Feeder-free differentiation of human iPSCs into natural killer cells with cytotoxic potential against malignant brain rhabdoid tumor cells.

Natural killer (NK) cells are cytotoxic immune cells that can eliminate target cells without prior stimulation. Human induced pluripotent stem cells (iPSCs) provide a robust source of NK cells for safe and effective cell-based immunotherapy against aggressive cancers. In this in vitro study, a feeder-free iPSC differentiation was performed to obtain iPSC-NK cells, and distinct maturational stages of iPSC-NK were characterized. Mature cells of CD56bright CD16bright phenotype showed upregulation of CD56, CD16, and NK cell activation markers NKG2D and NKp46 upon IL-15 exposure, while exposure to aggressive atypical teratoid/rhabdoid tumor (ATRT) cell lines enhanced NKG2D and NKp46 expression. Malignant cell exposure also increased CD107a degranulation markers and stimulated IFN-γ secretion in activated NK cells. CD56bright CD16bright iPSC-NK cells showed a ratio-dependent killing of ATRT cells, and the percentage lysis of CHLA-05-ATRT was higher than that of CHLA-02-ATRT. The iPSC-NK cells were also cytotoxic against other brain, kidney, and lung cancer cell lines. Further NK maturation yielded CD56-ve CD16bright cells, which lacked activation markers even after exposure to interleukins or ATRT cells - indicating diminished cytotoxicity. Generation and characterization of different NK phenotypes from iPSCs, coupled with their promising anti-tumor activity against ATRT in vitro, offer valuable insights into potential immunotherapeutic strategies for brain tumors.

Interconversion of Cancer Cells and Induced Pluripotent Stem Cells.

Cancer cells, especially cancer stem cells (CSCs), share many molecular features with induced pluripotent stem cells (iPSCs) that enable the derivation of induced pluripotent cancer cells by reprogramming malignant cells. Conversely, normal iPSCs can be converted into cancer stem-like cells with the help of tumor microenvironment components and genetic manipulation. These CSC models can be utilized in oncogenic initiation and progression studies, understanding drug resistance, and developing novel therapeutic strategies. This review summarizes the role of pluripotency factors in the stemness, tumorigenicity, and therapeutic resistance of cancer cells. Different methods to obtain iPSC-derived CSC models are described with an emphasis on exposure-based approaches. Culture in cancer cell-conditioned media or cocultures with cancer cells can convert normal iPSCs into cancer stem-like cells, aiding the examination of processes of oncogenesis. We further explored the potential of reprogramming cancer cells into cancer-iPSCs for mechanistic studies and cancer dependencies. The contributions of genetic, epigenetic, and tumor microenvironment factors can be evaluated using these models. Overall, integrating iPSC technology into cancer stem cell research holds significant promise for advancing our knowledge of cancer biology and accelerating the development of innovative and tailored therapeutic interventions.

TCGA RNA-Seq and Tumor-Infiltrating Lymphocyte Imaging Data Reveal Cold Tumor Signatures of Invasive Ductal Carcinomas and Estrogen Receptor-Positive Human Breast Tumors.

Comparative studies of immune-active hot and immune-deserted cold tumors are critical for identifying therapeutic targets and strategies to improve immunotherapy outcomes in cancer patients. Tumors with high tumor-infiltrating lymphocytes (TILs) are likely to respond to immunotherapy. We used the human breast cancer RNA-seq data from the cancer genome atlas (TCGA) and classified them into hot and cold tumors based on their lymphocyte infiltration scores. We compared the immune profiles of hot and cold tumors, their corresponding normal tissue adjacent to the tumor (NAT), and normal breast tissues from healthy individuals from the Genotype-Tissue Expression (GTEx) database. Cold tumors showed a significantly lower effector T cells, lower levels of antigen presentation, higher pro-tumorigenic M2 macrophages, and higher expression of extracellular matrix (ECM) stiffness-associated genes. Hot/cold dichotomy was further tested using TIL maps and H&E whole-slide pathology images from the cancer imaging archive (TCIA). Analysis of both datasets revealed that infiltrating ductal carcinoma and estrogen receptor ER-positive tumors were significantly associated with cold features. However, only TIL map analysis indicated lobular carcinomas as cold tumors and triple-negative breast cancers (TNBC) as hot tumors. Thus, RNA-seq data may be clinically relevant to tumor immune signatures when the results are supported by pathological evidence.

Modeling Human Brain Tumors and the Microenvironment Using Induced Pluripotent Stem Cells.

Brain cancer is a group of diverse and rapidly growing malignancies that originate in the central nervous system (CNS) and have a poor prognosis. The complexity of brain structure and function makes brain cancer modeling extremely difficult, limiting pathological studies and therapeutic developments. Advancements in human pluripotent stem cell technology have opened a window of opportunity for brain cancer modeling, providing a wealth of customizable methods to simulate the disease in vitro. This is achieved with the advent of genome editing and genetic engineering technologies that can simulate germline and somatic mutations found in human brain tumors. This review investigates induced pluripotent stem cell (iPSC)-based approaches to model human brain cancer. The applications of iPSCs as renewable sources of individual brain cell types, brain organoids, blood-brain barrier (BBB), and brain tumor models are discussed. The brain tumor models reviewed are glioblastoma and medulloblastoma. The iPSC-derived isogenic cells and three-dimensional (3D) brain cancer organoids combined with patient-derived xenografts will enhance future compound screening and drug development for these deadly human brain cancers.

Quantum Dot-Peptide Conjugates as Energy Transfer Probes for Sensing the Proteolytic Activity of Matrix Metalloproteinase-14.

We detail the assembly and characterization of quantum dot (QD)-dye conjugates constructed using a peptide bridge specifically designed to recognize and interact with a breast cancer biomarker─matrix metalloproteinase-14 (MMP-14). The assembled QD conjugates are then used as optically addressable probes, relying on Förster resonance energy transfer (FRET) interactions as a transduction mechanism to detect the activity of MMP-14 in solution phase. The QDs were first coated with dithiolane poly(ethylene glycol) (PEG) bearing a carboxyl group that allows coupling via amide bond formation with different dye-labeled peptides. The analytical capability of the conjugates is enabled by correlating changes in the FRET efficiency with the conjugate valence and/or QD-to-dye separation distance, triggered and modulated by enzymatic proteolysis of surface-tethered peptides. The FRET probe exhibits great sensitivity to enzyme digestion with sub-nanomolar limit of detection. We further analyze the proteolysis data within the framework of the Michaelis-Menten model, which considers the fact that surface-attached peptides have a slower diffusion coefficient than free peptides. This results in reduced collision frequency and lower catalytic efficiency, kcat/KM. Our results suggest that our conjugate design is promising, effective, and potentially useful for in vivo analysis.

Macrophage-Conditioned Media Promotes Adipocyte Cancer Association, Which in Turn Stimulates Breast Cancer Proliferation and Migration.

BACKGROUND: Breast cancer is the most common cancer in women and the leading cause of female cancer deaths worldwide. Obesity causes chronic inflammation and is a risk factor for post-menopausal breast cancer and poor prognosis. Obesity triggers increased infiltration of macrophages into adipose tissue, yet little research has focused on the effects of macrophages in early stages of breast tumor development in obese patients. In this study, the effects of pro-inflammatory macrophages on breast cancer-adipocyte crosstalk were investigated. METHODS: An innovative human cell co-culture system was built and used to model the paracrine interactions among adipocytes, macrophages, and breast cancer cells and how they facilitate tumor progression. The effects on cancer cells were examined using cell counts and migration assays. Quantitative reverse-transcription polymerase chain reaction was used to measure the expression levels of several cytokines and proteases to analyze adipocyte cancer association. RESULTS: Macrophage-conditioned media intensified the effects of breast cancer-adipocyte crosstalk. Adipocytes became delipidated and increased production of pro-inflammatory cytokines, even in the absence of cancer cells, although the expression levels were highest with all three cell components. As a result, co-cultured breast cancer cells became more aggressive, with increased proliferation and migration compared to adipocyte-breast cancer co-cultures treated with unconditioned media. CONCLUSIONS: A novel co-culture model was built to evaluate the crosstalk among human macrophages, adipocytes, and breast cancer cells. We found that macrophages may contribute to adipocyte inflammation and cancer association and thus promote breast cancer progression.

Effects of Polystyrene Microplastics on Human Kidney and Liver Cell Morphology, Cellular Proliferation, and Metabolism.

Microplastics have gained much attention due to their prevalence and abundance in our everyday lives. They have been detected in household items such as sugar, salt, honey, seafood, tap water, water bottles, and food items wrapped in plastic. Once ingested, these tiny particles can travel to internal organs such as the kidney and liver and cause adverse effects on the cellular level. Here, human embryonic kidney (HEK 293) cells and human hepatocellular (Hep G2) liver cells were used to examine the potential toxicological effects of 1 µm polystyrene microplastics (PS-MPs). Exposing cells to PS-MPs caused a major reduction in cellular proliferation but no significant decrease in cell viability as determined by the trypan blue assay in both cell lines. Cell viability remained at least 94% for both cell lines even at the highest concentration of 100 µg/mL of PS-MPs. Phase-contrast imaging of both kidney and liver cells exposed to PS-MPs at 72 h showed significant morphological changes and uptake of PS-MP particles. Confocal fluorescent microscopy confirmed the uptake of 1 µm PS-MPs at 72 h for both cell lines. Additionally, flow cytometry experiments verified that more than 70% of cells internalized 1 µm PS-MPs after 48 h of exposure for both kidney and liver cells. Reactive oxygen species (ROS) studies revealed kidney and liver cells exposed to PS-MPs had increased levels of ROS at each concentration and for every time point tested. Furthermore, quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis at 24 and 72 h revealed that both HEK 293 and Hep G2 cells exposed to PS-MPs lowered the gene expression levels of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and antioxidant enzymes superoxide dismutase 2 (SOD2) and catalase (CAT), thus reducing the potential of SOD2 and CAT to detoxify ROS. These adverse effects of PS-MPs on human kidney and liver cells suggest that ingesting microplastics may lead to toxicological problems on cell metabolism and cell-cell interactions. Because exposing human kidney and liver cells to microplastics results in morphological, metabolic, proliferative changes and cellular stress, these results indicate the potential undesirable effects of microplastics on human health.

Phenotypic, metabolic, and biogenesis properties of human stem cell-derived cerebellar spheroids.

Human cerebellum consists of high density and complexity of neurons. Thus, it is challenging to differentiate cerebellar-like organoids with similar cellular markers and function to the human brain. Our previous study showed that the combination of retinoic acid (RA), Wingless/integrated (Wnt) activator, and Sonic Hedgehog (SHH) activator promotes cerebellar differentiation from human induced pluripotent stem cells (hiPSCs). This study examined phenotypic, metabolic, and biogenesis in early cerebellar development. Cerebellum spheroids were differentiated from human iPSK3 cells. During day 7-14, RA and Wnt activator CHIR99021 were used and SHH activator purmorphamine (PMR) was added later to promote ventralization. Gene expression for early cerebellar layer markers, metabolism, and extracellular vesicle (EV) biogenesis were characterized. Zinc-induced neurotoxicity was investigated as a proof-of-concept of neurotoxicity study. Flow cytometry results showed that there was no significant difference in NEPH3, PTF1A, OLIG2, and MATH1 protein expression between RCP (RA-CHIR-PMR) versus the control condition. However, the expression of cerebellar genes for the molecular layer (BHLE22), the granule cell layer (GABRB2, PAX6, TMEM266, KCNIP4), the Bergmann glial cells (QK1, DAO), and the Purkinje cell layer (ARHGEF33, KIT, MX1, MYH10, PPP1R17, SCGN) was significantly higher in the RCP condition than the control. The shift in metabolic pathways toward glycolysis was observed for RCP condition. The EV biogenesis marker expression was retained. Mild zinc-induced neurotoxicity may exist when zinc exposure exceeds 1.0 µM. RCP treatment can promote specific cerebellar-like differentiation from hiPSCs indicated by gene expression of early cerebellar markers and regionally enriched genes. The higher cerebellar marker expression is accompanied by the elevated glycolysis with the retained EV biogenesis. This study should advance the understanding of biomarkers during early cerebellar development for cerebellum organoid engineering and neurotoxicity study.

Microplastics exposure affects neural development of human pluripotent stem cell-derived cortical spheroids.

Plastics have been part of our ecosystem for about a century and their degradation by different environmental factors produce secondary microplastics (MPs). To date, the impact of MPs on human health has not been well investigated. To understand the possible effects of polystyrene-MPs (PS-MPs) on the human brain, a 3D model of human forebrain cortical spheroids has been derived, which mimics early development of human cerebral cortex. The spheroids were exposed to 100, 50, and 5 µg/mL of 1 µm and 10 µm PS-MPs during day 4-10 and day 4-30. The short-term MP exposure showed the promoted proliferation and high gene expression of Nestin, PAX6, ATF4, HOXB4 and SOD2. For long-term exposure, reduced cell viability was observed. Moreover, changes in size and concentration of PS-MPs altered the gene expression of DNA damage and neural tissue patterning. In particular, ß-tubulin III, Nestin, and TBR1/TBR2 gene expression decreased in PS-MP treated conditions compare to the untreated control. The results of this study suggest that the size- and concentration-dependent exposure to PS-MPs can adversely affect embryonic brain-like tissue development in forebrain cerebral spheroids. This study has significance in assessing environmental factors in neurotoxicity and degeneration in human.

Long-Term Effects of Nanoscale Magnetite on Human Forebrain-like Tissue Development in Stem-Cell-Derived Cortical Spheroids.

The environmental nanoscale iron magnetite may contribute to the risk of developing neurodegenerative diseases. In addition, iron oxides can be used as the contrast agents in magnetic resonance imaging of neural tissues. The potential long-term impact of nanoscale iron oxides on cellular stress and neuro-inflammation remains unknown. The objective of this study is to evaluate the long-term effects of nanoscale iron oxide exposure on human pluripotent stem cell-derived cortical spheroids that mimic human forebrain-like tissue development. In particular, the cortical spheroids were treated with 8 nm and 15-20 nm magnetite at 0.023, 2.3, and 23 µg/mL for 4-30 days. The cell viability did not show significant differences among different test groups. The neuronal marker ß-tubulin III, cell proliferation marker Ki67, and antioxidant enzyme SOD2 did not show significant changes either. The molecular levels of cellular stress, inflammation, cell apoptosis, DNA damage and repair, and the reactive oxygen species (ROS) response were measured. A negative effect (i.e., increased inflammation and ROS response genes) of 8 nm iron oxide exposure and a positive effect (i.e., decreased inflammation, apoptosis, and ROS response and clean up genes) for 15-20 nm iron oxide exposure were observed. It is postulated that the intracellular iron content and the aggregation of iron oxides contribute to the observed differential response. Although our results demonstrate similar intracellular iron content for 8 nm and 15-20 nm groups, the aggregation is more severe for the 8 nm group (∼500 nm) than the 15 nm group (∼220-250 nm). Therefore, our data indicate an iron oxide aggregate size-dependent effects on cellular stress, inflammation, cell apoptosis, DNA damage, and the ROS response in the developing human forebrain-like tissue.

Advancements in Human Breast Cancer Targeted Therapy and Immunotherapy.

Human breast cancer treatment regimens have evolved greatly due to the significant advances in understanding the molecular mechanisms and pathways of the common subtypes of breast cancer. In this review, we discuss recent progress in breast cancer targeted therapy and immunotherapy as well as ongoing clinical trials. We also highlight the potential of combination therapies and personalized approaches to improve clinical outcomes. Targeted therapies have surpassed the hormone receptors and the human epidermal growth factor receptor 2 (HER2) to include many other molecules in targetable pathways such as the epidermal growth factor receptor (EGFR), poly (adenosine diphosphate-ribose) polymerase (PARP), and cyclin-dependent kinase 4/6 (CDK4/6). However, resistance to targeted therapy persists, underpinning the need for more efficacious therapies. Immunotherapy is considered a milestone in breast cancer treatments, including the engineered immune cells (CAR-T cell therapy) to better target the tumor cells, vaccines to stimulate the patient's immune system against tumor antigens, and checkpoint inhibitors (PD-1, PD-L1, and CTLA4) to block molecules that mediate immune inhibition. Targeted therapies and immunotherapy tested in breast cancer clinical trials are discussed here, with special emphasis on combinatorial approaches which are believed to maximize treatment efficacy and enhance patient survival.

Cytochrome P450-2D6: A novel biomarker in liver cancer health disparity.

Liver cancer morbidity and mortality rates differ among ethnic groups. In the United States, the burden of liver cancer in Asian Americans (AS) is higher compared to Caucasian Americans (CA). Research on liver cancer health disparities has mainly focused on environmental and socioeconomic factors yet has ignored the genotypic differences among various racial/ethnic groups. This lack of molecular level understanding has hindered the development of personalized medical approaches for liver cancer treatment. To understand the genetic heterogeneity of liver cancer between AS and CA, we performed a systematic analysis of RNA-seq data of AS and CA patients from The Cancer Genome Atlas (TCGA). We used four differential gene expression analysis packages; DESeq2, limma, edgeR, and Superdelta2, to identify the differentially expressed genes. Our analysis identified cytochrome P450-2D6 enzyme (CYP2D6) as the gene with the greatest differential expression with higher levels in AS compared to CA. To scrutinize the underlying mechanism of CYP2D6, Ingenuity Pathway Analysis (IPA) and Cytoscape were conducted and found hepatocyte nuclear factor-4α (HNF4A) and interleukin-6 (IL6) in direct association with CYP2D6. IL6 is downregulated in AS compared to CA, while HNF4A is not significantly different. Herein, we report that CYP2D6 may serve as a putative biomarker in liver cancer health disparities. Its negative association with IL6 proclaims an intricate relationship between CYP2D6 and inflammation in the ethnic differences seen in AS and CA liver cancer patients. The goal of the present study was to understand how genetic factors may contribute to the interethnic variability of liver cancer prevalence and outcomes in AS and CA patients. Identifying ethnic-specific genes may help ameliorate detection, diagnosis, surveillance, and treatments of liver cancer, as well as reduce disease-related incidence and mortality rates in the vulnerable population.

Studies of hormonal regulation, phenotype plasticity, bone metastasis, and experimental therapeutics in androgen-repressed human prostate cancer (ARCaP) model.

First established by Dr. Leland W. K. Chung's lab, the androgen-repressed prostate cancer cell (ARCaP) line is derived from the ascitic fluid of a prostate cancer (PCa) patient with widely metastatic disease. Based on its unique characteristic of growth suppression in the presence of androgen, ARCaP cell line has contributed to the research of PCa disease progression toward therapy- and castration-resistant PCa (t-CRPC). It has been widely applied in studies exploring experimental therapeutic reagents including Genistein, Vorinostat and Silibinin. ARCaP cells have showed increased metastatic potential to the bone and soft tissues. In addition, accumulating studies using ARCaP model have demonstrated the epithelial-to-mesenchymal transitional plasticity of PCa using epithelial-like ARCaPE line treated in vitro with growth factors derived from bone microenvironment. The resulting mesenchymal-like ARCaPM sub-clone derived from bone-metastasized tumor has high expression of several factors correlated with cancer metastasis, such as N-Cadherin, Vimentin, MCM3, Granzyme B, ß2-microglobulin and RANKL. In particular, the increased secretion of RANKL in ARCaPM further facilitates its capacity of inducing osteoclastogenesis at the bone microenvironment, leading to bone resorption and tumor colonization. Meanwhile, sphingosine kinase 1 (SphK1) acts as a key molecule driver in the neuroendocrine differentiation (NED) of ARCaP sublines, suggesting the unique facet of ARCaP cells for insightful studies in more malignant neuroendocrine prostate cancer (NEPC). Overall, the establishment of ARCaP line has provided a valuable model to explore the mechanisms underlying PCa progression toward metastatic t-CRPC. In this review, we will focus on the contribution of ARCaP model in PCa research covering hormone receptor activity, skeletal metastasis, plasticity of epithelial-to-mesenchymal transition (EMT) and application of therapeutic strategies.

Exposure of Human Lung Cells to Polystyrene Microplastics Significantly Retards Cell Proliferation and Triggers Morphological Changes.

Microplastics in the environment produced by decomposition of globally increasing waste plastics have become a dominant component of both water and air pollution. To examine the potential toxicological effects of microplastics on human cells, the cultured human alveolar A549 cells were exposed to polystyrene microplastics (PS-MPs) of 1 and 10 µm diameter as a model of the environmental contaminants. Both sizes caused a significant reduction in cell proliferation but exhibited little cytotoxicity, as measured by the maintenance of cell viabilities determined by trypan blue staining and by Calcein-AM staining. The cell viabilities did not drop below 93% even at concentrations of PS-MPs as high as 100 µg/mL. Despite these high viabilities, further assays revealed a population level decrease in metabolic activity parallel in time with a dramatic decrease in proliferation rate in PS-MP exposed cells. Furthermore, phase contrast imaging of live cells at 72 h revealed major changes in the morphology of cells exposed to microplastics, as well as the uptake of multiple 1 µm PS-MPs into the cells. Confocal fluorescent microscopy at 24 h of exposure confirmed the incorporation of 1 µm PS-MPs. These disturbances at the proliferative and cytoskeletal levels of human cells lead us to propose that airborne polystyrene microplastics may have toxicologic consequences. This is the first report of exposure of human cells to an environmental contaminant resulting in the dual effects of inhibition of cell proliferation and major changes in cell morphology. Our results make clear that human exposure to microplastic pollution has significant consequence and potential for harm to humans.

Biogenesis of Extracellular Vesicles Produced from Human-Stem-Cell-Derived Cortical Spheroids Exposed to Iron Oxides.

Stem-cell-derived extracellular vesicles (EVs) are promising tools for therapeutic delivery and imaging in the medical research fields. EVs that arise from endosomal compartments or plasma membrane budding consist of exosomes and microvesicles, which range between 30 and 200 nm and 100-1000 nm, respectively. Iron oxide nanoparticles can be used to label stem cells or possibly EVs for magnetic resonance imaging. This could be a novel way to visualize areas in the body that are affected by neurological disorders such as stroke. Human induced pluripotent stem cells (iPSK3 cells) were plated on low-attachment plates and treated with SB431542 and LDN193189 during the first week for the induction of cortical spheroid formation and grown with fibroblast growth factor 2 and cyclopamine during the second week for the neural progenitor cell (iNPC) differentiation. iNPCs were then grown on attachment plates and treated with iron oxide (Fe3O4) nanoparticles at different sizes (8, 15, and 30 nm in diameter) and concentrations (0.1, 10, and 100 µM). The spheroids and media collected from these cultures were used for iron oxide detection as well as EV isolation and characterizations, respectively. MTT assay demonstrated that the increased size and concentration of the iron oxide nanoparticles had little effect on the metabolic activity of iNPCs. In addition, the Live/Dead assay showed high viability in all the nanoparticle treated groups and the untreated control. The EVs isolated from these culture groups were analyzed and displayed similar or higher EV counts compared with control. The observed EV size averaged 200-250 nm, and electron microscopy revealed the expected exosome morphology for EVs from all groups. RT-PCR analysis of EV biogenesis markers (CD63, CD81, Alix, TSG101, Syntenin1, ADAM10, RAB27b, and Syndecan) showed differential expression between the iron-oxide-treated cultures and nontreated cultures, as well as between adherent and nonadherent 3D cultures. Iron oxide nanoparticles were detected inside the cortical spheroid cells but not EVs by MRI. The addition of iron oxide nanoparticles does not induce significant cytotoxic effects to cortical spheroids. In addition,, nanoparticles may stimulate the biogenesis of EVs when added to cortical spheroids in vitro.

Cerebellar Differentiation from Human Stem Cells Through Retinoid, Wnt, and Sonic Hedgehog Pathways.

Differentiating cerebellar organoids can be challenging due to complex cell organization and structure in the cerebellum. Different approaches were investigated to recapitulate differentiation process of the cerebellum from human-induced pluripotent stem cells (hiPSCs) without high efficiency. This study was carried out to test the hypothesis that the combination of different signaling factors including retinoic acid (RA), Wnt activator, and sonic hedgehog (SHH) activator promotes the cerebellar differentiation of hiPSCs. Wnt, RA, and SHH pathways were activated by CHIR99021 (CHIR), RA, and purmorphamine (PMR), respectively. Different combinations of the morphogens (RA/CHIR, RA/PMR, CHIR/PMR, and RA/CHIR/PMR) were utilized, and the spheroids (day 35) were characterized for the markers of three cerebellum layers (the molecular layer, the Purkinje cell layer, and the granule cell layer). Of all the combinations tested, RA/CHIR/PMR promoted both the Purkinje cell layer and the granule cell layer differentiation. The cells also exhibited electrophysiological characteristics using whole-cell patch clamp recording, especially demonstrating Purkinje cell electrophysiology. This study should advance the understanding of different signaling pathways during cerebellar development to engineer cerebellum organoids for drug screening and disease modeling. Impact statement This study investigated the synergistic effects of retinoic acid, Wnt activator, and sonic hedgehog activator on cerebellar patterning of human-induced pluripotent stem cell (hiPSC) spheroids and organoids. The results indicate that the combination promotes the differentiation of the Purkinje cell layer and the granule cell layer. The cells also exhibit electrophysiological characteristics using whole-cell patch clamp recording, especially demonstrating Purkinje cell electrophysiology. The findings are significant for understanding the biochemical signaling of three-dimensional microenvironment on neural patterning of hiPSCs for applications in organoid engineering, disease modeling, and drug screening.

Identifying county-level factors for female breast cancer incidence rate through a large-scale population study.

Female breast cancer (FBC) incidence rate (IR) varies greatly across counties in the United States (U.S.). Factors contributing to these geographic disparities have not been fully understood at the population level. In this study, we investigated the relationships between the county-level FBC IR and a diverse set of variables in demographics, socioeconomics, life style, health care accessibility, and environment. Our study included 1,277 counties in the U.S. where the female population was 10,000 or above for at least one race/ethnicity. After controlling for the racial/ethnic and other significant factors, percent of husband-wife family households (pHWFH) for a racial/ethnic group in a county is negatively associated with FBC IR (p < 0.001). A 10% increase in married family households may lower a county's IR by 5.2 cases per 100,000 females per year. We also found that PM2.5 (fine inhalable particles with a diameter of 2.5 micrometers or less) is positively associated with FBC IR (p < 0.001). Counties with the highest level of PM2.5 have approximately 4 additional FBC new cases per 100,000 females per year than counties with the lowest level of PM2.5. Furthermore, we found that the county-level factors contributing to FBC IR vary significantly for different racial groups using race-specific models. While confirming most of the previously known patient- and neighborhood-level risk factors (such as race/ethnicity, income, and health care accessibility), our study identified two significant county-level factors contributing to the spatial disparity of FBC IR across the U.S. The newly-identified beneficial factor (marriage) and risk factor (PM2.5), together with the verified known factors, may help provide insights to officials of health departments/organizations for them to make decisions on cancer intervention strategies.

Secretome analysis reveals upregulated granzyme B in human androgen-repressed prostate cancer cells with mesenchymal and invasive phenotype.

Epithelial-mesenchymal transition (EMT) is a critical early step in cancer metastasis and a complex process that involves multiple factors. In this study, we used proteomics approaches to investigate the secreted proteins (secretome) of paired human androgen-repressed prostate cancer (ARCaP) cell lines, representing the epithelial (ARCaP-E) and mesenchymal (ARCaP-M) phenotypes. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses showed high levels of proteins involved in bone remodeling and extracellular matrix degradation in the ARCaP-M cells, consistent with the bone metastasis phenotype. Furthermore, LC-MS/MS showed a significantly higher level of the serine protease granzyme B (GZMB) in ARCaP-M conditioned media (CM) compared to that of ARCaP-E. Using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to detect mRNA and Western blot to detect protein expression, we further demonstrated that the GZMB gene was expressed by ARCaP-M and the protein was secreted extracellularly. ARCaP-M cells with GZMB gene knockdown using small interfering RNA (siRNA) have markedly reduced invasiveness as demonstrated by the Matrigel invasion assay in comparison with the scrambled siRNA negative control. This study reports that GZMB secretion by mesenchymal-like androgen-repressed human prostate cancer cells promotes invasion, suggesting a possible extracellular role for GZMB in addition to its classic role in immune cell-mediated cytotoxicity.

Immune landscape of human prostate cancer: immune evasion mechanisms and biomarkers for personalized immunotherapy.

BACKGROUND: Despite recent advances in cancer immunotherapy, the efficacy of these therapies for the treatment of human prostate cancer patients is low due to the complex immune evasion mechanisms (IEMs) of prostate cancer and the lack of predictive biomarkers for patient responses. METHODS: To understand the IEMs in prostate cancer and apply such understanding to the design of personalized immunotherapies, we analyzed the RNA-seq data for prostate adenocarcinoma from The Cancer Genome Atlas (TCGA) using a combination of biclustering, differential expression analysis, immune cell typing, and machine learning methods. RESULTS: The integrative analysis identified eight clusters with different IEM combinations and predictive biomarkers for each immune evasion cluster. Prostate tumors employ different combinations of IEMs. The majority of prostate cancer patients were identified with immunological ignorance (89.8%), upregulated cytotoxic T lymphocyte-associated protein 4 (CTLA4) (58.8%), and upregulated decoy receptor 3 (DcR3) (51.6%). Among patients with immunologic ignorance, 41.4% displayed upregulated DcR3 expression, 43.26% had upregulated CTLA4, and 11.4% had a combination of all three mechanisms. Since upregulated programmed cell death 1 (PD-1) and/or CTLA4 often co-occur with other IEMs, these results provide a plausible explanation for the failure of immune checkpoint inhibitor monotherapy for prostate cancer. CONCLUSION: These findings indicate that human prostate cancer specimens are mostly immunologically cold tumors that do not respond well to mono-immunotherapy. With such identified biomarkers, more precise treatment strategies can be developed to improve therapeutic efficacy through a greater understanding of a patient's immune evasion mechanisms.

Human Stem Cell-derived Aggregates of Forebrain Astroglia Respond to Amyloid Beta Oligomers.

Astrocytes are vital components in neuronal circuitry and there is increasing evidence linking the dysfunction of these cells to a number of central nervous system diseases. Studying the role of these cells in human brain function in the past has been difficult due to limited access to the human brain. In this study, human induced pluripotent stem cells were differentiated into astrospheres using a hybrid plating method, with or without dual SMAD inhibition. The derived cells were assessed for astrocytic markers, brain regional identity, phagocytosis, calcium-transient signaling, reactive oxygen species production, and immune response. Neural degeneration was modeled by stimulation with amyloid-ß (Aß) 42 oligomers. Finally, co-culture was performed for the derived astrospheres with isogenic neurospheres. Results indicate that the derived astroglial cells express astrocyte markers with forebrain dorsal cortical identity, secrete extracellular matrix, and are capable of phagocytosing iron oxide particles and responding to Aß42 stimulation (higher oxidative stress, higher TNF-α, and IL-6 expression). RNA-sequencing results reveal the distinct transcriptome of the derived cells responding to Aß42 stimulation for astrocyte markers, chemokines, and brain regional identity. Co-culture experiments show the synaptic activities of neurons and the enhanced neural protection ability of the astroglial cells. This study provides knowledge about the roles of brain astroglial cells, heterotypic cell-cell interactions, and the formation of engineered neuronal synapses in vitro. The implications lie in neurological disease modeling, drug screening, and studying progression of neural degeneration and the role of stem cell microenvironment. Impact Statement Human pluripotent stem cell-derived astrocytes are a powerful tool for disease modeling and drug screening. However, the properties regarding brain regional identity and the immune response to neural degeneration stimulus have not been well characterized. Results of this study indicate that the derived astroglial cells express astrocyte markers with forebrain dorsal cortical identity, secrete extracellular matrix (ECM), and are capable of phagocytosing iron oxide particles and responding to amyloid-ß oligomers, showing the distinct transcriptome in astrocyte markers, chemokines, and brain regional identity. This study provides knowledge about the roles of brain astroglial cells, heterotypic cell-cell interactions, and engineering neural tissues in vitro.