Several studies demonstrated that: a) the expression of chemokine receptors in neoplastic cells is not random; b) CXCR4 is the most widely expressed chemokine receptor in most tumours; c) the effects of SDF-1 on CXCR4-expressing cancer cells are pleiotropic [31
]. In particular, SDF-1/CXCR4 axis is enrolled in many functional aspects of tumour progression, such as angiogenesis, site-specific metastasization, proliferation and survival of neoplastic cells [2
]. It is also recognized that metastastic behaviour of cancer cells may be reinforced by hypoxia, a condition that induce up-regulation of CXCR4 expression mediated by hypoxia-inducible factor-1a (HIF-1α) [33
]. SDF-1/CXCR4 axis is involved in the promotion of angiogenesis, local cell proliferation and migration of cancer cells to the metastatic sites in many different kinds of cancers as those of breast [21
], lung [36
], ovarian [1
], renal [38
], prostate [39
], and neuroblastoma [40
]. Moreover, it seems relevant to remind that CXCR4 expression has been associated to a worse prognosis in human tumours, and then, in the future, it could be also used as prognostic indicator [41
In contrast with the large number of studies in human oncology, there are only few scientific articles supporting the role of SDF-1/CXCR4 axis in spontaneous tumours of domestic animals. Tanabe and colleagues [26
] observed that the receptor's mRNA is not identifiable in mammary tissue of healthy cats. However, they found CXCR4 mRNA in area adjacent to necrotic tissue, surrounding blood vessels and in cells infiltrating the lymphatic tissue in 72.3% of 65 samples. In the same study, the authors observed a statistically significant relationship between infiltration of neoplastic cells in lymphatic and CXCR4 expression [26
], although no relationship was observed between CXCR4 expression and one-year survival time of the cats included in the study [26
]. In another study, higher expression of CXCR4 mRNA in metastatic cells as compared with cells from primary tumours was reported, altogether with the observation that neoplastic cells from feline mammary carcinomas express more CXCR4 than non-neoplastic mammary tissues [25
In the present study we demonstrated, for the first time, a high CXCR4 expression in several feline high grade mammary carcinoma, evaluated at protein level by immunohistochemistry. Interestingly, although the limited number of benign and metastatic lesions, but including normal tissues, we observed a statistically significant correlation between CXCR4 score levels and the increase of tumor grade or metastases. However, without drawing a definitive conclusion due to the need of higher numbers, the present results showing absence or low CXCR4 expression in normal and benign mammary tissues may suggest a specificity of CXCR4 expression for mammary carcinomas and metastases.
As far as the malignant primary tumours CXCR expression, although we observed some variability in the intensity of the anti-CXCR4 immunoreaction, only occasionally we detected variation in the percentage of positive cells. Moreover, although a large number of samples will be required to perform an appropriate statistical analysis, in this study the majority of metastastic lesions (5/6) displayed a higher expression compared to cells within the respective primary lesion. These observations, at least in part, support the results reported in the literature for breast cancer in women and in feline mammary carcinoma [42
The interest in researching on SDF-1/CXCR4 pathways is also oriented to discover 'new chemical entities' able to block this mechanism and specifically inhibit CXCR4 functions. Actually, there are some examples published in the international literature reporting the possible use of CXCR4 inhibitors. The latter have been used to reduce growth and metastasis of head and neck cancers and intracranial growth of brain tumours [43
]. These results have been also supported by in vitro
experiments that proved the efficacy of CXCR4 inhibitors in blocking SDF-1/CXCR4-mediated proliferation and migration in breast cancer and lymphoblastic leukaemia [19
]. In veterinary oncology, Oonuma and collaborators [25
] found a similar response in vitro
using established feline mammary carcinoma cell lines incubated with CXCR4 antagonists.
Here, besides the expression of CXCR4 in feline mammary tumours, we also investigated the functional role of this receptor in mediating proliferative signals. In vitro
, 5 out of 6 analyzed primary cultures of mammary carcinoma cells showed significant increased cell proliferation in response to CXCR4 activation by nanomolar concentrations of SDF-1. The effect was quantitatively variable but reached, on the average a values similar to that induced by FCS, thus confirming that SDF-1/CXCR4 axis is an important signalling pathway involved in feline mammary cell proliferation. Interestingly, the use of the CXCR4 antagonist AMD3100, while confirming the specificity of SDF-1 effects in vitro
through CXCR4, showed a reduction also in basal proliferation rate. Thus, it could be hypothesized that a basal SDF-1 secretion leads to autocrine CXCR4 activation in some tumor cell culture. This observation may also explain why in few cultures a lower proliferation is induced by exogenous SDF-1 that could be masked by the autocrine CXCR4 activation. A similar different response, related to constitutive autocrine SDF-1 effect, was recently demonstrated in human pituitary adenoma cell cultures in vitro
]. A larger number of tumors have to be analysed to confirm this hypothesis.
These data, although preliminary, strongly suggest that CXCR4 activity controls mammary carcinoma cell proliferation in cats as it does in humans, and propose that pharmacological inhibition of this receptor may represent an innovative approach for this kind of tumours. Moreover, our data propose that primary feline can reflect and maintain in vitro the phenotype of epithelial malignant carcinoma mammary cells and are potential suitable experimental models for assessing the biological activity of novel molecules with antitumour effects.
In the past, several studies reported the relevance of naturally occurring cancers in domestic animals as model for study human cancer biology and translational therapeutics [48
]. Among other tumours, mammary carcinomas show a relatively high similarity with the human counterpart as far biological behaviour, clinical course and responses to cytotoxic agents. The development of in vitro
models of feline mammary carcinomas may represent a relevant tool to identify novel molecular pharmacological targets to be used in veterinary setting and possibly extended to humans.