Assessment of the WAP-Myc mouse mammary tumor model for spontaneous metastasis.

Breast cancer is the most common form of cancer in women. Despite significant therapeutic advances in recent years, breast cancer also still causes the greatest number of cancer-related deaths in women, the vast majority of which (> 90%) are caused by metastases. However, very few mouse mammary cancer models exist that faithfully recapitulate the multistep metastatic process in human patients. Here we assessed the suitability of a syngrafting protocol for a Myc-driven mammary tumor model (WAP-Myc) to study autochthonous metastasis. A moderate but robust spontaneous lung metastasis rate of around 25% was attained. In addition, increased T cell infiltration was observed in metastatic tumors compared to donor and syngrafted primary tumors. Thus, the WAP-Myc syngrafting protocol is a suitable tool to study the mechanisms of metastasis in MYC-driven breast cancer.

Tumour-associated macrophages: Relation with progression and invasiveness, and assessment of M1/M2 macrophages in canine mammary tumours.

Macrophages represent a major component of the overall leucocyte population within neoplasms and are important for tumour behaviour in several cancers in human beings. However, little information regarding their role in canine mammary tumours (CMTs) is available. The aim of this study was to address the potential role of tumour-associated macrophages (TAMs) in CMTs. TAMs in CMTs excised from 82 female dogs were quantified at high power (400× magnification) and categorised as low (≤50) or high (>50) TAM counts. Higher TAM counts were associated with clinical stage (P<0.001), tumour type (P=0.016), tumour size (P=0.013), vascular invasion (P=0.031), lymph node metastasis (P=0.003), high proliferation rates (P=0.009), vascular microdensity (P=0.008), invasive tumour profile (P=0.002) and worse prognosis (P=0.018; hazard ratio=0.283). Almost all macrophages infiltrating malignant tumours with high TAM counts expressed CD206 (macrophage mannose receptor 1), while all benign tumours were infiltrated by macrophages expressing inducible nitric oxide synthase (NOS2), suggesting a phenotypic shift from classically activated macrophage (M1) subpopulations towards alternatively activated macrophage (M2) subpopulations in malignant tumours. A triple staining pattern revealed mixed M1/M2 profiles in some tumours, thus characterising an intermediate state. The results indicate that TAMs are associated with more aggressive types of mammary cancer in dogs.

A statistical assessment of the biological relationship between simultaneous canine mammary tumours.

Simultaneous canine mammary tumours (CMTs) are frequently reported in the literature, but few studies have addressed their biological relationship in detail or performed statistical assessments. In this study, 269 canine mammary gland tumours from 216 dogs were categorized using an extended histopathological classification, where semiquantitative and binomial scales enumerated morphological parameters of the tumours. The classification facilitated a statistical study of the biological relationship between simultaneous within-dog tumours. Seventy-seven percent of the dogs had single tumours and 23% had simultaneous tumours. Sixty-one percent of the neoplasias were benign, with complex adenoma as the most frequent diagnosis and 39% were malignant, with complex carcinoma as the most common malignancy. Simultaneous tumours within dogs more often had equal diagnoses and neoplastic level (benign or malignant) than would be expected by chance alone, as compared with random pairs of single tumours from different dogs. This statistically supported finding indicated the presence of a biological relationship between simultaneous tumours.

Pharmacologically Inactive Bisphosphonates as an Alternative Strategy for Targeting Osteoclasts: In Vivo Assessment of 5-Fluorodeoxyuridine-Alendronate in a Preclinical Model of Breast Cancer Bone Metastases.

Bisphosphonates have effects that are antiresorptive, antitumor, and antiapoptotic to osteoblasts and osteocytes, but an effective means of eliciting these multiple activities in the treatment of bone metastases has not been identified. Antimetabolite-bisphosphonate conjugates have potential for improved performance as a class of bone-specific antineoplastic drugs. The primary objective of the study was to determine whether an antimetabolite-bisphosphonate conjugate will preserve bone formation concomitant with antiresorptive and antitumor activity. 5-FdU-ale, a highly stable conjugate between the antimetabolite 5-fluoro-2'-deoxyuridine and the bisphosphonate alendronate, was tested for its therapeutic efficacy in a mouse model of MDA-MB231 breast cancer bone metastases. In vitro testing revealed osteoclasts to be highly sensitive to 5-FdU-ale. In contrast, osteoblasts had significantly reduced sensitivity. Tumor cells were resistant in vitro but in vivo tumor burden was nevertheless significantly reduced compared with untreated mice. Sensitivity to 5-FdU-ale was not mediated through inhibition of farnesyl diphosphate synthase activity, but cell cycle arrest was observed. Although serum tartrate-resistant acid phosphatase (TRAP) levels were greatly reduced by both drugs, there was no significant decrease in the serum bone formation marker osteocalcin with 5-FdU-ale treatment. In contrast, there was more than a fivefold decrease in serum osteocalcin levels with alendronate treatment (p < 0.001). This finding is supported by time-lapse micro-computed tomography analyses, which revealed bone formation volume to be on average 1.6-fold higher with 5-FdU-ale treatment compared with alendronate (p < 0.001). We conclude that 5-FdU-ale, which is a poor prenylation inhibitor but maintains potent antiresorptive activity, does not reduce bone formation and has cytostatic antitumor efficacy. These results document that conjugation of an antimetabolite with bisphosphonates offers flexibility in creating potent bone-targeting drugs with cytostatic, bone protection properties that show limited nephrotoxicity. This unique class of drugs may offer distinct advantages in the setting of targeted adjuvant therapy and chemoprevention of bone diseases. © 2016 American Society for Bone and Mineral Research.

Significance of rat mammary tumors for human risk assessment.

We have previously indicated that the ideal animal tumor model should mimic the human disease. This means that the investigator should be able to ascertain the influence of host factors on the initiation of tumorigenesis, mimic the susceptibility of tumor response based on age and reproductive history, and determine the response of the tumors induced to chemotherapy. The utilization of experimental models of mammary carcinogenesis in risk assessment requires that the influence of ovarian, pituitary, and placental hormones, among others, as well as overall reproductive events are taken into consideration, since they are important modifiers of the susceptibility of the organ to neoplastic development. Several species, such as rodents, dogs, cats, and monkeys, have been evaluated for these purposes; however, none of them fulfills all the criteria specified previously. Rodents, however, are the most widely used models; therefore, this work will concentrate on discussing the rat rodent model of mammary carcinogenesis.

Integrated multiplatform method for in vitro quantitative assessment of cellular uptake for fluorescent polymer nanoparticles.

Studies of cellular internalization of nanoparticles (NPs) play a paramount role for the design of efficient drug delivery systems, but so far they lack a robust experimental technique able to quantify the NP uptake in terms of number of NPs internalized in each cell. In this work we propose a novel method which provides a quantitative evaluation of fluorescent NP uptake by combining flow cytometry and plate fluorimetry with measurements of number of cells. Single cell fluorescence signals measured by flow cytometry were associated with the number of internalized NPs, exploiting the observed linearity between average flow cytometric fluorescence and overall plate fluorimeter measures, and previous calibration of the microplate reader with serial dilutions of NPs. This precise calibration has been made possible by using biocompatible fluorescent NPs in the range of 20-300 nm with a narrow particle size distribution, functionalized with a covalently bonded dye, Rhodamine B, and synthesized via emulsion free-radical polymerization. We report the absolute number of NPs internalized in mouse mammary tumor cells (4T1) as a function of time for different NP dimensions and surface charges and at several exposure concentrations. The obtained results indicate that 4T1 cells incorporated 10(3)-10(4) polymer NPs in a short time, reaching an intracellular concentration 15 times higher than the external one.

Assessment of the novel estrogen receptor PET tracer 4-fluoro-11ß-methoxy-16α-[(18)F]fluoroestradiol (4FMFES) by PET imaging in a breast cancer murine model.

PURPOSE: The aim of this study was to compare the in vivo stability, uptake, and positron emission tomography (PET) imaging performance of a novel estrogen receptor PET tracer, 4-fluoro-11ß-methoxy-16α-[(18)F]fluoroestradiol (4FMFES), with 16α-[(18)F]fluoroestradiol (FES). PROCEDURES: MC7-L1 and MC4-L2 (ER+) cell lines and their ERα-knockdown variants (ERαKD) were implanted subcutaneously in Balb/c mice. After 21 days, mice were imaged using either FES or 4FMFES. One hour post-injection, static images were acquired for 30 min and the tumor %ID/g uptake values were derived. Biodistribution data were also obtained 1 h following the injection of either FES or 4FMFES. Blood samples were taken at different times and analyzed on thin-layer chromatography to quantify the presence of radiometabolites for each radiotracer. To assess specific targeting to the estrogen receptors, mice bearing only ER+ tumors were treated with the competitive ER inhibitor fulvestrant 48 h prior to imaging with 4FMFES. RESULTS: Metabolic stability was found to be similar for both tracers in mice. Both FES and 4FMFES differentiated ER+ tumors from ERαKD tumors in biodistribution and PET imaging studies. 4FMFES achieved a significantly higher %ID/g uptake in ER+ tumors and MC4-L2 ERαKD tumors than FES in the PET imaging studies. Also, tumor-to-background ratio was higher in ER+ tumors using 4FMFES compared to FES. Dissection data showed a significantly higher %ID/g in all tested cell lines and ER-rich tissues using 4FMFES versus FES. Fulvestrant-treated mice had either low or undetectable tumor uptake. CONCLUSION: In a tumor-bearing mouse model, 4FMFES achieves better specific tumor uptake and better contrast than FES, making it a promising candidate for ER imaging.

Histopathological and immunohistochemical assessment of invasive micropapillary mammary carcinoma in dogs: a retrospective study.

Invasive micropapillary carcinoma (IMPC) of the mammary gland, despite its rare occurrence in humans and dogs, is an important neoplasm due to its aggressive behaviour. The aim of this study was to evaluate the clinicopathological and immunophenotypical characteristics of IMPC and to determine the overall survival of dogs with this tumour. Of the selected cases, the majority had >3 cm neoplasms (15/19, 78.95%) and lymph node metastases (16/16, 100%), but only two cases (2/9, 22.2%) had distant metastases. The IMPCs were classified as either pure (15/22, 68.18%) or mixed (7/22, 31.82%) types. There was a predominance of moderate histological grade tumours (16 grade II) and the average overall survival was 120 days. Positive immunohistochemical staining for epithelial membrane antigen and negative staining for CD-31, p63 and cytokeratin (CK) AE1AE3 in cystic formations confirmed the micropapillary nature of these neoplasms. A proportion of cases exhibited positive epithelial staining for p63 (4/20, 20%) and CK34ßE12 (20/22, 90.9%). Most cases were positive for oestrogen (19/20, 95%) and progesterone (19/20, 95%) receptors, but lacked HER-2 (16/22, 72.72%) and epidermal growth factor receptor (15/22, 68.18%) over-expression. The mean proliferation index was 14.8%. The findings demonstrate that, similar to humans, canine IMPCs behave aggressively with high rates of metastasis to regional lymph nodes and short overall survival times.

Correlation between 2- and 3-dimensional assessment of tumor volume and vascular density by ultrasonography in a transgenic mouse model of mammary carcinoma.

OBJECTIVE: Visualization and quantification of angiogenesis are instrumental in development of antiangiogenic therapy. Although both 2-dimensional (2D) and 3-dimensional (3D) ultrasonography have been used to monitor tumor growth and vasculature development, the correlation between them has not been sufficiently investigated. We hereby investigated the 2D and 3D sonographic correlation for tumor volume and vascular density confirmed by histologic assessment in the polyoma virus middle T antigen (PyMT) mouse model of mammary carcinoma. METHODS: Female PyMT mouse tumors were evaluated by ultrasonography in the 2D region of interest (ROI), 3D tumor volume, and 2D and 3D microvascular density after a bolus infusion of a nontargeted contrast-enhanced microbubble agent. Texas Red-dextran was used for quantitative histologic assessment of the tumor microvascular density. RESULTS: The individual 2D tumor ROI area correlated with the 3D tumor volume throughout the 2-week period. However, the extent of the increase in the 3D volume (380%; P < .01; n = 10) was higher than that of the 2D ROI area (72%; P < .01; n = 8-11). A significant and comparable increase in vascular density accessed by both 2D (87%; P < .05; n = 8) and 3D (64%; P < .05; n = 8) imaging was documented. Vascular density obtained through 3D imaging correlated significantly with 2D measurement. These data were confirmed by Texas Red-dextran quantification of vascular density. Conclusions. This study showed a valid application of sonographically based imaging technology in tumor volume and vascular density assessment as well as their 2D and 3D correlation, of which tumor vascular density measured by 2D ultrasonography appeared to be better correlated with the 3D data. Our data indicate that ultrasonography can be applied for real-time, accurate, noninvasive imaging of the tumor volume and vascular density in preclinical models.

EMS in Viracept--initial ('traditional') assessment of risk to patients based on linear dose response relations.

Prior to having performed in depth toxicological, genotoxicological and DMPK studies on ethyl methanesulfonate (EMS) providing solid evidence for a thresholded dose response relationship, we had prepared and shared with regulatory authorities a preliminary risk estimate based on standard linear dose-effect projections. We estimated that maximal lifetime cancer risk was in the order of 10(-3) (for lifetime ingestion of the maximally contaminated tablets) or 10(-4) for the exposure lasting for 3 months. This estimate was based on a lifetime cancer study with methyl methanesulfonate (MMS; as insufficient data were available for EMS) in rodents and default linear back extrapolation. Analogous estimates were made specifically for breast cancer based on short term tumorigenicity studies with EMS in rats, for the induction of heritable mutations based on specific locus and dominant lethal tests in mice and for the induction of birth defects based on teratogenicity studies in mice. We concluded that even under worst case assumptions of linear dose relations the chance of experiencing these adverse effects would be very small, comprising at most a minute additional burden among the background incidence of the patients.

An assessment of the clonality of the components of canine mixed mammary tumours by mitochondrial DNA analysis.

The aim of this study was to investigate if mutations in the mitochondrial DNA (mtDNA) D-loop fragment control region of canine mammary mixed tumours could be used as clonal markers that identified the cell population of origin. Ten benign mixed mammary tumours and nine carcinomas arising from benign mixed tumours were microdissected and DNA from epithelial and mesenchymal tumour cells and from normal mammary tissue was examined for sequence variations in a fragment of the hypervariable control region. Identical sequence variants in both the epithelial and mesenchymal components (as well as in the corresponding normal tissue) were found in 80% of the benign mixed tumours and in 89% of the carcinomas arising from benign mixed tumours suggesting a shared clonal origin. The distinctive sequence alterations identified in the epithelial and mesenchymal components of 15.8% of all 19 tumours examined, suggests the possibility that a minority of mammary tumours are polyclonal in origin or that early clonal divergence occurs. Increased mutation within the mtDNA D-loop fragment of mixed tumour components was not observed.

Assessment of cell proliferation and prognostic factors in canine mammary gland tumors / Avaliação da proliferação celular e fatores prognósticos em tumores mamários caninos

Three methods for the analysis of cell proliferation, mitotic index/10 high-power fields (10 HPF), mitotic index/four sets of 10 HPF (40 HPF), and MIB-1 index were evaluated in a series of canine mammary gland tumors, as well as the possible correlation between them. Fifty-six canine mammary gland tumors, including 23 benign and 33 malignant, were studied. In addition, the prognostic impact of mitotic index/10 HPF, and histological malignancy grade were evaluated in 17 malignant tumors, being seven ductal and 10 metaplastic carcinomas. The three methods used to evaluate cell proliferation were correlated with the prognostic impact of mitotic index/10 HPF and histological malignancy grade. The results showed a strong association between mitotic figure counts and MIB-1 index (P<0.0001). A correlation was observed between mitotic count per 40 HPF and MIB-1, and between mitotic index per 10 HPF and 40 HPF (P<0.05). Moreover, histological malignancy grade and mitotic figure counts were excellent prognostic factors during three-year follow-up (P<0.05). There was a correlation between the three methods used for the evaluation of cell proliferation and prognostic factors as observed in human breast cancer studies.

Avaliaram-se três métodos de proliferação celular, índice mitótico/10 campos de grande aumento (10 CGA), quatro vezes 10 CGA (40 CGA) e índice de marcação por MIB-1, em uma série de tumores mamários caninos, e as possíveis correlações entre estes métodos. Foram estudados 56 tumores mamários caninos, 23 benignos e 33 malignos. Foi também avaliado o impacto prognóstico do índice mitótico (10 CGA) e o grau histológico maligno em 17 tumores malignos, sete carcinomas ductais e 10 carcinomas metaplásicos. A correlação entre os três métodos para avaliar a proliferação celular e o impacto prognóstico do índice mitótico por 10 CGA e o grau histológico maligno foi realizada. Os resultados mostraram que existe uma forte associação entre contagem de mitose e o índice de marcação por MIB-1(P<0,0001) e correlação entre contagem de mitoses em 40 CGA e índice de marcação por MIB-1 e entre índice mitótico em 10 CGA e 40 CGA (P<0,05). Observou-se correlação entre os três métodos de avaliação da proliferação celular e os fatores prognósticos semelhante aos estudos de câncer de mama humano.

In vivo investigation of progressive alterations in rat mammary gland tumors by near-infrared spectroscopy.

We have investigated mammary gland tissues of female rats treated with 7,12-dimethylbenz[a]anthracene in sesame oil by a near infrared (NIR) spectroscopy finding that the DNA and water contents in the cancerous tissues were larger than those in the normal tissues but that the lipid content in the former was less than that in the latter. With protein contents, however, little difference was observed between the two. Thus, we used a lipid band around 1725 nm (the first overtone of n-alkane) and a protein band around 2054 nm (a combination band of amide A and amide II of polypeptides) for a quantitative evaluation of malignant changes in the mammary gland tissues. The lipid/protein band intensity ratios were calculated from the spectra of the mammary glands in the control animals and those of the noncancerous and cancerous sites in the treated animals. The lipid/protein ratios in the control animals, in the noncancerous sites, and in the cancerous sites were 1.452 +/- 0.221 (n = 5), 0.728 +/- 0.069 (n = 5), and 0.362 +/- 0.060 (n = 5), respectively. These values were significantly different from each other (P < 0.001). The lipid changes observed by near-infrared (NIR) spectroscopy were confirmed by the results obtained from chemical methods for the evaluation of lipid levels in the same samples. Thus, our NIR spectroscopic method would be able not only to discriminate between cancerous and normal tissues but also to distinguish animals with cancers from normal animals. In addition, as the cancer grew, the lipid band intensity decreased, this band was shifted to higher wavelengths, and collagen peaks appeared in the tissues. These findings were supported by histological examinations of the cancerous and normal tissues. The present study indicates that NIR spectroscopy has high specificity and sensitivity in discriminating cancerous tissues from normal mammary glands in animals and it may offer potential for noninvasive, in vivo diagnosis of female breast cancer in the near future.

Immunolocalization of the smooth muscle-specific protein calponin in complex and mixed tumors of the mammary gland of the dog: assessment of the morphogenetic role of the myoepithelium.

The immunohistochemical expression of the smooth muscle-specific protein calponin was studied to assess the contribution of myoepithelial cells to the histogenesis of spindle cells of complex and mixed tumors of the mammary gland of the dog and the origin of cartilage and bone in mixed tumors. Formalin-fixed tissues from 55 benign and malignant tumors (49 also containing surrounding normal mammary gland) were evaluated. Periacinar and periductal myoepithelial cells of all the 49 normal mammary glands were diffusely stained by the anti-human calponin monoclonal antibody. Calponin was found in 53 (98%) of the tumors studied, reacting with the myoepithelium-like cells of 86% of benign tumors and their remnants in 85% of malignant tumors. Five different types of calponin-immunoreactive myoepithelial cells were identified: hypertrophic myoepithelial cells. fusiform cells, stellate myoepithelial cells, rounded (myoepithelial) cells, and chondroblasts. Differences in staining intensity and staining pattern among these five types of cells suggested a transition of myoepithelial cells to chondroblasts. Stromal myofibroblasts also showed calponin immunoreactivity, but they did not react with a cytokeratin 14 monoclonal antibody, which recognizes myoepithelial cells in mammary gland. Calponin appears to be a very sensitive marker of normal and neoplastic myoepithelium in the canine mammary gland, and its identification in different cell types of complex and mixed tumors of the mammary gland of the dog suggests a major histogenetic role for myoepithelial cells.

Contrast-assisted destruction-replenishment ultrasound for the assessment of tumor microvasculature in a rat model.

Angiogenesis, the development of new blood vessels, is necessary for tumor growth. Anti-angiogenic therapies have recently received attention as a possible cancer treatment. The purpose of this study was to monitor the vascularity of induced tumors in rats using contrast-enhanced ultrasound during anti-angiogenic therapy. Six rats with subcutaneously implanted R3230 murine mammary adenocarcinomas were treated with an orally administered anti-angiogenic agent (SU11657) beginning 28 days after tumor implantation (20 mg/kg BW once daily). Three additional tumor-bearing control rats were treated with an equivalent volume of vehicle alone. Sonographic evaluation of tumor blood flow was performed using a modified Siemens Sonoline Elegra equipped with a 5.0 MHz linear transducer prior to drug administration, during the first 51 hours following initial drug administration, and on days 8 and 15 after initiation of therapy. Tumor volumes were estimated at each time point using a prolate ellipsoid method from linear dimensions measured on the B-mode ultrasound image in the three major axes. A destruction-replenishment technique was used for tumor blood flow evaluation using a constant rate infusion of intravenously delivered ultrasound contrast media (Definity). A destructive pulse was fired first, followed by a chain of non-destructive pulses that allowed for visualization of vascular contrast agent replenishment. Parametric maps of the time required for contrast agent replenishment and the time-integrated intensity were generated for both the tumor and kidney. Following ultrasound examination, contrast-enhanced computed tomography of each tumor was performed in the same imaging plane as that used to acquire the ultrasound images. Fifteen days after the start of treatment, tumors were excised, preserved in 10% formalin, and sectioned in a plane approximating the ultrasound and CT imaging planes. Sections were prepared for light microscopy with H & E, CD31 and factor VIII immunostain to evaluate overall morphology and vessel distribution. Ultrasound measurements of tumor volume, the spatial extent of contrast enhancement, and the time required for contrast replenishment within control tumors were significantly different from those of treated tumors. The time-integrated ultrasound contrast enhancement decreases and the time required for replenishment of the contrast agent within the tumor volume increases over the course of anti-angiogenic therapy. Parametric maps of integrated intensity are shown to correlate with the regions of viable tumor demonstrated on H & E and regions of elevated contrast intensity on CT. Contrast-enhanced ultrasound imaging of implanted tumors provides a tool to assess differences in the microcirculation of treated and control tumors in studies of anti-angiogenic agents.

Immunohistological assessment of fibrin deposition and thrombus formation in canine mammary neoplasia.

A commercially available monoclonal antibody against human fibrin was used to detect fibrin in canine formalin-fixed, paraffin wax-embedded tissue by applying a slightly modified alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. Twenty-eight mammary tumours from six bitches were examined for the presence of fibrin. Thrombi and extravascular fibrin deposits were detected in 15 tumours (12 complex adenocarcinomas, one adenocarcinoma, two solid carcinomas), and a single thrombus was detected in one adenoma; 12 tumours (three adenomas, one complex adenoma, four complex adenocarcinomas and four adenocarcinomas) did not show any staining reaction.