Optimal-Transport Analysis of Single-Cell Gene Expression Identifies Developmental Trajectories in Reprogramming.

Understanding the molecular programs that guide differentiation during development is a major challenge. Here, we introduce Waddington-OT, an approach for studying developmental time courses to infer ancestor-descendant fates and model the regulatory programs that underlie them. We apply the method to reconstruct the landscape of reprogramming from 315,000 single-cell RNA sequencing (scRNA-seq) profiles, collected at half-day intervals across 18 days. The results reveal a wider range of developmental programs than previously characterized. Cells gradually adopt either a terminal stromal state or a mesenchymal-to-epithelial transition state. The latter gives rise to populations related to pluripotent, extra-embryonic, and neural cells, with each harboring multiple finer subpopulations. The analysis predicts transcription factors and paracrine signals that affect fates and experiments validate that the TF Obox6 and the cytokine GDF9 enhance reprogramming efficiency. Our approach sheds light on the process and outcome of reprogramming and provides a framework applicable to diverse temporal processes in biology.

An Overview of the Molecular Cues and Their Intracellular Signaling Shared by Cancer and the Nervous System: From Neurotransmitters to Synaptic Proteins, Anatomy of an All-Inclusive Cooperation.

We propose an overview of the molecular cues and their intracellular signaling involved in the crosstalk between cancer and the nervous system. While "cancer neuroscience" as a field is still in its infancy, the relation between cancer and the nervous system has been known for a long time, and a huge body of experimental data provides evidence that tumor-nervous system connections are widespread. They encompass different mechanisms at different tumor progression steps, are multifaceted, and display some intriguing analogies with the nervous system's physiological processes. Overall, we can say that many of the paradigmatic "hallmarks of cancer" depicted by Weinberg and Hanahan are affected by the nervous system in a variety of manners.

Biomimetic analyses of interactions between macrophages and palmar fascia myofibroblasts derived from Dupuytren's disease reveal distinct inflammatory cytokine responses.

Dupuytren's disease (DD) is a common and heritable fibrosis of the hand. It is characterized by the shortening and thickening of the palmar fascia into myofibroblastic nodules that can progress to palmar-digital contractures and permanent loss of dexterity. Molecular analyses of DD tissues and the presence of inflammatory cell infiltrates suggest a pathogenesis initiated by a proinflammatory fascial milieu that promotes myofibroblast activation and palmar fascia contractures. However, the relative contributions of vascular and/or tissue derived immune system cells and cytokine-sensitive stromal myofibroblasts to the development of this proinflammatory microenvironment are poorly understood. To gain insights into this process, we have developed and tested a collagen-based 3D tissue biomimetic co-culture system to assess paracrine interactions between THP-1-derived pro-inflammatory macrophages and primary human palmar fascia myofibroblasts (PFMs). We observed significant and reproducible impacts of collagen-adherent macrophage and PFM co-cultures on the cytokine gene expression profiles of these cells compared to their respective monocultures, and significant changes to the resulting cytokine milieu in their shared culture media, notably TNF and IL-6. Our findings are consistent with central roles for PFMs in cytokine production and immunoregulation of the pro-inflammatory milieu hypothesized to promote DD development.

Mesenchymal Stem Cells as Regulators of Carcinogenesis.

Mesenchymal Stem Cells (MSCs) are adult stem cells; isolated from various body parts including bone marrow, adipose tissue and dental tissue, have been characterized well and used in regenerative medicine applications. The promising potential of MSCs makes them great candidates in many disorders. It has been well known in the literature that MSCs interact with cancer cells and regulate the carcinogenesis process at different stages. The dual role of MSCs in cancer progression should be clearly identified at the physiological and molecular level to identify clinical potential in cancer treatment. The promoting or suppressive role of MSCs in cancer is controlled by various growth factors, cytokines and chemokines which affect the cell proliferation, angiogenesis and metastasis. Although many studies have been conducted to explore MSC-cancer cell interactions, it is still unclear how MSCs communicate with cancer cells and tumor microenvironment. Further studies are required to investigate secreted factors and paracrine effects, tumor stroma environment, molecular regulators and downstream pathways that are involved in MSC-cancer interaction loop. MSC type, cancer type and stage specific phenotypic and transcriptomic profile changes should be identified in detail to improve clinical use of MSCs in cancer either as a target or as a tool.In the current book chapter, we review the literature to summarize current information about the MSC-cancer cell interactions in terms of soluble factors, angiogenesis, metastasis and drug resistance. The role of MSCs in tumor progression or suppression was discussed based on the current literature.

Altered TGF-α/ß signaling drives cooperation between breast cancer cell populations.

The role of tumor heterogeneity in regulating disease progression is poorly understood. We hypothesized that interactions between subpopulations of cancer cells can affect the progression of tumors selecting for a more aggressive phenotype. We developed an in vivo assay based on the immortalized nontumorigenic breast cell line MCF10A and its Ras-transformed derivatives AT1 (mildly tumorigenic) and CA1d (highly tumorigenic). CA1d cells outcompeted MCF10A, forming invasive tumors. AT1 grafts were approximately 1% the size of CA1d tumors when initiated using identical cell numbers. In contrast, CA1d/AT1 mixed tumors were larger than tumors composed of AT1 alone (100-fold) or CA1d (3-fold), suggesting cooperation in tumor growth. One of the mechanisms whereby CA1d and AT1 were found to cooperate was by modulation of TGF-α and TGF-ß signaling. Both of these molecules were sufficient to induce changes in AT1 proliferative potential in vitro. Reisolation of AT1 tumor-derived (AT1-TD) cells from these mixed tumors revealed that AT1-TD cells grew in vivo, forming tumors as large as tumorigenic CA1d cells. Cooperation between subpopulations of cancer epithelium is an understudied mechanism of tumor growth and invasion that may have implications on tumor resistance to current therapies.-Franco, O. E., Tyson, D. R., Konvinse, K. C., Udyavar, A. R., Estrada, L., Quaranta, V., Crawford, S. E., Hayward, S. W. Altered TGF-α/ß signaling drives cooperation between breast cancer cell populations.

The role of bone marrow mononuclear cell-conditioned medium in the proliferation and migration of human dermal fibroblasts.

BACKGROUND: Several recent studies have demonstrated the great potential of bone marrow cells in regenerative medicine, not only for their ability to differentiate to match a damaged cell type, but also because they synthesize and release various growth factors and cytokines.We examined the effect of bone marrow cell-conditioned medium in the healing process, especially in terms of fibroblast proliferation and migration. METHODS: These in vitro studies consisted of co-culture (without direct contact) of dermal fibroblasts with mononuclear bone marrow cells and the use of conditioned medium obtained from these cultures in a scratch wound model. RESULTS: Mononuclear cells were found to increase the proliferation of fibroblasts, and the conditioned medium showed a stimulatory effect on the migration of fibroblasts. CONCLUSION: When considered together with the observed increase in growth factor levels in conditioned medium, it appears that these cells act through a paracrine mechanism.

Biosensors for Cell Analysis.

Biosensors first appeared several decades ago to address the need for monitoring physiological parameters such as oxygen or glucose in biological fluids such as blood. More recently, a new wave of biosensors has emerged in order to provide more nuanced and granular information about the composition and function of living cells. Such biosensors exist at the confluence of technology and medicine and often strive to connect cell phenotype or function to physiological or pathophysiological processes. Our review aims to describe some of the key technological aspects of biosensors being developed for cell analysis. The technological aspects covered in our review include biorecognition elements used for biosensor construction, methods for integrating cells with biosensors, approaches to single-cell analysis, and the use of nanostructured biosensors for cell analysis. Our hope is that the spectrum of possibilities for cell analysis described in this review may pique the interest of biomedical scientists and engineers and may spur new collaborations in the area of using biosensors for cell analysis.

Macrophage-Conditioned Media Promotes Adipocyte Cancer Association, Which in Turn Stimulates Breast Cancer Proliferation and Migration.

BACKGROUND: Breast cancer is the most common cancer in women and the leading cause of female cancer deaths worldwide. Obesity causes chronic inflammation and is a risk factor for post-menopausal breast cancer and poor prognosis. Obesity triggers increased infiltration of macrophages into adipose tissue, yet little research has focused on the effects of macrophages in early stages of breast tumor development in obese patients. In this study, the effects of pro-inflammatory macrophages on breast cancer-adipocyte crosstalk were investigated. METHODS: An innovative human cell co-culture system was built and used to model the paracrine interactions among adipocytes, macrophages, and breast cancer cells and how they facilitate tumor progression. The effects on cancer cells were examined using cell counts and migration assays. Quantitative reverse-transcription polymerase chain reaction was used to measure the expression levels of several cytokines and proteases to analyze adipocyte cancer association. RESULTS: Macrophage-conditioned media intensified the effects of breast cancer-adipocyte crosstalk. Adipocytes became delipidated and increased production of pro-inflammatory cytokines, even in the absence of cancer cells, although the expression levels were highest with all three cell components. As a result, co-cultured breast cancer cells became more aggressive, with increased proliferation and migration compared to adipocyte-breast cancer co-cultures treated with unconditioned media. CONCLUSIONS: A novel co-culture model was built to evaluate the crosstalk among human macrophages, adipocytes, and breast cancer cells. We found that macrophages may contribute to adipocyte inflammation and cancer association and thus promote breast cancer progression.

Peripheral Nerve Single-Cell Analysis Identifies Mesenchymal Ligands that Promote Axonal Growth.

Peripheral nerves provide a supportive growth environment for developing and regenerating axons and are essential for maintenance and repair of many non-neural tissues. This capacity has largely been ascribed to paracrine factors secreted by nerve-resident Schwann cells. Here, we used single-cell transcriptional profiling to identify ligands made by different injured rodent nerve cell types and have combined this with cell-surface mass spectrometry to computationally model potential paracrine interactions with peripheral neurons. These analyses show that peripheral nerves make many ligands predicted to act on peripheral and CNS neurons, including known and previously uncharacterized ligands. While Schwann cells are an important ligand source within injured nerves, more than half of the predicted ligands are made by nerve-resident mesenchymal cells, including the endoneurial cells most closely associated with peripheral axons. At least three of these mesenchymal ligands, ANGPT1, CCL11, and VEGFC, promote growth when locally applied on sympathetic axons. These data therefore identify an unexpected paracrine role for nerve mesenchymal cells and suggest that multiple cell types contribute to creating a highly pro-growth environment for peripheral axons.

Paracrine Interactions between the Conjunctival and Corneal Epithelial Cells Regulate Microenvironmental Homeostasis during Artificially Induced Inflammation.

Purpose/Aim of the study: The corneal and conjunctival epithelium interact with each other and reciprocally modulate the levels of soluble mediators to maintain balance in the ocular surface. The aim of the present study was to analyze paracrine interactions between the corneal and conjunctival epithelium in an inflamed microenvironment (LPS or PMA induction) to test the levels of pro- and anti-inflammatory cytokines and nitric oxide released by the epithelia. MATERIALS AND METHODS: The corneal (pRSV-T) and conjunctival (HC0597) epithelial cell cultures and their indirect co-cultures were treated for 2 h with LPS (E. coli) or for 30 min with phorbol 12-myristate-13-acetate (PMA) to induce inflammation. Cytokine expression (IL-1ß, IL-6, IL-10) and the level of apoptosis were analyzed by ELISA, and the nitric oxide (NO) level by Griess reaction. Cells were incubated for 24 h. RESULTS: The apoptosis of the corneal and conjunctival epithelia decreased (by 43% and 53%, respectively) in co-cultures compared to corresponding monocultures. The conjunctival epithelium produced lower amounts (23%) of NO than the corneal epithelium. PMA and LPS had comparable effects on the levels of NO in mono- and co-cultures. The levels of the tested cytokines changed depending on the type of cell culture and culture conditions (mono- vs. co-cultures and inflammation). The most striking changes were observed for IL-6 expression. CONCLUSIONS: Paracrine interactions between the corneal and conjunctival epithelia may regulate microenvironmental homeostasis during artificially induced inflammation among others by balancing the levels of NO, cytokines, and the viability of cells.