Selective Inhibition of HIF1α Expression by ZnSO4 Has Antitumoral Effects in Human Melanoma.

Zinc as an essential trace metal is a ubiquitous component of various molecules of the cell. Studies indicated that it may modulate functions of various cancer cell types, and can even inhibit metastasis formation in experimental models. In melanoma, zinc was shown to affect melanin production and to induce apoptosis. Using human melanoma cell lines, we have tested the effects of ZnSO4 on cell proliferation, survival, migration as well as in vivo on experimental liver colony formation. We have found that ZnSO4 has antiproliferative and proapoptotic effects in vitro. In SCID mice intraperitoneal administration of ZnSO4 specifically inhibited liver colony formation without affecting primary tumor growth. To reveal the molecular mechanisms of action of zinc in human melanoma, we have tested mRNA expression of zinc finger transcription factors and found a strong inhibitory effect on HIF1α, as compared to WT1 whereas HIF2α and MTF1 expression was unaffected. Immunohistochemical detection of HIF1α protein in liver metastases confirmed its decreased nuclear expression after in vivo ZnSO4 treatment. These data indicate that in human melanoma zinc administration may have an antimetastatic effect due to a selective downregulation of HIF1α.

Molecular identification, expression and prognostic role of estrogen- and progesterone receptors in head and neck cancer.

The aims of this study were to assess the sex hormone receptor status of head and neck (HNC) cancers. Frozen surgical samples (n = 67) of HNC patients were analyzed. Protein expression of estrogen receptor (ER)alpha, ERbeta and progesterone receptor (PgR) of tumor cells was determined by immunocytochemistry. Data were confirmed at mRNA level by nested-PCR and sequencing. ER and PgR expressions confirmed by PCR analysis were frequent in HNC: 50.7 and 49.3% respectively. Concerning the ER isoforms, ERalpha expression was predominant over ERbeta in both of oral cavity- as well as laryngeal/hypopharyngeal (LH) cancers. The delta3 splice variant of ERalpha was detected at low frequency, while the delta5 splice variant of ERbeta was frequent in HNC. The incidence of functional receptor expression (coexpression of ER and PgR) was relatively frequent also in HNC (27/67, 40.3%) which was independent of the anatomical location of the tumor. Sex hormone receptor expressions did not affect survival of HNC patients, however, in the LH cancer subgroup ER expression was associated with a trend of shortened survival (p = 0.0636, Mantel-Cox generalized savage). ERalpha,beta and PgR expressions are frequent in HNC and may affect the prognosis of the disease, at least in case of LH cancers.

Melanoma genomics reveals signatures of sensitivity to bio- and targeted therapies.

Most of the melanoma markers used today are melanocytic markers or pigmentation pathway-associated genes driven by the microphthalmia transcription factor, MITF, and include among others, tyrosinase, dopachrome tautomerase, DCT, melan-A and S100B. Genomic studies repeatedly revealed several novel melanoma marker genes including those of the transcription factor NOTCH2, WNT5A, proliferation-associated genes TOPO2A and CDC2, membrane receptors FGFR and EphA3, adhesion molecules N-cadherin, beta3 integrin and syndecan-4, and the cell surface antigens CD59/protectin and MIA. Other genomic analyses tried to define the gene signature of the metastatic disease but failed to find a consistent one except the gold standard genes of beta3 integrin, syndecan-4 and WNT5a. Studies on the gene signatures of chemoresistance and cytokine sensitivity of melanoma clearly defined apoptosis-resistance as one of the key elements of the above biological properties, but the data are controversial, mostly because of the use of inappropriate model systems and the lack of confirmation on clinical samples. Accordingly, application of genomic technologies must be more "translational" to provide breakthrough in melanoma diagnosis and therapy.

Expression of CD44v3 protein in human endothelial cells in vitro and in tumoral microvessels in vivo.

The most universal angiogenic cytokines (VEGF, bFGF, HGF) are all heparin-binding proteins, the function of which is dependent on cell surface heparan sulfate proteoglycans (HSPG). Several proteoglycans have been demonstrated in endothelial cells, but only glypican-1 from the cell surface HSPG subfamily was documented at protein level. Here, we show that CD44v3 is expressed in human immortalized endothelial cells [anchorage-dependent human umbilical vein endothelial cells (HUVEC) and anchorage-independent Kaposi sarcoma (KS-Imm)] at mRNA and protein level, but is absent from the primary culture of human brain microvascular endothelial cells. We have shown that CD44v3 has a large cytoplasmic pool in endothelial cells, but a limited surface expression, mainly at filopodia, colocalized with MMP-2. Angiogenic factors like VEGF or bFGF did not affect surface detection of CD44v3 suggesting a constitutive expression. The putative functional role for endothelial cell surface CD44v3 was identified in chemotaxis assay when anti-CD44v3 antibody pretreatment proved to be inhibitory for HUVEC. Furthermore, we provided evidence for the CD44v3 protein expression in human endothelial cells in vivo in peritumoral microvessels of both human melanoma and glottic cancers, suggesting a role for this part-time heparan sulfate proteoglycan in tumor induced angiogenesis.

High-speed detection of occult tumor cells in peripheral blood.

Although detection of tumor cells in peripheral blood using imitiunocytochemistry and optical scanning is a promising method for screening and monitoring cancer, it poses a major technical challenge due to the extremely low tumor cell concentration in blood. The preferred detection method - digital microscopy - is far too slow for analysis of the large numbers of cells required for statistical validity. We describe here a novel prescan instrument that rapidly identifies a small number of candidates for subsequent examination by digital microscopy to determine if they are genuine tumor cells. The prescan is 500 times faster than digital microscopy and yet has a similar sensitivity. The high prescan speed is accomplished by trading resolution for field of view. The resolution of the prescan is determined by the laser spot size of about 10 microns. While this resolution is much coarser than the submicron resolution of microscopes, it is still sufficient for detecting fluorescent cells because it matches the size of a typical cell. The wide field of view and high scan rate are enabled by a novel application of fiber optics.

The effect of leukocyte interleukin injection (Multikine) treatment on the peritumoral and intratumoral subpopulation of mononuclear cells and on tumor epithelia: a possible new approach to augmenting sensitivity to radiation therapy and chemotherapy in oral cancer--a multicenter phase I/II clinical Trial.

OBJECTIVES/HYPOTHESIS: The main objective of this study was to investigate the effect of the administration of a novel immunoadjuvant, leukocyte interleukin injection, as part of an immuno-augmenting treatment regimen on the peritumoral and intratumoral subpopulations of the tumor infiltrating mononuclear cells and on the epithelial and stromal components, when administered to patients with advanced primary oral squamous cell carcinoma classified as T2-3N0-2M0, as compared with disease-matched control patients (not treated with leukocyte interleukin injection). STUDY DESIGN: Multicenter Phase I/II clinical trial. Fifty-four patients from four clinical centers were included in the dose-escalating study (27 in each group [leukocyte interleukin injection-treated and control groups]). Cumulative leukocyte inter-leukin injection doses were 2400, 4800, and 8000 IU (as interleukin-2 equivalent). METHODS: Paraffin-embedded tumor samples obtained at surgical resection of the residual tumor (between days 21 and 28 after treatment initiation) were used. Histological analysis, necrosis evaluation, and American Joint Committee on Cancer grading were performed from H&E-stained sections. Immunohistochemical analysis was performed on three different tumor regions (surface, zone 1; center, zone 2; and tumor-stroma interface, zone 3). Trichrome staining was used to evaluate connective tissue, and morphometric measurements were made using ImagePro analysis software. Cell cycling was determined by the use of Ki-67 marker. RESULTS: Leukocyte interleukin injection treatment induced a shift from stromal infiltrating T cells toward intraepithelial T cells and posted a significant (P <.05) increase in intraepithelial CD3-positive T cells independent of the leukocyte interleukin injection dose, whereas the increase in CD25 (interleukin-2 receptor alpha [IL-2Ralpha])-positive lymphoid cells was significant only at the lowest leukocyte interleukin injection dose (P <.05). Furthermore, both low- and medium-dose leukocyte interleukin injection treatment induced a significant (P <.05) increase in the number of cycling tumor cells, as compared with control values. CONCLUSION: The results could be highly beneficial for patients with oral squamous cell carcinoma. First, leukocyte interleukin injection treatment induces T-cell migration into cancer nests and, second, noncycling cancer cells may enter cell cycling on administration of leukocyte interleukin injection. This latter effect may modulate the susceptibility of cancer cells to radiation therapy and chemotherapy. The findings may indicate a need to re-evaluate the way in which follow-up treatment (with radiation therapy and chemotherapy) of patients with head and neck cancer is currently approached.

Reliability of quantitative reverse-transcriptase-PCR-based detection of tumour cells in the blood between different laboratories using a standardised protocol.

Differences in methods of reverse-transcriptase (RT)-polymerase chain reaction (PCR)-based detection of tumour cells in the blood gives rise to conflicting results, and standardisation is urgently needed. This pilot study aimed to assess the variation of RT-PCR-based detection of tumour cells in blood between four different laboratories using a commercially available kit with a standardised protocol. This kit allows comparison of results from different laboratories and facilitates the investigation of the influence of pre-analytical parameters. All laboratories analysed identical sets of blood samples spiked with tumour cells in a concentration range of 1-100 tumour cells/ml. To study at which level variation was introduced, three kinds of sample sets were generated in which (i) tumour cell RNA was spiked in the RNA of mononuclear cells (MNC), (ii) tumour cells were spiked in isolated MNC, and (iii) tumour cells were spiked in blood. Real-time quantitative RT-PCR was used to detect and quantify cytokeratin 20 (CK20) expression, which is indicative for the presence of epithelial tumour cells. All laboratories were able to detect CK20 expression in all spiked-RNA samples with limited variation in expression levels between laboratories. There was a positive correlation between the amount of spiked tumour cell RNA and CK20 expression level. RT-PCR analysis of spiked-MNC samples resulted in more variation in the CK20 expression levels between laboratories, however again all spiked samples were reported to be positive by all of the laboratories. The evaluation of spiked-blood samples gave rise to considerable quantitative and qualitative variation between the laboratories. Our results underline the importance and need for standardisation and extended quality control studies in the field of pre-analytics.

Expression and function of the AMF receptor by human melanoma in experimental and clinical systems.

Motility of tumor cells is the rate limiting potential of metastatic cells and is regulated by autocrine and paracrine factors. Autocrine motility factor/neuroleukin/phosphohexose isomerase (AMF) is one of the best characterized autocrine motogenic cytokines. Here we have studied its in vitro effects on several human melanoma cell lines and found that neither cell line exhibited mitogenic response to AMF at a concentration where motogenic response could be initiated. Similar to previous studies on murine melanoma, activation of the AMF receptor upregulated beta3 while it downregulated beta1 integrins at the cell surface, inducing an integrin phenotype characteristic for invasive/metastatic melanoma. The gp78/AMF receptor protein expression in human melanoma cell lines correlated to their in vivo spontaneous metastatic potential. Furthermore, in two out of three human melanoma lines the expression significantly increased in the primary tumor when spontaneous metastases developed (immunosuppressed newborn rat model versus SCID mice). In a prospective study we have also analyzed AMF receptor protein expression in primary tumors of 54 skin melanoma patients using IHC. These studies revealed three types of AMF receptor phenotype: weak, heterogenous and strong expression profile. While in thin tumors weak/heterogenous AMFR expression predominated, in thick tumors the strong expression profile was predominant. The connection between AMFR expression and the invasive/metastatic potential of melanoma was further supported by our observation that SSM melanoma in the vertical growth phase expressed this motility receptor more strongly than tumors in the radial growth phase.

Circulating tumor cell clusters in the peripheral blood of colorectal cancer patients.

PURPOSE: Recently several reverse transcription-PCR techniques have been proven to be useful for the detection of circulating micrometastases. However, this way intact cell clusters that were found in animal experiments of prognostic value could not be detected. In this study, evaluation and modification of a commercial, cytokeratin-based, immunomagnetic cell separation method was performed for the detection of intact cell clusters in colorectal carcinoma patients. EXPERIMENTAL DESIGN: Thirty-two colon cancer patients (6 were in Dukes stage B, 13 in stage C, and 13 in stage D) and 20 healthy donor samples were evaluated. Immunomagnetic cell separation was performed from the buffy coat of peripheral blood samples (20 ml) using the Carcinoma Cell Enrichment Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), avoiding any filtering steps. The enriched cell fraction was cytocentrifuged and immunocytochemically labeled using a pancytokeratin antibody (MNF116; Dako). RESULTS: Of 20 healthy samples, 2 contained one cytokeratin-positive cell. Of 32 single samples from malignant cases, 24 showed cytokeratin-positive cells. Tumor cell clusters, mixed-cell doublets (one cytokeratin-positive and -negative cell), and mixed-cell clusters were detected in 22 of 24 patients. In six cases, cytokeratin-positive dendritic-like cells were detected. Follow-up data indicate that chemotherapy cannot destroy all of the circulating tumor cell clusters. CONCLUSIONS: Using the methods presented, we could detect circulating colon cancer cells and cell clusters in colon carcinoma patients. Similar cellular structures were described previously only in rats. Present data prove that such structures are present in human colorectal cancer, too.

Induction of apoptosis in MDR1 expressing cells by daunorubicin with combinations of suboptimal concentrations of P-glycoprotein modulators.

The application of most agents with the capacity to reverse multidrug resistance (MDR) via modulation of the multidrug transporter P-glycoprotein (Pgp) was shown to be associated with toxic side-effects. For this reason, we have investigated the effect of combinations of suboptimal concentrations of Pgp blockers on the induction of apoptosis and growth arrest in daunorubicin (D) treated, MDR1 gene transfected cells. We used verapamil, PSC833 and Cremophor EL as Pgp modulators, which affect the function of Pgp by different mechanisms. Treatment of NIH3T3/MDR1 cells with combinations of suboptimal concentrations of Pgp modulators in the presence of D caused apoptosis and G(2) arrest to the same extent as optimal concentrations of singly used blockers. We conclude that combinations of suboptimal concentrations of Pgp modulators may cause effective sensitization of resistant tumor cells, and at the same time, may avoid the frequently observed toxic effects experienced in clinical trials with a single modifier applied at the optimal dose.

Constitutive intracellular expression and activation-induced cell surface up-regulation of CD44v3 in human T lymphocytes.

The cell adhesion molecule CD44 exists in multiple isoforms generated by alternative RNA splicing. Increased expression of CD44 isoforms containing exon v6 and v9 has been reported to be associated with the activated state of T lymphocytes. Using monoclonal antibodies against variant exon products we studied the expression of another variant exon, v3 on resting and in vitro activated human peripheral blood T cells. We found that CD44v3, in parallel with CD44v6, is up-regulated at the surface of normal T cells stimulated by anti-CD3 antibody or by the phorbol ester PMA, as well as on PMA-stimulated T cell leukemia lines CCRF-CEM and MOLT-4. Beside the cell surface, we demonstrated CD44v3 intracellularly in both resting and activated T cells by flow cytometry and immunomorphology. Reverse transcription-PCR and Western blot analyses confirmed the constitutive expression of CD44v3 in these cells. The increase in the cell surface expression of CD44v3 on stimulated T lymphocytes was inhibited by cycloheximide and brefeldin A, indicating the requirement of de novo protein synthesis and endoplasmic reticulum Golgi transport. Our studies establish CD44v3 as an additional activation marker for human T cells, with a yet unidentified function.

Expression of a decorin-like moleculein human melanoma.

Decorin, a member of the family of small leucin-rich proteoglycans, has originally been described as a secreted proteoglycan component of the connective tissues, and has been implicated in the negative regulation of cell proliferation directly or via interactions with TGF-beta. It was reported to be generally absent from tumor cells. Here we show that human melanoma cell lines express a decorin-like molecule. We detected decorin mRNA by RT-PCR in 7 out 7 human melanoma lines characterized by various metastatic potential. Using polyclonal antiserum against the core protein of decorin, the typical 80-120 kD glycanated form as well as a high molecular weight aberrant version (200-210 kD) of decorin were demonstrated by Western blot technique in the culture supernatants as well as in lysates of human melanoma cells. Finally, decorin epitope was also demonstrated immunohistochemically in human melanoma xenografts, as well as in tumor cells of surgically resected melanomas but not in melanocytes of nevi. The expression of this aberrant decorin did not inhibit the in vitroor in vivogrowth of human melanoma cells, and it was independent of their metastatic potential. Human melanoma cell lines expressing aberrant decorin retained sensitivity to the antiproliferative and gelatinase-stimulatory effects of exogenous TGF-beta.

Expression of CD44v3 splice variant is associated with the visceral metastatic phenotype of human melanoma.

We analyzed the immunohistochemical expression of the metastasis-associated protein, CD44v3, in 46 primary human malignant melanomas (MMs). This is the first time that the v3 splice variant of CD44 was found to be expressed in human melanomas (15 of 46), ranging from 3% to 35% of the cell population in the positive tumors. The expression of CD44v3 was observed in tumors thicker than 1.0 mm, and one-third of these tumors proved to be positive irrespective of the thickness. Patients were followed for a minimum of 61 months. The onset of lymph node or organ metastases occurred not later than 58 months and 60 months, respectively. Of the 15 CD44v3 positive tumors, 14 were observed in the organ metastatic tumor group, comprising the majority of those cases (14 of 21), and this association proved to be statistically significant compared with the non-metastatic (P<0.05) and lymph-node metastatic cases (P<0.01). CD44v3 expression in melanoma was also confirmed at the protein and messenger (mRNA) level in several human melanoma cell lines using flow cytometry and reverse transcriptase polymerase chain reaction analysis. In parallel to CD44v3, MMP-2 expression (determined using immunohistochemistry) was significantly elevated (P<0.05) but only in the organ metastatic group of MM. The 5-year survival of patients having thicker tumors than 1.0 mm (where v3 expression occurred) who had CD44v3+ tumors was significantly lower than those of the negative ones (35.7% versus 68.2%, respectively; P=0.025). Finally, we observed that the CD44v3-expressing tumors were characterized by significantly higher MMP-2 expression than the CD44v3-negative tumors (P<0.001), indicating a possible correlation between CD44v3- and MMP-2-positive phenotype and the organ metastatic potential of MM.

Tumorigenicity and immunogenicity of murine tumor cells expressing an MHC class II molecule with a covalently bound antigenic peptide.

The significance of CD4+ lymphocytes and major histocompatibility complex (MHC) class II-restricted antigens in antitumor immunity has been demonstrated in several animal models as well as in some human tumors. However, because of the lack of known class II-restricted antigens, the participation of CD4+ cells in antitumor responses has not been well characterized. Recent reports showed that class II proteins covalently linked to an antigenic peptide could be constructed and cells expressing these fusion proteins were recognized by specific TH cells. The aim of this study was to determine the effect of the expression of a class II-peptide construct on the tumorigenicity and immunogenicity of transfected murine tumor cells. We have constructed a gene for I-Ed beta chain covalently coupled to the I-Ed-restricted TH cell determinant of sperm whale myoglobin (SWM132-145). This class II fusion protein was recognized by a specific TH cell line on the surface of COS-7 cells or BALB/c sarcoma cells. The sarcoma cells expressing the MHC-peptide complex were rejected by immunocompetent BALB/c mice, and in vivo T-cell subset depletion experiments suggested the importance of CD4+ cells in the rejection. Moreover, splenocytes from mice immunized with tumor cells expressing the I-Ed-SWM complex showed specific peptide recognition in vitro. Such covalent MHC-peptide complexes could prove useful in studies on the role of CD4+ lymphocytes in antitumor immune responses, and also in designing new, more effective vaccine approaches to the immunotherapy of cancer, as class II-restricted tumor-associated antigens are identified for human cancers.

Liver metastatic ability of human melanoma cell line is associated with losses of chromosomes 4, 9p21-pter and 10p.

Genetic changes underlying the aggressive progression of human cutaneous melanoma are not completely understood. In order to characterise genetic alterations associated with the metastatic behaviour of this neoplasm we used comparative genomic hybridisation (CGH) in combination with fluorescence in situ hybridisation (FISH) on an experimental metastatic model of three related human melanoma cell lines. Tumour lines were selected based on their various metastatic capacity to liver in immunosuppressed mice. The parental cell line (A2058) was a human amelanotic melanoma cell line, adaptation of this line to in vivo growth as xenograft the HT168 tumour and its cell line was established. After intrasplenic transplantation of HT168 cells into immunosuppressed mice, a highly metastatic variant (HT168-M1) was selected. Several chromosomal aberrations common to all three lines indicating common clonal origin, as well as additional non-shared chromosomal changes were found. The original cell line (A2058) exhibited the highest number of genetic changes. Chromosomal alterations present only in the highly metastatic line (HT168-M1) involved losses on chromosome 4, 9p21.3-pter and 10p. Chromosome copy number patterns and the nature of chromosome 4 loss were further investigated by FISH using different centromeric probes and a chromosome 4 painting probe. According to our CGH and FISH results we assume that alterations present only in the aggressive metastatic subline are associated with the increased - metastatic potential. Our observations further support the hypothesis, based on some recently published data, that certain (so far unidentified) suppressor genes having an important role in tumour progression are located on these chromosomes.

Separation of distinct MUC2 mucin glycoforms using two anti-peptide monoclonal antibodies.

Two monoclonal antibodies (MAb996 and MAb994) were produced by immunisation with a synthetic peptide with a sequence based upon that of the protein core of the gastrointestinal MUC2 mucin. The epitopes were identified as T G T Q for MAb996 and P T G T Q for MAb994. Antibody competition tests also confirmed the overlapping nature of the epitopes for the two antibodies. MAb994 and MAb996 were employed in immunoadsorbent columns for the fractionation of human colorectal carcinoma tissue extracts. While the two antibodies displayed only relatively minor differences in immunological specificity and affinity for the immunising synthetic MUC2 mucin core related peptide, they had the capacity to separate antigenically distinct molecules when used as immunoadsorbents. The findings indicated that subfractions of MUC2 antibody-defined mucins exist in human carcinomas and that these may be distinguished by the differential exposure of determinants in the mucin protein core. The results are in accord with the view that aberrant patterns of glycosylation of mucins in human intestinal tumours produces a spectrum of variably glycosylated macromolecules.

[Morphological parameters of breast cancers detected by mammography with and without organized screening; a comparative study]. / Mammográfiával felismert rákok morphologiai paramétereinek összehasonlítása a területileg szervezett szürés során és alkalomszerüen vizsgáltaknál.

Some pathological findings and prognostic indices recorded in breast cancer cases, detected, on one hand, by a provider-initiated mammography screening program (Group 1), and, opportunistically, in self-referred symptomatic women (Group 2) on the other, are compared. In 8877 symptom-free women, aged 50-65 years, individually invited to attend the screening offered for the residents of the III., XII. and XIII. districts of Budapest, 67 cancer cases were detected (7.5 in 1000 screenees), in accordance with the cancer detection rate of the first, "prevalence" round of organised screening programmes. In the other group of 1593 symptomatic, self-referred women of the same age, 113 cancer cases were diagnosed by mammography. As far as the pathological parameters are concerned, the number of cases with invasive cancer less than 15 mm in diameter, and those with axillary nodes present was found to be significantly higher in the screened group as compared to the self-referred one (p < 0.01). In "small" cancers (i.e. less than 15 mm in diameter), no significant difference was found in the proportion of histologic grade III tumours among the two groups. In screen-detected cancers both the morphometric prognostic index (as calculated by Baak et al.) and the Nottingham Prognostic Index (NPI) proved to be more favourable, as compared to those in the self-referred group. The p-value as determined by Mann-Whithey test was 0.000003 in the screened group, and 0.000015 in the other one. These findings provide convincing evidence in support of the public health importance of provider-initiated, organised mammography screening for breast cancer, therefore, the introduction on service basis of organised breast screening into the health care system in Hungary is strongly recommended by the authors.

Cytokine sensitivity of metastatic human melanoma cell lines-- simultaneous inhibition of proliferation and enhancement of gelatinase activity.

The effect of a panel of cytokines on the proliferation and type IV collagenase production was studied in four melanoma cell lines of different origin, tumorigenicity and metastatic capacity. TGF-b, TNF-a and to a lesser extent, IL-1a exhibited antiproliferative effect on the cell lines, with some lines showing varying degree of resistance. The sensitivity did not correlate directly with the origin or the biological behavior of the tumor lines, suggesting that cytokine resistance of advanced stage melanoma cells may be relative. IL-2, IL-10 and IL-12 displayed little or no effect on proliferation. The effect of cytokines on metalloproteinase production showed a cell line dependent pattern. Interestingly, those cytokines that exhibited the most pronounced antiproliferative activity, also proved most effective in stimulating collagenase secretion, often simultaneously, in the same line. The results indicate that pleiotropic cytokines can have positive and negative effects simultaneously on various steps of tumor progression.

Role of sinusoidal heparan sulfate proteoglycan in liver metastasis formation.

Previous studies have indicated that the predominant sites of tumor cell extravasation in the liver are the sinusoidal vessels, where tumor cells contact the sinusoidal endothelium and the subendothelial extracellular matrix containing the basic components of the basement membrane. We studied the role of sinusoidal extracellular matrix in metastatsis formation by 3LL-HH murine tumor cells selected for their preferential liver colonization. 3LL-HH tumor cells did not efficiently adhere to cryosections of the liver, but they recognized the sinusoids and vessel walls. Pre-treatment of the mice with polyclonal anti-basement membrane antibodies [anti-laminin, anti-fibronectin and anti-heparan sulfate proteoglycan (HSPG)] significantly modulated the organ distribution of tumor cell colonies following intracardial injection: all 3 antibodies inhibited kidney colonization; anti-laminin and anti-fibronectin antibodies inhibited lung colonization; and only anti-HSPG antibody inhibited liver colonization. In several organs such as the heart, stomach, pancreas and bladder, anti-basement membrane antibody treatment did not alter the process of colonization. Immunofluorescence studies showed that anti-HSPG antibody recognized the basement membranes of sinusoids and blood vessels. Our data suggest a specific involvement of sinusoidal HSPG in the liver colonization of 3LL-HH cells.