Fig 1: Birinapant co-treatment sensitized CA125-negative primary HGSC cells to carboplatin.(a) DNA damage, detected by ?H2Ax immunostain, was examined in CA125-negative and -positive populations from three independent primary chemo-naive human HGSCs at 12, 24 and 36 h post-carboplatin treatment. The ?H2Ax foci appeared to resolve more quickly in CA125 negative compared with CA125-positive tumour cells (P=0.02 at 24 h and P<0.0001 at 36 h, unpaired two-sided t-test). Results are mean±s.e.m., n=3 HGSCs 5 fields of view each. Scale bars equal 100 µm. (b) Birinapant and carboplatin co-administration resulted in eradication of all tumour cells only in clinical samples with high cIAP levels in the CA125-negative population (results are mean±s.e.m., n=6 patients 1–4, 7 and 9 versus n=3 patients 5–6 and 8, sample run in triplicate). Cell survival was FACS quantitated based on propidium iodide and AnnexinV negativity. Western blot of pan-cIAP levels in CA125-negative and -positive subpopulations for patients 7–9 is shown. (c) Following in vitro treatment with birinapant and carboplatin co-therapy or monotherapy with either agent, primary chemo-naive human HGSC cells were re-plated in fresh media. No regrowth was observed from birinapant-sensitive HGSC (blue) following co-therapy. In contrast, growth was noted in all specimens after treatment with birinapant or carboplatin as a single agent, or after co-therapy in specimens with low levels of cIAP in their CA125-negative population (pink). Results are mean±s.e.m. (n=6 birinapant sensitive and n=3 birinapant less sensitive, samples run in triplicate). (d) CA125-negative, CA125-positive and bulk populations isolated from birinapant-sensitive primary chemo-naive human HGSC (n=3) were treated with carboplatin, birinapant or both drugs. Compared with vehicle-treated cells, carboplatin alone effectively eliminated the differentiated cells but did not kill the CA125-negative cells. On average birinapant as a single agent was equally effective against both the CA125-negative and -positive cells (P=0.085 unpaired two-sided t-test) but did not cause complete cell death in either population. Results are mean±s.e.m., n=3 HGSC run in triplicate.
Fig 2: MUC16/CA125-positive labeling of EOC cells in mouse tissues and tumor foci in IP-inoculated nude mice (MUC 16/CA 125, scale bar: 50 µm): (a) Negative tissue control (normal mouse liver). MUC16/CA125 immunolabeling, DAB chromogen, hematoxylin counterstain; (b) IP-ovarian carcinoma infiltrating skeletal myofibrils (stars) of the body wall. EOC cell-associated (yellow arrow) and extracellular (red arrows) MUC16-positive labeling. MUC16/CA125 immunolabeling, DAB chromogen, hematoxylin counterstain; (c) IP-inoculated ovarian carcinoma infiltrating the pancreas (stars) with MUC16-positive labeling of cytoplasmic membranes (arrows) and extracellular (* asterisks) labeling of neoplastic cells. MUC16/CA125 immunolabeling, DAB chromogen, hematoxylin counterstain.
Fig 3: A minor population of serous tumour cells that do not express CA125 are capable of in vivo growth.(a) Aggressive tumours not amenable to complete excision during surgery (sub-optimally cytoreduced, (n=6)) had a higher percentage of CA125-negative cells compared with tumours from patients where most disease could be removed (optimally cytoreduced, (n=10); P=0.004 unpaired two-sided t-test). (b) Schema for in vivo tumour initiation assay. Equal numbers of CA125-negative and -positive cells FACS isolated from fresh (n=5) or live-banked (n=7) HGSCs were injected in vivo and grown for 6 months. (c) The CA125-negative cells were the only population capable of generating tumours in eight solid and two matched solid tumour and ascites specimens. In two ascites samples, tumour outgrowth was noted from both populations; however larger tumours were generated from CA125-negative cells. (d) The weight distribution of xenografts arising from CA125-negative, CA125-positive and bulk tumour cells are shown for all 12 clinical samples tested. Results are median±IQR, P<0.01 repeated measure ANOVA. Scale bars are 5 mm.
Fig 4: (A) Scanned IP-implanted, AF(D)NMs-treated nude mouse tissue section showing a section delineated (brown line) of ovarian carcinoma expressing MUC16/CA125 antigens (brown staining). MUC16/CA125 IHC, DAB, chromogen, hematoxylin counterstain. (B) Shows the region of interest/ROI delineated (yellow line) and MUC16-positive signal (red) within the ROI for quantification using the Olympus Count and Measure function (Olympus-Cell Sens software).
Fig 5: Birinapant and carboplatin co-therapy was efficacious against intraperitoneal tumours.(a) Mice bearing intraperitoneal S1-GODL tumours were treated with vehicle, birinapant, carboplatin or birinapant and carboplatin for 4 weeks (n=8 per group). Half the mice were sacrificed at the completion of therapy, and half 6 weeks after cessation of treatment. (b) Immediately post therapy, organs of co-therapy treated mice were free of tumour in all except one (of four) where only small foci of tumours were detected. Six weeks after cessation of therapy, two of four co-therapy treated mice had no evidence of tumour implants, while remaining two mice had only microscopic evidence of disease. In contrast, multiple implanted tumours were detected on the organs of all mice in the vehicle and monotherapy treatment arms (n=4 per group). Scale bars are 500 µm. (c) Pelvic washings were collected, enumerated and concentrated into a cell pellet for each mouse. Immediately post therapy no tumour cells were detected in cell pellets from mice treated with birinapant and carboplatin co-therapy (n=4). In contrast, tumour was detected in pelvic washings of all vehicle or birinapant-treated mice and in one mouse treated with carboplatin (n=4 per group). Six weeks after cessation of therapy, only minute traces of tumour cells were detected in pelvic wash pellets from three of four co-therapy treated mice. Significant tumour burden was detected in all other cohorts. Insets of CA125-stained tumour cells are shown for each cell pellet (n=4 per group). Residual tumour was CA125 negative immediately following carboplatin therapy. Scale bars are 500 µm in hemotoxylin and eosin images, and 100 µm in CA125 insets. (d) Measurement of serum human CA125 in experimental mice demonstrated a correlation between tumour burden of S1-GODL cells and serum human CA125 in vehicle-treated mice. No human serum CA125 could be detected in carboplatin-treated mice immediately post therapy. In contrast, human CA125 was detected in serum of these mice 6 weeks after cessation of treatment. R2 calculated by linear regression.
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