The clinical relevance of cancer stem cells (CSC) remains a major challenge for current cancer therapies, but preliminary findings indicate that specific targeting may be possible. Recent studies have shown that these tumor subpopulations promote tumor angiogenesis through the increased production of VEGF, whereas the VEGF neutralizing antibody bevacizumab specifically inhibits CSC growth. Moreover, nimotuzumab, a monoclonal antibody against the epidermal growth factor receptor (EGFR) with a potent antiangiogenic activity, has been shown by our group to reduce the frequency of CSC-like subpopulations in mouse models of brain tumors when combined with ionizing radiation. These studies and subsequent reports from other groups support the relevance of approaches based on molecular-targeted therapies to selectively attack CSC. This review discusses the relevance of targeting both the EGFR and angiogenic pathways as valid approaches to this aim. We discuss the relevance of identifying better molecular markers to develop drug screening strategies that selectively target CSC.

Background: The poor prognosis of patients with high-grade glioma multiform (GBM) has led investigators to the search of new therapeutic strategies. Current treatment includes surgery when possible, radiotherapy and chemotherapy. Molecular-targeted therapies are in the process of clinical testing, and promising agents include monoclonal antibodies. Our study examined the antitumor activity of three different single therapies in nude mice bearing both subcutaneous and orthotopic brain xenografts of the U87MG human GBM cell line. Methods: Cell culture, Histology, Immunohistochemistry, Animal experiments, Statistical analysis. Results: Different groups of treatment included nimotuzumab, a humanized monoclonal antibody that inhibits the EGFR tyrosine kinase activity, or total body irradiation, or the chemotherapeutic agent temozolomide (TMZ). For the control group animals received saline solution instead of the antibody. For the subcutaneous model, only nimotuzumab or TMZ produced a significant delay in tumor growth. In the intracranial model, unlike TMZ, the systemic administration of the antibody did not reduce the tumor growth, despite both therapies inhibited the formation of microsatellites in the brain of mice. The antitumor activity of nimotuzumab was accompanied by a decrease in the microvessel density and the proliferative activity of tumor cells. TMZ only inhibited the tumor cell proliferation but not the formation of new tumor-associated microvessels in xenografts. For radiation therapy, neither antiproliferative nor antiangiogenic activity was found, in accordance with the lack of antitumor activity. Only nimotuzumab reduced the frequency of chemo and radioresistant CD133 + population. Conclusion: Our results illustrate the potential efficacy of nimotuzumab as a single agent against an EGFR-amplified human GBM, a tumor resistant to the therapy with all well-known forms of treatment.

Targeted therapy to the epidermal growth factor receptor (EGFR) seems to be related to its expression level on tumor cells. Interferon-alpha (IFN-alpha) induces growth inhibition but also may up-regulate the EGFR expression in some cancer cell lines. We aimed to determine whether the IFN-alpha combined with an EGFR-specific monoclonal antibody (nimotuzumab) may affect the growth of human tumor epithelial cell lines with different EGFR expression levels. H125, a lung adenosquamous carcinoma, and A431, a vulvar epidermoid carcinoma, cell lines express intermediate and high levels of EGFR, respectively, whereas MDA MB231, a breast adenocarcinoma cell line expresses undetectable levels of EGFR measured by flow cytometry/FACS. We found that IFN-alpha alone inhibited in a dose-dependent fashion the growth of all cell lines, but only up-regulated the EGFR expression in the lung carcinoma-derived cell line. Noteworthy, the combined treatment did not modify the complement-mediated cytotoxicity of the antibody although the antiproliferative activity of nimotuzumab in H125 cells in vitro increased when an IFN-alpha-conditioning treatment was used. In conclusion, this study may provide insights about the rational use of EGFR inhibitors into the immunopharmacological management of targeted therapies including the IFN-alpha for lung cancer.

Potential contribution of the Epidermal Growth Factor Receptor (EGFR) in melanoma immunobiology remains unclear, in part due to a lack of experimental models. We demonstrated previously that B16F10 melanoma transfected with the full length cDNA of the human EGFR increases the tumor cell proliferation in vitro. To further study its contribution in vivo, EGFR-transfected B16F10 cells were inoculated in syngenic C57BL/6 mice and its tumorigenic capacity was compared with the parental melanoma. Contrary to the observed in vitro effect, EGFR-transfected B16F10 cells displayed a delayed tumor growth rate in vivo, correlating inversely to the transgene expression. Interestingly, resulting tumors showed a downregulation of the EGFR transgene expression. Contrastingly, parental and EGFR-transfected B16F10 cells exhibited a similar tumorigenic potential in immunocompromised subjects, persisting the EGFR transgene expression. These results document the adaptability of melanoma to growth in immunocompetent individuals. Moreover, the potential EGFR expression during the melanoma outgrowth that would be downregulated by interacting with the host immune system during the tumor evolution is not excluded and which may be dissected in this model.

EGFR [EGF (epidermal growth factor) receptor] overexpression correlates with poor prognosis and bad outcomes in different tumours. However, evidence for EGFR contribution in melanoma immunobiology is limited. We have expressed the full-length human EGFR gene in a murine melanoma cell line. EGFR protein expression in stably trnasfected B16 cells in culture was defined by immunoblotting, immunohistochemistry and FACS. Additionally, transfected cells became sensitive to the lysis induced with an anti-EGFR monoclonal antibody in the presence of complement. Exogenous human EGF addition induced cell proliferation, validating the transfected receptor functionality. Thus we have developed a system to express a functional EGFR in order to evaluate the potential contribution of EGFR expression in melanoma biology and its resulting relevance as a target for immunointerventions in nonepithelial tumours.