Journal: Clinical and Translational Medicine
Article Title: Oncogenic driver and therapeutic target: Prolactin signalling axis in retroperitoneal sarcoma
doi: 10.1002/ctm2.70669
Figure Lengend Snippet: Functional characterisation of PRL‐mediated proliferation and chemoresistance in sarcoma models. (A and B) Secretory PRL quantification by ELISA confirming knockdown efficiency in the culture medium, n = 3. (C–F) Growth suppression following PRL depletion: CCK‐8 time‐course assay ( n = 5) and colony formation capacity ( n = 3) in PRL‐knockdown models. (G–J) Recombinant PRL (50 ng/mL)‐induced proliferative enhancement: (G and H) CCK‐8 ( n = 5) and (I and J) colony formation ( n = 3) in HT1080 and SW872 lines. (K–P) PRLR‐dependent proliferation modulation: (K–N) CCK‐8 dose‐response ( n = 5) and (O‐P) colony formation ( n = 3) analysis post‐PRLR perturbation. (Q and R) After treating SW872 and HT1080 cells with PRLR antibody rolinsatamab talirine (20 µg/mL), the effect on cell proliferation was detected by the CCK8 method, with n = 5. (S and T) Xenograft tumourigenesis assay demonstrating impaired SW872 growth with PRL knockdown ( n = 9). (U) A single SW872 clone exhibiting the lowest PRL expression among the pooled PRL‐knockout cells was isolated by limiting dilution cloning, expanded in culture and validated for PRL protein levels via ELISA. (V) Cell proliferation was assessed using the CCK‐8 assay following stable PRL knockout ( n = 5 biological replicates). (W) Bromocriptine‐mediated antiproliferative effects were evaluated in parallel in wild‐type and PRL‐knockout SW872 cell lines using the CCK‐8 assay ( n = 5). (X) In vivo efficacy was determined in a subcutaneous xenograft mouse model, wherein tumour growth derived from wild‐type or PRL‐knockout SW872 cells was monitored following bromocriptine treatment, n = 7. (Y and Z) Chemosensitisation effects: PRL pretreatment (50 ng/mL) enhances cytotoxicity of RG7112/abemaciclib/doxorubicin/gemcitabine, n = 5, RG7112 (10 µM), abemaciclib (5 µM), doxorubicin (2 µM), gemcitabine (10 µM). (a) Western blot analysis was performed to detect MDM2 expression in human adipocytes, liposarcoma cell lines (SW872, 93T449, 94T778), fibrosarcoma cell line HT1080 and clinically isolated liposarcoma cell lines established in our laboratory. (b) Western blot analysis was performed to detect MDM2 in 12 clinical retroperitoneal liposarcoma tissues and its corresponding paracancerous tissues, 6 clinical retroperitoneal fibrosarcoma tissues and corresponding paracancerous tissues. (c) Therapeutic synergy evaluation: bromocriptine combined with RG7112 in WEHI164 fibrosarcoma murine model, RG7112: 100 mg/kg per day, bromocriptine: 10 mg/kg, twice daily, ( n = 6). Data expressed as mean ± SD unless specified; * p < .05, ** p < .01, *** p < .001 by two‐tailed Student's t ‐test; ns: not significant.
Article Snippet: Antibodies were purchased from the following sources: PRLR (A4121; ABclonal, China), c‐MYC (A19032; ABclonal), c‐MYC (GB13076‐50; Servicebio, China), PRL (GB14138‐50; Servicebio), p‐STAT3 (AP0705; ABclonal), p‐STAT3 (AP0758; ABclonal), STAT3 (9139s; CST, USA), STAT3 (A21228; ABclonal) and GAPDH (TA08; ZSGB‐BIO, China).
Techniques: Functional Assay, Enzyme-linked Immunosorbent Assay, Knockdown, CCK-8 Assay, Recombinant, Expressing, Knock-Out, Isolation, Cloning, In Vivo, Derivative Assay, Western Blot, Two Tailed Test
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