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Interaction between epidermal growth factor receptor (EGFR) signaling with GM3 ganglioside

Interaction between epidermal growth factor receptor (EGFR) signaling with GM3 ganglioside expression has been previously described. Overall, our results claim that NeuGcGM3 and EGFR may coordinately donate to the tumor cell biology which restorative combinations against both of these targets may be a valid technique to explore. Keywords: EGFR, NeuGcGM3; Co-expression; Pulmonary metastasis; Mixture therapy Introduction Many epithelial tumors overexpress the EGFR and their activation can be related with cancers progression. Nevertheless, tumors show a varied response to anti-EGFR therapies, with level of resistance as common consequence of the procedure [1]. The N-Acetylneuramic acidity (NeuAcGM3) ganglioside, however, not the N-glycolylneuramic acidity (NeuGcGM3), can be detected in regular human being cells usually. However, many human being tumors communicate NeuGcGM3 ganglioside [2C7]. The manifestation of NeuGcGM3 have already been connected with a worse prognosis in digestive tract [8] and lung tumor [7, 9]. Differential association between EGFR signaling pathway and GM3 ganglioside manifestation continues to be reported [10C13]. Overexpression of GM3 raise the proliferation of carcinoma cells (A431) by ERK-independent signaling, in the current presence of urokinase plasminogen activator (uPA) and their receptor (uPAr) [14]. Conversely, GM3 depletion raise the EGFR phosphorylation as well as the ERK-dependent cell proliferation turns VX-950 into common [14]. These results provide a rational for a combined treatment targeting simultaneously both EGFR and GM3 mediated signaling pathways. The Center of Molecular Immunology (CIM, Havana, Cuba) have developed several immunotherapeutic projects targeting separately both EGFR [15, 16] and NeuGcGM3 [17, 18]. Therefore, we evaluate the frequency of co-expression of EGFR and NeuGcGM3 ganglioside in human tumors and in two spontaneous lung metastasis models of mice (Lewis lung carcinoma (3LL-D122) in C57BL/6 and mammary carcinoma (4T1) in BALB/c). Moreover, we perform an initial evaluation of the therapeutic implications of targeting simultaneously both molecules, in lung models. Materials and methods Patients samples Sections of formalin-fixed and paraffin-embedded tumor tissues from 92 patients were taken from the pathology department of the National Institute of Oncology and Radiobiology and Dr. Manuel Fajardo General Teaching Hospital after receiving approved consent by the Ethical Committee of the institute. Cell lines Lewis lung carcinoma (3LL-D122); mouse breast adenocarcinoma cells (4T1); human vulva epidermoid carcinoma (A431, ATCC, CRL-1555) and murine myeloma P3-X63-Ag8.653 (X63, ATCC, CRL-1580) were cultured in DMEM: F12 (Life Technologies Inc., Grand Islan, NY) supplemented with 10?% fetal bovine serum (FBS). Lung metastasis murine models Mice female of 6C8?week old female, were purchased from the Center for Laboratory Animal Production (CENPALAB, Havana, Cuba). Animals procedures were performed in accordance with the guidelines stipulated by Animal Subject Committee Review Board of the CIM and CIMs Institutional Animal Care and Use Committees. 3LL-D122-metastasis VX-950 model: C57BL/6 mice were injected into lateral tail veins (i.v.) with 2.5??105 of tumor cells. 4T1-metastasis model: BALB/c mice were transplanted subcutaneously (s.c). into the mammary gland with 1??104 of tumor cells. Primary tumor diameters were measured every 2C3?days with a caliper and tumor volume (mm3) was determined to the following formula?=?(minor diamenter)2??(major diameter)??/6. To study overall survival (OS), animals were monitored every day until the primary tumor exceeded 20?% of the body mass (4T1-model) and the signs of malignancy appeared. In parallel experiments, the mice were sacrificed 21?days (3LL-D122-model) and 25?days (4T1-model) after tumor implantation to evaluate lung metastases. Metastatic lung VX-950 CD160 were removed and metastases were quantified through lung weight, established as a surrogate of the number and size of metastasis. Control groups received PBS. Murine samples Tumor sections from the metastatic lungs were obtained by VX-950 cryostat (SLEE MEDICAL GMBH Co. Mainz, Germany) and mounted on plus slides. Afterwards, in both cases, the slides were kept at ?20?C until they were used for immunostaining. Monoclonal antibodies Ior egf/R3 (R3m) is a mAb against human EGFR [19]. 7A7 mAb for murine EGFR [20]. 14F7 mAb against NeuGcGM3 ganglioside and it was used in patients and murine samples [21]. Regarding the treatment: 14F7 mAb and 7A7.