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Movie S1 shows timelapse of cells undergoing failure of abscission and becoming multinucleated. Nuclei are in red.

Figures S1-S5 show how stiffness-mediated signaling downstream of Snail is affected by proliferation and expression of septin-6. Tables S1-S2 list primers for qPCR and binding sites in the septin-6 promoter. Legends for Movies S1-S2, which show abscission failure, are also included.

Metastasis, the leading cause of mortality in cancer patients, depends upon the ability of cancer cells to invade into the extracellular matrix that surrounds the primary tumor and to escape into the vasculature. To investigate the features of the microenvironment that regulate invasion and escape, we generated solid microtumors of MDA-MB-231 human breast carcinoma cells within gels of type I collagen. The microtumors were formed at defined distances adjacent to an empty cavity, which served as an artificial vessel into which the constituent tumor cells could escape. To define the relative contributions of matrix degradation and cell proliferation on invasion and escape, we used pharmacological approaches to block the activity of matrix metalloproteinases (MMPs) or to arrest the cell cycle. We found that blocking MMP activity prevents both invasion and escape of the breast cancer cells. Surprisingly, blocking proliferation increases the rate of invasion but has no effect on that of escape. We found that arresting the cell cycle increases the expression of MMPs, consistent with the increased rate of invasion. To gain additional insight into the role of cell proliferation in the invasion process, we generated microtumors from cells that express the fluorescent ubiquitination-based cell cycle indicator. We found that the cells that initiate invasions are preferentially quiescent, whereas cell proliferation is associated with the extension of invasions. These data suggest that matrix degradation and cell proliferation are coupled during the invasion and escape of human breast cancer cells and highlight the critical role of matrix proteolysis in governing tumor phenotype.

Multinucleation is found in more than one third of tumors and is linked to increased tolerance for mutation, resistance to chemotherapy, and invasive potential. The integrity of the genome depends on proper execution of the cell cycle, which can be altered through mechanotransduction pathways as the tumor microenvironment stiffens during tumorigenesis. Here, we show that signaling downstream of matrix metalloproteinase-3 (MMP3) or TGFβ, known inducers of epithelial–mesenchymal transition (EMT), also promotes multinucleation in stiff microenvironments through Snail-dependent expression of the filament-forming protein septin-6, resulting in midbody persistence, abscission failure, and multinucleation. Consistently, we observed elevated expression of Snail and septin-6 as well as multinucleation in a human patient sample of metaplastic carcinoma of the breast, a rare classification characterized by deposition of collagen fibers and active EMT. In contrast, a soft microenvironment protected mammary epithelial cells from becoming multinucleated by preventing Snail-induced upregulation of septin-6. Our data suggest that tissue stiffening during tumorigenesis synergizes with oncogenic signaling to promote genomic abnormalities that drive cancer progression. Significance: These findings reveal tissue stiffening during tumorigenesis synergizes with oncogenic signaling to promote genomic abnormalities that drive cancer progression. Cancer Res; 78(9); 2277–89. ©2018 AACR.

Cells communicate constantly with their surrounding extracellular matrix (ECM) to maintain homeostasis, using both mechanical and chemical signals. In cancer, abnormal signaling leads to stiffening of the ECM. A stiff microenvironment affects many aspects of the cell, including internal molecular signaling as well as behaviors such as motility and proliferation. Thus, cells and ECM interact in a feedback loop to drive matrix deposition and cross-linking, which alter the mechanical properties of the tissue. Stiffer tissue enhances the invasive potential of a tumor and decreases therapeutic efficacy. This chapter describes how specific molecular effects caused by an abnormally stiff tissue drive macroscopic changes that help determine disease outcome. A complete understanding may foster the generation of new cancer therapies.

Cells communicate constantly with their surrounding extracellular matrix (ECM) to maintain homeostasis, using both mechanical and chemical signals. In cancer, abnormal signaling leads to stiffening of the ECM. A stiff microenvironment affects many aspects of the cell, including internal molecular signaling as well as behaviors such as motility and proliferation. Thus, cells and ECM interact in a feedback loop to drive matrix deposition and cross-linking, which alter the mechanical properties of the tissue. Stiffer tissue enhances the invasive potential of a tumor and decreases therapeutic efficacy. This chapter describes how specific molecular effects caused by an abnormally stiff tissue drive macroscopic changes that help determine disease outcome. A complete understanding may foster the generation of new cancer therapies.

The mechanical properties of the tumor microenvironment (TME) play a critical role on the progression of breast cancer metastasis. However, the complex architecture of the TME conceals the individual effects of different biophysical and biochemical factors on tumor invasion and intravasation. To investigate this question, we engineered a robust breast tumor model of solid-like 3D aggregate of human breast cancer cells with interstitial fluid pressure (IFP), and further integrated it with an empty cavity to emulate the presence of an impaired capillary vessel. In brief, we embed MDA-MB-231 human breast cancer cells in one of two neighboring collagen type I cavities that are molded within polydimethylsiloxane (PDMS) channels. This multicellular aggregate is subject to selected gradients of hydrostatic pressure through opposing reservoirs of culture media that are located at the base (Pbase) and the tip (Ptip) of the tumor. We found that breast cancer cells disseminate from the multicellular aggregate and escape into the proximal cavity under Ptip > Pbase. The separation distance between the aggregate and the cavity influences the features of tumor escape. Tumor models that were seeded within a distance of less than 150 μm from the cavity demonstrated significantly shorter time (t1/2~ 4 days) for the escape of 50% of the tumor population than those seeded between 150 and 300 μm. In contrast, less than 50% of the tumors that were seeded longer than 300 μm apart of the cavity successfully escaped after ~ 2 weeks under Ptip > Pbase. In addition, we found that cells escaped into the cavity through three major modes: a) single-cell migration, b) multicellular invasion, and c) tumor growth. Single-cell migration was the dominant route of escape in collagen gels of low concentration (2.5 mg/ml). In contrast, tumor growth and multicellular invasion were the dominant modes of escape in collagen gels of high concentration (4mg/ml). Moreover, the tumor invasions were found to be preferentially directed normal to the surface of the tumor, and to be drastically eliminated in effect of pharmacological inhibition of matrix metalloproteinases (MMPs). These preliminary findings will be put together with additional quantitative studies to correlate tumor-cavity separation with the different modes of tumor escape. Overall, our engineered breast tumor model composes a unique platform to investigate the biophysical and biochemical mechanisms of the tumor microenvironment that drive tumor invasion and intravasation into the circulatory system. Citation Format: Andreas P. Kourouklis, Usman Ghani, Siyang Han, Yoseph Dance, Allison K. Simi, Joe Tien, Celeste M. Nelson. Tumor invasion and escape from an engineered solid-like aggregate of human breast cancer cells into a cavity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4526.

The purification of structurally similar compounds was investigated using selective impurity complex formation in solution followed by crystallization of the target compound. Two systems of structurally similar compounds, benzamide/benzoic acid (BAM/BA) and cinnamamide/cinnamic acid (CAM/CA), were chosen. Three reported co-formers that form co-crystals with both BA and CA were selected: isonicotinamide (INA), 2-amino-4,6-dimethylpyrimidine (DMP), and dimethylglyoxime (DMG). The addition of DMG to the BAM/BA system demonstrated the largest improvement of BAM purity. The amount of BA did not decrease with increasing amount of DMG added. For the CAM/CA system, adding DMP resulted in the largest CAM purity increase. Phase solubility diagrams were measured to calculate binding constants for 1:1 complexes. These binding constants were used as indications of the complexation level in the solution. For the CAM/CA system, the more complex formed in solution, the purer the CAM. This result was not seen in the BAM/B...

Cells are surrounded by mechanical stimuli in their microenvironment. It is important to determine how cells respond to the mechanical information that surrounds them in order to understand both development and disease progression, as well as to be able to predict cell behavior in response to physical stimuli. Here we describe a protocol to determine the effects of interstitial fluid flow on the migratory behavior of an aggregate of epithelial cells in a three-dimensional (3D) culture model. This protocol includes detailed methods for the fabrication of a 3D cell culture chamber with hydrostatic pressure control, the culture of epithelial cells as an aggregate in a collagen gel, and the analysis of collective cell behavior in response to pressure-driven flow.

Cells are surrounded by mechanical stimuli in their microenvironment. It is important to determine how cells respond to the mechanical information that surrounds them in order to understand both development and disease progression, as well as to be able to predict cell behavior in response to physical stimuli. Here we describe a protocol to determine the effects of interstitial fluid flow on the migratory behavior of an aggregate of epithelial cells in a three-dimensional (3D) culture model. This protocol includes detailed methods for the fabrication of a 3D cell culture chamber with hydrostatic pressure control, the culture of epithelial cells as an aggregate in a collagen gel, and the analysis of collective cell behavior in response to pressure-driven flow.

Collapsed blood or lymphatic vessels in the tumor microenvironment often cause fluid buildup, leading to heterogeneous flow throughout the tissue. Here, we used a three-dimensional (3D) engineered tumor model to investigate how fluid flow specifically influences invasion and chemoresistance of breast cancer cells. To mimic breast tumors, we cultured aggregates of MDA-MB-231 human breast cancer cells embedded in 3D collagen channels. Collagen channels were flanked on the ends by two media reservoirs. By changing the relative heights of media in the reservoirs, we controlled the pressure-induced flow experienced by the tumor cell aggregate. We found that the direction of flow through the collagen channel determined the invasive phenotype of the engineered tumor. These analyses will be repeated with the addition of chemotherapy drugs taxol or 5-fluorouracil in the media to determine the effect of fluid flow on chemotherapeutic response. Our engineered tumor model provides insight into how physical forces influence the invasive phenotype of cancer cells. Citation Format: Allison K. Simi, Andreas P. Kourouklis, Alexandra S. Piotrowski-Daspit, Joe Tien, Celeste M. Nelson. The role of pressure-driven flow in invasion and chemoresistance of cancer cells in an engineered breast tumor model [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 40.

This study investigates how increased stiffness of the tumor microenvironment can induce cellular multinucleation, an easily observable marker of polyploidy. Up to 37% percent of tumors exhibit whole-genome doubling, which typically precedes other somatic copy number alterations. Additionally, induction of tetraploidy in human cells promotes increased tolerance for mutation, resistance to chemotherapeutic drugs, and transformation in culture. Tumors are inherently stiffer than normal tissue, and this property has been shown to affect cell growth and proliferation. Similarly, cell cycle errors have long been linked to chromosomal abnormalities. Here, we used engineered two-dimensional substrata that mimic the stiffness of tumor and normal microenvironments to investigate how matrix stiffness regulates multinucleation in mammary epithelial cells. Multinucleation was quantified by staining with Hoescht to visualize the nuclei. Timelapse microscopy enabled visualization of the process by which cells become multinucleated. Changes in gene expression were determined by quantitative RT-PCR. Cells cultured on “stiff” substrata, representing tumor tissue, showed a nearly 14-fold increase in multinucleation compared to cells cultured on “soft” substrata, representing normal tissue. We found that multinucleation was regulated in part by signaling downstream of matrix metalloproteinase-3 (MMP3), which is commonly upregulated in cancer and known to induce epithelial-mesenchymal transition (EMT). This signaling depended on expression of the Rac1 splice variant, Rac1b, production of ROS, and expression of Snail. Under all conditions, cells cultured on soft substrata maintained a low frequency of multinucleation. Multinucleation on stiff substrata primarily resulted from midbody abscission failure. A soft microenvironment protected the stability of the genome in epithelial cells by preventing midbody stability, which depended on septin 4, a novel target of Snail. Importantly, we found that transforming growth factor-β (TGFβ), another EMT-inducer, also caused multinucleation downstream of Snail, which was prevented by culture on soft substrata. Our data thus suggest that tissue stiffening during tumorigenesis synergizes with oncogenic signaling to promote genomic abnormalities that drive cancer progression. Further, our results suggest that EMT-related signaling pathways are associated with disease progression not necessarily because they induce metastasis, but because they induce genomic instability. Note: This abstract was not presented at the meeting. Citation Format: Allison K. Simi, Alisya A. Anlas, Sherry X. Zhang, Tiffaney Hsia, Derek C. Radisky, Celeste M. Nelson. A soft microenvironment protects from failure of midbody abscission and multinucleation downstream of EMT initiators [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 5914. doi:10.1158/1538-7445.AM2017-5914

Cells translate mechanical forces in the environment into biochemical signals in a process called mechanotransduction. In this way, mechanical forces direct cell behavior, including motility, proliferation, and differentiation, and become important in physiological processes such as development and wound healing. Abnormalities in mechanotransduction can lead to aberrant cell behavior and disease, including cancer. Changes in extracellular mechanical forces or defects in mechanosensors can result in misregulation of signaling pathways inside the cell, and ultimately lead to malignancy. Here, we discuss the ways in which physical attributes of the tumor microenvironment can promote metastasis and genomic instability, two hallmark features of cancer.

Viral lethal mutagenesis is a strategy whereby the innate immune system or mutagenic pool nucleotides increase the error rate of viral replication above the error catastrophe limit. Lethal mutagenesis has been proposed as a mechanism for several antiviral compounds, including the drug candidate 5-aza-5,6-dihydro-2'-deoxycytidine (KP1212), which causes A-to-G and G-to-A mutations in the HIV genome, both in tissue culture and in HIV positive patients undergoing KP1212 monotherapy. This work explored the molecular mechanism(s) underlying the mutagenicity of KP1212, and specifically whether tautomerism, a previously proposed hypothesis, could explain the biological consequences of this nucleoside analog. Establishing tautomerism of nucleic acid bases under physiological conditions has been challenging because of the lack of sensitive methods. This study investigated tautomerism using an array of spectroscopic, theoretical, and chemical biology approaches. Variable temperature NMR and 2D infrared spectroscopic methods demonstrated that KP1212 existed as a broad ensemble of interconverting tautomers, among which enolic forms dominated. The mutagenic properties of KP1212 were determined empirically by in vitro and in vivo replication of a single-stranded vector containing a single KP1212. It was found that KP1212 paired with both A (10%) and G (90%), which is in accord with clinical observations. Moreover, this mutation frequency is sufficient for pushing a viral population over its error catastrophe limit, as observed before in cell culture studies. Finally, a model is proposed that correlates the mutagenicity of KP1212 with its tautomeric distribution in solution.

This study investigates how increased stiffness of the tumor microenvironment (TME) can facilitate cellular multinucleation. Multinucleation is an easily observable marker for polyploidy, facilitating the quantification of this phenotype. Polyploidy is commonly an intermediate to aneuploidy, defined as an abnormal number of chromosomes, a phenotype found in 85% of solid cancers that can drive tumorigenesis. Up to 37% percent of tumors exhibit whole-genome doubling, which typically precedes other somatic copy number alterations. Additionally, induction of tetraploidy in human cells promoted increased tolerance for mutation, resistance to chemotherapeutic drugs, and transformation in culture. Tumors are inherently stiffer than normal tissue, and this property has been shown to affect cell growth and proliferation. Similarly, cell cycle errors have long been linked to chromosomal abnormalities. Here, we used engineered two-dimensional substrata that mimic tumor and normal microenvironments to investigate how matrix stiffness regulates multinucleation in mammary epithelial cells. Cells cultured on “stiff” substrata, representing tumor tissue, showed a nearly 14-fold increase in multinucleation compared to cells cultured on “soft” substrata, representing normal tissue. We found that multinucleation was regulated in part by signaling downstream of MMP3, a protease commonly upregulated in cancer. This signaling depended on expression of the Rac1 splice variant, Rac1b, production of reactive oxygen species (ROS), and expression of Snail. Under all conditions, cells cultured on soft substrata maintained a low frequency of multinucleation. We also investigated biochemical effectors downstream of Snail. Aurora A kinase (Aurora A) is a mitotic regulator commonly elevated in cancer that has been shown to induce multinucleation in mammary epithelial cells and increase tumor incidence in mice. We found using microarray analysis that MMP3 increases the levels of Aurora A in mammary epithelial cells. We further discovered that upregulation of Aurora A in response to MMP3 signaling was limited to one transcript variant. These data suggest that an increase in multinucleation is driven by the stiffening of the tissue during tumorigenesis, in part by MMP3-induced signaling and regulation of Aurora A splicing. More broadly, these data suggest a key role for the mechanical properties of the tumor microenvironment in cancer progression. Citation Format: Allison K. Simi, Derek C. Radisky, Celeste M. Nelson. Tissue stiffness regulates multinucleation in mammary epithelial cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5097.