Integrative analysis of spatial and single-cell transcriptome data from human pancreatic cancer reveals an intermediate cancer cell population associated with poor prognosis.

BACKGROUND: Recent studies using single-cell transcriptomic analysis have reported several distinct clusters of neoplastic epithelial cells and cancer-associated fibroblasts in the pancreatic cancer tumor microenvironment. However, their molecular characteristics and biological significance have not been clearly elucidated due to intra- and inter-tumoral heterogeneity. METHODS: We performed single-cell RNA sequencing using enriched non-immune cell populations from 17 pancreatic tumor tissues (16 pancreatic cancer and one high-grade dysplasia) and generated paired spatial transcriptomic data from seven patient samples. RESULTS: We identified five distinct functional subclusters of pancreatic cancer cells and six distinct cancer-associated fibroblast subclusters. We deeply profiled their characteristics, and we found that these subclusters successfully deconvoluted most of the features suggested in bulk transcriptome analysis of pancreatic cancer. Among those subclusters, we identified a novel cancer cell subcluster, Ep_VGLL1, showing intermediate characteristics between the extremities of basal-like and classical dichotomy, despite its prognostic value. Molecular features of Ep_VGLL1 suggest its transitional properties between basal-like and classical subtypes, which is supported by spatial transcriptomic data. CONCLUSIONS: This integrative analysis not only provides a comprehensive landscape of pancreatic cancer and fibroblast population, but also suggests a novel insight to the dynamic states of pancreatic cancer cells and unveils potential therapeutic targets.

Relative infectivity of the SARS-CoV-2 Omicron variant in human alveolar cells.

With the continuous emergence of highly transmissible SARS-CoV-2 variants, the comparison of their infectivity has become a critical issue for public health. However, a direct assessment of the viral characteristic has been challenging because of the lack of appropriate experimental models and efficient methods. Here, we integrated human alveolar organoids and single-cell transcriptome sequencing to facilitate the evaluation. In a proof-of-concept study with four highly transmissible SARS-CoV-2 variants, including GR (B.1.1.119), Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (BA.1), a rapid evaluation of the relative infectivity was possible. Our system demonstrates that the Omicron variant is 5- to 7-fold more infectious to human alveolar cells than the other SARS-CoV-2 variants at the initial stage of infection. To our knowledge, for the first time, this study measures the relative infectivity of the Omicron variant under multiple virus co-infection and provides new experimental procedures that can be applied to monitor emerging viral variants.

Identifying Somatic Mitochondrial DNA Mutations.

Mitochondria are cellular organelles that play an essential role in eukaryotes, producing the energy needed for a cell to survive. Beyond the ~3.2 Gb of nuclear genomic DNA, each human cell has hundreds of mitochondria which carry one or a few copies of the 16.5 kb circular mitochondria DNA (mtDNA). Despite its small size, the circular genome encodes 37 genes, including 13 proteins that generate respiratory chain complexes together with other proteins of nuclear origin. Similar to nuclear genome, mtDNA in cancer cells frequently harbor somatically acquired alterations. Whole-genome or whole-exome sequencing of the tumor and its matched normal tissues (frequently blood or adjacent non-tumor tissues) enables sensitive and efficient detection of somatic mtDNA mutations. Because each cancer cell commonly carries hundreds to thousands of mtDNA copies, detection of mtDNA mutations is dependent on the heteroplasmic level of each mutation. Here, we describe strategies to accurately identify somatic mtDNA mutations in cancer genome studies.

Weight-bearing activity impairs nuclear membrane and genome integrity via YAP activation in plantar melanoma.

Acral melanoma commonly occurs in areas that are not exposed to much sunlight, such as the sole of the foot. Little is known about risk factors and mutational processes of plantar acral melanoma. Nuclear envelope rupture during interphase contributes to genome instability in cancer. Here, we show that the nuclear and micronuclear membranes of melanoma cells are frequently ruptured by macroscopic mechanical stress on the plantar surface due to weight-bearing activities. The marginal region of plantar melanoma nodules exhibits increased nuclear morphological abnormalities and collagen accumulations, and is more susceptible to mechanical stress than the tumor center. An increase in DNA damage coincides with nuclear membrane rupture in the tumor margin. Nuclear envelope integrity is compromised by the mechanosensitive transcriptional cofactor YAP activated in the tumor margin. Our results suggest a mutagenesis mechanism in melanoma and explain why plantar acral melanoma is frequent at higher mechanical stress points.

Estimation of intrafamilial DNA contamination in family trio genome sequencing using deviation from Mendelian inheritance.

With the increasing number of sequencing projects involving families, quality control tools optimized for family genome sequencing are needed. However, accurately quantifying contamination in a DNA mixture is particularly difficult when genetically related family members are the sources. We developed TrioMix, a maximum likelihood estimation (MLE) framework based on Mendel's law of inheritance, to quantify DNA mixture between family members in genome sequencing data of parent-offspring trios. TrioMix can accurately deconvolute any intrafamilial DNA contamination, including parent-offspring, sibling-sibling, parent-parent, and even multiple familial sources. In addition, TrioMix can be applied to detect genomic abnormalities that deviate from Mendelian inheritance patterns, such as uniparental disomy (UPD) and chimerism. A genome-wide depth and variant allele frequency plot generated by TrioMix facilitates tracing the origin of Mendelian inheritance deviations. We showed that TrioMix could accurately deconvolute genomes in both simulated and real data sets.

Enhanced polo-like kinase 1 expression in myelodysplastic syndromes.

BACKGROUND: Cancer is characterized by uncontrolled cellular proliferation, and Polo-like kinase 1 (PLK1), a key regulator of the cell cycle, is overexpressed in many cancers, including acute leukemia and lymphoma. However, the dynamics of PLK1 transcription in myelodysplastic syndromes (MDS) are unknown. This study aimed to investigate the transcript dynamics of PLK1 and determine its role in the pathophysiology of MDS. METHODS: PLK1 mRNA obtained from the bone marrow samples of 67 patients with MDS, 16 patients with secondary acute myeloid leukemia (sAML), and 10 healthy controls were analyzed using quantitative real-time PCR and compared according to various clinical parameters. RESULTS: The median PLK1 expression levels differed slightly, but not significantly, between MDS and sAML patients [661.21 (range, 29.38-8,987.31) vs. 1,462.05 (32.22-5,734.09), respectively], but were significantly higher (P<0.001) than the levels in the healthy controls [19.0 (1.60-49.90)]. Further analyses of PLK1 levels according to the WHO classification of MDS, prognostic risk groups, karyotype risk groups, marrow blast percentage, and depth of cytopenia did not reveal any significant associations. In patients progressing to sAML, PLK1 expression levels differed significantly according to the presence or absence of resistance to hypomethylation treatment (2,470.58 vs. 415.98, P=0.03). CONCLUSION: PLK1 is upregulated in MDS patients; however, its role in the pathophysiology of MDS is unclear. Gene upregulation in cases with pharmacotherapeutic resistance warrants further investigation.

Prognostic Prediction Model for Second Allogeneic Stem-Cell Transplantation in Patients With Relapsed Acute Myeloid Leukemia: Single-Center Report.

PURPOSE: To identify factors affecting survival outcomes and to develop a prognostic model for second allogeneic stem-cell transplantation (allo-SCT2) for relapsed acute myeloid leukemia (AML) after the first autologous or allogeneic stem-cell transplantation. PATIENTS AND METHODS: Seventy-eight consecutive adult AML patients who received allo-SCT2 were analyzed in this retrospective study. RESULTS: The 4-year overall survival (OS) rate was 28.7%. In multivariate analysis, poor cytogenetic risk at diagnosis, circulating blast ≥ 20% at relapse, duration from first transplantation to relapse < 9 months, and failure to achieve morphologic complete remission after allo-SCT2 were factors associated with poor OS. A prognostic model was developed with the following score system: intermediate and poor cytogenetic risk at diagnosis (0.5 and 1 point), peripheral blast ≥ 20% at relapse (1 point), duration from the first transplantation to relapse < 9 months (1 point), and failure to achieve morphologic complete remission after allo-SCT2 (1 point). The model identified 2 subgroups according to the 4-year OS rate: 51.3% in the low-risk group (score < 2) and 2.8% in the high-risk group (score ≥ 2) (P < .001). CONCLUSION: This prognostic model might be useful to make an appropriate decision for allo-SCT2 in relapsed AML after the first autologous or allogeneic stem-cell transplantation.

Ginseng treatment attenuates chronic cyclosporine nephropathy via reducing oxidative stress in an experimental mouse model.

BACKGROUND: This study was performed to investigate whether ginseng extract has a protective effect in an experimental mouse model of chronic cyclosporine (CsA) nephropathy. METHODS: Mice were treated with CsA (30 mg/kg/day, subcutaneously) with or without Korean red ginseng extract (KRG) (0.2, 0.4 g/kg/day, orally) on a 0.01% salt diet for 4 weeks. The effect of KRG on CsA-induced renal injury was evaluated by assessing renal function and pathology, mediators of inflammation, tubulointerstitial fibrosis and apoptotic cell death. Using an in vitro model, we also examined the effect of KRG on CsA-treated proximal tubular cells (HK-2). Oxidative stress was measured by assessing 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in 24-hour urine, tissue sections, and culture media. RESULTS: Four weeks of CsA treatment caused renal dysfunction, typical pathologic lesions and apoptotic cell death. KRG treatment reduced serum creatinine and blood urea nitrogen and histopathology and increased creatinine clearance. Proinflammatory and profibrotic molecules such as induced nitric oxide synthase, cytokines, transforming growth factor (TGF)-ß1 and TGF-ß1-inducible gene h3 and apoptotic cell death, also decreased with KRG treatment. Consistent with these results, in vitro studies showed that addition of KRG protected against CsA-induced morphological changes, cytotoxicity, inflammation, and apoptotic cell death as demonstrated by annexin V binding. These changes were accompanied by decrease in the level of 8-OHdG in urine and culture supernatant after KRG treatment. CONCLUSION: The results of our in vivo and in vitro studies demonstrate that KRG has a protective effect in CsA-induced renal injury via reducing oxidative stress.