CCN6 suppresses metaplastic breast carcinoma by antagonizing WNT/ß-catenin signaling to inhibit EZH2-driven EMT.

Metaplastic breast carcinomas (mBrCAs) are a highly aggressive subtype of triple negative breast cancer (TNBC) with histological evidence of epithelial to mesenchymal transition (EMT) and aberrant differentiation. Inactivation of the tumor suppressor gene CCN6 (also known as WISP3) is a feature of mBrCAs, and mice with conditional inactivation of Ccn6 in mammary epithelium (Ccn6-KO) develop spindle mBrCAs with EMT. Elucidation of the precise mechanistic details of how CCN6 acts as a tumor suppressor in mBrCA could help identify improved treatment strategies. Here, we showed that CCN6 interacts with the Wnt receptor FZD8 and co-receptor LRP6 on mBrCA cells to antagonize Wnt-induced activation of ß-catenin/TCF-mediated transcription. The histone methyltransferase EZH2 was identified as a ß-catenin/TCF transcriptional target in Ccn6-KO mBrCA cells. Inhibiting Wnt/ß-catenin/TCF signaling in Ccn6-KO mBrCa cells led to reduced EZH2 expression, decreased histone H3 lysine 27 trimethylation, and deregulation of specific target genes. Pharmacological inhibition of EZH2 reduced growth and metastasis of Ccn6-KO mBrCA mammary tumors in vivo. Low CCN6 is significantly associated with activated ß-catenin and high EZH2 in human spindle mBrCAs compared to other subtypes. Collectively, these findings establish CCN6 as a key negative regulator of a ß-catenin/TCF-EZH2 axis and highlight inhibition of ß-catenin or EZH2 as a potential therapeutic approach for patients with spindle mBrCAs.

A hybrid breast cancer/mesenchymal stem cell population enhances chemoresistance and metastasis.

Patients with triple-negative breast cancer remain at risk for metastatic disease despite treatment. The acquisition of chemoresistance is a major cause of tumor relapse and death, but the mechanisms are far from understood. We have demonstrated that breast cancer cells (BCCs) can engulf mesenchymal stem/stromal cells (MSCs), leading to enhanced dissemination. Here, we show that clinical samples of primary invasive carcinoma and chemoresistant breast cancer metastasis contain a unique hybrid cancer cell population coexpressing pancytokeratin and the MSC marker fibroblast activation protein-α. We show that hybrid cells form in primary tumors and that they promote breast cancer metastasis and chemoresistance. Using single-cell microfluidics and in vivo models, we found that there are polyploid senescent cells within the hybrid cell population that contribute to metastatic dissemination. Our data reveal that Wnt Family Member 5A (WNT5A) plays a crucial role in supporting the chemoresistance properties of hybrid cells. Furthermore, we identified that WNT5A mediates hybrid cell formation through a phagocytosis-like mechanism that requires BCC-derived IL-6 and MSC-derived C-C Motif Chemokine Ligand 2. These findings reveal hybrid cell formation as a mechanism of chemoresistance and suggest that interrupting this mechanism may be a strategy in overcoming breast cancer drug resistance.

Mesenchymal tumor cells drive adaptive resistance of Trp53-/- breast tumor cells to inactivated mutant Kras.

As precision medicine increases the response rate of treatment, tumors frequently bypass inhibition, and reoccur. In order for treatment to be effective long term, the mechanisms enabling treatment adaptation need to be understood. Here, we report a mouse model that, in the absence of p53 and the presence of oncogenic KrasG12D , develops breast tumors. Upon inactivation of KrasG12D , tumors initially regress and enter remission. Subsequently, the majority of tumors adapt to the withdrawal of KrasG12D expression and return. KrasG12D -independent tumor cells show a strong mesenchymal profile with active RAS-RAF-MEK-ERK (MAPK/ERK) signaling. Both KrasG12D -dependent and KrasG12D -independent tumors display a high level of genomic instability, and KrasG12D -independent tumors harbor numerous amplified genes that can activate the MAPK/ERK signaling pathway. Our study identifies both epithelial-mesenchymal transition (EMT) and active MAPK/ERK signaling in tumors that adapt to oncogenic KrasG12D withdrawal in a novel Trp53-/- breast cancer mouse model. To achieve long-lasting responses in the clinic to RAS-fueled cancer, treatment will need to focus in parallel on obstructing tumors from adapting to oncogene inhibition.

Cancer Cell Invasion of Mammary Organoids with Basal-In Phenotype.

This paper describes mammary organoids with a basal-in phenotype where the basement membrane is located on the interior surface of the organoid. A key materials consideration to induce this basal-in phenotype is the use of a minimal gel scaffold that the epithelial cells self-assemble around and encapsulate. When MDA-MB-231 breast cancer cells are co-cultured with epithelial cells from day 0 under these conditions, cells self-organize into patterns with distinct cancer cell populations both inside and at the periphery of the epithelial organoid. In another type of experiment, the robust formation of the basement membrane on the epithelial organoid interior enables convenient studies of MDA-MB-231 invasion in a tumor progression-relevant direction relative to epithelial cell-basement membrane positioning. That is, the study of cancer invasion through the epithelium first, followed by the basement membrane to the basal side, is realized in an experimentally convenient manner where the cancer cells are simply seeded on the outside of preformed organoids, and their invasion into the organoid is monitored. Interestingly, invasion is more prominent when tumor cells are added to day 7 organoids with less developed basement membranes compared to day 16 organoids with more defined ones.

Subcellular localization of EZH2 phosphorylated at T367 stratifies metaplastic breast carcinoma subtypes.

BACKGROUND: Metaplastic carcinoma is an aggressive, triple-negative breast cancer (TNBC) with differentiation towards squamous, spindle, or mesenchymal cell types. The molecular underpinnings of the histological subtypes are unclear. Our lab discovered a cytoplasmic function of EZH2, a transcriptional repressor, whereby pEZH2 T367 binds to cytoplasmic proteins in TNBC cells and enhances invasion and metastasis. Here, we investigated the expression and subcellular localization of pEZH2 T367 protein in metaplastic carcinomas. METHODS: Thirty-five metaplastic carcinomas (17 squamous, 10 mesenchymal, and 8 spindle) were evaluated and immunostained with anti-pEZH2 T367. We analyzed staining intensity (score 1-4), subcellular localization (nuclear/cytoplasmic), and localization within the tumor (center/invasive edge). Protein expression of pEZH2 T367-binding partners was measured from a quantitative multiplex proteomics analysis performed in our lab. RESULTS: Cytoplasmic pEZH2 T367 was significantly upregulated in squamous (14 of 17, 82%) compared to mesenchymal (4 of 10, 40%) and spindle (2 of 6, 33%) subtypes (p = 0.011). Twenty-five of 34 (73%) tumors with available tumor-normal interface showed accentuated cytoplasmic pEZH2 T367 at the infiltrative edge. Cytoplasmic pEZH2 T367 was upregulated in 9 of 10 (90%) tumors with lymph node metastasis (p = 0.05). Bioinformatics analyses identified an EZH2 protein network in metaplastic carcinomas (p value: < 1.0e-16). Using quantitative proteomics, we found significantly increased expression of cytoplasmic EZH2-binding partners in squamous compared to spindle and mesenchymal subtypes. CONCLUSIONS: pEZH2 T367 expression and subcellular localization may be useful to distinguish metaplastic carcinoma subtypes. pEZH2 T367 may play a role in the histological diversity and behavior of these tumors.

Quantitative proteomic landscape of metaplastic breast carcinoma pathological subtypes and their relationship to triple-negative tumors.

Metaplastic breast carcinoma (MBC) is a highly aggressive form of triple-negative cancer (TNBC), defined by the presence of metaplastic components of spindle, squamous, or sarcomatoid histology. The protein profiles underpinning the pathological subtypes and metastatic behavior of MBC are unknown. Using multiplex quantitative tandem mass tag-based proteomics we quantify 5798 proteins in MBC, TNBC, and normal breast from 27 patients. Comparing MBC and TNBC protein profiles we show MBC-specific increases related to epithelial-to-mesenchymal transition and extracellular matrix, and reduced metabolic pathways. MBC subtypes exhibit distinct upregulated profiles, including translation and ribosomal events in spindle, inflammation- and apical junction-related proteins in squamous, and extracellular matrix proteins in sarcomatoid subtypes. Comparison of the proteomes of human spindle MBC with mouse spindle (CCN6 knockout) MBC tumors reveals a shared spindle-specific signature of 17 upregulated proteins involved in translation and 19 downregulated proteins with roles in cell metabolism. These data identify potential subtype specific MBC biomarkers and therapeutic targets.

Next-generation sequencing identifies recurrent copy number variations in invasive breast carcinomas from Ghana.

African and African-American (AA) women have higher incidence of triple-negative breast cancers (TNBC) with high histological grade and aggressive clinical behavior, but the reasons are not fully understood. We recently found that the oncogenic protein EZH2 is overexpressed in Ghanaian breast cancer patients, with 16% of the tumors expressing cytoplasmic EZH2. Understanding the molecular underpinnings of these aggressive tumors may lead to the identification of potential targetable oncogenic drivers. We characterized the copy number variations of 11 Ghanaian breast tumor patients by targeted multiplexed PCR-based DNA next-generation sequencing (NGS) over 130 cancer-relevant genes. While the DNA quality was not optimal for mutation analysis, 90% of the tumors had frequent recurrent copy number alterations (CNAs) of 17 genes: SDHC, RECQL4, TFE3, BCL11A, BCL2L1, PDGFRA, DEK, SMUG1, AKT3, SMARCA4, VHL, KLF6, CCNE1, G6PD, FGF3, ABL1, and CCND1, with the top oncogenic functions being mitotic G1-G1/S-phase regulation, gene transcription, apoptosis, and PI3K/AKT pathway. The most common recurrent high-level CNAs were gains of RECQL4 and SDHC, in 50% and 60% of cases, respectively. Network analyses revealed a significant predicted interaction among 12 of the 17 (70.6%) genes with high-level CNAs (p = 5.7E-07), which was highly correlated with EZH2 expression (r = 0.4-0.75). By immunohistochemistry, RECQL4 and SDHC proteins were upregulated in 53 of 86 (61.6%) and 48 of 86 (56%) of Ghanaian invasive carcinoma tissue samples. In conclusion, our data show that invasive carcinomas from Ghana exhibit recurrent CNAs in 17 genes, with functions in oncogenic pathways, including PI3K/AKT and G1-G1/S regulation, which may have implications for the biology and treatment of invasive carcinomas in African and AA women.

A reproducible scaffold-free 3D organoid model to study neoplastic progression in breast cancer.

While 3D cellular models are useful to study biological processes, gel-embedded organoids have large variability. This paper describes high-yield production of large (~1 mm diameter), scaffold-free, highly-spherical organoids in a one drop-one organoid format using MCF10A cells, a non-tumorigenic breast cell line. These organoids display a hollow lumen and secondary acini, and express mammary gland-specific and progenitor markers, resembling normal human breast acini. When subjected to treatment with TGF-ß, the hypoxia-mimetic reagent CoCl2, or co-culture with mesenchymal stem/stromal cells (MSC), the organoids increase collagen I production and undergo large phenotypic and morphological changes of neoplastic progression, which were reproducible and quantifiable. Advantages of this scaffold-free, 3D breast organoid model include high consistency and reproducibility, ability to measure cellular collagen I production without noise from exogenous collagen, and capacity to subject the organoid to various stimuli from the microenvironment and exogenous treatments with precise timing without concern of matrix binding. Using this system, we generated organoids from primary metaplastic mammary carcinomas of MMTV-Cre;Ccn6fl/fl mice, which retained the high grade spindle cell morphology of the primary tumors. The platform is envisioned to be useful as a standardized 3D cellular model to study how microenvironmental factors influence breast tumorigenesis, and to potential therapeutics.

CCN6 regulates IGF2BP2 and HMGA2 signaling in metaplastic carcinomas of the breast.

PURPOSE: Metaplastic breast carcinomas are an aggressive subtype of triple-negative breast cancer (TNBC) in which part or all of the adenocarcinoma transforms into a non-glandular component (e.g., spindled, squamous, or heterologous). We discovered that mammary-specific Ccn6/Wisp3 knockout mice develop mammary carcinomas with spindle and squamous differentiation that share upregulation of the oncofetal proteins IGF2BP2 (IMP2) and HMGA2 with human metaplastic carcinomas. Here, we investigated the functional relationship between CCN6, IGF2BP2, and HMGA2 proteins in vitro and in vivo, and their expression in human tissue samples. METHODS: MMTV-cre;Ccn6fl/fl tumors and spindle TNBC cell lines were treated with recombinant CCN6 protein or vehicle. IGF2BP2 was downregulated using shRNAs in HME cells with stable CCN6 shRNA knockdown, and subjected to invasion and adhesion assays. Thirty-one human metaplastic carcinomas were arrayed in a tissue microarray (TMA) and immunostained for CCN6, IGF2BP2, and HMGA2. RESULTS: CCN6 regulates IGF2BP2 and HMGA2 protein expression in MMTV-cre;Ccn6fl/fl tumors, in MDA-MB-231 and - 468, and in HME cells. CCN6 recombinant protein reduced IGF2BP2 and HMGA2 protein expression, and decreased growth of MMTV-cre;Ccn6fl/fl tumors in vivo. IGF2BP2 shRNA knockdown was sufficient to reverse the invasive abilities conferred by CCN6 knockdown in HME cells. Analyses of the TCGA Breast Cancer Cohort (n = 1238) showed that IGF2BP2 and HMGA2 are significantly upregulated in metaplastic carcinoma compared to other breast cancer subtypes. In clinical samples, low CCN6 is frequent in tumors with high IGF2BP2/HMGA2 with spindle and squamous differentiation. CONCLUSIONS: These data shed light into the pathogenesis of metaplastic carcinoma and demonstrate a novel CCN6/IGF2BP2/HMGA2 oncogenic pathway with biomarker and therapeutic implications.

Strain-guided mineralization in the bone-PDL-cementum complex of a rat periodontium.

OBJECTIVE: The objective of this study was to investigate the effect of mechanical strain by mapping physicochemical properties at periodontal ligament (PDL)-bone and PDL-cementum attachment sites and within the tissues per se. DESIGN: Accentuated mechanical strain was induced by applying a unidirectional force of 0.06N for 14 days on molars in a rat model. The associated changes in functional space between tooth and bone, mineral formation and resorbing events at the PDL-bone and PDL-cementum attachment sites were identified by using micro-X-ray computed tomography (micro-XCT), atomic force microscopy (AFM), dynamic histomorphometry, Raman microspectroscopy, AFM-based nanoindentation technique, and were correlated with histochemical stains specific to low and high molecular weight GAGs, including biglycan, and osteoclast distribution through tartrate-resistant acid phosphatase (TRAP) staining. RESULTS: Unique chemical and mechanical qualities including heterogenous bony fingers with hygroscopic Sharpey's fibers contributing to a higher organic (amide III - 1240 cm-1) to inorganic (phosphate - 960 cm-1) ratio, with lower average elastic modulus of 8 GPa versus 12 GPa in unadapted regions were identified. Furthermore, an increased presence of elemental Zn in cement lines and mineralizing fronts of PDL-bone was observed. Adapted regions containing bony fingers exhibited woven bone-like architecture and these regions rich in biglycan (BGN) and bone sialoprotein (BSP) also contained high-molecular weight polysaccharides predominantly at the site of polarized bone growth. CONCLUSIONS: From a fundamental science perspective the shift in local properties due to strain amplification at the soft-hard tissue attachment sites is governed by semiautonomous cellular events at the PDL-bone and PDL-cementum sites. Over time, these strain-mediated events can alter the physicochemical properties of tissues per se, and consequently the overall biomechanics of the bone-PDL-tooth complex. From a clinical perspective, the shifts in magnitude and duration of forces on the periodontal ligament can prompt a shift in physiologic mineral apposition in cementum and alveolar bone albeit of an adapted quality owing to the rapid mechanical translation of the tooth.

Mineral density volume gradients in normal and diseased human tissues.

Clinical computed tomography provides a single mineral density (MD) value for heterogeneous calcified tissues containing early and late stage pathologic formations. The novel aspect of this study is that, it extends current quantitative methods of mapping mineral density gradients to three dimensions, discretizes early and late mineralized stages, identifies elemental distribution in discretized volumes, and correlates measured MD with respective calcium (Ca) to phosphorus (P) and Ca to zinc (Zn) elemental ratios. To accomplish this, MD variations identified using polychromatic radiation from a high resolution micro-computed tomography (micro-CT) benchtop unit were correlated with elemental mapping obtained from a microprobe X-ray fluorescence (XRF) using synchrotron monochromatic radiation. Digital segmentation of tomograms from normal and diseased tissues (N=5 per group; 40-60 year old males) contained significant mineral density variations (enamel: 2820-3095 mg/cc, bone: 570-1415 mg/cc, cementum: 1240-1340 mg/cc, dentin: 1480-1590 mg/cc, cementum affected by periodontitis: 1100-1220 mg/cc, hypomineralized carious dentin: 345-1450 mg/cc, hypermineralized carious dentin: 1815-2740 mg/cc, and dental calculus: 1290-1770 mg/cc). A plausible linear correlation between segmented MD volumes and elemental ratios within these volumes was established, and Ca/P ratios for dentin (1.49), hypomineralized dentin (0.32-0.46), cementum (1.51), and bone (1.68) were observed. Furthermore, varying Ca/Zn ratios were distinguished in adapted compared to normal tissues, such as in bone (855-2765) and in cementum (595-990), highlighting Zn as an influential element in prompting observed adaptive properties. Hence, results provide insights on mineral density gradients with elemental concentrations and elemental footprints that in turn could aid in elucidating mechanistic processes for pathologic formations.

Simulation and verification of macroscopic isotropy of hollow alginate-based microfibers.

A simulation of tensile strength of various alginate-based hollow microfibers using FEA analysis has been conducted with the hypothesis of macroscopic isotropy and linear elastic-plastic behavior. Results of student t-tests indicated that there was no significant difference between the experimental and simulated tensile strengths (p = 0.37, α = 0.05), while there was a significant reduction in elasticity as a result of chitosan coating (p = 0.024, α = 0.05). The hypothesis of macroscopic isotropy was verified by highly correlated (R(2) ≥ 0.92) theoretical and experimental elongation at break measurements, findings that could be extended to the failure analysis of alginate microfibers used in regenerative medicine.

The narwhal (Monodon monoceros) cementum-dentin junction: a functionally graded biointerphase.

In nature, an interface between dissimilar tissues is often bridged by a graded zone, and provides functional properties at a whole organ level. A perfect example is a "biological interphase" between stratified cementum and dentin of a narwhal tooth. This study highlights the graded structural, mechanical, and chemical natural characteristics of a biological interphase known as the cementum-dentin junction layer and their effect in resisting mechanical loads. From a structural perspective, light and electron microscopy techniques illustrated the layer as a wide 1000-2000 µm graded zone consisting of higher density continuous collagen fiber bundles from the surface of cementum to dentin, that parallels hygroscopic 50-100 µm wide collagenous region in human teeth. The role of collagen fibers was evident under compression testing during which the layer deformed more compared to cementum and dentin. This behavior is reflected through site-specific nanoindentation indicating a lower elastic modulus of 2.2 ± 0.5 GPa for collagen fiber bundle compared to 3 ± 0.4 GPa for mineralized regions in the layer. Similarly, microindentation technique illustrated lower hardness values of 0.36 ± 0.05 GPa, 0.33 ± 0.03 GPa, and 0.3 ± 0.07 GPa for cementum, dentin, and cementum-dentin layer, respectively. Biochemical analyses including Raman spectroscopy and synchrotron-source microprobe X-ray fluorescence demonstrated a graded composition across the interface, including a decrease in mineral-to-matrix and phosphate-to-carbonate ratios, as well as the presence of tidemark-like bands with Zn. Understanding the structure-function relationships of wider tissue interfaces can provide insights into natural tissue and organ function.

Fickian-Based Empirical Approach for Diffusivity Determination in Hollow Alginate-Based Microfibers Using 2D Fluorescence Microscopy and Comparison with Theoretical Predictions.

Hollow alginate microfibers (od = 1.3 mm, id = 0.9 mm, th = 400 µm, L = 3.5 cm) comprised of 2% (w/v) medium molecular weight alginate cross-linked with 0.9 M CaCl2 were fabricated to model outward diffusion capture by 2D fluorescent microscopy. A two-fold comparison of diffusivity determination based on real-time diffusion of Fluorescein isothiocyanate molecular weight (FITC MW) markers was conducted using a proposed Fickian-based approach in conjunction with a previously established numerical model developed based on spectrophotometric data. Computed empirical/numerical (Dempiricial/Dnumerical) diffusivities characterized by small standard deviations for the 4-, 70- and 500-kDa markers expressed in m²/s are (1.06 × 10-9 ± 1.96 × 10-10)/(2.03 × 10-11), (5.89 × 10-11 ± 2.83 × 10-12)/(4.6 × 10-12) and (4.89 × 10-12 ± 3.94 × 10-13)/(1.27 × 10-12), respectively, with the discrimination between the computation techniques narrowing down as a function of MW. The use of the numerical approach is recommended for fluorescence-based measurements as the standard computational method for effective diffusivity determination until capture rates (minimum 12 fps for the 4-kDa marker) and the use of linear instead of polynomial interpolating functions to model temporal intensity gradients have been proven to minimize the extent of systematic errors associated with the proposed empirical method.

Biomechanics of a bone-periodontal ligament-tooth fibrous joint.

This study investigates bone-tooth association under compression to identify strain amplified sites within the bone-periodontal ligament (PDL)-tooth fibrous joint. Our results indicate that the biomechanical response of the joint is due to a combinatorial response of the constitutive properties of organic, inorganic, and fluid components. Second maxillary molars within intact maxillae (N=8) of 5-month-old rats were loaded with a µ-XCT-compatible in situ loading device at various permutations of displacement rates (0.2, 0.5, 1.0, 1.5, 2.0 mm/min) and peak reactionary load responses (5, 10, 15, 20 N). Results indicated a nonlinear biomechanical response of the joint, in which the observed reactionary load rates were directly proportional to displacement rates (velocities). No significant differences in peak reactionary load rates at a displacement rate of 0.2mm/min were observed. However, for displacement rates greater than 0.2mm/min, an increasing trend in reactionary rate was observed for every peak reactionary load with significant increases at 2.0mm/min. Regardless of displacement rates, two distinct behaviors were identified with stiffness (S) and reactionary load rate (LR) values at a peak load of 5 N (S(5 N)=290-523 N/mm) being significantly lower than those at 10 N (LR(5 N)=1-10 N/s) and higher (S(10 N-20 N)=380-684 N/mm; LR(10 N-20 N)=1-19 N/s). Digital image correlation revealed the possibility of a screw-like motion of the tooth into the PDL-space, i.e., predominant vertical displacement of 35 µm at 5 N, followed by a slight increase to 40 µm at 10 N and 50 µm at 20 N of the tooth and potential tooth rotation at loads above 10 N. Narrowed and widened PDL spaces as a result of tooth displacement indicated areas of increased apparent strains within the complex. We propose that such highly strained regions are "hot spots" that can potentiate local tissue adaptation under physiological loading and adverse tissue adaptation under pathological loading conditions.

Age-related adaptation of bone-PDL-tooth complex: Rattus-Norvegicus as a model system.

Functional loads on an organ induce tissue adaptations by converting mechanical energy into chemical energy at a cell-level. The transducing capacity of cells alters physico-chemical properties of tissues, developing a positive feedback commonly recognized as the form-function relationship. In this study, organ and tissue adaptations were mapped in the bone-tooth complex by identifying and correlating biomolecular expressions to physico-chemical properties in rats from 1.5 to 15 months. However, future research using hard and soft chow over relevant age groups would decouple the function related effects from aging affects. Progressive curvature in the distal root with increased root resorption was observed using micro X-ray computed tomography. Resorption was correlated to the increased activity of multinucleated osteoclasts on the distal side of the molars until 6 months using tartrate resistant acid phosphatase (TRAP). Interestingly, mononucleated TRAP positive cells within PDL vasculature were observed in older rats. Higher levels of glycosaminoglycans were identified at PDL-bone and PDL-cementum entheses using alcian blue stain. Decreasing biochemical gradients from coronal to apical zones, specifically biomolecules that can induce osteogenic (biglycan) and fibrogenic (fibromodulin, decorin) phenotypes, and PDL-specific negative regulator of mineralization (asporin) were observed using immunohistochemistry. Heterogeneous distribution of Ca and P in alveolar bone, and relatively lower contents at the entheses, were observed using energy dispersive X-ray analysis. No correlation between age and microhardness of alveolar bone (0.7 ± 0.1 to 0.9 ± 0.2 GPa) and cementum (0.6 ± 0.1 to 0.8 ± 0.3 GPa) was observed using a microindenter. However, hardness of cementum and alveolar bone at any given age were significantly different (P<0.05). These observations should be taken into account as baseline parameters, during development (1.5 to 4 months), growth (4 to 10 months), followed by a senescent phase (10 to 15 months), from which deviations due to experimentally induced perturbations can be effectively investigated.