IDH mutations in G2-3 conventional central bone chondrosarcoma: a mono institutional experience.

BACKGROUND: Heterozygous isocitrate dehydrogenase (IDH) mutations occur in about half of conventional central bone chondrosarcomas (CCBC). Aim of this study was to assess the frequency and prognostic impact of IDH mutations in high grade CCBC patients. METHODS: 64 patients with G2 and G3 CCBC were included. DNA extraction, PCR amplification of IDH1/2 exon 4s, and sequencing analysis with Sanger were performed. RESULTS: IDH mutations were detected in 24/54 patients (44%): IDH1 in 18, IDH2 in 4, and both IDH1/2 in 2 patients. The frequency of mutations was 37% in G2 vs. 69% in G3 (p = 0.039), and 100% in three Ollier disease associated chondrosarcoma. 5-year overall survival (OS) at 124 months (range 1-166) was 51%, with no significant difference based on the IDH mutational status: 61% in IDHmut vs. 44% in IDH wild type (IDHwt). The 5-year relapse free survival (RFS) was 33% (95% CI:10-57) for IDHmut vs. 57% (95%CI: 30-77) for IDHwt. Progression free survival (PFS) was 25% (95%CI:1-65) IDHmut vs. 16% (95%CI: 0.7-52) IDHwt. 55% (5/9) of IDHmut G2 became higher grade at the recurrence, as compared with 25% (3/12) of G2 IDHwt. CONCLUSIONS: This study shows a higher frequency of IDH mutations in G3 CCBC as compared with G2. No significant differences in OS, RFS, and PFS by mutational status were detected. After relapse, a higher rate of G3 for IDH mutated CCBC was observed.

Human responses against HER-2-positive cancer cells in human immune system-engrafted mice.

BACKGROUND: Human immune system (HIS)-engrafted mice are new tools to investigate human immune responses. Here, we used HIS mice to study human immune responses against human HER-2-positive cancer cells and their ability to control tumour growth and metastasis. METHODS: BALB/c Rag2(-/-), Il2rg(-/-) mice were engrafted with CD34(+) or CD133(+) human cord blood hematopoietic stem cells (HSC) and vaccinated with human HER-2-positive cancer cells SK-OV-3 combined to human IL-12. RESULTS: Both CD34(+) or CD133(+) human HSC gave long-term engraftment and differentiation, both in peripheral blood and in lymphoid organs, and production of human antibodies. Vaccinated mice produced specific anti-HER-2 human IgG. An s.c. SK-OV-3 challenge was significantly inhibited (but not abolished) in both vaccinated and non-vaccinated HIS mice. Tumours were heavily infiltrated with human and murine cells, mice showed NK cells and production of human interferon-γ, that could contribute to tumour growth inhibition. Vaccinated HIS mice showed significantly inhibited lung metastases when compared with non-vaccinated HIS mice and to non-HIS mice, along with higher levels of tumour-infiltrating human dendritic cells. CONCLUSION: Anti-HER-2 responses were elicited through an adjuvanted allogeneic cancer cell vaccine in HIS mice. Human immune responses elicited in HIS mice effectively inhibited lung metastases.

Combined allogeneic tumor cell vaccination and systemic interleukin 12 prevents mammary carcinogenesis in HER-2/neu transgenic mice.

Transgenic Balb/c mice expressing the transforming rat HER-2/neu oncogene develop early and multifocal mammary carcinomas. Within the first 5 months of life the tissue-specific expression of HER-2/neu causes a progression in all their 10 mammary glands from atypical hyperplasia to invasive carcinoma. It was previously observed that chronic administration of interleukin (IL)-12 increased tumor latency, but every mouse eventually succumbed to multiple carcinomas. A significant improvement in tumor prevention was sought by administering allogeneic mammary carcinoma cells expressing HER-2/neu combined with systemic IL-12. This treatment reduced tumor incidence by 90% and more than doubled mouse lifetime. For the maximum prevention p185(neu) antigen must be expressed by allogeneic cells. IL-12 treatment strongly increased the cell vaccine efficacy. The mammary glands of mice receiving the combined treatment displayed a markedly reduced epithelial cell proliferation, angiogenesis, and HER-2/neu expression, while the few hyperplastic foci were heavily infiltrated by granulocytes, macrophages, and CD8(+) lymphocytes. Specific anti-HER-2/neu antibodies were produced and a nonpolarized activation of CD4(+) and CD8(+) cells secreting IL-4 and interferon (IFN)-gamma were evident. A central role for IFN-gamma in the preventive effect was proven by the lack of efficacy of vaccination in IFN-gamma gene knockout HER-2/neu transgenic Balb/c mice. A possible requirement for IFN-gamma is related to its effect on antibody production, in particular on IgG2a and IgG2b subclasses, that were not induced in IFN-gamma knockout HER-2/neu mice. In conclusion, our data show that an allogeneic HER-2/neu-expressing cell vaccine combined with IL-12 systemic treatment can prevent the onset of genetically determined tumors.

Interleukin 12-mediated prevention of spontaneous mammary adenocarcinomas in two lines of Her-2/neu transgenic mice.

The ability of interleukin (IL)-12 to prevent tumors when administered to individuals with a genetic risk of cancer was studied in two lines of transgenic mice expressing rat HER-2/neu oncogene in the mammary gland. Female BALB/c (H-2(d)) mice carrying the activated HER-2/ neu oncogene show no morphological abnormalities of the mammary gland until 3 wk of age. They then progress through atypical hyperplasia to in situ lobular carcinoma and at 33 wk of age all 10 mammary glands display invasive carcinomas. Adult FVB mice (H-2(q)) carrying the HER-2/neu protooncogene develop mammary carcinomas with a longer latency (38-49 wk) and a lower multiplicity (mean of 2.6 tumors/mice). Treatment with IL-12 (5 daily intraperitoneal injections, 1 wk on, 3 wk off; the first course with 50 ng IL-12/day, the second with 100 ng IL-12/day) begun at 2 wk of age in BALB/c mice and at 21 wk of age in FVB mice markedly delayed tumor onset and reduced tumor multiplicity. Analogous results were obtained in immunocompetent and permanently CD8(+) T lymphocyte-depleted mice. In both transgenic lines, tumor inhibition was associated with mammary infiltration of reactive cells, production of cytokines and inducible nitric oxide synthase, and reduction in microvessel number, in combination with a high degree of hemorrhagic necrosis.

Experimental results and related clinical implications of PET detection of epidermal growth factor receptor (EGFr) in cancer.

The epidermal growth factor receptor (EGFr) is one of the most studied molecules as a target for cancer therapy. Over these last few years, several studies attempting to identify predictive biomarkers of treatment response, such as the receptor status or other molecules related to the downstream signalling pathway, have been conducted. However, from a clinical point of view, the information obtained from ex vivo analyses still has various limitations that may be overcome by the combination with molecular imaging technologies which may provide a noninvasive, global, in vivo evaluation of the molecular tumour background. The aim of this review is to report the preclinical results of all positron emission tomography (PET) tracers synthesized until now for in vivo detection of EGFr in cancer. Two classes of PET compounds have been developed: labelled small molecules such as tyrosine kinase inhibitors and labelled monoclonal antibodies. The in vitro and in vivo results of these PET tracers are very different depending on the chemical properties, positron emission radionuclide, or animal models. As a consequence, various critical questions are still open, and the implications of a translation in the clinical setting for EGFr imaging in cancer patients is discussed.

CD99 isoforms dictate opposite functions in tumour malignancy and metastases by activating or repressing c-Src kinase activity.

CD99 gene encodes two distinct proteins, produced by alternative splicing of CD99 gene transcript. Full-length CD99 isoform (CD99wt) is formed by an extracellular domain, followed by a transmembrane domain and a 36 amino-acid intracytoplasmic domain, which is partially deleted in the truncated, short form (CD99sh). A differential expression of these two CD99 molecules can lead to distinct functional outcomes in lymphocytes. To investigate the functional effects of CD99 molecules on malignancy, forced overexpression of the two CD99 isoforms was induced in osteosarcoma and prostate cancer cells. The two isoforms exhibited opposite functions: the major form dramatically inhibits anchorage-independent growth, anoikis resistance, migration and metastasis, whereas the CD99sh remarkably favours the phenomena. A mechanistic analysis of CD99-transfected osteosarcoma cells points to involvement of c-Src family kinase activity in regulating CD99 functions in malignancy. Ser168 residue of CD99 plays a pivotal role in the reversion of the malignant phenotype. Our findings highlight the involvement of CD99 in crucial processes of cancer malignancy, serving as a curtain raiser for this, so far neglected molecule. In addition, a dualistic role for the two CD99 isoforms was shown in agreement with what was observed for other cell adhesion molecules.

Pathobiological implications of the d16HER2 splice variant for stemness and aggressiveness of HER2-positive breast cancer.

We have previously shown that the d16HER2 splice variant is linked to HER2-positive breast cancer (BC) tumorigenesis, progression and response to Trastuzumab. However, the mechanisms by which d16HER2 contributes to HER2-driven aggressiveness and targeted therapy susceptibility remain uncertain. Here, we report that the d16HER2-positive mammary tumor cell lines MI6 and MI7, derived from spontaneous lesions of d16HER2 transgenic (tg) mice and resembling the aggressive features of primary lesions, are enriched in the expression of Wnt, Notch and epithelial-mesenchymal transition pathways related genes compared with full-length wild-type (WT) HER2-positive cells (WTHER2_1 and WTHER2_2) derived from spontaneous tumors arising in WTHER2 tg mice. MI6 cells exhibited increased resistance to anoikis and significantly higher mammosphere-forming efficiency (MFE) and self-renewal capability than the WTHER2-positive counterpart. Furthermore, d16HER2-positive tumor cells expressed a higher fraction of CD29High/CD24+/SCA1Low cells and displayed greater in vivo tumor engraftment in serial dilution conditions than WTHER2_1 cells. Accordingly, NOTCH inhibitors impaired mammosphere formation only in MI6 cells. A comparative analysis of stemness-related features driven by d16HER2 and WTHER2 in ad hoc engineered human BC cells (MCF7 and T47D) revealed a higher MFE and aldehyde dehydrogenase-positive staining in d16HER2- vs WTHER2-infected cells, sustaining consistent BC-initiating cell enrichment in the human setting. Moreover, marked CD44 expression was found in MCF7_d16 and T47D_d16 cells vs their WTHER2 and Mock counterparts. Clinically, BC cases from two distinct HER2-positive cohorts characterized by high levels of expression of the activated-d16HER2 metagene were significantly enriched in the Notch family and signal transducer genes vs those with low levels of the metagene.

New target antigens for cancer immunoprevention.

Prevention of cancer through the activation of the immune system has been explored in recent years in preclinical systems thanks to the availability of several new transgenic mouse models that closely mimic the natural history of human tumors. The most thoroughly investigated model of cancer immunoprevention is the mammary carcinoma of HER-2/neu transgenic mouse. In this system it has clearly been shown that the activation of immune defences in healthy individuals can effectively prevent the subsequent onset of highly aggressive mammary carcinomas. A complete prevention was obtained using a combination of three signals (the so called "triplex" vaccine) that included the specific antigen (p185, the product of HER-2/neu) and nonspecific signals like allogeneic histocompatibility antigens and interleukin 12. The analysis of protective immune responses in models of cancer immunoprevention revealed some unexpected features, in particular the central role of antibodies in immunoprevention, at variance with conventional immuno-therapy which is firmly based on cytotoxic T cells. In the HER-2/neu system anti-p185 antibodies, in addition to immunological functions leading to tumor cell lysis, inhibit p185 dimerization and induce its internalization, resulting in the inhibition of mitogenic signaling. Most current tumor antigens appear to be unsuitable targets for cancer immunoprevention. An ideal antigen should have a crucial pathogenetic role in tumor growth to avoid the selection of antigen loss variants. Downregulation of major histocompatibility complex (MHC) expression during tumor progression frequently limits antigen recognition by MHC-restricted T cells. Thus an ideal antigen for cancer immunoprevention should be recognized both by T cells and by antibodies. Antibody binding to cell surface oncogenic determinants, in addition to complement- and cell-mediated tumor cell lysis, can block mitogenic signaling and induce internalization, resulting in tumor growth arrest. A search for new tumor antigens should be conducted among molecules that are directly involved in neoplastic transformation and are recognizable by the immune response also in MHC loss variants. Novel tumor antigens fulfilling both conditions will be crucial for the development of cancer immunoprevention and will provide new targets also for cancer immunotherapy.

Clones with different metastatic capacity and variant selection during metastasis: a problematic relationship.

Cells from TS/A, a metastasizing line derived from a spontaneous BALB/c mouse mammary adenocarcinoma, were injected either sc or iv in syngeneic mice, and the resulting lung metastases or lung colonies were briefly cultured in vitro and reinjected in mice by the same route; this procedure was repeated 10 times. All the variants obtained did not show a metastatic capacity higher than the parental cell line. Moreover, they gave a number of metastases significantly lower than that produced by high metastatic clones selected in vitro from TS/A. The number of lung colonies obtained with intravenously selected (COL) variants was significantly higher than that obtained with subcutaneously selected (META) variants, TS/A, or in vitro-selected clones; this was already observable after the first cycle of selection. Both COL and META variants did not show the in vitro growth properties of the in vitro-selected high metastatic clones. In conclusion, both intravenous and subcutaneous selection procedures did not lead to an enrichment in high metastatic populations, even if such populations were present in the parental line and had been cloned in vitro; only the intravenous procedure selected high-colonizing variants.

Immunoprevention of cancer: is the time ripe?

Immunotherapy applied to patients with established tumors rarely leads to an objective response, whereas patients apparently free from disease after conventional treatment and at risk of recurrence are beginning to receive vaccination. New classes of patients or not-yet patients are those with a high genetic or environmental risk of developing cancer. They may draw benefit from a "soft" treatment such as vaccination. This overview discusses the prospects of immune stimulation as a means of cancer prevention by inducing various forms of nonspecific or even specific immunity. Attainment of this goal provides the rationale and motivation for embarking on such a new and potentially rewarding enterprise.

Tamoxifen chemoprevention of a hormone-independent tumor in the proto-neu transgenic mice model.

We evaluated the effect of tamoxifen (TAM) on tumor development in proto-neu transgenic mice that spontaneously develop mammary carcinomas overexpressing the neu protein. These mammary carcinomas are hormone independent because superimposable growth of transplants was observed in females and males. Virgin transgenic mice treated with TAM from 24 weeks of age, ie., when subclinical mammary tumors are already present, showed a slightly accelerated tumor development. In contrast, transgenic mice treated with TAM starting at 12 weeks of age, when subclinical tumors are not yet present, showed a 50% reduction of tumor incidence. Light microscopy analysis of the mammary gland of these mice revealed an apparently normal ductal branching but a complete regression of the acini. In conclusion, TAM can prevent the occurrence of hormone-independent breast carcinoma if given early enough to inhibit normal cells.

Myogenic differentiation of human rhabdomyosarcoma cells induced in vitro by antineoplastic drugs.

The effect of various antineoplastic drugs (1-beta-D-arabinofuranosylcytosine, 5-azacytidine, cisplatin, dactinomycin, epirubicin, vincristine, and the activated metabolite of cyclophosphamide, mafosfamide) on cell differentiation in vitro was investigated using a human alveolar rhabdomyosarcoma cell clone, RMZ-RC2. These cells are able to differentiate spontaneously from small mononuclear proliferating elements to terminal, extremely elongated multinuclear structures resembling myotubes; morphological differentiation is accompanied by the expression of myosins, in particular the embryonic isoform, which was used in this study as a specific marker of myogenic differentiation. The proportion of differentiated myosin-positive cells, which was around 10-15% in control cultures 10-15 days after seeding, was increased by some drug treatments up to 30-40%; the proportion of multinuclear elements was also increased. 1-beta-D-arabinofuranosylcytosine and 5-azacytidine were the most effective drugs, while dactinomycin had no effect; other molecules ranked in between. Since significant increments were usually observed after treatment with drug doses inhibiting cell growth, the kinetic behavior of the absolute number of myosin-positive cells or nuclei was analyzed to assess whether some effects could be due to a negative selection of proliferating, undifferentiated cells. This appeared to be the case for vincristine and epirubicin, while 1-beta-D-arabinofuranosylcytosine, 5-azacytidine, and, to a lesser degree, mafosfamide and cisplatin actually seemed to increase differentiation ability.

Insulin-like growth factor I receptor-mediated circuit in Ewing's sarcoma/peripheral neuroectodermal tumor: a possible therapeutic target.

The disappointingly low survival rate observed in Ewing's sarcoma (ES)/peripheral neuroectodermal tumor (PNET) despite the adoption of aggressive multimodal treatments prompted us to study the existence of autocrine circuits to be used as innovative therapeutic targets. Of the several circuits analyzed, only the insulin-like growth factor receptor (IGF-IR)-mediated loop was found to be constantly present both in cell lines and clinical samples, suggesting a role for this autocrine circuit in the pathogenesis of ES/PNET. The in vitro inhibition of the IGF-IR-mediated circuit by the specific IGF-IR binding antibody alphaIR3 suppressed the growth of ES/PNET cells by decreasing the proliferative rate and increasing apoptosis. alphaIR3 also significantly inhibited the ability of ES/PNET cells to grow in soft agar and to migrate following a chemotactic stimulus. Inactivation of the IGF-IR signaling pathway may therefore be considered as an effective therapeutic modality for patients with ES/PNET.

Expression of transduced carcinoembryonic antigen gene in human rhabdomyosarcoma inhibits metastasis.

Carcinoembryonic antigen (CEA) is a highly glycosylated cell surface glycoprotein belonging to the immunoglobulin superfamily. CEA has been involved in vitro in adhesion mechanisms, but little is known about the function of this glycoprotein in vivo in normal tissue differentiation and malignancy. With regard to the relationship between CEA expression and tissue differentiation, it has been reported that transfection of the CEA gene in rat L6 myoblasts results in a complete block of myogenic differentiation. To extend investigations to the transformed myogenic counterpart and examine CEA effects on differentiation and malignancy outside the colon system, we have transfected the human CEA gene in human rhabdomyosarcoma cells. Human rhabdomyosarcoma cells transfected with the CEA gene correctly expressed membrane CEA anchored via glycosylphosphatidylinositol and secreted CEA in the medium. CEA gene transfer in human rhabdomyosarcoma cells, which display a limited differentiation ability, does not further inhibit myogenic differentiation or alter in vitro proliferation or natural killer sensitivity. CEA transfection does not affect s.c. growth in nude mice, but the ectopic expression of CEA in human rhabdomyosarcoma cells can strongly inhibit their metastatic ability to lungs and adrenals after i.v. injection. The impairment of metastatic potential correlates with a reduction in the homotypic adhesion properties of the cells. These data suggest that CEA, in some systems, can interfere with intercellular adhesion and, at least for cells not metastatic to the liver, can act as an anti-metastatic molecule.

Ability of systemic interleukin-12 to hamper progressive stages of mammary carcinogenesis in HER2/neu transgenic mice.

Previous studies in mice have shown that chronic administration of recombinant interleukin-12 (IL-12) hampers the progression of both chemical- and oncogene-dependent carcinogenesis. This suggests that a new preventive strategy may be envisaged for individuals with a genetic risk of cancer or carrying preneoplastic lesions. Starting at progressive stages of mammary carcinogenesis, female BALB/c and FVB mice carrying the activated rat HER2/neu oncogene (BALB-neuT) or the proto-oncogene (FVB-neuN) under the mouse mammary tumor virus promoter received multiple 5-day courses of different doses of IL-12. The times of tumor appearance, multiplicity, and histopathological features of the neoplastic lesions were evaluated. In both BALB-neuT and FVB-neuN mice, 5-day i.p. courses of 50/100 ng of IL-12/day inhibited mammary carcinogenesis when they coincided with the progression of early preneoplastic lesions. Inhibition appears to depend primarily on the ability of IL-12 to interfere with early tumor angiogenesis. Later treatments are much less effective, and daily doses of 10 and 2 ng are useless. The efficacy of early IL-12 courses suggests that they could be used to prevent mammary tumors in individuals at risk, whereas their lower efficacy in later stages of carcinogenesis and the dose range required pose some constraints on their use in the management of overt preneoplastic lesions. Precise understanding of tumor progression means that effective treatments can be commenced relatively late in the life of individuals at risk and that no lifetime administration is required.

CD99 engagement: an effective therapeutic strategy for Ewing tumors.

CD99 is a Mr 32,000 transmembrane molecule that shows a high level of expression on cells of the hemopoietic system as well as on Ewing tumor cells. Within the hematopoietic system, CD99 has been implicated in cell adhesion and cell death, participating in this way in the differentiation of T-cell precursors. In this study, we demonstrate that engagement of CD99 significantly inhibits the in vitro and in vivo growth ability of Ewing tumor cells by delivering an apoptotic stimulus and reducing the malignant potential of these cells. Moreover, we show that anti-CD99 monoclonal antibodies may be advantageously used in association with conventional anticancer agents. These results provide a novel entry site for therapeutic intervention, which may have application in the care of patients with Ewing tumor, and warrant additional studies to clarify the molecular mechanisms activated by CD99 engagement.

Immunizing and curative potential of replicating and nonreplicating murine mammary adenocarcinoma cells engineered with interleukin (IL)-2, IL-4, IL-6, IL-7, IL-10, tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, and gamma-interferon gene or admixed with conventional adjuvants.

To evaluate the efficacy of vaccinations with cytokine-gene-transduced tumor cells, BALB/c mice were challenged with 1 x 10(5) parental cells of a syngeneic adenocarcinoma cell line (TSA-pc). No protection was observed in mice immunized 30 days earlier with 1 x 10(5) nonreplicating mitomycin-C-treated TSA-pc alone, or with Corynebacterium parvum or Complete Freund Adjuvant (CFA). Ten to 30% of mice immunized with nonreplicating cells engineered to produce interleukin (IL)-2, IL-4, IL-6, IL-7, IL-10, tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, and gamma-interferon gene were protected. Fifty % of mice immunized with replicating TSA-pc admixed with C. parvum and 80-100% of mice immunized with replicating tumor cells transduced with IL-2, IL-4, IL-7, IL-10, or gamma-interferon gene were protected. No cure was afforded by TSA cells admixed with C. parvum or CFA, nor by TSA cells engineered with IL-6, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha gene injected starting 1 day after TSA-pc challenge. Complete tumor regression, however, was obtained in 10-20% of mice treated with TSA cells transduced with IL-2, IL-4, IL-7, or IL-10 and in 30% of those treated with TSA cells transduced with gamma-interferon gene.

Interleukin 12 gene therapy of MHC-negative murine melanoma metastases.

Immunological gene therapy of cancer relies heavily on the activation of T cells, but tumors with defects in MHC gene expression are not recognized by MHC-restricted T cells. To investigate the potential of cytokine genes for the therapy of MHC-negative tumors, we transduced B78H1, a class I-negative murine melanoma clone, with a polycistronic vector carrying murine interleukin (IL)-12 genes. The clones studied produced 400-25,000 pg/ml IL-12; their in vitro growth properties were similar to those of parental cells. A complete inhibition of growth was observed in vivo both after s.c. and i.v. administration of all IL-12 clones. IL-12-transduced cells were also used as a therapeutic vaccine in mice bearing micrometastases by nontransduced parental cells. A significant (80-90%) reduction in the number of lung nodules was obtained. Immunohistochemical analysis and studies in immunocompromised hosts showed that T cells and natural killer cells had a significant role in the elimination of IL-12-releasing cells. In situ hybridization with cytokine probes detected a strong increase in the proportion of leukocytes positive for IFN-gamma, tumor necrosis factor alpha, IL-1beta, and IFN-inducible protein 10 at the site of rejection of IL-12-engineered tumor cells. However, it was clear that the loss of in vivo growth was also due to T-cell- and natural killer cell-independent factors, possibly related to the antiangiogenic properties of IL-12. In conclusion, tumor therapy based on IL-12 gene transduction was effective on a MHC-negative metastatic tumor, suggesting a possible application to MHC-defective human neoplasms.

Blockage of insulin-like growth factor-I receptor inhibits the growth of Ewing's sarcoma in athymic mice.

Innovative, more effective treatment modalities are needed for Ewing's sarcoma (ES), a neoplasm with a disappointingly low survival rate despite the use of aggressive multimodal therapeutic approaches. We have previously shown (K. Scotlandi et al, Cancer Res., 56: 4570-4574, 1996) the existence and the pathogenetic relevance of an autocrine loop that is mediated by the insulin-like growth factor-I receptor (IGF-IR) and is crucial for the survival and proliferation of ES cells in vitro. In this study, we report that the IGF-IR-blocking monoclonal antibody alphaIR3 may also significantly inhibit ES cell growth in vivo. In particular, in almost one-half of the animals tested, after s.c. inoculation with TC-71 ES cells, the blockage of IGF-IR by alphaIR3 induced a complete regression of tumors that developed, which suggests that IGF-IR is valuable as a specific target for novel therapeutic strategies. In addition, suramin, a drug that can interfere with growth factor binding with their receptors, inhibited the tumorigenic and the metastatic ability of TC-71 cells and, therefore, is a promising agent to be combined with conventional cytotoxic drugs for the design of more effective therapeutic regimens.

Antisense epidermal growth factor receptor transfection impairs the proliferative ability of human rhabdomyosarcoma cells.

Human rhabdomyosarcoma cells express membrane epidermal growth factor receptor (ECF-R), which could confer responsiveness to EGF and transforming growth factor-alpha (TGF-alpha) of autocrine or paracrine origin. To study the role played by this growth factor circuit in the proliferation and differentiation of myogenic neoplastic cells, human rhabdomyosarcoma EGF-R-expressing cells (RD/18 clone) have been transfected with a plasmid containing a fragment of the EGF-R cDNA in the antisense orientation. In vitro growth and differentiative ability were studied on six antisense-transfected clones (AS) in comparison to parental RD/18 cells and to cells transfected with the plasmid containing only the neomycin resistance gene (NEO). A reduced EGF-R membrane expression was found in AS clones by decreased immunofluorescence with an anti-EGF-R monoclonal antibody. All AS transfectants had a greatly impaired proliferative ability, even when cultured in fetal bovine serum-containing medium. Proliferation of AS clones was completely blocked in medium supplemented with 2% horse serum. The differentiation ability of AS clones was heterogeneous, ranging from clones with a percentage of myosin-positive cells higher than controls to clones with a negligible myosin expression. Therefore, the growth impairment determined by the loop interruption is not sufficient to switch on the differentiation program. The role played by EGF-R in the proliferation of human rhabdomyosarcoma cells suggests that this receptor could constitute a target for a therapeutic approach.