Fig 2: Parasite homing and extravascular BM localization during mouse infection.(A) Synchronous infections of mice with mCherryHsp70-FLucef1a merozoites, rings, trophozoites, schizonts, or gametocytes. Data show that merozoites, rings, and gametocytes are significantly enriched in BM and spleen (red arrows, ***P < 0.001) compared to mean across all organs (dashed line). Data are expressed relative to parasite numbers quantified in the brain (normalized to 100) and correspond to intravascular and extravascular compartments in all organs. (B) Representative diagram of hematopoietic development. Hematopoietic stem cells (HSCs) undergo a series of developmental steps until formation of the final nucleated stage termed orthochromatic erythroblast (OCE), which upon enucleation gives rise to reticulocytes (Retic). Reticulocytes intravasate into circulation and rapidly mature to terminal normocytes. Extravascular RBC precursor cells are marked by CD44, whereas CD71 is lost shortly after intravastion. (C) Presence of stage-specific reporter parasites in CD44+ RBCs following injection of merozoites. At 24 hpi, EG-CFP parasites, but not GNP-CFP and Con-CFP parasites, are largely confined to parenchyma and sinusoids where most are present in CD44+ RBCs (left panel), reflecting distribution of CD44+ RBCs (mid left panel). No parasites are detected in CD44+ cells in peripheral blood (mid right panel). Representative images of CD44+ cells in a BM section (magenta) are shown, upon injection of fluorescein isothiocyanate (FITC)–dextran (cyan) to mark vasculature (top panel). Bottom image: Close-up of an infected CD44+ RBC in BM. (D) Presence of uninfected UBC-GFP RBCs in the BM extravascular niche. UBC-GFP RBCs were obtained from transgenic donor mice and injected into naïve, uninfected recipient mice and quantified in BM. A small proportion of RBCs accumulates in BM extravasculature but not in the brain extravascular niche (left panel). Representative image is shown (left) and a control with mCherryHsp70-FLucef1a P. berghei parasites (right). (E) Inhibition of parasite extravasation using receptor antibodies. Mice were pretreated for 24 to 48 hours with P-selectin antibodies before injection of mCherryHsp70-FLucef1a merozoites and compared to untreated and nonviable (4% paraformaldehyde-fixed) control parasites (left panel). Parasite abundance in BM but not in brain or peripheral circulation is reduced upon P-selectin pretreatment compared to controls. Injection of merozoites from stage-specific reporter parasites upon P-selectin pretreatment reveals greatest reduction in BM parenchyma with EG-CFP and Con-CFP parasites, whereas intravascular localization is increased (right panel). Error bars represent SEs from five separate experiments in three mice per condition. (F) Quantification of peripheral parasitemia upon treatment with receptor antibodies. UBC-GFP mice were pretreated for 24 to 48 hours with P-selectin before injection of mCherryHsp70-FLucef1a merozoites (right panel) and compared to untreated control parasites (left panel). Peripheral parasitemia and tissue burden were quantified before and after cardiac perfusion to remove circulating parasites—leaving only those in the extravascular spaces. Extravascular parasite load decreases significantly in BM and spleen compared to control animals, whereas peripheral parasitemia (obtained before perfusion) increases. P values are shown as **P < 0.05 and ***P < 0.005. All error bars represent SDs of the mean.

Fig 3: Mixed inoculation of TME-enriched CD44+/CD24low/- and CD44+/ß1+ cells further supports the key roles of CD44+/CD24low/- cells in metastasisCD44+/CD24low/- cells and CD44+/ß1+ cells were sorted out from TME-Stimulated mCherry- or mPlum-expressing MCF-7 cells, respectively. Three groups of cells were injected (procedure as in Figure 8) to mice: Group 1 - CD44+/CD24low/-cells; Group 2 - CD44+/ß1+ cells; Group 3 - mixture of both cell types in a 1:1 ratio. In all three groups, the total number of inoculated tumor cells was the same. Primary tumors and metastases were followed for 12 weeks, as described in Figure 8. (A) Development of primary tumors. (A1) An image of a resected primary tumor from a mouse of Group 3, demonstrating mCherry-expressing cells derived from sorted CD44+/CD24low/- cells and mPlum-expressing cells derived from sorted CD44+/ß1+ cells. (A2) Average sizes of primary tumors ± SEM are presented in mm3, followed every two weeks by caliper measurements (p > 0.05, ANOVA). (B) Metastases detected ex vivo by the CRi Maestro™ device. (B1) Proportions of mice that developed metastases in all excised organs. A significant difference was not expected in this cohort size, yet the differences between the three groups were close-to-significant (p = 0.056, Fisher's Exact Test). (B2) Proportions of mice that developed metastases in bones (left) and LNs (right). The differences between the three groups for bone metastases were close-to significant (p = 0.056, Fisher's Exact Test), and the differences between the three groups for LN metastases were significant (p = 0.042, Fisher's exact test). (C) Type of metastatic cells found in bones and LNs of mice of Group 3 (inoculated with a 1:1 mix of both sub-populations). (C1) Bone metastases. (C2) LN metastases. All panels sum up results obtained in n = 2 experimental repeats in which tumor cells were inoculated to a total of n = 8–11 mice (please see comments on mice numbers and statistics in “Materials and methods”).

Fig 4: TME Stimulation enriches the CD44+/CD24low/- sub-population in MCF-7 and T47D Luminal-A breast tumor cellsMCF-7 or T47D breast tumor cells were exposed to TME Stimulation in culture (estrogen - 10-8 M, TNFa - 50 ng/ml, EGF - 30 ng/ml) for three days. No stimulation = Cells grown with vehicles only. Expression of CD44 and CD24 on the surface of the cells was determined by FACS analyses, using fluorescently-labeled specific Abs. Isotype-matched control Abs were used in order to determine baseline staining and to set location of axes (Data not shown; Please see “Materials and methods” for more details). (A) MCF-7 cells. (B) T47D cells. (A1, B1) Non-stimulated cells. (A2, B2) Cells exposed to TME Stimulation. In both panels, the results are from a representative experiment of n = 3, showing similar results. Averages ± SD obtained in n = 3 independent experimental repeats are demonstrated in Supplementary Figure S2A.

Fig 5: TME stimulation does not enrich for ALDH1+ cells but selects for doxorubicin-resistant CD44+/CD24low/- cells(A) MCF-7 breast tumor cells were exposed to TME Stimulation (as in Figure 1). No stimulation = Cells grown with vehicles only. ALDH1 activity was determined using the ALDEFLUOR™ kit, where controls with DEAB inhibitor were used to set polygon gate location. (A1, A2) Non-stimulated cells. (B1, B2) Cells exposed to TME Stimulation. The results are from a representative experiment of n = 3, showing similar results. Averages ± SD obtained in n = 3 independent repeats are demonstrated in Supplementary Figure S3A and positive technical control of SKBR3 cells is shown in Supplementary Figure S3B. (B) Proportions of CD44+/CD24low/- cells following treatment by doxorubicin. MCF-7 cells were concomitantly exposed to TME Stimulation (as in Figure 1) and to 0.1 µM doxorubicin (or vehicles as controls). The proportion of CD44+/CD24low/- cells was determined by FACS analyses, using fluorescently-labeled specific Abs. Isotype-matched Abs were used as control (Data not shown). *p < 0.05 for the difference between TME-stimulated and non-stimulated cells. The panel sums up the results obtained in n = 3 independent repeats. Results of a representative experiment out of n = 3 experiments, showing similar findings, are presented in Supplementary Figure S4.