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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.

Supplementary Methods from Recruitment of Myeloid but not Endothelial Precursor Cells Facilitates Tumor Regrowth after Local Irradiation

Supplementary Figures 1-4 from Recruitment of Myeloid but not Endothelial Precursor Cells Facilitates Tumor Regrowth after Local Irradiation

Tumor neovascularization and growth might be promoted by the recruitment of bone marrow–derived cells (BMDC), which include endothelial precursor cells and “vascular modulatory” myelomonocytic (CD11b+) cells. BMDCs may also drive tumor regrowth after certain chemotherapeutic and vascular disruption treatments. In this study, we evaluated the role of BMDC recruitment in breast and lung carcinoma xenograft models after local irradiation (LI). We depleted the bone marrow by including whole-body irradiation (WBI) of 6 Gy as part of a total tumor dose of 21 Gy, and compared the growth delay with the one achieved after LI of 21 Gy. In both models, the inclusion of WBI induced longer tumor growth delays. Moreover, WBI increased lung tumor control probability by LI. Exogenous delivery of BMDCs from radiation-naïve donors partially abrogated the WBI effect. Myeloid BMDCs, primarily macrophages, rapidly accumulated in tumors after LI. Intratumoral expression of stromal-derived factor 1α (SDF-1α), a chemokine that promotes tissue retention of BMDCs, was noted 2 days after LI. Conversely, treatment with an inhibitor of SDF-1α receptor CXCR4 (AMD3100) with LI significantly delayed tumor regrowth. However, when administered starting from 5 days post-LI, AMD3100 treatment was ineffective. Lastly, with restorative bone marrow transplantation of Tie2-GFP–labeled BMDC population, we observed an increased number of monocytes but not endothelial precursor cells in tumors that recurred following LI. Our results suggest that an increase in intratumoral SDF-1α triggered by LI recruits myelomonocytes/macrophages which promotes tumor regrowth. Cancer Res; 70(14); 5679–85. ©2010 AACR.

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.

Exosomes are cell-secreted microvesicles that play important roles in epithelial ovarian cancer (EOC) progression, as they are constantly secreted into ascites fluids. While cells spontaneously release exosomes, alterations in intracellular calcium or extracellular pH can release additional exosomes. Yet, little is known about how these exosomes compare to those that are continuously released without stimulation and how they mediate cellular activities important in cancer progression. Here, we demonstrate that chelation of extracellular calcium leads to release of chelation-induced exosomes (CI-exosomes) from OVCAR-3 EOC cells. CI-exosomes display a unique miRNA profile compared to naturally secreted exosomes (SEC-exosomes). Furthermore, treatment with CI- and SEC-exosomes leads to differential biophysical and functional changes including, adhesion and migration in EOC-derived fibroblasts that suggest the development of a malignant tumor microenvironment. This result highlights how tumor environmental factors contribute to heterogeneity in exosome populations and how different exosome populations mediate diversity in stromal cell behavior.

This abstract is being presented as a short talk in the scientific program. A full abstract is available in the Short Talks from Proffered Abstracts section (PR007) of the Conference Proceedings. Citation Format: Michelle R. Dawson, Deepraj Ghosh. Physical and metabolic aspects of therapy induced senescence and polyploidy in an evolving tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr A002.

Radiation and chemotherapy are highly effective at killing cancer cells but cells that survive treatment often develop therapy-induced senescence (TIS). Since growth is arrested in TIS, this has been considered a positive treatment outcome; however, senescent cells that remain metabolically active and develop a senescence associated secretory phenotype (SASP) can also promote cancer progression. In addition, a small number of cancer cells are able to escape this dormant state and contribute to cancer progression. Polyploid giant cancer cells (PGCCs) represent a small subpopulation of non-mitotic cancer cells that evades treatment through periods of transient dormancy and then relapses into full-blown disease through amitotic budding into chemoresistant progenitor cells. The PGCC phenotype mimics senescent cells in multiple ways, including enlarged size, increased beta-galactosidase expression, increased metabolic activity, and pro-inflammatory SASP; yet, there is a significant gap in our understanding of how PGCCs contribute to TIS escape and subsequent chemoresistance. In addition, increased numbers of large PGCCs are seen in late stage and metastatic cancers; yet, their role in tumor recurrence and metastasis has not been established. We previously showed PGCCs have a unique actin cytoskeletal organization, giving rise to elevated stiffness and migratory persistence. These findings prompted us to further examine vimentin intermediate filaments (VIFs) that act as shock absorbers in the cell, protecting cells from compressive loads. Based on this, we postulated that PGCCs have unique adaptations in their vimentin structure to help support their enlarged morphology and drive their persistent migration. Indeed, we showed that PGCCs have increased levels of cytoplasmic vimentin and evenly distributed VIFs compared to non-PGCCs. This dispersed network of VIFs often polarized at the leading-edge during migration, and was necessary for PGCCs phenotype, as disruption of VIFs decreased PGCC volume and blocked migratory persistence. The VIF network also scaffolds lysosomes and autophagosomes in the cytosol to regulate their fusion during autophagy. Our new data clearly shows that inhibiting autophagy with Bafilomycin limits PGCC migration. In addition, targeting VIF structure also blocks autophagy. Thus, the structure of VIFs and autophagic flux are both critical in directing PGCCs migratory persistence. We have developed a novel 3D tumor microenvironment model that allows us to monitor cancer cell behavior over 4-6 weeks, which allows us to monitor cell recovery from TIS and dormancy. Using this model, we show that PGCC’s unique biophysical properties are directly linked to their dysregulated metabolism and altered cell structure. These studies provide critical information about how aging, TIS, and polyploidy affect evolving tumor microenvironments. Citation Format: Michelle R. Dawson, Deepraj Ghosh. Physical and metabolic aspects of therapy induced senescence and polyploidy in an evolving tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr PR007.

High-grade serous ovarian cancer (HGSOC) constitutes the majority of all ovarian cancer cases and has staggering rates of both refractory and recurrent disease. While most patients respond to the initial treatment with paclitaxel and platinum-based drugs, up to 25% do not, and of the remaining that do, 75% experience disease recurrence within the subsequent two years. Intrinsic resistance in refractory cases is driven by environmental stressors like tumor hypoxia which alter the tumor microenvironment to promote cancer progression and resistance to anticancer drugs. Recurrent disease describes the acquisition of chemoresistance whereby cancer cells survive the initial exposure to chemotherapy and develop adaptations to enhance their chances of surviving subsequent treatments. Of the environmental stressors cancer cells endure, exposure to hypoxia has been identified as a potent trigger and priming agent for the development of chemoresistance. Both in the presence of the stress of hypoxia or the therapeutic stress of chemotherapy, cancer cells manage to cope and develop adaptations which prime populations to survive in future stress. One adaptation is the modification in the secretome. Chemoresistance is associated with translational reprogramming for increased protein synthesis, ribosome biogenesis, and vesicle trafficking. This leads to increased production of soluble proteins and extracellular vesicles (EVs) involved in autocrine and paracrine signaling processes. Numerous studies have demonstrated that these factors are largely altered between the secretomes of chemosensitive and chemoresistant patients. Such factors include cytokines, growth factors, EVs, and EV-encapsulated microRNAs (miRNAs), which serve to induce invasive molecular, biophysical, and chemoresistant phenotypes in neighboring normal and cancer cells. This review examines the modifications in the secretome of distinct chemoresistant ovarian cancer cell populations and specific secreted factors, which may serve as candidate biomarkers for aggressive and chemoresistant cancers.

Recent studies suggest that glypican-1 (GPC-1) is a biomarker for prostate cancer, but there are few studies elucidating the role of GPC-1 in prostate cancer progression. We observed high expression of GPC-1 in more aggressive prostate cancer cell lines such as PC-3 and DU-145. While inhibition of GPC-1 expression in PC-3 cells decreased cell growth and migration in vitro, it surprisingly increased cell proliferation and migration in DU-145 cells, suggesting that the role of GPC-1 is cell type-dependent. Further, GPC-1 inhibition increased PC-3 tumor size in NCr nude mice xenografts. We hypothesized that the discrepancy between the in vitro and in vivo data is mediated by stromal cells in the tumor microenvironment. Thus, we tested the effect of tumor conditioned media (TCM) on gene expression in human mesenchymal stem cells and fibroblasts. Treatment of stromal cells with TCM from PC-3 cells transfected with GPC-1 shRNA increased the expression of migration markers, endocrine/paracrine biomolecules, and extracellular matrix components. Additionally, the decreased cell growth in GPC-1 knockdown PC-3 cells was rescued by coculturing with stromal cells. These data demonstrate the paradoxical role that GPC-1 plays in prostate cancer cell growth by interacting with stromal cells and through ECM remodeling and endocrine/paracrine signaling.

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 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.

The microenvironment in a solid tumor includes a multitude of cell types, matrix proteins, and growth factors that profoundly influence cancer cell mechanics by providing both physical and chemical stimulation. This tumor microenvironment, which is both dynamic and heterogeneous in nature, plays a critical role in cancer progression from the growth of the primary tumor to the development of metastatic and drug-resistant tumors. This chapter provides an overview of the biophysical tools used to study cancer cell mechanics and mechanical changes in the tumor microenvironment at different stages of cancer progression, including growth of the primary tumor, local invasion, and metastasis. Quantitative single cell biophysical analysis of intracellular mechanics, cell traction forces, and cell motility can easily be combined with analysis of critical cell fate processes, including adhesion, proliferation, and drug resistance, to determine how changes in mechanics contribute to cancer progression. This biophysical approach can be used to systematically investigate the parameters in the tumor that control cancer cell interactions with the stroma and to identify specific conditions that induce tumor-promoting behavior, along with strategies for inhibiting these conditions to treat cancer. Increased understanding of the underlying biophysical mechanisms that drive cancer progression may provide insight into novel therapeutic approaches in the fight against cancer.

The microenvironment in a solid tumor includes a multitude of cell types, matrix proteins, and growth factors that profoundly influence cancer cell mechanics by providing both physical and chemical stimulation. This tumor microenvironment, which is both dynamic and heterogeneous in nature, plays a critical role in cancer progression from the growth of the primary tumor to the development of metastatic and drug-resistant tumors. This chapter provides an overview of the biophysical tools used to study cancer cell mechanics and mechanical changes in the tumor microenvironment at different stages of cancer progression, including growth of the primary tumor, local invasion, and metastasis. Quantitative single cell biophysical analysis of intracellular mechanics, cell traction forces, and cell motility can easily be combined with analysis of critical cell fate processes, including adhesion, proliferation, and drug resistance, to determine how changes in mechanics contribute to cancer progression. This biophysical approach can be used to systematically investigate the parameters in the tumor that control cancer cell interactions with the stroma and to identify specific conditions that induce tumor-promoting behavior, along with strategies for inhibiting these conditions to treat cancer. Increased understanding of the underlying biophysical mechanisms that drive cancer progression may provide insight into novel therapeutic approaches in the fight against cancer.

For a solid tumor to grow, it must be able to support the compressive stress that is generated as it presses against the surrounding tissue. Although the literature suggests a role for the cytoskeleton in counteracting these stresses, there has been no systematic evaluation of which filaments are responsible or to what degree. Here, using a three-dimensional spheroid model, we show that cytoskeletal filaments do not actively support compressive loads in breast, ovarian, and prostate cancer. However, modulation of tonicity can induce alterations in spheroid size. We find that under compression, tumor cells actively efflux sodium to decrease their intracellular tonicity, and that this is reversible by blockade of sodium channel NHE1. Moreover, although polymerized actin does not actively support the compressive load, it is required for sodium efflux. Compression-induced cell death is increased by both sodium blockade and actin depolymerization, whereas increased actin polymerization offers protective effects and increases sodium efflux. Taken together, these results demonstrate that cancer cells modulate their tonicity to survive under compressive solid stress.

Cancer-associated fibroblasts (CAFs) are known to promote tumor growth and metastasis; however their differential accumulation in invasive and noninvasive tumors is not fully understood. We hypothesized that differences in cell adhesion may contribute to this phenomenon. To test this, we analyzed the adhesion of CAF-precursor fibroblasts and mesenchymal stem cells to invasive and noninvasive cancers originating from the the breast, ovaries, and prostate. In all cases, stromal cells preferentially adhered to more invasive cancer cells. Modulating integrin and cadherin binding affinities with calcium chelation revealed that adhesion was independent of integrin activity but required cadherin function. Invasive cancer cells had increased expression of mesenchymal markers cadherin 2 and 11 that localized with stromal cell cadherin 11, suggesting that these molecules are involved in stromal cell engraftment. Blockade of cadherin 11 on stromal cells inhibited adhesion and may serve as a target for metastatic disease.

Though survival rates for women with stage I breast cancer have radically improved, treatment options remain poor for the 40% of women diagnosed with later-stage disease. For these patients, improved chemotherapeutic treatment strategies are critical to eradicate any disseminated tumor cells. Despite many promising new drugs in vitro, most ultimately fail in the clinic. One aspect often lost during testing is in vivo circulation half-lives rarely exceed 24 hours, whereas in vitro studies involve drug exposure for 2-3 days. Here, we show how mimicking these exposure times alters efficacy. Next, using this model we show how drug response is highly time-dependent by extending analysis of cell viability out to two weeks. Variations in response both with feeding and time were dependent on drug mechanism of action. Finally, we show that by implementing this temporal knowledge of drug effects to optimize scheduling of drug administration we are able to regain chemosensitivity in a Carboplatin-resistant cell line.

Naturally derived biomaterials have emerged as modulators of cell function and tissue substitutes. Here, we developed crosslinked glutaraldehyde (GTA) scaffolds for the expansion and differentiation of mesenchymal stem cells (MSCs). The mechanical and architectural properties of the scaffolds were altered by varying the concentration of gelatin and GTA. Higher GTA concentrations were associated with an increase in more confined pores and osteogenic differentiation. In addition, myogenic potential varied with crosslinking degree, although bulk mechanical properties were unaltered. Correlation analysis revealed that ALP activity of differentiated MSCs on higher gelatin concentration scaffolds was dependent on traditional effectors, including environment elasticity and spread area. In contrast, the differentiation capacity of cells cultured on lower gelatin concentration scaffolds did not correlate with these factors, instead it was dependent on the hydrated pore structure. These results suggest that scaffold composition can determine what factors direct differentiation and may have critical implications for biomaterial design.

The wound healing process initiates after injury to a tissue and involves a series of orchestrated events to minimize the invasion of foreign matters such as bacteria and efficiently regenerate the damaged tissue. A variety of cells must be recruited to the tissue during wound healing. However, this process is severely disrupted in patients suffering from chronic illness, including diabetes, leading to impaired healing or non-healing wounds. Current avenues of treatment include negative-pressure therapy, wound debridement, growth factor replacement, and cell-based therapies. Among these therapies, mesenchymal stem cells (MSCs) delivery to the wound holds a very high promise due to the innate abilities of MSCs that include immunogenicity, plasticity, and self-renewal. Bone marrow derived MSCs have been shown to promote more rapid wound healing by increased cytokine production in diabetic mice. However, the lack of understanding of the mechanical and chemical interaction of the transplanted MSCs with the factors present in the regenerative niches limits their efficacy in the wound bed. In this study, we sought to understand how the changes in MSC biochemical and biophysical properties can affect their function in vitro and in vivo. We demonstrate that pretreatment of MSCs with the mechano-stimulatory soluble factor transforming growth factor (TGF-β1), which is highly expressed in injury sites, improves wound closure in a syngeneic murine wound model. This improved wound closure correlated with increased invasion into the wound bed. In vitro studies demonstrated that TGF-β1 pretreatment expedited wound closure by increasing adhesion, traction force, and migration even after removal of the stimulus. Furthermore, this response was mediated by the cytoskeletal protein focal adhesion kinase. Taken together, this study suggests that defined chemical stimuli can benefit site specific adaptability of MSCs to improve their function and therapeutic usefulness.

Most cancer patients die from metastasis. Recent studies have shown that gold nanoparticles (AuNPs) can slow down the migration/invasion speed of cancer cells and suppress metastasis. Since nuclear stiffness of the cell largely decreases cell migration, our hypothesis is that targeting AuNPs to the cell nucleus region could enhance nuclear stiffness, and therefore inhibit cell migration and invasion. Our results showed that upon nuclear targeting of AuNPs, the ovarian cancer cell motilities decrease significantly, compared with nontargeted AuNPs. Furthermore, using atomic force microscopy, we observed an enhanced cell nuclear stiffness. In order to understand the mechanism of cancer cell migration/invasion inhibition, the exact locations of the targeted AuNPs were clearly imaged using a high-resolution three-dimensional imaging microscope, which showed that the AuNPs were trapped at the nuclear membrane. In addition, we observed a greatly increased expression level of lamin A/C protein, which is located in the inner nuclear membrane and functions as a structural component of the nuclear lamina to enhance nuclear stiffness. We propose that the AuNPs that are trapped at the nuclear membrane both (1) add to the mechanical stiffness of the nucleus and (2) stimulate the overexpression of lamin A/C located around the nuclear membrane, thus increasing nuclear stiffness and slowing cancer cell migration and invasion.

Multipotent human mesenchymal stem cells (hMSCs) are uniquely suited for the growing field of regenerative medicine due to their ease of isolation, expansion, and transplantation. However, during ex vivo expansion necessary to obtain clinically relevant cell quantities, hMSCs undergo fundamental changes culminating in cellular senescence. The molecular changes as hMSCs transition into senescence have been well characterized, but few studies have focused on the mechanical properties that govern many processes necessary for therapeutic efficacy. We show that before detectable differences in classical senescence markers emerge, single-cell mechanical and cytoskeletal properties reveal a subpopulation of 'non-functioning' hMSCs that appears after even limited expansion. This subpopulation, characterized by a loss of dynamic cytoskeletal stiffening and morphological flexibility in response to chemotactic signals grows with extended culture resulting in overall decreased hMSC motility and ability to contract collagen gels. These changes were mitigated with cytoskeletal inhibition. Finally, a xenographic wound healing model was used to demonstrate that these in vitro differences correlate with decreased ability of hMSCs to aid in wound closure in vivo. These data illustrate the importance of analyzing not only the molecular markers, but also mechanical markers of hMSCs as they are investigated for potential therapeutics.

Aims: Sputum poses a critical diffusional barrier that strongly limits the efficacy of drug and gene carriers in the airways of individuals with cystic fibrosis (CF). Previous attempts to enhance particle penetration of CF sputum have focused on either reducing its barrier properties via mucolytics, or decreasing particle adhesion to sputum constituents by coating the particle surface with non-mucoadhesive polymers, including polyethylene glycol (PEG). Neither approach has enabled particles to penetrate expectorated sputum at rates previously observed for non-mucoadhesive nanoparticles in human cervicovaginal mucus. Here, we sought to investigate whether a common mucolytic, N-acetyl cysteine (NAC), in combination with dense PEG coatings on particles, can synergistically enhance particle penetration across fresh undiluted CF sputum. Materials & methods: We used high-resolution multiple particle tracking to measure the diffusion of uncoated and PEG-coated nanoparticles in native and NAC-treated CF sputum. Results: We discovered that 200 nm particles, if densely coated with PEG, were able to penetrate CF sputum pretreated with NAC with average speeds approaching their theoretical speeds in water. Based on the rapid penetration of PEG-coated particles in NAC-treated sputum, we determined that the average spacing between sputum mesh elements was increased from 145 ± 50 nm to 230 ± 50 nm upon NAC treatment. Mathematical models based on particle transport rates suggest as much as 75 and 30% of 200 and 500 nm PEG-coated particles, respectively, may penetrate a physiologically thick NAC-treated CF sputum layer within 20 min. Uncoated particles were trapped in CF sputum pretreated with NAC nearly to the same extent as in native sputum, suggesting that NAC treatment alone offered little improvement to particle penetration. Conclusion: NAC facilitated rapid diffusion of PEG-coated, muco-inert nanoparticles in CF sputum. Our results provide a promising strategy to improve drug and gene carrier penetration in CF sputum, offering hope for improved therapies for CF.

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.

Tumor neovascularization and growth might be promoted by the recruitment of bone marrow–derived cells (BMDC), which include endothelial precursor cells and “vascular modulatory” myelomonocytic (CD11b+) cells. BMDCs may also drive tumor regrowth after certain chemotherapeutic and vascular disruption treatments. In this study, we evaluated the role of BMDC recruitment in breast and lung carcinoma xenograft models after local irradiation (LI). We depleted the bone marrow by including whole-body irradiation (WBI) of 6 Gy as part of a total tumor dose of 21 Gy, and compared the growth delay with the one achieved after LI of 21 Gy. In both models, the inclusion of WBI induced longer tumor growth delays. Moreover, WBI increased lung tumor control probability by LI. Exogenous delivery of BMDCs from radiation-naïve donors partially abrogated the WBI effect. Myeloid BMDCs, primarily macrophages, rapidly accumulated in tumors after LI. Intratumoral expression of stromal-derived factor 1α (SDF-1α), a chemokine that promotes tissue retention of BMDCs, was noted 2 days after LI. Conversely, treatment with an inhibitor of SDF-1α receptor CXCR4 (AMD3100) with LI significantly delayed tumor regrowth. However, when administered starting from 5 days post-LI, AMD3100 treatment was ineffective. Lastly, with restorative bone marrow transplantation of Tie2-GFP–labeled BMDC population, we observed an increased number of monocytes but not endothelial precursor cells in tumors that recurred following LI. Our results suggest that an increase in intratumoral SDF-1α triggered by LI recruits myelomonocytes/macrophages which promotes tumor regrowth. Cancer Res; 70(14); 5679–85. ©2010 AACR.

Mesenchymal stem cells (MSCs) are recruited to the tumor microenvironment and influence tumor progression; however, how MSCs induce the invasion of cancer cells is not completely understood. Here, we used a 3D coculture model to determine how MSCs affect the migration of invasive breast cancer cells. Coculture with MSCs increases the elongation, directional migration and traction generation of breast cancer cells. MSC-induced directional migration directly correlates with traction generation and is mediated by transforming growth factor β (TGF-β) and the migratory proteins rho-associated kinase, focal adhesion kinase and matrix metalloproteinases. Treatment with MSC conditioned media or recombinant TGF-β1 elicits a similar migration response to coculture. Taken together, this work suggests TGF-β is secreted by MSCs, leading to force-dependent directional migration of invasive breast cancer cells. These pathways may be potential targets for blocking cancer cell invasion and subsequent metastasis.

Chemorefractory ovarian cancer patients show extremely poor prognosis. Microtubule-stabilizing Taxol (paclitaxel) is a first-line treatment against ovarian cancer. Despite the close interplay between microtubules and cell adhesion, it remains unknown if chemoresistance alters the way cells adhere to their extracellular environment, a process critical for cancer metastasis. To investigate this, we isolated Taxol-resistant populations of OVCAR3 and SKOV3 ovarian cancer cell lines. Though Taxol-resistant cells neither effluxed more drug nor gained resistance to other chemotherapeutics, they did display increased microtubule dynamics. These changes in microtubule dynamics coincided with faster attachment rates and decreased adhesion strength, which correlated with increased surface β1-integrin expression and decreased focal adhesion formation, respectively. Adhesion strength correlated best with Taxol-sensitivity and was found to be independent of microtubule polymerization but dependent on focal adhesion kinase (FAK), which was up-regulated in Taxol-resistant cells. FAK inhibition also decreased microtubule dynamics to equal levels in both populations, indicating alterations in adhesive signaling are up-stream of microtubule dynamics. Taken together, this work demonstrates that Taxol-resistance dramatically alters how ovarian cancer cells adhere to their extracellular environment causing down-stream increases in microtubule dynamics, providing a therapeutic target that may improve prognosis by not only recovering drug sensitivity, but also decreasing metastasis.

Despite major advances in the characterization of molecular regulators of cancer growth and metastasis, patient survival rates have largely stagnated. Recent studies have shown that mechanical cues from the extracellular matrix can drive the transition to a malignant phenotype. Moreover, it is also known that the metastatic process, which results in over 90% of cancer-related deaths, is governed by intracellular mechanical forces. To better understand these processes, we identified metastatic tumor cells originating from different locations which undergo inverse responses to altered matrix elasticity: MDA-MB-231 breast cancer cells that prefer rigid matrices and SKOV-3 ovarian cancer cells that prefer compliant matrices as characterized by parameters such as tumor cell proliferation, chemoresistance, and migration. Transcriptomic analysis revealed higher expression of genes associated with cytoskeletal tension and contractility in cells that prefer stiff environments, both when comparing MDA-MB-231 to SKOV-3 cells as well as when comparing bone-metastatic to lung-metastatic MDA-MB-231 subclones. Using small molecule inhibitors, we found that blocking the activity of these pathways mitigated rigidity-dependent behavior in both cell lines. Probing the physical forces exerted by cells on the underlying substrates revealed that though force magnitude may not directly correlate with functional outcomes, other parameters such as force polarization do correlate directly with cell motility. Finally, this biophysical analysis demonstrates that intrinsic levels of cell contractility determine the matrix rigidity for maximal cell function, possibly influencing tissue sites for metastatic cancer cell engraftment during dissemination. By increasing our understanding of the physical interactions of cancer cells with their microenvironment, these studies may help develop novel therapeutic strategies.

Current biochemical characterizations of cystic fibrosis (CF) sputum do not address the high degree of microheterogeneity in the rheological properties of the mucosal matrix and only provide bulk-average particle diffusion coefficients. The viscoelasticity of CF sputum greatly reduces the diffusion rates of colloidal particles, limiting the effectiveness of gene delivery to underlying lung cells. We determine diffusion coefficients of hundreds of individual amine-modified and carboxylated polystyrene particles (diameter 100-500 nm) embedded in human CF sputum with 5 nm and 33 ms of spatiotemporal resolution. High resolution multiple particle tracking is used to calculate the effective viscoelastic properties of CF sputum at the micron scale, which we relate to its macroscopic viscoelasticity. CF sputum microviscosity, as probed by 100- and 200-nm particles, is an order of magnitude lower than its macroviscosity, suggesting that nanoparticles dispersed in CF sputum are transported primarily through lower viscosity pores within a highly elastic matrix. Multiple particle tracking provides a non-destructive, highly sensitive method to quantify the high heterogeneity of the mucus pore network. The mean diffusion coefficient becomes dominated by relatively few but fast-moving particles as particle size is reduced from 500 to 100 nm. Neutrally charged particles with a diameter

Glypican-1 (GPC-1) is a cell surface heparan sulfate proteoglycan that mediates cell proliferation, differentiation and migration. It also possess oncogenic functions in breast, pancreatic and lung cancer cells, and is a proposed biomarker for prostate cancer. Despite these data the function of GPC-1 in prostate cancer is not fully known. We addressed this gap-in-knowledge using in vitro and in vivo mouse models. We first showed that GPC-1 was overexpressed in bone metastatic prostate cancer cells (PC-3) but was not detected in lymph node metastatic cancer cells (LNCaP) and non-cancerous prostate cells (RWPE-1). Next, we inhibited GPC-1 expression using shRNA in bone metastatic cancer cells (PC-3), and verified inhibition using western blot analysis and qRT-PCR. Crystal violet staining, trans-well assays, matrigel colony cultures and adhesion assays were used to determine the effect of GPC-1 inhibition on cancer cell growth, migration, spheroid formation and cell adhesion. Inhibition of GPC-1 reduced prostate cancer cell growth, migration and spheroid formation; however, there were no changes in cell adhesion. These in vitro data suggested that GPC-1 played a role in prostate cancer progression by acting as an oncogene. We further investigated this possibility in vivo using athymic (NCr) mice xenografts. Interestingly, inhibition of GPC-1 in PC-3 cells appeared to increase tumor size as compared to scrambled controls. Further analysis showed that that inhibition of GPC-1 increased the tumor expression of MMP-9 and N-cadherin, but not E-cadherin. We hypothesized that the discrepancy between the in vitro and in vivo results may be mediated by interaction of PC-3 cells with the tumor microenvironment. We addressed this hypothesis by testing the effect of tumor conditioned media (TCM) from control and GPC-1 knockdown cells on human bone marrow derived mesenchymal stem cells (hMSC) and fibroblast (HS-27). Treatment of both hMSC and HS-27 cells with TCM from GPC-1 knockdown PC-3 cells increased MMP-9 and interestingly MMP-2 expression and activity as compared to control cells. We also detected increases in the expression of N-Cadherin in a co-culture model. Collectively, these data suggest that GPC-1 acts as an oncogene to enhance prostate cancer cell growth in vitro; however, these data also suggest the GPC-1 may act as a tumor suppressor in vivo. This discrepancy may be due to the interaction off GPC-1 with the tumor microenvironment, which would not be detected in single-cell culture models. Citation Format: Nhat D. Quach, Matthew Eggert, Deepraj Ghosh, Michelle R. Dawson, Robert Arnold, Brian S. Cummings. Glypican-1: A tumor suppressor or an oncogene in human bone metastatic prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4465. doi:10.1158/1538-7445.AM2017-4465

The mechanical properties within the cell are regulated by the organization of the actin cytoskeleton, which is linked to the extracellular environment through focal adhesion proteins that transmit force. Chemical and mechanical stimuli alter the organization of cytoskeletal actin, which results in changes in cell shape, adhesion, and differentiation. By combining particle-tracking microrheology and traction force cytometry, we can monitor the mechanical properties of the actin meshwork and determine how changes in the intracellular network contribute to force generation. In this study, we investigated the effects of chemical (differentiation factors) and mechanical (substrate rigidity) stimuli important in mesenchymal stem cell (MSC) differentiation on the intracellular mechanics and traction stress generation. We found the presence of adipogenic factors resulted in stiffening of the actin meshwork regardless of substrate rigidity. In contrast, these factors increased traction stresses on hard substrates, which was associated with increased expression of contractility genes. Furthermore, MSCs cultured on hard substrates expressed both adipogenic and osteogenic markers indicative of mixed differentiation. On hard substrates, heterogeneity in the local elastic modulus-traction stress correlation was also increased in response to adipogenic factors, indicating that these mechanical properties may be reflective of differences in the level of MSC differentiation. These results suggest intracellular rheology and traction stress generation are spatially regulated and contribute insight into how single cell mechanical forces contribute to MSC differentiation.

A growing body of evidence suggests that the developmental process of epithelial-to-mesenchymal transition (EMT) is co-opted by cancer cells to metastasize to distant sites. This transition is associated with morphologic elongation and loss of cell-cell adhesions, though little is known about how it alters cell biophysical properties critical for migration. Here, we use multiple-particle tracking (MPT) microrheology and traction force cytometry to probe how genetic induction of EMT in epithelial MCF7 breast cancer cells changes their intracellular stiffness and extracellular force exertion, respectively, relative to an empty vector control. This analysis demonstrated that EMT alone was sufficient to produce dramatic cytoskeletal softening coupled with increases in cell-exerted traction forces. Microarray analysis revealed that these changes corresponded with down-regulation of genes associated with actin cross-linking and up-regulation of genes associated with actomyosin contraction. Finally, we show that this loss of structural integrity to expedite migration could inhibit mesenchymal cell proliferation in a secondary tumor as it accumulates solid stress. This work demonstrates that not only does EMT enable escape from the primary tumor through loss of cell adhesions but it also induces a concerted series of biophysical changes enabling enhanced migration of cancer cells after detachment from the primary tumor.

Although current treatments for localized ovarian cancer are highly effective, this cancer still remains the most lethal gynecological malignancy, largely owing to the fact that it is often detected only after tumor cells leave the primary tumor. Clinicians have long noted a clear predilection for ovarian cancer to metastasize to the soft omentum. Here, we show that this tropism is due not only to chemical signals but also mechanical cues. Metastatic ovarian cancer cells (OCCs) preferentially adhere to soft microenvironments and display an enhanced malignant phenotype, including increased migration, proliferation and chemoresistance. To understand the cell–matrix interactions that are used to sense the substrate rigidity, we utilized traction force microscopy (TFM) and found that, on soft substrates, human OCCs increased both the magnitude of traction forces as well as their degree of polarization. After culture on soft substrates, cells underwent morphological elongation characteristic of epithelial-to-mesenchymal transition (EMT), which was confirmed by molecular analysis. Consistent with the idea that mechanical cues are a key determinant in the spread of ovarian cancer, the observed mechanosensitivity was greatly decreased in less-metastatic OCCs. Finally, we demonstrate that this mechanical tropism is governed through a Rho–ROCK signaling pathway.

Although current treatments for localized ovarian cancer are highly effective, this cancer still remains the most lethal gynecological malignancy, largely owing to the fact that it is often detected only after tumor cells leave the primary tumor. Clinicians have long noted a clear predilection for ovarian cancer to metastasize to the soft omentum. Here, we show that this tropism is due not only to chemical signals but also mechanical cues. Metastatic ovarian cancer cells (OCCs) preferentially adhere to soft microenvironments and display an enhanced malignant phenotype, including increased migration, proliferation and chemoresistance. To understand the cell–matrix interactions that are used to sense the substrate rigidity, we utilized traction force microscopy (TFM) and found that, on soft substrates, human OCCs increased both the magnitude of traction forces as well as their degree of polarization. After culture on soft substrates, cells underwent morphological elongation characteristic of epithelial-to-mesenchymal transition (EMT), which was confirmed by molecular analysis. Consistent with the idea that mechanical cues are a key determinant in the spread of ovarian cancer, the observed mechanosensitivity was greatly decreased in less-metastatic OCCs. Finally, we demonstrate that this mechanical tropism is governed through a Rho–ROCK signaling pathway.

Here we integrated multiphoton laser scanning microscopy and the registration of second harmonic generation images of collagen fibers to overcome difficulties in tracking stromal cell-matrix interactions for several days in live mice. We show that the matrix-modifying hormone relaxin increased tumor-associated fibroblast (TAF) interaction with collagen fibers by stimulating beta1-integrin activity, which is necessary for fiber remodeling by matrix metalloproteinases.

Mesenchymal stem cells (MSCs) play an important role in matrix remodeling, fibroblast activation, angiogenesis, and immunomodulation and are an integral part of fibrovascular networks that form in developing tissues and tumors. The engraftment and function of MSCs in tissue niches is regulated by a multitude of soluble proteins. Transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor-BB (PDGF) have previously been recognized for their role in MSC biology; thus, we sought to investigate their function in mediating MSC mechanics and matrix interactions. Cytoskeletal organization, characterized by cell elongation, stress fiber formation, and condensation of actin and microtubules, was dramatically affected by TGF-β1, individually and in combination with PDGF. The intracellular mechanical response to these stimuli was measured with particle tracking microrheology. MSCs stiffened in response to TGF-β1 (their elastic moduli was ninefold higher than control cells), a result that was enhanced by the addition of PDGF (100-fold change). Blocking TGF-β1 or PDGF signaling with inhibitors SB-505124 or JNJ-10198409, respectively, reversed soluble-factor-induced stiffening, indicating that crosstalk between these two pathways is essential for stiffening response. A genome-wide microarray analysis revealed TGF-β1-dependent regulation of cytoskeletal actin-binding protein genes. Actin crosslinking and bundling protein genes, which regulate cytosolic rheology through changes in semiflexible actin polymer meshwork, were upregulated with TGF-β1 treatment. TGF-β1 alone and in combination with PDGF also amplified surface integrin expression and adhesivity of MSCs with extracellular matrix proteins. These findings will provide a more mechanistic insight for modeling tissue-level rigidity in fibrotic tissues and tumors.

The progression of neoplastic malignancies is a complex process resulting not only from the accumulation of mutations within tumor cells, but also modulation of the tumor microenvironment. Recent advances have shown that the recruitment and subsequent heterotypic interactions of stromal cells--including fibroblasts and bone marrow-derived mesenchymal stem cells (MSCs)--are crucial for carcinogenesis. Though extensive work has been done analyzing the signals that recruit these cells, the governing mechanical properties have not been fully investigated. Here, we report that despite their initial similarities, MSCs respond not only faster but also more dramatically to pro-migratory tumor-secreted soluble factors. Utilizing multiple particle tracking microrheology to probe the cytoskeletal mechanical properties, we show that MSCs stiffen completely within one hour, three times faster than fibroblasts. In addition, unlike fibroblasts, MSCs exposed to tumor-secreted soluble factors display a functionally different phenotype characterized by morphological elongation, decreased actin stress fiber density, and decreased adhesion. Quantitative real-time PCR indicates these phenomena occur based on differential expression of small GTPases RhoA and Cdc42, but not Rac1. These findings demonstrate a fundamental difference in the recruitment of fibroblasts and MSCs.

We sought to characterize the function of bone marrow stromal cell (BMSC) populations in tumor progression. Because this function may depend on the cell-lineage and mouse strain heterogeneity, we first characterized ex vivo the BMSCs harvested from C57BL/6 versus FVB mice and established their in vivo function in tumor growth and metastasis experiments. All plastic-adherent BMSCs expressed platelet-derived growth factor receptor beta (PDGFRβ) and stem cell antigen 1 (Sca1), consistent with a mesenchymal precursor phenotype, as well as CD80. Moreover, these BMSCs were capable of differentiation along mesenchymal lineage into adipocytes, osteoblasts, chondrocytes or myofibroblasts. However, further phenotypic analysis detected a distinct populations of myeloid (CD11b(+)) precursor cells amongst the ex vivo expanded BMSCs -with specific surface marker phenotypes and gene expression pattern. When co-implanted with metastatic cancer cells, all the BMSCs persisted and integrated into tumor stroma, but only myeloid BMSCs significantly promoted tumor growth and metastasis. These data demonstrate the differential effect of BMSCs sub-populations on tumor progression. These results may have important implications for anti-tumor therapy and for the use of mesenchymal BMSCs as cell-based therapies.

Mesenchymal stem cells (MSCs) are excellent candidates for the development of cell-based gene delivery systems; however, extended cell culture, required for therapeutic development, alters MSC morphology, reducing MSC migration upon reinfusion. Spontaneous migration of MSCs to tumors is mediated by tumor secretion of proangiogenic chemokines. Multiple particle tracking microrheology was used to investigate the effect of tumor-secreted molecules on MSC viscoelasticity, which was correlated with MSC migration and morphology. Within 24 hours after MSC treatment with tumor-conditioned media (TCM), MSCs were elongated, with more than 5-fold difference in the length of lamellipodia. Within 24 hours, the migration of MSCs, measured using a Boyden chamber assay, toward TCM was increased 10-fold over control media. The mean squared displacements (MSDs) of 100-nm carboxylated polystyrene particles, injected into the cytoplasm of human MSCs using the Biolistic Particle Injection System, were determined with 33 ms temporal and 5 nm spatial resolution using multiple particle tracking. The frequency dependent elastic and viscous moduli were calculated from the complex shear moduli, which were determined from the Fourier transform of the time-dependent MSDs, using the frequency-dependent Stokes-Einstein equation. Pretreatment of MSCs with TCM resulted in rapid changes in cytoplasmic viscoelasticity with a 9.8-fold increase in the average elastic moduli, which increased from 35 to 344 dyn/cm2, and a 3.5-fold decrease in the average viscous moduli, which was reduced from 99 to 28 dyn/cm2, within 1 hour (n = 6-8 cells per group). We hypothesize that tumor-secreted molecules increase MSC mobility by altering cytoskeletal organization. Changes in MSC viscosity may be in part to reduced actin cross-linking during cytoskeletal reorganization. Increased MSC rigidity may be due to MSC elongation, which leads to the formation of polymer entanglements as the ratio of cell length to width is greatly increased.