Fig. 1: T-ALL and B-ALL PDX mice produced EVs with membrane-anchored HSP70 expression.

A Flow cytometry showing that few ALL cells (<1%) were expressing high level of HSP70. Immunostaining for HSP70 expression performed without permeabilization on BM cells from T-ALL and B-ALL PDX models. Flow cytometry is shown on hCD45⁺ cells for T-ALL and B-ALL viable cells (FVS450 negative). B Immunostaining on cells without permeabilization. Cells expressing HSP70 (HSP70 high) or negative for expression (HSP70 neg) were purified by FACS and observed by microscopy. EVs with membrane-anchored HSP70 expression were observed among the population of cells expressing HSP70. Example of microscopy for B-ALL cells. Microscopy with magnification ×63, scale bar represents 10 µm. C Procedure followed to isolate EVs in BM of PDX mice developing ALL. D Quantification and size of EVs isolated from BM with the NTA. Data are shown as means ± SD; n = 9 mice. P value measured by one-way Anova with Tukey’s multiple comparison test; ****P < 0.0001; ns, non-significant. E Procedure followed to isolate stain EVs prior to pull-down and analysis. F Flow cytometry confirming expression of HSP70 by EVs produced in T-ALL and B-ALL PDX mice, compared with control EVs produced by an NSG mouse. Data are representative of two independent experiments. G Microscopy on EVs purified from control NSG and ALL PDX models showing fluorescent EVs stained with HSP70-ATTO488, microscopy with magnification ×63, scale bar represents 5 µm.