In Vivo Longitudinal Tracking of Lymphangiogenesis and Angiogenesis in Cutaneous Melanoma Mouse Model Using Multifunctional Optical Coherence Tomography.

Melanoma is a high-risk skin cancer because it tends to metastasize early and ultimately leads to death. In this study, we introduced a noninvasive multifunctional optical coherence tomography (MFOCT) for the early detection of premetastatic pathogenesis in cutaneous melanoma by label-free imaging of microstructures (i.e., providing the thickness and the scattering information) and microcirculation (i.e., providing depth-resolved angiography and lymphangiography). Using MFOCT-based approaches, we presented an in vivo longitudinal observation of the tumor microenvironment in Braf V600E/V600E ;Pten -/- mice with inducible melanoma monitored for 42 days. Quantitative analysis of MFOCT images identified an increased number of lymphatic and vascular vessels during tumor progression and faster lymphangiogenesis (beginning on day 21) than angiogenesis (beginning on day 28) in the melanoma microenvironment. We further observed lymphatic vessel enlargement from the first week of melanoma development, implying tumor cells interacting with the vessels and increased likelihood of metastasis. MFOCT identified cutaneous melanoma‒associated angiogenesis and lymphangiogenesis before the possible visual perception of the tumor (≥42 days) and before metastasis could be diagnosed using micropositron emission tomography (35 days). Thus, the proposed quantitative analysis using MFOCT has the potential for early detection of cutaneous melanoma progression or prediction of metastatic melanoma in a mouse model. However, retrospective and extensive experiments still need to be performed in the future to confirm the value of MFOCT in clinical application.

Combined methyl aminolevulinate-based photodynamic therapy and imiquimod in a patient with perianal extramammary Paget's disease.

Extramammary Paget's disease (EMPD) is a rare form of intraepithelial adenocarcinoma. Complete surgical removal of localized disease is the standard treatment for EMPD but carries anaesthesia-related risks and possible postoperative functional deficits. Herein, we present a case of perianal EMPD successfully treated with topical methyl aminolevulinate-based photodynamic therapy (MAL-PDT), followed by topical imiquimod. Immunohistochemical analysis after PDT revealed high expression of Toll-like receptor 7 on keratinocytes, Paget's cells, and dermal inflammatory cells, as well as increased expression of intraepidermal Langerhans cells, dermal macrophages, and T cells. We propose that MAL-PDT may prime the enhancing effects of topical imiquimod. Combined local treatment with PDT and imiquimod may provide an alternative and non-invasive strategy for perianal EMPD.

A Functional Assay to Assess Toxicity During Murine B Cell Development In Vitro.

B lymphocytes, or B cells, are important players in immunity that produce antigen-specific immunoglobulins. As a result, they are involved in various immune-linked pathologies. To better understand, prevent, or treat B cell-associated disease and immunotoxicity, we developed an in vitro assay to model early murine B cell differentiation within the bone marrow. This model uses sorted B cell precursors cultured on a supporting stromal cell layer, which over time acquire markers of further differentiated B cells, such as surface antigens and rearranged immunoglobulin light chain. Importantly, we utilized our in vitro model to validate our previous observations that xenobiotics, such as tungsten and organotins, alter B cell development in vivo. Furthermore, gene expression can be modulated in this model using retroviral transduction, making it amenable to investigating signaling pathways involved in disruption of B cell differentiation. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Assessment of early B lymphocyte differentiation in vitro Support Protocol: Isolation of murine bone marrow Alternate Protocol 1: Addition of recombinant interleukin-7 Alternate Protocol 2: Genetic manipulation via retroviral transduction.

Tungsten Blocks Murine B Lymphocyte Differentiation and Proliferation Through Downregulation of IL-7 Receptor/Pax5 Signaling.

Tungsten is an emerging environmental toxicant associated with several pediatric leukemia clusters, although a causal association has not been established. Our previous work demonstrated that tungsten exposure resulted in an accumulation of pre-B cells in the bone marrow, the same cell type that accumulates in pediatric acute lymphoblastic leukemia (ALL). To better understand the relevant molecular mechanisms, we performed RNA-sequencing on flow sorted pre-B cells from control and tungsten-exposed mice. Tungsten decreased the expression of multiple genes critical for B cell development, including members of the interleukin-7 receptor (IL-7R) and pre-B cell receptor signaling pathways, such as Jak1, Stat5a, Pax5, Syk, and Ikzf3. These results were confirmed in an in vitro model of B cell differentiation, where tungsten arrested differentiation at the pro-B cell stage and inhibited proliferation. These changes were associated with decreased expression of multiple genes in the IL-7R signaling pathway and decreased percentage of IL-7R, phosphorylated STAT5 double-positive cells. Supplementation with IL-7 or overexpression of Pax5, the transcription factor downstream of IL-7R, rescued the tungsten-induced differentiation block. Together, these data support the hypothesis that IL-7R/Pax5 signaling axis is critical to tungsten-mediated effects on pre-B cell development. Importantly, many of these molecules are modulated in ALL.

From the Cover: Tributyltin Alters the Bone Marrow Microenvironment and Suppresses B Cell Development.

Organotins are industrial chemicals and agricultural pesticides, and they contaminate both outdoor and indoor environments. Organotins are detectable in human sera at biologically active concentrations and are immuno-and neuro-toxicants. Triphenyltin, tributyltin (TBT) and dibutyltin activate peroxisome proliferator-activated receptor γ in bone marrow multipotent mesenchymal stromal cells and promote adipogenesis. TBT also has been shown to suppress osteogenesis; osteoblasts not only support bone homeostasis but also support B lymphopoiesis. In addition, developing B cells are highly sensitive to exogenous insults. Thus, we hypothesized that bone marrow B cells may be negatively affected by TBT exposure both directly, through activation of apoptosis, and indirectly, through alterations of the bone marrow microenvironment. TBT activated apoptosis in developing B cells at environmentally relevant concentrations (as low as 80 nM) in vitro, via a mechanism that is distinct from that induced by high dose (µM) TBT and that requires p53. TBT suppressed the proliferation of hematopoietic cells in an ex vivo bone marrow model. Concurrent treatment of stromal cells and B cells or pretreatment of stromal cells with TBT induced adipogenesis in the stromal cells and reduced the progression of B cells from the early pro B (Hardy fraction B) to the pre B stage (Hardy fraction D). In vivo, TBT induced adipogenesis in bone marrow, reduced "aging-sensitive" AA4+CD19+ B cells in bone marrow, and reduced splenic B cell numbers. Immunosenescence and osteoporosis are adverse health effects of aging, we postulate that TBT exposure may mimic, and possibly intensify, these pathologies.

Tungsten Promotes Sex-Specific Adipogenesis in the Bone by Altering Differentiation of Bone Marrow-Resident Mesenchymal Stromal Cells.

Tungsten is a naturally occurring metal that increasingly is being incorporated into industrial goods and medical devices, and is recognized as an emerging contaminant. Tungsten preferentially and rapidly accumulates in murine bone in a concentration-dependent manner; however the effect of tungsten deposition on bone biology is unknown. Other metals alter bone homeostasis by targeting bone marrow-derived mesenchymal stromal cell (MSC) differentiation, thus, we investigated the effects of tungsten on MSCsin vitroandin vivoIn vitro, tungsten shifted the balance of MSC differentiation by enhancing rosiglitazone-induced adipogenesis, which correlated with an increase in adipocyte content in the bone of tungsten-exposed, young, male mice. Conversely, tungsten inhibited osteogenesis of MSCsin vitro; however, we found no evidence that tungsten inhibited osteogenesisin vivo Interestingly, two factors known to influence adipogenesis are sex and age of mice. Both female and older mice have enhanced adipogenesis. We extended our study and exposed young female and adult (9-month) male and female mice to tungsten for 4 weeks. Although tungsten accumulated to a similar extent in young female mice, it did not promote adipogenesis. Interestingly, tungsten did not accumulate in the bone of older mice; it was undetectable in adult male mice, and just above the limit of detect in adult female mice. Surprisingly, tungsten enhanced adipogenesis in adult female mice. In summary, we found that tungsten alters bone homeostasis by altering differentiation of MSCs, which could have significant implications for bone quality, but is highly dependent upon sex and age.