John D. Landua

STAT3 Signaling Is Activated Preferentially in Tumor-Initiating Cells in Claudin-Low Models of Human Breast Cancer

In breast cancer, a subset of tumor‐initiating cells (TIC) or “cancer stem cells” are thought to be responsible for tumor maintenance, treatment resistance, and disease recurrence. While current breast cancer stem cell markers (e.g., CD44high/CD24low/neg, ALDH positive) have allowed enrichment for such cells, they are not universally expressed and may actually identify distinct TIC subpopulations in the same tumor. Thus, additional markers of functional stem cells are needed. The STAT3 pathway is a critical regulator of the function of normal stem cells, and evidence is accumulating for its important role in breast cancer stem cells. However, due to the lack of a method for separating live cells based on their level of STAT3 activity, it remains unknown whether STAT3 functions in the cancer stem cells themselves, or in surrounding niche cells, or in both. To approach this question, we constructed a series of lentiviral fluorescent (enhanced green fluorescent protein, EGFP) reporters that enabled flow cytometric enrichment of cells differing in STAT3‐mediated transcriptional activity, as well as in vivo/in situ localization of STAT3 responsive cells. Using in vivo claudin‐low cell line xenograft models of human breast cancer, we found that STAT3 signaling reporter activity (EGFP+) is associated with a subpopulation of cancer cells enriched for mammosphere‐forming efficiency, as well as TIC function in limiting dilution transplantation assays compared to negative or unsorted populations. Our results support STAT3 signaling activity as another functional marker for human breast cancer stem cells thus making it an attractive therapeutic target for stem‐cell‐directed therapy in some breast cancer subtypes. Stem Cells 2014;32:2571–2582

Ptch1 is required locally for mammary gland morphogenesis and systemically for ductal elongation

Systemic hormones and local growth factor-mediated tissue interactions are essential for mammary gland development. Using phenotypic and transplantation analyses of mice carrying the mesenchymal dysplasia (mes) allele of patched 1 (Ptch1mes), we found that Ptch1mes homozygosity led to either complete failure of gland development, failure of post-pubertal ductal elongation, or delayed growth with ductal dysplasia. All ductal phenotypes could be present in the same animal. Whole gland and epithelial fragment transplantation each yielded unique morphological defects indicating both epithelial and stromal functions for Ptch1. However, ductal elongation was rescued in all cases, suggesting an additional systemic function. Epithelial function was confirmed using a conditional null Ptch1 allele via MMTV-Cre-mediated disruption. In Ptch1mes homozygotes, failure of ductal elongation correlated with diminished estrogen and progesterone receptor expression, but could not be rescued by exogenous ovarian hormone treatment. By contrast, pituitary isografts were able to rescue the ductal elongation phenotype. Thus, Ptch1 functions in the mammary epithelium and stroma to regulate ductal morphogenesis, and in the pituitary to regulate ductal elongation and ovarian hormone responsiveness.

Methods for Preparing Fluorescent and Neutral Red-Stained Whole Mounts of Mouse Mammary Glands

Whole mount preparations of mouse mammary glands are useful for evaluating overall changes in growth and morphology, and are essential for detecting and evaluating focal or regionally-localized phenotypes that would be difficult to detect or analyze using other techniques. We present three newly developed methods for preparing whole mounts of mammary glands from genetically-engineered mice expressing fluorescent proteins, as well as using either neutral red or a variety of fluorescent dyes. Unlike traditional hematoxylin- or carmine-stained preparations, neutral red-stained and some fluorescent preparations can be used for several common downstream analyses.

A Geometrically-Constrained Mathematical Model of Mammary Gland Ductal Elongation Reveals Novel Cellular Dynamics within the Terminal End Bud.

Mathematics is often used to model biological systems. In mammary gland development, mathematical modeling has been limited to acinar and branching morphogenesis and breast cancer, without reference to normal duct formation. We present a model of ductal elongation that exploits the geometrically-constrained shape of the terminal end bud (TEB), the growing tip of the duct, and incorporates morphometrics, region-specific proliferation and apoptosis rates. Iterative model refinement and behavior analysis, compared with biological data, indicated that the traditional metric of nipple to the ductal front distance, or percent fat pad filled to evaluate ductal elongation rate can be misleading, as it disregards branching events that can reduce its magnitude. Further, model driven investigations of the fates of specific TEB cell types confirmed migration of cap cells into the body cell layer, but showed their subsequent preferential elimination by apoptosis, thus minimizing their contribution to the luminal lineage and the mature duct.

Wnt-Responsive Cancer Stem Cells Are Located Close to Distorted Blood Vessels and Not in Hypoxic Regions in a p53-Null Mouse Model of Human Breast Cancer

Cancer stem cells (CSCs, or tumor‐initiating cells) may be responsible for tumor formation in many types of cancer, including breast cancer. Using high‐resolution imaging techniques, we analyzed the relationship between a Wnt‐responsive, CSC‐enriched population and the tumor vasculature using p53‐null mouse mammary tumors transduced with a lentiviral Wnt signaling reporter. Consistent with their localization in the normal mammary gland, Wnt‐responsive cells in tumors were enriched in the basal/myoepithelial population and generally located in close proximity to blood vessels. The Wnt‐responsive CSCs did not colocalize with the hypoxia‐inducible factor 1α‐positive cells in these p53‐null basal‐like tumors. Average vessel diameter and vessel tortuosity were increased in p53‐null mouse tumors, as well as in a human tumor xenograft as compared with the normal mammary gland. The combined strategy of monitoring the fluorescently labeled CSCs and vasculature using high‐resolution imaging techniques provides a unique opportunity to study the CSC and its surrounding vasculature.

Three-dimensional vasculature reconstruction of tumour microenvironment via local clustering and classification

The vasculature inside breast cancers is one important component of the tumour microenvironment. The investigation of its spatial morphology, distribution and interactions with cancer cells, including cancer stem cells, is essential for elucidating mechanisms of tumour development and treatment response. Using confocal microscopy and fluorescent markers, we have acquired three-dimensional images of vasculature within mammary tumours and normal mammary gland of mouse models. However, it is difficult to segment and reconstruct complex vasculature accurately from the in vivo three-dimensional images owing to the existence of uneven intensity and regions with low signal-to-noise ratios (SNR). To overcome these challenges, we have developed a novel three-dimensional vasculature segmentation method based on local clustering and classification. First, images of vasculature are clustered into local regions, whose boundaries well delineate vasculature even in low SNR and uneven intensity regions. Then local regions belonging to vasculature are identified by applying a semi-supervised classification method based on three informative features of the local regions. Comparison of results using simulated and real vasculature images, from mouse mammary tumours and normal mammary gland, shows that the new method outperforms existing methods, and can be used for three-dimensional images with uneven background and low SNR to achieve accurate vasculature reconstruction.

A mystery wrapped in an enigma: Matrigel enhancement of mammary cell growth and morphogenesis.

The analysis of normal mammary morphogenesis is facilitated by the use of mammary fat pad transplantation. The recent experiments on analysis of normal mammary epithelial stem cell activity rely heavily on this technique. In this review, we discuss the known and unknown attributes of using Matrigel in the injection of the mammary epithelial cell suspension. Matrigel greatly increases the “take” frequency of the injected cell suspension; however, there is some uncertainty regarding the interpretation of some of the results. After consideration of these issues, our conclusion is that Matrigel is important in order to obtain rigorous and reproducible results.

Carbon Nanotube Capsules Enhance the In vivo Efficacy of Cisplatin

Over the past few years, numerous nanotechnology-based drug delivery systems have been developed in an effort to maximize therapeutic effectiveness of conventional drug delivery, while limiting undesirable side effects. Among these, carbon nanotubes (CNTs) are of special interest as potential drug delivery agents due to their numerous unique and advantageous physical and chemical properties. Here, we show in vivo favorable biodistribution and enhanced therapeutic efficacy of cisplatin (CDDP) encapsulated within ultra-short single-walled carbon nanotube capsules (CDDP@US-tubes) using three different human breast cancer xenograft models. In general, the CDDP@US-tubes demonstrated greater efficacy in suppressing tumor growth than free CDDP in both MCF-7 cell line xenograft and BCM-4272 patient-derived xenograft (PDX) models. The CDDP@US-tubes also demonstrated a prolonged circulation time compared to free CDDP which enhanced permeability and retention (EPR) effects resulting in significantly more CDDP accumulation in tumors, as determined by platinum (Pt) analysis via inductively-coupled plasma mass spectrometry (ICP-MS). STATEMENT OF SIGNIFICANCE Over the past decade, drug-loaded nanocarriers have been widely fabricated and studied to enhance tumor specific delivery. Among the diverse classes of nanomaterials, carbon nanotubes (CNTs), or more specifically ultra-short single-walled carbon nanocapsules (US-tubes), have been shown to be a popular, new platform for the delivery of various medical agents for both imaging and therapeutic purposes. Here, for the first time, we have shown that US-tubes can be utilized as a drug delivery platform in vivo to deliver the chemotherapeutic drug, cisplatin (CDDP) as CDDP@US-tubes. The studies have demonstrated the ability of the US-tube platform to promote the delivery of encapsulated CDDP by increasing the accumulation of drug in breast cancer resistance cells, which reveals how CDDP@US-tubes help overcome CDDP resistance.

Coupling Oriented Hidden Markov Random Field Model with Local Clustering for Segmenting Blood Vessels and Measuring Spatial Structures in Images of Tumor Microenvironment

Interactions between cancer cells and factors within the tumor microenvironment (mE) are essential for understanding tumor development. The spatial relationships between blood vessel cells and cancer cells, e.g. tumor initiating cells (TICs), are an important parameter. Accurate segmentation of blood vessel is necessary for the quantization of their spatial relationships. However, this remains an open problem due to uneven intensity and low signal to noise ratio (SNR). To overcome these challenges, we propose a novel approach that integrates an oriented hidden Markov random field model (Ori-HMRF) with local clustering. The local clustering delineates boundaries of blood vessel segments with low SNR. Then blood vessel segments are viewed as random variables in the Ori-HMRF and their spatial dependence is defined based on directional information. The Ori-HMRF model suppresses noise and generates accurate blood vessel segmentation results. Experimental validations were conducted on both normal mammary and breast cancer tissues.

Epithelial and non-epithelial Patched-1 (Ptch1) play opposing roles to regulate proliferation and morphogenesis of the mouse mammary gland

Patched 1 (Ptch1) has epithelial, stromal and systemic roles in murine mammary gland organogenesis, yet specific functions remain undefined. Cre-recombinase-mediated Ptch1 ablation in mammary epithelium increased proliferation and branching, but did not phenocopy transgenic expression of activated smoothened (SmoM2). The epithelium showed no evidence of canonical hedgehog signaling, and hyperproliferation was not blocked by smoothened (SMO) inhibition, suggesting a non-canonical function of PTCH1. Consistent with this possibility, nuclear localization of cyclin B1 was increased. In non-epithelial cells, heterozygous Fsp-Cre-mediated Ptch1 ablation increased proliferation and branching, with dysplastic terminal end buds (TEB) and ducts. By contrast, homozygous Ptch1 ablation decreased proliferation and branching, producing stunted ducts filled with luminal cells showing altered ovarian hormone receptor expression. Whole-gland transplantation into wild-type hosts or estrogen/progesterone treatment rescued outgrowth and hormone receptor expression, but not the histological changes. Bone marrow transplantation failed to rescue outgrowth. Ducts of Fsp-Cre;Ptch1fl/fl mice were similar to Fsp-Cre;SmoM2 ducts, but Fsp-Cre;SmoM2 outgrowths were not stunted, suggesting that the histology might be mediated by Smo in the local stroma, with systemic Ptch1 required for ductal outgrowth and proper hormone receptor expression in the mammary epithelium. Summary: Systemic and tissue-specific depletion of patched 1 in epithelial and stromal compartments of the mammary gland defines functions in ductal patterning, proliferation and gene expression.