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STAT1 is a critical effector and a target gene of interferon (IFN) signaling, and thus a central mediator of antiviral responses. As both a mediator and a target of IFN signals, STAT1 expression reports on, and determines IFN activity. Gene expression analyses of melanoma patient samples revealed varied levels of STAT1 expression, which highly correlated with expression of >700 genes. The ability of oncolytic viruses to exploit tumor-induced defects to antiviral responses suggests that oncolytic viruses may efficiently target a subset of melanomas, yet these should be defined. We modeled this scenario with murine B16F10 melanomas, immortalized skin fibroblasts as controls and a novel oncolytic virus, EHDV-TAU. In B16F10 cells, constitutive low expression of STAT1 and its target genes, which included intracellular pattern recognition receptors (PRRs), correlated with their inability to mount IFN-based antiviral responses upon EHDV-TAU challenge, and with potency of EHDV-TAU-induced oncolysis. This underexpression of interferon stimulated genes (ISGs) and PRRs, and the inability of EHDV-TAU to induce their expression, were reversed by epigenetic modifiers, suggesting epigenetic silencing as a basis for their underexpression. Despite their inability to mount IFN/STAT-based responses upon viral infection, EHDV-TAU infected B16F10 cells secreted immune-stimulatory chemokines. Accordingly, in vivo, EHDV-TAU enhanced intratumoral infiltration of cytotoxic T-cells and reduced growth of local and distant tumors. We propose that "STAT1 signatures" should guide melanoma virotherapy treatments, and that oncolytic viruses such as EHDV-TAU have the potential to exploit the cellular context of low-STAT1 tumors.

The melanoma‐microglia cross‐talk upregulates the expression of aldolase C (ALDOC) in melanoma cells from several patients. The response of melanoma cells from 2 different patients to ALDOC upregulation was diametrically divergent. Whereas several metastasis‐associated functions including brain metastasis formation were augmented in one melanoma cell line (Mel 1), these functions were attenuated in the other melanoma cell line (Mel 2)., Previous studies indicated that microglia cells upregulate the expression of aldolase C (ALDOC) in melanoma cells. The present study using brain‐metastasizing variants from three human melanomas explores the functional role of ALDOC in the formation and maintenance of melanoma brain metastasis (MBM). ALDOC overexpression impacted differentially the malignant phenotype of these three variants. In the first variant, ALDOC overexpression promoted cell viability, adhesion to and transmigration through a layer of brain endothelial cells, and amplified brain micrometastasis formation. The cross‐talk between this MBM variant and microglia cells promoted the proliferation and migration of the latter cells. In sharp contrast, ALDOC overexpression in the second brain‐metastasizing melanoma variant reduced or did not affect the same malignancy features. In the third melanoma variant, ALDOC overexpression augmented certain characteristics of malignancy and reduced others. The analysis of biological functions and disease pathways in the ALDOC overexpressing variants clearly indicated that ALDOC induced the expression of tumor progression promoting genes in the first variant and antitumor progression properties in the second variant. Overall, these results accentuate the complex microenvironment interactions between microglia cells and MBM, and the functional impact of intertumor heterogeneity. Since intertumor heterogeneity imposes a challenge in the planning of cancer treatment, we propose to employ the functional response of tumors with an identical histology, to a particular drug or the molecular signature of this response, as a predictive indicator of response/nonresponse to this drug.

Background: Intersecting a genome-wide expression profile of metastatic and nonmetastatic human neuroblastoma xenograft variants with expression profiles of tumours from stage 1 and 4 neuroblastoma patients, we previously characterised hexokinase 2 (HK2) as a gene whose expression was upregulated in both metastatic neuroblastoma variants and tumours from stage 4 neuroblastoma patients. Methods: Local and metastatic neuroblastoma cell variants as well as metastatic neuroblastoma cells genetically manipulated to downregulate the expression of HK2 were utilised for in vitro and in vivo examinations of the involvement of HK2 in neuroblastoma. Results: Hexokinase 2 expression and its activity levels were increased in neuroblastoma metastatic variants as compared with the local variants. The upregulation of HK2 confers upon the metastatic cells high resistance to the antiproliferative effect of the HK2 inhibitor 3-BrPa and to the chemotherapy agent Deferoxamine. The inhibition of HK2 transcript lowered the proliferation and motility of sh-HK2 cells as compared with sh-control cells. Mice that were inoculated with sh-HK2 cells had a lower incidence of local tumours, smaller tumour volumes and a diminished load of lung metastasis compared with mice inoculated with sh-control cells. Conclusions: Hexokinase 2 plays a significant role in shaping the malignant phenotype of neuroblastoma and influences the progression of this disease.

Paired like homeobox 2B (PHOX2B) is a minimal residual disease (MRD) marker of neuroblastoma. The presence of MRD, also referred to as micro-metastases, is a powerful marker of poor prognosis in neuroblastoma. Lung metastasis is considered a terminal event in neuroblastoma. Lung micro-metastatic neuroblastoma (MicroNB) cells show high expression levels of PHOX2B and possess a less malignant and metastatic phenotype than lung macro metastatic neuroblastoma (MacroNB) cells, which hardly express PHOX2B. In vitro assays showed that PHOX2B knockdown in MicroNB cells did not affect cell viability; however it decreased the migratory capacity of the MicroNB-shPHOX2B cells. An orthotopic inoculation of MicroNB-shPHOX2B cells into the adrenal gland of nude mice resulted in significantly larger primary tumors and a heavier micro-metastatic load in the lungs and bone-marrow, than when control cells were inoculated. PHOX2B expression was found to be regulated by methylation. The PHOX2B promoter in MacroNB cells is significantly more methylated than in MicroNB cells. Demethylation assays using 5-azacytidine demonstrated that methylation can indeed inhibit PHOX2B transcription in MacroNB cells. These pre-clinical data strongly suggest that PHOX2B functions as a suppressor of neuroblastoma progression.

The development of melanoma brain metastasis is largely dependent on mutual interactions between the melanoma cells and cells in the brain microenvironment. Here, we report that the extracellular cysteine protease inhibitor cystatin C (CysC) is involved in these interactions. Microglia-derived factors upregulated CysC secretion by melanoma. Similarly, melanoma-derived factors upregulated CysC secretion by microglia. Whereas CysC enhanced melanoma cell migration through a layer of brain endothelial cells, it inhibited the migration of microglia cells toward melanoma cells. CysC was also found to promote the formation of melanoma three-dimensional structures in matrigel. IHC analysis revealed increased expression levels of CysC in the brain of immune-deficient mice bearing xenografted human melanoma brain metastasis compared to the brain of control mice. Based on these in vitro and in vivo experiments we hypothesize that CysC promotes melanoma brain metastasis. Increased expression levels of CysC were detected in the regenerating brain of mice after stroke. Post-stroke brain with melanoma brain metastasis showed an even stronger expression of CysC. The in vitro induction of stroke-like conditions in brain microenvironmental cells increased the levels of CysC in the secretome of microglia cells, but not in the secretome of brain endothelial cells. The similarities between melanoma brain metastasis and stroke with respect to CysC expression by and secretion from microglia cells suggest that CysC may be involved in shared pathways between brain metastasis and post-stroke regeneration. This manifests the tendency of tumor cells to highjack physiological molecular pathways in their progression.

International audience; We previously identified the chemokine receptor CCR4 as part of the molecular signature of melanoma brain metastasis. The aim of this study was to determine the functional significance of CCR4 in melanoma brain metastasis. We show that CCR4 is more highly expressed by brain metastasizing melanoma cells than by local cutaneous cells from the same melanoma. Moreover, we found that the expression of CCR4 is significantly higher in paired clinical specimens of melanoma metastases than in samples of primary tumors from the same patients. Notably, the expression of the CCR4 ligands, Ccl22 and Ccl17 is upregulated at the earliest stages of brain metastasis, and precedes the infiltration of melanoma cells to the brain. In-vitro, CCL17 induced migration and transendothelial migration of melanoma cells. Functionally, human melanoma cells over-expressing CCR4 were more tumorigenic and produced a higher load of spontaneous brain micrometastasis than control cells. Blocking CCR4 with a small molecule CCR4 antagonist in-vivo, reduced the tumorigenicity and micrometastasis formation of melanoma cells. Taken together, these findings implicate CCR4 as a driver of melanoma brain metastasis.

Brain metastasis confers an extremely unfavorable prognosis upon melanoma patients. The mechanisms underlying the homing of metastatic melanoma to the brain and survival of metastatic melanoma cells in the brain are unknown. Tumor cells, including melanoma, use chemokine receptor-ligand axes to home to specific organ sites. To identify chemokine receptors that might be involved in brain-targeted melanoma metastasis, we first established a chemokine receptor profile of cultured melanoma cells (3 cell lines of cutaneous melanoma and 5 cell lines of melanoma brain metastasis). The expression of the membrane-bound chemokine CX3CL1 by these lines was also determined. We show that out of 19 receptors tested, cultured melanoma cells express CCR3, CCR4, CXCR3, CXCR7, CX3CR1 and membrane CX3CL1. Utilizing cells from newly created variants of human melanoma xenografts, we found that the expression of CCR4 was significantly higher in one brain metastatic variant compared to its expression in the corresponding local variant. Local and metastatic variants stimulated with the CCR4 ligand, CCL22, showed a differential AKT phosphorylation pattern. These findings may suggest the involvement of CCR4 in the process of brain metastasis in human melanoma, and that CCR4 may be a novel molecular biomarker for the identification of melanoma cells likely to metastasize to the brain.

CXCL10 was recently shown to exert antimalignancy functions by influencing the tumor microenvironment. Here, we have taken a different approach, investigating the effects of CXCL10 directly on tumor-promoting functions in colorectal carcinoma (CRC) cells. CXCL10 expression was detected in preferred metastatic sites of CRC (liver, lungs, and lymph nodes), and its CXCR3 receptor was expressed by eight CRC cell lines (detected: reverse transcription-PCR and/or flow cytometry). Detailed analysis was done on two cell lines derived from primary CRC tumors (SW480, KM12C) and their metastatic descendents (SW620 and KM12SM). The three known variants of CXCR3 (CXCR3-A, CXCR3-B, and CXCR3-alt) were detected in all four cell lines. CXCR3 expression was also observed on colorectal tumor cells in biopsies of CRC patients (immunohistochemistry). CXCL10 and CXCR3 expression were potently induced in CRC cells by Interferon γ and all four CRC cell lines responded to CXCL10 by extracellular signal-regulated kinase 1/2 dephosphorylation. The chemokine did not affect tumor cell growth or angiogenesis-related functions in the tumor cells, such as CXCL8 and vascular endothelial growth factor secretion. Importantly, CXCL10 significantly up-regulated invasion-related properties in CRC cells: It promoted matrix metalloproteinase 9 expression and induced CRC cell migration. Of note, CXCL10-induced migration was detected only in the two metastatic cells and not in their primary counterparts. Also, CXCL10 promoted the adhesion of metastatic cells to laminin. These results suggest that CXCL10 can be exploited by CRC cells toward their progression, thus possibly antagonizing the antimalignancy effects of the chemokine on the tumor microenvironment. Therefore, care should be taken when considering CXCL10 as a therapeutic antitumor modality for CRC treatment. [Cancer Res 2007;67(7):3396–405]

Soluble pulmonary factors have been reported to be capable of inhibiting the viability of cancer cells that metastasize to the lung, but the molecular identity was obscure. Here we report the isolation and characterization of the beta subunit of hemoglobin as a lung-derived antimetastatic factor. Peptide mapping in the beta subunit of human hemoglobin (HBB) defined a short C-terminal region (termed Metox) as responsible for activity. In tissue culture, both HBB and murine HBB2 mediated growth arrest and apoptosis of lung-metastasizing neuroblastoma cells, along with a variety of other human cancer cell lines. Metox acted similarly and its administration in human tumor xenograft models limited the development of adrenal neuroblastoma tumors as well as spontaneous lung and bone marrow metastases. Expression studies in mice indicated that HBB2 is produced by alveolar epithelial and endothelial cells and is upregulated in mice bearing undetectable metastasis. Our work suggested a novel function for HBB as a theranostic molecule: an innate antimetastasis factor with potential utility as an anticancer drug and a biomarker signaling the presence of clinically undetectable metastasis. Cancer Res; 77(1); 14–26. ©2016 AACR.

V600E being the most common mutation in BRAF, leads to constitutive activation of the MAPK signaling pathway. The majority of V600E BRAF positive melanoma patients treated with the BRAF inhibitor vemurafenib showed initial good clinical responses but relapsed due to acquired resistance to the drug. The aim of the present study was to identify possible biomarkers associated with the emergence of drug resistant melanoma cells. To this end we analyzed the differential gene expression of vemurafenib-sensitive and vemurafenib resistant brain and lung metastasizing melanoma cells. The major finding of this study is that the in vitro induction of vemurafenib resistance in melanoma cells is associated with an increased malignancy phenotype of these cells. Resistant cells expressed higher levels of genes coding for cancer stem cell markers (JARID1B, CD271 and Fibronectin) as well as genes involved in drug resistance (ABCG2), cell invasion and promotion of metastasis (MMP-1 and MMP-2). We also showed that drug-resistant melanoma cells adhere better to and transmigrate more efficiently through lung endothelial cells than drug-sensitive cells. The former cells also alter their microenvironment in a different manner from that of drug-sensitive cells. Biomarkers and molecular mechanisms associated with drug resistance may serve as targets for therapy of drug-resistant cancer.

Ly-6E.1 is highly expressed in murine tumor cells with a high malignancy phenotype and may serve as a marker for such a phenotype. In this study, we examined the effects of various growth conditions and stress on the expression levels of Ly-6E.1 by tumor cells. Previous preliminary results have shown that murine DA3 mammary tumor cells expressing high levels of Ly-6E.1 (Ly-6(hi)) are more highly tumorigenic than the same tumor cells expressing low levels of this membrane protein (Ly-6(lo)). In this study, we demonstrate that mice bearing Ly-6(hi) DA3 tumors have a significantly higher burden of spontaneous pulmonary metastasis than mice bearing Ly-6(lo) DA3 tumors. Furthermore, the survival time of the former mice was significantly shorter than that of the latter ones. We further show that certain other members of the Ly-6 gene family such as Ly-6C.1 and Ly-6G.1 are coregulated with Ly-6E.1. This was shown to occur with respect to both DA3 cells as well as A3 tumor cells which are of fibroblast origin. However, these 2 cells differ with respect to regulation of Sca-2 (TSA1, another member of the Ly-6 family) expression on these cells. Levels of Sca-2 on A3 cells appear to be coregulated with Ly-6E.1 (i.e., Ly-6(hi) A3 cells express high levels of Sca-2 and Ly-6(lo) A3 cells express low levels of Sca-2). These 2 Ly-6 proteins were, however, not coregulated on DA3 cells. Both Ly-6(hi) as well as Ly-6(lo) DA3 cells express equal levels of Sca-2. Levels of Thy-1, another glycosylphosphatidylinositol (GPI)-anchored protein expressed by A3 tumor cells, were equally expressed by both Ly-6(hi) and Ly-6(lo) A3 tumor cells. Levels of Ly-6 (but not those of CD44) on A3 tumor cells were upregulated on cells from dense cultures but were not influenced by the position of the cells in the cell cycle. Stress conditions such as serum starvation or heat shock upregulated the expression of Ly-6 by the 2 types of tumor cells but did not induce apoptosis in these cells. The kinetics of the stress-dependent upregulation of Ly-6 expression differed, however, between the epithelial and fibroblastic tumor cells.

Brain metastases occur frequently in melanoma patients with advanced disease whereby the prognosis is dismal. The underlying mechanisms of melanoma brain metastasis development are not well understood. Identification of molecular determinants regulating melanoma brain metastasis would advance the development of prevention and therapy strategies for this disease. Gene expression profiles of cutaneous and brain-metastasizing melanoma variants from three xenograft tumor models established in our laboratory revealed that expression of tight junction component CLDN1 was lower in the brain-metastasizing variants than in cutaneous variants from the same melanoma. The objective of our study was to determine the significance of CLDN1 downregulation/loss in metastatic melanoma and its role in melanoma brain metastasis. An immunohistochemical analysis of human cells of the melanocyte lineage indicated a significant CLDN1 downregulation in metastatic melanomas. Transduction of melanoma brain metastatic cells expressing low levels of CLDN1 with a CLDN1 retrovirus suppressed their metastatic phenotype. CLDN1-overexpressing melanoma cells expressed a lower ability to migrate and adhere to extracellular matrix, reduced tumor aggressiveness in nude mice and, most importantly, eliminated the formation of micrometastases in the brain. In sharp contrast, the ability of the CLDN1-overexpressing cells to form lung micrometastases was not impaired. CLDN1-mediated interactions between these cells and brain endothelial cells constitute the mechanism underlying these results. Taken together, we demonstrated that downregulation or loss of CLDN1 supports the formation of melanoma brain metastasis, and that CLDN1 expression could be a useful prognostic predictor for melanoma patients with a high risk of brain metastasis.

Recent data suggest that the mechanisms determining whether a tumor cell reaching a secondary organ will enter a dormant state, progress toward metastasis, or go through apoptosis are regulated by the microenvironment of the distant organ. In neuroblastoma, 60-70% of children with high-risk disease will ultimately experience relapse due to the presence of micrometastases. The main goal of this study is to evaluate the role of the lung microenvironment in determining the fate of neuroblastoma lung metastases and micrometastases. Utilizing an orthotopic mouse model for human neuroblastoma metastasis, we were able to generate two neuroblastoma cell populations-lung micrometastatic (MicroNB) cells and lung macrometastatic (MacroNB) cells. These two types of cells share the same genetic background, invade the same distant organ, but differ in their ability to create metastasis in the lungs. We hypothesize that factors present in the lung microenvironment inhibit the propagation of MicroNB cells preventing them from forming overt lung metastasis. This study indeed shows that lung-derived factors significantly reduce the viability of MicroNB cells by up regulating the expression of pro-apoptotic genes, inducing cell cycle arrest and decreasing ERK and FAK phosphorylation. Lung-derived factors affected various additional progression-linked cellular characteristics of neuroblastoma cells, such as the expression of stem-cell markers, morphology, and migratory capacity. An insight into the microenvironmental effects governing neuroblastoma recurrence and progression would be of pivotal importance as they could have a therapeutic potential for the treatment of neuroblastoma residual disease.

We have previously shown that Fc gamma receptor type II B1 (Fc(gamma)RIIB1), when expressed on non-lymphoid tumor cells, significantly enhanced their tumorigenic phenotype. This study elucidates the role of the intracellular domain of Fc(gamma)RIIB1 in the enhancement of the malignant phenotype of polyoma-transformed 3T3 cells. We investigated the tumorigenic potential conferred by different variants of the receptor: Fc(gamma)RIIB1, a full-length receptor (B1) whose intracellular region is encoded by exons 8, 9 and 10; Fc(gamma)RIIB2, a spliced variant (B2) whose cytoplasmic domain comprises exons 9 and 10 and lacks exon 8; and Fc(gamma)RIIB1-CT53, a deleted mutant whose cytoplasmic domain contains the fragment encoded by exon 8 alone. We have investigated various properties of cells transfected with each of the above variants: tumorigenicity in syngeneic mice, formation of colonies in soft agar, growth rate, production of soluble receptor and capping of the ligand-bound receptor. Results show that while the presence of exon 8 did not enhance growth rate in vitro or production of soluble Fc(gamma)R, it did enhance the tumorigenic phenotype of transfected cells (both in vivo and in vitro growth in soft agar). B1-expressing cells exhibited a significantly higher tumorigenic phenotype than B2 cells. The presence of exon 8 alone (CT53 mutant) conferred the transfected cells a higher tumorigenic phenotype than Fc(gamma)R-negative control cells but lower than intact B1 or B2 cells, indicating that the presence of B1-specific exon 8 is not sufficient but that the presence of an intact B1 intracellular domain is essential, for conferring the high tumorigenicity phenotype upon cells. We conclude that the capping, following ligand binding contributed by exon 8, and the function contributed by the specific localization of exons 9 and 10 in B1 cells may determine their malignant phenotype.

The working hypothesis of this study is that the interactions between the brain microenvironment and melanoma cells determine metastasis formation at this organ site. The aim of the study was to evaluate the contribution of such interactions to the formation of brain metastasis in nude mice xenografted with human melanoma cells. An insight into these interactions is an essential prerequisite for the development of effective targeted therapy for melanoma brain metastasis. We assessed the effects of soluble factors present in supernatants of short-term cultures of normal mouse brain (referred here after as brain-derived soluble factors) on several characteristics linked to melanoma brain metastasis. It was found that brain-derived soluble factors affect differentially cutaneous and brain-metastasizing melanoma cells variants in vitro. Such factors enhanced the viability of cutaneous melanoma cells but caused an S phase arrest followed by apoptosis of brain-metastasizing cells. Brain-derived soluble factors enhanced migration of melanoma cells metastasizing to the brain, but did not affect the migration of the cutaneous variants. Such factors upregulated the expression of the chemokine receptor CCR4 in both cutaneous and brain-metastasizing melanoma cells. It is not unlikely that CCR4 ligands expressed in the brain interact with the CCR4-expressing melanoma cells thereby directing them to the brain. Brain-derived soluble factors enhanced the transmigration, across human brain endothelial cells of cutaneous but not of brain-metastasizing melanoma variants. This activity could promote the capacity of the cutaneous cells to metastasize to the brain.

MOPC-315 plasmacytoma-bearing BALB/c mice were treated with high doses of melphalan, causing a permanent and complete regression of the tumor. In the present study we analyzed plasmacytoma-regressor mice (PRM) 3–6 months after plasma-cytoma regression. A second group of otherwise untreated normal mice was treated with melphalan (M—control group). A third group of mice remained untreated and served as an age-and sex-matched control group. PRM were cachectic and had an increased mortality rate compared to the M and the C control groups. Histopathological examination indicated that the spleen of PRM showed pronounced abnormalities, primarily in the red pulp. These abnormalities consisted of extramedullary hematopoiesis and myeloid-granulocytic hyperplasia. Spleens of M mice showed similar abnormalities but to a much lesser extent. Flow cytometric analysis of cellular surface markers of PRM splenocytes indicated a high number of large MAC-1-and GR-1-positive cells compared to splenocytes of M or C controls. These large cells also expressed For receptors (FcτRII), stained positively with non-specific esterase and adhered to plastic dishes; a certain percentage expressed MAC-2 and MAC-3 antigens. A quantitative suppression of CD4+ T cells and of B cells was also shown. Circulating levels of TNF were higher in PRM than in M or C mice. The capacity of splenocytes from PRM to secrete factors that stimulated CFU-GM colony formation in soft agar by bone-marrow cells from normal mice was significantly up-regulated compared to that of splenocytes from M or C mice. PRM-derived splenocytes also reacted significantly better to rIL-3 and rGM-CSF than splenocytes from M or C controls. We conclude that the splenic myeloproliferation in PRM was not caused by melphalan chemotherapy alone and is an abnormality related to the primary tumor, possibly in conjunction with chemotherapy. No evidence of a secondary overt malignancy was obtained.

Brain metastasis occurs frequently in melanoma patients with advanced disease whereby the prognosis is dismal. The underlying mechanisms of melanoma brain metastasis development are not well understood. We generated a reproducible melanoma brain metastasis model, consisting of brain-metastasizing variants and local, subdermal variants that originate from the same melanomas thus sharing a common genetic background. The brain-metastasizing variants were obtained by intracardiac inoculation. Brain metastasis variants when inoculated subdermally yielded spontaneous brain dormant micrometastasis. Cultured cells from the spontaneous brain micrometastasis grew very well in vitro and generated subdermal tumors after an orthotopic inoculation. Expression analysis assays indicated that the brain metastasis and micrometastasis cells expressed higher levels of angiopoietin-like 4, prostaglandin-synthesizing enzyme cyclooxygenase-2, matrix metalloproteinase-1 and preferentially expressed antigen in melanoma and lower levels of claudin-1 and cysteine-rich protein 61 than the corresponding cutaneous variants. The reproducible models of human melanoma metastasizing experimentally and spontaneously to the brain will facilitate the identification of novel biomarkers and targets for therapy and contribute to the deciphering of mechanisms underlying melanoma metastasis.

The mechanism by which dormant tumor cells can begin growing after long periods of inactivity and accelerate disease recurrence is poorly understood. The present study characterizes dormant neuroblastoma (NB) cells, as well as metastatic cells, which reside in the same organ microenvironment. A xenograft model of human NB consisting of variants that generate nonmetastatic local tumors in the orthotopic inoculation site and variants that generate lung metastatic NB (MetNB) cells was developed in our laboratory. The present study shows that lungs of mice inoculated with nonmetastatic NB variants contain disseminated neuroblastoma (DisNB) human cells. Both DisNB and MetNB variants expressed a similar tumorigenicty phenotype in vivo , whereas the MetNB variants produced a heavy metastatic load and the DisNB variants produced no or little metastasis. A comparative in vitro characterization of MetNB and DisNB cells revealed similarities and differences. DisNB, but not MetNB cells, expressed the minimal residual disease markers PHOX2B and TH. MetNB cells demonstrated higher migratory capacity, an elevated matrix metalloproteinase (MMP) secretion, and a higher constitutive phosphorylation of extracellular signal-regulated kinase (ERK) than DisNB cells. We suggest that characteristics common to both MetNB and DisNB cells were acquired relatively early in the metastatic process and the characteristics that differ between these variants were acquired later. We hypothesize that the DisNB cells are metastasis precursors, which may progress toward metastasis under certain microenvironmental conditions.

Cellular subclones of high and low tumorigenicity obtained from a mouse c-Ha-ras-transformed clone, were examined for their sensitivity to tumor-necrosis-factor (TNF)-mediated cytotoxicity. Cells of the highly tumorigenic subclones showed a significantly enhanced resistance to the cytotoxic effect of TNF plus cyclohexamide (CHI) as compared to cells of the low-tumorigenic subclones. The enhanced resistance to TNF+CHI was not due to a lower expression of TNF receptors on the cells. The c-Ha-ras-transfected cells were transformed and maintained in culture only (C cells). In vivo passage of cells of the initially low-tumorigenic c-Ha-ras subclones through the mouse significantly enhanced the tumorigenic potential of these CTC cells (culture/tumor/culture). In correlation with their enhanced tumorigenicity, the CTC cells were highly resistant to TNF-mediated cytotoxicity as compared to C cells of the same subclone. Furthermore, the involvement of TNF in determining the tumorigenic phenotype of the c-Ha-ras-transformed cells was demonstrated in a more direct manner. Cells of a c-Ha-ras-transformed low-tumorigenic, highly TNF-sensitive subclone were selected by repeated cycles of in vitro exposure to TNF alpha. As a result, a stable, highly TNF-resistant population of cells emerged. These TNF-resistant cells caused more tumors in mice as compared to their original TNF-sensitive cells. These results show that the resistance to the cytotoxic effect of TNF plus cyclohexamide may be involved, at least partially, in the tumorigenic potential of c-Ha-ras-transformed cells and suggest a possible role for TNF in the enhancement of the tumorigenic potential of these cells in mice.

Cloned BALB/c 3T3 cells transformed in vitro with polyoma virus (PyV) acquired a higher tumorigenicity phenotype after a single in vivo passage. Some of the in vivo passaged cells (CTC cells) exhibited also a higher metastatic phenotype than cells from the same clones that were maintained only in culture (C cells). A phenotypic comparison between CTC and C cells was performed. It was found that most CTC lines exhibited a higher binding to laminin compared to their clonal C cell ancestors. Some CTC cells were less sensitive to the cytotoxic effects of TNF-alpha than the corresponding C cells. CTC cells originating from tumors which appeared after a long latency period (late tumors) tended to express Fc gamma RII while CTC cells originating from tumors which appeared after a short latency period (early tumors) as well as the corresponding C cells tended not to express Fc gamma RII. The expression of a membrane epitope recognized by a monoclonal antibody expressing specificity towards PyV transformed cells, was down-regulated on late tumor cells compared to early tumor cells. Transfection of cloned PyV-transformed BALB/c 3T3 cells with the beta 1Fc gamma RII gene augmented the tumorigenicity and metastatic phenotype of the transfectants compared to control transfectants.

Metastasis is the primary cause of mortality in Neuroblastoma (NB) patients, but the metastatic process in NB is poorly understood. Metastsis is a multistep process that requires the coordinated action of many genes. The identification of genes that promote or suppress tumor metastasis can advance our understanding of this process. In the present study, we utilized a human NB xenograft model comprising local and metastatic NB variants, which was recently developed in our laboratory. We set out to identify molecular correlates of NB metastasis and to determine the clinical relevance of these molecules. We first performed genome-wide expression profiles of metastatic and nonmetastatic NB variants that have an identical genetic background. We found that some of the proteins highly expressed in the metastatic NB variants are localized in the cytoplasm and endoplasmic reticulum. Other proteins are linked to metabolic processes and signaling pathways, thereby supporting the invasive and metastatic state of the cells. Subsequently, we intersected the differentially expressed genes in the human xenografted variants with genes differentially expressed in Stage 1 and Stage 4 primary tumors of NB patients. By using the same gene-expression platform, molecular correlates associated with metastatic progression in primary NB tumors were identified. The resulting smaller gene set was clinically relevant as it discriminated between high- and low-risk NB patients.

The extravasation of tumor cells is a pivotal stage in the formation of hematogenous metastasis. An interaction of selectins expressed on endothelial cells and selectin ligands expressed by tumor cells has been implicated to play a role in extravasation. In the present study we used a human-mouse model to prove the hypothesis that the selectin ligand sialyl Lewis-a (sLe-a) is indeed involved in the in vivo extravasation of colorectal carcinoma (CRC) cells. The results indicated that highly metastatic CRC cells expressing high levels of sLe-a extravasate more efficiently than non-metastatic CRC cells expressing low levels of sLe-a. It was also demonstrated that down regulating the expression levels of sLe-a in CRC cells by genetic manipulations, significantly reduced CRC extravasation. Non-specific effects of these manipulations were ruled out. The results of this study indicate that the arrest and adhesion of CRC cells, and possibly of other types of cancer cells as well, to endothelium depend on the expression of the selectin ligand sLe-a by the tumor cells.

Bone marrow stroma cells secrete the chemokine CXCL12 that may support bone marrow metastasis formation by neuroblastoma cells. The present study demonstrates that bone marrow stroma cell lines also secrete CXCL10, a chemokine that was shown in the past to have anti-malignancy functions. A receptor recognized by antibodies against CXCR3 was shown to be expressed by six neuroblastoma cell lines. Further detailed analysis was performed on the NUB6 and SK-NMC neuroblastoma cells, showing that CXCL10 induced potent Erk phosphorylation in a G(alpha)i-dependent manner. The role of a CXCR3-like receptor in Erk phosphorylation was substantiated by the ability of CXCL11, another potent CXCR3 ligand, to induce Erk phosphorylation in the NUB6 and SK-NMC cells. Further characterization of CXCL10 activities indicated that CXCL10 partly inhibited the growth of the NUB6 and SK-NMC cells. Both NUB6 and SK-NMC cells did not migrate to CXCL10, although their migratory machinery was intact, as evidenced by their migration to bone marrow constituents. Altogether, these results suggest that CXCL10 interacts with a CXCR3-like receptor in neuroblastoma cell lines, raising the possibility that following the homing of the tumor cells to the bone marrow (through a CXCL10-independent mechanism), CXCL10 may partly inhibit neuroblastoma cell growth at this site.

The progression of breast cancer is affected by multiple cellular and microenvironmental components. The monocyte chemoattractant MCP-1, IL-6 and matrix metalloproteinases (MMP) were suggested to promote, each on its own, breast cancer progression. We recently demonstrated that the high-tumorigenicity phenotype of the DA3 and CSML murine mammary adenocarcinoma cells is correlated with a high expression of MCP-1, IL-6 and MMP. This raised the possibility that common intrinsic tumor-derived factors regulate the concordant expression of these 3 components. The aim of the present study was to gain insight into the mode by which the secretion of MCP-1, IL-6 and MMP from murine mammary adenocarcinoma cells is regulated. This was investigated in cellular clones established from a highly malignant variant of the DA3 tumor (DA3-high). We also determined the secretion of the antimalignancy chemokine IP-10 from these cells. The results indicate that the secretion levels of IL-6, MMP and IP-10 varied between the clones. In contrast, all the clones secreted uniformly high levels of MCP-1, suggesting that MCP-1 constitutes an important feature of the malignancy phenotype of mammary carcinoma. In most of the clones, elevated levels of 1 of the 3 promalignancy factors did not correlate with a high expression of the other 2 factors and vice versa. These findings indicate that the 3 promalignancy factors are not coregulated by a common intrinsic tumor-derived factor. Rather, these results suggest that the individual capacities of the different clones to secrete these factors are summed up in the high-malignancy DA3 parental tumor population, which secretes relatively high levels of MCP-1, IL-6 and MMP as compared to DA3 cells expressing a low-malignancy phenotype. In contrast to the lack of coordinated intrinsic regulation of MCP-1, IL-6 and MMP, it was found that recombinant TNFα, a product of tumor-associated macrophages contributing to breast cancer progression, upregulated the secretion of MCP-1, IL-6 and MMP from all the clones. These results suggest a key role for this microenvironmental, monocyte-derived cytokine in the coordinated regulation of these 3 molecules. Furthermore, additional results demonstrated that monocytic cell-derived TNFα upregulated MCP-1 secretion from the tumor cells and that MCP-1 in turn promoted the secretion of TNFα from monocytic cells. This may result in a positive feedback loop, whereby the tumor cells and the monocytic cells at tumor site promote each other's ability to express and secrete promalignancy factors. We next attempted to assess the contribution of the promalignancy factors MCP-1, IL-6 and MMP and of the antimalignancy factor IP-10 to mammary adenocarcinoma progression. To this end, a preliminary formula was developed in which the net balance between secretion levels of the promalignancy factors and that of the antimalignancy IP-10 chemokine from different clones was related to their in vivo tumorigenicity profile. This formula suggests that a balance between the secretion levels of these factors plays an important role in determining the malignancy phenotype of mammary carcinomas. In all, our findings demonstrate that the mammary tumor cell population is composed of a heterogeneous assortment of clones whose individual characteristics are averaged in the whole population. The malignancy potential of such tumors is thus determined, inter alia, by a combinatorial effect of several promalignancy and antimalignancy factors secreted from each of the clones comprising these tumors. Our results also suggest that the expression of such factors is determined by several nonmutually exclusive regulatory mechanisms. © 2003 Wiley-Liss, Inc.

Tumor progression is a multistep process in which alterations in the expression of numerous gene products may give rise to highly malignant cellular variants. In the present study, we analyzed the differential expression of several genes in cellular variants of mammary adenocarcinomas with high or low malignancy potential, which originated in a common ancestor. To assess the generality of our findings, high and low malignancy variants were derived from two different mammary adenocarcinoma cell lines, namely DA3 and CSML cells. Of major importance is the fact that the differences between high- and low-malignancy variants observed in one system of mammary adenocarcinoma cells (DA3 cells) were identically reproduced in the other system of mammary adenocarcinoma cells (CSML cells). The high malignancy variants of tumors both DA3-high and CSML-high (previously called CSML-100), expressed higher levels of factors that induce monocyte migration than the low malignancy DA3-low and CSML-low (previously called CSML-0) variants. In addition, it was found that DA3-high and CSML-high cell variants expressed higher levels of monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) and matrix metalloproteinases (MMPs) than the low malignancy variants (DA3-low and CSML-low). These results suggest that MCP-1, IL-6 and MMPs potentially contribute to mammary adenocarcinoma progression and that their expression is regulated by a common pathway. The expression of MCP-1, IL-6 and MMPs in both DA3-high and CSML-high cells was up-regulated by tumor necrosis factor alpha (TNFalpha). The fact that TNFalpha exerted similar effects on the expression of these three factors in both cell systems raises the possibility of a coordinated co-regulation of tumor-promoting factors.

The homing of hemopoietic stem cells to the bone marrow is mediated by specific interactions occurring between CXCR4, which is expressed on hemopoietic stem cells, and its ligand, stromal cell-derived factor-1 (SDF-1), a CXC chemokine secreted by bone marrow stromal cells. In the present study we evaluated the possibility that neuroblastoma cells use a mechanism similar to that used by hemopoietic stem cells to home to the bone marrow and adhere to bone marrow stromal cells. Our study suggests that CXCR4 expression may be a general characteristic of neuroblastoma cells. SH-SY5Y neuroblastoma cells express not only CXCR4, but also its ligand, SDF-1. CXCR4 expression on SH-SY5Y neuroblastoma cells is tightly regulated by tumor cell-derived SDF-1, as demonstrated by the ability of neutralizing Abs against human SDF-1α to up-regulate CXCR4 expression on the tumor cells. The reduction in CXCR4 expression following short term exposure to recombinant human SDF-1α can be recovered as a result of de novo receptor synthesis. Recombinant human SDF-1α induces the migration of CXCR4-expressing SH-SY5Y neuroblastoma cells in CXCR4- and heterotrimeric G protein-dependent manners. Furthermore, SH-SY5Y cells interact at multiple levels with bone marrow components, as evidenced by the fact that bone marrow-derived constituents promote SH-SY5Y cell migration, adhesion to bone marrow stromal cells, and proliferation. These results suggest that SH-SY5Y neuroblastoma cells are equipped with adequate machinery to support their homing to the bone marrow. Therefore, the ability of neuroblastoma tumors to preferentially form metastases in the bone marrow may be influenced by a set of complex CXCR4-SDF-1 interactions.

The search for mechanisms that regulate tumor progression has motivated the authors' laboratory to establish a unique murine model system, consisting of two lines of DA3 mammary adenocarcinoma cells that were derived originally from a common ancestor but differed in their malignant potential. Studies indicated that the highly malignant phenotype manifested by one of the cell lines (termed Ly-6hi DA3 cells) was associated with high expression of the Ly-6E.1 antigen. To characterize the mechanisms controlling the high malignancy phenotype expressed by Ly-6hi DA3 cells, the study was focussed on the potential contribution of tumor-derived factors to the high malignancy phenotype expressed by these cells. To this end, the expression of CC chemokines, major chemoattractants of monocytes and T cells, by the highly malignant Ly-6hi DA3 cells as compared to the low malignancy Ly-6lo DA3 cells was evaluated. The results indicate that the highly malignant cells express higher levels of factors that induce monocyte migration than the low malignancy cells. Two CC chemokines were shown to be highly produced by Ly-6hi DA3 cells, MIP-1alpha and MCP-1, of which only the latter was shown to contribute to the high migratory activity expressed by the high malignancy Ly-6hi DA3 cells. Since MCP-1 may attract monocytes to tumor sites, these findings suggest that monocyte-derived mediators, such as growth factors or angiogenic cytokines, have pro-malignancy effects that contribute to the high malignancy phenotype expressed by Ly-6hi DA3 cells.

Angiogenic and poorly angiogenic tumor variants were obtained by an intraperitoneal inoculation of cells from clones of polyoma-virus transformed BALB c 3T3 cells into syngeneic mice. The angiogenic tumor cells expressed a higher tumorigenicity phenotype and a higher capacity to produce artificial pulmonary metastases than cells from the poorly angiogenic tumors. The former cells expressed also significantly higher levels of the lymphocyte activation protein Ly-6E.1 than the former cells. The two types of cells did not differ in expression levels of CD44 and of a polyoma-virus specific membrane antigen. These results raise the possibility that the angiogenic phenotype is coregulated with Ly-6. The effect on Ly-6 expression of signal transduction through TNF receptors, functioning as pivotal regulators of angiogenesis was therefore studied. It was found that TNFα and more so antibodies against Fas down-regulate expression levels of Ly-6. This down-regulation seemed to be selective as expression levels of CD44 were not affected by this treatment.

Murine Ly-6 is a molecule expressed by various cells, including several types of hematopoietic cells such as pluripotent stem cells, and activated T cells. Ly-6 is also expressed on tumor cells originating from a variety of tissues. Preliminary observations suggested that the expression of Ly-6A/E is up-regulated on highly tumorigenic variants of polyoma-virus(PyV)-transformed BALB/c 3T3 cells as compared with weakly tumorigenic variants. On the basis of these observations, we sorted PyV-transformed A3C cells or DA3 mammary adenocarcinoma cells into stable sub-populations expressing high or low levels of membrane or mRNA Ly-6A/E. In vivo studies indicated that the high-Ly-6A/E-expressing cells in both tumor systems expressed a considerably more malignant phenotype (higher efficiency in local tumor production as well as in lung colonization) than low-Ly-6A/E expressors. Since the high-Ly-6A/E expressors did not exhibit any growth advantage in vitro over low Ly-6A/E expressors, we concluded that interactions of the former cells with micro-environmental factors operating in vivo (e.g., Ly-6A/E ligands) conferred upon these cells a highly malignant phenotype. Apart from the difference in Ly-6A/E expression, no other phenotypic characteristics distinguished highly from weakly malignant tumor cells. Similarly to T cells, where antibodies to Ly-6 transduce (or co-transduce) a proliferative signal, antibodies to Ly-6A/E were found to transduce a mitogenic signal to high-Ly-6A/E-expressing tumor cells but not to low-Ly-6A/E expressors. Taken together, these results show that Ly-6A/E expression is directly or indirectly associated in vivo with a highly malignant phenotype of 2 types of non-lymphoid murine tumors.

The ectopic expression of Fc gamma RII by PyV transformed 3T3 cells derived from tumors of long latency has been established. It was suggested that this expression is one of several changes conferring upon the cells an increased capacity for survival. We found that in one case cells expressing a very high level of Fc gamma RII had also a very high metastatic phenotype as compared to FcR negative cells. Direct evidence that Fc gamma RIIbl functions as a progression factor was provided by transfection experiments. The transfected gene conferred an increased malignancy and invasive phenotype upon PyV or c-Ha-ras transformed cells. In the present study we tested the possibility that Fc gamma RII expressing tumor cells could interfere with the immune system. The following subjects were investigated: 1) The ability of Fc gamma R on the tumor cells to bind the ligand and/or release IBF. 2) The effect of a local accumulation of ligand and/or IBF (assumed to take place in situ in the tumor) on Fc gamma RII expressing T cells. It was found that both tumor-derived receptor positive and beta l transfected PyV transformed cells were capable of binding aggregated mouse IgG. The binding of bivalent ligand was followed by an increase in membrane Fc gamma RII expression. Also both types of cells were capable of releasing IBF. We then tested the possibility that a local accumulation of IgG within the tumor could effect Fc gamma R expressing T cells. It was found that aggregated mouse IgG (as well as IgGl) could stimulate the proliferation of the T cell hybridoma (T2D4) and other Fc gamma RII expressing T cells. We also found that the expression of beta Fc gamma RII specific mRNA peaked at the logarithmic phase of T2D4 cultures, in parallel with their maximal potential to release IBF. Several pathways for interference with the immune system are suggested.

We analyzed several cellular and molecular properties of BALB/c 3T3 cellular clones transformed in vitro with polyoma virus and exhibiting a high or low tumorigenicity phenotype. We also analyzed the same clones after a single in vivo passage in syngeneic mice. This passage invariably induced and/or selected variants exhibiting a very high tumorigenicity phenotype. BALB/c mice bearing tumors induced by the inoculation of the above cells, regardless of their tumorigenicity phenotype, have a lower number of L3T4 positive splenocytes than appropriate controls. The response to Con-A of spleen cells from such mice was also suppressed. Concomitantly, an increase in Mac-1 positive splenocytes could be measured. In spite of the non-specific suppression of T cells, spleen cells from tumor-bearers showed a specific proliferative response to polyoma antigens. Molecular analysis of polyoma transformed cells showed no differences between the various cells with respect to integration of the polyoma viral genes or with respect to src , myc and fos proto-oncogenes. In vitro maintained cells and in vivo passaged cells seemed to differ, however, in the content of polyoma middle T. Whereas polyoma virus transformed cells maintained only in culture never expressed low affinity receptors for IgG (FcγRII), certain in vivo passaged cells did. This expression could be measured both at the protein and the mRNA level. Those in vivo passaged cells which expressed Fα RII gave tumors following a long latency period. Ongoing experiments will indicate whether or not FcγRII expression is linked to long latency of tumor development.

In this study we report on some lines of ongoing research performed in our laboratory, in relation to the increased expression of FcR on tumor cells, as well as on cells present in the tumor-bearing host, and its possible role in tumor progression. In a previous study we have shown that a Polyoma virus (PyV)-induced anaplastic carcinoma (SEYF-a tumor) contained an FcR-expressing subpopulation of tumorigenic cells. We tested the effect of in vivo passaging of FcR-expressing and of non-FcR-expressing sub-populations of SEYF-a tumor cells on the expression of FcR, as revealed by the ability of these cells to bind the 2.4G2 monoclonal antibody, which is directed against mouse Fc gamma 2b/gamma 1R. It was found that upon in vivo passaging these two sub-populations became practically identical in their ability to bind anti-Fc gamma R antibody. On the other hand, in vitro passaging of FcR-expressing SEYF-a cells resulted in a gradual decrease in the expression of Fc gamma R. These results, indicating that the expression of Fc gamma R on tumor cells, per se, is dependent on a factor present in the in vivo environment were confirmed using 3T3 cells transformed in vitro by PyV (C) and forming tumors at first injection to mice (CTC). C cultures of various clones did not express Fc gamma R, while CTC cultures (cultures from tumors) became positive. We also detected an increase in the level of a soluble form of Fc gamma 2b/gamma 1R in the circulation of mice bearing PyV induced tumors. This increase paralleled the appearance of palpable tumors. A similar pattern of increase was observed in mice inoculated with the c-H-ras transformed tumorigenic clone 8/F/5, but not in mice inoculated with non-tumorigenic 3T3 cells. Data published by us show that metastatic breast cancer patients had significantly elevated Fc gamma R levels on their peripheral blood mononuclear cells (PBMC). Experiments presented here indicate a direct correlation between increased Fc gamma R levels on PBMC and tumor mass in colon, ovary and lung metastatic carcinoma patients. The possibility that malignantly transformed cells have the potential to cause proliferation of Fc gamma R expressing T cells was tested. It was found that extract derived from r-H-ras transformed 3T3 cells triggers the proliferation of a T cell hybridoma expressing Fc gamma R.

The frequency of rosette-forming cells (RFC) in spleens of mice immunized with sheep red blood cells (SRBC) was significantly reduced by pretreatment with preparations obtained from tumor extracts. This effect was not produced by preparations obtained by similar methods from normal tissue extracts. Significant suppression of RFC frequency was also found in spleens of mice immunized with SRBC at late stages of tumor growth, as compared to that in immunized controls and in mice bearing tumors at early stages. The results suggest that at late stages, a tumor cell component is released by tumor cells which may be similar to the nondialyzable immune suppressive factor(s) separated by us from tumor extracts, factors found in ascites fluid of tumor-bearing mice and those known to be released from tumor cells in vitro.

Murine B16 melanoma cells and metastasis variants of this tumor are resistant to NK activity mediated by normal splenocytes. The B16 cells are, however, sensitive to splenocyte-mediated cytostasis. Cytostasis was measured by a [125I]UDR incorporation-inhibition [(125I]UDR I-I) assay. The main characteristics of the [125I[UDR I-I assay and of the cells mediating it are as follows: The activity is mediated by splenocytes but not by thymocytes, it is not syngeneically restricted and it does not decrease with age. The presence of effector cells is required as splenocyte supernatants or supernatants of effector-target cell mixtures do not cause [125I]UDR I-I. The activity is probably mediated by at least 2 populations of non-phagocytic splenocytes. The first population adheres to plastic surfaces and to Sephadex G-10 columns while the other does not. The sensitivity to [125I]UDR I-I of the high metastasis B16 variant was similar to that of the low metastasis variant.

Using the polyoma virus-induced ascitic SEYF-a tumor, we evaluated isopycnic and velocity sedimentation gradients of Percoll as methods for separating tumor-seeking lymphocytes and tumor cells. It was established that the velocity sedimentation method is suitable for separation of small lymphocytes lodging within the SEYF-a tumor. This was confirmed by a serological analysis of the separated SEYF-a cell population. The results of this study strongly support our previously reported data demonstrating the in vivo coating of the tumor cells proper with potentially cytotoxic antibodies.

Three types of natural immune responses against malignant cells were studied in vitro: Cytotoxicity mediated by splenic NK cells; cytostasis mediated by splenocytes and binding of naturally occurring antibodies to various tumour targets. These responses were studied in untreated 3 and 12 month old mice and in mice of both age groups inoculated with B16 melanoma cells. The results showed that in normal mice NK activity decreases with age, cytostatic activity remains unchanged and the titre of natural antibodies increases. Twelve-month old mice were shown to be appreciably more resistant than 3 month old mice to the development of tumours from subthreshold numbers of B16 tumour cells. In mice injected with threshold amounts of the B16 tumour, there was no change in any of the responses in the tumour-free period, but there was a decrease in NK activity and an increase in cytostatic activity when a large tumour mass developed. An increase in the titre of natural antibodies in young mice injected with the tumour was also seen. The correlation between these changes and tumour appearance and development is discussed.

Syngeneic antisera raised against three polyoma tumors, SEYF-a SESO and SEWE, of strain ABY, A and ASW origin, respectively, contain antibodies directed against multiple specificities. One specificity is cross-reactive for the three polyoma tumors, but appears to be absent from a large variety of other tumors tested. Other, more "public" antigens are shared with a variety of other tumors of viral or non-viral origin. Five different public specificities of this type have been demonstrated and to some extent defined. Since the SEWE ascites tumor, with the shortest passage history, is much less liable to induce antibodies against the public specificities than the long-passaged SEYF-a and SESO, it is likely that the corresponding antigenic determinants arise as a result of passenger virus pick-up during continued passage, or are due to secondary cytogenetic changes.

In this study we tested whether or not a correlation exists between the immunochemical character, the biological activity and the binding patterns of IgG prepared from low pH eluates of tumor cells harvested at various stages of tumor growth. The experiments were carried out using SEYF-a cells, a polyoma virus-induced ascites tumor, propagated in syngeneic A.BY mice. It was found that IgG eluted from tumor cells consisted mainly of IgG2a molecules. The specific cytotoxic activity of these IgG preparations was highest in eluates from 11–15-day-old tumor cells, and it decreased with propagation time of the tumor. This indicated the fixation onto old cells either of IgG2a molecules with no cytotoxic activity, or of molecules with the capacity to inhibit such an activity. Simultaneously the binding patterns of these IgG preparations also changed with propagation time of the tumors. IgG molecules eluted from SEYF-a cells 11–15 days after inoculation of the tumor were homogeneous with respect to their binding constant to the corresponding tumor cells. Thus, a correlation was indicated between the exclusive presence of antibodies with a high-binding constant and a high-specific cytotoxic activity. On the other hand, IgG molecules eluted from SEYF-a cells 26–30 or 38–42 days after inoculation of the tumor were heterogeneous with respect to their binding patterns to tumor cells. Such preparations contain IgG molecules with high-binding constants as well as those with low-binding constants. IgG preparations from serum or from ascitic fluid of tumor-bearing mice demonstrated at all stages of tumor propagation heterogeneity with respect to their binding pattern to the cells and low levels of cytotoxic activity compared with IgG from tumor eluates.

Eight different syngeneic murine ascites tumor preparations were tested for their ability to lyse antibody-coated chicken erythrocytes in an assay for antibody-dependent cell-mediated cytotoxicity (ADCC)1 . All of the tumor preparations demonstrated significant ADCC effector activity, even at effector-cell:target-cell ratios of less than 1:1. In contrast, none of eight in vitro murine tumor-cell lines showed any consistent ability to function as ADCC effector cells. Several cell fractionation experiments were performed to ascertain the nature of the effector cells in the ascites tumors. Adherent cells were removed from tumor preparations by passage of cells over columns of Degalan plastic beads and 19S Fc-receptor-bearing cells were removed by sedimentation of EA rosette-forming cells. Although each of these procedures depleted only a minority of the total cells, all the ADCC activity was simultaneously removed in each case. Depletion of phagocytic cells by treatment of tumor preparations with carbonyl iron and magnetism only slightly diminished the ADCC activity. These results indicate that the ADCC activity associated with ascites tumors is due to effector cells most probably of host origin, but possibly representing an atypical sub-population of tumor cells. The characteristics of the effector cell closely parallel those defined for the major nonphagocytic ADCC effector cell in normal mouse spleen and peritoneal exudate.

Several low pH buffers were used in order to dissociate radioiodinated antibodies from sensitized living tumor cells. Three buffers were further tested for their dissociation abilities under different conditions of time and temperature, and for their influence on the eluted antibodies. The cytotoxicity mediated by these low pH buffers was also determined by viability assays. Optimal results were obtained with 0.1 M citrate buffer at pH 3.5.

The IgG fraction from rabbit antisera against a mouse plasmacytoma (RaPCT-IgG) was incubated, under acidic conditions, with lysosomal extracts (LE) derived from the corresponding tumors. The LE-treated IgG demonstrated a significant decrease in its complement-dependent cytotoxic potential when compared to RaPCT-IgG incubated without LE under the same conditions (control IgG). Ammonium sulfate (AS) precipitation of the control IgG and the LE-treated IgG preparations at 40% saturation of AS revealed that some of the IgG lost its precipitability at this AS concentration after LE treatment, but could be precipitated at 60% or 70% saturation. The LE-treated IgG and control IgG fractions that precipitated by 40% saturated AS showed similar gel filtration profiles. The LE-treated IgG fraction precipitating at 70% saturation of AS (but not at 40%) showed a different gel filtration profile. This fraction was separated into several subfractions, some with an apparent lower molecular weight. Some of these subfractions which still contained antigenic IgG lacked any expression of antigenic Fc, and were not cytotoxic to tumor cells. They could, however, rebind to tumor cells and block the complement-dependent cytotoxic activity of native RaPCT-IgG. Lysosomal enzymes of tumor origin seem, therefore, to convert cytotoxic antitumor antibodies into molecules devoid of Fc but still retaining their antigen binding activity. Such an activity, if occurring in vivo might enhance tumor growth.

Previous results obtained in our laboratory suggested that natural antibodies reactive with L5178Y lymphoma cells play a role in the induction of lung tumors by the chemical carcinogen urethane. In order to characterize some of the naturally-occurring L5178Y reactive antibodies we prepared hybridomas that secreted natural monoclonal IgM antibodies reactive with L5178Y lymphoma cells. In the present study we characterized some of these antibodies and provided further proof as to their role in urethane carcinogenesis. One hybridoma secreted a cytotoxic antibody that reacted only with mouse lymphoma cell lines. Other non-cytotoxic monoclonal L5178Y-reactive antibodies showed various degrees of cross-reactivity with syngeneic, allogeneic and xenogeneic cells of normal or malignant origin. One of these antibodies reacted much better with activated T cells than with resting ones. Four groups of mice were treated with urethane. Three groups were injected twice a week during 5 months with different IgM preparations of natural monoclonal antibodies. The mice in the fourth group were not treated with IgM and served as controls. Five months after the urethane treatment the mice were sacrificed and the number of tumor foci in the lungs of each mouse was determined. The results show that the group treated with the cytotoxic monoclonal antibody 1.80 had a significant decrease, while the group treated with the IgM myeloma protein 104E had a significant increase in the number of tumor foci compared to urethane-treated mice that did not receive any IgM treatment. The tumor incidence in the group treated with the 104E IgM was significantly higher than that in the control group.

A spontaneous lymphoma (141) producing monoclonal IgM is established in NZB/NZW F1 (B/W) mice who spontaneously develop an autoimmune disease. Idiotypic determinants of 141 IgM are present on the lymphoma cell surface as shown by indirect immunofluorescence and specific cytotoxicity with rabbit anti-idiotypic antiserum. Fluorescence and cytotoxicity are inhibited by 141 IgM but not by 104E IgM, a monoclonal IgM produced by a BALB/c plasmacytoma. Immunization of B/W mice with 141 IgM before transplantation of lymphoma 141 confers protective immunity. No such protection occurs after immunization with 104E IgM or other unrelated proteins. Protected mice contain spleen cells cytotoxic for 141 lymphoma cells. This cytotoxicity is blocked by incubation of spleen cells with 141 IgM but not with 104E IgM. Moreover, splenic lymphocytes from protected mice are stimulated to synthesize DNA by 141 IgM but not by 104E IgM. These results suggest that specific cellular immune responses to idiotypic determinants may participate in the observed protection against challenge with the corresponding B-cell tumor.

We have studied the ability of various antisera to induce resistance to complement-dependent lysis (CdL) (lysostripping) of surface antigens of B and T lymphoma cells. Alloantisera, xenoantisera, as well as anti-Rauscher-leukemia virus (RLV) antisera, were used. Whereas all antisera could induce resistance to CdL mediated by the same serum that served for lysostripping (autolysostrip), only some of the tested antisera could induce resistance to CdL by other antisera (co-lysostrip). In some combinations co-lysostrip was unidirectional, i.e. in a pair of antisera, one induced autolysostrip as well as co-lysostrip, whereas the other antiserum induced autolysostrip only. In other combinations co-lysostrip was reciprocal. These investigations do not support the possibility that viral components and alloantigenic determinants are associated on mouse lymphoma cells.

Different clones of mouse hybridomas, derived from the fusion of unstimulated mouse peritoneal cells with mouse myeloma cells, producing IgM monoclonal antibodies directed against the membrane of bromelain-treated mouse erythrocytes (MRBC(Br)) have been previously established. We have recently shown that one of these hybridomas produce, in ascites, antibodies cross-reacting with phosphorylcholine derivatives (trimethylammonium (TMA) derivatives). In this work the cross-reactivity for TMA derivatives of the monoclonal antibodies produced by 4 anti-MRBC(Br) hybridomas have been studied at the cell level (plaque-forming cells). Phosphorylcholine, choline bromide and p -aminophenyl-trimethylammonium were found to be potent specific inhibitors of plaque formation (anti MRBC(Br)). The hemolytic activities of ascites and tissue culture supernatants were studied and their inhibition by TMA derivatives was determined. Immunoglobulins from ascites purified on TMA immunoadsorbant column were analyzed by two-dimensional gel electrophoresis, their spectrotype was compared to the spectrotype of immunoglobulins from tissue culture supernatants from the same hybridoma radioactively tagged by internal incorporation of [ 14 C]leucine. It could be shown without ambiguity that the PTMA column retained an IgM with the same characteristics as the IgM secreted in vitro.

Monoclonal IgM anti-erythrocyte autoantibody produced by a NZB-derived hybridoma has been found to react with trimethylammonium-containing compounds. Such compounds are able to prevent the lysis of bromelain-treated mouse erythrocytes (BrMRBC) by those autoantibodies. Using a column of insolubilized betaine hydrazide (BH) the monoclonal anti-erythrocyte antibody has been specifically retained. Elution of this antibody was accomplished by 0.15 M choline (Ch).

With the aid of an assay measuring complement dependent cytotoxicity mediated by syngeneic antibodies, we performed a serological analysis of surface antigens of a polyoma-virus-induced murine tumor (SEYF-a). In vivo propagated SEYF-a ascites tumor cells expressed a specific membrane antigen in addition to various other cross-reacting antigens. Among these we could identify at least four separate specificities. Two of these were present on Mu LV-induced lymphoma cells, the first on Moloney-virus-induced YAC cells and the second on Gross-virus-induced GHA cells. The third cross-reacting antigen was detected on EL-4 cells. At least one additional specificity was present on two methylcholanthreneinduced murine sarcomas. Normal syngeneic lymphoid cells were insensitive to cytotoxicity mediated by the anti-tumor antisera. Quantitative and perhaps also qualitative differences between the antigenic expression of in vivo propagated and cultured SEYF-a cells were indicated. These studies show that hyperimmune sera produced in syngeneic mice against transplanted tumors may contain a considerable number of antibody specificities, only some of which are specific for the tumor. Furthermore the results also suggest that polyoma-virus-induced tumors may possess individually distinct antigenic specificities, over and above the known cross-reacting TSTA or TSSA type antigen. Analyse Serologique Des Antigenes Specifiques Et A Reactions Croisees De La Membrane Des Cellules D'une Tumeur Murine Induite Par Le Virus Polyome Nous avons effectue une analyse serologique des antigenes de la surface des cellules d'une tumeur murine induite par le virus polyome (SEYF-a) en mesurant la cytotoxicite des anticorps syngeniques liee au complement. Les cellules de la tumeur ascitique SEYF-a propagees in vitro expriment un antigene membranaire specifique ainsi que divers autres antigenes a reactions croisees. Parmi ces derniers, nous avons pu identifier au moins quatre specificites distinctes. Deux de ces antigenes etaient presents sur des cellules de lymphome induit par le MuLV, le premier sur des cellules YAC induites par le virus de Moloney et le second sur des cellules GHA induites par le virus de Gross. Le troisieme a ete detecte sur les cellules EL-4; il y en avait au moins un quatrieme sur des sarcomes murins induits par le methylcholanthrene. Les cellules lymphoides syngeniques normales sont insensibles a la cytotoxicite mediee par les antiserums antitumoraux. Des differences d'expression antigenique, non seulement quantitatives mais peut-ětre aussi qualitatives, sont apparues entre les cellules SEYF-a propagees in vitro et les měmes cellules en culture. Ces etudes montrent que les serums hyperimmuns produits chez des souris syngeniques contre les tumeurs transplantees peuvent contenir un nombre considerable de specificites dont quelques-unes seulement sont specifiques pour la tumeur. De surcroit, les resultats conduisent a penser que les tumeurs induites par le virus polyome peuvent posseder des specificites antigeniques individuellement uniques en plus des antigenes du type TSTA ou TSSA, dont on connait les reactions croisees.

Alloantisera prepared in inbred mice and directed against H-2 histocompatibility differences or against the thymic antigens θC3H and TL (thymus leukemia) were assayed for their immunosuppressive action when injected in mice against whom the alloantisera were directed. There was a significant depression of the reactions to bovine serum albumin (BSA) and sheep erythrocytes (SRBC) as determined for BSA by passive hemagglutination and by direct hemagglutination of SRBC. Plaque production (measured by the Jerne plaque assay) against SRBC was also inhibited. The mice exhibited leukopenia, and in one experiment there was a significant increase in the number of takes of syngeneic tumor transplants in antiserum-treated mice given a subthreshold inoculum of tumor cells.

The present study was intended to establish whether IgG2-containing eluates, dissociated from carcinogen-induced tumors by a low pH buffer, have tumor-enhancing properties. Threshold amounts of cells from mouse tumor lines originally induced by benzo (a)-pyrene or by methylcholanthrene were mixed and incubated, prior to inoculation, with IgG2-containing eluates, originating either from the corresponding tumors (autologous eluates) or from other syngeneic tumors (isologous eluates). Tumor cells incubated and injected together with autologous eluates gave in some experiments a higher yield of tumors in syngeneic mice than did cells not subjected to this treatment. Some isologous eluates originating from several primary tumors or from syngeneic tumors belonging to a different line from the one tested, were also capable of enhancing tumor growth.