into NOD scid gamma mice. CN34-TE2 cells yielded tumors in 19/20 sites compared to 12/20 sites for the CN34-parental cells after 10 weeks (left). Gross tumor explants (right). n = 20 independent mammary fat pad injections (pooled from 5 mice with 4 injections each per condition and represented as open squares, right). **P<0.01, ***P<0.001 were obtained using one-sided Fisher’s exact test (c–d).