Victoria Virador

In Vitro Three-Dimensional (3D) Models in Cancer Research: An Update

Tissues are three‐dimensional (3D) entities as is the tumor that arises within them. Though disaggregated cancerous tissues have produced numerous cell lines for basic and applied research, it is generally agreed that these lines are poor models of in vivo phenomena. In this review we focus on in vitro 3D models used in cancer research, particularly their contribution to molecular studies of the early stages of metastasis, angiogenesis, the tumor microenvironment, and cancer stem cells. We present a summary of the various formats used in the field of tissue bioengineering as they apply to mechanistic modeling of cancer stages or processes. In addition we list studies that model specific types of malignancies, highlight drastic differences in results between 3D in vitro models and classical monolayer culturing techniques, and establish the need for standardization of 3D models for meaningful preclinical and therapeutic testing.

Influence of α-melanocyte-stimulating hormone and ultraviolet radiation on the transfer of melanosomes to keratinocytes

The epidermal melanin unit in human skin is composed of melanocytes and keratinocytes. Melanocytes, located in the basal layer of the epidermis, manufacture melanin‐loaded organelles called melanosomes. Through their dendritic processes, melanocytes distribute melanosomes to neighboring keratinocytes, where their presence confers to the skin its characteristic color and photoprotective properties. In this study, we used murine melanocytes and keratinocytes alone and in coculture to characterize the processes involved in melanosome transfer. Ultraviolet (UV) radiation induced an accumulation of melanosomes in melanocytes, whereas treatment with α‐melanocyte‐stimulating hormone (MSH) induced exocytosis of melanosomes accompanied by ruffling of the melanocyte membrane. We found that keratinocytes phagocytose melanosomes and latex beads equally well and that this phagocytic process was increased by exposure of keratinocytes to UV radiation or to MSH. Coculture of melanocytes and keratinocytes resulted in an increase in MSH released to the medium. Gene array analysis of MSH‐treated melanocytes showed up‐regulation of many genes associated with exocytosis. In our studies, we never observed cytophagocytosis of melanosome‐filled processes. This result, together with the other findings, suggests that a combination of signals that increase melanosome production and release by melanocytes and that stimulate phagocytosis by keratinocytes are the most relevant mechanisms involved in skin tanning.

Isolation of a mesenchymal cell population from murine dermis that contains progenitors of multiple cell lineages

The skin contains two known subpopula–tions of stem cells/epidermal progenitors: a basal kera–tinocyte population found in the interfollicular epithelium and cells residing in the bulge region of the hair follicle. The major role of the interfollicular basal kera–tinocyte population may be epidermal renewal, whereas the bulge population may only be activated and recruited to form a cutaneous epithelium in case of trauma. Using 3–dimensional cultures of murine skin under stress conditions in which only reserve epithelial cells would be expected to survive and expand, we demonstrate that a mesenchymal population resident in neonatal murine dermis has the unique potential to develop an epidermis in vitro. In monolayer culture, this dermal subpopulation has long–term survival capabilities in restricted serum and an inducible capacity to evolve into multiple cell lineages, both epithelial and mesenchymal, depending on culture conditions. When grafted subcutaneously, this dermal subpopulation gave rise to fusiform structures, reminiscent of disorganized muscle, that stained positive for smooth muscle actin and desmin; on typical epidermal grafts, abundant melanocytes appeared throughout the dermis that were not associated with hair follicles. The multipotential cells can be repeatedly isolated from neonatal murine dermis by a sequence of differential centrif–ugation and selective culture conditions. These results suggest that progenitors capable of epidermal differentiation exist in the mesenchymal compartment of an abundant tissue source and may have a function in mesenchy–mal–epithelial transition upon insult. Moreover, these cells could be available in sufficient quantities for lineage determination or tissue engineering applications.–Crigler, L., Kazhanie, A., Yoon, T.‐J., Zakhari, J., Anders, J., Taylor, B., Virador, V. M. Isolation of a mesenchymal cell population from murine dermis that contains progenitors of multiple cell lineages. FASEB J. 21, 2050–2063 (2007)

MT‐1 melatonin receptor expression increases the antiproliferative effect of melatonin on S‐91 murine melanoma cells

Abstract:  Melatonin, a derivative of tryptophan that is present in all vertebrates, was first described in bovine pineal gland. It is known that melatonin is a highly conserved molecule, present also in unicellular organisms and plants. Several effects of melatonin have been described, including receptor‐ and non‐receptor‐mediated actions. Herein, we studied the effects of melatonin on in vitro and in vivo cell proliferation of Cloudman S‐91 murine melanoma cells. We demonstrated that melatonin treatment significantly inhibits S‐91 melanoma cell proliferation in vitro (EC50 = 10−7 m) as well as reduces tumor growth in vivo. We also demonstrated that melatonin directly increases the activity of the antioxidant enzymes catalase and glutathione peroxidase. These effects are most likely triggered through the direct intracellular action of melatonin, since the presence of receptors could not be demonstrated in this cell line. Expression of MT‐1 melatonin receptor by stable transfection, mediated a dramatic antiproliferative melatonin effect (EC50 = 10−10 m) in S‐91 cells. The expressed receptor is negatively coupled to the adenylyl cyclase/cyclic AMP signaling pathway via Gi protein. These results suggest that expression of the MT‐1 melatonin receptor in melanoma cells is a potential alternative approach to specifically target cells in cancer therapeutic treatment.

3D in vitro tissue models and their potential for drug screening

Introduction: The development of one standard, simplified in vitro three-dimensional tissue model suitable to biological and pathological investigation and drug-discovery may not yet be feasible, but standardized models for individual tissues or organs are a possibility. Tissue bioengineering, while concerned with finding methods of restoring functionality in disease, is developing technology that can be miniaturized for high throughput screening (HTS) of putative drugs. Through collaboration between biologists, physicists and engineers, cell-based assays are expanding into the realm of tissue analysis. Accordingly, three-dimensional (3D) micro-organoid systems will play an increasing role in drug testing and therapeutics over the next decade. Nevertheless, important hurdles remain before these models are fully developed for HTS. Areas covered: We highlight advances in the field of tissue bioengineering aimed at enhancing the success of drug candidates through pre-clinical optimization. We discuss models that are most amenable to high throughput screening with emphasis on detection platforms and data modeling. Expert opinion: Modeling 3D tissues to mimic in-vivo architecture remains a major challenge. As technology advances to provide novel methods of HTS analysis, so do potential pitfalls associated with such models and methods. We remain hopeful that integration of biofabrication with HTS will significantly reduce attrition rates in drug development.

L‐asparaginase inhibits invasive and angiogenic activity and induces autophagy in ovarian cancer

Recent work identified L‐asparaginase (L‐ASP) as a putative therapeutic target for ovarian cancer. We suggest that L‐ASP, a dysregulator of glycosylation, would interrupt the local microenvironment, affecting the ovarian cancer cell—endothelial cell interaction and thus angiogenesis without cytotoxic effects. Ovarian cancer cell lines and human microvascular endothelial cells (HMVEC) were exposed to L‐ASP at physiologically attainable concentrations and subjected to analyses of endothelial tube formation, invasion, adhesion and the assessment of sialylated proteins involved in matrix‐associated and heterotypic cell adhesion. Marked reduction in HMVEC tube formation in vitro, HMVEC and ovarian cancer cell invasion, and heterotypic cell‐cell and cell‐matrix adhesion was observed (P < 0.05–0.0001). These effects were associated with reduced binding to ß1integrin, activation of FAK, and cell surface sialyl LewisX (sLex) expression. No reduction in HMVEC E‐selectin expression was seen consistent with the unidirectional inhibitory actions observed. L‐ASP concentrations were non‐toxic to either ovarian cancer or HMVEC lines in the time frame of the assays. However, early changes of autophagy were observed in both cell types with induction of ATG12, beclin‐1, and cleavage of LC‐3, indicating cell injury did occur. These data and the known mechanism of action of L‐ASP on glycosylation of nascent proteins suggest that L‐ASP reduces of ovarian cancer dissemination and progression through modification of its microenvironment. The reduction of ovarian cancer cell surface sLex inhibits interaction with HMVEC and thus HMVEC differentiation into tubes, inhibits interaction with the local matrix reducing invasive behaviour, and causes cell injury initiating autophagy in tumour and vascular cells.

Genomic and phenotypic analysis reveals a key role for CCN1 (CYR61) in BAG3-modulated adhesion and invasion.

Chaperone protein quantity may regulate the balance of proteins involved in invasion and malignancy. BAG3 is a co‐chaperone and pro‐survival protein that has been implicated in adhesion, migration, and metastasis. We reported that BAG3 overexpression in MDA435 human breast cancer cells results in a significant decrease in migration and adhesion to matrix molecules that is reversed upon deletion of the BAG3 proline‐rich domain (dPXXP). We now hypothesize that transcriptional analysis would identify proteins involved in matrix‐related processes that are regulated by BAG3 and/or its PXXP domain mutant. Expression array analysis of MDA435 cells overexpressing either wild‐type BAG3 (FL) or dPXXP identified CCN1 as a BAG3 target protein. CCN1 is a known AP‐1 target. Increased AP‐1 transcriptional activity and AP‐1 DNA‐binding was found in MDA435 dPXXP cells. Consistent with these findings, CCN1 quantity and secretion were increased in dPXXP mutants but suppressed in FL cells; both BAG3 forms resulted in up‐regulated CCN1 in HeLa cells. CCN1 silencing in the BAG3 FL overexpressors reduced the already low phospho‐integrin β1 in response to attachment on collagen IV. Matrigel invasion of HeLa cells engineered with the BAG3 constructs was enhanced in FL cells and minimal in dPXXP cells. CCN1 silencing blocked a greater percentage of the serum‐induced invasion in FL cells than in dPXXP cells. This implies a context‐dependent function of BAG3 on CCN1 and thus mesenchymal behaviour. CCN1 may be necessary for adhesion and matrix‐related signalling in FL cells, abrogating a negative signal of the PXXP domain when BAG3 is intact. We propose that BAG3 regulates CCN1 expression to regulate tumour cell adhesion and migration. Published in 2009 by John Wiley & Sons, Ltd.

The anti-apoptotic activity of BAG3 is restricted by caspases and the proteasome.

Background Caspase-mediated cleavage and proteasomal degradation of ubiquitinated proteins are two independent mechanisms for the regulation of protein stability and cellular function. We previously reported BAG3 overexpression protected ubiquitinated clients, such as AKT, from proteasomal degradation and conferred cytoprotection against heat shock. We hypothesized that the BAG3 protein is regulated by proteolysis. Methodology/Principal Findings Staurosporine (STS) was used as a tool to test for caspase involvement in BAG3 degradation. MDA435 and HeLa human cancer cell lines exposed to STS underwent apoptosis with a concomitant time and dose-dependent loss of BAG3, suggesting the survival role of BAG3 was subject to STS regulation. zVAD-fmk or caspase 3 and 9 inhibitors provided a strong but incomplete protection of both cells and BAG3 protein. Two putative caspase cleavage sites were tested: KEVD (BAG3E345A/D347A) within the proline-rich center of BAG3 (PXXP) and the C-terminal LEAD site (BAG3E516A/D518A). PXXP deletion mutant and BAG3E345A/D347A, or BAG3E516A/D518A respectively slowed or stalled STS-mediated BAG3 loss. BAG3, ubiquitinated under basal growth conditions, underwent augmented ubiquitination upon STS treatment, while there was no increase in ubiquitination of the BAG3E516A/D518A caspase-resistant mutant. Caspase and proteasome inhibition resulted in partial and independent protection of BAG3 whereas inhibitors of both blocked BAG3 degradation. STS-induced apoptosis was increased when BAG3 was silenced, and retention of BAG3 was associated with cytoprotection. Conclusions/Significance BAG3 is tightly controlled by selective degradation during STS exposure. Loss of BAG3 under STS injury required sequential caspase cleavage followed by polyubiquitination and proteasomal degradation. The need for dual regulation of BAG3 in apoptosis suggests a key role for BAG3 in cancer cell resistance to apoptosis.

The human promyelocytic leukemia protein is a tumor suppressor for murine skin carcinogenesis.

Expression of the PMLRARα fusion dominant‐negative oncogene in the epidermis of transgenic mice resulted in spontaneous skin tumors attributed to changes in both the PML and RAR pathways [Hansen et al., Cancer Res 2003; 63:5257–5265]. To determine the contribution of PML to skin tumor susceptibility, transgenic mice were generated on an FVB/N background, that overexpressed the human PML protein in epidermis and hair follicles under the control of the bovine keratin 5 promoter. PML was highly expressed in the epidermis and hair follicles of these mice and was also increased in cultured keratinocytes where it was confined to nuclear bodies. While an overt skin phenotype was not detected in young transgenic mice, expression of keratin 10 (K10) was increased in epidermis and hair follicles and cultured keratinocytes. As mice aged, they exhibited extensive alopecia that was accentuated on the C57BL/6J background. Following skin tumor induction with 7, 12‐dimethylbenz[a]anthracene (DMBA) as initiator and 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) as promoter, papilloma multiplicity and size were decreased in the transgenic mice by 35%, and the conversion of papillomas to carcinomas was delayed. Cultured transgenic keratinocytes underwent premature senescence and upregulated transcripts for p16 and Rb but not p19 and p53. Together, these changes suggest that PML participates in regulating the growth and differentiation of keratinocytes that likely influence its activity as a suppressor for tumor development. Published 2008 Wiley‐Liss, Inc.

Cellular populations isolated from newborn mouse skin including mesenchymal stem cells.

We developed protocols for isolation and characterization of mesenchymal progenitors from murine dermis. Our protocols are part of a more general isolation procedure starting with neonatal murine skin, which has been described in detail by U. Lichti and coauthors (Nat Protoc 3(5):799-810, 2008). We list Lichtis procedures in an abbreviated form as part of this methods section. Our methods to isolate mesenchymal stem cells are presented as a continuous workflow of isolation and characterization, including flow cytometry, cell survival assays, colony formation assays, immunoblotting, immunostaining, multipotential differentiation assays, and in vivo engraftment. In most cases, the protocols are standard; in others, they were adapted to our particular purpose. We made special emphasis on the use of in vitro three-dimensional cultures to cue mesenchymal progenitors into epidermal cells.