Fig. 2

ARV-1502 increases anti-cancer activity of cisplatin and 5FU in cells from a murine primary cancer constitutively expressing DnaK. A Hematoxylin and Eosin (H&E) staining of a spontaneous mass removed from the abdomen of a DnaK positive mouse. The normal architecture is effaced by unencapsulated, poorly demarcated, densely cellular neoplasm composed of round cells arranged in sheets. Neoplastic cells have variably distinct cell borders, a scant amount of eosinophilic cytoplasm, a round, occasionally indented nucleus with finely stippled chromatin and one variably prominent nucleolus. Anisocytosis and anisokaryosis are moderate, and mitotic count is up to 7 in a single high-power field (2.32 mm²). These findings are consistent with a round cell neoplasia. The images of the section were taken at 4× (top) and 40× (bottom). B Western Blot analysis confirms expression of DnaK-V5 in the murine primary cancer cells isolated from the spontaneous tumor. Both eM-DnaK and DnaK expressed in cancer cells were fused to a V5 peptide sequence for convenient detection. eM-DnaK has been used as positive control for antibody detection. Cells isolated from a spleen of a DnaK^−/− mouse were used as negative control. Upper part: membrane probed with anti-V5 antibody recognizing DnaK-V5. Lower part: membrane probed with an anti-GAPDH antibody. Markers specifying the molecular weight (MW) are indicated. C Viability assay of primary murine cancer cells treated with anti-cancer drugs with or without ARV-1502. Cells from the spontaneous tumor mass (round cell neoplasia) detected in a DnaK positive mouse were isolated and then treated with the anti-cancer drugs, cisplatin (25 µM) or 5FU (75 µM). In parallel, the cells were also treated with ARV-1502. We assessed cell viability by using the trypan blue assay. Percentage of alive cells for each treatment are calculated as percentage using untreated cell as reference. The results are representative of two independent experiments using primary cells from two different spontaneous tumors. Statistical differences were tested using Student’s t test. All statistical tests were two sided. ***p < 0.001, **p < 0.01