Fig 1: CD10 supports CSCs by cleavage of OGP. A,B) Indicated tumor cell lines after prolonged mammosphere culture were treated with 5 × 10-9 m OGP, OGP (L2A), or OGP (Y10A). Representative images of mammosphere formation in A) BT-474mammo cells and B) MCF-7mammo cells. Scale bar, 100 µm. C) MCF-7 and D) BT-549 cells pretreated with 100 × 10-9 m biotinylated OGP, due to an undetectable signal of fluorescence-labeled peptide at a lower concentration,[ 124 , 125 , 126 , 127 , 128 , 129 ] without (-) or with 10 × 10-6 m non-biotinylated OGP (L2A) or OGP (Y10A) were incubated with neutravidin-Texas red. The cell surface-bound biotinylated OGP was evaluated by fluorescence microscopy. Scale bar, 50 µm. E–J) Indicated cell lines were cultured alone (-) or cocultured with the indicated CAFs in the presence of 500 × 10-9 m OGP (L2A) or OGP (Y10A). Unlabeled peptides were used at 100 times more than the physiological concentration for the competitive binding assay as previously reported.[ 130 , 131 ] E,F) Percentage of ALDH1+ cells in E) MCF-7 and F) BT-549 cell (mean ± SEM, n = 4). G,H) Representative immunofluorescent images of G) PKH26 and H) Numb in MCF-7 cells. Scale bar, 50 µm. I,J) Representative images of mammosphere formation in I) MCF-7 and J) BT-474 cells. Scale bar, 100 µm. K,L) After coculture with (E–J) cancer cells were treated with docetaxel. Apoptosis of tumor cells was determined after 12 h. K) Percentage of apoptotic MCF-7 and BT-549 cells evaluated by flow cytometry (mean ± SEM, n = 4). L) Apoptosis of SK-BR3 cells determined by western blotting for cleaved caspase-3 and PARP (n = 3).
Fig 2: OGP suppresses CSCs via the YGFGG domain that can be cleaved by CD10. A–E) Indicated tumor cell lines after prolonged mammosphere culture were treated with 5× 10-9 m OGP1–14, OGP10–14, or OGP1–9, which is approximately equivalent to the OGP concentration of 10% FBS used in conventional cell culture. A,B) Representative images of mammosphere formation in A) MCF-7mammo cells and B) BT-474mammo cells. Scale bar, 100 µm. C,D) Percentage of ALDH1+ cells in C) MCF-7mammo cells and D) BT-549mammo cells (mean ± SEM, n = 3). E) Percentage of CD44highCD24low cells in MCF-7mammo cells (mean ± SEM, n = 3). F–J) Indicated tumor cell lines after prolonged mammosphere culture were treated with 5 × 10-9 m OGP1–14 with or without 5 µg mL-1 rhCD10. F,G) Representative images of mammosphere formation in F) MCF-7mammo cells and G) BT-474mammo cells. Scale bar, 100 µm. H,I) Percentage of ALDH1+ cells in H) MCF-7mammo cells and I) BT-549mammo cells (mean ± SEM, n = 3). J) Percentage of CD44highCD24low cells in MCF-7mammo cells (mean ± SEM, n = 3).
Fig 3: Hydrolyzation of OGP by CD10 provides a potential therapeutic target for cancer treatments. A–E) MCF-7 cells alone (-) or with indicated CAFs were injected into fat-pads of 6-weeks-old NOD/SCID mice. Docetaxel was administrated when tumors were palpable. A) Representative images of xenograft growth monitored by PET-CT (n = 6 per group). The circles indicate xenografts. B) Tumor growth curves (n = 6 per group). Mean ± SEM, ***P < 0.001 by one-way ANOVA at week 6. C) Representative images of a-SMA, CD10, and ALDH1 immunofluorescence staining in the sections of xenografts (n = 6 per group). Scale bar, 50 µm. D) The proportions of ALDH1+ tumor cells isolated from harvested xenografts were determined by flow cytometry (mean ± SEM, n = 5 per group). E) Representative images of immunofluorescence staining for apoptotic tumor cells (TUNEL+CK+) in the sections of xenografts (n = 6 per group). F) OGP levels in tumor interstitial fluid of CD10+ CAFhigh (n = 29) and CD10+ CAFlow/- (n = 35) human breast cancer samples were evaluated by ELISA (mean ± SEM). ***P < 0.001 by Mann-Whitney U tests. G) Biopsies from patients with different responses to neoadjuvant chemotherapy were obtained before treatments. OGP levels in tumor interstitial fluid were evaluated by ELISA. pCR, pathological complete response, no invasive carcinoma or ductal carcinoma in situ in breast or axilla, n = 13; B- or A-only pCR, pCR in breast or axilla only, n = 4; PR, partial remission, n = 62; SD, stable disease, n = 37; PD, progressive disease, n = 8. Mean ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001 by Mann-Whitney U tests. H) Schematics highlighting the primary findings of this study.
Fig 4: CD10 in CAFs sustains cancer stemness and chemoresistance. A–E) Indicated tumor cell lines were cultured alone (-) or cocultured with CD10+GPR77+-depleted CAFs (CD10+GPR77+-d) or paired CD10+GPR77+ CAFs transduced without (-) or with shGFP or shCD10. A) Representative images of mammosphere formation in MCF-7 cells. Scale bar, 100 µm. B) Representative images of PKH26 and Numb immunofluorescence staining of MCF-7 cells. Scale bar, 50 µm. C,D) Percentage of ALDH1+ cells in C) MCF-7 and D) BT-549 cells detected by flow cytometry. Numerical values are presented as percentage (mean ± SEM, n = 3). E) Percentage of CD44highCD24low cells in MCF-7 cells cocultured with indicated CAFs was detected by flow cytometry. Numerical values are presented as percentage (mean ± SEM, n = 3). F,G) Representative flow cytometry plots for F) MCF-7 and G) BT-549 cells treated with docetaxel after being cultured alone (-) or cocultured with the indicated CAFs. The proportion of Annexin V+/Propidium iodide- (early apoptosis) and Annexin V+/Propidium iodide+ (late apoptosis) cells is shown. The numerical values indicate Annexin V+ percentage. Data are represented as the mean ± SEM of F) n = 4 or G) n = 3 independent experiments. H) Representative immunoblots for cleaved/total caspase-3 and PARP in SK-BR3 cells treated with docetaxel after being cultured alone (-) or cocultured with indicated CAFs (n = 3). I–L) MCF-7 cells were cocultured with CD10+GPR77+ CAFs or CD10+GPR77+-depleted CAFs transduced with lentiviral empty vectors (vector) or CD10-expressing vectors (CD10 overexpression). I) Representative images of mammosphere formation. Scale bar, 100 µm. J) Percentage of ALDH1+ cells detected by flow cytometry. Numerical values are presented as percentage (mean ± SEM, n = 3). K) Pcercentage of CD44highCD24low cells detected by flow cytometry. Numerical values are presented as percentage (mean ± SEM, n = 3). L) After the coculture, MCF-7 cells were treated with docetaxel. The proportion of Annexin V+/Propidium iodide- and Annexin V+/Propidium iodide+ was detected by flow cytometry. The numerical values indicate Annexin V+ percentage (mean ± SEM, n = 3).
Fig 5: CD10 expression in CAFs. A) Representative flow cytometry plot of gating strategy to identify the CD10+GPR77+ CAF subset in breast cancer clinical samples. B) Representative images of CD10, GPR77, and a-SMA immunofluorescence staining in human breast cancer sections. White arrows indicate CD10+GPR77+ CAFs. Scale bar, 50 µm. C) CD10 levels in indicated mammospheres and CD10+GPR77+ CAFs (n = 3). D) CD10 levels in indicated tumor cells treated with docetaxel and CD10+GPR77+ CAFs (n = 3).
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