Morphological and immunohistochemical characterization and molecular classification of spontaneous mammary gland tumors in macropods

Mammary gland neoplasms in macropods are uncommonly reported, and the morphological and immunohistochemical characteristics are incompletely described. The goal of this study was to describe the morphologic features of macropod mammary neoplasms and to determine the molecular subtypes of mammary carcinomas using a panel of antibodies against estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (Her-2), p63, smooth muscle actin (SMA), and epidermal growth factor receptor (EGFR). Biopsy and necropsy specimens were examined from 21 macropods with mammary tumors submitted to Northwest ZooPath from 1996 to 2019. In accordance with the histologic classification of canine mammary tumors proposed by Goldschmidt and colleagues, tubulopapillary (2), tubular (10), and comedo-carcinomas (2), adenoma (1), lobular hyperplasia (3), fibroadenomatous hyperplasia (1), and mastitis (2) were diagnosed. Red kangaroos ( Osphranter rufus) were most commonly diagnosed with mammary carcinomas (79% of all carcinomas). Seven carcinomas had lymphovascular invasion and 2 also had pulmonary metastases. Six of these 7 carcinomas were classified as grade 3. Immunohistochemistry (IHC) for all antibodies was performed on 9/14 carcinomas, and partial IHC was performed for 3 cases. All 12 carcinomas were immunoreactive for PR, 5 for ER, 9 for EGFR, and none for Her-2. Five of the 9 mammary carcinomas with complete IHC data were classified as luminal A subtype, and 4 were normal-like subtype. Accurate classification of mammary tumors in macropods based on morphology, immunohistological characteristics, and molecular subtype may be helpful in guiding clinical management, prognosis, and potential therapeutic targets.

Cancer Influences the Elemental Composition of the Myocardium More Strongly than Conjugated Linoleic Acids-Chemometric Approach to Cardio-Oncological Studies

The aim of the study was to verify in a cardio-oncological model experiment if conjugated linoleic acids (CLA) fed to rats with mammary tumors affect the content of selected macro- and microelements in their myocardium. The diet of Sprague–Dawley females was supplemented either with CLA isomers or with safflower oil. In hearts of rats suffering from breast cancer, selected elements were analyzed with a quadrupole mass spectrometer with inductively coupled plasma ionization (ICP-MS). In order to better understand the data trends, cluster analysis, principal component analysis and linear discriminant analysis were applied. Mammary tumors influenced macro- and microelements content in the myocardium to a greater extent than applied diet supplementation. Significant influences of diet (p = 0.0192), mammary tumors (p = 0.0200) and interactions of both factors (p = 0.0151) were documented in terms of Fe content. CLA significantly decreased the contents of Cu and Mn (p = 0.0158 and p = 0.0265, respectively). The level of Ni was significantly higher (p = 0.0073), which was more pronounced in groups supplemented with CLA. The obtained results confirmed antioxidant properties of CLA and the relationship with Se deposition. Chemometric techniques distinctly showed that the coexisting pathological process induced differences to the greater extent than diet supplementation in the elemental content in the myocardium, which may impinge on cardiac tissue’s susceptibility to injuries.

Local, multimodal intralesional therapy renders distant brain metastases susceptible to PD-L1 blockade in a preclinical model of triple-negative breast cancer

Despite recent progress in therapeutic strategies, prognosis of metastatic triple-negative breast cancer (TNBC) remains dismal. Evidence suggests that the induction and activation of tumor-residing conventional type-1 dendritic cells (cDC1s) is critical for the generation of CD8+ T cells that mediate the regression of mammary tumors and potentiate anti-PD-1/PD-L1 therapeutic efficacy. However, it remains unknown whether this strategy is effective against metastatic TNBC, which is poorly responsive to immunotherapy. Here, using a mouse model of TNBC, we established orthotopic mammary tumors and brain metastases, and treated mammary tumors with in situ immunomodulation (ISIM) consisting of intratumoral injections of Flt3L to mobilize cDC1s, local irradiation to induce immunogenic tumor cell death, and TLR3/CD40 stimulation to activate cDC1s. ISIM treatment of the mammary tumor increased circulating T cells with effector phenotypes, and infiltration of CD8+ T cells into the metastatic brain lesions, resulting in delayed progression of brain metastases and improved survival. Furthermore, although anti-PD-L1 therapy alone was ineffective against brain metastases, ISIM overcame resistance to anti-PD-L1 therapy, which rendered these tumor-bearing mice responsive to anti-PD-L1 therapy and further improved survival. Collectively, these results illustrate the therapeutic potential of multimodal intralesional therapy for patients with unresectable and metastatic TNBC.

Evolution of HER2-positive mammary carcinoma: HER2 loss reveals claudin-low traits in cancer progression

HER2-positive breast cancers may lose HER2 expression in recurrences and metastases. In this work, we studied cell lines derived from two transgenic mammary tumors driven by human HER2 that showed different dynamics of HER2 status. MamBo89HER2stable cell line displayed high and stable HER2 expression, which was maintained upon in vivo passages, whereas MamBo43HER2labile cell line gave rise to HER2-negative tumors from which MamBo38HER2loss cell line was derived. Both low-density seeding and in vitro trastuzumab treatment of MamBo43HER2labile cells induced the loss of HER2 expression. MamBo38HER2loss cells showed a spindle-like morphology, high stemness and acquired in vivo malignancy. A comprehensive molecular profile confirmed the loss of addiction to HER2 signaling and acquisition of an EMT signature, together with increased angiogenesis and migration ability. We identified PDGFR-B among the newly expressed determinants of MamBo38HER2loss cell tumorigenic ability. Sunitinib inhibited MamBo38HER2loss tumor growth in vivo and reduced stemness and IL6 production in vitro. In conclusion, HER2-positive mammary tumors can evolve into tumors that display distinctive traits of claudin-low tumors. Our dynamic model of HER2 status can lead to the identification of new druggable targets, such as PDGFR-B, in order to counteract the resistance to HER2-targeted therapy that is caused by HER2 loss.

Prognostic impact of alterations in E-cadherin and P-cadherin expression in canine mammary tumors

Expression of cadherins has been correlated to the development and aggressiveness of epithelial neoplasms, however, in canine mammary tumors, their significance prognostic is uncertain. Due to this fact, the expression of the intracellular adhesion molecules E and P-cadherin and correlation with overall survival were analyzed in 25 canine mammary gland tumors. E-cadherin expression reduction was correlated with histological type, high histological grade, and overall survival rate. P-cadherin staining was higher in malignant tumors, unrelated to other clinicopathological features of aggressiveness. The results of this study suggest a relationship between lower expression of E and P-cadherin and worse prognostic in canine mammary tumors, including shorter overall survival.

Dual recombinase action in the normal and neoplastic mammary gland epithelium

We developed a transgenic mouse line that expresses the codon-optimized Flp recombinase under the control of the MMTV promoter in luminal epithelial cells of the mammary gland. In this report, we demonstrate the versatile applicability of the new MMTV-Flp strain to manipulate genes in a temporally and spatially controlled manner in the normal mammary gland, in luminal-type mammary tumors that overexpress ERBB2, and in a new KRAS-associated mammary cancer model. Although the MMTV-Flp is expressed in a mosaic pattern in the luminal epithelium, the Flp-mediated activation of a mutant KrasG12D allele resulted in basal-like mammary tumors that progressively acquired mesenchymal features. Besides its applicability as a tool for gene activation and cell lineage tracing to validate the cellular origin of primary and metastatic tumor cells, we employed the MMTV-Flp transgene together with the tamoxifen-inducible Cre recombinase to demonstrate that the combinatorial action of both recombinases can be used to delete or to activate genes in established tumors. In a proof-of-principle experiment, we conditionally deleted the JAK1 tyrosine kinase in KRAS-transformed mammary cancer cells using the dual recombinase approach and found that lack of JAK1 was sufficient to block the constitutive activation of STAT3. The collective results from the various lines of investigation showed that it is, in principle, feasible to manipulate genes in a ligand-controlled manner in neoplastic mammary epithelial cells, even when cancer cells acquire a state of cellular plasticity that may no longer support the expression of the MMTV-Flp transgene.