Illustrated Protocols to Improve Undergraduate Student Research Independence.

One big challenge for undergraduate research students is gaining independence in the laboratory. In this curricular project, undergraduate students transformed research protocols developed for experienced scientists into protocols understandable to someone new to a laboratory. This process enabled themselves and other students to more quickly learn and master new techniques and advance to independent projects. Typically, students started with an original research protocol that assumed basic knowledge, such as instructions that came with a kit (i.e. plasmid purification kit instructions). Students created notes that explained the purpose of each step and reagent and provided example calculations. Then students illustrated the protocols with photos of materials needed, equipment used, action shots of difficult steps and screenshots of software programs. This approach has been used by students in laboratory courses and by new independent research students learning laboratory techniques. In the laboratory courses where students contributed to this project as part of a writing assignment, additional professional experience was gained by presenting a talk about their completed Illustrated Protocols to their classmates and by creating group posters that were presented at an undergraduate research symposium. After completion of this activity, undergraduate students gained confidence by applying their new knowledge to create user-friendly protocols. Students reported increased understanding of what is happening in each step, while instructors reported increased student independence and confidence that the protocol was being applied correctly and consistently. Thus, designing Illustrated Protocols enhanced learning and independence for the students creating the protocol and provided valuable help for future students.

SIX1 reprograms myogenic transcription factors to maintain the rhabdomyosarcoma undifferentiated state.

Rhabdomyosarcoma (RMS) is a pediatric muscle sarcoma characterized by expression of the myogenic lineage transcription factors (TFs) MYOD1 and MYOG. Despite high expression of these TFs, RMS cells fail to terminally differentiate, suggesting the presence of factors that alter their functions. Here, we demonstrate that the developmental TF SIX1 is highly expressed in RMS and critical for maintaining a muscle progenitor-like state. SIX1 loss induces differentiation of RMS cells into myotube-like cells and impedes tumor growth in vivo. We show that SIX1 maintains the RMS undifferentiated state by controlling enhancer activity and MYOD1 occupancy at loci more permissive to tumor growth over muscle differentiation. Finally, we demonstrate that a gene signature derived from SIX1 loss correlates with differentiation status and predicts RMS progression in human disease. Our findings demonstrate a master regulatory role of SIX1 in repression of RMS differentiation via genome-wide alterations in MYOD1 and MYOG-mediated transcription.

Cdon promotes neural crest migration by regulating N-cadherin localization.

Neural crest cells (NCCs) are essential embryonic progenitor cells that are unique to vertebrates and form a remarkably complex and coordinated system of highly motile cells. Migration of NCCs occurs along specific pathways within the embryo in response to both environmental cues and cell-cell interactions within the neural crest population. Here, we demonstrate a novel role for the putative Sonic hedgehog (Shh) receptor and cell adhesion regulator, cdon, in zebrafish neural crest migration. cdon is expressed in developing premigratory NCCs but is downregulated once the cells become migratory. Knockdown of cdon results in aberrant migration of trunk NCCs: crestin positive cells can emigrate out of the neural tube but stall shortly after the initiation of migration. Live cell imaging analysis demonstrates reduced directedness of migration, increased velocity and mispositioned cell protrusions. In addition, transplantation analysis suggests that cdon is required cell-autonomously for directed NCC migration in the trunk. Interestingly, N-cadherin is mislocalized following cdon knockdown suggesting that the role of cdon in NCCs is to regulate N-cadherin localization. Our results reveal a novel role for cdon in zebrafish neural crest migration, and suggest a mechanism by which Cdon is required to localize N-cadherin to the cell membrane in migratory NCCs for directed migration.

MicroRNA-30a regulates zebrafish myogenesis through targeting the transcription factor Six1.

Precise spatiotemporal regulation of the SIX1 homeoprotein is required to coordinate vital tissue development, including myogenesis. Whereas SIX1 is downregulated in most tissues following embryogenesis, it is re-expressed in numerous cancers, including tumors derived from muscle progenitors. Despite crucial roles in development and disease, the upstream regulation of SIX1 expression has remained elusive. Here, we identify the first direct mechanism for Six1 regulation in embryogenesis, through microRNA30a (miR30a)-mediated repression. In zebrafish somites, we show that miR30a and six1a and six1b (hereafter six1a/b) are expressed in an inverse temporal pattern. Overexpression of miR30a leads to a reduction in six1a/b levels, and results in increased apoptosis and altered somite morphology, which phenocopies six1a/b knockdown. Conversely, miR30a inhibition leads to increased Six1 expression and abnormal somite morphology, revealing a role for endogenous miR30a as a muscle-specific miRNA (myomiR). Importantly, restoration of six1a in miR30a-overexpressing embryos restores proper myogenesis. These data demonstrate a new role for miR30a at a key node in the myogenic regulatory gene network through controlling Six1 expression.

Genomic signatures of pregnancy-associated breast cancer epithelia and stroma and their regulation by estrogens and progesterone.

Pregnancy-associated breast cancers (PABC) generally present at advanced stages and have a poor prognosis. The reasons are unclear but we hypothesized that the continuous high levels of estrogens and progesterone were involved. We have now carried out a detailed analysis of PABC compared to tumors of age-matched nonpregnant (non-PABC) women. Malignant epithelia and tumor-associated stroma of PABC and non-PABC were isolated by laser capture microdissection and gene expression profiled. Additionally, normal breast epithelia and stroma adjacent to the two tumor types were analyzed. Lastly, subsets of previously identified E- and P-regulated genes were defined in all tissues. We find that PABC signatures cluster with established breast cancer subtypes. Major hormone-regulated genes whose expression correlated with epithelia of PABC dealt with regulation of cell proliferation, metabolism, and tumor aggressiveness, including genes used to predict tumor recurrence. Compared to normal epithelia, a significant number of genes associated with cell cycle processes were enriched in PABC, many of which are hormone regulated. Thus, compared to normal epithelia, many of the genes that were differentially expressed in epithelia of PABC were distinct from those differentially expressed in non-PABC. With regard to the tumor microenvironment, immune-related genes were enriched in tumor-associated stroma of PABC. Compared to normal stroma, PABC-associated stroma overexpressed immune response genes, while genes involved in angiogenesis and extracellular matrix deposition were more commonly downregulated. This suggests that the heightened aggressiveness of PABC may involve a predisposition to metastasis through extracellular matrix degradation, plus angiogenesis independence. Moreover, genes encoding cell proliferative factors, signaling, immunomodulators and cell death, were hormone regulated in stroma. In sum, these analyses demonstrate complex patterns of enrichment and hormonal regulation of genes in PABC and suggest that it may have a distinct biological nature.

Rat mammary extracellular matrix composition and response to ibuprofen treatment during postpartum involution by differential GeLC-MS/MS analysis.

Breast cancer patients diagnosed within five years following pregnancy have increased metastasis and decreased survival. A hallmark of postpartum biology that may contribute to this poor prognosis is mammary gland involution, involving massive epithelial cell death and dramatic stromal remodeling. Previous studies show pro-tumorigenic properties of extracellular matrix (ECM) isolated from rodent mammary glands undergoing postpartum involution. More recent work demonstrates systemic ibuprofen treatment during involution decreases its tumor-promotional nature. Utilizing a proteomics approach, we identified relative differences in the composition of mammary ECM isolated from nulliparous rats and those undergoing postpartum involution, with and without ibuprofen treatment. GeLC-MS/MS experiments resulted in 20327 peptide identifications that mapped to 884 proteins with a <0.02% false discovery rate. Label-free quantification yielded several ECM differences between nulliparous and involuting glands related to collagen-fiber organization, cell motility and attachment, and cytokine regulation. Increases in known pro-tumorigenic ECM proteins osteopontin, tenascin-C, and laminin-α1 and pro-inflammatory proteins STAT3 and CD68 further identify candidate mediators of breast cancer progression specific to the involution window. With postpartum ibuprofen treatment, decreases in tenascin-C and three laminin chains were revealed. Our data suggest novel ECM mediators of breast cancer progression and demonstrate a protective influence of ibuprofen on mammary ECM composition.

Macrophages are crucial for epithelial cell death and adipocyte repopulation during mammary gland involution.

Mammary gland development is dependent on macrophages, as demonstrated by their requirement during the expansion phases of puberty and pregnancy. Equally dramatic tissue restructuring occurs following lactation, when the gland regresses to a state that histologically resembles pre-pregnancy through massive programmed epithelial cell death and stromal repopulation. Postpartum involution is characterized by wound healing-like events, including an influx of macrophages with M2 characteristics. Macrophage levels peak after the initial wave of epithelial cell death, suggesting that initiation and execution of cell death are macrophage independent. To address the role of macrophages during weaning-induced mammary gland involution, conditional systemic deletion of macrophages expressing colony stimulating factor 1 receptor (CSF1R) was initiated just prior to weaning in the Mafia mouse model. Depletion of CSF1R(+) macrophages resulted in delayed mammary involution as evidenced by loss of lysosomal-mediated and apoptotic epithelial cell death, lack of alveolar regression and absence of adipocyte repopulation 7 days post-weaning. Failure to execute involution occurred in the presence of milk stasis and STAT3 activation, indicating that neither is sufficient to initiate involution in the absence of CSF1R(+) macrophages. Injection of wild-type bone marrow-derived macrophages (BMDMs) or M2-differentiated macrophages into macrophage-depleted mammary glands was sufficient to rescue involution, including apoptosis, alveolar regression and adipocyte repopulation. BMDMs exposed to the postpartum mammary involution environment upregulated the M2 markers arginase 1 and mannose receptor. These data demonstrate the necessity of macrophages, and implicate M2-polarized macrophages, for epithelial cell death during normal postpartum mammary gland involution.

Non-steroidal anti-inflammatory drugs target the pro-tumorigenic extracellular matrix of the postpartum mammary gland.

Breast cancer patients diagnosed postpartum have poor prognosis. The postpartum mammary gland undergoes tissue regression to return to the pre-pregnant state. This involution is characterized by wound healing programs known to be tumor promotional in other contexts. Previous studies have shown that mammary extracellular matrix (ECM) from nulliparous rats has tumor suppressive attributes, while mammary ECM from involuting mammary glands is promotional. In models of pregnancy-associated breast cancer, non-steroidal anti-inflammatory drug (NSAID) treatment targeted to postpartum involution inhibits tumor progression, in part by suppressing COX-2 dependent collagen deposition. Because mammary ECM proteins are coordinately regulated, NSAID treatment is anticipated to result in additional protective changes in the mammary extracellular matrix. Here, systemic NSAID treatment was utilized during postpartum involution to reduce mammary COX-2 activity. ECM was isolated from actively involuting glands of rats treated with NSAIDs and compared to ECM isolated from control-involution and nulliparous rats in 3D cell culture and xenograft assays. Compositional changes in ECM between groups were identified by proteomics. In four distinct 3D culture assays, normal and transformed mammary epithelial cells plated in NSAID-involution ECM, phenocopied cells plated in ECM from nulliparous rats rather than ECM from control-involution rats. Tumor cells mixed with NSAID-involution ECM and injected orthotopically in mice formed smaller tumors than cells mixed with control-involution ECM. Proteomic analyses identified and 3D culture assays implicated the ECM protein tenascin-C as a potential mediator of tumor progression during involution that is decreased by NSAID treatment. In summary, NSAID treatment decreases tumor-promotional attributes of postpartum involution mammary ECM.

Postpartum mammary gland involution drives progression of ductal carcinoma in situ through collagen and COX-2.

The prognosis of breast cancer in young women is influenced by reproductive history. Women diagnosed within 5 years postpartum have worse prognosis than nulliparous women or women diagnosed during pregnancy. Here we describe a mouse model of postpartum breast cancer that identifies mammary gland involution as a driving force of tumor progression. In this model, human breast cancer cells exposed to the involuting mammary microenvironment form large tumors that are characterized by abundant fibrillar collagen, high cyclooxygenase-2 (COX-2) expression and an invasive phenotype. In culture, tumor cells are invasive in a fibrillar collagen and COX-2-dependent manner. In the involuting mammary gland, inhibition of COX-2 reduces the collagen fibrillogenesis associated with involution, as well as tumor growth and tumor cell infiltration to the lung. These data support further research to determine whether women at high risk for postpartum breast cancer would benefit from treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) during postpartum involution.

Isolation of mammary-specific extracellular matrix to assess acute cell-ECM interactions in 3D culture.

Studies of mammary epithelial cells (MECs) cultured with reconstituted basement membrane proteins derived from EHS tumors have contributed greatly to the understanding of both normal physiology and transformation. Only when plated on such biologically relevant substratum are MECs able to form morphologically correct, differentiated structures, highlighting a critical role for extracellular matrix (ECM) proteins in MEC organization and function. Here, we describe methods modified from the original EHS matrix protocol for isolating tissue-specific ECM from rat mammary glands, and for subsequent use in short-term 3D cell culture models designed to assess acute cell-ECM interactions. Using this protocol, the final matrix is enriched up to 58-fold for ECM proteins such as fibronectin and laminin, while cellular proteins such as GAPDH are reduced 98-fold. We have previously shown that MECs plated in mammary-specific ECM form more elaborate duct-like and alveolar-like structures compared to MECs plated in Matrigel™, demonstrating the biological relevance of tissue-specific ECM. Use of mammary-specific ECM in 3D cell culture models will further our ability to study the intricate interplay between a cell and its microenvironment, and permit identification of modifying factors.

Alternatively activated macrophages and collagen remodeling characterize the postpartum involuting mammary gland across species.

Recent pregnancy correlates with decreased survival for breast cancer patients compared with non-pregnancy-associated breast cancer. We hypothesize that postpartum mammary involution induces metastasis through wound-healing programs known to promote cancer. It is unknown whether alternatively activated M2 macrophages, immune cells important in wound-healing and experimental tumorigenesis that also predict poor prognosis for breast cancer patients, are recruited to the normal involuting gland. Macrophage markers CD68, CSF-1R, and F4/80 were examined across the pregnancy and involution cycle in rodent and human mammary tissues. Quantitative immunohistochemistry revealed up to an eightfold increase in macrophage number during involution, which returned to nulliparous levels with full regression. The involution macrophages exhibit an M2 phenotype as determined by high arginase-1 and low inducible nitric oxide synthase staining in rodent tissue, and by mannose receptor expression in human breast tissue. M2 cytokines IL-4 and IL-13 also peaked during involution. Extracellular matrix (ECM) isolated from involuting rat mammary glands was chemotactic for macrophages compared with nulliparous mammary ECM. Fibrillar collagen levels and proteolysis increased dramatically during involution, and denatured collagen I acted as a strong chemoattractant for macrophages in cell culture, suggesting proteolyzed fibrillar collagen as a candidate ECM mediator of macrophage recruitment. M2 macrophages, IL-4, IL-13, fibrillar collagen accumulation, and proteolysis of collagen are all components of tumor promotional microenvironments, and thus may mediate promotion of breast cancers arising in the postpartum setting.

Macrophages in breast cancer: do involution macrophages account for the poor prognosis of pregnancy-associated breast cancer?

Macrophage influx is associated with negative outcomes for women with breast cancer and has been demonstrated to be required for metastasis of mammary tumors in mouse models. Pregnancy-associated breast cancer is characterized by particularly poor outcomes, however the reasons remain obscure. Recently, post-pregnancy mammary involution has been characterized as having a wound healing signature. We have proposed the involution-hypothesis, which states that the wound healing microenvironment of the involuting gland is tumor promotional. Macrophage influx is one of the prominent features of the involuting gland, identifying the macrophage a potential instigator of tumor progression and a novel target for breast cancer treatment and prevention.

An in-solution ultrasonication-assisted digestion method for improved extracellular matrix proteome coverage.

Epithelial cell behavior is coordinated by the composition of the surrounding extracellular matrix (ECM); thus ECM protein identification is critical for understanding normal biology and disease states. Proteomic analyses of ECM proteins have been hindered by the insoluble and digestion-resistant nature of ECM. Here we explore the utility of combining rapid ultrasonication- and surfactant-assisted digestion for the detailed proteomics analysis of ECM samples. When compared with traditional overnight digestion, this optimized method dramatically improved the sequence coverage for collagen I, revealed the presence of hundreds of previously unidentified proteins in Matrigel, and identified a protein profile for ECM isolated from rat mammary glands that was substantially different from that found in Matrigel. In a three-dimensional culture assay to investigate epithelial cell-ECM interactions, mammary epithelial cells were found to undergo extensive branching morphogenesis when plated with mammary gland-derived matrix in comparison with Matrigel. Cumulatively these data highlight the tissue-specific nature of ECM composition and function and underscore the need for optimized techniques, such as those described here, for the proteomics characterization of ECM samples.

Microenvironment of the involuting mammary gland mediates mammary cancer progression.

Breast cancer diagnosed after a completed pregnancy has higher metastatic potential and therefore a much poorer prognosis. We hypothesize that following pregnancy the process of mammary gland involution, which returns the gland to its pre-pregnant state, co-opts some of the programs of wound healing. The pro-inflammatory milieu that results, while physiologically normal, promotes tumor progression. In this review, the similarities between mammary gland involution after cessation of milk-production and pathological tissue remodeling are discussed in light of emerging data demonstrating a role for pathological tissue remodeling in cancer.