RESUMEN
Antibodies are essential reagents that are increasingly used in diagnostics and therapy. Their specificity and capacity to recognize their native antigen are critical characteristics for their in vivo application. Follicle-stimulating hormone receptor is a GPCR protein regulating ovarian follicular maturation and spermatogenesis. Recently, its potentiality as a cancer biomarker has been demonstrated but no antibody suitable for in vivo tumor targeting and treatment has been characterized so far. In this paper we describe the first successful attempt to recover recombinant antibodies against the FSHR and that: i) are directly panned from a pre-immune library using whole cells expressing the target receptor at their surface; ii) show inhibitory activity towards the FSH-induced cAMP accumulation; iii) do not share the same epitope with the natural binder FSH; iv) can be produced inexpensively as mono- or bivalent functional molecules in the bacterial cytoplasm. We expect that the proposed biopanning strategy will be profitable to identify useful functional antibodies for further members of the GPCR class.
Asunto(s)
Biblioteca de Péptidos , Receptores de HFE/antagonistas & inhibidores , Receptores de HFE/inmunología , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunología , Animales , Especificidad de Anticuerpos , AMP Cíclico/metabolismo , Femenino , Hormona Folículo Estimulante/farmacología , Células HEK293 , Humanos , Inmunización , Células L , Masculino , Ratones , Dominios Proteicos , Receptores de HFE/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Transducción de Señal , SolubilidadRESUMEN
Follicle-stimulating hormone receptor (FSHR) is present on endothelial cells of blood vessels and endometrial glands of the proliferative and secretory endometrium. So far, the expression of FSHR in endometriosis has not been studied. We evaluated FSHR expression in 194 tissue specimens representing 3 relevant types of endometriosis: rectovaginal endometriotic nodules, ovarian endometriotic cysts, and peritoneal endometriotic implants. Specimens of normal endometrium were used as controls. Archival formalin-fixed and paraffin-embedded material was analyzed immunohistochemically with a highly specific monoclonal antihuman FSHR antibody using the peroxidase method. A robust vascular FSHR expression was found in all 194 patients, irrespective of the endometriosis lesion location. Follicle-stimulating hormone receptor was not detected in normal host tissues located more than 5 mm from the lesions. The endometriotic lymphatic vessels do not express FSHR. The density of FSHR-positive vessels in patients with rectovaginal endometriotic nodules was 46.0 ± 5.7 vessels/mm(2) Similar values were obtained for ovarian endometriotic cysts and peritoneal endometriosis. The density of FSHR-positive vessels associated with the core of rectovaginal endometriotic nodules was 2-fold higher than that of the perilesional, adjacent normal host tissue (64.2 ± 8.2 vs 27.2 ± 3.2 vessels/mm(2), respectively). Expression of FSHR was also detected either in endometriotic glandular epithelial cells, endometriotic stromal cells, or in both cell types (23%, 25%, and 21% of patients, respectively). Normal endometrium expressed FSHR predominately in basalis, in a cellular distribution dependent on hormonal environment. In conclusion, our data suggest novel FSHR expression in endometriotic lesions, qualitatively and quantitatively different from that of normal endometrium.
Asunto(s)
Vasos Sanguíneos/metabolismo , Endometriosis/metabolismo , Endometriosis/patología , Receptores de HFE/metabolismo , Endometrio/irrigación sanguínea , Endometrio/citología , Endometrio/metabolismo , Células Endoteliales/metabolismo , Femenino , Humanos , InmunohistoquímicaRESUMEN
BACKGROUND: Follicle-stimulating hormone receptor (FSHR) is expressed on the endothelial surface of blood vessels associated with solid tumor periphery, where angiogenesis is known to occur. The correlation between FSHR expression and formation of new peritumoral vessels has not been previously investigated. METHODS: We used immunohistochemical techniques involving specific antibodies to detect FSHR and the endothelial markers (CD34, VEGFR2, and D2-40) in tissue samples from 83 patients with lymph node-negative, invasive breast cancer representing four main clinical treatment groups: HR+/HER2-, HR+/HER2+, HR-/HER2+ and triple-negative. RESULTS: The FSHR+ vessels were exclusively located at breast cancer periphery, in a layer that extended 2 mm into and 5 mm outside of the tumor. The percentage of blood vessels expressing FSHR reached a maximum of 100% at the demarcation line between the tumor and the normal tissue. Common among FSHR+ vessels, regardless of breast cancer type, were the high densities of arterioles and venules (6.4 ± 1.4 and 13.9 ± 2.1 vessels/mm(2), respectively). These values were 3-fold higher that those noticed for CD34+ arterioles and venules associated with normal breast tissue located at a distance greater than 10 mm outside the tumors. The average density of FSHR+ and CD34+ blood vessels as well as of D2-40+ lymphatic vessels did not differ significantly among breast cancer subgroups. FSHR+ vessels did not express VEGFR2. The endothelial FSHR expression correlated significantly with the peritumoral CD34+ vessels' density (p < 0.001) and tumor size (p = 0.01). CONCLUSION: Endothelial FSHR expression in breast cancer is associated with vascular remodeling at tumor periphery.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neovascularización Patológica/metabolismo , Receptores de HFE/metabolismo , Remodelación Vascular , Biomarcadores , Células Endoteliales/metabolismo , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Clasificación del Tumor , Neovascularización Patológica/genética , Receptores de HFE/genéticaRESUMEN
BACKGROUND: The Follicle Stimulating Hormone receptor (FSHR) is expressed by the vascular endothelium in a wide range of human tumors. It was not determined however if FSHR is present in metastases which are responsible for the terminal illness. METHODS: We used immunohistochemistry based on a highly FSHR-specific monoclonal antibody to detect FSHR in cancer metastases from 6 major tumor types (lung, breast, prostate, colon, kidney, and leiomyosarcoma ) to 6 frequent locations (bone, liver, lymph node, brain, lung, and pleura) of 209 patients. RESULTS: In 166 patients examined (79%), FSHR was expressed by blood vessels associated with metastatic tissue. FSHR-positive vessels were present in the interior of the tumors and some few millimeters outside, in the normally appearing tissue. In the interior of the metastases, the density of the FSHR-positive vessels was constant up to 7 mm, the maximum depth available in the analyzed sections. No significant differences were noticed between the density of FSHR-positive vessels inside vs. outside tumors for metastases from lung, breast, colon, and kidney cancers. In contrast, for prostate cancer metastases, the density of FSHR-positive vessels was about 3-fold higher at the exterior of the tumor compared to the interior. Among brain metastases, the density of FSHR-positive vessels was highest in lung and kidney cancer, and lowest in prostate and colon cancer. In metastases of breast cancer to the lung pleura, the percentage of blood vessels expressing FSHR was positively correlated with the progesterone receptor level, but not with either HER-2 or estrogen receptors. In normal tissues corresponding to the host organs for the analyzed metastases, obtained from patients not known to have cancer, FSHR staining was absent, with the exception of approx. 1% of the vessels in non tumoral temporal lobe epilepsy samples. CONCLUSION: FSHR is expressed by the endothelium of blood vessels in the majority of metastatic tumors.