BACKGROUND
chemokines are small messengers with chemoattractant function .
they belong to a large superfamily of peptides produced and secreted by different cell types and classified in four groups  accordingly to structural determinants  <cit> .
the complex chemokine system is involved in a wide range of cell functions ranging from organogenesis to malignancy.
chemokine activity is mediated by the activation of a family of specific g protein coupled receptors.
chemokine receptor activation is mediated by coupling to intracellular heterotrimeric g-proteins associated with the inner surface of the plasma membrane  <cit> .
upon ligand binding, chemokine receptors promote g protein activation leading to the inhibition of camp synthesis and the activation of phospholipase c that cleaves phosphatidylinositol -bisphosphate  into the second messengers inositol triphosphate  and diacylglycerol .
dag activates protein kinase-c , while ip <dig> induces the release of calcium ions from intracellular stores.
several studies supported the role of chemokinergic axis in physiological activities including organogenesis  <cit> , haematopoiesis  <cit> , angiogenesis  <cit> , homing of lymphocytes  <cit> , immune response and inflammation  <cit> .
among cxc chemokine receptors, in the past years, cxcr <dig> attracted great attention for its pleiotropic activity outside the immune system  <cit> .
cxcr <dig> activity is dependent on its interaction with its unique ligand: stromal cell-derived factor- <dig>   <cit> .
sdf- <dig> was recently reported to bind also a second receptor cxcr <dig>  which regulates very different cellular activities  <cit>  including tumor angiogenesis  <cit> .
beside regulation of ca++ homeostasis and pkc activation, cxcr <dig> also modulate erk1/ <dig> map kinase and akt activities through a paracrine/autocrine mechanism  <cit> .
the activation of all these signalling cascades generates specific biological responses, such as chemotaxis, degranulation, release of superoxide anions, and cell proliferation.
over-expression of cxcr <dig> is considered a key regulatory step in several human malignancies included breast cancer, resulting in a poor prognosis  <cit> .
in detail, cxcr <dig> activity was involved in the oestrogen resistance of breast cancer  <cit> .
moreover, high cxcr <dig> expression has been related to the metastatic potential of breast cancer cells, since.
in vitro experiments showed that cxcr <dig> activation by sdf- <dig> regulates motility and metastatic potential of neoplastic epithelial cell lines  <cit> , while cxcr <dig> inhibitors are effective in reducing chemotactic and metastatic potential of this receptor  <cit> .
in cats, most studies are focused on the characterization of the cxcr <dig> role associated with fiv infection  <cit> , because of the role of this receptor as mediator of virus cell entry, as also observed in humans  <cit> .
conversely, few studies have been targeted on the role of cxcr <dig> in feline mammary gland neoplasia  <cit> , although cxcr <dig> may have a role in development of mammary carcinoma in cats as in women.
feline mammary tumors show age, incidence, histopathology and pattern of metastasis similar to human breast cancer  <cit> .
in addition, the lack of estrogens dependence in most of these tumours, suggests that this cancer may represent a suitable animal model for estrogen receptor  negative breast cancer  <cit> .
recently, feline mammary carcinoma subtypes have been described to share features with human inflammatory mammary carcinoma  <cit> .
at the molecular level her <dig> overexpressing malignancies in cats may be considered homologue of the her <dig> positive- poor prognosis-human counterpart  <cit> .
thus, research in this field may be relevant to better understand the biology of feline mammary tumour, also as a comparative model for human breast cancer, and to evaluate the cxcr <dig> role in cancer mechanisms.
moreover, immunohistochemistry screening of feline mammary tumours may have interesting applications in clinical practice as prognostic factor and to evaluate the possible response to cxcr <dig> inhibitors.
the aim of the present study was to investigate, by immunohistochemistry, the levels of cxcr <dig> expression in feline mammary tumours and metastases, and the proliferative activity induced by sdf- <dig> on feline carcinoma primary cultures.
RESULTS
feline mammary tumours and histological diagnoses
the clinical and pathological data of the feline mammary carcinomas analysed are summarised in table  <dig>  the  <dig> cats under study included  <dig> domestic short-hairs and  <dig> persian;  <dig> were intact females, while  <dig> were spayed.
the age ranged from  <dig> to  <dig> years .
in one case the age was not available.
primary malignant tumours were observed in 24/ <dig> cats , 7/ <dig> animals beard two neoplastic mammary glands thus we collected a total of  <dig> primary tumour samples.
only 2/ <dig> benign tumours were observed .
the primary tumor samples under evaluation included  <dig> simple tubulopapillary primary carcinomas , of which four of them gave metastasis .
the other samples were diagnosed as solid carcinomas  of which one gave lymph node metastasis,  <dig> cribriform carcinomas ,  <dig> simple tubular carcinomas  and  <dig> metastatic adenosquamous carcinoma .
.
twenty-seven out of  <dig> primary malignant lesions were classified as high grade carcinomas  and  <dig> as intermediate grade .
no grade i tumours were observed in this study.
only  <dig> benign tumours  were diagnosed in this group of animals.
four normal tissue samples, derived from non-neoplastic mammary glands resected during tumor surgery, were included in the study as cxcr <dig> expression reference.
cat
f, female, fs, female spayed; dsh, domestic short-hair; pc, persian; stpc, simple tubulopapillary carcinoma; sc, solid carcinoma; stc, simple tubular carcinoma; cc, cribriform carcinoma; ba, basaloid adenoma; ca, complex adenoma; asc, adenosquamous carcinoma; n.a., not available.
for normal tissues the mammary gland topography is indicated.
*a and b indicate samples from two glands of the same cat, m labels metastatic tissue.
primary tumours and corresponding metastases of the same cat are reported in bold.
cxcr <dig> expression in feline mammary carcinomas
the expression of cxcr <dig>  analyzed by immunohistochemistry, was observed in  <dig> out of  <dig>  of primary carcinoma samples  although the staining pattern and intensity differed between tumors as evaluated by cxcr <dig> scores  reported in table  <dig>  in  <dig> out of  <dig> carcinoma-bearing animals we analyzed tumors taken from two different mammary glands of the same cat ; in this subgroup 4/ <dig> tumors showed different score of cxcr <dig> expression among carcinomas derived from gland a and b from the same cat, possibly reflecting heterogeneity across tumour lesions.
the expression of cxcr <dig> was mostly confined to the membrane and cytoplasm of neoplastic cells  and differed in intensity and staining pattern  and among primary  and, in most cases, the corresponding metastatic lesions .
negative controls and cxcr <dig> antibody specificity were performed as reported in supplementary figure s <dig>
to evaluate the specificity of cxcr <dig> expression in mammary carcinomas and metastasis, we analyzed the level of cxcr <dig> immunopositivity in normal mammary tissues and benign lesions .
as depicted in figure  <dig> , normal mammary gland tissue did not express detectable level of cxcr <dig>  while epithelial cells of basaloid adenoma appeared weakly positive for cxcr <dig>  in scattered areas throughout the section, and the complex adenoma was essentially negative .
interestingly,  <dig> out of  <dig> metastatic tumours appeared to have a stronger cxcr <dig> expression as compared to the respective primary tumours .
nevertheless, in two metastatic sites, the neoplastic cells showed either a lighter immunoreaction if compared with the respective primary tumour  or no changes .
sdf-1/cxcr <dig> role in primary cultures of feline mammary carcinoma
to investigate the biological role of cxcr <dig> expression in feline mammary carcinoma, fibroblast-free, primary cell cultures were obtained from  <dig> primary carcinomas .
in vitro feline mammary cancer cells appeared as adherent monolayers with spindle-like shape .
the expression of cxcr <dig> in each culture was verified by if staining, confirming the immunopositivity of mammary carcinoma primary cells , as in the tissue of origin.
the absence of contaminant fibroblasts, which can interfere with both immunofluorescence and proliferation studies, was demonstrated by the lack of pro-collagen expression in the mammary carcinoma cell cultures, while isolated tumour fibroblasts, used as positive controls, showed a strong staining .
beside cxcr <dig> expression, a phenotypic characterization of the cells from the primary cultures was performed, by analyzing the expression of common mammary lineage markers , or proteins  overexpressed in mammary carcinoma conferring malignant behaviour .
as expected, feline mammary carcinoma cells from all tumour tested, homogeneously expressed the mammary epithelial marker ema.
moreover, in vitro cultures abundantly expressed ck <dig>  and, in variable proportion of cells, ck <dig> .
in addition, we include the evaluation of er-α immunocytofluorescence to define its pattern of expression in feline mammary cancer cells, detecting different levels of positivity, but generally low or weak .
as far as her2/neu and egfr expression in feline primary cultures, both proteins were expressed in all mammary carcinoma cultures.
representative results from immunocytofluorescence are reported in figure  <dig>  including the cxcr <dig> positivity.
in order to analyze the proliferative effects induced by cxcr <dig> activation, the obtained mammary carcinoma primary cultures were starved for  <dig> h and treated with  <dig> nm sdf- <dig> for further  <dig> h , and then mtt cell viability assays were performed.
sdf- <dig> exposure induced a statistically significant proliferative stimulus in  <dig> out of  <dig> feline mammary cultures as compared to serum-starved untreated controls, with a mean increase of cell growth of +30%, p <  <dig>  .
to discriminate the level of growth-arrest induced by serum deprivation, in a subgroup of  <dig> cultures the sdf- <dig> stimulus was compared to complete medium  effects: serum was able to significantly recover the proliferative rate of cells of about +50% over the corresponding starved cultures.
thus, sdf- <dig> significantly promotes feline mammary carcinoma cell growth, although with individual differences among cultures from different tumors, that is in the same order of magnitude, although lower, than that induced by fcs.
to characterize the mitogenic effects of sdf- <dig>  a dose-response curve  was performed in one primary culture .
the chemokine showed a dose-dependent effect with all the concentrations tested able to induce a significant growth stimulation, that was maximal at  <dig> nm.
to demonstrate that sdf- <dig> effects occurred through cxcr <dig> expressed in feline mammary carcinoma cells, we exposed cells to the cxcr <dig> antagonist amd <dig>  amd <dig> pre-treatment, was able to revert the sdf- <dig> increase in cell proliferation, confirming that cxcr <dig> is mediating sdf- <dig> effects in these cells.
in addition, we observed that amd <dig> significantly inhibits basal cell growth, possibly implying the occurrence of a constitutive role of cxcr <dig> in cell proliferation.
discussion
several studies demonstrated that: a) the expression of chemokine receptors in neoplastic cells is not random; b) cxcr <dig> is the most widely expressed chemokine receptor in most tumours; c) the effects of sdf- <dig> on cxcr4-expressing cancer cells are pleiotropic  <cit> .
in particular, sdf-1/cxcr <dig> axis is enrolled in many functional aspects of tumour progression, such as angiogenesis, site-specific metastasization, proliferation and survival of neoplastic cells  <cit> .
it is also recognized that metastastic behaviour of cancer cells may be reinforced by hypoxia, a condition that induce up-regulation of cxcr <dig> expression mediated by hypoxia-inducible factor-1a   <cit> .
sdf-1/cxcr <dig> 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  <cit> , lung  <cit> , ovarian  <cit> , renal  <cit> , prostate  <cit> , and neuroblastoma  <cit> .
moreover, it seems relevant to remind that cxcr <dig> expression has been associated to a worse prognosis in human tumours, and then, in the future, it could be also used as prognostic indicator  <cit> .
in contrast with the large number of studies in human oncology, there are only few scientific articles supporting the role of sdf-1/cxcr <dig> axis in spontaneous tumours of domestic animals.
tanabe and colleagues  <cit>  observed that the receptor's mrna is not identifiable in mammary tissue of healthy cats.
however, they found cxcr <dig> mrna in area adjacent to necrotic tissue, surrounding blood vessels and in cells infiltrating the lymphatic tissue in  <dig> % of  <dig> samples.
in the same study, the authors observed a statistically significant relationship between infiltration of neoplastic cells in lymphatic and cxcr <dig> expression  <cit> , although no relationship was observed between cxcr <dig> expression and one-year survival time of the cats included in the study  <cit> .
in another study, higher expression of cxcr <dig> 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 cxcr <dig> than non-neoplastic mammary tissues  <cit> .
in the present study we demonstrated, for the first time, a high cxcr <dig> 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 cxcr <dig> 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 cxcr <dig> expression in normal and benign mammary tissues may suggest a specificity of cxcr <dig> 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-cxcr <dig> 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  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  <cit> .
the interest in researching on sdf-1/cxcr <dig> pathways is also oriented to discover 'new chemical entities' able to block this mechanism and specifically inhibit cxcr <dig> functions.
actually, there are some examples published in the international literature reporting the possible use of cxcr <dig> inhibitors.
the latter have been used to reduce growth and metastasis of head and neck cancers and intracranial growth of brain tumours  <cit> .
these results have been also supported by in vitro experiments that proved the efficacy of cxcr <dig> inhibitors in blocking sdf-1/cxcr4-mediated proliferation and migration in breast cancer and lymphoblastic leukaemia  <cit> .
in veterinary oncology, oonuma and collaborators  <cit>  found a similar response in vitro using established feline mammary carcinoma cell lines incubated with cxcr <dig> antagonists.
here, besides the expression of cxcr <dig> in feline mammary tumours, we also investigated the functional role of this receptor in mediating proliferative signals.
in vitro,  <dig> out of  <dig> analyzed primary cultures of mammary carcinoma cells showed significant increased cell proliferation in response to cxcr <dig> activation by nanomolar concentrations of sdf- <dig>  the effect was quantitatively variable but reached, on the average a values similar to that induced by fcs, thus confirming that sdf-1/cxcr <dig> axis is an important signalling pathway involved in feline mammary cell proliferation.
interestingly, the use of the cxcr <dig> antagonist amd <dig>  while confirming the specificity of sdf- <dig> effects in vitro through cxcr <dig>  showed a reduction also in basal proliferation rate.
thus, it could be hypothesized that a basal sdf- <dig> secretion leads to autocrine cxcr <dig> activation in some tumor cell culture.
this observation may also explain why in few cultures a lower proliferation is induced by exogenous sdf- <dig> that could be masked by the autocrine cxcr <dig> activation.
a similar different response, related to constitutive autocrine sdf- <dig> effect, was recently demonstrated in human pituitary adenoma cell cultures in vitro  <cit> .
a larger number of tumors have to be analysed to confirm this hypothesis.
these data, although preliminary, strongly suggest that cxcr <dig> 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  <cit> .
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.
CONCLUSIONS
with the increase in the development of novel therapeutic agents feline mammary carcinomas can provide a useful model to test new drugs, as far as efficacy and toxicity, including novel cxcr <dig> antagonists.
in conclusion, our results suggest that routine evaluation of cxcr <dig> in feline mammary neoplastic lesions might be useful for selection of cases which may be treated with targeted chemotherapy.
however, further investigations are required to better evaluate the potential role of cxcr <dig> as prognostic factor and as a target for novel chemotherapeutic agents.

 chemokines are small messengers with chemoattractant function. they belong to a large superfamily of peptides produced and secreted by different cell types and classified in four groups accordingly to structural determinants. the complex chemokine system is involved in a wide range of cell functions ranging from organogenesis to malignancy. chemokine activity is mediated by the activation of a family of specific g protein coupled receptors. chemokine receptor activation is mediated by coupling to intracellular heterotrimeric g-proteins associated with the inner surface of the plasma membrane. upon ligand binding, chemokine receptors promote g protein activation leading to the inhibition of camp synthesis and the activation of phospholipase c that cleaves phosphatidylinositol -bisphosphate into the second messengers inositol triphosphate and diacylglycerol. dag activates protein kinase-c, while ip induces the release of calcium ions from intracellular stores. several studies supported the role of chemokinergic axis in physiological activities including organogenesis, haematopoiesis, angiogenesis, homing of lymphocytes, immune response and inflammation. among cxc chemokine receptors, in the past years, cxcr attracted great attention for its pleiotropic activity outside the immune system. cxcr activity is dependent on its interaction with its unique ligand: stromal cell-derived factor-. sdf- was recently reported to bind also a second receptor cxcr which regulates very different cellular activities including tumor angiogenesis. beside regulation of ca++ homeostasis and pkc activation, cxcr also modulate erk1/ map kinase and akt activities through a paracrine/autocrine mechanism. the activation of all these signalling cascades generates specific biological responses, such as chemotaxis, degranulation, release of superoxide anions, and cell proliferation. over-expression of cxcr is considered a key regulatory step in several human malignancies included breast cancer, resulting in a poor prognosis. in detail, cxcr activity was involved in the oestrogen resistance of breast cancer. moreover, high cxcr expression has been related to the metastatic potential of breast cancer cells, since. in vitro experiments showed that cxcr activation by sdf- regulates motility and metastatic potential of neoplastic epithelial cell lines, while cxcr inhibitors are effective in reducing chemotactic and metastatic potential of this receptor. in cats, most studies are focused on the characterization of the cxcr role associated with fiv infection, because of the role of this receptor as mediator of virus cell entry, as also observed in humans. conversely, few studies have been targeted on the role of cxcr in feline mammary gland neoplasia, although cxcr may have a role in development of mammary carcinoma in cats as in women. feline mammary tumors show age, incidence, histopathology and pattern of metastasis similar to human breast cancer. in addition, the lack of estrogens dependence in most of these tumours, suggests that this cancer may represent a suitable animal model for estrogen receptor negative breast cancer. recently, feline mammary carcinoma subtypes have been described to share features with human inflammatory mammary carcinoma. at the molecular level her overexpressing malignancies in cats may be considered homologue of the her positive- poor prognosis-human counterpart. thus, research in this field may be relevant to better understand the biology of feline mammary tumour, also as a comparative model for human breast cancer, and to evaluate the cxcr role in cancer mechanisms. moreover, immunohistochemistry screening of feline mammary tumours may have interesting applications in clinical practice as prognostic factor and to evaluate the possible response to cxcr inhibitors. the aim of the present study was to investigate, by immunohistochemistry, the levels of cxcr expression in feline mammary tumours and metastases, and the proliferative activity induced by sdf- on feline carcinoma primary cultures.  feline mammary tumours and histological diagnoses the clinical and pathological data of the feline mammary carcinomas analysed are summarised in table the cats under study included domestic short-hairs and persian; were intact females, while were spayed. the age ranged from to years. in one case the age was not available. primary malignant tumours were observed in 24/ cats, 7/ animals beard two neoplastic mammary glands thus we collected a total of primary tumour samples. only 2/ benign tumours were observed. the primary tumor samples under evaluation included simple tubulopapillary primary carcinomas, of which four of them gave metastasis. the other samples were diagnosed as solid carcinomas of which one gave lymph node metastasis, cribriform carcinomas, simple tubular carcinomas and metastatic adenosquamous carcinoma.. twenty-seven out of primary malignant lesions were classified as high grade carcinomas and as intermediate grade. no grade i tumours were observed in this study. only benign tumours were diagnosed in this group of animals. four normal tissue samples, derived from non-neoplastic mammary glands resected during tumor surgery, were included in the study as cxcr expression reference. cat f, female, fs, female spayed; dsh, domestic short-hair; pc, persian; stpc, simple tubulopapillary carcinoma; sc, solid carcinoma; stc, simple tubular carcinoma; cc, cribriform carcinoma; ba, basaloid adenoma; ca, complex adenoma; asc, adenosquamous carcinoma; n.a., not available. for normal tissues the mammary gland topography is indicated. *a and b indicate samples from two glands of the same cat, m labels metastatic tissue. primary tumours and corresponding metastases of the same cat are reported in bold. cxcr expression in feline mammary carcinomas the expression of cxcr analyzed by immunohistochemistry, was observed in out of of primary carcinoma samples although the staining pattern and intensity differed between tumors as evaluated by cxcr scores reported in table in out of carcinoma-bearing animals we analyzed tumors taken from two different mammary glands of the same cat; in this subgroup 4/ tumors showed different score of cxcr expression among carcinomas derived from gland a and b from the same cat, possibly reflecting heterogeneity across tumour lesions. the expression of cxcr was mostly confined to the membrane and cytoplasm of neoplastic cells and differed in intensity and staining pattern and among primary and, in most cases, the corresponding metastatic lesions. negative controls and cxcr antibody specificity were performed as reported in supplementary figure s to evaluate the specificity of cxcr expression in mammary carcinomas and metastasis, we analyzed the level of cxcr immunopositivity in normal mammary tissues and benign lesions. as depicted in figure, normal mammary gland tissue did not express detectable level of cxcr while epithelial cells of basaloid adenoma appeared weakly positive for cxcr in scattered areas throughout the section, and the complex adenoma was essentially negative. interestingly, out of metastatic tumours appeared to have a stronger cxcr expression as compared to the respective primary tumours. nevertheless, in two metastatic sites, the neoplastic cells showed either a lighter immunoreaction if compared with the respective primary tumour or no changes. sdf-1/cxcr role in primary cultures of feline mammary carcinoma to investigate the biological role of cxcr expression in feline mammary carcinoma, fibroblast-free, primary cell cultures were obtained from primary carcinomas. in vitro feline mammary cancer cells appeared as adherent monolayers with spindle-like shape. the expression of cxcr in each culture was verified by if staining, confirming the immunopositivity of mammary carcinoma primary cells, as in the tissue of origin. the absence of contaminant fibroblasts, which can interfere with both immunofluorescence and proliferation studies, was demonstrated by the lack of pro-collagen expression in the mammary carcinoma cell cultures, while isolated tumour fibroblasts, used as positive controls, showed a strong staining. beside cxcr expression, a phenotypic characterization of the cells from the primary cultures was performed, by analyzing the expression of common mammary lineage markers, or proteins overexpressed in mammary carcinoma conferring malignant behaviour. as expected, feline mammary carcinoma cells from all tumour tested, homogeneously expressed the mammary epithelial marker ema. moreover, in vitro cultures abundantly expressed ck and, in variable proportion of cells, ck. in addition, we include the evaluation of er-α immunocytofluorescence to define its pattern of expression in feline mammary cancer cells, detecting different levels of positivity, but generally low or weak. as far as her2/neu and egfr expression in feline primary cultures, both proteins were expressed in all mammary carcinoma cultures. representative results from immunocytofluorescence are reported in figure including the cxcr positivity. in order to analyze the proliferative effects induced by cxcr activation, the obtained mammary carcinoma primary cultures were starved for h and treated with nm sdf- for further h, and then mtt cell viability assays were performed. sdf- exposure induced a statistically significant proliferative stimulus in out of feline mammary cultures as compared to serum-starved untreated controls, with a mean increase of cell growth of +30%, p <. to discriminate the level of growth-arrest induced by serum deprivation, in a subgroup of cultures the sdf- stimulus was compared to complete medium effects: serum was able to significantly recover the proliferative rate of cells of about +50% over the corresponding starved cultures. thus, sdf- significantly promotes feline mammary carcinoma cell growth, although with individual differences among cultures from different tumors, that is in the same order of magnitude, although lower, than that induced by fcs. to characterize the mitogenic effects of sdf- a dose-response curve was performed in one primary culture. the chemokine showed a dose-dependent effect with all the concentrations tested able to induce a significant growth stimulation, that was maximal at nm. to demonstrate that sdf- effects occurred through cxcr expressed in feline mammary carcinoma cells, we exposed cells to the cxcr antagonist amd amd pre-treatment, was able to revert the sdf- increase in cell proliferation, confirming that cxcr is mediating sdf- effects in these cells. in addition, we observed that amd significantly inhibits basal cell growth, possibly implying the occurrence of a constitutive role of cxcr in cell proliferation. discussion several studies demonstrated that: a) the expression of chemokine receptors in neoplastic cells is not random; b) cxcr is the most widely expressed chemokine receptor in most tumours; c) the effects of sdf- on cxcr4-expressing cancer cells are pleiotropic. in particular, sdf-1/cxcr axis is enrolled in many functional aspects of tumour progression, such as angiogenesis, site-specific metastasization, proliferation and survival of neoplastic cells. it is also recognized that metastastic behaviour of cancer cells may be reinforced by hypoxia, a condition that induce up-regulation of cxcr expression mediated by hypoxia-inducible factor-1a. sdf-1/cxcr 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, lung, ovarian, renal, prostate, and neuroblastoma. moreover, it seems relevant to remind that cxcr expression has been associated to a worse prognosis in human tumours, and then, in the future, it could be also used as prognostic indicator. in contrast with the large number of studies in human oncology, there are only few scientific articles supporting the role of sdf-1/cxcr axis in spontaneous tumours of domestic animals. tanabe and colleagues observed that the receptor's mrna is not identifiable in mammary tissue of healthy cats. however, they found cxcr mrna in area adjacent to necrotic tissue, surrounding blood vessels and in cells infiltrating the lymphatic tissue in % of samples. in the same study, the authors observed a statistically significant relationship between infiltration of neoplastic cells in lymphatic and cxcr expression, although no relationship was observed between cxcr expression and one-year survival time of the cats included in the study. in another study, higher expression of cxcr 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 cxcr than non-neoplastic mammary tissues. in the present study we demonstrated, for the first time, a high cxcr 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 cxcr 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 cxcr expression in normal and benign mammary tissues may suggest a specificity of cxcr 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-cxcr 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 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. the interest in researching on sdf-1/cxcr pathways is also oriented to discover'new chemical entities' able to block this mechanism and specifically inhibit cxcr functions. actually, there are some examples published in the international literature reporting the possible use of cxcr inhibitors. the latter have been used to reduce growth and metastasis of head and neck cancers and intracranial growth of brain tumours. these results have been also supported by in vitro experiments that proved the efficacy of cxcr inhibitors in blocking sdf-1/cxcr4-mediated proliferation and migration in breast cancer and lymphoblastic leukaemia. in veterinary oncology, oonuma and collaborators found a similar response in vitro using established feline mammary carcinoma cell lines incubated with cxcr antagonists. here, besides the expression of cxcr in feline mammary tumours, we also investigated the functional role of this receptor in mediating proliferative signals. in vitro, out of analyzed primary cultures of mammary carcinoma cells showed significant increased cell proliferation in response to cxcr activation by nanomolar concentrations of sdf- the effect was quantitatively variable but reached, on the average a values similar to that induced by fcs, thus confirming that sdf-1/cxcr axis is an important signalling pathway involved in feline mammary cell proliferation. interestingly, the use of the cxcr antagonist amd while confirming the specificity of sdf- effects in vitro through cxcr showed a reduction also in basal proliferation rate. thus, it could be hypothesized that a basal sdf- secretion leads to autocrine cxcr activation in some tumor cell culture. this observation may also explain why in few cultures a lower proliferation is induced by exogenous sdf- that could be masked by the autocrine cxcr activation. a similar different response, related to constitutive autocrine sdf- 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 cxcr 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. 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.  with the increase in the development of novel therapeutic agents feline mammary carcinomas can provide a useful model to test new drugs, as far as efficacy and toxicity, including novel cxcr antagonists. in conclusion, our results suggest that routine evaluation of cxcr in feline mammary neoplastic lesions might be useful for selection of cases which may be treated with targeted chemotherapy. however, further investigations are required to better evaluate the potential role of cxcr as prognostic factor and as a target for novel chemotherapeutic agents. 
chemokines regulate many physiological and pathological processes including organogenesis, chemotaxis of inflammatory cells, as well as tumour progression and metastasization. in particular, the chemokine/receptor pair sdf-1/cxcr has been involved in the regulation of metastatic potential of neoplastic cells, including breast cancer. cxcr expression in neoplastic cells was evaluated by immunohistochemistry. moreover, cxcr activation in primary cultures of feline mammary carcinomas causes increase in the proliferative rate. the aim of this study was the immunohistochemical defininition of the expression profile of cxcr in primary and metastatic feline mammary carcinomas and the evaluation of the role of sdf- in feline mammary tumour cell proliferation. six primary, fibroblast-free primary cultures were obtained from fresh feline mammary carcinomas and characterized by immunofluorescence for cxcr and malignant mammary cell marker expression. finally, in the primary cultures obtained from primary feline mammary carcinomas cxcr expression was detected in all cells and its activation by sdf- in vitro treatment caused a significant increase in the proliferation rate in out of tumours. thus, sdf-1/cxcr system seems to play a tumorigenic in feline mammary gland malignancy and in vitro cultures from these tumour samples may represent an experimental model to investigate the biological and pharmacological role of this chemokinergic axis. the level of cxcr immunoreactivity was semi-quantitatively estimated as cxcr score evaluating both the number of positive cells and the intensity of staining. these results indicate that malignant feline mammary tumours commonly express cxcr with a higher level in malignant tumours, and, in most of the cases analysed, metastatic cells display stronger immunoreactivity for cxcr than the corresponding primary tumours. a total of mammary surgical samples, including primary tumours, metastases, and normal mammary tissues were anlyzed. in benign lesions analyzed, only the single basaloid adenoma showed a mild positive immunostaining against cxcr normal tissue did not show cxcr immunoreactivity. cxcr expression was observed in out of malignant tissues with a higher cxcr score observed in out of metastatic lesions than in the respective primary tumours.
 mammary tumours frequently develop in female domestic cats being highly malignant in a large percentage of cases. chemokines regulate many physiological and pathological processes including organogenesis, chemotaxis of inflammatory cells, as well as tumour progression and metastasization. in particular, the chemokine/receptor pair sdf-1/cxcr has been involved in the regulation of metastatic potential of neoplastic cells, including breast cancer. the aim of this study was the immunohistochemical defininition of the expression profile of cxcr in primary and metastatic feline mammary carcinomas and the evaluation of the role of sdf- in feline mammary tumour cell proliferation.  a total of mammary surgical samples, including primary tumours, metastases, and normal mammary tissues were anlyzed. tumor samples were collected from a total number of animals, as in some cases concurrent occurrence of neoplasm in more than one mammary gland was observed. tissues were processed for standard histological examination, and all lesions were classified according to the world health organization criteria. cxcr expression in neoplastic cells was evaluated by immunohistochemistry. the level of cxcr immunoreactivity was semi-quantitatively estimated as cxcr score evaluating both the number of positive cells and the intensity of staining. six primary, fibroblast-free primary cultures were obtained from fresh feline mammary carcinomas and characterized by immunofluorescence for cxcr and malignant mammary cell marker expression. sdf-1-dependent in vitro proliferative effects were also assayed. cxcr expression was observed in out of malignant tissues with a higher cxcr score observed in out of metastatic lesions than in the respective primary tumours. in benign lesions analyzed, only the single basaloid adenoma showed a mild positive immunostaining against cxcr normal tissue did not show cxcr immunoreactivity. cxcr score was statistically significantly associated with the histological features of the samples, showing an increase accordingly with the degree of neoplastic transformation. finally, in the primary cultures obtained from primary feline mammary carcinomas cxcr expression was detected in all cells and its activation by sdf- in vitro treatment caused a significant increase in the proliferation rate in out of tumours.  these results indicate that malignant feline mammary tumours commonly express cxcr with a higher level in malignant tumours, and, in most of the cases analysed, metastatic cells display stronger immunoreactivity for cxcr than the corresponding primary tumours. moreover, cxcr activation in primary cultures of feline mammary carcinomas causes increase in the proliferative rate. thus, sdf-1/cxcr system seems to play a tumorigenic in feline mammary gland malignancy and in vitro cultures from these tumour samples may represent an experimental model to investigate the biological and pharmacological role of this chemokinergic axis. 