Nuclear morphology predicts cell survival to cisplatin chemotherapy

Abstract

The emergence of chemotherapy resistance drives cancer lethality in cancer patients, with treatment initially reducing overall tumor burden followed by resistant recurrent disease. While molecular mechanisms underlying resistance phenotypes have been explored, less is known about the cell biological characteristics of cancer cells that survive to eventually seed the recurrence. To identify the unique phenotypic characteristics associated with survival upon chemotherapy exposure, we characterized nuclear morphology and function as prostate cancer cells recovered following cisplatin treatment. Cells that survived in the days and weeks after treatment and resisted therapy-induced cell death showed increasing cell size and nuclear size, enabled by continuous endocycling resulting in repeated whole genome doubling. We further found that cells that survive after therapy release were predominantly mononucleated and likely employ more efficient DNA damage repair. Finally, we show that surviving cancer cells exhibit a distinct nucleolar phenotype and increased rRNA levels. These data support a paradigm where soon after therapy release, the treated population mostly contains cells with a high level of widespread and catastrophic DNA damage that leads to apoptosis, while the minority of cells that have successful DDR are more likely to access a pro-survival state. These findings are consistent with accession of the polyaneuploid cancer cell (PACC) state, a recently described mechanism of therapy resistance and tumor recurrence. Our findings demonstrate the fate of cancer cells following cisplatin treatment and define key cell phenotypic characteristics of the PACC state. This work is essential for understanding and, ultimately, targeting cancer resistance and recurrence.

Keywords: Cancer therapy resistance; Nuclear morphology; Polyaneuploid cancer cell (PACC) state; Polyploidy.

Fig. 1

Cell death and survival after cisplatin treatment. Relative cell number (percent normalized to population count at 0 days post-treatment removal) as a function of recovery time (A) was determined for treated PC3 cells generated with 72 h 6 µM [LD50] cisplatin treatment. From this data, we also determined (B) the percent change in the death rate for each of the recovery days we investigated. (C) Flow cytometry was used to measure surface expression of annexin V as a marker of apoptosis in treated PC3s generated with 72 h [LD50] cisplatin treatment at days 1, 2, and 3 post-treatment removal compared to parental PC3 prostate cancer cells.

Fig. 2

Increasing cellular and nuclear volume in surviving cells over time. Natural log-transformed (A) cellular volume and (B) nuclear volume of live parental (n = 214) and treated PC3 cells, generated with 72h 6 µM cisplatin treatment, at various timepoints in recovery, day 1 (n = 117), day 3 (n = 184), day 5 (n = 115), day 10 (n = 161), and day 15 (n = 120), was determined by suspending cells in Matrigel and staining DNA with Vybrant™ DyeCycle™ Green Stain (for DNA) and CellTracker™ Orange (for cytoplasm). 3D images were obtained via confocal microscopy and images were rendered for volumetric determination using Imaris 9.8 software. Data was analyzed for differences between groups using a one-way ANOVA, a post-hoc Tukey's multiple comparisons tests was used to detect between-group differences. (C) Nuclear and cytoplasmic volumes were used to calculate a nuclear to cytoplasmic volume ratio of treated PC3s at various timepoints past treatment removal. A Kruskal-Wallis test was used to analyze if there were any significant differences among the samples, Dunn's multiple comparison's test used to test for comparisons between groups. (D) Nuclei were isolated from parental and treated PC3 cells before mounting on PLL-coated dishes. Nuclei were stained for lamin A/C and covered with ProLong^TM Diamond Antifade Mountant with DAPI before imaging on a Zeiss Observer Z1 microscope. (E) The size of isolated nuclei of parental (n = 825) and treated PC3 cells, generated with 72h 6 µM cisplatin treatment, at various timepoints in recovery, day 1 (n = 456), day 5 (n = 536), day 10 (n = 866), was quantitated via a custom CellProfiler 4.0 pipeline. A Kruskal-Wallis test was used to analyze if there were any significant differences among the samples, Dunn's multiple comparison's test used to test for comparisons between groups. For all tests: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Fig. 3

Repeated whole genome doubling in the absence of cell division. PC3 cells were transduced with a (A) Ki67p-T2A-FUCCI construct, gifted from Alexander Zambon. (B, C) Treated PC3s were generated from this reporter cell line with 72 h 6 µM cisplatin treatment, and timelapse microscopy was performed with an Incucyte SX5 with images taken every 30 min after release from treatment, scale bar = 200 µm. Two representative cells are shown. (D) Cisplatin-treated PC3s were tested for de novo DNA synthesis via a Click-It Plus EdU assay according to the manufacturer's protocol. Images were acquired using the 10x objective on a Zeiss Observer Z1 microscope. A custom CellProfiler pipeline was developed and used to quantify DAPI+Edu+ nuclei. (E) PC3 cells were treated with 6 µM cisplatin for 72 h, and immunofluorescence imaging was used to visualize genomic contents as a measure of integrated DAPI intensity for parental PC3 cells (n = 529) and day 1 (n = 635), day 3 (n = 226), day 5 (n = 210), day 10 (n = 167), day 15 (n = 162) post-treatment removal. This was analyzed for differences between groups using a one-way ANOVA, a post-hoc Games-Howell's multiple comparisons tests was used to detect between-group differences. For these tests: *** = p < 0.001, **** = p < 0.0001.

Fig. 4

Characterization of the nuclear morphology of cisplatin-treated prostate cancer cells. Representative immunofluorescence images (A) to demonstrate the nuclear morphology of treated PC3 cells generated with 72 h 6 µM cisplatin treatment following removal from chemotherapy. (B) Live treated PC3 cells at various timepoints in recovery were suspended in Matrigel and staining DNA with Vybrant™ DyeCycle™ Green Stain (for DNA) and CellTracker™ Orange (for cytoplasm). 3D images were obtained via confocal microscopy and images were rendered and volumes determined using Imaris 9.8 software. (C) Population distribution of mononucleated and multinucleated PC3s, derived from images described in (A). Nuclear morphology (mononucleated vs. multinucleated) was manually discriminated and counted using Fiji. At least 250 cells were analyzed per condition. (D) Nuclear morphology was also determined using the 3D images described in (B). The population distribution of mononucleated and multinucleated treated PC3 cells generated with 72h (E) 25 µM etoposide treatment or (F) 5 nM docetaxel treatment was also determined, derived from immunofluorescent images of cells stained for lamin A/C and mounted with DAPI. Nuclear morphology (mononucleated vs. multinucleated) was discriminated and counted in Fiji software.

Fig. 5

The DNA damage response in cisplatin-treated prostate cancer cells. PC3 cells were treated with 6 µM cisplatin for 72h. (A) Immunofluorescence staining and imaging was used to visualize genomic contents (DAPI), sites of DNA damage (gH2AX), and DDR machinery recruitment (53BP1). From these images, a custom CellProfiler 4.0 pipeline was used to determine raw count per unit cellular area of the number of (B) gH2AX foci and (C) 53BP1 foci, as well as (D) the colocalization ratio of gH2AX with 53BP1. (E) The relative proportion of cells with (Y) and without (N) gH2AX pan-nuclear stained nuclei from was determined. (F) For each day post-treatment recovery, cells were classified as mononucleates and multinucleates, and the colocalization ratio of gH2AX with 53BP1 was determined for each cell class. Mononucleated, multinucleated, and mitotically catastrophic cells were classified via a custom CellProfiler 4.0 pipeline, and (G-H) the proportion of multinucleated cells to those experiencing mitotic catastrophe were plotted across the different days post-treatment removal. Mann-Whitney tests were used to detect differences between groups in (B), (C), (D), and (F). For all tests: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Fig. 6

Nucleolar counts, size, and molecular signatures. Treated PC3 cancer cells generated with 72h 6 µM cisplatin treatment were stained for lamin A/C and nucleolin, and were mounted with DAPI, (A) representative images of nucleoli are shown. A custom CellProfiler 4.0 pipeline was used to then determine the (B) number and (C) area of the nucleoli of the treated PC3s throughout the recovery period to post-treatment removal. Nucleolar area was analyzed for differences between groups using a one-way ANOVA, and a post-hoc Tukey's multiple comparisons test was used to detect between-group differences. The lamin A/C stain was used to calculate nuclear area, and thus (D) the nucleolar to nuclear area ratio at each timepoint post-treatment removal could be calculated (E) Expression of phosphorylated nucleophosmin (P-NPM) and total nucleophosmin (NPM) in PC3 cells and PC3s post-treatment removal; a Kruskal-Wallis test was used to analyze if there were any significant differences among the samples, with a Dunn's multiple comparison's post-hoc test. For all statistical tests: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.