Fig 2: Conventional and unconventional approaches in tissue evaluation are shown. As illustrated at the top of this figure, tissues may be stained with dyes to reveal structural features, as illustrated in the H&E stained tissue sample on the right hand side. This image shows a DCIS lesion exhibiting the solid architecture. On the lower portion of this figure we illustrate the digital pathology approach used in the procedures described below. In this case separate images of CD44 and CD24 immunofluorescence are collected, then ratioed in silico to reveal a computed image wherein the presence of CD44hi/CD24lo cells are highlighted. Thus, a subpopulation of tumor cells can be identified. In this case, an invasive breast disease was studied. Note the presence of a subset of highly positive CD44hi/CD24lo cells at the periphery of the duct.

Fig 3: A three-dimensional rendering of all BRIM data for DCIS patients.Values of the parameters CD74/CD59 (z axis), CD44/CD24 (x axis) and N-cadherin/E-cadherin (y axis) are plotted for each DCIS patient. Some samples18 had BRIM values far from the origin (0, 0, 0). Of the samples near the origin, 5 had scores of <50 for CD74/CD59 and 0–2 for the other two parameters. Note that DCIS samples near the walls of the three-dimensional plot could be interpreted as false-negatives if only one BRIM parameter was measured.

Fig 4: Illustration of BRIM.A DCIS section was labeled with anti-CD44 (A) and anti-CD24 (B). (Panels A,B) were prepared identically. (Panel C) reveals intraductal CD44hi/CD24lo cells at high contrast. The white arrows identify a region of CD44hi/CD24lo cells that are included in the quantitative line profile analyses of (panels D–F). (Panels D–F) show quantitative line profile analyses (the line profile extends from the right to left hand sides of the image at the level of the arrow). Noise reduction and contrast enhancement are seen in the ratio image of (panel F). The pseudocolor image in (panel C) is scaled as indicated by the bar on the right side. (Distance scale range is shown on the lower left side of (panel A).

Fig 5: Frequency of CD44+CD24-pSTAT3+ cells and dependency on JAK/STAT3 signaling in IBC. A, Representative immunofluorescence analysis of CD44, CD24, and pSTAT3 in IBC samples. Scale bars, 10 µm. Nuclei were stained with DAPI. B, Percent of the four cell types in each IBC tumor of the indicated subtype. C, Percent pSTAT3+ cells in the four cell types (n = 33 patients). D, Percent pSTAT3+ cells within the four cell types in each tumor classified by subtype. E, Graphs depicting SUM149 (top) and SUM190 (bottom) xenograft tumor volume in mice treated with vehicle, paclitaxel (PTX), ruxolitinib (RUX), or the combination, with green arrow depicting treatment start when tumors were first palpable (~20 mm3 for SUM149 and ~60 mm3 for SUM190). Error bars, SEM; n = 5 mice with 2 tumors/mouse. P values were calculated by two-way ANOVA. F, Graph of tumor weights at endpoint from experiment shown in E. Error bars, SD. G, Immunoblot analysis of pSTAT3 and STAT3 in xenografts. ACTB was used as a loading control. Dot plot depicts quantification of pSTAT3/STAT3 ratios. P values for B–D and F–G calculated by one-way ANOVA with Tukey multiple comparisons.