Micro blood analysis technology (µBAT): multiplexed analysis of neutrophil phenotype and function from microliter whole blood samples.

There is an ongoing need to do more with less and provide highly multiplexed analysis from limited sample volumes. Improved "sample sparing" assays would have a broad impact across pediatric and other rare sample type studies in addition to enabling sequential sampling. This capability would advance both clinical and basic research applications. Here we report the micro blood analysis technology (µBAT), a microfluidic platform that supports multiplexed analysis of neutrophils from a single drop of blood. We demonstrate the multiplexed orthogonal capabilities of µBAT including functional assays (phagocytosis, neutrophil extracellular traps, optical metabolic imaging) and molecular assays (gene expression, cytokine secretion). Importantly we validate our microscale platform using a macroscale benchmark assay. µBAT is compatible with lancet puncture or microdraw devices, and its design facilitates rapid operations without the need for specialized equipment. µBAT offers a new method for investigating neutrophil function in populations with restricted sample amounts.

The role of satellite cell-derived TRIM28 in mechanical load- and injury-induced myogenesis.

Satellite cells are skeletal muscle stem cells that contribute to postnatal muscle growth, and they endow skeletal muscle with the ability to regenerate after a severe injury. Here we discovered that this myogenic potential of satellite cells requires a protein called tripartite motif-containing 28 (TRIM28). Unexpectedly, multiple lines of both in vitro and in vivo evidence revealed that the myogenic function of TRIM28 is not dependent on changes in the phosphorylation of its serine 473 residue. Moreover, the functions of TRIM28 were not mediated through the regulation of satellite cell proliferation or differentiation. Instead, our findings indicate that TRIM28 regulates the ability of satellite cells to progress through the process of fusion. Specifically, we discovered that TRIM28 controls the expression of a fusogenic protein called myomixer and concomitant fusion pore formation. Collectively, the outcomes of this study expose the framework of a novel regulatory pathway that is essential for myogenesis.

Role of dual specificity phosphatases (DUSPs) in melanoma cellular plasticity and drug resistance.

Melanoma cells exhibit phenotypic plasticity that allows transition from a proliferative and differentiated phenotype to a more invasive and undifferentiated or transdifferentiated phenotype often associated with drug resistance. The mechanisms that control melanoma phenotype plasticity and its role in drug resistance are not fully understood. We previously demonstrated that emergence of MAPK inhibitor (MAPKi)-resistance phenotype is associated with decreased expression of stem cell proliferation genes and increased expression of MAPK inactivation genes, including dual specificity phosphatases (DUSPs). Several members of the DUSP family genes, specifically DUSP1, -3, -8 and -9, are expressed in primary and metastatic melanoma cell lines and pre-and post BRAFi treated melanoma cells. Here, we show that knockdown of DUSP1 or DUSP8 or treatment with BCI, a pharmacological inhibitor of DUSP1/6 decrease the survival of MAPKi-resistant cells and sensitizes them to BRAFi and MEKi. Pharmacological inhibition of DUSP1/6 upregulated nestin, a neural crest stem cell marker, in both MAPKi-sensitive cells and cells with acquired MAPKi-resistance. In contrast, treatment with BCI resulted in upregulation of MAP2, a neuronal differentiation marker, only in MAPKi-sensitive cells but caused downregulation of both MAP2 and GFAP, a glial marker, in all MAPKi-resistant cell lines. These data suggest that DUSP proteins are involved in the regulation of cellular plasticity cells and melanoma drug resistance and are potential targets for treatment of MAPKi-resistant melanoma.

EPAC Regulates Melanoma Growth by Stimulating mTORC1 Signaling and Loss of EPAC Signaling Dependence Correlates with Melanoma Progression.

Exchange proteins directly activated by cAMP (EPAC) belong to a family of RAP guanine nucleotide exchange factors (RAPGEF). EPAC1/2 (RAPGEF3/4) activates RAP1 and the alternative cAMP signaling pathway. We previously showed that the differential growth response of primary and metastatic melanoma cells to cAMP is mediated by EPAC. However, the mechanisms responsible for this differential response to EPAC signaling are not understood. In this study, we show that pharmacologic inhibition or siRNA-mediated knockdown of EPAC selectively inhibits the growth and survival of primary melanoma cells by downregulation of cell-cycle proteins and inhibiting the cell-cycle progression independent of ERK1/2 phosphorylation. EPAC inhibition results in upregulation of AKT phosphorylation but a downregulation of mTORC1 activity and its downstream effectors. We also show that EPAC regulates both glycolysis and oxidative phosphorylation, and production of mitochondrial reactive oxygen species, preferentially in primary melanoma cells. Employing a series of genetically matched primary and lymph node metastatic (LNM) melanoma cells, and distant organ metastatic melanoma cells, we show that the LNM and metastatic melanoma cells become progressively less responsive and refractory to EPAC inhibition suggesting loss of dependency on EPAC signaling correlates with melanoma progression. Analysis of The Cancer Genome Atlas dataset showed that lower RAPGEF3, RAPGEF4 mRNA expression in primary tumor is a predictor of better disease-free survival of patients diagnosed with primary melanoma suggesting that EPAC signaling facilitates tumor progression and EPAC is a useful prognostic marker. These data highlight EPAC signaling as a potential target for prevention of melanoma progression. IMPLICATIONS: This study establishes loss of dependency on EPAC-mTORC1 signaling as hallmark of primary melanoma evolution and targeting this escape mechanism is a promising strategy for metastatic melanoma.

Role of the Skin Microenvironment in Melanomagenesis: Epidermal Keratinocytes and Dermal Fibroblasts Promote BRAF Oncogene-Induced Senescence Escape in Melanocytes.

BRAFV600E is the most common mutation driver in melanoma. This mutation is known to cause a brief burst of proliferation followed by growth arrest and senescence, which prevent an uncontrolled cell proliferation. This phenomenon is known as oncogene-induced senescence (OIS) and OIS escape is thought to lead to melanomagenesis. Much attention has been focused on the melanocyte-intrinsic mechanisms that contribute to senescence escape. Additional genetic events such as the loss of tumor suppressor PTEN and/or epigenetic changes that contribute to senescence escape have been described. However, the role of the skin microenvironment-specifically, the role of epidermal keratinocytes-on melanomagenesis is not fully understood. In this study, we employ a microfluidic platform to study the interaction between melanocytes expressing the BRAFV600E mutation as well as keratinocytes and dermal fibroblasts. We demonstrate that keratinocytes suppress senescence-related genes and promote the proliferation of transformed melanocytes. We also show that a keratinocyte-conditioned medium can alter the secretion of both pro- and anti-tumorigenic factors by transformed melanocytes. In addition, we show that melanocytes and keratinocytes from donors of white European and black African ancestry display different crosstalks; i.e., white keratinocytes appear to promote a more pro-tumorigenic phenotype compared with black keratinocytes. These data suggest that keratinocytes exert their influence on melanomagenesis both by suppressing senescence-related genes in melanocytes and by affecting the balance of the melanocyte-secreted factors that favor tumorigenesis.

Microfluidic model with air-walls reveals fibroblasts and keratinocytes modulate melanoma cell phenotype, migration, and metabolism.

Melanoma evolution is a complex process. The role epidermal keratinocytes and dermal fibroblasts play in this process and the mechanisms involved in tumor-stroma interactions remain poorly understood. Here, we used a microfluidic platform to evaluate the cross-talk between human primary melanoma cells, keratinocytes and dermal fibroblasts. The microfluidic device included multiple circular chambers separated by a series of narrow connection channels. The microdevice design allowed us to develop a new cell patterning method based on air-walls, removing the need for hydrogel barriers, porous membranes, or external equipment. Using this method, we co-cultured melanoma cells in the presence of keratinocytes and/or dermal fibroblasts. The results demonstrated that the presence of dermal fibroblasts and keratinocytes led to changes in melanoma cell morphology and growth pattern. Molecular analysis revealed changes in the chemokine secretion pattern, identifying multiple secreted factors involved in tumor progression. Finally, optical metabolic imaging showed that melanoma cells, fibroblasts, and keratinocytes exhibited different metabolic features. Additionally, the presence of stromal cells led to a metabolic shift in melanoma cells, highlighting the role the skin microenvironment on melanoma evolution.