CXCR4 expression in feline mammary carcinoma cells: evidence of a proliferative role for the SDF-1/CXCR4 axis

Abstract

BackgroundMammary 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/CXCR4 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 CXCR4 in primary and metastatic feline mammary carcinomas and the evaluation of the role of SDF-1 in feline mammary tumour cell proliferation.ResultsA total of 45 mammary surgical samples, including 33 primary tumours (31 carcinomas and 2 adenomas), 6 metastases, and 4 normal mammary tissues were anlyzed. Tumor samples were collected from a total number of 26 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. CXCR4 expression in neoplastic cells was evaluated by immunohistochemistry. The level of CXCR4 immunoreactivity was semi-quantitatively estimated as CXCR4 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 CXCR4 and malignant mammary cell marker expression. SDF-1-dependent in vitro proliferative effects were also assayed. CXCR4 expression was observed in 29 out of 31 malignant tissues with a higher CXCR4 score observed in 4 out of 6 metastatic lesions than in the respective primary tumours. In 2 benign lesions analyzed, only the single basaloid adenoma showed a mild positive immunostaining against CXCR4. Normal tissue did not show CXCR4 immunoreactivity. CXCR4 score was statistically significantly associated with the histological features of the samples, showing an increase accordingly with the degree of neoplastic transformation (from normal tissue to metastatic lesions). Finally, in the primary cultures obtained from 6 primary feline mammary carcinomas CXCR4 expression was detected in all cells and its activation by SDF-1 in vitro treatment caused a significant increase in the proliferation rate in 5 out of 6 tumours.ConclusionsThese results indicate that malignant feline mammary tumours commonly express CXCR4, with a higher level in malignant tumours, and, in most of the cases analysed, metastatic cells display stronger immunoreactivity for CXCR4 than the corresponding primary tumours. Moreover, CXCR4 activation in primary cultures of feline mammary carcinomas causes increase in the proliferative rate. Thus, SDF-1/CXCR4 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 biologi...

In vitro and in vivo characterization of stem-like cells from canine osteosarcoma and assessment of drug sensitivity

“…Among proliferation-and metastasis-associated proteins in CSCs, the cytokine CXCL12 and its receptor CXCR4 contribute to bone sarcoma progression [90], in which the metastatic diffusion represents a major life-limiting factor for both humans and dogs. Few studies explored the biological relevance of the CXCL12/CXCR4 axis in canine OSA [91], showing high expression similarly to other canine solid tumors [55]. In the present study, CXCL12/CXCR4 expression within sarcospheres well-matches with their diffuse detection in tumor xenografts and original OSA tissues of the corresponding dogs (see Tables), suggesting that the regulation of CSC invasive behavior by this chemokine system involve autocrine/paracrine activity besides the chemokinergic stimuli produced by tumor microenvironment.…”

“…IHC was performed on 4-μm sections of formalin-fixed paraffin-embedded tissue derived from 12 OSA (9 males and 3 females with mean age 7.7 years, range 0.5-14). As previously described [55], deparaffinized/rehydrated sections were incubated in citrate-antigen retrieval buffer, nonspecific immunoreactivity was blocked with 10% normal goat serum (Sigma-Aldrich) and primary antibodies applied overnight at 4 °C. The following antibodies were used: Oct4 (Abcam), CXCR4 (Sigma-Aldrich), CXCL12 (Abcam), CD117 and STAT3 (SantaCruz).…”

“…OSA1, OSA2 and OSA3 CSC express, although not homogeneously, all the above mentioned markers, thus indicating the prevalence of cells with stem-like phenotype within spheroids ( Figure 2). In addition, we tested the immunoreactivity for the chemokine receptor CXCR4 and its ligand CXCL12, both overexpressed in several human [63][64][65] and canine tumors [55] including bone sarcomas, and contributing to CSC maintenance, migration, and homing in metastatic sites [66]. IF analysis revealed that both proteins are expressed in the majority of sarcosphere cells (although CXCL12 was detected in less OSA2 cells) ( Figure 2) confirming the relevance of the CXCL12/CXCR4 axis in OSA likely involved in the control and/or modulation of cell stemness.…”

In vitro and in vivo antiproliferative activity of metformin on stem-like cells isolated from spontaneous canine mammary carcinomas: translational implications for human tumors

“…Immunohistochemistry (IHC) was as described previously [ 54 ]. Antibodies used were as follows: anti-EGFR (rabbit polyclonal; Cell Signaling Technology), anti-ER-α clone 1D5, anti-CD44, clone DF1485 and anti-Ki-67, clone MIB-1, (mouse monoclonal, Dako, Glostrup, Denmark) and anti-CD24 (goat polyclonal; SantaCruz Biothechnology).…”

Emerging Targets in Pituitary Adenomas: Role of the CXCL12/CXCR4-R7 System

“…Furthermore, CXCL12 is also indirectly implicated in tumor pathogenesis, acting as chemoattractant for CXCR4-positive cells, directing tumor cell migration [ 155 – 157 ] and controlling invasive and metastatic properties of CXCR4-expressing cancer cells to distant organs [ 158 , 159 ]. The invasive behavior of cancer cells might indirectly depend on locally released CXCL2 that, via an autocrine mechanism, binds to CXCR4 impairing chemotaxis towards CXCL12-producing target organs and metastatic spread [ 160 ].…”