Spatially organized multicellular immune hubs in human colorectal cancer.

Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd individuals. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across tumors from affected individuals and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T cell-attracting chemokines. By identifying interacting cellular programs, we reveal the logic underlying spatially organized immune-malignant cell networks.

Transcriptional mediators of treatment resistance in lethal prostate cancer.

Metastatic castration-resistant prostate cancer is typically lethal, exhibiting intrinsic or acquired resistance to second-generation androgen-targeting therapies and minimal response to immune checkpoint inhibitors1. Cellular programs driving resistance in both cancer and immune cells remain poorly understood. We present single-cell transcriptomes from 14 patients with advanced prostate cancer, spanning all common metastatic sites. Irrespective of treatment exposure, adenocarcinoma cells pervasively coexpressed multiple androgen receptor isoforms, including truncated isoforms hypothesized to mediate resistance to androgen-targeting therapies2,3. Resistance to enzalutamide was associated with cancer cell-intrinsic epithelial-mesenchymal transition and transforming growth factor-ß signaling. Small cell carcinoma cells exhibited divergent expression programs driven by transcriptional regulators promoting lineage plasticity and HOXB5, HOXB6 and NR1D2 (refs. 4-6). Additionally, a subset of patients had high expression of dysfunction markers on cytotoxic CD8+ T cells undergoing clonal expansion following enzalutamide treatment. Collectively, the transcriptional characterization of cancer and immune cells from human metastatic castration-resistant prostate cancer provides a basis for the development of therapeutic approaches complementing androgen signaling inhibition.

A single-cell landscape of high-grade serous ovarian cancer.

Malignant abdominal fluid (ascites) frequently develops in women with advanced high-grade serous ovarian cancer (HGSOC) and is associated with drug resistance and a poor prognosis1. To comprehensively characterize the HGSOC ascites ecosystem, we used single-cell RNA sequencing to profile ~11,000 cells from 22 ascites specimens from 11 patients with HGSOC. We found significant inter-patient variability in the composition and functional programs of ascites cells, including immunomodulatory fibroblast sub-populations and dichotomous macrophage populations. We found that the previously described immunoreactive and mesenchymal subtypes of HGSOC, which have prognostic implications, reflect the abundance of immune infiltrates and fibroblasts rather than distinct subsets of malignant cells2. Malignant cell variability was partly explained by heterogeneous copy number alteration patterns or expression of a stemness program. Malignant cells shared expression of inflammatory programs that were largely recapitulated in single-cell RNA sequencing of ~35,000 cells from additionally collected samples, including three ascites, two primary HGSOC tumors and three patient ascites-derived xenograft models. Inhibition of the JAK/STAT pathway, which was expressed in both malignant cells and cancer-associated fibroblasts, had potent anti-tumor activity in primary short-term cultures and patient-derived xenograft models. Our work contributes to resolving the HSGOC landscape3-5 and provides a resource for the development of novel therapeutic approaches.

Author Correction: A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors.

Single-cell genomics is essential to chart tumor ecosystems. Although single-cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumors, single-nucleus RNA-Seq (snRNA-Seq) is needed to profile frozen or hard-to-dissociate tumors. Each requires customization to different tissue and tumor types, posing a barrier to adoption. Here, we have developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We analyzed 216,490 cells and nuclei from 40 samples across 23 specimens spanning eight tumor types of varying tissue and sample characteristics. We evaluated protocols by cell and nucleus quality, recovery rate and cellular composition. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types, but at different proportions. Our work provides guidance for studies in a broad range of tumors, including criteria for testing and selecting methods from the toolbox for other tumors, thus paving the way for charting tumor atlases.

Intrinsic Resistance to Immune Checkpoint Blockade in a Mismatch Repair-Deficient Colorectal Cancer.

Immunotherapy with checkpoint inhibitors, such as the programmed death-1 (PD-1) antibodies pembrolizumab and nivolumab, are effective in a variety of tumors, yet not all patients respond. Tumor microsatellite instability-high (MSI-H) has emerged as a biomarker of response to checkpoint blockade, leading to the tissue agnostic approval of pembrolizumab in MSI-H cancers. Here we describe a patient with MSI-H colorectal cancer that was treated with this immune checkpoint inhibitor and exhibited progression of disease. We examined this intrinsic resistance through genomic, transcriptional, and pathologic characterization of the patient's tumor and the associated immune microenvironment. The tumor had typical MSI-H molecular features, including a high neoantigen load. We also identified biallelic loss of the gene for ß2-microglobulin (B2M), whose product is critical for antigen presentation. Immune infiltration deconvolution analysis of bulk transcriptome data from this anti-PD-1-resistant tumor and hundreds of other colorectal cancer specimens revealed a high natural killer cell and M2 macrophage infiltration in the patient's cancer. This was confirmed by single-cell transcriptome analysis and multiplex immunofluorescence. Our study provides insight into resistance in MSI-H tumors and suggests immunotherapeutic strategies in additional genomic contexts of colorectal cancer.

A Cancer Cell Program Promotes T Cell Exclusion and Resistance to Checkpoint Blockade.

Immune checkpoint inhibitors (ICIs) produce durable responses in some melanoma patients, but many patients derive no clinical benefit, and the molecular underpinnings of such resistance remain elusive. Here, we leveraged single-cell RNA sequencing (scRNA-seq) from 33 melanoma tumors and computational analyses to interrogate malignant cell states that promote immune evasion. We identified a resistance program expressed by malignant cells that is associated with T cell exclusion and immune evasion. The program is expressed prior to immunotherapy, characterizes cold niches in situ, and predicts clinical responses to anti-PD-1 therapy in an independent cohort of 112 melanoma patients. CDK4/6-inhibition represses this program in individual malignant cells, induces senescence, and reduces melanoma tumor outgrowth in mouse models in vivo when given in combination with immunotherapy. Our study provides a high-resolution landscape of ICI-resistant cell states, identifies clinically predictive signatures, and suggests new therapeutic strategies to overcome immunotherapy resistance.

Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids.

Ex vivo systems that incorporate features of the tumor microenvironment and model the dynamic response to immune checkpoint blockade (ICB) may facilitate efforts in precision immuno-oncology and the development of effective combination therapies. Here, we demonstrate the ability to interrogate ex vivo response to ICB using murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS). MDOTS/PDOTS isolated from mouse and human tumors retain autologous lymphoid and myeloid cell populations and respond to ICB in short-term three-dimensional microfluidic culture. Response and resistance to ICB was recapitulated using MDOTS derived from established immunocompetent mouse tumor models. MDOTS profiling demonstrated that TBK1/IKKε inhibition enhanced response to PD-1 blockade, which effectively predicted tumor response in vivo Systematic profiling of secreted cytokines in PDOTS captured key features associated with response and resistance to PD-1 blockade. Thus, MDOTS/PDOTS profiling represents a novel platform to evaluate ICB using established murine models as well as clinically relevant patient specimens.Significance: Resistance to PD-1 blockade remains a challenge for many patients, and biomarkers to guide treatment are lacking. Here, we demonstrate feasibility of ex vivo profiling of PD-1 blockade to interrogate the tumor immune microenvironment, develop therapeutic combinations, and facilitate precision immuno-oncology efforts. Cancer Discov; 8(2); 196-215. ©2017 AACR.See related commentary by Balko and Sosman, p. 143See related article by Deng et al., p. 216This article is highlighted in the In This Issue feature, p. 127.

Dicer loss and recovery induce an oncogenic switch driven by transcriptional activation of the oncofetal Imp1-3 family.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression critical for organismal viability. Changes in miRNA activity are common in cancer, but how these changes relate to subsequent alterations in transcription and the process of tumorigenesis is not well understood. Here, we report a deep transcriptional, oncogenic network regulated by miRNAs. We present analysis of the gene expression and phenotypic changes associated with global miRNA restoration in miRNA-deficient fibroblasts. This analysis uncovers a miRNA-repressed network containing oncofetal genes Imp1, Imp2, and Imp3 (Imp1-3) that is up-regulated primarily transcriptionally >100-fold upon Dicer loss and is resistant to resilencing by complete restoration of miRNA activity. This Dicer-resistant epigenetic switch confers tumorigenicity to these cells. Let-7 targets Imp1-3 are required for this tumorigenicity and feed back to reinforce and sustain expression of the oncogenic network. Together, these Dicer-resistant genes constitute an mRNA expression signature that is present in numerous human cancers and is associated with poor survival.

Pancreatic Cancer Cell Exosome-Mediated Macrophage Reprogramming and the Role of MicroRNAs 155 and 125b2 Transfection using Nanoparticle Delivery Systems.

Exosomes are nano-sized endosome-derived small intraluminal vesicles, which are important facilitators of intercellular communication by transporting contents, such as protein, mRNA, and microRNAs, between neighboring cells, such as in the tumor microenvironment. The purpose of this study was to understand the mechanisms of exosomes-mediated cellular communication between human pancreatic cancer (Panc-1) cells and macrophages (J771.A1) using a Transwell co-culture system. Following characterization of exosome-mediated cellular communication and pro-tumoral baseline M2 macrophage polarization, the Panc-1 cells were transfected with microRNA-155 (miR-155) and microRNA-125b-2 (miR-125b2) expressing plasmid DNA using hyaluronic acid-poly(ethylene imine)/hyaluronic acid-poly(ethylene glycol) (HA-PEI/HA-PEG) self-assembling nanoparticle-based non-viral vectors. Our results show that upon successful transfection of Panc-1 cells, the exosome content was altered leading to differential communication and reprogramming of the J774.A1 cells to an M1 phenotype. Based on these results, genetic therapies targeted towards selective manipulation of tumor cell-derived exosome content may be very promising for cancer therapy.

Direct Conversion of CH3NH3PbI3 from Electrodeposited PbO for Highly Efficient Planar Perovskite Solar Cells.

Organic-inorganic hybrid perovskite materials have recently been identified as a promising light absorber for solar cells. In the efficient solar cells, the perovskite active layer has generally been fabricated by either vapor deposition or two-step sequential deposition process. Herein, electrochemically deposited PbO film is in situ converted into CH3NH3PbI3 through solid-state reaction with adjacent CH3NH3I layer, exhibiting a large-scale flat and uniform thin film with fully substrate coverage. The resultant planar heterojunction photovoltaic device yields a best power conversion efficiency of 14.59% and an average power conversion efficiency of 13.12 ± 1.08% under standard AM 1.5 conditions. This technique affords a facile and environment-friendly method for the fabrication of the perovskite based solar cells with high reproducibility, paving the way for the practical application.

Temporal and Spatial Denoising of Depth Maps.

This work presents a procedure for refining depth maps acquired using RGB-D (depth) cameras. With numerous new structured-light RGB-D cameras, acquiring high-resolution depth maps has become easy. However, there are problems such as undesired occlusion, inaccurate depth values, and temporal variation of pixel values when using these cameras. In this paper, a proposed method based on an exemplar-based inpainting method is proposed to remove artefacts in depth maps obtained using RGB-D cameras. Exemplar-based inpainting has been used to repair an object-removed image. The concept underlying this inpainting method is similar to that underlying the procedure for padding the occlusions in the depth data obtained using RGB-D cameras. Therefore, our proposed method enhances and modifies the inpainting method for application in and the refinement of RGB-D depth data image quality. For evaluating the experimental results of the proposed method, our proposed method was tested on the Tsukuba Stereo Dataset, which contains a 3D video with the ground truths of depth maps, occlusion maps, RGB images, the peak signal-to-noise ratio, and the computational time as the evaluation metrics. Moreover, a set of self-recorded RGB-D depth maps and their refined versions are presented to show the effectiveness of the proposed method.

Induction of survivin inhibition, G2/M cell cycle arrest and autophagic on cell death in human malignant glioblastoma cells.

Chemotherapy efficacy is limited by intrinsic and acquired resistance in glioblastoma (GBM); hence, novel tactics are crucial. Survivin has been demonstrated as a key resistant factor in GBM because of its function in inhibiting apoptosis, regulating autophagy, and in promoting G2/M cell cycle transition. Parthenolide has been reported to be an effective antitumor agent in a variety of tumor cells and decreases survivin level in leukemia cells. But the effect of parthenolide on survivin and the cell death process in GBM is still unknown. The aim of this study was to examine whether parthenolide had the potential to inhibit cell proliferation in the GBM cell line U373. The parthenolide-induced effects in relation to survivin suppression and cell death were further investigated. Our results showed that parthenolide substantially inhibited cell viability with IC50 values of approximate 16 µM. Treatment with parthenolide at the dose of 16 µM led to considerable downregulation of survivin, G2/M cell cycle arrest and Chk2 upregulation in cells. Parthenolide induced apoptosis in only a few cells and a slight increase in activated caspases 3 levels. By contrast, parthenolide induced a significant increase of intracellular autophagosomes and the expression of autophagy related proteins, including ULK1 and LC3 I/LC3 II, in the treated cells. These results suggested that parthenolide induced survivin inhibition, G2/M cell cycle arrest, and triggered cell death through autophagic cell death in the GBM cell line.

Electrodeposition of PbO and its in situ conversion to CH3NH3PbI3 for mesoscopic perovskite solar cells.

The perovskite CH3NH3PbI3 was prepared on a mesoscopic TiO2 film, starting from electrodepositing PbO, to iodination to PbI2, and then interdiffusion reaction with CH3NH3I. The as-prepared film was used as a light absorber for the perovskite solar cells, exhibiting a high PCE of 12.5% under standard AM 1.5 conditions.

Effectiveness of an e-learning curriculum on occupational health for music performers.

OBJECTIVE: The purpose of this study was (1) to evaluate the effectiveness of the e-learning curriculum and (2) to explore the type of questions raised by students through the "Health Promotion for Music Performers" (HPMP) e-learning curriculum. MATERIALS AND METHODS: This study was primarily a pedagogical research composed of a pre- and postintervention design coupled with a 1-month longitudinal knowledge retention measurement. The intervention, the HPMP e-learning curriculum, was implemented over 14 weeks, once a week, for a total of 14 classes. Each class consisted of a 60-min prerecorded lecture followed by a 40-min real-time interactive discussion. The interdisciplinary faculty panel consisted of experts from the field of music and medicine. The Self-Assessment Questionnaire (SAQ) was used to evaluate knowledge changes concerning (1) Practice and Performance issues and (2) Health and Life Style issues. RESULTS: Fifteen graduate-level music students participated in the study. The SAQ scores on the 1-month follow-up test for Practice and Performance issues were significantly higher than the pretest (t=2.731, p<0.05). On the other hand, no significant differences were found between the posttest and pretest or between the follow-up test and posttest. Regarding Health and Life Style issues, comparison at all three measurement points did not reveal any significant difference. Questions raised by students fell into four major categories: performance injury (45%), performance anxiety (22%), general physiology (22%), and general psychology (11%). CONCLUSIONS: The findings suggest that the HPMP e-learning course enhanced student awareness of Practice and Performance issues but did not have as significant an impact on student awareness of Health and Lifestyle issues.

Macrophage migration inhibitory factor increases cell motility and up-regulates αvß3 integrin in human chondrosarcoma cells.

The macrophage migration-inhibitory factor (MIF) is a pro-inflammatory cytokine first known for its effect on macrophage migration and activation. Recent studies have shown that MIP plays a critical role in tumor growth, angiogenesis, and metastasis. Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. However, the effects of MIF on human chondrosarcoma cells are largely unknown. In the present study, MIF was found to increase the migration and the expression of αvß3 integrin in human chondrosarcoma cells. The phosphatidylinositol 3-kinase (PI3K), Akt, and NF-κB pathways were activated by MIF treatment, and the MIF-induced expression of integrin and migration activity were inhibited by the specific inhibitors and mutant forms of PI3K, Akt, and NF-κB cascades. In addition, migration-prone sublines demonstrated that increased cell migration ability was correlated with increased expression of MIF and αvß3 integrin. Taken together, our results indicate that MIF enhanced the migration of the chondrosarcoma cells by increasing αvß3 integrin expression through the PI3K/Akt/NF-κB signal transduction pathway.

An evaluation of a weight-loss program incorporating E-learning for obese junior high school students.

Obesity in adolescents is continuing to rise at an alarming rate and is becoming an important public health problem in Taiwan. Therefore, the aims of this study were (1) to evaluate the effectiveness of a Weight-loss E-learning Program (WEP) on obese Chinese adolescents and (2) to gauge this group's satisfaction with the WEP. The design was quasi-experimental, using purposive samples from two junior high schools in Taipei, Taiwan. Obese adolescents between 12 and 14 years of age with body mass indexes (BMI) over 25 kg/m2 were recruited. A 14-week WEP was developed to expedite weight loss for the selected adolescents. Data such as BMI, waist-to-hip ratio, waist circumference, hip circumference, mid-arm circumference, triceps skinfold, blood pressure, and physical fitness were collected through standardized instruments and methods before and after the WEP. The satisfaction of the subjects and four psychosocial variables were evaluated and taken into account by authoritative scales and questionnaires. In total, 37 adolescents participated in this study. After the WEP, we found significant decreases in BMI (p < 0.05), waist circumference (p < 0.05), and triceps skinfold (p < 0.001) in the sample population. Improvements were found in three of four tests of physical fitness (p < 0.001, p < 0.05, and p < 0.01, respectively). All psychosocial variables showed significant favorable changes (p < 0.01 for self-esteem scores, p < 0.001 for the other three variables) and satisfaction levels for the WEP ranged from 56.6% to 83.8% in four different criteria. The WEP was effective in helping obese Chinese adolescents lose weight. However, there is still room for improvement.

Application of tele-ultrasound in emergency medical services.

In emergency medical services, portable ultrasound scanners have the potential to become new-age stethoscopes for emergency physicians. For trauma cases in particular, portable ultrasound scanners can scan the chest and abdomen of emergency patients both rapidly and conveniently. This study describes the development of tele-ultrasound for pre-diagnosis in a medical emergency setting as a part of the updated Mobile Hospital Emergency Medical System (MHEMS). An emergency medical technician can provide an emergency physician with a patient's ultrasound images and medical information during the patient's pre-hospitalization and transportation period using a combination of the MHEMS, the portable ultrasound scanner, and the onboard 3G communication capabilities. The MHEMS includes a Dispatch and Mission Control Center that facilitates the communication between the Emergency Department of a specified hospital, the systems aboard the ambulance. Early receipt of information relevant to the patient will enhance pre-diagnosis options for on-duty emergency physicians and allow for a hospital's emergency department to promptly prepare necessary surgical instruments or beds. Furthermore, emergency medical technicians can also obtain instructions from on-duty physicians to enhance damage and disaster control ability in critical moments.

A conceptual persistent healthcare quality improvement process for software development management.

This paper illustrates a sustained conceptual service quality improvement process for the management of software development within a healthcare enterprise. Our proposed process is revised from Niland's healthcare quality information system (HQIS). This process includes functions to survey the satisfaction of system functions, describe the operation bylaws on-line, and provide on-demand training. To achieve these goals, we integrate five information systems in National Taiwan University Hospital, including healthcare information systems, health quality information system, requirement management system, executive information system, and digital learning system, to form a full Deming cycle. A preliminary user satisfaction survey showed that our outpatient information system scored an average of 71.31 in 2006.