Cultured epithelial autografts for the treatment of large-to-giant congenital melanocytic nevus in 31 patients

Introduction: Giant congenital melanocytic nevus (GCMN) is a large melanocytic nevus, and its full-thickness removal is usually difficult due to the lack of skin available for reconstruction. Curettage is an alternative approach in cases of GCMN to remove the superficial dermis above the cleavage plane with a curette in the neonatal period, and its major complications include repigmentation, retarded epithelization, and hypertrophic scar formation. In Japan, the JACE® cultured epidermal autograft (CEA) was approved and covered by public healthcare insurance for the treatment of congenital melanocytic nevus (CMN) that is difficult to treat with conventional methods in 2016. We have used CEA for wounds after curettage in the neonatal period or following ablation after the neonatal period in combination with laser therapies to reduce the above-mentioned complications. Methods: In this study, we summarized all consecutive CMN patients treated using CEA from December 2016 to April 2019 and evaluated the duration required for epithelialization, incidence of hypertrophic scar, and color change in the target nevus by comparing the L∗ values one year later between the Curettage group, the non-Curettage group with initial treatment or the subsequent group. Results: No significant differences were seen in the epithelization period or incidence of hypertrophic scars among the groups, but the color of the target nevus was improved significantly in the Curettage group (p < 0.01) and non-Curettage group with initial treatment (p < 0.01). Conclusions: In conclusion, CEA seems to accelerate epithelization after curettage or ablation of CMN, and this treatment could improve the color of CMN when applied initially

Ex vivo Induction of Apoptotic Mesenchymal Stem Cell by High Hydrostatic Pressure

Among promising solutions for tissue repair and wound healing, mesenchymal stem (or stromal) cells (MSCs) have been a focus of attention and have become the most clinically studied experimental cell therapy. Recent studies reported the importance of apoptosis in MSC-mediated immunomodulation, in which apoptotic MSCs (apoMSCs) were shown to be superior to living MSCs. Nowadays, high hydrostatic pressure (HHP), a physical technique that uses only fluid pressure, has been developed and applied in various bioscience fields, including biotechnology, biomaterials, and regenerative medicine, as its safe and simply operation. In the current study, we investigated the impact of HHP treatment on human bone marrow-MSC survival and proliferation. Based on the detection of executioner caspase activation, phosphatidylserine exposure, DNA fragmentation (TUNEL) and irrefutable ultrastructural morphological changes on transmission electron microscopy (TEM), our data revealed that HHP treatment induced complete apoptosis in MSCs. Notably, this technique might provide manipulated products for use in cell-based therapies as manufacturing capability expands. We hope that our findings will contribute to the improvement of MSCs or EVs in translational research development. Graphical Abstract

High Hydrostatic Pressure Therapy Annihilates Squamous Cell Carcinoma in a Murine Model

Cutaneous squamous cell carcinoma (cSCC) is one of the most common skin cancers. In the treatment of cSCC, it is necessary to remove it completely, and reconstructive surgery, such as a skin graft or a local or free flap, will be required, depending on the size, with donor-site morbidity posing a burden to the patient. The high hydrostatic pressure (HHP) technique has been developed as a physical method of decellularizing various tissues. We previously reported that HHP at 200 MPa for 10 min could inactivate all cells in the giant congenital melanocytic nevus, and we have already started a clinical trial using this technique. In the present study, we explored the critical pressurization condition for annihilating cSCC cells in vitro and confirmed that this condition could also annihilate cSCC in vivo. We prepared 5 pressurization conditions in this study (150, 160, 170, 180, and 190 MPa for 10 min) and confirmed that cSCC cells were killed by pressurization at ≥160 MPa for 10 min. In the in vivo study, the cSCC cells inactivated by HHP at 200 MPa for 10 min were unable to proliferate after injection into the intradermal space of mice, and transplanted cSCC tissues that had been inactivated by HHP showed a decreased weight at 5 weeks after implantation. These results suggested that HHP at 200 MPa for 10 min was able to annihilate SCC, so HHP technology may be a novel treatment of skin cancer

Response Predictors of DCS Therapy in Gastric Cancer

Objectives: The aim of this study was to identify biomarkers for predicting the efficacy of docetaxel, cisplatin, and S-1 (DCS) therapy for advanced gastric cancer using microarrays of biopsy specimens before chemotherapy. Methods: Nineteen samples were taken from 19 patients with unresectable metastatic gastric cancer who received DCS as a first-line therapy. Laser capture microdissection was performed, and total cellular RNA was extracted from each microdissected sample. Whole-gene expression was analyzed by microarray, and the difference in mRNA expression observed with the microarrays was confirmed by quantitative real-time PCR. Immunohistochemical staining was performed using clinical tissue sections obtained by endoscopic biopsy. Results: Eleven patients were identified as early responders and 8 patients as nonresponders to DCS therapy. Twenty-nine genes showed significant differences in relative expression ratios between tumor and normal tissues. A classifier set of 29 genes had high accuracy (94.7%) for distinguishing gene expression between 11 early responders and 8 nonresponders. Decreasing the size of the classifier set to 4 genes (PDGFB, PCGF3, CISH, and ANXA5) increased the accuracy to 100%. Expression levels by real-time PCR for validation were well correlated with those 4 genes in microarrays. Conclusion: The genes identified may serve as efficient biomarkers for personalized cancer-targeted therapy

Sessile serrated adenoma/polyp-cancer sequenceにおいてS100Pは遺伝子脱メチル化により発現し、細胞増殖を促進する

Background/Aims: Sessile serrated adenomas/polyps (SSA/Ps) are a putative precursor lesion of colon cancer. Although the relevance of DNA hypermethylation in the SSA/P-cancer sequence is well documented, the role of DNA hypomethylation is unknown. We investigated the biological relevance of DNA hypomethylation in the SSA/P-cancer sequence by using 3-dimensional organoids of SSA/P. Methods: We first analyzed hypomethylated genes using datasets from our previous DNA methylation array analysis on 7 SSA/P and 2 cancer in SSA/P specimens. Expression levels of hypomethylated genes in SSA/P specimens were determined by RT-PCR and immunohistochemistry. We established 3-dimensional SSA/P organoids and performed knockdown experiments using a lentiviral shRNA vector. DNA hypomethylation at CpG sites of the gene was quantitated by MassARRAY analysis. Results: The mean number of hypomethylated genes in SSA/P and cancer in SSA/P was 41.6 ± 27.5 and 214 ± 19.8, respectively, showing a stepwise increment in hypomethylation during the SSA/P-cancer sequence. S100P, S100α2, PKP3, and MUC2 were most commonly hypomethylated in SSA/P specimens. The mRNA and protein expression levels of S100P, S100α2, and MUC2 were significantly elevated in SSA/P compared with normal colon tissues, as revealed by RT-PCR and immunohistochemistry, respectively. Among these, mRNA and protein levels were highest for S100P. Knockdown of the S100P gene using a lentiviral shRNA vector in 3-dimensional SSA/P organoids inhibited cell growth by >50% (p < 0.01). The mean diameter of SSA/P organoids with S100P gene knockdown was significantly smaller compared with control organoids. MassARRAY analysis of DNA hypomethylation in the S100P gene revealed significant hypomethylation at specific CpG sites in intron 1, exon 1, and the 5′-flanking promoter region. Conclusion: These results suggest that DNA hypomethylation, including S100P hypomethylation, is supposedly associated with the SSA/P-cancer sequence. S100P overexpression via DNA hypomethylation plays an important role in promoting cell growth in the SSA/P-cancer sequence

Exploration of the Pressurization Condition for Killing Human Skin Cells and Skin Tumor Cells by High Hydrostatic Pressure

High hydrostatic pressure (HHP) is a physical method for inactivating cells or tissues without using chemicals such as detergents. We previously reported that HHP at 200 MPa for 10 min was able to inactivate all cells in skin and giant congenital melanocytic nevus (GCMN) without damaging the extracellular matrix. We also reported that HHP at 150 MPa for 10 min was not sufficient to inactivate them completely, while HHP at 200 MPa for 10 min was able to inactivate them completely. We intend to apply HHP to treat malignant skin tumor as the next step; however, the conditions necessary to kill each kind of cell have not been explored. In this work, we have performed a detailed experimental study on the critical pressure and pressurization time using five kinds of human skin cells and skin tumor cells, including keratinocytes (HEKas), dermal fibroblasts (HDFas), adipose tissue-derived stem cells (ASCs), epidermal melanocytes (HEMa-LPs), and malignant melanoma cells (MMs), using pressures between 150 and 200 MPa. We pressurized cells at 150, 160, 170, 180, or 190 MPa for 1 s, 2 min, and 10 min and evaluated the cellular activity using live/dead staining and proliferation assays. The proliferation assay revealed that HEKas were inactivated at a pressure higher than 150 MPa and a time period longer than 2 min, HDFas and MMs were inactivated at a pressure higher than 160 MPa and for 10 min, and ASCs and HEMa-LPs were inactivated at a pressure higher than 150 MPa and for 10 min. However, some HEMa-LPs were observed alive after HHP at 170 MPa for 10 min, so we concluded that HHP at a pressure higher than 180 MPa for 10 min was able to inactivate five kinds of cells completely