Modeling Collective Invasion and Single-Cell Mesenchymal Invasion in Three-Dimensional Matrigel-Collagen I Cultures.

Understanding the modes and mechanisms of tumor cell invasion is key to developing targeted therapies against metastatic disease. In vitro assays modeling tumor progression have primarily been optimized for studying classical single-cell migration through an epithelial-mesenchymal transition (EMT). Although experimental and clinical histopathological evidence has revealed that tumor invasion is plastic and that epithelial carcinomas can invade by a range of modes that vary from single, mesenchyme-like cells, all the way to cohesive, collective units, few in vitro assays have been designed to assess these modes specifically. Thus, we have developed a Matrigel-Collagen I overlay assay that is suitable for identifying and quantifying both collective and mesenchymal invasion. This three-dimensional (3D) culture assay utilizes the features of Matrigel and Collagen I to mimic the laminin-rich basement membrane and the stiff, fibrillar Collagen I tumor microenvironment allowing for spheroid invasion to be assessed at the interface between these two matrix components.

Applied stretch initiates directional invasion through the action of Rap1 GTPase as a tension sensor.

Although it is known that a stiffening of the stroma and the rearrangement of collagen fibers within the extracellular matrix facilitate the movement of tumor cells away from the primary lesion, the underlying mechanisms responsible are not fully understood. We now show that this invasion, which can be initiated by applying tensional loads to a three-dimensional collagen gel matrix in culture, is dependent on the Rap1 GTPases (Rap1a and Rap1b, referred to collectively as Rap1). Under these conditions Rap1 activity stimulates the formation of focal adhesion structures that align with the tensional axis as single tumor cells move into the matrix. These effects are mediated by the ability of Rap1 to induce the polarized polymerization and retrograde flow of actin, which stabilizes integrins and recruits vinculin to preformed adhesions, particularly those near the leading edge of invasive cells. Rap1 activity also contributes to the tension-induced collective invasive elongation of tumor cell clusters and it enhances tumor cell growth in vivo Thus, Rap1 mediates the effects of increased extracellular tension in multiple ways that are capable of contributing to tumor progression when dysregulated.

The cell surface mucin podocalyxin regulates collective breast tumor budding.

BACKGROUND: Overexpression of the transmembrane sialomucin podocalyxin, which is known to play a role in lumen formation during polarized epithelial morphogenesis, is an independent indicator of poor prognosis in a number of epithelial cancers, including those that arise in the breast. Therefore, we set out to determine if podocalyxin plays a functional role in breast tumor progression. METHODS: MCF-7 breast cancer cells, which express little endogenous podocalyxin, were stably transfected with wild type podocalyxin for forced overexpression. 4T1 mammary tumor cells, which express considerable endogenous podocalyxin, were retrovirally transduced with a short hairpin ribonucleic acid (shRNA) targeting podocalyxin for stable knockdown. In vitro, the effects of podocalyxin on collective cellular migration and invasion were assessed in two-dimensional monolayer and three-dimensional basement membrane/collagen gel culture, respectively. In vivo, local invasion was assessed after orthotopic transplantation in immunocompromised mice. RESULTS: Forced overexpression of podocalyxin caused cohesive clusters of epithelial MCF-7 breast tumor cells to bud off from the primary tumor and collectively invade the stroma of the mouse mammary gland in vivo. This budding was not associated with any obvious changes in histoarchitecture, matrix deposition or proliferation in the primary tumour. In vitro, podocalyxin overexpression induced a collective migration of MCF-7 tumor cells in two-dimensional (2-D) monolayer culture that was dependent on the activity of the actin scaffolding protein ezrin, a cytoplasmic binding partner of podocalyxin. In three-dimensional (3-D) culture, podocalyxin overexpression induced a collective budding and invasion that was dependent on actomyosin contractility. Interestingly, the collectively invasive cell aggregates often contained expanded microlumens that were also observed in vivo. Conversely, when endogenous podocalyxin was removed from highly metastatic, but cohesive, 4T1 mammary tumor cells there was a decrease in collective invasion in three-dimensional culture. CONCLUSIONS: Podocalyxin is a tumor cell-intrinsic regulator of experimental collective tumor cell invasion and tumor budding.

Podocalyxin enhances breast tumor growth and metastasis and is a target for monoclonal antibody therapy.

INTRODUCTION: Podocalyxin (gene name PODXL) is a CD34-related sialomucin implicated in the regulation of cell adhesion, migration and polarity. Upregulated expression of podocalyxin is linked to poor patient survival in epithelial cancers. However, it is not known if podocalyxin has a functional role in tumor progression. METHODS: We silenced podocalyxin expression in the aggressive basal-like human (MDA-MB-231) and mouse (4T1) breast cancer cell lines and also overexpressed podocalyxin in the more benign human breast cancer cell line, MCF7. We evaluated how podocalyxin affects tumorsphere formation in vitro and compared the ability of podocalyxin-deficient and podocalyxin-replete cell lines to form tumors and metastasize using xenogenic or syngeneic transplant models in mice. Finally, in an effort to develop therapeutic treatments for systemic cancers, we generated a series of antihuman podocalyxin antibodies and screened these for their ability to inhibit tumor progression in xenografted mice. RESULTS: Although deletion of podocalyxin does not alter gross cell morphology and growth under standard (adherent) culture conditions, expression of PODXL is required for efficient formation of tumorspheres in vitro. Correspondingly, silencing podocalyxin resulted in attenuated primary tumor growth and invasiveness in mice and severely impaired the formation of distant metastases. Likewise, in competitive tumor engraftment assays where we injected a 50:50 mixture of control and shPODXL (short-hairpin RNA targeting PODXL)-expressing cells, we found that podocalyxin-deficient cells exhibited a striking decrease in the ability to form clonal tumors in the lung, liver and bone marrow. Finally, to validate podocalyxin as a viable target for immunotherapy, we screened a series of novel antihuman podocalyxin antibodies for their ability to inhibit tumor progression in vivo. One of these antibodies, PODOC1, potently blocked tumor growth and metastasis. CONCLUSIONS: We show that podocalyxin plays a key role in the formation of primary tumors and distant tumor metastasis. In addition, we validate podocalyxin as potential target for monoclonal antibody therapy to inhibit primary tumor growth and systemic dissemination.

The anti-adhesive mucin podocalyxin may help initiate the transperitoneal metastasis of high grade serous ovarian carcinoma.

High grade serous ovarian tumors often metastasize transperitoneally, a process that begins when small tumor nodules de-adhere and are released into the fluid of the abdominal cavity where they float freely to reach new sites on the peritoneal wall. Podocalyxin, a small anti-adhesive sialomucin, has been shown to contribute to non-adhesive membrane domain formation in some epithelia and is overexpressed in a variety of cancers. We therefore assessed podocalyxin expression on a previously characterized tissue microarray and found that 87% (169/194) of high grade serous epithelial ovarian carcinomas were positive for podocalyxin. In addition, cell surface localization of podocalyxin was associated with a significant decrease in disease-free survival in these tumors. When podocalyxin was force-expressed in serous ovarian carcinoma-derived OVCAR-3 cells it was targeted to the cell surface and it decreased the adhesion of these cells to mesothelial monolayers, fibronectin and immobilized ß1 integrin-binding antibodies. This decrease in adhesion was associated with a modest decrease in cell surface ß1 integrin. In monolayer culture, podocalyxin was targeted to the free, apical domains of OVCAR-3 cells and it appeared to decrease ß1 integrin levels on the attached basolateral domains of the same cells. Furthermore, in 3-dimensional basement membrane gel culture, the cells formed small, cohesive nodules and podocalyxin localized to membrane domains at the cell-basement membrane interface. Therefore, podocalyxin's ability to facilitate the formation of non-adhesive membrane domains may contribute to the formation of free-floating high grade serous tumor nodules during the initial steps of transperitoneal metastasis.

Regulation of the BRCA1 promoter in ovarian surface epithelial cells and ovarian carcinoma cells.

As BRCA1 expression is often suppressed in sporadic ovarian carcinoma we characterized the regulation of the 231nt proximal 'L6' fragment of the BRCA1 promoter in two human ovarian surface epithelial cell and two sporadic ovarian carcinoma cell lines. Two individual regulatory elements within L6, the 'RIBS' element and the potential 'CRE' element were each necessary, but alone not sufficient for L6 activation in all four cell lines. The latter element showed some affinity for the CREB transcription factor, but cAMP pathway stimulation failed to promote its activation. This element did, however, interact with, and was activated by, c-Jun and Fra2 which suggests that it can interact with AP1-like transcription factors and that it may act co-operatively with RIBS-binding factors to regulate BRCA1 transcription in ovarian cells.

The CD34-related molecule podocalyxin is a potent inducer of microvillus formation.

BACKGROUND: Podocalyxin is a CD34-related transmembrane protein involved in hematopoietic cell homing, kidney morphogenesis, breast cancer progression, and epithelial cell polarization. Although this sialomucin has been shown to block cell adhesion, the mechanisms involved remain enigmatic. It has, however, been postulated that the adaptor proteins NHERF-1 and 2 could regulate apical targeting of Podocalyxin by linking it to the actin cytoskeleton. PRINCIPAL FINDINGS: Here, in contrast, we find that full-length Podocalyxin acts to recruit NHERF-1 to the apical domain. Moreover, we show that ectopic expression of Podocalyxin in epithelial cells leads to microvillus formation along an expanded apical domain that extends laterally to the junctional complexes. Removal of the C-terminal PDZ-binding domain of Podocalyxin abolishes NHERF-1 recruitment but, surprisingly, has no effect on the formation of microvilli. Instead, we find that the extracellular domain and transmembrane region of Podocalyxin are sufficient to direct recruitment of filamentous actin and ezrin to the plasma membrane and induce microvillus formation. CONCLUSIONS/SIGNIFICANCE: Our data suggest that this single molecule can modulate NHERF localization and, independently, act as a key orchestrator of apical cell morphology, thereby lending mechanistic insights into its multiple roles as a polarity regulator, tumor progression marker, and anti-adhesin.