Subpopulation of breast cancer cells exhibits aberrant nuclear morphology and chemoresistance

Despite the relevance of PGCCs in the context of chemoresistance and disease relapse, they remain poorly characterized. PGCCs have previously been observed in cancer cell lines as a small but significant subpopulation that is amplified upon Paclitaxel (PTX) treatment11,12,15. We confirmed and quantified this observation in MDA-MB-231 cells through immunostaining and cell cycle analysis via flow cytometry. Cells containing more than 2.5 times (250 µm2+ for MDA-MB-231 cells) normal DNA content (as characterized by nuclear area or PI fluorescence) were found to comprise between 2 to 5 percent of total cell population (Figs 1A–C and S1E). Our polyploidal population contained a wide range of chromosomal content in excess of 4N, suggesting multiple mechanisms contribute to their formation, leading to levels of excess DNA duplication (4N, 8N, 16N+). To ensure the increase in nuclear size is indicative of increased DNA content and not differences in binding affinity of DAPI dye to DNA, we analyzed the fluorescence intensity as a function of nuclear area and found no statistical differences between intensity per area for PGCCs and non-PGCCs (p = 0.182) (Fig. S1). Morphological quantification of cell area revealed a strong correlation between nuclear area and cell size (Fig. S1A). However, this trend was significantly weaker for PGCCs, suggesting a dysregulation in mechanisms that govern the ratio of nuclear to cytosplasmic volume (Fig. 1G,H). The change of cell area as a function of nuclear area was lower for PGCCs than non-PGCCs, as evidenced by the reduced slope for the correlation. The reduced slope shows PGCC cell area did not increase at the same rate with nuclear area, which could indicate a shorter growth phase in the cell cycle (less time spent in G1/G2). Alternatively, an increase in cell thickness may be responsible for this difference. Volume analysis show a 13-fold increase in volume for PGCCs compared to non-PGCCs (p = 0.0006), but only a 6-fold increase in average cell area (p = 0.0009) (Fig. S1B,C), pointing towards increased thickness as a factor in the lower rate of correlation, at least in part. Upon treatment with PTX, the PGCC subpopulation was significantly increased in a concentration dependent manner (Fig. 1C,F), ranging from a 3-fold increase at 25 nM PTX (p = 0.032) up to a 10-fold increase at 100 nM PTX treatment for 48 hours (p = 0.007). We hypothesized that this increase in PGCC population was due to their abnormal phenotype, which conferred a survival advantage to these cells during PTX treatment. To confirm our hypothesis, we captured 24 hour time-lapse videos (Fig. 1D) of MDA-MB-231 cells treated with 100 nM PTX and analyzed the number of apoptotic cells. Apoptotic cells were identified by rapid changes in cell shape from adherent and spread to detached and rounded, as described previously16. Non-PGCCs show significant cell death, while PGCCs were able to survive the 24-hour treatment (Fig. 1E). To determine how PGCCs withstand the mechanical stress associated with their increased size and the chemical stress from PTX treatment, a single cell biophysical approach was used to investigate fundamental differences in PGCCs and non-PGCCs.

Figure 1 Characterizing PGCC occurrence and PTX resistance in MDA-MB-231 cancer cells. (A) Nuclear area distribution of MDA-MB-231 cells stained with DAPI (n = 5, 5000 + cells). (B) Cell cycle analysis via flow cytometry of DMSO and 48-hr 100 nM PTX treated cells show different stages of cell cycle and polyploidy. (C) Quantification of cell cycle analysis for DMSO and 100 nM PTX treated cells reveal increased PGCC occurrence. (D) Time lapse images of MDA cells before and after 24-hr treatment with 100 nM PTX (PGCCs indicated by white arrow). (E) Quantification of time lapse cell survival (500 + cells) show PGCC resistance to treatment. (F) PGCC incidence rate rises with increasing doses of 48-hr PTX treatment (n = 4). (G) Nuclear and cell area correlation of non-PGCCs and (H) PGCCs (40 + cells) show decreased correlation in the PGCC subpopulation. Cell cycle analysis and time lapse experiments were performed in triplicate. Results are reported as the mean ± SEM. Significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001. Full size image

PGCCs demonstrate altered cytoskeletal mechanics

First, multiple particle tracking microrheology (MPT) was used to determine cell mechanical properties from the thermal motion of ballistically injected fluorescent nanoparticles17. This technique was used to probe the local mechanical properties in individual cells (PGCCs and non-PGCCs) based on the thermal displacements of 200-nm particles embedded in the cell cytoplasm (10–20 particles per cell). For tracking studies, PGCCs and non-PGCCs were identified based on differences in nuclear size. For each condition we report the ensemble averaged MSD <<r2(τ)>> of individual particle traces (n~30 cells and ~100 traces per condition). The logarithmic slope of MSD for PGCCs and non-PGCCs was similar (0.69 < α < 0.76), in the range indicating viscoelastic behavior (Fig. 2A,B). However, ensemble averaged PGCC particle traces show a 2-fold reduction in amplitude across all time lags, indicating inherent stiffness. This inherently stiffer cytoplasm would be required to withstand increased mechanical stresses associated with the increased size of PGCCs. The lower amplitude of particle traces observed in the PGCC subpopulation represent increased constraint of particles within the cytoskeletal network, as individual particle displacements are dependent on their interactions with surrounding cytoskeletal elements. Since each particle only probes the mechanical properties of their local environment, we can analyze the variation in particle MSDs to gain insight on the heterogeneity of mechanical properties. Overall heterogeneity of PGCCs was significantly higher than non-PGCCs. The coefficient of variation (standard deviation divided by the mean) of individual particle MSDs at a time lag of 0.1 seconds (Fig. 2C) in addition to ensemble averaged MSD per cell (Fig. S2C) was higher, suggesting higher heterogeneity not only at the population scale but also on the local regional level within the cell. Due to the nature of MPT, we suspected that the altered mechanical phenotype exhibited by the PGCCs as well as associated heterogeneity was tightly linked to the cytoskeletal network of the cell, as proposed by previous studies17. To confirm, we visualized the actin and microtubule networks of PGCCs and non-PGCCs via immunostaining (Fig. 2D). The images show increased appearance of actin stress fibers and abnormal microtubule organization. Further quantification of actin stress filaments showed a ~20% (p = 0.003) and ~12% (p = 0.04) increase in actin stress fiber length and width (Fig. 2E,F), respectively. This suggested differential organization and coverage increase of actin stress fibers contributed to localized regions and overall stiffness within the PGCCs.

Figure 2 Altered cytoskeletal organization drive increased stiffness in PGCCs. (A) Ensemble averaged MSDs derived from particle motion embedded within cell cytoplasm of non-PGCCs and (B) PGCCs show decreased particle motion and increased stiffness in PGCCs (over 100 traces). (C) Coefficient of variation analysis for ensemble MSDs of non-PGCC and PGCCs reveal increased heterogeneity in PGCCs. (D) Fluorescent images of non-PGCC and PGCCs (white arrow) stained with Phalloidin (F-actin, red), Anti-α-tubulin (Microtubule, green), and DAPI (Nucleus, blue). (E) Actin stress fiber quantification show increased stress fiber length and (F) width in PGCCs compared to non-PGCCs. Results are reported as the mean ± SEM. Significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001. Full size image

Mechanical properties of PGCC nuclei are characterized by elevated stiffness

Given the close interplay between cytoskeletal and nuclear mechanics, next we wanted to investigate if the stiffer cytoplasmic mechanics of PGCCs and upregulation of F-actin bundled elements would manifest within nuclear behavior in similar fashion. Multiple particle analysis was conducted on Hoescht-stained chromatin granules within PGCC and non-PGCCs as described previously17. Consistent with previous studies, both PGCCs and non-PGCCs initially exhibited behavior akin to an elastic solid, represented by a MSD slope close to 0, but at longer time lags nuclear behavior transitioned to a more viscous nature, corresponding with an increasing MSD slope tending towards 1 (representing a perfectly viscous fluid). However, similar to cytoplasmic mechanics, the amplitude of PGCC nuclear particle MSDs shows a significant decrease compared to non-PGCCs (Fig. 3A,B), suggesting an inherently stiffer nuclear structure at lower time lags. Relaxation time quantifies the transition from elastic to viscous character and is defined as when the slope of the MSD reaches 0.5. Surprisingly, PGCCs demonstrated a faster transition to viscous character, with a relaxation time of 5.73 seconds compared to non-PGCCs at 6.52 seconds. This highlights differences in the dynamics of PGCC nuclear mechanics, allowing a rapid transition from an initially elastic state to a viscous one. Heterogeneity analysis of individual particle MSDs further show a greater variation in nuclear mechanical properties in PGCCs compared to non-PGCCs at for all time lags on an individual particle level (Fig. 3C), indicating uneven regions of differing stiffnesses present within the nucleus. To determine if this difference in overall nuclear stiffness is due to a dysregulation in nuclear lamina, we fluorescently stained for nuclear envelope associated protein lamin A/C (Fig. 3D) and quantified expression around the nucleus. We found that lamin A/C expression as a function of nuclear area remains unchanged between PGCCs and non-PGCCs (Fig. 3E). However, quantification of intense chromatin granules (representative of heterochromatic foci18) in high magnification fluorescent images show a significantly increased number of foci as a function of nuclear area (Fig. 3F), which could comprise the inherently stiff regions of the nuclei, containing highly condensed chromatin18.

Figure 3 Nuclear biomechanical analysis reveal stiffer nuclei in PGCCs. Ensemble averaged MSDs derived from chromatin granule motion within nuclei of (A) non-PGCCs and (B) PGCCs show decreased particle motion and increased nuclear stiffness in PGCCs (100 + traces, 30 + cells). (C) Coefficient of variation analysis for ensemble MSDs of non-PGCC and PGCCs reveal increased heterogeneity in nuclear mechanics for PGCCs. (D) Fluorescent images of non-PGCC and PGCCs (white arrow) stained with anti-lamin A/C (nuclear lamin, green) and DAPI (nucleus, blue). (E) Fluorescence intensity quantification of lamin A/C expression in MDA cells reveal no significant differences between PGCCs and non-PGCCs. (F) Quantification of heterochromatic foci density show increased number of foci as a function of area in PGCCs. Results are reported as the mean ± SEM. Significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001. Full size image

PGCCs migrate slowly but with more persistence

Previous studies have shown cell mechanics and migration is directly correlated19,20. To examine the migration of PGCCs, MDA-MB-231 cells were seeded on collagen coated 24 well plates to evaluate their motility. At lower time lags, non-PGCCs exhibited ~57% higher speeds of migration compared to PGCCs (p = 0.0056) (Fig. 4B). This difference in average speed can primarily be attributed to an incongruity of time spent in the fast versus slow modes of migration (Fig. 4A) (cutoff between slow and fast modes defined by movement of at least 1 normal cell length within 1 hour) of these MDA-MB-231 cells. On average, PGCCs spent 56% more time in the slow mode of movement (p = 0.0003) as opposed to non-PGCCs (Fig. 4C). However, despite the slower cell velocity, the directional persistence of PGCCs was significantly higher. This manifested in the MSD traces of cell tracks during the longer time lags of 150 minutes or more, at which point the PGCCs surprisingly covered a larger net distance than non-PGCCs (Fig. 4D). This observation is further reinforced by persistence analysis (determined by the logarithmic slope of the MSD, with 1 being a randomly moving cell and 2 being a cell moving in a straight line) of PGCC and non-PGCC motility, where PGCCs on average were ~35% more persistent in their migration (p = 0.0042) (Fig. 4E). Increased persistence has been previously associated with cancer invasiveness21,22, leading to metastatic dissemination into surrounding tissue. Overall, PGCCs exhibit a slow but steady migratory phenotype, dictated by their abnormal cytoskeletal organization and large size.

Figure 4 PGCCs exhibit a more persistent migratory phenotype. (A) Schematic depicting the two modes of movement during cell migration. The slow and fast mode cutoff is defined as movement of at least 1 average cell length per hour. (B) Average speed of random migration and (C) Fraction of time spent in the slow mode of movement for non-PGCCs and PGCCs. (D) Cell motion derived MSDs of PGCCs non-PGCCs during 12-hr migration assay (n = 5, 50+ cells). (E) Persistence analysis of non-PGCCs and PGCCs show increased directional persistence during migration. Significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001. Full size image

PGCCs nuclei deform more readily in response to scratch

Nuclear deformation during cell migration is an essential part of the migratory process, not only for nuclear relocation during migration but also during regions of confinement23. To investigate the dynamics of nuclear deformation in PGCCs, we looked at their nuclear deformation during induced directional movement via scratch assay (Fig. 5A). True to their lower nuclear relaxation time, PGCCs were able to alter their nuclear shape factor at a faster rate than non-PGCCs, with increased rate of nuclear shape factor change (shape factor of 1 defined as circular and 0 as a line) measured at 1 to 8 hours after scratch (Fig. 5B). To further probe the behavior of the PGCC nuclei during directed migration, we examined the nuclear mechanics of PGCCs at each hour after the initial scratch was made. Averaged ensemble MSD of all cells taken at short time scales (time lag < 1 second) revealed a softening of PGCC but not non-PGCC nuclei in response to induced migration (Fig. 5C,D) as marked by increasing amplitudes in MSDs. However, PGCC relaxation times were also increased in response to the scratch, pointing towards a reorganization of chromatin structure that allowed PGCCs to soften their nuclei but at the same time limited the dynamics of their relaxation. Taken together, these results suggest PGCCs alter their chromatin organization in order to facilitate periods of rapid migration, contributing to their invasiveness.

Figure 5 PGCCs have more deformable nuclei and alter nuclear mechanics during migration. (A) Time lapse images of confluent monolayer with scratch closing over the course of 16 hours. (B) Rate of nuclear deformation measured as the change in nuclear shape factor from 0 to 8 hours after initial scratch (30 + cells). (C) Total averaged ensemble MSDs of tracked chromatin granules in non-PGCCs and (D) PGCCs hours after scratch (10 + cells). (E) MSD at 1 second time lag of non-PGCC and PGCCs hours after scratch show increased softening of PGCC nuclei compared to non-PGCCs. Full size image

RhoA-ROCK1 and actin cytoskeleton regulates cytoplasmic and nuclear mechanics

The underlying actin cytoskeleton of cells not only provide structural support but also defines the mechanical properties of the cell. A key regulator of the organization and dynamics of the actin network is the RhoA-ROCK1 mediated pathway24. Given the differential upregulation of actin stress bundle size and increased stiffness of PGCCs, involvement of the RhoA-ROCK1 and downstream targets is likely. By using latrunculin (actin cytoskeleton inhibitor), H1152 (ROCK inhibitor) and ML7 (MLCK inhibitor), we disrupted overall actin organization and dynamics of MDA-MB-231 cells. Again, particle and chromatin granule tracking was used to probe the biomechanical properties of PGCCs and non-PGCCs under treatment. When we treated PGCCs and non-PGCCs with latrunculin, H1152 and ML7, we observed no significant change in the slope of the ensemble averaged MSDs, but a reduction in amplitude, representing overall cytoplasmic softening in both PGCCs and non-PGCCs (Fig. 6A,B), as expected and in agreement with previous studies25. When we examined the nuclear mechanical properties after treatment with the inhibitors, non-PGCCs show a slight decrease in amplitude, as well as a decrease in relaxation time (Fig. 6C). In contrast, PGCCs responded significantly only for latrunculin treatment by increasing their amplitude and decreasing relaxation time, but had little response to H1152 and ML7 (Fig. 6D). When we examined the degree of cytoplasmic softening (as measured by a reduction in amplitude of ensemble MSDs at 1 second time lag normalized to respective controls), the effect of the inhibitors was much greater for PGCCs and increased compliance to levels similar to non-PGCCs (Figs 6E, 7B). This suggests PGCCs obtain their cytoplasmic biophysical phenotype via dysregulation in the RhoA-ROCK1 pathway and maintain it through upregulation of the actin cytoskeletal network. After this upregulation was disrupted, the PGCCs lost the underlying mechanisms that drove their stiffness, resulting in softening to levels closer to non-PGCCs (Fig. 7B). The same trend can be seen in the heterogeneity within these cells. After latrunculin inhibition, coefficients of variation of PGCCs normalized to non-PGCC cells reduced from 1.64-fold down to 1.08 (Fig. 7B), as the actin cytoskeletal elements causing localized regions of stiffness was disrupted. Similar to the cytoplasmic results, this effect of latrunculin on nuclear mechanics was more pronounced in PGCCs, rescuing nuclear compliance to levels close to non-PGCCs (Figs 6E, 7B), which suggests involvement of the actin network in maintaining nuclear stiffness, perhaps in the form of the perinuclear actin cap (Fig. S4). However, in contrast to the cytoplasmic results, the PGCCs had a significantly weaker response in terms of a reduction in relaxation time and MSD amplitude when treated with H1152 and ML7 compared to non-PGCCs (Fig. 6E), pointing towards a disruption in the RhoA-ROCK1 pathway mediated coupling of cytoskeletal and nuclear mechanics.

Figure 6 Biophysical phenotype of PGCCs is driven by actin cytoskeletal organization and mediated via the RhoA-ROCK1 pathway. (A) Total averaged MSD ensembles of particles embedded in non-PGCCs and (B) PGCCs tracked after 2-hour pretreatment with ML7 (MLCK inhibitor), H1152 (ROCK inhibitor) and latrunculin A (Actin cytoskeleton disruptor). (C) Total averaged MSD ensembles of chromatin granule motion in non-PGCCs and (D) PGCCs tracked after 2-hour pretreatment with ML7, H1152 and latrunculin A. (E) Summary table of inhibitor studies detailing MSD (at 1 second time lag), CV (coefficient of variation) and alpha (logarithmic slope of MSD as a function of time, for cytoplasmic mechanics) and RT (relaxation time, for nuclear mechanics). All results are normalized to either non-PGCC or PGCC untreated counterparts. Experiments were performed in triplicate. Full size image