Fig 1: Hh-activated CAFs form a reversible, chemo-resistant CSC niche via FGF pathway activity. a RT-qPCR analysis of Fgf5 expression in M6 whole tumors. n = 3 biological replicates per treatment group with three technical replicates per assay. Statistical significance was determined using unpaired two-tailed Student’s t test with equal s.d. b Expression of Fgf5 at single-cell resolution in M6 tumor models. Freshly isolated M6-Ctrl (blue), M6-Hh (magenta), and M6-Hh tumors + SMOi (100 mg/kg/bid; violin dots) were captured using 10x Chromium technology. t-SNE plot represents the subcellular clusters present in the breast TME of M6 tumors. A sole subset of Hh-activated CAFs exhibits robust expression of Fgf5 in M6-Hh tumors. c Representative immunohistochemistry staining for phospho-FGFR on M6 tumors. Scale bars: 100 µm. Quantification of phospho-FGFR-positive cancer cells at the tumor-stromal interface. n = 4 biological replicates per treatment group; statistical significance was determined using unpaired two-tailed Student’s t test with equal s.d. d Relative mRNA expression of CSC markers Id3 and Sox10 in M6-Ctrl cells treated with DMSO (vehicle; blue) or recombinant FGF5 (red) in vitro. n = 3 biological replicates with three technical replicates per experiment; statistical significance was determined using unpaired two-tailed Student’s t test. e Primary and secondary tumorsphere formation of M6-Ctrl cells treated with DMSO (vehicle; blue) or recombinant FGF5 (red). Sphere Formation Efficiency (SFE) values in % are mean ± s.e.m.; n = 3 biological replicates with three technical replicates per tumorsphere assay. Representative phase contrast micrographs of M6-Ctrl spheres upon recombinant FGF5 stimulation. Scale bars: 100 µm. f Cell viability of M6-Ctrl and M6-Hh cells treated with indicated agents (n = 5 biological replicates with six technical replicates each). Statistical significance was determined using unpaired two-tailed Student’s t test with equal s.d.; *P < 0.05; **P < 0.01; ***P < 0.001. Bars represent mean ± s.e.m.
Fig 2: Phase I clinical trial of docetaxel and SMO inhibitor, NVP-LDE225 (EDALINE) in patients with advanced TNBC. a Representative computed tomography (CT) images from a patient with complete radiological response. The 54-year-old postmenopausal woman was diagnosed with recurrent metastatic TNBC in the lungs with one measurable lesion on the right upper lobe (red arrow) and several nonmeasurable lesions (blue arrows). Therapeutic response was evaluated according to RECIST criteria version 1.1. The remaining structure seen in the right upper lobe corresponds to the azygos vein (violet arrow). b Representative HH and GLI1 immunostaining of treatment-naive tumor specimens from patients enrolled in the EDALINE trial. The left panel is representative of patients with high HPAS (characterized by high epithelial HH ligand and high stromal GLI1 expression) while the right panel represents low/intermediate epithelial HH and low stromal GLI1 expression. Scale bars: 100 µm. c Representative immunohistochemistry staining for phospho-FGFR, collagen deposition depicted by SHG imaging and concomitant phospho-FAK and ALDH1 stem cell marker expression in treatment-naive tumor specimens with high HPAS from the EDALINE trial. The left panel represents tissue derived from the patient who experienced a complete clinical response, the middle is from the patient with stable disease and the right panel represents tissue from the patient with high HPAS who progressed on the prescribed regimen. Scale bars: 100 µm. The white arrows illustrate examples of co-staining. d Graphical summary: Paracrine Hh signaling in TNBC drives a reversible stem-like, chemo-resistant phenotype via FGF signaling and ECM remodeling. CAFs represent the primary cells of the breast TME that respond to Hh ligand stimulation. Hh-activated CAFs enhance ECM collagen deposition and express FGF5 to establish a supportive niche for chemo-resistant cancer stem cell maintenance. This study strongly highlights a novel rational approach targeting both the tumor cells and their surrounding signaling support using SMO inhibitors to overcome chemoresistance in patients with TNBC
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