Targeting melanoma with front-line therapy does not abrogate Nodal-expressing tumor cells.

Metastatic melanoma is a highly aggressive skin cancer with a poor prognosis. It is the leading cause of skin cancer deaths with a median overall survival for advanced-stage metastatic disease of <6 months. Despite advances in the field with conventional and targeted therapies, the heterogeneity of melanoma poses the greatest ongoing challenge, ultimately leading to relapse and progression to a more drug-resistant tumor in most patients. Particularly noteworthy are recent findings, indicating that these therapies exert selective pressure on tumors resulting in the activation of pathways associated with cancer stem cells that are unresponsive to current therapy. Our previous studies have shown how Nodal, an embryonic morphogen of the transforming growth factor-beta superfamily, is one of these critical factors that is reactivated in aggressive melanoma and resistant to conventional chemotherapy, such as dacarbazine. In the current study, we sought to determine whether BRAF inhibitor (BRAFi) therapy targeted Nodal-expressing tumor cells in uniquely matched unresectable stage III and IV melanoma patient samples before and after therapy that preceded their eventual death due to disease. The results demonstrate that BRAFi treatment failed to affect Nodal levels in melanoma tissues. Accompanying experiments in soft agar and in nude mice showed the advantage of using combinatorial treatment with BRAFi plus anti-Nodal monoclonal antibody to suppress tumor growth and metastasis. These data provide a promising new approach using front-line therapy combined with targeting a cancer stem cell-associated molecule-producing a more efficacious response than monotherapy.

Nodal as a biomarker for melanoma progression and a new therapeutic target for clinical intervention.

Nodal, an embryonic morphogen belonging to the TGF-ß superfamily, is an important regulator of embryonic stem cell fate. We have recently demonstrated that Nodal is expressed significantly in aggressive melanoma. Surprisingly, expression of the Nodal coreceptor, Cripto-1, was detected in only a small fraction of the melanoma tumor cell population, indicating a primary role for Cripto-1-independent signaling of Nodal in melanoma. In this review, we discuss how regulatory factors present in an embryonic environment, such as Lefty, can downregulate Nodal expression and inhibit tumorigenicity and plasticity of melanoma cells. Our translational studies show that antibodies against Nodal are capable of repressing melanoma vasculogenic mimicry and of inducing apoptosis in melanoma tumors in an in vivo lung-colonization assay. Our previous work and ongoing studies suggest that Nodal may represent a novel diagnostic marker and therapeutic target in melanoma.

Heterogeneous Expression of Invasive and Metastatic Properties in a Prostate Tumor Model.

Cellular heterogeneity of neoplasia is well demonstrated in the Dunning R-3327 rat prostate adenocarcinoma. In this study, we measured the differential expression of invasive and metastatic properties of this prostate model by cloning from a heterogeneous parental cell line. Four cell clones were derived and characterized by morphological studies, E-cadherin expression, and invasive and metastatic potential. Three of the clones (clones 5A, 5C, and 5D) demonstrated a fibroblastic morphology and were anchored to the substrate by loose microvillous processes. The fourth clone (clone 5B) grew in tight clusters and displayed many closely spaced microvilli, long overlapping cytoplasmic regions with well-defined junctional complexes. The parental line (R3327-5) demonstrated a combination of both these growth patterns. E-cadherin expression was absent in clones 5A, 5C, and 5D and very prominent in clone 5B, when compared to the parental line. The absence of E-cadherin expression correlated with increased invasiveness, as measured in an in vitro invasion assay. Subcutaneous injections of clones 5A, 5C, and 5D yielded lung metastases and no primary tumors at the site of inoculation while clone 5B was tumorigenic and produced fewer lung metastases in vivo. These clones, therefore, provide a potential for studying a variety of molecules involved in prostate cancer invasion and metastasis, especially for the direct testing of the significance of E-cadherin expresssion in prostate cancer progression.

An in vitro Model for Studying Mechanisms Underlying Synoviocyte-Mediated Cartilage Invasion in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease of joints involving the pathological development of an invasive and destructive pannus tissue which contributes to the loss of cartilage and bone. To further analyze the process of cartilage degradation and invasion, we have developed an in vitro model composed of cartilage matrix and synoviocytes (isolated from RA pannus tissue, as well as normal synovial membrane). The matrix is derived from pig articular cartilage and contains collagen type II and proteoglycans and is similar in composition to human cartilage. Data generated from this model reveal that synoviocytes isolated from RA pannus tissue invaded cartilage matrix in a manner which directly correlated with the severity of the disease. Analysis of mechanisms associated with the invasive process demonstrate that highly invasive RA synoviocytes maintain a round morphology during attachment and spreading on cartilage matrix, compared with their normal counterparts. Furthermore, the level of secretion of matrix metalloproteinase (MMP) activity was shown to correlate with the RA phenotype, which could be modulated with a novel MMP inhibitor. Normal synoviocytes could be "converted" to an RA phenotype by specific inflammatory cytokines, such that invasion of cartilage matrix was augmented by culturing these cells in the presence of 5 U/ml IL-1b or 18 U/ml TGFb. Invasion was inhibited by 150 U/ml TNFa, and unaffected by 100 ng/ml PDGF. In addition, synovial fluid from RA patients induced invasion of normal synoviocytes, in a concentration dependent manner, from 150% to 460%; however, synovial fluid from another inflammatory arthritidy (Crohn's) did not augment invasion to the same degree. Moreover, this "conversion effect" appears to be specific for synoviocytes, since similar effects could not be achieved with human skin fibroblasts. This in vitro model of synoviocyte-mediated cartilage invasion allows for further molecular characterization of the invasive properties of the synoviocyte which contribute to RA.

Fibroblast Enhancement of Tumor Invasion in a Tumor-Host Interface Recapitulated in-vitro.

Tumor cells and fibroblasts were isolated from the tumor-host interface of a colon 4047 tumor growing subcutaneously in a Fischer 344 rat. The populations were co-cultured to recapitulate the tumor-host interface in vitro. The co-cultured populations grew in a predictable pattern with tumor cells forming nodules surrounded by fibroblasts. Population dynamic experiments demonstrated the fibroblasts enhanced the growth of the tumor cells but tumor inhibited and ultimately destroyed the fibroblasts. Video microscopic examination of the fibroblasts demonstrated intense membrane ruffling adjacent to the tumor nodules followed by membrane fragmentation and detachment. Immunohistochemical staining for gelatinase A was markedly positive within the fibroblasts surrounding the tumor nodules; but negative within the tumor and in fibroblasts when tumor was absent. This technique recapitulates many aspects of the tumor-host interface in vitro and may be a useful model for evaluating several aspects of tumor-host interaction.