Fig. 3. EV^AML incites inflammatory responses in HSPCs.

A A schematic of iMLL-AF9 chimeric mouse model generation. B Assessment of leukemic hematopoietic fraction (CD45.1+) in the PB over the course of doxycycline induction. BM was assessed after 18 days of induction (n = 3) for leukemic fraction chimerism (C), the frequency of MLL-AF9-expressing cells (based on NGFR expression; cell expresses MLL-AF9-IRES-NGFR) (D), and myeloid cell frequency (E) in the leukemic fraction of the BM. F Spleen size and (G) LT-HSC (Lin⁻ cKit⁺ Sca1⁺ Flk2^- Cd150⁺ Cd48⁻), ST-HSC (Lin⁻ cKit⁺ Sca1⁺ Flk2⁻ Cd150⁻ Cd48⁻), MPP-2 (Lin⁻ cKit⁺ Sca1⁺ Flk2⁻ Cd150⁺ Cd48⁺), MPP-3 (Lin⁻ cKit⁺ Sca1⁺ Flk2⁻ Cd150⁻ Cd48⁺), MPP-4 (Lin⁻ cKit⁺ Sca1⁺ Flk2⁺ Cd150⁻ Cd48⁺) frequency were assessed (n = 3). H The role for EV^AML in inciting inflammatory responses in HSPCs were carried out by challenging FACS-sorted HSPCs (HSPCs sorted and pool from n = 3 mice per experiment) with EV^AML. I, L Comparison of HSPC gene expression following 2 h exposure with EV derived from C1498 (C1498-EV^AML) and healthy BM (BM-EV^Healthy) (n = 3). M, P Comparison of HSPC gene expression following exposure with EV^AML from both C1498 and iMLL-AF9 blasts (AF9-EV^AML) (n = 3). Q HSPC-secreted Cxcl10 were assessed 72 h following EV^AML exposure using ELISA. (R) Methylcellulose assay analysis of HSPC colony forming unit counts following 72 h challenge with either C1498-EV^AML (n = 3), or (S) EVs from the BM plasma of C1498-engrafted mice and PBS-injected mice (n = 3). Values expressed as mean ± s.d., Statistical significance calculated using ANOVA and Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.