Canine Mammary Carcinoma Cell Research Articles

Effect of Melatonin on Chemoresistance Exhibited by Spheres Derived from Canine Mammary Carcinoma Cells.

Mammary cancer is a frequent disease in female dogs, where a high proportion of cases correspond to malignant tumors that may exhibit drug resistance. Within the mammary tumor microenvironment, there is a cell subpopulation called cancer stem cells (CSCs), which are capable of forming spheres in vitro and resisting anti-tumor treatments, partly explaining the recurrence of some tumors. Previously, it has been described that spheres derived from canine mammary carcinoma cells CF41.Mg and REM 134 exhibit stemness characteristics. Melatonin has shown anti-tumor effects on mammary tumor cells; however, its effects have been poorly evaluated in canine mammary CSCs. This study aimed to analyze the effect of melatonin on the chemoresistance exhibited by stem-like neoplastic cells derived from canine mammary carcinoma to cytotoxic drugs such as doxorubicin and mitoxantrone. CF41.Mg and REM 134 cells were cultured in high-glucose DMEM supplemented with fetal bovine serum and L-glutamine. The spheres were cultured in ultra-low attachment plates in DMEM/F12 medium without fetal bovine serum and with different growth factors. The CD44+/CD24-/low phenotype was analyzed by flow cytometry. The viability of sphere-derived cells (MTS reduction) was studied in the presence of melatonin (0.1 or 1 mM), doxorubicin, mitoxantrone, and luzindole. In addition, the gene (RT-qPCR) of the multidrug resistance bombs MDR1 and ABCG2 were analyzed in the presence of melatonin. Both cell types expressed the MT1 gene, which encodes the melatonin receptor MT1. Melatonin 1 mM does not modify the CD44+/CD24-/low phenotype; however, the hormone reduced viability (p < 0.0001) only in CF41.Mg spheres, without inducing an additive effect when co-incubated with cytotoxic drugs. These effects were independent of the binding of the hormone to its receptor MT1, since, by pharmacologically inhibiting them, the effect of melatonin was not blocked. In CF41.Mg spheres, the relative gene expression of ABCG2 and MDR1 was decreased in response to the hormone (p < 0.001). These results indicate that melatonin negatively modulates the cell survival of spheres derived from CF41.Mg cells, in a way that is independent of its MT1 receptor. These effects did not counteract the resistance to doxorubicin and mitoxantrone, even though the hormone negatively regulates the gene expression of MDR1 and ABCG2.

Cytotoxic effects of crotoxin from Crotalus durissus terrificus snake in canine mammary tumor cell lines.

Mammary gland tumors are the most prevalent neoplasm in intact female dogs, and they are good natural models to study comparative oncology. Most canine mammary malignancies, as in women, are commonly refractory to conventional therapies and demand continuous new therapeutic approaches. Crotalus durissus terrificus, also called rattlesnake, has more than 60 different proteins in its venom with multiple pharmaceutical uses, such as antitumor, antiviral, and antimicrobial action. Crotoxin, a potent β-neurotoxin formed by the junction of two subunits, a basic subunit (CB-PLA2) and an acidic subunit (crotapotin), has already been reported to have anticancer properties in different types of cancers. In this work, we describe the cytotoxic potential of crotoxin and its subunits compared to doxorubicin (drug of choice) in two canine mammary carcinoma cell lines. Crotoxin, CB-PLA2, crotalic venom, and doxorubicin decreased cell viability and the ability to migrate in a dose-dependent manner, and crotapotin did not present an antitumoral effect. For all compounds, the predominant cell death mechanism was apoptosis. In addition, crotoxin did not show toxicity in normal canine mammary gland cells. Therefore, this work showed that crotoxin and CB-PLA2 had cytotoxic activity, migration inhibition, and pro-apoptotic potential in canine mammary gland carcinoma cell lines, making their possible use in cancer research.

Antitumor Effect of Berberine Analogs in a Canine Mammary Tumor Cell Line and in Zebrafish Reporters via Wnt/β-Catenin and Hippo Pathways.

The heterogeneous nature of human breast cancer (HBC) can still lead to therapy inefficacy and high lethality, and new therapeutics as well as new spontaneous animal models are needed to benefit translational HBC research. Dogs are primarily investigated since they spontaneously develop tumors that share many features with human cancers. In recent years, different natural phytochemicals including berberine, a plant alkaloid, have been reported to have antiproliferative activity in vitro in human cancers and rodent animal models. In this study, we report the antiproliferative activity and mechanism of action of berberine, its active metabolite berberrubine, and eight analogs, on a canine mammary carcinoma cell line and in transgenic zebrafish models. We demonstrate both in vitro and in vivo the significant effects of specific analogs on cell viability via the induction of apoptosis, also identifying their role in inhibiting the Wnt/β-catenin pathway and activating the Hippo signals with a downstream reduction in CTGF expression. In particular, the berberine analogs NAX035 and NAX057 show the highest therapeutic efficacy, deserving further analyses to elucidate their mechanism of action more in detail, and in vivo studies on spontaneous neoplastic diseases are needed, aiming at improving veterinary treatments of cancer as well as translational cancer research.

Newcastle disease virus LaSota strain induces apoptosis and activates the TNFα/NF-κB pathway in canine mammary carcinoma cells.

Spontaneous canine mammary carcinomas (CMCs) have been widely considered a good research model for human breast cancers, which brings much attention to CMCs. In recent years, the oncolytic effect of Newcastle disease virus (NDV) on cancer cells has been widely studied, but its effect on CMCs is still unclear. This study aims to investigate the oncolytic effect of NDV LaSota strain on canine mammary carcinoma cell line (CMT-U27) in vivo and in vitro. The in vitro cytotoxicity and immunocytochemistry experiments showed that NDV selectively replicated in CMT-U27 cells, and inhibited cell proliferation and migration but not in MDCK cells. KEGG analysis of transcriptome sequencing indicated the importance of the TNFα and NF-κB signalling pathways in the anti-tumour effect of NDV. Subsequently, the significantly increased expression of TNFα, p65, phospho-p65, caspase-8, caspase-3 and cleaved-PARP proteins in the NDV group suggested that NDV induced CMT-U27 cells apoptosis by activating the caspase-8/caspase-3 pathway and the TNFα/NF-κB signalling pathway. Nude mice tumour-bearing experiments showed that NDV could significantly decrease the growth rate of CMC in vivo. In conclusion, our study demonstrates the effective oncolytic effects of NDV on CMT-U27 cells in vivo and in vitro, and suggests NDV as a promising candidate for oncolytic therapy.

Tetramethylpyrazine-Rhein Derivative inhibits the migration of canine inflammatory mammary carcinoma cells by mitochondrial damage-mediated apoptosis and cadherins downregulation

BackgroundCanine inflammatory mammary carcinoma (CIMC) has a high incidence of metastasis, high lethality, and poor prognosis, which needs novel adjuvant agents. Tetramethylpyrazine-Rhein Derivative (TRD) has been shown to have antitumor activity, which is a potential research direction for CIMC. PurposeThis study evaluated the efficacy of TRD on CIMC in vitro and in vivo, and provided possibilities for the application of active compounds in traditional Chinese medicine. MethodsIn vitro, TRD cytotoxicity was measured with CCK-8. Flow cytometry and transmission electron microscope were used to detect the cell cycle, cell death, and changes in mitochondria. Wound-healing assay, cell invasion assay, and scanning electron microscope were used to evaluate the suppression of cell migration and invasion. Expression changes were detected by RT-qPCR and western blot assay. In vivo, the lung metastasis models were randomly divided into control, low-dose TRD, high-dose TRD, and positive groups. Each group was administered orally once a day for 18 days and took in vivo imaging photos. ResultsThe IC50 of TRD in CHMp and MDCK were 42.59 and 79.37 μM, respectively. TRD mediated cell apoptosis by mitochondrial damage and caused S and G2/M phase arrest by downregulating cyclin B1. Moreover, TRD reduced filopodia and inhibited cell migration by downregulating cadherins. In CIMC lung metastasis models, TRD could effectively inhibit tumor growth (P < 0.001) in the lungs without significant toxicity. ConclusionTRD showed potential activity to inhibit CIMC lung metastasis with multi-target and low toxicity.

Synthesis of structurally diverse derivatives of aconitine-type diterpenoid alkaloids and their anti-proliferative effects on canine breast cancer cells

As one of the most common malignancies in female dogs, no drugs have been developed specifically for the treatment of canine mammary carcinoma. In our previous study, a series of diterpenoid alkaloids derivatives were synthesized and exhibited good anti-proliferative activity in vitro against both normal and adriamycin-resistant human breast cancer cells lines. In this study, a series of structurally diverse aconitine-type alkaloids derivatives were also synthesized basing on the minimal modification principle, by modifying on A-ring, C-ring, D-ring, N-atom or salt formation on aconitine skeleton. Their anti-proliferative effects and mechanism on canine mammary cancer cells were investigated, exhibiting the importance of the substitution at A ring, the long chain ester at the C8, the hydroxyl group at the C13, the phenyl ring at the C14 and the N-ethyl group, while the methoxy group at the C1 and C16 showed little effect on the activity. The results of the proliferation, apoptosis and ultrastructure tests of the treated canine mammary carcinoma cells referred that the representative compound, aconitine linoleate (25) could block the cell cycle of canine mammary carcinoma cells in the G0/G1 phase, and exhibit the anti-proliferative effect by inducing apoptosis.

MTOR pathway as a potential therapeutic target for cancer stem cells in canine mammary carcinoma.

Mammary adenocarcinoma, the most common cancer in female dogs, often exhibits the lymph node and lung metastases and has a higher mortality rate. However, mammary adenocarcinoma has no established treatment, except early surgical excision. Canine mammary carcinoma has many common features with human mammary carcinoma, including clinical characteristics, heterogeneity, and genetic aberrations, making it an excellent spontaneous tumor model for human breast cancer. Diverse cancers comprised heterogeneous cell populations originating from cancer stem cells (CSCs) with self-renewal ability. Therefore, in addition to conventional therapy, therapeutic strategies targeting CSCs are essential for cancer eradication. The present study aimed to extract inhibitors of canine mammary CSCs that suppress their self-renewal ability. Sphere-formation assay, which evaluates self-renewal ability, was performed for the canine mammary cancer cell lines CTBp and CNMp. The spheres formed in this assay were used in inhibitor library screening, which identified various signaling pathways such as proteosome, stress inducer, and mammalian target of rapamycin (mTOR). The present study focused on the mTOR signaling pathway. Western blotting showed higher levels of phosphorylated mTOR in sphere-forming CTBp and CNMp cells than in adherent cells. Drug sensitivity examination using the mTOR inhibitors everolimus and temsirolimus revealed dose-dependent reductions in viability among both sphere-forming cells and adherent cells. Expression of phosphorylated mTOR in adherent and sphere-forming cells decreased by everolimus and temsirolimus treatment. In mice transplanted with CTBp-derived spheres, everolimus treatment significantly decreased tumor volume compared to control. These results reveal that the mTOR signaling pathway may be a potential to be a therapeutic target in both cancer cells and CSCs. Novel therapeutic strategies for canine mammary carcinoma are expected to benefit to human breast carcinoma as well.

Pretreatment with tadalafil attenuates cardiotoxicity induced by combretastatin A4 disodium phosphate in rats.

Combretastatin A4 disodium phosphate (CA4DP) is a prodrug of combretastatin A4 (CA4), a microtubule-disassembling agent that exhibits antitumor effects by inhibiting tumor cell proliferation and inducing morphological changes and apoptosis in vascular endothelial cells in tumors. However, cardiotoxicity induced by ischemia and hypertension is a severe adverse event. In this study, we focused on the fact that phosphodiesterase (PDE) 5 inhibitors dilate the heart and peripheral blood vessels and aimed to investigate whether co-administration of tadalafil, a PDE5 inhibitor, can attenuate cardiotoxicity without altering the antitumor effect of CA4DP. To investigate cardiotoxicity, CA4DP and/or tadalafil were administered to rats, and blood pressure, echocardiography, histopathology, and cGMP concentration in the myocardium were examined. Administration of CA4DP increased systolic blood pressure, decreased cardiac function, lowered cGMP levels in the myocardium, and led to necrosis of myocardial cells. Co-administration of tadalafil attenuated these CA4DP-induced changes. To investigate the antitumor effect, canine mammary carcinoma cell lines (CHMp-13a) and human umbilical vein endothelial cells were cultured with CA4 and/or tadalafil, and cell proliferation and endothelial vascular tube disruption were examined. CHMp-13a cells were transplanted into nude mice and treated with CA4DP and/or tadalafil. CA4-induced inhibition of cell proliferation and disruption of the endothelial vascular tube were not affected by co-treatment with tadalafil, and the antitumor effects of CA4DP in xenograft mice were not reduced by co-administration of tadalafil. These results revealed that myocardial damage induced by CA4DP was attenuated by co-administration of tadalafil while maintaining antitumor efficacy.

Molecular Characterization of CF33 Canine Cell Line and Evaluation of Its Ability to Respond against Infective Stressors in Sight of Anticancer Approaches.

Simple SummaryCanine mammary cancer is very common and has many similarities with human breast cancer. Risk factors, physiological and pathological behaviors, and the clinical course in dogs are very similar to humans. Several molecular similarities have also been reported, such as overexpression of EGF, proliferation markers, metalloproteinase and cyclooxygenase, TP53 mutations, and CXCR4/SDF1 axis activation. These common characteristics make these breast tumors resistant to conventional therapies. It is therefore necessary to study therapeutic alternatives. Cell lines could be helpful to test in vitro immunomodulant anti-cancer therapies, allowing a reduction of laboratory animals’ involvement in the preliminary tests and achieving results in a shorter time. Although the canine mammary carcinoma cell line CF33 has been widely used in many studies on dog mammary cancer, characterization of its gene expression profile and of the influence of infective stressors of this cell line is poor. Our study shows the interaction of CF33 and Salmonella Typhimurium (ST) as an infective stressor, indicating that these cells may represent an in vitro model for assessing novel therapeutic approaches using bacteria.Spontaneous mammary tumors are the most frequent neoplasms in bitches and show similarities with human breast cancer in risk factors, clinical course, and histopathology. The poor prognosis of some cancer subtypes, both in human and dog, demands more effective therapeutic approaches. A possible strategy is the new anticancer therapy based on immune response modulation through bacteria or their derivatives on canine mammary carcinoma cell lines. The aim of the present study was to analyze the CF33 cell line in terms of basal expression of immune innate genes, CXCR4 expression, and interaction with infectious stressors. Our results highlight that CF33 maintains gene expression parameters typical of mammary cancer, and provides the basal gene expression of CF33, which is characterized by overexpression of CXCR4, CD44, RAD51, LY96, and a non-continuous expression of TP53 and PTEN. No mutations appeared in the CXCR4 gene until the 58th passage; this may represent important information for studying the CXCR4 pathway as a therapeutic target. Moreover, the CF33 cell line was shown to be able to interact with Salmonella Typhimurium (ST) (an infective stressor), indicating that these cells could be used as an in vitro model for developing innovative therapeutic approaches involving bacteria.

The response of the canine mammary simple carcinoma and carcinosarcoma cells to 5-aminolaevulinic acid-based photodynamic therapy: An in vitro study.

Canine mammary gland tumors (CMGTs) are heterogeneous tumors and share many similar features with human breast cancer. Despite the improvement of current treatment options, new treatment modalities are required to effectively kill tumor cells without general toxicity in the treatment of CMGTs. Photodynamic therapy (PDT) is a promising method for cancer treatment. However, there is a limited study evaluating the therapeutic efficacy of PDT in the treatment of CMGTs. In this context, we, for the first time, investigated the therapeutic potential of 5-aminolaevulinic acid (5-ALA) mediated PDT at 6 and 12J/cm2 in two different subtypes [Tubulopapillary carcinoma (TPC) and carcinosarcoma (CS)] cells via different molecular analysis. The cytotoxic effects of 5-ALA/PDT on these cells were analyzed by intracellular PpIX level, WST-1 and ROS analysis. Furthermore, the underlying moleculer mechanism of 5-ALA/PDT mediated apoptotic effects on TPC and CS cells were evaluated Annexin V, AO/PI, RT-PCR and western blot analysis. The 5-ALA/PDT treatment upon irradiation considerably inhibited the viability of both TPC and CS cells (p<0.01) and caused apoptotic death through elevated ROS levels, the activation of Caspase-9, and Caspase-3, and the overexpression of Bax. However, the response of TPC and CS cells to 5-ALA/PDT was different. Our preliminary in vitro findings provide novel insights into the molecular mechanisms underlying 5-ALA/PDT mediated apoptosis in both TPC and CS cells. However, the therapeutic response of CMGT cells to 5-ALA/PDT is limited.

Establishment of stable expression of firefly luciferase and EGFP in a canine inflammatory mammary carcinoma cell line and tumor-bearing model in nude mice.

Canine inflammatory mammary carcinoma (CIMC) is a type of canine malignant mammary tumor with a poor prognosis and high mortality. We transduced firefly luciferase and enhanced green fluorescent protein (EGFP) into CHMp, a CIMC cell line, and established CHMp-Luc-EGFP cells. We investigated the characteristics of this cell line in vitro and in vivo. CHMp-Luc-EGFP was passaged continuously 75 times, with stable expression of luciferase and EGFP. Compared with the wild-type, CHMp-Luc-EGFP had similar proliferation, metastasis, histopathology characteristics, and expression of E-cadherin, N-cadherin, and Ki-67. A tumor-bearing model was established by implantation of CHMp-Luc-EGFP cells, and the dynamic changes of tumors were visualized and quantified using the IVIS imaging system. In summary, the cell line we established could reflect the biological characteristics of CHMp cells, visualize the tumor progression in vivo, and provide a powerful tool for the study of CIMC.

Establishment of a New Canine Inflammatory Mammary Carcinoma Cell Line and Analysis of its Cystine-glutamate Transporter Subunit Expression.

IntroductionInflammatory mammary carcinoma (IMC) is a rare disease with a poor prognosis and one affecting dogs. Inflammatory breast carcinoma (IBC) is a subtype of malignant breast cancer in humans with a high degree of malignancy and a similarly poor prognosis. Since the clinical symptoms and prognoses of both are similar, canine IMC has been considered as a model of human IBC. In this study, we newly established a stable IMC-derived cell line from a patient at the Yamaguchi University Animal Medical Center in Japan.Material and MethodsThe patient was a female toy poodle presenting with an inflamed mammary gland, which was diagnosed as IMC. The cell line was established from a tissue biopsy. Surface antigen marker (CD24 and CD44) expression was determined. Cystine/glutamate antiporter (xCT) expression was determined by Western blotting, flow cytometry and fluorescence immunostaining, and sulfasalazine was administered to ascertain if it suppressed xCT expression. Stem cell marker (Nanog, Sox2, Myc and Klf4) expression and aldehyde dehydrogenase (ALDH) activity were also investigated.ResultsThe cultured cells showed xCT, and its suppression showed downregulation of stem cell markers and ALDH activity. Stable cell proliferation was verified.ConclusionA new canine IMC-derived cell line was established. In the future, we aim to study the effect of xCT on the maintenance of cancer stem cell properties in canine tumours, and propose a new therapeutic method for the treatment of canine IMC by targeting xCT.

MicroRNA-124 inhibits canine mammary carcinoma cell proliferation, migration and invasion by targeting CDH2

Canine mammary carcinoma (CMC) is the most common malignant tumor and the second leading cause of cancer-related mortality of dogs worldwide. MicroRNA-124 (miR-124) is an important tumor suppressor implicated in various aspects of carcinogenesis. However, the roles and mechanisms of miR-124 in CMC development remains to be determined. We used the quantitative real-time polymerase chain reaction (qRT-PCR) assay to evaluate the expression levels of miR-124 in CMC tissues obtained from 20 CMC cases and CHMm and CHMp cells. CMC cell lines were transfected with lipfactormine™2000, and the cell proliferation was measured by Cell Counting Kit-8 (CCK-8). Transwell assay were employed for evaluating the cell invasion and migration, while western blot assay was used to detect the protein changes in epithelial-mesenchymal transition (EMT) and CDH2 protein levels. The relationship between miR-124 and the 3′-untranslated region (3'-UTR) of CDH2 was predicted via bioinformatics analysis and verified by dual-luciferase reporter assay. The results revealed that miR-124 was reduced in CMC tissues and cell lines. Besides, observed high histological grade and tumor metastasis were associated with the down-regulation of miR-124 and up-regulation of CDH2. Functional analyses showed that in vitro transfection of CHMm and CHMp cells with miR-124 mimics inhibited their proliferation, migration, invasion, and EMT; however, transfection with miR-124 inhibitor resulted in the reversed effect. Besides, we showed that miR-124 directly suppressed the expression of CDH2, leading to the inhibition of CHMm cell proliferation and EMT. In conclusion, miR-124 regulates CMC tumor growth and EMT by targeting CDH2, maybe a potential therapeutic strategy against CMC.

Pathways Involved in the Development of Vasculogenic Mimicry in Canine Mammary Carcinoma Cell Cultures

Vasculogenic mimicry (VM) is the ability of highly aggressive cancer cells to form fluid-conducting channels that facilitate the nutrition and metastasis of cancer cells. Considering the importance of VM in the prognosis of canine mammary gland tumours, this study aimed to investigate global gene expression in two canine mammary carcinoma cell cultures associated with the capacity for VM invitro. The cell lines were subjected to an in-vitro assay to form VM channels (3D culture). Each cell line was then used in 2D conditions as controls and we compared the global gene expression with that of the 3D cultures. A total of 1,217 differentially expressed genes (DEGs) (P<0.05, fold change >2.0 or <2.0) were observed in 3D conditions compared with 2D culture in the UNESP-CM9 cell line, of which 677 were upregulated genes and 540 were downregulated. In contrast, the UNESP-CM60cell line had only one upregulated and two downregulated genes. Overall, we identified several genes and pathways involved in the development of VM and these molecular data will be useful for future studies aimed at identifying diagnostic and therapeutic targets for VM in canine mammary carcinoma.

Canine Cancer Cells Activate Platelets via the Platelet P2Y12 Receptor

In addition to their well-known functions in haemostasis, anucleated platelets have a critical role in cancer biology. Many human and non-human cancer types can directly interact with and activate platelets, promoting cancer malignancy and progression. Although naturally occurring canine neoplastic diseases mimic the biologically complex conditions of human cancers more closely than laboratory-bred mice, studies evaluating the relationship between cancer cells and platelets in dogs are scarce, and the effects of tumour cells on platelets in these animals are unknown. To evaluate whether cancer cells could activate canine platelets, we assessed the response of platelet-rich plasma to cultured canine cancer cells using light transmittance aggregometry. Similar to human and murine cancer cell research, we demonstrated that both canine osteosarcoma and mammary carcinoma cells activated canine platelets invitro, resulting in platelet aggregation. The degree of aggregation was most pronounced at a cancer cell to platelet ratio of 1:200 for most cell lines. Mechanistic studies revealed that the platelet adenosine diphosphate (ADP) receptor P2Y12 is essential for canine platelet aggregation induced by canine cancer. ADP receptor blockage on platelets inhibited >50% of cancer cell-induced maximum platelet aggregation in all cell lines evaluated. As in other species, our results suggest that canine cancers may activate canine platelets invivo. This mechanism is likely relevant for the biology and progression of cancer in the dog.

Exosomes Derived from Radioresistant Breast Cancer Cells Promote Therapeutic Resistance in Naïve Recipient Cells.

Radiation resistance is a significant challenge in the treatment of breast cancer in humans. Human breast cancer is commonly treated with surgery and adjuvant chemotherapy/radiotherapy, but recurrence and metastasis upon the development of therapy resistance results in treatment failure. Exosomes are extracellular vesicles secreted by most cell types and contain biologically active cargo that, when transferred to recipient cells, can influence the cells’ genome and proteome. We propose that exosomes secreted by radioresistant (RR) cells may be able to disseminate the RR phenotype throughout the tumour. Here, we isolated exosomes from the human breast cancer cell line, MDA-MB-231, and the canine mammary carcinoma cell line, REM134, and their RR counterparts to investigate the effects of exosomes derived from RR cells on non-RR recipient cells. Canine mammary cancer cells lines have previously been shown to be excellent translational models of human breast cancer. This is consistent with our current data showing that exosomes derived from RR cells can increase cell viability and colony formation in naïve recipient cells and increase chemotherapy and radiotherapy resistance, in both species. These results are consistent in cancer stem cell and non-cancer stem cell populations. Significantly, exosomes derived from RR cells increased the tumoursphere-forming ability of recipient cells compared to exosomes derived from non-RR cells. Our results show that exosomes are potential mediators of radiation resistance that could be therapeutically targeted.

Canine Natural Killer Cell-Derived Exosomes Exhibit Antitumor Activity in a Mouse Model of Canine Mammary Tumor.

Natural killer (NK) cells are key immune cells engaged in fighting infection and malignant transformation. In this study, we found that canine NK cell-derived exosomes (NK-exosomes) separated from activated cytotoxic NK cell supernatants express specific markers including CD63, CD81, Alix, HSP70, TSG101, Perforin 1, and Granzyme B. We examined the antitumor effects of NK-exosomes in an experimental murine mammary tumor model using REM134 canine mammary carcinoma cell line. We observed changes in tumor size, tumor initiation, progression, and recurrence-related markers in the control, tumor group, and NK-exosome-treated tumor group. We found that the tumor size in the NK-exosome-treated tumor group decreased compared with that of the tumor group in the REM134-driven tumorigenic mouse model. We observed significant changes including the expression of tumorigenesis-related markers, such as B cell-specific Moloney murine leukemia virus insertion site 1 (Bmi-1), vascular endothelial growth factor (VEGF), matrix metallopeptidase-3 (MMP-3), interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), multidrug resistance protein (MDR), tumor suppressor protein p53 (p53), proliferating cell nuclear antigen (PCNA), and the apoptotic markers, B cell lymphoma-2 associated X (Bax) and B cell lymphoma-extra large (Bcl-xL) belonging to the Bcl-2 family, in the tumor group compared with those in the control group. The expression of CD133, a potent cancer stem cell marker, was significantly higher than that of the control. By contrast, the NK-exosome-treated tumor group exhibited a significant reduction in Bmi-1, MMP-3, IL-1β, IL-6, TNF-α, Bax, Bcl-xL, and PCNA expression compared with that in the tumor group. Furthermore, the expression of CD133, which mediates tumorigenesis, was significantly decreased in the NK-exosome-treated tumor group compared with that in the tumor group. These findings indicate that canine NK-exosomes represent a promising therapeutic tool against canine solid tumors, including mammary carcinoma.

Effects of Lapatinib on HER2-Positive and HER2-Negative Canine Mammary Carcinoma Cells Cultured In Vitro.

HER2 is a prognostic and predictive marker widely used in breast cancer. Lapatinib is a tyrosine kinase inhibitor that works by blocking the phosphorylation of the receptor HER2. Its use is related to relatively good results in the treatment of women with HER2+ breast cancer. Thus, this study aimed to verify the effects of lapatinib on four canine primary mammary gland carcinoma cell cultures and two paired metastatic cell cultures. Cultures were treated with lapatinib at concentrations of 100, 500, 1000 and 3000 nM for 24 h and the 50% inhibitory concentration (IC50) for each cell culture was determined. In addition, a transwell assay was performed to assess the ability of lapatinib to inhibit cell migration. Furthermore, we verified HER2 expression by RT-qPCR analysis of cell cultures and formalin-fixed paraffin-embedded tissues from samples corresponding to those used in cell culture. Lapatinib was able to inhibit cell proliferation in all cell cultures, but it was not able to inhibit migration in all cell cultures. The higher the expression of HER2 in a culture, the more sensitive the culture was to treatment. This relationship may be an indication that the expression of HER2 may be a predictive factor and opens a new perspective for the treatment of primary and metastatic mammary gland cancer.

Involvement of Nestin in the Progression of Canine Mammary Carcinoma

Nestin, a class VI intermediate filament protein, is known to be expressed in various types of human neoplasms, including breast cancer, and is associated with their progression. However, its expression and role in canine mammary tumors remain unknown. We analyzed nestin expression in canine mammary tumors using in situ hybridization and immunohistochemistry. We also investigated its role in a canine mammary carcinoma cell line using RNA interference. Nestin expression was not observed in luminal epithelial cells of any of the 62 cases of benign mammary lesions examined, although myoepithelial cells showed its expression in most cases. In 16/50 (32%) primary mammary carcinomas and 6/15 (40%) metastases of mammary carcinomas, cytoplasmic nestin expression was detected in luminal epithelial cells. In luminal cells of primary mammary carcinomas, its expression was positively related to several pathological parameters that indicate high-grade malignancy, including histological grading (P < .01), vascular/lymphatic invasion (P < .01), Ki-67 index (P < .01), and metastasis (P < .05). Immunohistochemistry revealed that nestin expression was related to vimentin expression in mammary carcinomas (P < .01). This relationship was confirmed using reverse transcription-quantitative polymerase chain reaction using 9 cell lines derived from canine mammary carcinoma (P < .01). Finally, nestin knockdown in canine mammary carcinoma cells using small interfering RNA inhibited cell proliferation and migration based on WST-8, Boyden chamber, and cell-tracking assays. These findings suggest that nestin may at least partially mediate these behaviors of canine mammary carcinoma cells.

Maltotriose-Chlorin e6 Conjugate Linked via Tetraethyleneglycol as an Advanced Photosensitizer for Photodynamic Therapy. Synthesis and Antitumor Activities against Canine and Mouse Mammary Carcinoma Cells.

Glycoconjugated chlorins represent a promising class of compounds that meet the requirements for the third-generation photosensitizer (PS) for photodynamic therapy (PDT). We have focused on the use of glucose (Glc) to improve the performance of the PS based on the Warburg effect—a phenomenon where tumors consume higher Glc levels than normal cells. However, as a matter of fact, Glc-conjugation has a poor efficacy in hydrophilic modification; thus, the resultant PS is not suitable for intravenous injection. In this study, a Glc-based oligosaccharide, such as maltotriose (Mal3), is conjugated to chlorin e6 (Ce6). The conjugation is assisted by two additional molecular tools, such as propargyl amine and a tetraethylene glycol (TEG) derivative. This route produced the target Mal3–Ce6 conjugate linked via the TEG spacer (Mal3–TEG–Ce6), which shows the required photoabsorption properties in the physiological media. The PDT test using canine mammary carcinoma (SNP) cells suggested that the antitumor activity of Mal3–TEG–Ce6 is extremely high. Furthermore, in vitro tests against mouse mammary carcinoma (EMT6) cells have been demonstrated, providing insights into the photocytotoxicity, subcellular localization, and analysis of cell death and reactive oxygen species (ROS) generation for the PDT system with Mal3–TEG–Ce6. Both apoptosis and necrosis of the EMT6 cells occur by ROS that is generated via the photochemical reaction between Mal3–TEG–Ce6 and molecular oxygen. Consequently, Mal3–TEG–Ce6 is shown to be a PS showing the currently desired properties.