3A).38, learn more 39 Because CSCs are a subpopulation of the SP, we conclude that they too may carry markers of normal progenitors. When unsorted tumor cells were exposed to media that favors the survival and growth of hepatic progenitor cells, the percentage of SP cells increased (Fig. 4A). In contrast, non-SP cells failed to propagate or even survive in progenitor media (Supporting Fig. 3), in accord with the view that they are more differentiated than

SP cells. The SP population was reduced when tumor cells were incubated in media that elicits differentiation of hepatic progenitors into mature hepatocytes40 (Fig. 4B). Previous work has shown that MYC tumor cells can differentiate into mature hepatic cells upon the inactivation of MYC in vivo.31

We found that concurrent repression of the MYC transgene by doxycycline enhanced the effect of differentiation media on the MYC-driven tumor cells, as manifested by complete loss of the progenitor marker AFP and an increase in C/EBPα, a marker for mature hepatocytes (Fig. 4C, Supporting Fig. 4D). We conclude that SP cells from the MYC-driven hepatic tumors possess properties similar to normal progenitor cells, and that the same is likely to be true of the CSC subset of SP cells. The ABC transporter proteins MDR1 and BCRP have been shown previously to efflux Hoechst 33342 dye.19, 20 Sorted tumor cells were Deforolimus analyzed for the mRNA of ABC transporters as well as MRP1 (Abcc1a and Abcc1b). Only Mdr1a and Mdr1b mRNAs were more highly expressed in SP cells than in non-SP cells (Fig. 5A). These

results were also confirmed by western blot analysis (Fig. 5B). Notably, expression of BCRP was not detected by either means. Exposure of LT2-MYC tumor cells to progenitor media enriched for MDR1 expression, whereas differentiation media did not (Supporting Fig. 4D). Because MDR1 was more highly expressed than BCRP in SP cells, we used a functional analysis to determine whether it was MDR1 that mediated SP formation. To this end, we used hydrodynamic transfection of MYC to elicit hepatic tumors in mice that were deficient in either Mdr1a/1b or Bcrp and analyzed the resulting tumors for SP cells. Hydrodynamic C-X-C chemokine receptor type 7 (CXCR-7) transfection of MYC elicited hepatic tumors in mice of all genotypes by 90 days (Supporting Fig. 4C). MYC induction of tumors in wildtype and Bcrp−/− mice resulted in the formation of an SP population, whereas hepatic tumors in Mdr1a/1b−/− mice did not have an SP population (Fig. 5C). The role of MDR1 in mediating the SP phenotype was further verified in vitro: overexpression of MDR1 enhanced the SP phenotype, whereas partial knockdown reduced it (Supporting Fig. 4A,B). These data demonstrate that, whereas MDR1 does not affect tumorigenesis, it is responsible for the SP phenotype seen in our tumor model. MDR1 and BCRP efflux a number of similar chemotherapeutics, including doxorubicin (Dox), which is utilized in the treatment of primary hepatic tumors.