A lysosome-targeted dextran-doxorubicin nanodrug overcomes doxorubicin-induced chemoresistance of myeloid leukemia

The hypoxic microenvironment is presumed to be a sanctuary for myeloid leukemia cells that causes relapse following chemotherapy, but the underlying mechanism remains elusive. Using a zebrafish xenograft model, we observed that the hypoxic hematopoietic tissue preserved most of the chemoresistant leukemic cells following the doxorubicin (Dox) treatment. And hypoxia upregulated TFEB, a master regulator of lysosomal biogenesis, and increased lysosomes in leukemic cells. Specimens from relapsed myeloid leukemia patients also harbored excessive lysosomes, which trapped Dox and prevented drug nuclear influx leading to leukemia chemoresistance. Pharmaceutical inhibition of lysosomes enhanced Dox-induced cytotoxicity against leukemic cells under hypoxia circumstance. To overcome lysosome associated chemoresistance, we developed a pH-sensitive dextran-doxorubicin nanomedicine (Dex-Dox) that efficiently released Dox from lysosomes and increased drug nuclear influx. More importantly, Dex-Dox treatment significantly improved the chemotherapy outcome in the zebrafish xenografts transplanted with cultured leukemic cells or relapsed patient specimens. Overall, we developed a novel lysosome targeting nanomedicine that is promising to overcome the myeloid leukemia chemoresistance. Supplementary Information The online version contains supplementary material available at 10.1186/s13045-021-01199-8.

the zebrafish xenograft model, we identified the hypoxic caudal hematopoietic tissue (CHT) were enriched with lysosome-abundant chemoresistant leukemic cells and further developed a lysosome targeting nanomedicine to enhance the chemotherapy efficacy.
The two days post-fertilization (2dpf ) zebrafish embryos are immunodeficient due to the absence of adaptive immune system [4] and were used for xenografting myeloid leukemia cells, including Kasumi-1, K562 and OA3, to investigate the chemoresistance mechanism. The accumulated leukemic cells in CHT increased from 3-h post-injection (hpi) to 16 hpi, but the total leukemic cell number were comparable ( Fig. 1A-C, Additional file 1: Fig. S1A-F). Moreover, the CHT-localized leukemic cells were mainly distributed in the caudal vein plexus of CHT (Fig. 1H). To explore the chemosensitivity Open Access *Correspondence: zhaom38@mail.sysu.edu.cn; wujun29@mail.sysu.edu.cn; jianglj7@mail.sysu.edu.cn of leukemic cells in CHT, we treated K562- (Fig. 1D-G) and Kasumi-1-(Additional file 1: Fig. S1G-J) xenografted zebrafish with Dox. The fluorescence intensity, cell number and the expression of human ribosome gene L32 did not significantly reduce upon Dox treatment. The leukemia cells resided in CHT were negative with the apoptosis marker TUNEL, confirming that the cells were chemoresistant (Additional file 1: Fig. S1K, L).
We then tested the 2dpf zebrafish embryos for hypoxia markers, and found the hypoxia indicator pimonidazole (PIM) and the hypoxia-associated genes hif1al were highly enriched in CHT ( Fig. 1I and Additional file 2: Fig. S2A). Besides the lysosome-related genes TFEB, LAMP1 and LC3B also increased in K562 under hypoxia (Additional file 2: Fig. S2B). TFEB is a master regulator of lysosome biogenesis [6,7], we then assumed that hypoxia might increase TFEB expression to activate lysosome biogenesis. Indeed, we found the lysosome-high cells and expressions of lysosome genes such as V-ATPase, LAMP1 and LAMP2 were highly enriched in hypoxic K562 and Kasumi-1 cells (Additional file 2: Fig. S2C-H). Furthermore, more CHT-localized leukemic cells were stained positive with LysoTracker compared with cells in other tissues of leukemia-zebrafish xenografts (Fig. 1J, K), indicating the hypoxic CHT preserved leukemic cells with enriched lysosomes.
We next explored the role of lysosome in regulating leukemia chemoresistance. Lysosome inhibitor bafilomycin (Baf ) or chloroquine (CQ) efficiently decreased the ratio of LysoTracker-or LysoSensor-high K562 cells (Additional file 3: Fig. S3A-D). We examined the intracellular location of Dox using its autonomous red fluorescence. Dox was mainly located in lysosomes but transported into the nucleus when treated with Baf or CQ (Fig. 1L). Baf or CQ also enhanced the Dox-induced cytotoxicity against chemoresistant cells in hypoxia-cultured cells (Additional file 3: Fig. S3E) and in xenografted zebrafish (Fig. 1M, Additional file 3: Fig. S3F).
Mechanistically Dex-Dox induced apoptosis in chemoresistant leukemia cells as we found more TUNEL + K562 cells in Dex 5k -Dox treated CHT (Additional file 6: Fig.  S6A, B). Futhermore, Dex 5k/150k -Dox released Dox from lysosomes to enter the nuclei (Fig. 2C), but had no effect on lysosome pH as compared to Dox (Additional file 6: Fig. S6C-H), suggesting Dex 5k/150k -Dox might exhibit antileukemic effect through facilitating Dox nuclear influx. In addition, Dox released from Dex-Dox nanomedicine was highly accumulated in zebrafish and transported into the CHT localized leukemic cells more efficiently than Dox alone (Additional file 7: Fig. S7A-D).
We further explored the therapeutic effect of Dex-Dox with myeloid leukemia patient samples. The leukemic cells from the relapsed patient had increased Fig. 1 Hypoxic CHT harbored chemoresistant leukemic cells with increased lysosomes that sequestered Dox to prevent its nuclear entry and cytotoxicity. A-C K562 cells were microinjected into 2dpf embryos. The cell number and fluorescent intensity of leukemic cells localized in CHT at different time points post-injection were counted. D-G K562-xenografted zebrafish were treated with Dox from one-day-post injection (1 dpi) to 3 dpi, and the leukemic cells in CHT were quantified with the fluorescent intensity (E), the cell number (F) and the mRNA expression of human ribosome gene L32 (G). H Dox-resistant Kasumi-1 cells were mainly localized in the CVP vessels visualized by flk:GFP fish. CVP-caudal vein plexus, CA-caudal aorta, ISV-intersegmental vessel. I The 2dpf zebrafish embryos were stained with the PIM antibody to detect hypoxia tissue. The CHT was labeled with yellow rectangles. J, K The Kasumi-1-xenografted zebrafish were stained with LysoTracker and quantified for lysosome enrichment in CHT and non-CHT in vivo. The entire CHT in the left panel was labeled with yellow rectangles, and the region in blue rectangles were magnified in the right panels to show details. L The subcellular localization of Dox in K562 cells was visualized by its autonomous red fluorescence in Dox, Dox + Baf or Dox + CQ. M The chemoresistant K562 cells that localized in CHT were more sensitive to Dox + CQ while CQ alone has no toxicity. Bar plots are shown as average ± SEM. The statistical significance between groups was determined using Student's t-test or ANOVA analysis. *Indicates p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001  ratio of LysoTracker-high cells than the primary patient cells (Fig. 2D), and Dex 5k -Dox efficiently eliminated the relapsed cells than Dox (Fig. 2E). Similarly, the hypoxia-incubated leukemic cells had increased ratio of LysoTracker-high cells, and more resistant to Dox, but they were susceptible to Dex 150k -Dox treatment (Fig. 2F,  G). We also found that Dex 5k/150k -Dox efficiently eliminated these relapsed patient cells in xenografted zebrafish (Fig. 2H, Additional file 7: Fig. S7E, F). Overall, our data reveal that the hypoxia-lysosome axis controls the myeloid leukemia chemoresistance, and the newly developed lysosome targeting nanomedicine is a promising strategy to eliminate chemoresistant leukemic cells (Additional file 8: Fig. S8).

Supplementary Information
The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s13045-021-01199-8. Additional file 8. Figure S8. Graphic abstract. By visualizing in a zebrafish xenograft model, we found that the chemoresistant leukemic cells were mainly accumulated in the hypoxic hematopoietic tissue (A). The hypoxic microenvironment characterized leukemic cells with excessive lysosomes, thereby sequestering the chemotherapeutics inside to reduce its cytotoxicity (left panel in C). We developed the pH-sensitive Dex-Dox nanomedicine to release Dox from lysosomes and enter the nucleus, thereby efficiently eliminating the chemoresistant leukemic cells in vivo (B, right panel in C).

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