Begoña Alburquerque González¹, Silvia Montoro García², DAVID GARCIA BERNAL³, MANUEL BERNABE GARCIA⁴, María Jesús Rodríguez Arcas⁵, Fernando Feliciano López Calderón⁶, Daniel Torres Moreno⁷, Victoria Pérez Parra⁸, Verónica Castillo Guardiola⁹, Rubén Cárdenas Gámez¹⁰, David Sánchez Gutierrez¹¹, JOSE GARCIA SOLANO¹², JUAN CABEZAS HERRERA¹³, Pablo Conesa¹²

(1) Universidad Católica San Antonio de Murcia (UCAM). Grupo IMIB: Patología Molecular y Farmacogenética, 30003, España (Región de Murcia)
(2) Cell Culture Lab, Facultad de Ciencias de la Salud, UCAM Universidad Católica San Antonio de Murcia, Campus de los Jerónimos, s/n, Guadalupe, 30107, España (Región de Murcia)
(3) TRASPLANTE HEMATOPOYÉTICO / TERAPIA CELULAR, IMIB-Arrixaca, España
(4) CIRUGÍA DIGESTIVA, ENDOCRINA Y TRASPLANTE DE ÓRGANOS ABDOMINALES, IMIB-Arrixaca, España
(5) Directora técnica en Farmacia Arcas, España (Región de Murcia)
(6) Universidad Católica San Antonio de Murcia (UCAM). Grupo IMIB: Patología Molecular y Famracogenética., España (Región de Murcia), 30204, España (Región de Murcia)
(7) Group of Molecular Pathology and Pharmacogenetics, IMIB, Hospital Universitario Santa Lucía., 30202, España (Región de Murcia)
(8) Técnico Anatomía Patológica. Hospital Universitario Santa Lucía. Cartagena., 30202, España (Región de Murcia)
(9) Facultativo Especialista Análisis Clínicos. Hospital Universitario Santa Lucía. Cartagena., 30202, España (Región de Murcia)
(10) Residente de Análisis Clínicos. Hospital Universitario Santa Lucía. Cartagena., 30202, España (Región de Murcia)
(11) Residente de Anatomía Patológica. Hospital Universitario Santa Lucía de Cartagena., 30202, España (Región de Murcia)
(12) PATOLOGÍA MOLECULAR Y FARMACOGENÉTICA, IMIB-Arrixaca, España
(13) TERAPIAS MOLECULARES Y BIOMARCADORES DE TUMORES SÓLIDOS, IMIB-Arrixaca, España

The formation of metastases is the result of invasion and migration processes in which rearrangement of the actin cytoskeleton plays a crucial role. Mouse models have proven to be an important tool for unraveling the biological complexity of the metastatic cascade and are essential for testing novel therapies. Previously, our group identified with in vitro approaches the potential role for the antidepressant Tofranil® (FDA approved) through Fascin-1 (FSCN1) inhibition in colorectal cancer cell lines. Therefore, we hypothesized that Tofranil® may be clinically applicable to treat colorectal carcinoma cells before and after escaping from the primary tumour. The current study is divided in two procedures. Firstly, we investigated the effect of Tofranil® on tumour growth using a primary tumour model (Procedure-I) of immunosuppressed mice induced by xenographt of human HCT 116 bioluminescent cells. Secondly, we established a model of metastatic colorectal cancer (Procedure-III) by intravenous infusion of tumor cells and further explore the therapeutic potential of the drug.

HCT 116 cells were stably transfected with pGL4.51[luc2/CMV/Neo] vector using turbofectin reagent protocol. Male-female (60%-40%) athymic nude mice (5 weeks old) were obtained from Charles River Laboratories. Animals were maintained under specific pathogen free conditions based on the guidelines established by the IMIB ethical committee. A toxicity study (n=4) was carried out to confirm the oral doses of the drug. Following, in Procedure-I, HT 116 cells were suspended in DMEM and subcutaneously inoculated with 100 μL matrigel into the left flank of each mouse. Mice were divided into two groups: (a) vehicle group (n = 9); (b) treatment group (n = 12). Tumour volume was measured three times per week by using a caliper. Tumour weights were recorded after excision at the final time point. In Procedure-III, HCT 116 cells were intravenously inoculated into the tail vain and mice were divided into three groups: (a) vehicle group (n = 6); (b) treatment initialized one month later the xenograft (n = 4); and (c) treatment initialized the day of the xenograft (n= 4). Tofranil® solution was administrated by oral gavage at 95 mg/kg for 15 days (Procedure-I) and 86, 1 mg/kg (Procedure III). Bioluminescence was monitored in live mice using In Vivo Imagining System (IVIS).

The effect of Tofranil® treatment has been demonstrated in a model of xenograft tumor of HCT116 cells (Figure 1). Mice receiving Tofranil® therapy showed lower tumor burden as measured by bioluminescence (Total flux [p/s] p<0.05. Figure 2). Resected tumors were weighed at the final time-point groups (a, b) (p<0.0001, Figure 2). For Procedure-III, bioluminescence signal appeared shortly after injection and metastasis was observed. The effect of Tofranil® treatment in this Procedure showed that mice in “group c” had a lower metastatic bioluminescence scope compared to “group b”. Mice receiving treatment one month after tumor inoculation, did not exhibit reduced tumor burden compared to vehicle (a). These outcomes were supported by the immunohistochemical FSCN1 staining of the mice tumors. Euthanasia was often required in group c (severe metastasis).

These two models with human tumor cell lines might be useful for innovative drug repositioning and mechanism studies. We also show preliminary data for testing the effectiveness of Tofranil® in colorectal carcinogenesis with encouraging results.