Fig 2: FAP(hF1) UCAR T-cells successfully deplete CAFs and accumulate in TNBC tumor stroma in vivo (A) Schematic of UCAR T-cell treatment and analysis of orthotopic TNBC tumors implanted in NSG mice. (B) Bar graph representing weights of tumor harvested from mice treated with control UT, FAP(hF1) UCAR, FAP(mF3) UCAR or Meso UCAR T-cells as per schematic in (A). Bars show the means ± SEM; P-values determined by Student t test (two-tailed, unpaired), n=2. (C) Flow cytometry plot of human CD45+ UCAR T-cell population in orthotopic TNBC tumors harvested from mice treated as indicated. (D) Box and whiskers plot representing quantitation of total number of CD45+ cells per gram tumor, calculated using (C). P-values determined by Student t test (two-tailed, unpaired), n=2. (E) Stacked bars plot representing CD4+ and CD8+ population, as percentage of total human CD45+ cells, in orthotopic TNBC tumors from mice treated with indicated UCAR T-cells. Bars show the means ± SEM, n=2. (F) Immunohistochemistry for detection of human CD8+ cells in orthotopic TNBC tumors from mice treated with indicated UCAR T-cells. Yellow arrows indicate human CD8 stained cells. (G) Bar graph representing quantitation of (F), each section divided into 4 fields of view. Bars show the means ± SEM; P-values determined by Student t test (two-tailed, unpaired), n=2. (H) Box and whiskers plot representing quantitation of total number of human FAP+ cells per gram tumor, as determined by flow cytometry analysis of orthotopic TNBC tumors from mice treated with indicated UCAR T-cells. P-values determined by Student t test (two-tailed, unpaired), n=2. (I) Trichrome staining of orthotopic TNBC tumor sections from mice treated with indicated UCAR T-cells. Collagen network indicated by blue staining. (J) Bar graph representing quantitation of blue stained Collagen area per section (F), measured using ImageJ. Bars show the means ± SEM; P-values determined by Student t test (two-tailed, unpaired), n=2. *P < 0.05; **P < 0.01; ns, not significant.

Fig 3: TSCs I and II and TNCs do not express hematopoietic markers. The panel shows CD14, CD19, CD34, CD45, and HLA-DR surface marker expression for all cell types through flow cytometry analysis. Cell count bar charts display marker expressions (% of positive events) on single cells.

Fig 4: Flow cytometry characterization of hSCs from schwannoma. A) Representative cytofluorimetric diagram dot-plot forward scatter (FSC-H) vs. side scatter (SSC-H; 106) of pre-purified cell population obtained from primary cell preparation. The P2 area (highlighted in red) represents the total cell population analysed (87.32 %), from which cell debris has been excluded; B) Representative cytofluorimetric dot-histogram of CD271+ cells (red peak), showing that just after post-tumour digestion only the 6.84 % of cells were immunopositive; C) The CD271+ cells (red peak) rose to 57.13 % one week after culturing; D) Then following purification the CD271+ cells (red peak) increased to 83.30 %; E) Before purification, the 38.39 % of the mixed culture was composed by CD45+ cells (violet peak). In all dot-histograms (B, C, D, E), results are expressed as fluorescent intensity vs. number of events; for each analysis 104 event were counted; APC-H, allophycocyanine fluorescent label; Qdot 800-H, fluorescent label (Thermo Fischer); F) Chart representing the percentage of cells of different origin in the mixed culture, before purification: 57.13 % CD271+ cells; 38.39 % CD45+ cells; 4.48 % unknown cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig 5: Characterization of hPDLSCs and changes in the cell cytoskeleton and viability after tension force application. (A) Morphology of the 3th generation hPDLSCs. Original magnification ×40. (B) After 3 weeks of osteogenic induction, hPDLSCs formed calcified nodules with a reddish coloration. Original magnification ×100. (C) After 2 weeks of adipogenic induction, hPDLSCs developed spherical lipid accumulations. Original magnification ×100. (D) The expression rates of positive markers (Strol-1, CD44, CD146, and CD90) were 98.2%, 99.2%, 99.8% and 99.6%, respectively, while the negative markers CD45 and CD34 showed positive expression rates of 0.51% and 0.94%, respectively. (E) The growth curve was represented as a “S shape.” (F) The cell viability in the 0, 1, 3, 6, 9, and 12 h groups showed no significant differences, as shown by CCK-8 assays. (G) Immunofluorescence staining of F-actin in hPDLSCs in the 0, 1, 3, 6, 9, 12 h groups. (red, F-actin; blue, DAPI). Scale bar = 10 μm. Data are presented as “mean ± standard deviation”