The side population enriches for leukemia-propagating cell activity and Wnt pathway expression in zebrafish acute lymphoblastic leukemia

Abstract

Cancer stem cells have been strongly linked to resistance and relapse in many malignancies. However, purifying them from within the bulk tumor has been challenging, so their precise genetic and functional characteristics are not well defined. The side population assay exploits the ability of some cells to efflux Hoechst dye via ATP-binding cassette transporters. Stem cells have increased expression of these transporters and this assay has been shown to enrich for stem cells in various tissues and cancers. This study identifies the side population within a zebrafish model of acute lymphoblastic leukemia and correlates the frequency of side population cells with the frequency of leukemia stem cells (more precisely referred to as leukemia-propagating cells within our transplantation model). In addition, the side population within the leukemia evolves with serial transplantation, increasing in tandem with leukemia-propagating cell frequency over subsequent generations. Sorted side population cells from these tumors are enriched for leukemia-propagating cells and have enhanced engraftment compared to sorted non-side population cells when transplanted into syngeneic recipients. RNA-sequencing analysis of sorted side population cells compared to non-side population cells identified a shared expression profile within the side population and pathway analysis yielded Wnt-signaling as the most overrepresented. Gene set enrichment analysis showed that stem cell differentiation and canonical Wnt-signaling were significantly upregulated in the side population. Overall, these results demonstrate that the side population in zebrafish acute lymphoblastic leukemia significantly enriches for leukemia-propagating cells and identifies the Wnt pathway as a likely genetic driver of leukemia stem cell fate.

Rps15a Imparts Chemoresistance by Regulating Cd44 Expression and Cell Stemness in Esophageal Squamous Cell Carcinoma

“…Regulation of p-gp by RPS15A could further affect the number and proportion of stemlike cancer cell side-population (SP) cells (Fig.6) through the e ux mechanism for chemoresistance. [33] In CDDP-treated ESCC cells, the expression level of CD44 signi cantly decreased and apoptosis increased concurrently upon RPS15A deletion (Fig.3). Our ndings are similar to a previous report that showed that CD44 inhibition reduced the adhesion of multiple myeloma cells and reversed the resistance to lenalidomide.…”

“…6) through the e ux mechanism for chemoresistance. [33] In CDDP-treated ESCC cells, the expression level of CD44 signi cantly decreased and apoptosis increased concurrently upon RPS15A deletion (Fig. 3).…”

The multiple ways Wnt signaling contributes to acute leukemia pathogenesis

“…T‐ALL can be induced by the injection of a plasmid containing mouse Myc under control of the zebrafish Rag2 promoter into zebrafish embryos 125 . Although LSC markers have not been established in ALL, Baeten and colleagues 126 isolated T‐ALL LSCs in this model. The authors used the side‐population assay and confirmed the stemness potential of the isolated cells in limiting‐dilution assays.…”

Zebrafish B cell acute lymphoblastic leukemia: new findings in an old model

“…myeloid leukemia (AML) and T-ALL [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37]. For T-ALL in particular, zebrafish models have been highly informative, advancing our understanding of T-ALL genetics, pro-and anti-oncogenic interactions between different genes and pathways, tumor heterogeneity, leukemia stem cells, and in screens for new therapeutics [28,[38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53]. However, despite the fact that zebrafish T-ALL models had proven to be fertile grounds for study, B-ALL modeling in D. rerio had not been fruitful, with only one low penetrance and long latency line reported [54].…”