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Polyploid giant cancer cells (PGCCs) are a commonly observed histological feature of human tumors and are particularly prominent in late stage and drug resistant cancers. The chromosomal duplication conferred by their aneuploidy gives rise to DNA damage resistance and complex tumor cell karyotypes, a driving factor in chemotherapy resistance and disease relapse. Furthermore, PGCCs also exhibit key cytoskeletal features that give rise to a distinct biophysical phenotype, including increased density of polymerized actin and vimentin intermediate filaments, nuclear and cytoskeletal stiffening, increased traction force, and migratory persistence. Despite recent research highlighting the role PGCCs play in cancer progression, this population of tumor cells remains poorly characterized in terms of their biophysical properties. In this review, we will discuss the various aspects of their biomolecular phenotype, such as increased stemness as well as a mixed EMT signature. These features have been extensively associated with tumorigenesis and recurrence, and aggressive cancers. Additionally, we will also examine the distinct PGCC cytoskeletal features of actin and filamentous vimentin. Specifically, how the differential organization of these networks serve to support their increased size and drive migratory persistence. These findings could shed light on potential therapeutic strategies that allow for specific elimination or mitigation of the invasive potential of these polyploid cancer cells. Lastly, we will examine how the biophysical and molecular phenotype of PGCCs combine to tip the scale in favor of promoting cancer progression, presenting an important target in the clinical treatment of cancer.

The essential bacterial division protein in Escherichia coli, FtsZ, assembles into the FtsZ‐ring at midcell and recruits other proteins to the division site to promote septation. A region of the FtsZ amino acid sequence that links the conserved polymerization domain to a C‐terminal protein interaction site was predicted to be intrinsically disordered and has been implicated in modulating spacing and architectural arrangements of FtsZ filaments. While the majority of cell division proteins that directly bind to FtsZ engage either the polymerization domain or the C‐terminal interaction site, ClpX, the recognition and unfolding component of the bacterial ClpXP proteasome, has a secondary interaction with the predicted intrinsically disordered region (IDR) of FtsZ when FtsZ is polymerized. Here, we use NMR spectroscopy and reconstituted degradation reactions in vitro to demonstrate that this linker region is indeed disordered in solution and, further, that amino acids in the IDR of FtsZ enhance the degradation in polymer‐guided interactions.

Polyploidal giant cancer cells (PGCCs) are multinucleated chemoresistant cancer cells found in heterogeneous solid tumors. Due in part to their apparent dormancy, the effect of PGCCs on cancer progression has remained largely unstudied. Recent studies have highlighted the critical role of PGCCs as aggressive and chemoresistant cancer cells, as well as their ability to undergo amitotic budding to escape dormancy. Our recent study demonstrated the unique biophysical properties of PGCCs, as well as their unusual migratory persistence. Here we unveil the critical function of vimentin intermediate filaments (VIFs) in maintaining the structural integrity of PGCCs and enhancing their migratory persistence. We performed in-depth single-cell analysis to examine the distribution of VIFs and their role in migratory persistence. We found that PGCCs rely heavily on their uniquely distributed and polarized VIF network to enhance their transition from a jammed to an unjammed state to allow for directional migration. Both the inhibition of VIFs with acrylamide and small interfering RNA knockdown of vimentin significantly decreased PGCC migration and resulted in a loss of PGCC volume. Because PGCCs rely on their VIF network to direct migration and to maintain their enlarged morphology, targeting vimentin or vimentin cross-linking proteins could provide a therapeutic approach to mitigate the impact of these chemoresistant cells in cancer progression and to improve patient outcomes with chemotherapy.

The tumor microenvironment is a complex milieu that dictates the growth, invasion, and metastasis of cancer cells. Both cancer and stromal cells in the tumor tissue encounter and adapt to a variety of extracellular factors, and subsequently contribute and drive the progression of the disease to more advanced stages. As the disease progresses, a small population of cancer cells becomes more invasive through a complex process known as epithelial-mesenchymal transition, and nearby stromal cells assume a carcinoma associated fibroblast phenotype characterized by enhanced migration, cell contractility, and matrix secretion with the ability to reorganize extracellular matrices. As cells transition into more malignant phenotypes their biophysical properties, controlled by the organization of cytoskeletal proteins, are altered. Actin and its associated proteins are essential modulators and facilitators of these changes. As the cells respond to the cues in the microenvironment, actin driven mechanical forces inside and outside the cells also evolve. Recent advances in biophysical techniques have enabled us to probe these actin driven changes in cancer and stromal cells and demarcate their role in driving changes in the microenvironment. Understanding the underlying biophysical mechanisms that drive cancer progression could provide critical insight on novel therapeutic approaches in the fight against cancer.

The essential bacterial division protein in Escherichia coli, FtsZ, assembles into the FtsZ-ring at midcell and recruits other proteins to the division site to promote septation. A region of the FtsZ amino acid sequence that links the conserved polymerization domain to a C-terminal protein interaction site was predicted to be intrinsically disordered and has been implicated in modulating spacing and architectural arrangements of FtsZ filaments. While the majority of cell division proteins that directly bind to FtsZ engage either the polymerization domain or the C-terminal interaction site, ClpX, the recognition and unfolding component of the bacterial ClpXP proteasome, has a secondary interaction with the predicted intrinsically disordered region (IDR) of FtsZ when FtsZ is polymerized. Here, we use NMR spectroscopy and reconstituted degradation reactions in vitro to demonstrate that this linker region is indeed disordered in solution and, further, that amino acids in the IDR of FtsZ enhance the degradation by conformationally-guided interactions.

The tumor microenvironment is a complex milieu that dictates the growth, invasion, and metastasis of cancer cells. Both cancer and stromal cells in the tumor tissue encounter and adapt to a variety of extracellular factors, and subsequently contribute and drive the progression of the disease to more advanced stages. As the disease progresses, a small population of cancer cells becomes more invasive through a complex process known as epithelial-mesenchymal transition, and nearby stromal cells assume a carcinoma associated fibroblast phenotype characterized by enhanced migration, cell contractility, and matrix secretion with the ability to reorganize extracellular matrices. As cells transition into more malignant phenotypes their biophysical properties, controlled by the organization of cytoskeletal proteins, are altered. Actin and its associated proteins are essential modulators and facilitators of these changes. As the cells respond to the cues in the microenvironment, actin driven mechanical forces inside and outside the cells also evolve. Recent advances in biophysical techniques have enabled us to probe these actin driven changes in cancer and stromal cells and demarcate their role in driving changes in the microenvironment. Understanding the underlying biophysical mechanisms that drive cancer progression could provide critical insight on novel therapeutic approaches in the fight against cancer.

Mesenchymal stem cells accumulated in tissue specific sites are essential for the regenerative process; however, biological aging and environmental stress can induce senescence - an irreversible state of growth arrest - that not only affects the behavior of cells but also disrupts their ability to restore tissue integrity. While abnormal tissue properties including increased extracellular matrix stiffness are linked with the risk of developing breast cancer, the role and contribution of senescent MSCs to the disease progression to malignancy are not well understood. Here, we investigated senescence associated biophysical changes in MSCs and how they influence cancer cell behavior in a 3D matrix interface model. Although senescent MSCs were far less motile than pre-senescent MSCs, they induced an invasive breast cancer phenotype, characterized by increased spheroid growth and cell invasion in collagen gels. Further analysis of collagen gels using second harmonic generation showed increased collagen density when senescent MSCs were present, suggesting that senescent MSCs actively remodel the surrounding matrix. This study provides direct evidence of the pro-malignant effects of senescent MSCs in tumors.

Senescence is a potent tumor-suppressive mechanism that irreversibly arrests the growth of damaged cells. However, senescent cells that accumulate in tissues eventually develop a senescence-associated secretory phenotype (SASP) that alters the microenvironment to promote cancer. Paracrine factors in the SASP may also contribute to the formation of rare giant polyploidal cancer cells (GPCCs). A single-cell mechanical approach was used to profile cytoskeletal and nuclear mechanics, morphology, motility, and adhesion for breast cancer cells treated with conditioned media from senescent fibroblasts. Our study showed that a small but significant population of MDA-MB-231 breast cancer cells (less than 5%) treated with conditioned media from senescent LF-1 fibroblasts develop an enlarged morphology, chromosomal instability, and polyploidy, a phenotype associated with GPCCs. Although GPCCs are highly invasive and chemoresistant, little is known about their biophysical properties. First, we developed a method for identifying the small subpopulation of GPCCs in a heterogeneous population of cancer cells based on increased nuclear area and confirmed that GPCCs are more resistant to paclitaxel than normal-size MDA-MB-231 cells (NCCs). We then compared critical biophysical properties of NCCs and GPCCs, including cytoskeletal and nuclear mechanics, cell and nuclear morphology, motility, and adhesion. Cells were stained for cytoskeletal proteins actin, tubulin, and vinculin. Cytoskeletal organization was dramatically altered in GPCCs compared to NCCs. GPCCs displayed more disorganized microtubule structure, dense actin stress fibers, and mature focal adhesions. Intracellular particle tracking microrheology was used to measure cytoskeletal and nuclear mechanics. These studies demonstrated that although GPCCs are thought to be highly invasive cancer cells, they are inherently stiffer than NCCs, in terms of both their cytoskeletal and nuclear mechanics. This was surprising since more invasive cancer cells are often more compliant than less invasive cancer cells. This result may be in part to the ability for GPCCs to behave like activated stromal cells that stiffen in the tumor; we confirmed that GPCCs display similar adhesive behavior as activated stromal cells. To determine how mechanics correlates with cell migration, we used time-lapse nuclear tracking to measure cell motility. The average cell speed was higher for NCCs than for GPCCs; however, GPCCs moved longer distances over time because their motion was more directional. These findings highlight the unusual biophysical behavior of GPCCs. To develop pharmacologic tools that target GPCCs, it is imperative to understand their biophysical properties. Citation Format: Michelle Dawson, Botai Xuan, Deepraj Ghosh. Biophysics of polyploidal cancer cells in an aging stroma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1315.