Quantitative Proteomic and Mutational Landscape of Metaplastic Breast Carcinoma and Generation of a 3D Organoid Model of Neoplastic Progression

Triple-negative breast cancer (TNBC) is considered to be the most aggressive and has worse prognosis compared to other breast cancers and accounts for roughly 18% of all epithelial cancers of the breast, or carcinomas. TNBC exhibits complex molecular heterogeneity both inter- and intratumorally and likely consists of several distinct molecular subgroups that are currently unknown. Metaplastic breast carcinoma (MBC) is even more aggressive than triple-negative breast cancer (TNBC) but also typically presents as triple-negative histologically, and is defined by the admixture of both invasive glandular and non-glandular “metaplastic” heterologous elements of spindle, squamous or sarcomatoid subtypes. The protein profiles underpinning the phenotypic diversity and metastatic behavior of MBC are unknown. We present a quantitative multi-subtype proteomic landscape of MBC, non-metaplastic TNBC, and normal breast from small yet well-annotated cohort of 27 patients, and also present the somatic mutational landscape on the same cohort. We used multiplex isobaric tandem mass tag (TMT) labeling for proteomics and quantified 5,798 proteins, and from whole-exome sequencing for genomics analysis we found 980 total somatic mutational variants. MBCs displayed increased epithelial-to-mesenchymal transition (EMT) and extracellular matrix (ECM) signaling, and reduced metabolic pathways compared to TNBC. We discovered subtype-specific profiles among MBCs including distinct upregulated profiles; translation and ribosomal events in spindle, inflammation and apical junctions in squamous, and extracellular matrix in sarcomatoid. Comparison of the proteomes of spindle MBC with MMTV-cre;Ccn6fl/fl spindle MBC mouse tumors revealed a shared spindle-specific signature of 17 upregulated proteins involved in translation (e.g. RPL4,6,18, P3H1, PYCR1). The somatic mutational landscape also revealed MBCs share common TP53 mutations, and in PLEC, MUC17, CRYBG2, and ZNF681. We identified that spindle and squamous MBC exhibit overlapping mutational profiles of genes involved in transcription, RNA metabolic processes and actin filament binding, while sarcomatoid tumors harbor distinct mutations in MAPK, WNT, protocadherin cluster genes, calcium binding and ECM organization. These data identify subtype-specific MBC protein profiles and mutational signatures that identified novel biomarkers for therapy. Three-dimensional (3D) cell culture has been widely used in recent decades, compared with monolayer (2D) culture, because they better mimic the in vivo state. 3D systems utilize different types of gels critical for their success, such as collagen or the reconstituted basement membrane, Matrigel, which has enabled recapitulation of tissue architecture and function that is more physiologic compared to 2D. However, conventional 3D models using gel-embedded platforms have large variability and slow transport of biomolecules to the matrix-encapsulated cells. Here, we developed a highly reproducible, 3D scaffold-free hanging drop method amenable for primary tissues including mouse and human tumors, and our analyses describe a one drop-one organoid format using MCF10A cells, a non-tumorigenic breast cell line. We attained high-yield production of uniform organoids that resemble normal human breast acini, express both mammary gland-specific and progenitor markers, and we developed treatment assays for EMT induction and neoplastic progression delivering rapid quantification of phenotypic and morphological changes. Integration of 3D methods with omics analyses is envisioned to enhance the study of neoplastic progression and generate novel targets of both MBC and TNBC tumors.PHDMolecular & Cellular PathologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/155234/1/djomehri_1.pd

Modeling Diffusivity Through Alginate-Based Microfibers: A Comparison of Numerical and Analytical Models Based on Empirical Spectrophotometric Data

Abstract: The study of mass transport across hollow and solid 3D microfibers to study metabolic profiles is a key aspect of tissue engineering approach. A new modified numerical mathematical model based on Fickian equations in cylindrical coordinates has been proposed for determining the membrane diffusivity of 2% (w/v) alginate-based stents cross-linked with 10% CaCl2. Based on the economical and direct spectrophotometric measurements, using this model, inward diffusivities ranging from 5.2x10-14 m2/s 2.93x10-12m2/s were computed for solutes with Stokes radii ranging between 0.36 to 3.5 nm, diffusing through bare alginate and alginate-chitosan-alginate microfibers. In parallel an analytical solution to the cylindrical Fickian equation was derived to validate the numerical solution using experimental diffusion data from a solid stent. Excellent agreement was found between the numerical and analytical models with a maximum calculated residual value of 4%. Using these models, a flexible computational platform is proposed to conduct custom diffusion and MW cut-off characterization across micro-porous microfibers not limited to alginate in composition

Definition of a Twelve-Point Polygonal SAA Boundaryfor the GLAST Mission

The Gamma-Ray Large Area Space Telescope (GLAST), set to launch in early 2008, detects gamma rays within a huge energy range of 100 MeV - 300 GeV. Background cosmic radiation interferes with such detection resulting in confusion over distinguishing cosmic from gamma rays encountered. This quandary is resolved by encasing GLAST's Large Area Telescope (LAT) with an Anti-Coincidence Detector (ACD), a device which identifies and vetoes charged particles. The ACD accomplishes this through plastic scintillator tiles; when cosmic rays strike, photons produced induce currents in Photomultiplier Tubes (PMTs) attached to these tiles. However, as GLAST orbits Earth at altitudes {approx}550km and latitudes between -26 degree and 26 degree, it will confront the South Atlantic Anomaly (SAA), a region of high particle flux caused by trapped radiation in the geomagnetic field. Since the SAA flux would degrade the sensitivity of the ACD's PMTs over time, a determined boundary enclosing this region need be attained, signaling when to lower the voltage on the PMTs as a protective measure. The operational constraints on such a boundary require a convex SAA polygon with twelve edges, whose area is minimal ensuring GLAST has maximum observation time. The AP8 and PSB97 models describing the behavior of trapped radiation were used in analyzing the SAA and defining a convex SAA boundary of twelve sides. The smallest possible boundary was found to cover 14.58% of GLAST's observation time. Further analysis of defining a boundary safety margin to account for inaccuracies in the models reveals if the total SAA hull area is increased by {approx}20%, the loss of total observational area is < 5%. These twelve coordinates defining the SAA flux region are ready for implementation by the GLAST satellite

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

Diffusive and Mechanical Properties of Biodegradable Alginate Stents

Hydrogels like alginate and chitosan have proven potential in drug delivery systems. Their advantageous qualities such as biocompatibility, biodegradability, non-toxicity, and structural tunability make them excellent drug carriers. Alginate-chitosan-alginate (ACA) hollow fibers (i.e., stents) were fabricated with a semi-permeable membrane to allow release of drugs by diffusion into a desired lumen of the body. A solution of 2% (w/v) medium molecular weight alginate crosslinked with 10% CaCl2 was used to fabricate hollow stents (od~1300 µm, id~900 µm, t~300 µm, L=3.5cm). For empty stents, solutes with Stokes radii ranging from 0.36 to 3.5 nm were determined to have diffusivities between 2.93x10-12 and 5.2x10-14 m2/s for uncoated and ACA membranes, as determined by spectrophotometry and mathematical modeling in Matlab 7.10.0(R2010a). A comparative technique with fluorescence microscopy was used to validate the diffusivity range of 3.78x10-10 - 3.94x10-13 m2/s for FITC-dextrans with molecular weights ranging from 4-500 kDa. Mechanical testing on bare hollow alginate stents showed that as CaCl2 concentration and cross-linking duration was increased, the tensile strength and elastic modulus increased. Bare alginate and ACA stents cross-linked with 10% CaCl2 for 60 min had the following tensile properties: E=0.24 MPa, UTS=0.097MPa and E=0.053MPa, UTS=1.03x10-01 MPa

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

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 (R2 ≥ 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

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&lt;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

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

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