Journal: BMC Cancer

Article Title: Autophagy modulating therapeutics inhibit ovarian cancer colony generation by polyploid giant cancer cells (PGCCs)

doi: 10.1186/s12885-022-09503-6

Figure Lengend Snippet: Polyploid Giant Cancer Cell (PGCC) life cycle and experimental setup for CAOV3 and OVCAR3 PGCC induction and subsequent progeny generation. A PGCC life cycle depicting a diploid cancer cell undergoing chemotherapy-induced polyploidization followed by neosis – the generation of diploid progeny through depolyploidization. B Timeline for assessing the effect of the autophagy modulators hydroxychloroquine (HCQ), nelfinavir mesylate (NFV), and rapamycin (Rapa) on carboplatin- (CPt) or docetaxel- (DTx) induced PGCC formation and subsequent progeny generation. CAOV3 or OVCAR3 cells were seeded on day 0, treated with chemotherapy drugs CPt or DTx from day 1 through day 4, allowed to recover for three days. To assess the effect of the autophagy modulators on PGCC formation, cells were treated with HCQ, NFV, or Rapa concurrently with CPt or DTx, then fixed, stained, and imaged on day 7 as described in . C To assess the effect of the autophagy modulators on PGCC progeny development, cells were treated with CPt or DTx from day 1 through day 4 and allowed to recover from day 4 – day 7 as above, but then PGCCs were separated based on cell size and re-plated, allowed to adhere overnight, then treated with HCQ, NFV, or Rapa on day 8 through day 14 – a time during which PGCCs form progeny. Finally, PGCC progeny were fixed, stained with crystal violet, imaged, and quantified as described in . D Clinically available autophagy drugs used in this study are briefly diagrammed for their mechanisms in terms of proteotoxic stress and modulation of functional autophagy

Article Snippet: Specifically, CAOV3 and OVCAR3 cells were trypsinized and filtered through 30 µm size-exclusion cell strainers for CAOV3 cells (pluriSelect, #43-50030-03) and with cell strainers of 10 µm size-exclusion for OVCAR3 cells (pluriSelect, #43-50010-03).

Techniques: Staining, Functional Assay

Journal: BMC Cancer

Article Title: Autophagy modulating therapeutics inhibit ovarian cancer colony generation by polyploid giant cancer cells (PGCCs)

doi: 10.1186/s12885-022-09503-6

Figure Lengend Snippet: Carboplatin (CPt) and docetaxel (DTx) induce the formation of CAOV3 and OVCAR3 PGCCs. A Representative photomicrographs of CAOV3 and OVCAR3 PGCCs formed by 10 µM CPt or 5 nM DTx are shown. E-Cadherin stain (green) was used to allow cytoplasmic determination, while Hoescht 33342 (blue) was used for nuclei quantitation. Note E-Cadherin well delineates cell–cell junctions in bound OVCAR3 cells. Scale bars are 10 µm. B CAOV3 cells were treated with 10 µM CPt or vehicle control (72 h treatment) and assessed for PGCC formation (72 h after CPt removal, by Hoescht 33342 quantitation). Threshold for PGCC identification is indicated by dashed gray line. C CAOV3 cells were similarly assessed for PGCC formation induced by 5 nM DTx. D , E OVCAR3 cells were similarly assessed for PGCC formation rates. P -values indicated Fisher’s exact test result, and 100 cells quantified from two independent experiments are shown

Article Snippet: Specifically, CAOV3 and OVCAR3 cells were trypsinized and filtered through 30 µm size-exclusion cell strainers for CAOV3 cells (pluriSelect, #43-50030-03) and with cell strainers of 10 µm size-exclusion for OVCAR3 cells (pluriSelect, #43-50010-03).

Techniques: Staining, Quantitation Assay

Journal: BMC Cancer

Article Title: Autophagy modulating therapeutics inhibit ovarian cancer colony generation by polyploid giant cancer cells (PGCCs)

doi: 10.1186/s12885-022-09503-6

Figure Lengend Snippet: Hydroxychloroquine (HCQ) does not prevent CPt-or DTx–induced PGCC formation in CAOV3 and OVCAR3 cells. A CAOV3 cells were treated with 33 µM HCQ or vehicle control for 72 h and allowed to recover in fresh drug-free media for 72 h prior to PGCC quantitation by Hoescht 33342 staining. B CAOV3 cells were co-treated with CPt and HCQ or vehicle control for 72 h and allowed to recover in fresh drug-free media for 72 h prior to PGCC quantitation. C CAOV3 cells were co-treated with DTx and HCQ or vehicle control for 72 h and allowed to recover in fresh drug-free media for 72 h prior to PGCC quantitation. D - F Identical experiments as in ( A-C ) were performed using OVCAR3 cells. P -values indicated Fisher’s exact test result, and 100 cells quantified from two independent experiments are shown

Article Snippet: Specifically, CAOV3 and OVCAR3 cells were trypsinized and filtered through 30 µm size-exclusion cell strainers for CAOV3 cells (pluriSelect, #43-50030-03) and with cell strainers of 10 µm size-exclusion for OVCAR3 cells (pluriSelect, #43-50010-03).

Techniques: Quantitation Assay, Staining

Journal: BMC Cancer

Article Title: Autophagy modulating therapeutics inhibit ovarian cancer colony generation by polyploid giant cancer cells (PGCCs)

doi: 10.1186/s12885-022-09503-6

Figure Lengend Snippet: Nelfinavir mesylate (NFV) does not prevent CPt- or DTx–induced PGCC formation in CAOV3 or OVCAR3 cells. A CAOV3 cells were treated with 10 µM NFV or vehicle control for 72 h and allowed to recover in fresh drug-free media for 72 h prior to PGCC quantitation by Hoescht 33342 staining. B CAOV3 cells were co-treated with CPt and NFV or vehicle control for 72 h and allowed to recover in fresh drug-free media for 72 h prior to PGCC quantitation. C CAOV3 cells were co-treated with DTx and NFV or vehicle control for 72 h and allowed to recover in fresh drug-free media for 72 h prior to PGCC quantitation. D-F Identical experiments as in ( A-C ) were performed using OVCAR3 cells. P -values indicated Fisher’s exact test result, and 100 cells quantified from two independent experiments are shown

Article Snippet: Specifically, CAOV3 and OVCAR3 cells were trypsinized and filtered through 30 µm size-exclusion cell strainers for CAOV3 cells (pluriSelect, #43-50030-03) and with cell strainers of 10 µm size-exclusion for OVCAR3 cells (pluriSelect, #43-50010-03).

Techniques: Quantitation Assay, Staining

Journal: BMC Cancer

Article Title: Autophagy modulating therapeutics inhibit ovarian cancer colony generation by polyploid giant cancer cells (PGCCs)

doi: 10.1186/s12885-022-09503-6

Figure Lengend Snippet: Rapamycin (Rapa) does not increase PGCC formation in CAOV3 or OVCAR3 cells. A CAOV3 cells were treated with 10 nM Rapa or vehicle control for 72 h and allowed to recover in fresh drug-free media for 72 h prior to PGCC quantitation by Hoescht 33342 staining. B CAOV3 cells were co-treated with CPt and Rapa or vehicle control for 72 h and allowed to recover in fresh drug-free media for 72 h prior to PGCC quantitation. C CAOV3 cells were co-treated with DTx and Rapa or vehicle control for 72 h and allowed to recover in fresh drug-free media for 72 h prior to PGCC quantitation. D-F Identical experiments as in ( A-C ) were performed using OVCAR3 cells. P -values indicated Fisher’s exact test result, and 100 cells quantified from two independent experiments are shown

Article Snippet: Specifically, CAOV3 and OVCAR3 cells were trypsinized and filtered through 30 µm size-exclusion cell strainers for CAOV3 cells (pluriSelect, #43-50030-03) and with cell strainers of 10 µm size-exclusion for OVCAR3 cells (pluriSelect, #43-50010-03).

Techniques: Quantitation Assay, Staining

Journal: BMC Cancer

Article Title: Autophagy modulating therapeutics inhibit ovarian cancer colony generation by polyploid giant cancer cells (PGCCs)

doi: 10.1186/s12885-022-09503-6

Figure Lengend Snippet: HCQ, NFV, and Rapa prevent colony outgrowth from chemotherapy-induced PGCCs in CAOV3 and OVCAR3 cells. A CAOV3 PGCCs were initially formed using the 72 h CPt treatments followed by fresh drug-free media for 72 h. These PGCCs were isolated using size-exclusion filters and replated. 33 µM HCQ, 10 µM NFV, or 10 nM Rapa was then added and replenished 72 h after first drug addition. Resultant colonies originating from isolated PGCCs were then stained and quantified using crystal violet. Representative stained colonies are shown. B A similar assay was used to query CAOV3 PGCCs formed by 5 nM DTx. C, D. Identical assays as in ( A , B ) were performed for OVCAR3 cells. Data are mean ± standard deviation of three independent experiments. Scale bar indicates 2 mm. * P < 0.05 and ** P < 0.01 by Student’s t-test comparing drug-treated to vehicle control

Article Snippet: Specifically, CAOV3 and OVCAR3 cells were trypsinized and filtered through 30 µm size-exclusion cell strainers for CAOV3 cells (pluriSelect, #43-50030-03) and with cell strainers of 10 µm size-exclusion for OVCAR3 cells (pluriSelect, #43-50010-03).

Techniques: Isolation, Staining, Standard Deviation