Fig 2: Inhibition of the insulin signaling pathway enhanced the immune response and prolonged the survival time of TNBC-bearing mice. a Schematic representation of the dosing regimen. b KM analysis showing the survival of the negative control (NC) (n = 6), DOX (n = 6), HNMPA (n = 7), and combination (DOX + HNMPA, n = 7) groups. The statistical significance of the differences between the four treatment groups was evaluated via the log-rank test. c HE staining; i, representative immune cell aggregation in HNMPA (2 mm, orange circle); ii, macrophages (50 µm, black arrow) and neutrophils (50 µm, yellow circle); iii, lymphocyte infiltration (50 µm, yellow arrow) in tumors (50 µm, red arrow); d HE staining, tumor necrosis (50 µm) and lymphocyte infiltration in the non-necrotic zone of tumors (50 µm, yellow arrow and green circle). e, f Expression level of CD45 (red) detected by immunofluorescence staining in the HNMPA-treated and combination-treated groups; n = 30 fields of view per group. g Expression level of Ki67 detected by IHC in the four groups (each group, n = 30 fields of view). h–j Activation of factors downstream of the insulin signaling pathway (2 µm). p-AKT (red), p-ERK (green), every group, n = 30 fields of view. The data are presented as the means ± s.t.d. Compared with those of the NC group, the statistical significance was determined via one-way ANOVA followed by Dunnett’s T3 multiple comparison test

Fig 3: Differentiation of NCP5s and NCP6s into CD66+ cells. A Representative flow cytometry gating strategy used to analyze CD66b+ cells derived from NCP5s and NCP6s cultured with SFGc for 5 days (n = 10). Plots showing the identification of basophils (bordeaux gate), monocytes (light blue gate), eosinophils (orange gate) and undifferentiated cells (gray gate), as well as PMs (beige gate), MYs (pink gate), MMs (light red gate), BCs (red gate) and SNs (dark red gate) within the CD66b+ cells (green gate). B Bar graphs showing the percentages of CD66b+ cells (green contour, mean ± s.e.m. refers to total CD45+ cells, n = 10), eosinophils (orange contour), basophils (bordeaux contour), monocytes (light blue contour) and undifferentiated cells (gray contour) derived from NCP5s and NCP6s cultured for 5 days with SFGc. C Bar graphs showing the percentages of CD66b+PMs (beige contour), MYs (pink contour), MMs (light red contour), BCs (red contour) and SNs (dark red contour) derived from NCP3s, NCP4s, NCP5s and NCP6s cultured for 5 days with SFGc (mean ± s.e.m., n = 5 for NCP3s and NCP4s; n = 10 for NCP5s and NCP6s). D Bar graph representing the generation of SNs (alias CD10+ cells) from NCP3s, NCP4s, NCP5s and NCP6s treated with SFGc for 5 days (mean ± s.e.m., n = 5 for NCP3s and NCP4s; n = 10 for NCP5s and NCP6s). E Bar graph displaying the fold expansion of purified NCP3s, NCP4s, NCP5s and NCP6s treated with SFGc for 5 days (mean ± s.e.m., n = 5 for NCP3s and NCP4s; n = 10 for NCP5s and NCP6s). D, E Statistical analysis was performed via one-way ANOVA and Tukey’s post hoc test. * = p < 0.05, ** = p < 0.01, *** = p < 0.001

Fig 4: Identification of NCP5s and NCP6s in BM-LDCs. A Flow cytometry gating strategy illustrating how to identify Lin-SSChiCD66b-CD11b-CD16-CD64dimCD115-CD117+CD71hiCD45RA+NCP5 (panel VII, light green gate) and Lin-SSChiCD66b-CD11b-CD16-CD64dimCD115-CD117+CD71hiCD45RA-NCP6 (panel VII, red gate) within the SSChiCD45+ cells in BM-LDCs (panel I), after the exclusion of mature CD16-eosinophils (panel II), mature CD11b+neutrophils (panel III), immature CD64+monocytes (panel IV), CD117-CD71dim/hiPM w/o eNePs (panel V, yellow gate) and CD71+CD117+CD66b+eNePs (panel VI, blue gate). The bottom panels show the identification of SSClowCD66b-CD34+CD64dimCD115-CD45RA- NCP1s (panel VIII, orange gate), SSClowCD66b-CD34+CD64dimCD115-CD45RA+ NCP2s (panel IX, green gate), SSClowCD66b-CD34dim/-CD64dimCD115-CD45RA+ NCP3s (panel X, magenta gate) and SSClowCD66b-CD34dim/-CD64dimCD115-CD45RA- NCP4s (panel XI, light blue gate). One representative experiment (out of 10 performed, with similar results) is shown. B Histograms depicting the expression of CD15, CD38, CD49d, CD71, and CD117, as well as that of SSC-A, by total CD45+BM-LDC cells, NCP3s, NCP4s, NCP5s, NCP6s, eNePs and PMs w/o eNePs, as defined in panel (A). The data are representative of 1 of 5 independent experiments, with similar results. C Morphology of purified NCPs and PMs. Sorted NCP5s, NCP6, and PMs were stained via the May-Grunwald procedure

Fig 5: Flow cytometry and immunohistochemistry analysis of neutrophil progenitors in the BM-LDCs of HDs, CP-CML patients, and SM patients. A Frequencies of NCP1s, NCP2s, NCP3s, NCP4s, NCP5s, NCP6s, PMs, MYs, MMs and BCs in CD45+ bone marrow-low density cells (BM-LDCs) from HDs (n = 8, gray line), CP-CML patients (n = 13, blue line) and SM patients (n = 6, light yellow). B Bar graph highlighting NCP5s and NCP6s, as reported in panel (A), from HDs (gray contour) and CP-CML patients (blue contour) in CD45+BM-LDCs. C Bar graph showing the frequency of NCP1s, NCP2s, NCP3s, and NCP4s in the narrower area defined as CD34+/CD34dim/- cells in HDs (n = 8, gray contour) compared with those in CP-CML patients (n = 15, blue contour). D, E Bar graph showing the frequency of cGMPs, cMoPs and MDPs in CD34+/CD34dim/- cells from HDs (n = 8, gray contours) compared with those from CP-CML patients (n = 14, blue contours). A, C, D Data are presented as the means ± s.e.m.s. Statistical analysis was performed via the Mann‒Whitney test. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. Human FFPE BM sections from (F) HDs (n = 3) and (G) CP-CML patients (n = 3) were immunostained as indicated. Fewer neutrophil precursors (based on larger and round nuclei) are found in normal tissue than in CP-CML tissue. Numerous NCP4s and NCP6s are instead identifiable in BM CP-CML, as indicated by the colored asterisks (turquoise for NCP4, green for NCP5 and red for NCP6)