Increased Blood Levels of Human S100 in Melanoma Chick Embryo Xenografts’ Circulation

The chorioallantoic membrane (CAM) of the fertilized egg allows grafting of human melanomas for short‐term investigations and offers the opportunity to investigate the behavior of metastasizing cells and the release of S100β into peripheral blood. Tissue from one primary melanoma as well as cutaneous and subcutaneous metastases of 10 melanoma patients with elevated levels of S100 in the peripheral blood before surgery were transplanted onto the CAM of chick embryos at day 5/6 of development. Grafts were nourished by the host blood supply 2 days after transplantation. Histologically, 3 days after grafting, metastasizing melanoma cells could be found near the vessels of the host membrane, penetrating the endothelial layer and entering the blood system. Growth conditions remained stable for 6 days after transplantation. Blood samples were taken from a larger CAM vessel before collecting the xenografts 5 days after grafting. Measurement of human S100 in peripheral blood was performed in a blinded manner. No negative control showed elevated levels of human S100 protein. Samples deriving from melanoma xenografts contained highly elevated levels of S100 protein in 80% of cases. The data strongly support the concept of graft–host interaction concerning adherence of tumors and extravasation of human melanoma cells.     

memA/DRS, a putative mediator of multiprotein complexes, is overexpressed in the metastasizing human melanoma cell lines BLM and MV3

memA was isolated by subtractive hybridization in which the mRNA repertoire was compared in a panel of human melanoma cell lines with different metastasizing potential. Expression of memA mRNA is elevated in the highly metastasizing human melanoma cell lines and derived xenografts, as compared with the non-metastasizing ones. In a collection of human tumor cell lines and melanoma metastasis lesions, memA mRNA expression could be detected in the A-431 (epidermoid carcinoma), HT-1080 (fibrosarcoma), JEG-3 and JAR (choriocarcinomas) cell lines and in three out of 11 melanoma metastasis lesions. The distribution of memA mRNA in a collection of healthy human organs is also tissue restricted. Sequence analysis revealed that the MEMA protein is identical with a 160 kDa nuclear 'domain rich in serines' (DRS) protein occurring free in the nucleoplasm and in U2-ribonucleoprotein structures. MEMA is also homologous to pinin, a 140 kDa protein associated with the desmosome-intermediate filament complex, and to a 32 kDa porcine neutrophilic protein that was copurified with components of the NADPH-oxidase enzyme complex. The encoded amino acid sequence predicts that the MEMA protein has three coiled-coil domains, one glycine loop domain, is very hydrophilic and contains regions rich in glutamine/proline, glutamic acid and serine residues.     

Chick embryo chorioallantoic membrane model systems to study and visualize human tumor cell metastasis

Since their introduction almost a century ago, chick embryo model systems involving the technique of chorioallantoic grafting have proved invaluable in the in vivo studies of tumor development and angiogenesis and tumor cell dissemination. The ability of the chick embryo’s chorioallantoic membrane (CAM) to efficiently support the growth of inoculated xenogenic tumor cells greatly facilitates analysis of human tumor cell metastasis. During spontaneous metastasis, the highly vascularized CAM sustains rapid tumor formation within several days following cell grafting. The dense capillary network of the CAM also serves as a repository of aggressive tumor cells that escaped from the primary tumor and intravasated into the host vasculature. This spontaneous metastasis setting provides a unique experimental model to study in vivo the intravasation step of the metastatic cascade. During experimental metastasis when tumor cells are inoculated intravenously, the CAM capillary system serves as a place for initial arrest and then, for tumor cell extravasation and colonization. The tissue composition and accessibility of the CAM for experimental interventions makes chick embryo CAM systems attractive models to follow the fate and visualize microscopically the behavior of grafted tumor cells in both spontaneous and experimental metastasis settings.     

Alternative Immunomodulatory Strategies for Xenotransplantation: CD80/CD86-CTLA4 Pathway-Modified Immature Dendritic Cells Promote Xenograft Survival

Background

Xenotransplantation is a promising approach to circumventing the current organ shortage. However, T-cell-dependent anti-xenoresponses are a major challenge to successful xenografts. Given the advantages of the use of CTLA4-Ig in the survival of allografts, the purpose of the study was to investigate the therapeutic potential of CTLA4-IgG4 modified immature dendritic cells (imDCs) in the prevention of islets xenograft rejection.

Methods

CTLA4-IgG4 was constructed by the fusion of the extracellular regions of porcine CTLA4 to human the hIgG4 Fc region. The imDCs were induced and cultured from porcine peripheral blood mononuclear cells (PBMC). The CTLA4-IgG4 modified imDCs were delivered via the portal vein to the liver of diabetic mice (insulin-dependent diabetes mellitus) before islet xenografting, and mCTLA4-Ig was administered intravenously after xenotransplantation.

Results

The xenograft survival of mice receiving unmodified imDCs was approximately 30 days. However, following administration of CTLA4-IgG4 modified imDCs before grafting and mCTLA4-Ig after grafting, xenografts survived for more than 100 days. Flow cytometric analysis showed that the CD4⁺CD25⁺Foxp3⁺ Treg population was increased in spleens. The efficacy of donor CTLA4-IgG4 modified imDCs correlated partially with the amplification of Tregs.

Conclusions

These results confirm that selective inhibition of the direct and indirect pathways of T-cell activation by donor CTLA4-IgG4 modified imDCs and receptor CTLA4-Ig is a highly effective strategy to promote survival of xenografts.     

Increased levels of DUSP6 phosphatase stimulate tumourigenesis in a molecularly distinct melanoma subtype

The mitogen-activated protein kinase (MAPK) pathway is important in melanoma. In this pathway, DUSP6 phosphatase negatively controls the activation of extracellular signal-regulated (ERK) kinase. Through comparison of melanoma signalling pathways between immortal mouse melanocytes and their tumourigenic derivatives, retrieved from mouse xenografts, we identified a molecularly distinct subtype of melanoma, characterized by reduced ERK activity and increased DUSP6 expression. Overexpression of DUSP6 enhanced anchorage-independent growth and invasive ability of immortal mouse melanocytes, suggesting that increased DUSP6 expression contributes to melanoma formation in the mouse xenografts. In contrast, reduced tumourigenicity was observed after DUSP6 overexpression in human melanoma cells. A minority of thick human primary melanomas had high DUSP6 expression and the same poor melanoma-specific survival as the majority of thick primaries with low DUSP6 levels. We have demonstrated that DUSP6 is important in melanoma and that it plays a different role in our distinct subtype of mouse melanoma compared with that in classic human melanoma.     

MDA-7/IL-24: novel cancer growth suppressing and apoptosis inducing cytokine

The melanoma differentiation-associated gene-7 (mda-7) was cloned by subtraction hybridization as a molecule whose expression is elevated in terminally differentiated human melanoma cells. Current information based on structural and sequence homology, has led to the recognition of MDA-7 as an IL-10 family cytokine member and its renaming as IL-24. Northern blot analysis revealed mda-7/IL-24 expression in human tissues associated with the immune system such as spleen, thymus, peripheral blood leukocytes and normal melanocytes. The MDA-7/IL-24 mouse counterpart, FISP, appears to be a Th2-specific protein and the rat counterpart, C49A/MOB-5, is associated with wound healing and is also induced as a consequence of ras-transformation. A notable property of MDA-7/IL-24 is its ability to induce apoptosis in a large spectrum of human cancer derived cell lines, in mouse xenografts and upon intratumoral injection in human tumors (phase I clinical trials). Various aspects of this intriguing molecule including its cytokine and anti-tumoral effects are described and discussed.     

Identification of DNA repair gene Ercc1 as a novel target in melanoma

Increased expression of DNA repair genes contributes to the extreme resistance shown by melanoma to conventional DNA-damaging chemotherapeutics. One such chemotherapeutic effective against a range of other cancers, but not melanoma, is cisplatin. The DNA repair protein, ERCC1, is needed to remove cisplatin-induced DNA damage. We have shown that ERCC1 is essential for melanoma growth and resistance to cisplatin in a mouse xenograft model. Untreated xenografts of our transformed Ercc1-proficient melanocyte cell line grew very rapidly as malignant melanoma. Cisplatin treatment caused initial shrinkage of xenografts, but cisplatin-resistant regrowth soon followed. Cells reisolated into culture had twofold elevated levels of ERCC1 compared to both input cells and cells reisolated from untreated xenografts. An isogenic Ercc1-deficient derivative grew equally well in vitro as the Ercc1-proficient melanocyte cell line. However, in xenografts, the Ercc1-deficient melanomas were much slower to establish and were completely cured by just two cisplatin treatments.     

In vitro sensitization of human lymphoid cells to antigens on cultured melanoma cells: II. Sensitization against melanoma-associated antigens

Peripheral blood lymphoid cells from patients with malignant melanoma can be sensitized on allogeneic or autochthonous melanoma monolayers. Peak cytotoxicity occurred after 5 days of sensitization. Sensitization appeared to be directed against melanoma-associated antigens, as judged by the pattern of cytotoxic reactivity. Sensitized cells were cytotoxic against autochthonous or allogeneic melanoma cells, but not against autochthonous fibroblasts or allogeneic tumor cells of different histologic types. Sensitization of responder lymphoid cells from melanoma patients on allogeneic melanoma cells usually resulted in more pronounced cytotoxicity against autochthonous melanoma target cells than did sensitization on autochthonous melanoma monolayers. These results indicate that cell cultures of human malignant melanoma contain tumor-associated antigens which can sensitize human peripheral blood lymphoid cells in vitro. These results also support the concept that there are cross-reactive tumor-associated antigens in human malignant melanomas.     

S100 protein expression in human melanoma cells: comparison of levels of expression among different cell lines and individual cells in different phases of the cell cycle

The synthesis of S100 protein in cultured human melanoma cells was examined using metabolic labeling with [35S]methionine, immunoprecipitation with anti-S100 protein antiserum, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Six of seven cell lines derived from melanomas synthesized relatively large amounts of S100 protein, whereas three cell lines derived from normal melanocytes synthesized lesser amounts. Synthesis of S100 protein was not detected in 10 human cell lines of nonneuroectodermal origin. Analysis of poly(A+) RNA from one melanoma cell line by Northern blot hybridization with a probe specific for the beta subunit of rat S100 protein revealed a single mRNA species of 1.0 kb coding for the human protein. Flow cytometric analysis of individual cells of two melanoma cell lines and the rat glioma cell line C6 indicated that G0/G1 cells were heterogeneous with respect to S100 protein expression, while almost all the cells in S + G2 + M expressed S100 protein. These results suggest that expression of S100 protein in G0/G1 could be a prerequisite for progression of the cells through the cell cycle.     

绿色荧光蛋白基因修饰的人视网膜色素上皮细胞异种移植的长期观察

探讨利用视网膜色素上皮 (retinalpigmentepithelium ,RPE)细胞移植介导目的基因转移至视网膜的可行性。人的RPE细胞在体外经携带绿色荧光蛋白 (greenfluorescentprotein ,GFP)基因的逆转录病毒感染及G4 1 8筛选后 ,手术显微镜直视下经睫状体平坦部注射到兔眼视网膜下间隙。术后通过活体荧光眼底照相 ,荧光显微镜、共聚焦显微镜及透射电镜等观察眼球铺片及切片 ,发现经 gfp基因修饰的人RPE细胞在兔眼视网膜下间隙可存活一年以上。移植的RPE gfp细胞不仅仅局限于移植部位 ,大多扩散到超过 2～ 3个象限的眼底 ,镶嵌于宿主RPE细胞之间或呈单层排列在宿主色素上皮与神经视网膜之间 ,并持续高水平表达GFP。移植后早期玻璃体内每周注射免疫抑制剂普乐可复 (FK5 0 6 )可明显改善移植细胞的存活状态     

mGPDH Deficiency leads to melanoma metastasis via induced NRF2

Oxidative stress critically influences carcinogenesis and the progression of melanoma, and aggressive malignant melanoma activity is due to its high metastatic ability. Some findings in several cancer cell lines have indicated that mGPDH, a component of the mitochondrial respiratory chain, also modulates oxidative stress. However, the role of mGPDH in melanoma remains elusive. Here, we report that the mGPDH protein level is decreased in human skin melanoma compared to normal skin and decreased in metastatic melanoma compared to primary melanoma. Our in vivo and in vitro experiments indicated that mGPDH depletion accelerated melanoma migration and invasion without affecting proliferation or apoptosis. Mechanistically, we found elevated NRF2 protein levels in human skin melanoma and mGPDH-knockout (ko) metastatic xenografts in the lungs of nude mice. Moreover, in A375 melanoma cells, the loss of mGPDH-induced NRF2 expression but did not affect NRF2 protein degradation. Additionally, melanoma metastasis induced by the loss of mGPDH was rescued by the further down-regulation of NRF2 in vivo and in vitro. Consistently, mGPDH overexpression (oe) depressed NRF2 expression and attenuated the malignant properties of melanoma cells. In conclusion, our findings suggest that mGPDH suppresses melanoma metastasis by inhibiting NRF2 and downstream oxidative signals, highlighting the therapeutic potential of mGPDH for melanoma treatment.     

Tumor size,leukocyte adherence inhibition and serum levels of tumor antigen in dogs with the canine transmissible venereal sarcoma

Summary Tumor antigen (TA) associated with the canine transmissible venereal sarcoma (CTVS) was detected in the sera of dogs bearing the tumor. Rabbit antisera specific for tumor antigen and 3 M KCl extracts of CTVS cells were used in both a competitive enzyme-linked immunosorbent assay (ELISA) and antigen-capture ELISA to quantify levels of circulating TA. In a study of 29 dogs bearing the transplanted CTVS, levels of circulating TA correlated positively with tumor volume. In a longitudinal study of four dogs receiving a transplant of 10⁸ viable CTVS cells, circulating CTVS antigen was detected transiently 2 days after transplantation, while persistent levels of TA associated with increasing tumor volume were demonstrable 19–34 days after transplantation. In three of four tumor-bearing dogs, levels of serum TA correlated inversely with values obtained with peripheral blood leukocytes in the leukocyte adherence inhibition (LAI) assay; elevated levels of circulating TA found in dogs with large (>7 cm³) tumors were associated with decreased LAI reactivity of peripheral blood leukocytes. TA could not be detected in sera 48–72 h after surgical removal of CTVS whereas LAI reactivity of peripheral blood leukocytes to CTVS antigen rebounded 1–3 weeks following tumor excision. Results of this study support the use of the competitive ELISA and LAI techniques in assessing levels of circulating tumor antigen, tumor burden and tumor-specific immunity.Supported by grants from the American Kennel Club, National Institutes of Health (CA23 469) and Funds for Research on Canine Diseases provided by the 1963 Connecticut Legislature     

Kinetics of IL-7 and IL-15 Levels after Allogeneic Peripheral Blood Stem Cell Transplantation following Nonmyeloablative Conditioning

Background

We analysed kinetics of IL-7 and IL-15 levels in 70 patients given peripheral blood stem cells after nonmyeloablative conditioning.

Methods

EDTA-anticoagulated plasma and serum samples were obtained before conditioning and about once per week after transplantation until day 100. Samples were aliquoted and stored at −80°C within 3 hours after collection until measurement of cytokines. IL-7 and IL-15 levels were measured by ELISAs.

Results

Median IL-7 plasma levels remained below 6 pg/L throughout the first 100 days, although IL-7 plasma levels were significantly higher on days 7 (5.1 pg/mL, P = 0.002), 14 (5.2 pg/mL, P<0.001), and 28 (5.1 pg/mL, P = 0.03) (but not thereafter) than before transplantation (median value of 3.8 pg/mL). Median IL-15 serum levels were significantly higher on days 7 (12.5 pg/mL, P<0.001), 14 (10.5 pg/mL, P<0.001), and 28 (6.2 pg/mL, P<0.001) than before transplantation (median value of 2.4 pg/mL). Importantly, IL-7 and IL-15 levels on days 7 or 14 after transplantation did not predict grade II–IV acute GVHD.

Conclusions

These data suggest that IL-7 and IL-15 levels remain relatively low after nonmyeloablative transplantation, and that IL-7 and IL-15 levels early after nonmyeloablative transplantation do not predict for acute GVHD.     

Growth characteristics of human melanoma multicellular spheroids in liquid-overlay culture: comparisons with the parent tumour xenografts

The growth characteristics of multicellular spheroids, derived from human melanoma xenografts and cultivated in liquid-overlay culture, were studied and compared with those of the parent tumours. Six of the seven melanomas investigated formed spheroids, which grew exponentially up to a volume of 1-2 X 10(7) microns 3 (a diameter of 270-340 microns) before the growth rate tapered off. The morphology of the spheroids varied considerably among the melanomas; some spheroids grew as densely packed, spherical structures of cells whereas others were loosely packed and showed an irregular shape. Central necrosis developed when the spheroids attained a diameter of 150-200 microns. The histological and cytological appearance of the spheroids was remarkably similar to that of the parent xenograft in five of the six cases. The sixth melanoma contained two subpopulations with distinctly different DNA content, one of which was predominant in the spheroids, the other in the tumours. This gave rise to clear histological and cytological differences. The volume-doubling time of the spheroids during the exponential growth phase ranged from 1.7 +/- 0.2 to 2.7 +/- 0.4 days and the fraction of cells in S from 13 +/- 1 to 28 +/- 2%. The volume-doubling time decreased with increasing fraction of cells in S, indicating that the differences in growth rate were due mainly to differences in the growth fraction or to differences in the duration of G1. The spheroid volume-doubling times did not correlate with those of the parent xenografts (Td = 4.2-22.5 days at V = 200 mm3), possibly because the cell loss factors of the xenografts were large and varied among the melanomas. The fractions of cells in G1/G0, S and G2 + M in the spheroids and the xenografts did not correlate either, but were found to be within the same narrow ranges in the spheroids and the xenografts--i.e. 50-80% (G1/G0), 10-30% (S) and 10-20% (G2 + M).     

Active interaction of human A375 melanoma cells with the lymphatics in vivo

We have used the avian chorioallantoic membrane (CAM) to study the interaction of tumor cells with the lymphatics in vivo. The vascular endothelial growth factor-C (VEGF-C) has been shown to be lymphangiogenic. We have therefore grown VEGF-C-expressing human A375 melanoma cells on the CAM. These tumors induced numerous lymphatics at the invasive front, and compressed or destroyed VEGF receptor (R)-3-positive lymphatics were observed within the solid tumors. The lymphatics in the CAM and in the A375 melanomas could also be demonstrated with an antibody against Prox 1, a highly specific marker of lymphatic endothelial cells. Proliferation studies revealed a BrdU labeling index of 11.6% of the lymphatic endothelial cells in the tumors and at their margins. A great number of melanoma cells invaded the lymphatics. Such interactions were not observed with VEGF-C-negative Malme 3 M melanoma cells. Lymphangiogenesis was inhibited to some extent when A375 melanoma cells were transfected with cDNA encoding soluble VEGFR-3 (sflt4), and the BrdU labeling index of the lymphatics in these tumors was 3.9%. Invasion of lymphatics and growth of blood vascular capillaries were not inhibited by the transfection. Therefore, tumor-induced lymphangiogenesis seems to be dependent to some extent on VEGF-C/flt4 interactions, but invasion of lymphatics seems to be a distinct mechanism.     

The chick chorioallantoic membrane as a model system for the study of tissue invasion by viral transformed cells.

The chick chorioallantoic membrane (CAM) was used as an assay system to investigate the the invasive properties of viral transformed NIH/3Y3 cells. Scanning electron microscopy demonstrated that single Kirsten sarcoma virus (KiSV)-transformed cells passed between the epithelial cells of the CAM ectoderm within 6 hr of application, while viable NIH/3T3 cells did not penetrate the ectoderm within 24 hr. The transformed cells entered the mesoderm of the CAM and formed tumors of proliferating cells. The application of 5 X 10(5) KiSV-transformed cells resulted in the formation of donor cells resulted in the formation of the donor cell tumors within 5 days in 43% of the membranes. No tumors were formed when as many as 5 X 10(6) NIH/3T3 cells were applied to the membrane. NIH/3T3 cells transformed by the Abelson leukemia virus or the Moloney sarcoma virus also ivaded the CAM and formed tumors of proliferating cells within the mesoderm, while cells infected with the Moloney leukemia virus did not. NIH/3T3 cells inoculated onto the CAM 8 days after infection and transformation with KiSV formed tumors with a frequency similar to that of KiSV transformed cells that have been passaged in culture for many generations. Cells that formed invasive tumors within the mesoderm also attracted loops of host blood vessels.