Cells in the polyaneuploid cancer cell (PACC) state have increased metastatic potential

Abstract

Although metastasis is the leading cause of cancer deaths, it is quite rare at the cellular level. Only a rare subset of cancer cells (~ 1 in 1.5 billion) can complete the entire metastatic cascade: invasion, intravasation, survival in the circulation, extravasation, and colonization (i.e. are metastasis competent). We propose that cells engaging a Polyaneuploid Cancer Cell (PACC) phenotype are metastasis competent. Cells in the PACC state are enlarged, endocycling (i.e. non-dividing) cells with increased genomic content that form in response to stress. Single-cell tracking using time lapse microscopy reveals that PACC state cells have increased motility. Additionally, cells in the PACC state exhibit increased capacity for environment-sensing and directional migration in chemotactic environments, predicting successful invasion. Magnetic Twisting Cytometry and Atomic Force Microscopy reveal that cells in the PACC state display hyper-elastic properties like increased peripheral deformability and maintained peri-nuclear cortical integrity that predict successful intravasation and extravasation. Furthermore, four orthogonal methods reveal that cells in the PACC state have increased expression of vimentin, a hyper-elastic biomolecule known to modulate biomechanical properties and induce mesenchymal-like motility. Taken together, these data indicate that cells in the PACC state have increased metastatic potential and are worthy of further in vivo analysis.

Keywords: Chemotaxis; Deformability; Motility; Polyaneuploid cancer cells (PACCs); Polyploid giant cancer cells (PGCCs); Vimentin.

Fig. 1

Cancer cells undergo a Polyaneuploid Transition in response to applied stress: a 2D morphology of PACC state induction in PC3 cells using a 72 h dose of 6 μM cisplatin. Cell populations are untreated, undergoing a PAT 1 day post-treatment, or have entered a definitive PACC state 10 days post-treatment. Acquired by 20X phase microscopy. PACC cell borders are demarcated in white. b Relative DNA content of untreated cells vs. treated cells 1 day post-treatment. Acquired by flow cytometry

Fig. 2

Cells in the PACC state are more motile: a, b Quantification of the (A) accumulated distance and (B) Euclidean distance travelled by non-PACC parental cells or PACCs in uniform 20% FBS-supplemented media conditions (+ / +) throughout a 24-h time lapse. c Spider plots depicting the motility tracks of single non-PACC parental cells (n = 50) or PACCs (n = 49) in uniform 20% FBS-supplemented media conditions (+ / +) throughout a 24-h time lapse. d Directness of travel demonstrated by non-PACC parental cells or PACCs in uniform 20% FBS-supplemented media conditions (+ / +) throughout a 24-h time lapse. e Linear regression comparing the Euclidean distance travelled and 2D cell surface area of non-PACC parental cells and PACCs. f Linear regression comparing the Directness and 2D cell surface area of non-PACC parental cells and PACCs

Fig. 3

Cells in the PACC state demonstrate a directional response to a chemotactic FBS gradient: a, b Quantification of the (A) Euclidean distance travelled by or (B) directness of movement of non-PACC parental cells or PACCs in either uniform 20% FBS-supplemented media conditions (+ / +), uniform serum-free media conditions (−/−), or a chemotactic gradient of 0–20% FBS-supplemented media (−/ +) throughout a 24-h time lapse. c Spider plots depicting the motility tracks of single non-PACC parental cells (n = 50) or PACCs (n = 60) in a chemotactic gradient of 0–20% FBS-supplemented media (−/ +) throughout a 24-h time lapse. Red tracks indicate cells that had a net migration toward up the FBS gradient. d Results of the Rayleigh Test and Rayleigh Test with Vector Data for approximation of chemotactic behavior in non-PACC parental cells and PACCs. e Parallel (to FBS gradient) and Perpendicular (to FBS gradient) Forward Migration Indices for assessment of chemotactic behavior/chemotaxis in non-PACC parental cells and PACCs

Fig. 4

Cells in the PACC state have altered cytoskeletal stiffness and increased cytoskeletal dynamics: a Intracellular network cytoskeletal stiffness of non-PACC parental cells PACCs acquired via MTC using ferromagnetic RGD-linked beads. b Young’s Modulus (or cortical stiffness) of the peri-nuclear region of non-PACC parental cells and PACCs acquired using AFM. c Demonstration of functional deformability in PACCs: Evidence of simultaneous migration and deformation of PACCs through 10 μm and 20 μm wide channels of a custom invasion channel microfluidic device throughout a 36-h time lapse. d Schematic of the microfluidic device. e Mean Squared Displacement of spontaneous movement of RGD-linked beads attached to the cytoskeleton of non-PACC parental cells and PACCs throughout a 5-min time lapse

Fig. 5

PACCs have increased expression of vimentin at both the RNA and Protein level: a mRNA Nanostring analysis comparing normalized counts of VIM mRNA transcripts in untreated, cisplatin-treated, docetaxel-treated, and etoposide-treated populations. b RT-qPCR analysis of relative VIM expression between untreated cells, cells undergoing a PAT 1 day post-treatment, cells undergoing a PAT 5 days post-treatment, and PACC cells 10 days post-treatment. c Western blot and quantitative densitometry of VIM expression in untreated cells, cells undergoing a PAT 1 day post-treatment, cells undergoing a PAT 5 days post-treatment, and PACC cells 10 days post-treatment. d Integrated density of single-cell VIM signal in untreated cells, cells undergoing a PAT 1 day post-treatment, cells undergoing a PAT 5 days post-treatment, and PACC cells 10 days post-treatment using either a 1500 ms exposure time or a 500 ms exposure time. e Percentage of VIM positive cells by immunofluorescence tiled imaging in non-PACC parental and PACC populations. f Mean Fluorescent Intensity of single-cell VIM signal in untreated cells, cells undergoing a PAT 1 day post-treatment, cells undergoing a PAT 5 days post-treatment, and PACC cells 10 days post-treatment using either a 1500 ms exposure time or a 500 ms exposure time

Fig. 6

Vimentin ablation causes decreased motility in PACCs: a Spider plots depicting the motility tracks of single untreated non-PACC parental cells (n = 60), 3 mM acrylamide pre-treated non-PACC parental cells (n = 61), untreated PACCs (n = 61), or 3 mM acrylamide pre-treated PACCs (n = 61) throughout a 24-h time lapse. b, c Quantification of the b) Euclidean distance and c) directness travelled by untreated or treated non-PACC parental cells and untreated or treated PACCs throughout a 24-h time lapse