Correction: High-dose drug heat map analysis for drug safety and efficacy in multi-spheroid brain normal cells and GBM patient-derived cells.

[This corrects the article DOI: 10.1371/journal.pone.0251998.].

Analysis of T cell receptor clonotypes in tumor microenvironment identifies shared cancer-type-specific signatures.

Despite the conventional view that a truly random V(D)J recombination process should generate a highly diverse immune repertoire, emerging reports suggest that there is a certain bias toward the generation of shared/public immune receptor chains. These studies were performed in viral diseases where public T cell receptors (TCR) appear to confer better protective responses. Selective pressures generating common TCR clonotypes are currently not well understood, but it is believed that they confer a growth advantage. As very little is known about public TCR clonotypes in cancer, here we set out to determine the extent of shared TCR clonotypes in the intra-tumor microenvironments of virus- and non-virus-driven head and neck cancers using TCR sequencing. We report that tumor-infiltrating T cell clonotypes were indeed shared across individuals with the same cancer type, where the majority of shared sequences were specific to the cancer type (i.e., viral versus non-viral). These shared clonotypes were not particularly enriched in EBV-associated nasopharynx cancer but, in both cancers, exhibited distinct characteristics, namely shorter CDR3 lengths, restricted V- and J-gene usages, and also demonstrated convergent V(D)J recombination. Many of these shared TCRs were expressed in patients with a shared HLA background. Pattern recognition of CDR3 amino acid sequences revealed strong convergence to specific pattern motifs, and these motifs were uniquely found to each cancer type. This suggests that they may be enriched for specificity to common antigens found in the tumor microenvironment of different cancers. The identification of shared TCRs in infiltrating tumor T cells not only adds to our understanding of the tumor-adaptive immune recognition but could also serve as disease-specific biomarkers and guide the development of future immunotherapies.

High-dose drug heat map analysis for drug safety and efficacy in multi-spheroid brain normal cells and GBM patient-derived cells.

To test the safety and efficacy of drugs via a high does drug heat map, a multi-spheroids array chip was developed by adopting a micropillar and microwell structure. In the chip, patient-derived cells were encapsulated in alginate and grown to maturity for more than 7 days to form cancer multi-spheroids. Multi-spheroids grown in conventional well plates require many cells and are easily damaged as a result of multiple pipetting during maintenance culture or experimental procedures. To address these issues, we applied a micropillar and microwell structure to the multi-spheroids array. Patient-derived cells from patients with Glioblastoma (GBM), the most common and lethal form of central nervous system cancer, were used to validate the array chip performance. After forming multi-spheroids with a diameter greater than 100µm in a 12×36 pillar array chip (25mm × 75mm), we tested 70 drug compounds (6 replicates) using a high-dose to determine safety and efficacy for drug candidates. Comparing the drug response of multi-spheroids derived from normal cells and cancer cells, we found that four compounds (Dacomitinib, Cediranib, LY2835219, BGJ398) did not show toxicity to astrocyte cell and were efficacious to patient-derived GBM cells.

Three-Dimensional Aggregated Spheroid Model of Hepatocellular Carcinoma Using a 96-Pillar/Well Plate.

A common method of three-dimensional (3D) cell cultures is embedding single cells in Matrigel. Separated cells in Matrigel migrate or grow to form spheroids but lack cell-to-cell interaction, which causes difficulty or delay in forming mature spheroids. To address this issue, we proposed a 3D aggregated spheroid model (ASM) to create large single spheroids by aggregating cells in Matrigel attached to the surface of 96-pillar plates. Before gelling the Matrigel, we placed the pillar inserts into blank wells where gravity allowed the cells to gather at the curved end. In a drug screening assay, the ASM with Hepatocellular carcinoma (HCC) cell lines showed higher drug resistance compared to both a conventional spheroid model (CSM) and a two-dimensional (2D) cell culture model. With protein expression, cytokine activation, and penetration analysis, the ASM showed higher expression of cancer markers associated with proliferation (p-AKT, p-Erk), tight junction formation (Fibronectin, ZO-1, Occludin), and epithelial cell identity (E-cadherin) in HCC cells. Furthermore, cytokine factors were increased, which were associated with immune cell recruitment/activation (MIF-3α), extracellular matrix regulation (TIMP-2), cancer interaction (IL-8, TGF-ß2), and angiogenesis regulation (VEGF-A). Compared to CSM, the ASM also showed limited drug penetration in doxorubicin, which appears in tissues in vivo. Thus, the proposed ASM better recapitulated the tumor microenvironment and can provide for more instructive data during in vitro drug screening assays of tumor cells and improved prediction of efficacious drugs in HCC patients.

Integrative Profiling of T790M-Negative EGFR-Mutated NSCLC Reveals Pervasive Lineage Transition and Therapeutic Opportunities.

PURPOSE: Despite the established role of EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutated NSCLC, drug resistance inevitably ensues, with a paucity of treatment options especially in EGFR T790M-negative resistance. EXPERIMENTAL DESIGN: We performed whole-exome and transcriptome analysis of 59 patients with first- and second-generation EGFR TKI-resistant metastatic EGFR-mutated NSCLC to characterize and compare molecular alterations mediating resistance in T790M-positive (T790M+) and -negative (T790M-) disease. RESULTS: Transcriptomic analysis revealed ubiquitous loss of adenocarcinoma lineage gene expression in T790M- tumors, orthogonally validated using multiplex IHC. There was enrichment of genomic features such as TP53 alterations, 3q chromosomal amplifications, whole-genome doubling and nonaging mutational signatures in T790M- tumors. Almost half of resistant tumors were further classified as immunehot, with clinical outcomes conditional on immune cell-infiltration state and T790M status. Finally, using a Bayesian statistical approach, we explored how T790M- and T790M+ disease might be predicted using comprehensive genomic and transcriptomic profiles of treatment-naïve patients. CONCLUSIONS: Our results illustrate the interplay between genetic alterations, cell lineage plasticity, and immune microenvironment in shaping divergent TKI resistance and outcome trajectories in EGFR-mutated NSCLC. Genomic and transcriptomic profiling may facilitate the design of bespoke therapeutic approaches tailored to a tumor's adaptive potential.

Impact of poverty reduction on access to water and sanitation in low- and lower-middle-income countries: country-specific Bayesian projections to 2030.

BACKGROUND: In 2017, 785 million people globally lacked access to basic services of drinking water and 2 billion people lived without basic sanitation services. Most of these people live in low- and lower-middle-income countries in South Asia, Southeast Asia and sub-Saharan Africa. To monitor the progress towards universal access to water and sanitation, this study aimed to predict the coverage of access to basic drinking water supply and sanitation (WSS) services as well as the reduction in the practice of open defecation by 2030, under two assumptions: following the current trends and accelerated poverty reduction. METHODS AND FINDINGS: Households reporting access to basic WSS services and those practising open defecation were extracted from 210 nationally representative Demographic Health Surveys and Multiple Cluster Indicator Surveys (1994-2016) from 51 countries. A Bayesian hierarchical mixed effect linear regression model was developed to predict the indicators in 2030 at national, urban-rural and wealth-specific levels. A Bayesian regression model with accelerated reduction in poverty by 2030 was applied to assess the impact of poverty reduction on these indicators. Out of 51 countries, only nine (Bangladesh, Bhutan, Ghana, India, Nepal, Pakistan, The Philippines, Togo and Vietnam) were predicted to reach over 90% coverage in access to basic services of drinking water by 2030. However, none of the countries were projected to achieve equivalent coverage for access to basic sanitation services. By 2030, 21 countries were projected to achieve the target of less than 10% households practising open defecation. Urban-rural and wealth-derived disparities in access to basic WSS services, especially sanitation, were more pronounced in sub-Saharan Africa than South Asia and Southeast Asia. Access to basic sanitation services was projected to benefit more from poverty reduction than access to basic drinking water services. Households residing in rural settings were predicted to receive greater benefit from poverty reduction than urban populations in access to both basic WSS services. CONCLUSION: Achieving poverty eradication targets may have a substantial positive impact on access to basic water supply and sanitation services. However, many low- and lower-middle-income countries will struggle to achieve the goal of universal access to basic services, especially in the sanitation sector.

The Genomic and Immune Landscapes of Lethal Metastatic Breast Cancer.

The detailed molecular characterization of lethal cancers is a prerequisite to understanding resistance to therapy and escape from cancer immunoediting. We performed extensive multi-platform profiling of multi-regional metastases in autopsies from 10 patients with therapy-resistant breast cancer. The integrated genomic and immune landscapes show that metastases propagate and evolve as communities of clones, reveal their predicted neo-antigen landscapes, and show that they can accumulate HLA loss of heterozygosity (LOH). The data further identify variable tumor microenvironments and reveal, through analyses of T cell receptor repertoires, that adaptive immune responses appear to co-evolve with the metastatic genomes. These findings reveal in fine detail the landscapes of lethal metastatic breast cancer.

Glycobiology of syndecan-1 in bacterial infections.

Syndecan-1 (Sdc1) is a major cell surface heparan sulfate (HS) proteoglycan of epithelial cells, a cell type targeted by many bacterial pathogens early in their pathogenesis. Loss of Sdc1 in mice is a gain-of-function mutation that significantly decreases the susceptibility to several bacterial infections, suggesting that subversion of Sdc1 is an important virulence strategy. HS glycosaminoglycan (GAG) chains of cell surface Sdc1 promote bacterial pathogenesis by facilitating the attachment of bacteria to host cells. Engagement of cell surface Sdc1 HS chains by bacterial adhesins transmits signal through the highly conserved Sdc1 cytoplasmic domain, which can lead to uptake of intracellular bacterial pathogens. On the other hand, several bacteria that do not require Sdc1 for their attachment and invasion stimulate Sdc1 shedding and exploit the capacity of Sdc1 ectodomain HS GAGs to disarm innate defense mechanisms to evade immune clearance. Recent data suggest that select HS sulfate motifs, and not the overall charge of HS, are important in the inhibition of innate immune mechanisms. Here, we discuss several examples of Sdc1 subversion in bacterial infections.

Female reproductive factors, exogenous hormone use, and pancreatic cancer risk: the Japan Public Health Center-based prospective study.

An association between female reproductive factors, exogenous hormone use, and pancreatic cancer risk has long been suggested in laboratory settings, but epidemiological findings remain mixed and inconclusive. Studies carried out on Asian populations are also limited. In this study, 45 617 women aged 40-69 years were followed for an average of 18.4 years in the Japan Public Health Center-based prospective cohort and 211 pancreatic cases were identified as of 31 December 2011. We applied multivariate-adjusted Cox proportional hazards regression models using age as a time-scale to assess the association between female reproductive factors (menstrual status, menarche age, menopause age, number of births, age at first birth, total years of fertility, history of breastfeeding) and exogenous hormone use with the incidence of pancreatic cancer through hazard ratios and confidence intervals. No significant associations were found between our examined female reproductive factors and pancreatic cancer incidence. The use of exogenous hormones was found to be associated with an increased risk of pancreatic cancer in a multivariate-adjusted model (hazard ratio: 1.47; 95%; confidence interval: 1.00-2.14) in the Japanese female population. Our results suggest that exogenous hormones may play a role in the formation of pancreatic cancer, and further prospective studies are warranted for clarification.

Molecular functions of syndecan-1 in disease.

Syndecan-1 is a cell surface heparan sulfate proteoglycan that binds to many mediators of disease pathogenesis. Through these molecular interactions, syndecan-1 can modulate leukocyte recruitment, cancer cell proliferation and invasion, angiogenesis, microbial attachment and entry, host defense mechanisms, and matrix remodeling. The significance of syndecan-1 interactions in disease is underscored by the striking pathological phenotypes seen in the syndecan-1 null mice when they are challenged with disease-instigating agents or conditions. This review discusses the key molecular functions of syndecan-1 in modulating the onset, progression, and resolution of inflammatory diseases, cancer, and infection.

Mutations in the epidermal growth factor receptor (EGFR) gene in triple negative breast cancer: possible implications for targeted therapy.

INTRODUCTION: Triple negative breast cancer is associated with poorer prognosis and unresponsiveness to endocrine and anti-HER2 directed agents. Despite emerging data supporting the use of polyADP-ribose polymerase (PARP) inhibitors, complete and durable responses are rare and exploration of additional targeted therapies is needed. Epidermal growth factor receptor (EGFR) is expressed in triple negative breast cancer and several clinical trials are testing the role of anti-EGFR directed therapy. However, the rate of EGFR mutations is poorly defined. We, therefore, sought to characterize EGFR mutations in triple negative breast cancers. METHODS: Seventy samples were randomly chosen from a cohort of 653 triple negative breast tumours for EGFR mutation analysis. These samples were immunostained for EGFR protein expression and consisted of negatively stained and positively stained cases. DNA was extracted from paraffin blocks and polymerase chain reaction was performed to amplify exon regions 18 to 21 of the EGFR gene. Direct sequencing of the purified PCR products was performed. RESULTS: EGFR mutations were found in 8 of 70 samples (11.4%). Mutations were predominantly exon 19 deletions (4 of 70 samples, 5.7%), which clustered in the region spanning codons 746 to 759 within the kinase domain of EGFR. Two types of exon 19 deletions were seen: a 15 nucleotide deletion (del E746-A750) (2 of 70 samples) and a 24 nucleotide deletion (del S752 - I759) (2 of 70 samples). Other exon 19 mutations observed were the inversion of the complementary strand (1 of 70 samples). Exon 21 mutations included missense substitution, L858R (1 of 70 samples) and T847I (2 of 70 samples). Mutations observed were independent of EGFR protein expression determined by immunohistochemical staining. CONCLUSIONS: This study is among the first to document the presence and estimate the prevalence of EGFR mutations in triple negative breast cancer. These findings have potential implications for the design of clinical trials involving anti-EGFR directed therapy which currently do not select for patients based on presence of activating EGFR mutations, which may hence be underpowered to detect significant benefit in unselected populations. More complete sampling of EGFR mutation status in triple negative breast cancer is needed to determine the true mutation rate.

Therapeutic targets in triple negative breast cancer - where are we now?

Triple negative breast cancers, defined by lack of expression of estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (Her2/EGFR2/ERBB2), are increasingly gaining attention for its intricate relationship with basal-like and BRCA1-linked breast carcinomas, and its lack of effective tailored therapies. Triple negative breast tumors usually account for 10-20% of all breast cancers in Asian populations, similarly in Caucasian populations (10-23%), but occur at much higher frequencies in individuals of African descent (20-47%). In addition, triple negative breast cancers are usually of high histological grade and are accompanied by aggressive clinical behavior with shorter time to recurrences and death, and preference for metastasis to the brain and lungs. In this report, we would like to review recent patents and the relationship between triple negative and basal-like breast cancers, as well as BRCA1-linked breast carcinomas. Specifically, potential treatment modalities and current innovations that are designed for the predictive and prognostic values of triple negative breast tumors will be discussed.

Glomerular basement membrane thickness in an Asian population using a novel image analysis software.

AIMS: We aimed to develop an image analysis software that enabled measurement of glomerular basement membrane (GBM) thickness. METHODS: With this software, we evaluated the range of GBM widths found in a cohort of Asian patients diagnosed with a spectrum of renal diseases including minimal change/IgM nephropathy, focal and segmental glomerulosclerosis, IgA nephropathy, systemic lupus erythematosus nephritis, diabetic nephropathy, pauci-immune crescentic glomerulonephritis, thin basement membrane disease, and tubulointerstitial nephritis. Measurements were taken from a minimum of five glomerular capillary loops of each glomerulus. For each loop, at least 10 different points of the GBM were measured. RESULTS: The average GBM width measured for minimal change disease was 347.4 +/- 9.0 nm, with the highest value being 403.9 nm and lowest being 214.7 nm. No association was found between GBM width and gender. We found a significant increase in GBM width in pathological states like lupus nephropathy (p < 0.0001), diabetic nephritis (p < 0.001) and tubulointerstitial nephritis (p < 0.01) as compared with minimal change disease. Only one case of thin membrane nephropathy (198.7 nm) was available for analysis and we found a significant thinning of the GBM. CONCLUSIONS: These observations provide insights into the range of GBM thickness in several disease states and support the use of this novel software in the daily diagnostic laboratory setting.

Increased expression of non-sulfated chondroitin correlates with adverse clinicopathological parameters in prostate cancer.

Chondroitin sulfate is a structurally diverse glycosaminoglycan, which contains a variable degree of sulfation that helps to determine its biological function. It is involved in the regulation of cellular activity and has been implicated in carcinogenesis. To determine if the non-sulfated chondroitin backbone has a functional role in prostate cancer, we analyzed its expression by immunohistochemistry using the 1B5 monoclonal antibody and a set of tissue microarrays constructed with 227 prostate specimen cores from 81 cases of benign prostate tissue and 77 cases of prostate cancer, of which 69 of these cases are matched. Non-sulfated chondroitin was found in the secretory epithelial cells and stromal regions of both prostatic adenocarcinoma and benign prostatic tissues, as well as in the basal cells of benign glands. A higher percentage of cancerous cells were stained positively for non-sulfated chondroitin as compared with benign secretory cells of the same patient. Cancerous cells stained more intensely for non-sulfated chondroitin. This increase in percentage of cells stained and increase in staining intensity were associated with higher pathological T stage and extraprostatic extension. Non-sulfated chondroitin expression (either staining intensity or percentage of cells stained) in adenocarcinoma and its peritumoral stroma correlated significantly with several clinicopathological parameters of unfavorable outcome, including higher pathological T stage and Gleason score, presence of tumor in both prostatic lobes, extraprostatic extension, seminal vesicle involvement and preoperative prostate-specific antigen levels. These data suggest that non-sulfated chondroitin is a potentially useful biomarker for prostate cancer, and may be involved in regulating prostate cancer behavior.