[Show abstract][Hide abstract] ABSTRACT: Cleared mammary fat pad (MFP) transplantation has been a standard technique for studies of mammary development and cancer for several decades. The mammary gland is comprised of several fundamental components: The epithelial compartment contains basal/myoepithelial cells and luminal cells, and the stromal compartment (called the MFP) contains adipocytes, smooth muscle cells, fibroblasts, and immune cells. In 3- to 4-wk-old female mice, the mammary epithelium is concentrated very close to the nipple and has not yet grown beyond the mammary lymph node to penetrate the bulk of the MFP. This developmental feature provides an anatomical fixed point, and enables one to cut away the portion of the MFP from the nipple to the lymph node, leaving behind the majority of the MFP free of epithelium. The “cleared” MFP can serve as a supportive native microenvironment fully sufficient for the organogenesis of injected donor epithelium. Normal mammary epithelial donor cells will produce histologically and functionally normal mammary ductal epithelium several weeks posttransplant, with the exception that the ducts will not be connected to the nipple. The assay described here provides a powerful platform for assessing the developmental and tumorigenic potential of engineered cells of interest.
[Show abstract][Hide abstract] ABSTRACT: Tubulogenesis is fundamental to the development of many epithelial organs. Although lumen formation in cysts has received considerable attention, less is known about lumenogenesis in tubes. Here we utilized hepatocyte growth factor induced tubulogenesis in MDCK cells, which form tubes enclosing a single lumen. We report the mechanism that controls tubular lumenogenesis and limits each tube to a single lumen. Knockdown of 114RhoGEF, a guanine nucleotide exchange factor for RhoA, did not perturb the early stages of tubulogenesis induced by hepatocyte growth factor. However, this knockdown impaired later stages of tubulogenesis, resulting in multiple lumens in a tube. Inhibition of Rho Kinase or myosin IIA, which are downstream of RhoA, led to formation of multiple lumens. We studied lumen formation by live cell imaging, which revealed that inhibition of this pathway blocked cell movement, suggesting that cell movement is necessary for consolidating multiple lumens into a single lumen. Lumen formation in tubules is mechanistically quite different from lumenogenesis in cysts. Thus, we demonstrate a novel pathway that regulates directed cell migration and single lumen formation in epithelial tube morphogenesis.
No preview · Article · Oct 2015 · Journal of Cell Science
[Show abstract][Hide abstract] ABSTRACT: Despite major advances in understanding the molecular and genetic basis of cancer, metastasis remains the cause of >90% of cancer-related mortality. Understanding metastasis initiation and progression is critical to developing new therapeutic strategies to treat and prevent metastatic disease. Prevailing theories hypothesize that metastases are seeded by rare tumour cells with unique properties, which may function like stem cells in their ability to initiate and propagate metastatic tumours. However, the identity of metastasis-initiating cells in human breast cancer remains elusive, and whether metastases are hierarchically organized is unknown. Here we show at the single-cell level that early stage metastatic cells possess a distinct stem-like gene expression signature. To identify and isolate metastatic cells from patient-derived xenograft models of human breast cancer, we developed a highly sensitive fluorescence-activated cell sorting (FACS)-based assay, which allowed us to enumerate metastatic cells in mouse peripheral tissues. We compared gene signatures in metastatic cells from tissues with low versus high metastatic burden. Metastatic cells from low-burden tissues were distinct owing to their increased expression of stem cell, epithelial-to-mesenchymal transition, pro-survival, and dormancy-associated genes. By contrast, metastatic cells from high-burden tissues were similar to primary tumour cells, which were more heterogeneous and expressed higher levels of luminal differentiation genes. Transplantation of stem-like metastatic cells from low-burden tissues showed that they have considerable tumour-initiating capacity, and can differentiate to produce luminal-like cancer cells. Progression to high metastatic burden was associated with increased proliferation and MYC expression, which could be attenuated by treatment with cyclin-dependent kinase (CDK) inhibitors. These findings support a hierarchical model for metastasis, in which metastases are initiated by stem-like cells that proliferate and differentiate to produce advanced metastatic disease.
[Show abstract][Hide abstract] ABSTRACT: Postnatal organogenesis occurs in an immune competent environment and is tightly controlled by interplay between positive and negative regulators. Innate immune cells have beneficial roles in postnatal tissue remodeling, but roles for the adaptive immune system are currently unexplored. Here we show that adaptive immune responses participate in the normal postnatal development of a non-lymphoid epithelial tissue. Since the mammary gland (MG) is the only organ developing predominantly after birth, we utilized it as a powerful system to study adaptive immune regulation of organogenesis. We found that antigen-mediated interactions between mammary antigen-presenting cells and interferon-γ (IFNγ)-producing CD4+ T helper 1 cells participate in MG postnatal organogenesis as negative regulators, locally orchestrating epithelial rearrangement. IFNγ then affects luminal lineage differentiation. This function of adaptive immune responses, regulating normal development, changes the paradigm for studying players of postnatal organogenesis and provides insights into immune surveillance and cancer transformation.
Full-text · Article · Aug 2015 · Developmental Cell
[Show abstract][Hide abstract] ABSTRACT: Purpose To develop a strategy of achieving targeted collection of endothelial cells (ECs) by endovascular methods and analyzing the gene expression profiles of collected single ECs. Methods and results 134 ECs and 37 leukocytes were collected from four patients' intra-iliac artery endovascular guide wires by fluorescence activated cell sorting (FACS) and analyzed by single-cell quantitative RT-PCR for expression profile of 48 genes. Compared to CD45+ leukocytes, the ECs expressed higher levels (p < 0.05) of EC surface markers used on FACS and other EC related genes. The gene expression profile showed that these isolated ECs fell into two clusters, A and B, that differentially expressed 19 genes related to angiogenesis, inflammation and extracellular matrix remodeling, with cluster B ECs have demonstrating similarities to senescent or aging ECs. Conclusion Combination of endovascular device sampling, FACS and single-cell quantitative RT-PCR is a feasible method for analyzing EC gene expression profile in vascular lesions.
[Show abstract][Hide abstract] ABSTRACT: The accessibility and transparency of the cornea makes it a good tissue model for monitoring immunological responses using in vivo real time imaging analysis (Lee et al., 2010; Tan et al., 2013). These corneal qualities have also allowed for high-resolution in vivo imaging of non-ocular tissue transplanted into the anterior chamber of the mouse eye (Speier et al., 2008a; Speier et al., 2008b). This protocol was adapted from Speier (2008) to successfully assess real-time in vivo myeloid cell dynamics in wounded corneas of c-fms-EGFP mice (Chan et al., 2013). Macrophage colony-stimulating factor (CSF-1) regulates the differentiation and proliferation of cells of the mononuclear phagocyte system. The activity of CSF-1 is mediated by the CSF-1 receptor that is encoded by c-fms (Csf1r) protooncogene. The c-fms gene is expressed in macrophage, trophoblast cell lineages, and to some extent granulocytes. In the c-fms-EGFP mice EGFP, enhanced green fluorescent protein, is driven under the Csf1r, colony stimulating factor 1 receptor, promoter and highlights myeloid cells (Sasmono et al., 2003). This protocol can be further adapted to image other transgenic mice expressing fluorescent proteins.
[Show abstract][Hide abstract] ABSTRACT: The cornea is an excellent model system to use for the analysis of wound repair because of its accessibility, lack of vascularization, and simple anatomy. Corneal injuries may involve only the superficial epithelial layer or may penetrate deeper to involve both the epithelial and stromal layers. Here we describe two well-established in vivo corneal wound models: a mechanical wound model that allows for the study of re-epithelialization and a chemical wound model that may be used to study stromal activation in response to injury (Stepp et al., 2014; Carlson et al., 2003).
[Show abstract][Hide abstract] ABSTRACT: The tumor microenvironment plays an essential role in various stages of cancer development. This environment, composed of the extracellular matrix, fibroblasts, endothelial cells, and cells of the immune system regulates the behavior of and co-evolve with tumor cells. Many of the components, including the innate and adaptive immune cells, play multifaceted roles during cancer progression and can promote or inhibit tumor development, depending on local and systemic conditions. Interestingly, a strategy by which tumor cells gain drug resistance is by modifying the tumor microenvironment. Together, understanding the mechanisms by which the tumor microenvironment functions should greatly facilitate the development of new therapeutic interventions by targeting the tumor niche.
No preview · Article · Jul 2015 · The Cancer Journal
[Show abstract][Hide abstract] ABSTRACT: The character of EGFR signals can influence cell fate but mechanistic insights into intestinal EGFR-Ras signalling are limited. Here we show that two distinct Ras nucleotide exchange factors, RasGRP1 and SOS1, lie downstream of EGFR but act in functional opposition. RasGRP1 is expressed in intestinal crypts where it restricts epithelial growth. High RasGRP1 expression in colorectal cancer (CRC) patient samples correlates with a better clinical outcome. Biochemically, we find that RasGRP1 creates a negative feedback loop that limits proliferative EGFR-SOS1-Ras signals in CRC cells. Genetic Rasgrp1 depletion from mice with either an activating mutation in KRas or with aberrant Wnt signalling due to a mutation in Apc resulted in both cases in exacerbated Ras-ERK signalling and cell proliferation. The unexpected opposing cell biological effects of EGFR-RasGRP1 and EGFR-SOS1 signals in the same cell shed light on the intricacy of EGFR-Ras signalling in normal epithelium and carcinoma.
No preview · Article · May 2015 · Nature Cell Biology
[Show abstract][Hide abstract] ABSTRACT: IntroductionMost cancer deaths are due to metastasis—the spread of cancer from its site of origin to distant, vital organs—and the physiological damage caused by tumor growth in those organs. While the broad outlines of the process of metastatic spread are known, much of the details of the process remain poorly understood. To continue to improve cancer survival rates, we must face and tackle the problems inherent to metastatic disease. Cancers that are detected early, before they are believed to have spread to other organs, are generally treated with more success than cancers that are metastatic at diagnosis. However, even cancers that are detected early will recur in some patients, but our ability to predict which individuals will have recurrences is limited. Thus, adjuvant therapy is often given to patients with early-stage disease who are believed as a group to be at risk for recurrence, leading to overtreatment of some patients to benefit a subset of them and possibly failing to tr ...
No preview · Article · Mar 2015 · Journal of Molecular Medicine
[Show abstract][Hide abstract] ABSTRACT: Myeloid cells contribute to increased malignancy and poor prognosis in breast cancer. We demonstrate that anti-CSF-1R therapy depletes a cell population sharing characteristics of tumor-associated macrophages (TAMs) and dendritic cells (DCs). Intravital imaging combined with cellular characterization has refined our understanding of anti-CSF-1R therapy on the tumor microenvironment.
[Show abstract][Hide abstract] ABSTRACT: Since Gross and Lapiere firstly discovered matrix metalloproteinases (MMPs) as important collagenolytic enzymes during amphibian tadpole morphogenesis in 1962, this intriguing family of extracellular proteinases has been implicated in various processes of developmental biology. However, the pathogenic roles of MMPs in human diseases such as cancer has also garnered widespread attention. The most straightforward explanation for their role in cancer is that MMPs, through extracellular matrix degradation, pave the way for tumor cell invasion and metastasis. While this notion may be true for many circumstances, we now know that, depending on the context, MMPs may employ additional modes of functionality. Here, we will give an update on the function of MMPs in development and cancer, which may directly regulate signaling pathways that control tissue homeostasis and may even work in a non-proteolytic manner. These novel findings about the functionality of MMPs have important implications for MMP inhibitor design and may allow us to revisit MMPs as drug targets in the context of cancer and other diseases.
[Show abstract][Hide abstract] ABSTRACT: Expansion of myeloid cells associated with solid tumor development is a key contributor to neoplastic progression. Despite their clinical relevance, the mechanisms controlling myeloid cell production and activity in cancer remains poorly understood. Using a multistage mouse model of breast cancer, we show that production of atypical T cell-suppressive neutrophils occurs during early tumor progression, at the onset of malignant conversion, and that these cells preferentially accumulate in peripheral tissues but not in the primary tumor. Production of these cells results from activation of a myeloid differentiation program in bone marrow (BM) by a novel mechanism in which tumor-derived granulocyte-colony stimulating factor (G-CSF) directs expansion and differentiation of hematopoietic stem cells to skew hematopoiesis toward the myeloid lineage. Chronic skewing of myeloid production occurred in parallel to a decrease in erythropoiesis in BM in mice with progressive disease. Significantly, we reveal that prolonged G-CSF stimulation is both necessary and sufficient for the distinguishing characteristics of tumor-induced immunosuppressive neutrophils. These results demonstrate that prolonged G-CSF may be responsible for both the development and activity of immunosuppressive neutrophils in cancer.
Full-text · Article · Jan 2015 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Cells of the innate immune system have a dual role in
cancer development in both tumor initiation and progression.
Innate immune cells can, on the one hand, aid
malignant transformation and tumor outgrowth and, on
the other hand, prevent tumor progression. The innate
immune system has the ability to tune the inflammatory
response and is a key player in cancer-related inflammation,
which can precede the development of malignancy
or be induced by oncogenic changes promoting a protumor
inflammatory milieu. In this review, we discuss
the emerging cellular and molecular mechanisms of the
innate immune system and inflammation in tumor initiation
and progression, and point to the outstanding
questions that remain.
Full-text · Article · Nov 2014 · Trends in Cell Biology
[Show abstract][Hide abstract] ABSTRACT: The extracellular matrix (ECM) is a highly dynamic structure that is present in all tissues and continuously undergoes controlled remodelling. This process involves quantitative and qualitative changes in the ECM, mediated by specific enzymes that are responsible for ECM degradation, such as metalloproteinases. The ECM interacts with cells to regulate diverse functions, including proliferation, migration and differentiation. ECM remodelling is crucial for regulating the morphogenesis of the intestine and lungs, as well as of the mammary and submandibular glands. Dysregulation of ECM composition, structure, stiffness and abundance contributes to several pathological conditions, such as fibrosis and invasive cancer. A better understanding of how the ECM regulates organ structure and function and of how ECM remodelling affects disease progression will contribute to the development of new therapeutics.
No preview · Article · Nov 2014 · Nature Reviews Molecular Cell Biology