Associação Portuguesa de Investigação em Cancro
Inoculated Cell Density as a Determinant Factor of the Growth Dynamics and Metastatic Efficiency of a Breast Cancer Murine Model
Inoculated Cell Density as a Determinant Factor of the Growth Dynamics and Metastatic Efficiency of a Breast Cancer Murine Model
Treatment and management of breast cancer imposes a heavy burden on the public health care. However, in spite of the considerable advancements in the treatment of this malignancy in the recent years, many patients continue to progress to metastatic disease and, for those with advanced breast cancer, palliative care is oftentimes the endpoint. The manifestation of metastases is predictive of poor clinical outcome, and prevails one of the most challenging issues faced by cancer treatment today. A continuous effort in dissecting the biological processes behind cancer cell dissemination has been pushing forward our understanding of the metastatic disease and uncovering vulnerabilities that may be exploited for the development of novel agents to treat metastatic cancer. In this respect, mouse models are crucial to our comprehensive knowledge on the molecular basis and pathogenesis of cancer. Moreover, the use of translatable and predictive tumor models is a recognized need for the successful drug development. Yet, the unavailability of suitable mouse models that accurately recapitulate the complexity of human tumor progression has been a handicap. For example, the ectopic location of cancer cells usually fails to produce metastases, owing to the limited tumor microenvironment generated, and the surgical resection of primary tumors is often required in order to prolong mice survival and enable the development of spontaneous metastases. The use of genetic engineered mouse models surpass some of these constrains, offering the possibility of orthotopic neoplastic generation in immune competent hosts. However, metastatic lesions may appear only upon long latency periods and generally their incidence is low. Therefore, the setting up of spontaneous metastases faces several challenges, and improvement of its modeling remains of major importance. The 4T1 murine cells spontaneously generate tumors and are described to metastasize to the lungs, liver, lymph nodes, brain and bones, in a way that closely resembles human breast cancer. Owing to its characteristics, these cells have been widely used to study stage IV human breast cancer. Nonetheless, 4T1 metastatic breast cancer model suffers from the liability of fast growing tumors, enhanced by the frequent inoculation of a large number of cells, rendering a tumor microenvironment that does not recapitulate human breast tumors and early mice euthanasia. These factors limit its usefulness to understand the biology of metastatic breast cancer and therefore the identification of novel therapeutic opportunities and the corresponding proof of concept. In this work we established the conditions enabling high metastatic take rate of the widespread triple-negative murine 4T1 syngeneic breast cancer model, towards a more reliable pre-clinical screening of anticancer drugs. It was demonstrated that 4T1 tumors grew in the mammary fat pad of mice when as few as 500 cancer cells were implanted, with 87% tumor incidence. The lungs were the main organs colonized with 100% efficiency, though distant lesions were also commonly identified in other tissues, such as the mesentery and pancreas. This significant reduction in the number of inoculated cells also resulted in increased tumor doubling times and decreased specific growth rates, following a Gompertzian tumor expansion. Extending the time frame of primary tumor development, without requiring its excision, was beneficial for metastatic progression, and set the ground to better assess the effect of antimetastatic therapies.
Ana C. Gregório1,2, Nuno A. Fonseca1,3, Vera Moura1,4, Manuela Lacerda5, Paulo Figueiredo6, Sérgio Simões1,3, Sérgio Dias7, João Nuno Moreira1,3*
1CNC - Center for Neurosciences and Cell Biology, University of Coimbra, Coimbra, Portugal
2IIIUC – Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal
3FFUC - Faculty of Pharmacy, Pólo das Ciências da Saúde, University of Coimbra, Coimbra, Portugal
4TREAT U, SA, Coimbra, Portugal
5IPATIMUP – Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal
6POFG-EPE – Portuguese Institute of Oncology Francisco Gentil, Coimbra, Portugal
7IMM – Institute of Molecular Medicine, Faculty of Medicine, University of Lisbon, Lisbon, Portugal
4T1 metastatic breast cancer model have been widely used to study stage IV human breast cancer. However, the frequent inoculation of a large number of cells, gives rise to fast growing tumors, as well as to a surprisingly low metastatic take rate. The present work aimed at establishing the conditions enabling high metastatic take rate of the triple-negative murine 4T1 syngeneic breast cancer model. An 87% 4T1 tumor incidence was observed when as few as 500 cancer cells were implanted. 4T1 cancer cells colonized primarily the lungs with 100% efficiency, and distant lesions were also commonly identified in the mesentery and pancreas. The drastic reduction of the number of inoculated cells resulted in increased tumor doubling times and decreased specific growth rates, following a Gompertzian tumor expansion. The established conditions for the 4T1 mouse model were further validated in a therapeutic study with peguilated liposomal doxorubicin, in clinical used in the setting of metastatic breast cancer. Inoculated cell density was proven to be a key methodological aspect towards the reproducible development of macrometastases in the 4T1 mouse model and a more reliable pre-clinical assessment of antimetastatic therapies.
Plos One
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0165817
