Susanna Virolainen

Photodynamic Ablation of Lymphatic Vessels and Intralymphatic Cancer Cells Prevents Metastasis

Destruction of lymphatic vessels by photodynamic therapy suppresses metastasis by eradicating both the in-transit tumor cells and their conduit to distant tissues. Shining a Light on Tumor Metastasis Tumor cells have several routes that enable them to move from the primary tumor to distant tissues, a process called metastasis. It is metastasis of the primary tumor that kills most cancer patients. One of the least studied routes of metastasis is the lymphatic system. Many tumors produce vascular endothelial growth factor–C (VEGF-C) or VEGF-D, factors that promote the formation of new lymphatic vessels (lymphangiogenesis). The newly formed lymphatic vessels enable tumor cells to travel from the primary tumor to the regional lymph nodes from whence they can spread throughout the body. In a new study, Tammela and colleagues investigate whether eradicating tumor-associated lymphatic vessels and the tumor cells they contain using photodynamic therapy (PDT) with a light-activated cytotoxic compound could reduce or eliminate tumor metastasis. They selected the mouse ear as their model system because it is easy to image both the lymphatic vessels and the in-transit tumor cells. The mouse ear is also amenable to PDT because it is very thin, thus enabling sufficient tissue penetration by the infrared light that activates the cytotoxic compound (verteporfin). The authors implanted mouse melanoma cells or human lung tumor cells into the mouse ear tip and waited 2 weeks for the primary tumors to become established and to induce lymphangiogenesis. Using immunohistochemistry, they observed that the newly formed lymphatic vessels contained in-transit tumor cells as well as small tumor nodules. Using pathology sections taken from a patient with recurrent melanoma, they confirmed that the tumor-associated lymphatic vessels of cancer patients contain in-transit tumor cells and tumor nodules. But could PDT eliminate both tumor-associated lymphatic vessels and the tumor cells inside them? The authors injected verteporfin in a liposomal preparation intradermally into the mouse ear and showed that this cytotoxic dye accumulated specifically in the lymphatic vessels. When they illuminated the mouse ear with infrared laser light, the lymphatic vessels started to shrink and fragment, and became leaky. Could this treatment prevent relapse when combined with surgery? The authors implanted mouse melanoma cells into the flanks of mice and waited 2 weeks until the tumor cells had become established and had metastasized via the lymphatic vessels to the axillary lymph nodes. They then injected verteporfin into the primary tumor and surrounding tissue and illuminated the whole flank with infrared laser light before surgically removing the primary tumor and lymph nodes. They found that the mice receiving PDT and surgery had a much lower relapse rate than those that underwent surgery alone. But will PDT be useful for eradicating tumor-associated lymphatic vessels deep within the tissues of human cancer patients? Using a pig model, Tammela and colleagues demonstrated that this should be feasible. They injected verteporfin into pig hoofs and then used a special laser catheter inserted at the knee to activate the dye. They showed that the lymphatic system ceased to drain as the vessels fragmented and became clogged, indicating that PDT can target lymphatic vessels deep within the body. PDT using verteporfin is currently used clinically to destroy overgrown blood vessels in the retina of patients with macular degeneration. So it should certainly be feasible to use PDT to destroy tumor-associated lymphatic vessels in cancer patients undergoing surgery. Shining a light deep within the body should ensure that an oft-overlooked conduit for tumor metastasis will remain illuminated. The dissemination of tumor cells to sites far from the primary tumor (metastasis) is the principal cause of death in cancer patients. Tumor-associated lymphatic vessels are a key conduit for metastatic tumor cells, which typically first colonize the lymph nodes. Although the primary tumor and affected lymph nodes can be removed during surgery, tumor cells inside lymphatic vessels are left behind. Here, we show that in-transit tumor cells inside lymphatic vessels in mice bearing mouse melanomas or human lung tumors give rise to metastases. Using photodynamic therapy with the benzoporphyrin derivative verteporfin, we selectively destroyed lymphatic vessels in mice and pigs. Destruction of tumor-associated lymphatic vessels also eradicated intralymphatic tumor cells and prevented metastasis of mouse melanoma cells and subsequent relapse. Photodynamic therapy, when combined with anti-lymphangiogenic therapy, prevented further tumor invasion of lymphatic vessels. These findings highlight the potential of targeting in-transit tumor cells in patients.

Metastatic Outgrowth Encompasses COL-I, FN1, and POSTN Up-Regulation and Assembly to Fibrillar Networks Regulating Cell Adhesion, Migration, and Growth

Although the outgrowth of micrometastases into macrometastases is the rate-limiting step in metastatic progression and the main determinant of cancer fatality, the molecular mechanisms involved have been little studied. Here, we compared the gene expression profiles of melanoma lymph node micro- and macrometastases and unexpectedly found no common up-regulation of any single growth factor/cytokine, except for the cytokine-like SPP1. Importantly, metastatic outgrowth was found to be consistently associated with activation of the transforming growth factor-beta signaling pathway (confirmed by phospho-SMAD2 staining) and concerted up-regulation of POSTN, FN1, COL-I, and VCAN genes-all inducible by transforming growth factor-beta. The encoded extracellular matrix proteins were found to together form intricate fibrillar networks around tumor cell nests in melanoma and breast cancer metastases from various organs. Functional analyses suggested that these newly synthesized protein networks regulate adhesion, migration, and growth of tumor cells, fibroblasts, and endothelial cells. POSTN acted as an anti-adhesive molecule counteracting the adhesive functions of FN1 and COL-I. Further, cellular FN and POSTN were specifically overexpressed in the newly forming/formed tumor blood vessels. Transforming growth factor-beta receptors and the metastasis-related matrix proteins, POSTN and FN1, in particular, may thus provide attractive targets for development of new therapies against disseminated melanoma, breast cancer, and possibly other tumors, by affecting key processes of metastasis: tumor/stromal cell migration, growth, and angiogenesis.

Switch to an invasive growth phase in melanoma is associated with tenascin-C, fibronectin, and procollagen-I forming specific channel structures for invasion

Malignant melanomas are characterized by their high propensity to invade and metastasize, but the molecular mechanisms of these traits have remained elusive. Our DNA microarray analyses of benign nevi and melanoma tissue specimens revealed that the genes encoding extracellular matrix proteins tenascin‐C (TN‐C), fibronectin (FN), and procollagen‐I (PCOL‐I) are highly upregulated in invasive and metastatic melanomas. The expression and distribution of these proteins were further studied by immunohistochemistry in benign nevi, radially and vertically growing melanomas, sentinel node micrometastases, and macrometastases. TN‐C was increased in all invasive tumours and metastases, especially at invasion fronts, but not in benign nevi or non‐invasive melanomas. Significantly, the intensity of TN‐C staining correlated with metastasis to sentinel lymph nodes, better than tumour thickness (Breslow). Moreover, TN‐C, FN, and PCOL‐I appeared to co‐localize in the tumours and form tubular meshworks and channels ensheathing the melanoma cells. Our data suggest that melanoma invasion is associated with the formation of special channel‐like structures, providing a new concept, structured tumour cell spreading. Altogether, these data provide potential new prognostic markers and therapeutic targets/strategies for preventing melanoma dissemination. Copyright

TGF-β Signaling, Activated Stromal Fibroblasts, and Cysteine Cathepsins B and L Drive the Invasive Growth of Human Melanoma Cells

Accumulating evidence indicates that interactions between cancer cells and stromal cells are important for the development/progression of many cancers. Herein, we found that the invasive growth of melanoma cells in three-dimensional-Matrigel/collagen-I matrices is dramatically increased on their co-culture with embryonic or adult skin fibroblasts. Studies with fluorescent-labeled cells revealed that the melanoma cells first activate the fibroblasts, which then take the lead in invasion. To identify the physiologically relevant invasion-related proteases involved, we performed genome-wide microarray analyses of invasive human melanomas and benign nevi; we found up-regulation of cysteine cathepsins B and L, matrix metalloproteinase (MMP)-1 and -9, and urokinase- and tissue-type plasminogen activators. The mRNA levels of cathepsins B/L and plasminogen activators, but not MMPs, correlated with metastasis. The invasiveness/growth of the melanoma cells with fibroblasts was inhibited by cell membrane-permeable inhibitors of cathepsins B/L, but not by wide-spectrum inhibitors of MMPs. The IHC analysis of primary melanomas and benign nevi revealed cathepsin B to be predominantly expressed by melanoma cells and cathepsin L to be predominantly expressed by the tumor-associated fibroblasts surrounding the invading melanoma cells. Finally, cathepsin B regulated TGF-β production/signaling, which was required for the activation of fibroblasts and their promotion of the invasive growth of melanoma cells. These data provide a basis for testing inhibitors of TGF-β signaling and cathepsins B/L in the therapy of invasive/metastatic melanomas.

Systematic search for the best gene expression markers for melanoma micrometastasis detection

Melanoma is notorious for its high tendency to metastasize and its refractoriness to treatment thereafter. Metastasis is believed to occur mostly through the lymphatic system, and the status of sentinel lymph nodes is currently recognized as the best prognostic indicator. Unfortunately, the lymphatic metastatic process is still poorly understood and the occurrence of sentinel node metastases (micrometastases) may be underestimated. We performed genome‐wide gene expression analyses of melanoma lymph node micrometastases and macrometastases, and of primary melanomas and benign naevi, to characterize the early metastatic cells molecularly and to disclose the best diagnostic markers and rational targets for therapy. Significance analysis of microarrays identified 22 over‐ and five under‐expressed genes with ≥ four‐fold changes in the micrometastases. Of these genes, MLANA, TYR, MIA, ERBB3, PRAME, and SPP1 were tested as potential markers by RT‐PCR and immunohistochemistry. In a prospective study of 160 patients, our graded MLANA and TYR RT‐PCR analyses disclosed clinically significant metastases, as assessed by disease recurrence, better than histological and immunohistochemical examinations. These results strongly suggest the clinical implementation of quantifiable RT‐PCR assays to confirm and complement the pathological examination of sentinel node metastases. Furthermore, SPP1 and PRAME proved valuable as melanoma‐specific markers capable of differentiating melanoma cells from benign naevi in the sentinel lymph nodes. Importantly, these two genes may also prove to be ideal targets for drug development and therapy. Most molecular traits of the micrometastases were already present in the primary tumours, suggesting that micrometastasis to sentinel lymph nodes is a fairly non‐selective process. Copyright

Expression of antizyme inhibitor 2 in mast cells and role of polyamines as selective regulators of serotonin secretion.

Background Upon IgE-mediated activation, mast cells (MC) exocytose their cytoplasmic secretory granules and release a variety of bioactive substances that trigger inflammatory responses. Polyamines mediate numerous cellular and physiological functions. We report here that MCs express antizyme inhibitor 2 (AZIN2), an activator of polyamine biosynthesis, previously reported to be exclusively expressed in the brain and testis. We have investigated the intracellular localization of AZIN2 both in resting and activated MCs. In addition, we have examined the functional role of polyamines, downstream effectors of AZIN2, as potential regulators of MC activity. Methodology/Principal Findings Immunostainings show that AZIN2 is expressed in primary and neoplastic human and rodent MCs. We demonstrate that AZIN2 localizes in the Vamp-8 positive, serotonin-containing subset of MC granules, but not in tryptase-containing granules, as revealed by double immunofluorescence stainings. Furthermore, activation of MCs induces rapid upregulation of AZIN2 expression and its redistribution, suggesting a role for AZIN2 in secretory granule exocytosis. We also demonstrate that release of serotonin from activated MCs is polyamine-dependent whereas release of histamine and β-hexosaminidase is not, indicating a granule subtype-specific function for polyamines. Conclusions/Significance The study reports for the first time the expression of AZIN2 outside the brain and testis, and demonstrates the intracellular localization of endogenous AZIN2 in MCs. The granule subtype-specific expression and its induction after MC activation suggest a role for AZIN2 as a local, in situ regulator of polyamine biosynthesis in association with serotonin-containing granules of MCs. Furthermore, our data indicates a novel function for polyamines as selective regulators of serotonin release from MCs.

Increased expression of matrix metalloproteinases-21 and -26 and TIMP-4 in pancreatic adenocarcinoma.

Pancreatic adenocarcinoma is known for early aggressive local invasion, high metastatic potential, and a low 5-year survival rate. Matrix metalloproteinases (MMPs) play important roles in tumor growth and invasion. Earlier studies on pancreatic cancer have found increased expression of certain MMPs to correlate with poorer prognosis, short survival time or presence of metastases. We studied the expression of MMP-21, -26, and tissue inhibitor of matrix metalloproteinases (TIMP)-4 in 50 tissue samples, including 25 adenocarcinomas, seven other malignant pancreatic tumors, and 18 control samples of non-neoplastic pancreatic tissue with immunohistochemistry. The regulation of MMP-21, -26, and TIMP-4 mRNAs by cytokines was studied with RT-PCR in pancreatic cancer cell lines PANC-1, BxPC-3, and AsPC-1. MMP-21, -26, and TIMP-4 were detected in cancer cells in 64, 40, and 60% of tumors, respectively. MMP-21 expressed in well-differentiated cancer cells and occasional fibroblasts, like TIMP-4, tended to diminish in intensity from grade I to grade III tumors. Patients with metastatic lymph nodes had increased expression of MMP-26 in actual tumor samples. All cultured cancer cell lines expressed MMP-21 basally at low levels, and presence of the protein was confirmed immunohistochemically in cultured cells. MMP-21 expression was induced by epidermal growth factor (EGF) in PANC-1 cells. MMP-26 was neither expressed basally nor induced by tumor necrosis factor α, transforming growth factor β-1 (TGFβ1), EGF, or interferon γ. Basal TIMP-4 expression was lowest in the poorly differentiated cancer cell line PANC-1 compared to better-differentiated BxPC-3 and AsPC-1 cells. TIMP-4 expression was induced by TGFβ1 in PANC-1 cells and by EGF in BxPC-3 cells. Our findings suggest that MMP-21 is not a marker of invasiveness, but rather of differentiation, in pancreatic cancer and it may be upregulated by EGF. The putative role of MMP-26 as a marker of metastases warrants further studies. Unlike other TIMPs, TIMP-4 was not upregulated in relation to aggressiveness of pancreatic cancer.

Melkersson-Rosenthal syndrome:

OBJECTIVES: To study characteristics of Melkersson-Rosenthal syndrome (MRS) patients with facial palsy (FP) and differences in patients treated at the Departments of Otorhinolaryngology and Dermatology. METHODS: Clinical picture of MRS was studied from patient charts at two departments. Patients with FP received a questionnaire and were examined. Tissue biopsies were searched for non-necrotizing granulomatous infiltrations typical of MRS and blood DNA for UNC-93B1 gene mutations predisposing to herpesvirus infection. RESULTS: At the Department of Otorhinolaryngology, all 18 MRS patients had FP, 9 the triad form. Two patients revealed non-necrotizing granulomatous infiltrations during acute edema episodes; another two had association with uveitis. Edema was rarely persistent and did not dominate the clinical picture. No UNC-93B1 mutations were found. At the Department of Dermatology, 2 patients had triad MRS and 15 had monosymptomatic granulomatous cheilitis with persistent edema and typical MRS histology. CONCLUSION: The clinical picture of MRS with FP differed from the current knowledge of edema-dominated MRS. More studies focusing on MRS with FP would broaden our understanding of the syndrome.

MMP-21 is upregulated at early stages of melanoma progression but disappears with more aggressive phenotype

The expression of matrix metalloproteinases (MMPs) is frequently altered during malignant transformation. We examined the profile of three recently cloned MMPs, MMP-21, MMP-26, and MMP-28, in melanomas in vivo and in culture. Immunohistochemistry for MMPs-21, -26, -28, and -13 in melanoma specimens (27 nonmetastatic, 26 with nodal micrometastases, and 10 in situ melanomas) from 63 patients was performed. MMP-21 was expressed in melanoma cells in 29/53 cases, being more frequent in melanoma samples without micrometastases. Six out of ten in situ melanomas were positive, while five nevus samples were negative. MMP-26 and -28 were not generally expressed in melanoma cells. MMP-13 was detected in melanoma cells in 36/53 samples. MMP-21 was not found in sentinel nodes with metastases, while MMP-13 was seen in all of them. MMP-21 messenger RNA was variably expressed in all five melanoma cell lines investigated using reverse transcriptase–polymerase chain reaction. Our results suggest that expression of MMP-21 may serve as a marker of malignant transformation of melanocytes and does not associate with the presence of micrometastases.

Prospective immunohistochemical analysis of BRAF V600E mutation in melanoma

The v-raf murine sarcoma viral oncogene homolog B1 (BRAF) V600E mutation is the most common activating genetic alteration of this oncogene and a predictive marker for the therapeutic use of BRAF inhibitors in melanoma. Our aim was to evaluate the performance of BRAF V600E mutation-specific monoclonal antibody (VE1) in a prospective diagnostic setting of melanoma patients (n = 102). All 41 cases (40.2%) that showed a V600E mutation in the cyclic minisequencing analysis of the DNA were also initially scored immunopositive. Two cases that were scored as BRAF V600E mutation positive by immunohistochemistry were negative in the DNA-based mutation analysis and determined to be immunonegative in a repeated staining with more representative specimens. Thus, BRAF V600E mutation detection using immunohistochemistry was 100% sensitive and 96.8% specific, when compared with the analysis of the DNA. None of the BRAF V600K mutations was detected by the VE1 antibody (n = 7). However, the VE1 antibody detected a rare V600E2 mutation. We also studied the role of BRAF V600E mutation in a set of melanoma patients who had been investigated for sentinel node metastasis. Melanoma lymph node metastases were diagnosed in 21.8% (12/55) of the sentinel nodes, and BRAF V600E immunopositivity was detected in 34.5% (19/55) of the cases. BRAF V600E mutation status did not correlate with any clinicopathological parameters. In conclusion, analysis of BRAF V600E mutation in melanoma by immunohistochemistry is a sensitive and specific method, which can be used to identify BRAF inhibitor-sensitive melanoma patients as a first-line method due to its rapid and affordable nature.