Multiple Features of Advanced Melanoma Recapitulated in Tumorigenic Variants of Early Stage (Radial Growth Phase) Human Melanoma Cell Lines: Evidence For a Dominant Phenotype.

The vast majority of primary human cutaneous melanomas undergo a slow and gradual progression from a clinically indolent, curable radial growth phase (RGP) to a malignant vertical growth phase. We sought to develop a way of isolating genetically related malignant variants from a benign RGP human melanoma, called WM35. The parent and variants were then used as a model system to examine to what extent the expression of clinically and biologically relevant phenotypic features characteristic of advanced melanomas are associated with (and thus perhaps causative of) such a malignant conversion. Such a model system could also be used as a means of eventually identifying genetic alterations and cellular changes involved in the malignant switch in melanoma progression. To develop such a model, we subjected WM35 cells to retroviral insertional mutagenesis, which was followed by selection for progressive growth of solid tumors in nude mice. Highly aggressive and phenotypically stable tumorigenic variants were derived which contained at least four integrated proviruses. In contrast to the parental WM35 cells, these cell lines expressed several phenotypic features characteristic of naturally derived, advanced-stage malignant melanoma cells. Thus, in addition to tumor-forming ability in nude mice, the variants were growth factor and anchorage independent, overexpressed the MUC18 adhesion molecule, and lost responsiveness to the growth-inhibitory effect of several cytokines, including interleukin 6, transforming growth factor beta, interleukin 1beta, and tumor necrosis factor-alpha. Tumorigenicity and multicytokine resistance were dominant traits since in somatic cell hybrids between the parental cells and a tumorigenic subline no suppressive effect of the former cell population was observed. These findings suggest that one or more dominantly acting genetic alterations might be involved in this progression of RGP melanoma cells. The identity of such alterations remains to be determined

Soluble stroma‐related biomarkers of pancreatic cancer

Abstract The clinical management of pancreatic ductal adenocarcinoma (PDAC) is hampered by the lack of reliable biomarkers. This study investigated the value of soluble stroma‐related molecules as PDAC biomarkers. In the first exploratory phase, 12 out of 38 molecules were associated with PDAC in a cohort of 25 PDAC patients and 16 healthy subjects. A second confirmatory phase on an independent cohort of 131 PDAC patients, 30 chronic pancreatitis patients, and 131 healthy subjects confirmed the PDAC association for MMP7, CCN2, IGFBP2, TSP2, sICAM1, TIMP1, and PLG. Multivariable logistic regression model identified biomarker panels discriminating respectively PDAC versus healthy subjects (MMP7 + CA19.9, AUC = 0.99, 99% CI = 0.98–1.00) (CCN2 + CA19.9, AUC = 0.96, 99% CI = 0.92–0.99) and PDAC versus chronic pancreatitis (CCN2 + PLG+FN+Col4 + CA19.9, AUC = 0.94, 99% CI = 0.88–0.99). Five molecules were associated with PanIN development in two GEM models of PDAC (PdxCre/LSL‐KrasG12D and PdxCre/LSL‐KrasG12D/+/LSL‐Trp53R172H/+), suggesting their potential for detecting early disease. These markers were also elevated in patient‐derived orthotopic PDAC xenografts and associated with response to chemotherapy. The identified stroma‐related soluble biomarkers represent potential tools for PDAC diagnosis and for monitoring treatment response of PDAC patients

Chemotherapy-induced neutropenia elicits metastasis formation in mice by promoting proliferation of disseminated tumor cells

ABSTRACTChemotherapy is the standard of care for most malignancies. Its tumor debulking effect in adjuvant or neoadjuvant settings is unquestionable, although secondary effects have been reported that paradoxically promote metastasis. Chemotherapy affects the hematopoietic precursors leading to myelosuppression, with neutropenia being the main hematological toxicity induced by cytotoxic therapy. We used renal and lung murine tumor models metastatic to the lung to study chemotherapy-induced neutropenia (CIN) in the metastatic process. Cyclophosphamide and doxorubicin, two myelosuppressive drugs, but not cisplatin, increased the burden of artificial metastases to the lung, by reducing neutrophils. This effect was recapitulated by treatment with anti-Ly6G, the selective antibody-mediated neutrophil depletion that unleashed the formation of lung metastases in both artificial and spontaneous metastasis settings. The increased cancer dissemination was reversed by granulocyte-colony stimulating factor-mediated boosting of neutrophils in combination with chemotherapy. CIN affected the early metastatic colonization of the lung, quite likely promoting the proliferation of tumor cells extravasated into the lung at 24–72 hours. CIN did not affect the late events of the metastatic process, with established metastasis to the lung, nor was there any effect on the release of cancer cells from the primary, whose growth was, in fact, somewhat inhibited. This work suggests a role of neutrophils associated to a common cancer treatment side effect and claims a deep dive into the relationship between chemotherapy-induced neutropenia and metastasis

Expression of thrombospondin-1 by tumor cells in patient-derived ovarian carcinoma xenografts

<div><p></p><p><i>Purpose</i>: Thrombospondin-1 (TSP-1), a major regulator of cell interaction with the environment, is often deregulated in cancers, including ovarian carcinoma. Both the tumor and the host cells can release TSP-1 in the tumor microenvironment. The relative contribution of the two sources in determining TSP-1 levels in ovarian cancer remains to be elucidated. This study was designed to investigate the expression of tumor TSP-1 in a panel of 29 patient-derived ovarian adenocarcinoma xenografts (PDX), using analytical tools specific for human (tumor-derived) rather than murine (host-derived) TSP-1.</p><p><i>Methodology</i>: Human-specific microarray and ELISA were used to measure tumor TSP-1 expression and plasma levels.</p><p><i>Results</i>: Tumor-derived TSP-1 was heterogeneously expressed in PDX. Expression was higher in the corresponding original patient's tumor, where stroma-derived TSP-1 is also analyzed, indicating that both the tumor and the host contribute to TSP-1 production. TSP-1 was differentially expressed according to tumor grade, but not affected by p53 expression or mutational status. Findings were confirmed in an external gene expression dataset (101 patients). In a functional enrichment analysis, TSP-1 correlated with genes related to angiogenesis, cell motility, communication and shape. Plasma TSP-1, detectable in 10/11 PDX, was not associated to its expression in the tumor. The possible association of plasma TSP-1 with p53 mutations and response to chemotherapy warrants further investigation.</p><p><i>Conclusions</i>: Ovarian carcinoma PDX are a useful tool to investigate the relative contribution of stroma and tumor cells in the production of tumor associated factors, in relation to the tumor behavior, molecular properties and response to therapy.</p></div

Fibronectin fragments generated by pancreatic trypsin act as endogenous inhibitors of pancreatic tumor growth

Abstract Background The pancreatic microenvironment has a defensive role against cancer but it can acquire tumor-promoting properties triggered by multiple mechanisms including alterations in the equilibrium between proteases and their inhibitors. The identification of proteolytic events, targets and pathways would set the basis for the design of new therapeutic approaches. Methods and results Here we demonstrate that spheroids isolated from human and murine healthy pancreas and co-transplanted orthotopically with pancreatic ductal adenocarcinoma (PDAC) in mouse pancreas inhibited tumor growth. The effect was mediated by trypsin-generated fibronectin (FN) fragments released by pancreatic spheroids. Tumor inhibition was observed also in a model of acute pancreatitis associated with trypsin activation. Mass spectrometry proteomic analysis of fragments and mAb against different FN epitopes identified the FN type III domain as responsible for the activity. By inhibiting integrin α5β1, FAK and FGFR1 signaling, the fragments induced tumor cell detachment and reduced cell proliferation. Consistent with the mutual relationship between the two pathways, FGF2 restored both FGFR1 and FAK signaling and promoted PDAC cell adhesion and proliferation. FAK and FGFR inhibitors additively inhibited PDAC growth in vitro and in orthotopic in vivo models. Conclusions This study identifies a novel role for pancreatic trypsin and fibronectin cleavage as a mechanism of protection against cancer by the pancreatic microenvironment. The finding of a FAK-FGFR cross-talk in PDAC support the combination of FAK and FGFR inhibitors for PDAC treatment to emulate the protective effect of the normal pancreas against cancer

Protease-activated receptor-1 (PAR-1) promotes the motility of human melanomas and is associated to their metastatic phenotype

Protease-activated receptor-1 (PAR-1) is a unique G-protein-coupled receptor belonging to the protease-activated receptor family. Its activation leads to downstream signaling events that launch a variety of cellular responses related to tumor progression. PAR-1 expression has been associated to a variety of human cancers, and our previous studies reveal a high PAR-1 expression in melanoma specimens as compared to common nevi. In the present study, we investigated the contribution of PAR-1 to the malignant phenotype of human melanoma cell lines obtained from cutaneous primary lesions, capable of different metastatic behaviors in the patients from which they have been derived. We found that melanoma cells isolated from lesions giving rise to metastases in patients (WM115, WM278A, WM1361A, WM983A), had higher PAR-1 mRNA and protein expression, as compared to those obtained from lesions that did not develop metastatic disease (WM793, WM35). The cells isolated from metastatic primary lesions were able to colonize the lungs of immunodeficient SCID mice while those isolated from non-metastatic lesions were not. Additionally, cells expressing elevated PAR-1 had higher migratory and invasive abilities than those holding minimal PAR-1 expression. The migration and invasion capabilities of the melanoma cells expressing high PAR-1 were hampered by genetic and pharmacological interventions. The reduction of PAR-1 expression by siRNA and the inhibition of PAR-1 function by the SCH79797 specific antagonist significantly decreased the melanoma cell motility and invasiveness, down to an extent similar to that of the non-metastatic and low PAR-1 expressing cells. Our results provide strong evidence supporting the implication of PAR-1 in the malignant progression of human melanoma