Case selection, histopathological analysis, and overall survival analysis
Samples from eight cases of IMPC and one control normal canine mammary gland were selected at the Laboratory of Comparative Pathology of the Institute of Biological Sciences, Universidade Federal de Minas Gerais (UFMG), after approval by the Animal Experimentation Ethics Committee (CEUA protocol number: 362/2016). The human IMPC sample used as a positive control was donated by the Laboratory of Clinical Analysis “Hermes Pardini”, Belo Horizonte, Minas Gerais, and was approved by the Research Ethics Committee (CAAE-0002.0.204.203-11).
For histopathological analysis (grading, histotype and margins), primary tumor specimens were fixed with 10% neutral buffered formalin, embedded in paraffin (to generate formalin-fixed, paraffin-embedded (FFPE) samples), sliced into 4 µm thick histological sections, and stained with hematoxylin and eosin (Sigma-Aldrich, Carlsbad, CA, USA). All cases were reviewed and reclassified independently by two pathologists (GDC and TS). In brief, carcinomas with cystic formations containing nests of epithelial cells with a moruliform appearance (an infiltrating micropapillary pattern) were diagnosed as IMPC, regardless of whether they were associated with in situ micropapillary areas [25] (Fig. 1). The invasive areas of canine IMPCs were graded according to the Nottingham grading system [26, 27]. The overall survival time was defined as the period (in days) between surgery and death due to the tumor. The follow-up period was 400 days. Seven of the dogs evaluated died due to the disease; however, in one case, the dog died because of hemorrhagic diathesis. The survival rate was calculated using the Kaplan–Meier method [6, 9, 28].
Immunohistochemistry
IHC was performed as previously described with minor modifications [9, 29]. Sections (4 μm) of primary tumors were mounted on silanized slides, and a peroxidase-based detection system, Novolink™ Polymer (Leica Biosystems Newcastle Ltd., Newcastle, UK), was applied. Slides were dewaxed in xylene, and endogenous peroxidase activity was quenched with 3% H2O2 in methanol. The reagents were applied manually, and immunoreactivity was visualized by incubating the slides with 3,3'-diaminobenzidine (Lab Vision DAB substrate system; Lab Vision, Fremont, California, USA) for 5 minutes. The antibodies used in this study are described in detail in Table 2. Negative controls were established using normal serum (Lab Vision Ultra V Block) instead of the primary antibody. Normal canine mammary gland tissue was used as a positive control for all immunohistochemical staining procedures (see Supplementary Fig. 1).
IF staining for phenotypic markers and imaging via superresolution microscopy
IF staining was performed as previously described by Rodrigues M.A. et al. (2016) [12, 30–32]. In brief, FFPE tissue sections were dewaxed, rehydrated, and unmasked in Trilogy solution (Cell Marque, Koclin, CA, USA) with pressurized heating (125°C) for 20 minutes according to the manufacturer’s instructions. Next, samples were rinsed with phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl and 10 mM phosphate buffer solution (pH 7.4)) (Sigma-Aldrich), incubated with PBS containing 0.2% Triton X-100 (Sigma-Aldrich) for another 20 minutes, and blocked with PBS containing 1% bovine serum albumin (BSA, Sigma-Aldrich) for 30 minutes. Next, sections were incubated with primary antibodies against the nuclear envelope markers Lamin B1/B2, aSMA, Vimentin, CD31, von Willebrand factor, p63, S100A4 and MUC1. Detailed information about each antibody is presented in Table 2. The immunoreactivity of these antibodies has already been validated in canine species, as described in previous studies and by the manufacturer [6, 24, 33–39]. One sample of human IMPC was used as a positive control for all labeling performed in the present study (see Supplementary Figs. 4 and 5). Then, sections were rinsed three times with PBS for 5 minutes each. Subsequently, sections were incubated with an Alexa Fluor® 488-conjugated goat anti-rabbit IgG antibody (1:1000, Life Technologies, Carlsbad, CA, USA), an Alexa Fluor® 555-conjugated goat anti-mouse IgG antibody (1:1000, Life Technologies) and Hoechst 33258 (1 mg/mL, Life Technologies) for 1 hour at room temperature. Next, the samples were washed 3x with PBS for 10 minutes each and were then mounted using Prolong Gold Antifade Reagent (Life Technologies). A negative control was included in all reactions by omitting the primary antibodies (see Supplementary Fig. 3e). Images were acquired with a Zeiss LSM 880 connected to an Airyscan detector (Carl Zeiss, Jena, Germany) using an 40× 1.3 NA oil objective. In this study, we used the Airyscan system to increase the signal-to-noise ratio and resolution. This system is a 32-channel GaAsP-PMT area detector that is used for superresolution microscopy to resolve structures beyond the diffraction-limited resolution of conventional light microscopes [40]. Samples were excited at 405 nm and observed at 420-480 nm to detect Hoechst, excited at 488 nm and observed at 500-525 nm to detect Alexa Fluor 488, and excited at 543 nm and observed using a longpass (LP) 570 nm filter to detect the Alexa Fluor 555 signal. Zeiss Efficient Navigation (ZEN) software was used to display orthogonal projections (XY, XZ, and YZ). The fluorescence microscopy results were evaluated in invasive areas of IMPCs, and five to ten images were acquired from each sample. Seven markers were analyzed (n = 300 images).
Tissue processing for ultrastructural evaluation
For TEM, one normal and one tumor biopsy sample (five fragments of each) fixed with 10% neutral buffered formalin were cut into pieces of approximately 2 mm (length x width) and subsequently postfixed with 5% glutaraldehyde (biological grade; Electron Microscopy Sciences, Hatfield, PA, USA) in 0.05 M phosphate buffer (pH 7.3) for 24 hours. Then, the fragments were postfixed with reduced osmium (osmium tetroxide 1% and potassium ferrocyanide in distilled water) for 90 minutes and dehydrated in ethanol and acetone before embedding in epoxy Araldite resin (Electron Microscopy Sciences, Hatfield, PA, USA). After preparation of ultrathin sections at a thickness of 60 nm, images were acquired using a Tecnai G-12 FEI–120 KV microscope. The images were adjusted for resolution, sharpness, and contrast using Adobe Photoshop software (Adobe System, Inc., Mountain View, CA, USA).