Rational approach to design gold nanoparticles for photothermal therapy: the effect of gold salt on physicochemical, optical and biological properties.

Among the unique characteristics associated to gold nanoparticles (AuNPs) in biomedicine, their ability to convert light energy into heat opens ventures for improved cancer therapeutic options, such as photothermal therapy (PTT). PTT relies on the local hyperthermia of tumor cells upon irradiation with light beams, and the association of AuNPs with radiation within the near infrared (NIR) range constitutes an advantageous strategy to potentially improve PTT efficacy. Herein, it was explored the effect of the gold salt on the AuNPs' physicochemical and optical properties. Mostly spherical-like negatively charged AuNPs with variable sizes and absorbance spectra were obtained. In addition, photothermal features were assessed using in vitro phantom models. The best formulation showed the ability to increase their temperature in aqueous solution up to 19 °C when irradiated with a NIR laser for 20 min. Moreover, scanning transmission electron microscopy confirmed the rearrangement of the gold atoms in a face-centered cubic structure, which further allowed to calculate the photothermal conversion efficiency upon combination of theoretical and experimental data. AuNPs also showed local retention after being locally administered in in vivo models. These last results obtained by computerized tomography allow to consider these AuNPs as promising elements for a PTT system. Moreover, AuNPs showed high potential for PTT by resulting in in vitro cancer cells' viability reductions superior to 70 % once combine with 5 min of NIR irradiation.

Safety of Gold Nanoparticles: From In Vitro to In Vivo Testing Array Checklist.

In recent years, gold nanoparticles (AuNPs) have aroused the interest of many researchers due to their unique physicochemical and optical properties. AuNPs are being explored in a variety of biomedical fields, either in diagnostics or therapy, particularly for localized thermal ablation of cancer cells after light irradiation. Besides the promising therapeutic potential of AuNPs, their safety constitutes a highly important issue for any medicine or medical device. For this reason, in the present work, the production and characterization of physicochemical properties and morphology of AuNPs coated with two different materials (hyaluronic and oleic acids (HAOA) and bovine serum albumin (BSA)) were firstly performed. Based on the above importantly referred issue, the in vitro safety of developed AuNPs was evaluated in healthy keratinocytes, human melanoma, breast, pancreatic and glioblastoma cancer cells, as well as in a three-dimensional human skin model. Ex vivo and in vivo biosafety assays using, respectively, human red blood cells and Artemia salina were also carried out. HAOA-AuNPs were selected for in vivo acute toxicity and biodistribution studies in healthy Balb/c mice. Histopathological analysis showed no significant signs of toxicity for the tested formulations. Overall, several techniques were developed in order to characterize the AuNPs and evaluate their safety. All these results support their use for biomedical applications.

Presumptive hemophagocytic syndrome associated with co-infections with FIV, Toxoplasma gondii, and Candidatus mycoplasma haemominutum in an adult cat.

A 9-year-old neutered male cat, previously test-positive for feline immunodeficiency virus (FIV), was presented with an history of vomiting, hyporexia, and weight loss. Panleukopenia was identified on complete blood counts, and bone marrow evaluation revealed ineffective granulocytic hyperplasia and rare neutro-, erythro-, and rubriphagocytosis. Prednisolone was initiated with no response, and progression to pancytopenia occurred. On abdominal ultrasonographic examination, splenomegaly was present. PCR testing was positive for Candidatus Mycoplasma haemominutum and IgG antibodies against Toxoplasma gondii were detected (titer 1:2560). Treatment with antibiotics, feline recombinant interferon-ω, chlorambucil, mycophenolate, and raltegravir was implemented with no clinical improvement, and splenectomy was performed. Cytologic evaluation of splenic aspirates revealed exuberant neutro-, erythro-, and rubriphagocytosis. Histopathology of the spleen also showed many erythrophagocytic macrophages with no evidence of malignancy, and a diagnosis of hemophagocytic syndrome (HS) was made. The WBC count and hematocrit reached reference values 1 day and 3 months, respectively, after splenectomy. The cat was treated with cyclosporine and lomustine. Disease progression led to the development of septic hepatitis, and the cat was euthanized. To our knowledge, this is the first case of presumptive HS in cats that might have been associated with FIV, Toxoplasma gondii, and Candidatus Mycoplasma haemominutum co-infection.

The ADAM17 sheddase complex regulator iTAP/Frmd8 modulates inflammation and tumor growth.

The metalloprotease ADAM17 is a sheddase of key molecules, including TNF and epidermal growth factor receptor ligands. ADAM17 exists within an assemblage, the "sheddase complex," containing a rhomboid pseudoprotease (iRhom1 or iRhom2). iRhoms control multiple aspects of ADAM17 biology. The FERM domain-containing protein iTAP/Frmd8 is an iRhom-binding protein that prevents the precocious shunting of ADAM17 and iRhom2 to lysosomes and their consequent degradation. As pathophysiological role(s) of iTAP/Frmd8 have not been addressed, we characterized the impact of iTAP/Frmd8 loss on ADAM17-associated phenotypes in mice. We show that iTAP/Frmd8 KO mice exhibit defects in inflammatory and intestinal epithelial barrier repair functions, but not the collateral defects associated with global ADAM17 loss. Furthermore, we show that iTAP/Frmd8 regulates cancer cell growth in a cell-autonomous manner and by modulating the tumor microenvironment. Our work suggests that pharmacological intervention at the level of iTAP/Frmd8 may be beneficial to target ADAM17 activity in specific compartments during chronic inflammatory diseases or cancer, while avoiding the collateral impact on the vital functions associated with the widespread inhibition of ADAM17.

Current Insights and Progress in the Clinical Management of Head and Neck Cancer.

Head and neck cancer (HNC), also known as the cancer that can affect the structures between the dura mater and the pleura, is the 6th most common type of cancer. This heterogeneous group of malignancies is usually treated with a combination of surgery and radio- and chemotherapy, depending on if the disease is localized or at an advanced stage. However, most HNC patients are diagnosed at an advanced stage, resulting in the death of half of these patients. Thus, the prognosis of advanced or recurrent/metastatic HNC, especially HNC squamous cell carcinoma (HNSCC), is notably poorer than the prognosis of patients diagnosed with localized HNC. This review explores the epidemiology and etiologic factors of HNC, the histopathology of this heterogeneous cancer, and the diagnosis methods and treatment approaches currently available. Moreover, special interest is given to the novel therapies used to treat HNC subtypes with worse prognosis, exploring immunotherapies and targeted/multi-targeted drugs undergoing clinical trials, as well as light-based therapies (i.e., photodynamic and photothermal therapies).

Vet-OncoNet: Malignancy Analysis of Neoplasms in Dogs and Cats.

Analysis of canine and feline tumor malignancy data can help clinicians identify high-risk patients and make more accurate decisions. Based on a sample of 16,272 cancer records, including 3266 cats and 13,006 dogs, collected from January 2019 to December 2021 in the Vet-OncoNet Network database, this study aimed to compare the tumor malignancy profile between cats and dogs, considering animal-related factors (sex, age, and breed), topography, and geographic location using a mixed-effects logistic regression model. Cats had a higher proportion of malignant tumors (78.7%) than dogs (46.2%), and the malignancy profile was very different regarding tumors' topographies. The mean age of malignant tumors occurred eight months later than benign ones (9.1, SD = 3.4; 9.8, SD = 3.2), in general. Species (OR = 3.96, 95%CI 3.57: 4.39) and topography (MOR = 4.10) were the two most important determinants of malignancy risk. Female dogs had a higher risk than male dogs (OR = 1.19, 95%CI 1.08: 1.31), which does not appear to be the case in cats (OR = 0.98, 95%CI 0.77: 1.23). Breed contributed significantly to differences in malignancy risk in dogs (MOR = 1.56), particularly in pit bulls and boxers. District of residence was not so relevant in predicting malignancy risk (MOR = 1.14). In both species, the risk of malignancy increased by approximately 20% every three years. It could be hypothesized that species differences in genetic structure may contribute to tumor malignancy.

Biological Thermal Performance of Organic and Inorganic Aerogels as Patches for Photothermal Therapy.

Aerogels are materials with unique properties, among which are low density and thermal conductivity. They are also known for their exquisite biocompatibility and biodegradability. All these features make them attractive for biomedical applications, such as their potential use in photothermal therapy (PTT). This technique is, yet, still associated with undesirable effects on surrounding tissues which emphasizes the need to minimize the exposure of healthy regions. One way to do so relies on the use of materials able to block the radiation and the heat generated. Aerogels might be potentially useful for this purpose by acting as insulators. Silica- and pectin-based aerogels are reported as the best inorganic and organic thermal insulators, respectively; thus, the aim of this work relies on assessing the possibility of using these materials as light and thermal insulators and delimiters for PTT. Silica- and pectin-based aerogels were prepared and fully characterized. The thermal protection efficacy of the aerogels when irradiated with a near-infrared laser was assessed using phantoms and ex vivo grafts. Lastly, safety was assessed in human volunteers. Both types presented good textural properties and safe profiles. Moreover, thermal activation unveils the better performance of silica-based aerogels, confirming the potential of this material for PTT.

Tumor-infiltrating lymphocytes in early breast cancer: an exploratory analysis focused on HER2+ subtype in Portuguese patients.

Tumor-infiltrating lymphocytes (TILs) have shown prognostic value in breast cancer. This study evaluated the TILs scores in 186 Portuguese patients diagnosed with early breast cancer, with special focus on HER2 subtype. Stromal TILs were scored on the core needle biopsies, as well as in the resected specimen in HER2+ patients submitted to neoadjuvant treatment with trastuzumab and pertuzumab. TILs were higher in tumors with negative hormone receptor status and HER2 amplifications, and in triple-negative breast cancer. In HER2+ patients treated with dual anti-HER neoadjuvant therapy, the TILs score on the surgical specimen was generally lower than in the biopsy.

Cross Species Analysis and Comparison of Tumors in Dogs and Cats, by Age, Sex, Topography and Main Morphologies. Data from Vet-OncoNet.

The animal cancer burden is essential for the translational value of companion animals in comparative oncology. The present work aims to describe, analyze, and compare frequencies and associations of tumors in dogs and cats based on the Animal Cancer Registry created by Vet-OncoNet. With 9079 registries, regarding 2019 and 2020, 81% (n = 7355) belonged to dogs. In comparison, cats have a general one-year right advance in the mean age of cancer diagnosis compared to dogs. The multivariate topography group analysis shows a distinct pattern between the two species: dogs have higher odds of cancer in the genito-urinary system, spleen, soft tissue tumors and skin, while cats show higher odds for tumors in the eyes, digestive organs, nasal cavity, lymph nodes, bones and mammary glands. Regarding morphologies, dogs are overrepresented in mast cell tumors (MCT), melanomas, and hemangiosarcomas. While cats are overrepresented in fibrosarcomas, lymphomas (T and B-cell), in malignant mammary tumors, and squamous cell carcinoma (SCC). Females have greater odds only in the mammary gland, with males having greater odds in six of twelve topographies. This study is the first outcome of continuous animal cancer registration studies in Portugal.

A randomized controlled preclinical trial on 3 interposal temporomandibular joint disc implants: TEMPOJIMS-Phase 2.

The effort to develop an effective and safe temporomandibular joint (TMJ) disc substitute has been one of the mainstreams of tissue engineering. Biodegradable customized scaffolds could approach safety and effectiveness to regenerate a new autologous disc, rather than using non-biodegradable materials. However, it is still technically challenging to mimic the biomechanical properties of the native disc with biodegradable polymers. In this study, new 3D tailored TMJ disc implants were developed: (1) Poly(glycerol sebacate) (PGS) scaffold reinforced with electrospun Poly(εcaprolactone) (PCL) fibers on the outer surface (PGS+PCL); (2) PCL and polyethylene glycol diacrylate (PEGDA) (PCL+PEGDA); and (3) PCL. The TMJ implants were tested in a randomized preclinical trial, conducted in 24 black Merino sheep TMJ, perfoming bilateral interventions. Histologic, imaging, and kinematics analysis was performed. No statistical changes were observed between the PGS+PCL disc and the control group. The PCL+PEGDA and PCL groups were associated with statistical changes in histology (p = 0.004 for articular cartilage mid-layer; p = 0.019 for structure changes and p = 0.017 for cell shape changes), imaging (p = 0.027 for global appreciation) and dangerous material fragmentation was observed. No biomaterial particles were observed in the multi-organ analysis in the different groups. The sheep confirmed to be a relevant animal model for TMJ disc surgery and regenerative approaches. The PCL and PCL+PEGDA discs presented a higher risk to increase degenerative changes, due to material fragmentation. None of the tested discs regenerate a new autologous disc, however, PGS+PCL was safe, demonstrated rapid resorption, and was capable to prevent condyle degenerative changes.

Proof-of-Concept Study of Multifunctional Hybrid Nanoparticle System Combined with NIR Laser Irradiation for the Treatment of Melanoma.

The global impact of cancer emphasizes the importance of developing innovative, effective and minimally invasive therapies. In the context of superficial cancers, the development of a multifunctional nanoparticle-based system and its in vitro and in vivo safety and efficacy characterization are, herein, proposed as a proof-of-concept. This multifunctional system consists of gold nanoparticles coated with hyaluronic and oleic acids, and functionalized with epidermal growth factor for greater specificity towards cutaneous melanoma cells. This nanoparticle system is activated by a near-infrared laser. The characterization of this nanoparticle system included several phases, with in vitro assays being firstly performed to assess the safety of gold nanoparticles without laser irradiation. Then, hairless immunocompromised mice were selected for a xenograft model upon inoculation of A375 human melanoma cells. Treatment with near-infrared laser irradiation for five minutes combined with in situ administration of the nanoparticles showed a tumor volume reduction of approximately 80% and, in some cases, led to the formation of several necrotic foci, observed histologically. No significant skin erythema at the irradiation zone was verified, nor other harmful effects on the excised organs. In conclusion, these assays suggest that this system is safe and shows promising results for the treatment of superficial melanoma.

Gold-Based Nanoplataform for the Treatment of Anaplastic Thyroid Carcinoma: A Step Forward.

Anaplastic thyroid carcinoma (ATC) is a very rare subtype of thyroid carcinoma and one of the most lethal malignancies. Poor prognosis is mainly associated with its undifferentiated nature, inoperability, and failing to respond to the typically used therapies for thyroid cancer. Photothermal Therapy (PTT) entails using light to increase tissues' temperature, leading to hyperthermia-mediated cell death. Tumours are more susceptible to heat as they are unable to dissipate it. By using functionalized gold nanoparticles (AuNPs) that transform light energy into heat, it is possible to target the heat to the tumour. This study aims to formulate ATC-targeted AuNPs able to convert near-infrared light into heat, for PTT of ATC. Different AuNPs were synthetized and coated. Size, morphology, and surface plasmon resonances band were determined. The optimized coated-AuNPs were then functionalized with ligands to assess ATC's specificity. Safety, efficacy, and selectivity were assessed in vitro. The formulations were deemed safe when not irradiated (>70% cell viability) and selective for ATC. However, when irradiated, holo-transferrin-AuNPs were the most cytotoxic (22% of cell viability). The biodistribution and safety of this formulation was assessed in vivo. Overall, this novel formulation appears to be a highly promising approach to evaluate in a very near future.

Stereology in Grading and Prognosis of Canine Cutaneous Mast Cell Tumors.

Canine cutaneous mast cell tumors (ccMCTs) are currently graded according to Patnaik and Kiupel grading schemes. The qualitative and semiquantitative parameters applied in these schemes may lead to inter- and intraobserver variability. This study investigates the prognostic value of volume-weighted mean nuclear volume (vv¯), a stereological estimation that provides information about nuclear size and its variability. vv¯ of 55 ccMCTs was estimated using the "point-sampled intercept" method and compared with histological grade and clinical outcome. The clinical history of dogs treated with surgical excision alone was available for 30 ccMCTs. Statistical differences in vv¯ were found between grade II (x¯ = 115 ± 29 µm3) and grade III ccMCTs (x ¯= 197 ± 63 µm3), as well as between low-grade (x ¯= 113 ± 28 µm3) and high-grade ccMCTs (x¯ = 184 ± 63 µm3). An optimal cutoff value of vv¯ ≥ 150 µm3 and vv¯ ≥ 140 µm3 was determined for grade III and high-grade ccMCTs, respectively. In terms of prognosis, vv¯  was not able to predict the clinical outcome in 42% of the cases; however, cases with vv¯ <125 µm3 had a favorable outcome. These results indicate that, despite having limited prognostic value when used as a solitary parameter, vv¯ is highly reproducible and is associated with histological grade as well as with benign behavior.

Tetracycline Antibiotics Induce Host-Dependent Disease Tolerance to Infection.

Several classes of antibiotics have long been known to have beneficial effects that cannot be explained strictly on the basis of their capacity to control the infectious agent. Here, we report that tetracycline antibiotics, which target the mitoribosome, protected against sepsis without affecting the pathogen load. Mechanistically, we found that mitochondrial inhibition of protein synthesis perturbed the electron transport chain (ETC) decreasing tissue damage in the lung and increasing fatty acid oxidation and glucocorticoid sensitivity in the liver. Using a liver-specific partial and acute deletion of Crif1, a critical mitoribosomal component for protein synthesis, we found that mice were protected against sepsis, an observation that was phenocopied by the transient inhibition of complex I of the ETC by phenformin. Together, we demonstrate that mitoribosome-targeting antibiotics are beneficial beyond their antibacterial activity and that mitochondrial protein synthesis inhibition leading to ETC perturbation is a mechanism for the induction of disease tolerance.

Evaluation of hematology, general serum biochemistry, bone turnover markers and bone marrow cytology in a glucocorticoid treated ovariectomized sheep model for osteoporosis research.

Osteoporosis is a metabolic disorder characterized by a loss of bone mass and structure and increasing the risk of fragility fractures, mostly among postmenopausal women. Sheep is a recognized large animal model for osteoporosis research. An experimental group of ewes (3-4 years old) was subjected to ovariectomy (OVX) and weekly glucocorticoid (GC) application for 24 weeks and compared with a sham control group. Blood and bone marrow parameters were analyzed before and 24 weeks after OVX and GC administration. Osteopenia was confirmed through micro-computed tomography and histomorphometric analysis of L4 vertebra in the study end. A statistically significant increase was observed in mean corpuscular volume, mean cell hemoglobin and monocytes and a decrease in red blood count and eosinophils (p<0.05). Alkaline phosphatase (ALP), gamma-glutamyl transpeptidase, magnesium and α1-globulin increased, and creatinine, albumin, sodium and estradiol decreased (p<0.05). A slight decrease of bone formation markers (bone ALP and osteocalcin) and an increase of bone resorption markers (C-terminal telopeptides of collagen type 1 and tartrate-resistant acid phosphatase) were observed, but without statistical significance. This study aims to contribute to better knowledge of sheep as a model for osteoporosis research and the consequences that a performed induction protocol may impose on organic metabolism.

A Step Forward in Breast Cancer Research: From a Natural-Like Experimental Model to a Preliminary Photothermal Approach.

Breast cancer is one of the most frequently diagnosed malignancies and common causes of cancer death in women. Recent studies suggest that environmental exposures to certain chemicals, such as 7,12-Dimethylbenzanthracene (DMBA), a chemical present in tobacco, may increase the risk of developing breast cancer later in life. The first-line treatments for breast cancer (surgery, chemotherapy or a combination of both) are generally invasive and frequently associated with severe side effects and high comorbidity. Consequently, novel approaches are strongly required to find more natural-like experimental models that better reflect the tumors' etiology, physiopathology and response to treatments, as well as to find more targeted, efficient and minimally invasive treatments. This study proposes the development and an in deep biological characterization of an experimental model using DMBA-tumor-induction in Sprague-Dawley female rats. Moreover, a photothermal therapy approach using a near-infrared laser coupled with gold nanoparticles was preliminarily assessed. The gold nanoparticles were functionalized with Epidermal Growth Factor, and their physicochemical properties and in vitro effects were characterized. DMBA proved to be a very good and selective inductor of breast cancer, with 100% incidence and inducing an average of 4.7 tumors per animal. Epigenetic analysis showed that tumors classified with worst prognosis were hypomethylated. The tumor-induced rats were then subjected to a preliminary treatment using functionalized gold nanoparticles and its activation by laser (650-900 nm). The treatment outcomes presented very promising alterations in terms of tumor histology, confirming the presence of necrosis in most of the cases. Although this study revealed encouraging results as a breast cancer therapy, it is important to define tumor eligibility and specific efficiency criteria to further assess its application in breast cancer treatment on other species.

Discrimination of Benign and Malignant Lesions in Canine Mammary Tissue Samples Using Raman Spectroscopy: A Pilot Study.

Breast cancer is a health problem that affects individual life quality and the family system. It is the most frequent type of cancer in women, but men are also affected. As an integrative approach, comparative oncology offers an opportunity to learn more about natural cancers in different species. Methods based on Raman spectroscopy have shown significant potential in the study of the human breast through the fingerprinting of biological tissue, which provides valuable information that can be used to identify, characterize and discriminate structures in breast tissue, in both healthy and carcinogenic environments. One of the most important applications of Raman spectroscopy in medical diagnosis is the characterization of microcalcifications, which are highly important diagnostic indicators of breast tissue diseases. Raman spectroscopy has been used to analyze the chemical composition of microcalcifications. These occur in benign and malignant lesions in the human breast, and Raman helps to discriminate microcalcifications as type I and type II according to their composition. This paper demonstrates the recent progress in understanding how this vibrational technique can discriminate through the fingerprint regions of lesions in unstained histology sections from canine mammary glands.

Feline oral cavity lesions diagnosed by histopathology: a 6-year retrospective study in Portugal.

OBJECTIVES: The aim of this study was to analyse the frequency of oral cavity lesions in cats, their anatomical location and histological diagnosis, and the effect of life stage, breed and sex on different diagnoses. METHODS: For this purpose, a retrospective study comprising 297 feline oral cavity lesions was performed over a 6-year period between 2010 and 2015. Histopathological records from the DNAtech Pathology Laboratory (Lisbon, Portugal) were analysed. RESULTS: The incidence of oral disease was higher in male cats (n = 173; 58.4%), mature adults (ranging from 7 to 10 years old [n = 88; 33.0%]) and in the European Shorthair breed (n = 206; 73.6%). The gingiva was the site where oral lesions were most commonly found, with 128 samples (43.1%). Incisional biopsies were used to obtain the majority of samples (n = 256; 86.2%), while excisional biopsies and punch biopsies were performed in 36 (12.1%) and five (1.7%) cases, respectively. Inflammatory and neoplastic lesions accounted for 187 (63%) and 110 (37%) of the studied cases, respectively. Malignancies were found in >80% of neoplastic cases. Feline chronic gingivostomatitis was the most common histological diagnosis (n = 116; 39.1%), followed by squamous cell carcinoma (n = 49; 16.5%) and eosinophilic granuloma complex (n = 33; 11.1%). CONCLUSIONS AND RELEVANCE: The present work, involving a large series of samples of feline oral cavity lesions, from numerous geographically scattered practices and all examined at a reference veterinary pathology laboratory, adds important new understanding of the epidemiology of feline oral disease.

Deletion of iRhom2 protects against diet-induced obesity by increasing thermogenesis.

OBJECTIVE: Obesity is the result of positive energy balance. It can be caused by excessive energy consumption but also by decreased energy dissipation, which occurs under several conditions including when the development or activation of brown adipose tissue (BAT) is impaired. Here we evaluated whether iRhom2, the essential cofactor for the Tumour Necrosis Factor (TNF) sheddase ADAM17/TACE, plays a role in the pathophysiology of metabolic syndrome. METHODS: We challenged WT versus iRhom2 KO mice to positive energy balance by chronic exposure to a high fat diet and then compared their metabolic phenotypes. We also carried out ex vivo assays with primary and immortalized mouse brown adipocytes to establish the autonomy of the effect of loss of iRhom2 on thermogenesis and respiration. RESULTS: Deletion of iRhom2 protected mice from weight gain, dyslipidemia, adipose tissue inflammation, and hepatic steatosis and improved insulin sensitivity when challenged by a high fat diet. Crucially, the loss of iRhom2 promotes thermogenesis via BAT activation and beige adipocyte recruitment, enabling iRhom2 KO mice to dissipate excess energy more efficiently than WT animals. This effect on enhanced thermogenesis is cell-autonomous in brown adipocytes as iRhom2 KOs exhibit elevated UCP1 levels and increased mitochondrial proton leak. CONCLUSION: Our data suggest that iRhom2 is a negative regulator of thermogenesis and plays a role in the control of adipose tissue homeostasis during metabolic disease.

Microtomographic characterization of calcifications in canine mammary tumours.

The present work describes the microtomographic characterization of macro- and microcalcifications present in excised canine mammary glands. In human breast cancer, microcalcifications are highly relevant for diagnosis and prognosis, often being the sole element determining biopsy. Canine mammary tumours are considered a model for human breast cancer, but the morphological features of calcifications had still to be studied in this species. The objective of this research is to contribute to the characterization of the mineralization features of the canine mammary gland. In the present study, the excised mammary glands of 33 bitches underwent fluoroscopic examination. In 30 of the samples, the presence of calcification was suspected, and multiple biopsies were taken of these areas. Biopsy fragments underwent microtomographic scanning. Microcalcifications were found in non-neoplastic glandular tissue, benign and malign lesions, as it is known to happen in humans. Qualitative evaluation regarding morphology of the imaged calcifications showed similarities to breast cancer findings, based on the BI-RADS 2013 classification, such as pleomorphism and shape. No differences in the quantitative morphological parameters of volume, surface, surface/volume, SMI and structure thickness were found when macrocalcifications were considered. However, although significant differences existed in these parameters between microcalcifications from malignant canine mammary tumours and the two other groups, none were found between non-neoplastic and benign tumours. Findings further support the use of this spontaneous animal model for the study of human breast cancer, considering how clinically relevant microcalcifications are in humans.