Emerging Trends in Gastrointestinal Cancer Targeted Therapies: Harnessing Tumor Microenvironment, Immune Factors, and Metabolomics Insights.

Gastrointestinal (GI) cancers are the leading cause of new cancer cases and cancer-related deaths worldwide. The treatment strategies for patients with GI tumors have focused on oncogenic molecular profiles associated with tumor cells. Recent evidence has demonstrated that the tumor cell functions are modulated by its microenvironment, compromising fibroblasts, extracellular matrices, microbiome, immune cells, and the enteric nervous system. Along with the tumor microenvironment components, alterations in key metabolic pathways have emerged as a hallmark of tumor cells. From these perspectives, this review will highlight the functions of different cellular components of the GI tumor microenvironment and their implications for treatment. Furthermore, we discuss the major metabolic reprogramming in GI tumor cells and how understanding metabolic rewiring could lead to new therapeutic strategies. Finally, we briefly summarize the targeted agents currently being studied in GI cancers. Understanding the complex interplay between tumor cell-intrinsic and -extrinsic factors during tumor progression is critical for developing new therapeutic strategies.

Cancer-Associated Fibroblast Induces Acinar-to-Ductal Cell Transdifferentiation and Pancreatic Cancer Initiation Via LAMA5/ITGA4 Axis.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is characterized by desmoplastic stroma surrounding most tumors. Activated stromal fibroblasts, namely cancer-associated fibroblasts (CAFs), play a major role in PDAC progression. We analyzed whether CAFs influence acinar cells and impact PDAC initiation, that is, acinar-to-ductal metaplasia (ADM). ADM connection with PDAC pathophysiology is indicated, but not yet established. We hypothesized that CAF secretome might play a significant role in ADM in PDAC initiation. METHODS: Mouse and human acinar cell organoids, acinar cells cocultured with CAFs and exposed to CAF-conditioned media, acinar cell explants, and CAF cocultures were examined by means of quantitative reverse transcription polymerase chain reaction, RNA sequencing, immunoblotting, and confocal microscopy. Data from liquid chromatography with tandem mass spectrometry analysis of CAF-conditioned medium and RNA sequencing data of acinar cells post-conditioned medium exposure were integrated using bioinformatics tools to identify the molecular mechanism for CAF-induced ADM. Using confocal microscopy, immunoblotting, and quantitative reverse transcription polymerase chain reaction analysis, we validated the depletion of a key signaling axis in the cell line, acinar explant coculture, and mouse cancer-associated fibroblasts (mCAFs). RESULTS: A close association of acino-ductal markers (Ulex europaeus agglutinin 1, amylase, cytokeratin-19) and mCAFs (α-smooth muscle actin) in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1Cre (KPC) and LSL-KrasG12D/+; Pdx1Cre (KC) autochthonous progression tumor tissue was observed. Caerulein treatment-induced mCAFs increased cytokeratin-19 and decreased amylase in wild-type and KC pancreas. Likewise, acinar-mCAF cocultures revealed the induction of ductal transdifferentiation in cell line, acinar-organoid, and explant coculture formats in WT and KC mice pancreas. Proteomic and transcriptomic data integration revealed a novel laminin α5/integrinα4/stat3 axis responsible for CAF-mediated acinar-to-ductal cell transdifferentiation. CONCLUSIONS: Results collectively suggest the first evidence for CAF-influenced acino-ductal phenotypic switchover, thus highlighting the tumor microenvironment role in pancreatic carcinogenesis inception.

Metabolic Rewiring and Stemness: A Critical Attribute of Pancreatic Cancer Progression.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive diseases with a poor 5-year survival rate. PDAC cells rely on various metabolic pathways to fuel their unlimited proliferation and metastasis. Reprogramming glucose, fatty acid, amino acid, and nucleic acid metabolisms contributes to PDAC cell growth. Cancer stem cells are the primary cell types that play a critical role in the progression and aggressiveness of PDAC. Emerging studies indicate that the cancer stem cells in PDAC tumors are heterogeneous and show specific metabolic dependencies. In addition, understanding specific metabolic signatures and factors that regulate these metabolic alterations in the cancer stem cells of PDAC paves the way for developing novel therapeutic strategies targeting CSCs. In this review, we discuss the current understanding of PDAC metabolism by specifically exploring the metabolic dependencies of cancer stem cells. We also review the current knowledge of targeting these metabolic factors that regulate CSC maintenance and PDAC progression.

Chemokine-mucinome interplay in shaping the heterogeneous tumor microenvironment of pancreatic cancer.

Pancreatic cancer (PC) is exemplified by a complex immune-suppressive, fibrotic tumor microenvironment (TME), and aberrant expression of mucins. The constant crosstalk between cancer cells, cancer-associated fibroblasts (CAFs), and the immune cells mediated by the soluble factors and inflammatory mediators including cytokines, chemokines, reactive oxygen species (ROS) promote the dynamic temporal switch towards an immune-escape phenotype in the neoplastic cells and its microenvironment that bolsters disease progression. Chemokines have been studied in PC pathogenesis, albeit poorly in the context of mucins, tumor glycocalyx, and TME heterogeneity (CAFs and immune cells). With correlative analysis from PC patients' transcriptome data, support from available literature, and scientific arguments-based speculative extrapolations in terms of disease pathogenesis, we have summarized in this review a comprehensive understanding of chemokine-mucinome interplay during stromal modulation and immune-suppression in PC. Future studies should focus on deciphering the complexities of chemokine-mediated control of glycocalyx maturation, immune infiltration, and CAF-associated immune suppression. Knowledge extracted from such studies will be beneficial to mechanistically correlate the mucin-chemokine abundance in serum versus pancreatic tumors of patients, which may aid in prognostication and stratification of PC patients for immunotherapy.

MUC16 promotes triple-negative breast cancer lung metastasis by modulating RNA-binding protein ELAVL1/HUR.

BACKGROUND: Triple-negative breast cancer (TNBC) is highly aggressive with an increased metastatic incidence compared to other breast cancer subtypes. However, due to the absence of clinically reliable biomarkers and targeted therapy in TNBC, outcomes are suboptimal. Hence, there is an urgent need to understand biological mechanisms that lead to identifying novel therapeutic targets for managing metastatic TNBC. METHODS: The clinical significance of MUC16 and ELAVL1 or Hu antigen R (HuR) was examined using breast cancer TCGA data. Microarray was performed on MUC16 knockdown and scramble TNBC cells and MUC16-associated genes were identified using RNA immunoprecipitation and metastatic cDNA array. Metastatic properties of MUC16 were evaluated using tail vein experiment. MUC16 and HuR downstream pathways were confirmed by ectopic overexpression of MUC16-carboxyl-terminal (MUC16-Cter), HuR and cMyc as well as HuR inhibitors (MS-444 and CMLD-2) in TNBC cells. RESULTS: MUC16 was highly expressed in TNBC and correlated with its target HuR. Depletion of MUC16 showed decreased invasion, migration, and colony formation abilities of human and mouse TNBC cells. Mice injected with MUC16 depleted cells were less likely to develop lung metastasis (P = 0.001). Notably, MUC16 and HuR were highly expressed in the lung tropic TNBC cells and lung metastases. Mechanistically, we identified cMyc as a HuR target in TNBC using RNA immunoprecipitation and metastatic cDNA array. Furthermore, MUC16 knockdown and pharmacological inhibition of HuR (MS-444 and CMLD-2) in TNBC cells showed a reduction in cMyc expression. MUC16-Cter or HuR overexpression models indicated MUC16/HuR/cMyc axis in TNBC cell migration. CONCLUSIONS: Our study identified MUC16 as a TNBC lung metastasis promoter that acts through HuR/cMyc axis. This study will form the basis of future studies to evaluate the targeting of both MUC16 and HuR in TNBC patients.

Tumor microenvironment enriches the stemness features: the architectural event of therapy resistance and metastasis.

Cancer divergence has many facets other than being considered a genetic term. It is a tremendous challenge to understand the metastasis and therapy response in cancer biology; however, it postulates the opportunity to explore the possible mechanism in the surrounding tumor environment. Most deadly solid malignancies are distinctly characterized by their tumor microenvironment (TME). TME consists of stromal components such as immune, inflammatory, endothelial, adipocytes, and fibroblast cells. Cancer stem cells (CSCs) or cancer stem-like cells are a small sub-set of the population within cancer cells believed to be a responsible player in the self-renewal, metastasis, and therapy response of cancer cells. The correlation between TME and CSCs remains an enigma in understanding the events of metastasis and therapy resistance in cancer biology. Recent evidence suggests that TME dictates the CSCs maintenance to arbitrate cancer progression and metastasis. The immune, inflammatory, endothelial, adipocyte, and fibroblast cells in the TME release growth factors, cytokines, chemokines, microRNAs, and exosomes that provide cues for the gain and maintenance of CSC features. These intricate cross-talks are fueled to evolve into aggressive, invasive, migratory phenotypes for cancer development. In this review, we have abridged the recent developments in the role of the TME factors in CSC maintenance and how these events influence the transition of tumor progression to further translate into metastasis and therapy resistance in cancer.

Mucins reprogram stemness, metabolism and promote chemoresistance during cancer progression.

Mucins are high-molecular-weight glycoproteins dysregulated in aggressive cancers. The role of mucins in disease progression, tumor proliferation, and chemotherapy resistance has been studied extensively. This article provides a comprehensive review of mucin's function as a physical barrier and the implication of mucin overexpression in impeded drug delivery to solid tumors. Mucins regulate the epithelial to mesenchymal transition (EMT) of cancer cells via several canonical and non-canonical oncogenic signaling pathways. Furthermore, mucins play an extensive role in enriching and maintaining the cancer stem cell (CSC) population, thereby sustaining the self-renewing and chemoresistant cellular pool in the bulk tumor. It has recently been demonstrated that mucins regulate the metabolic reprogramming during oncogenesis and cancer progression, which account for tumor cell survival, proliferation, and drug-resistance. This review article focuses on delineating mucin's role in oncogenic signaling and aberrant regulation of gene expressions, culminating in CSC maintenance, metabolic rewiring, and development of chemoresistance, tumor progression, and metastasis.

Pancreatic Tumor Microenvironment Factor Promotes Cancer Stemness via SPP1-CD44 Axis.

BACKGROUND & AIMS: Tumor-microenvironment factors and cancer stem cells (CSCs) play a critical role in the aggressiveness of pancreatic cancer (PC). However, the degree to which tumor-microenvironment factors promote stemness remains unexplored. Here, we examined whether cancer-associated fibroblasts (CAFs) promote CSC features in PC. METHODS: PC cells were treated long-term (30, 60, and 90 days) with conditioned media (CM)-derived from normal human fibroblasts (NFs) and CAFs. The stemness features of tumorsphere formation and stemness populations, along with CSCs markers, were analyzed using 2-dimensional and 3-dimensional sodium alginate bead-based co-culture models. Immunohistochemistry and immunofluorescence staining were performed for CSCs and fibroblast markers in autochthonous KrasG12D/+; Trp53R172H/+; Pdx1-Cre mice and human pancreatic tumors. Polymerase chain reaction array and gene knockdown were performed to identify the mechanism of stemness enrichment. RESULTS: Long-term treatment of PC cells with CAF-CM enriched stemness, as indicated by significantly higher CD44+, ALDH+, and AF+ populations in PC cells. Increased tumorsphere formation and elevated CSC, self-renewal, and drug-resistance markers in CAF-CM-treated PC cells were observed. In addition, CAFs co-cultured with PC cells in the 3-dimensional model showed a substantial increase in stemness features. CD44 and α-smooth muscle actin were positively correlated and their expressions progressively increased from the early to late stages of KrasG12D/+; Trp53R172H/+; Pdx1-Cre mouse and human pancreatic tumors. Osteopontin/secreted phosphoprotein 1 was identified as the top differentially overexpressed gene in CAF-CM-treated PC cells and knockdown of osteopontin/secreted phosphoprotein 1 significantly reduced stemness characteristics in CAF-CM-treated PC cells. CONCLUSIONS: Our data uncovered novel insight into the interplay between CAF and enrichment of stemness population through the osteopontin/secreted phosphoprotein 1-CD44 axis in PC.

RNA Polymerase II-Associated Factor 1 Regulates Stem Cell Features of Pancreatic Cancer Cells, Independently of the PAF1 Complex, via Interactions With PHF5A and DDX3.

BACKGROUND & AIMS: It is not clear how pancreatic cancer stem cells (CSCs) are regulated, resulting in ineffective treatments for pancreatic cancer. PAF1, a RNA polymerase II-associated factor 1 complex (PAF1C) component, maintains pluripotency of stem cells, by unclear mechanisms, and is a marker of CSCs. We investigated mechanisms by which PAF1 maintains CSCs and contributes to development of pancreatic tumors. METHODS: Pancreatic cancer cell lines were engineered to knockdown PAF1 using inducible small hairpin RNAs. These cells were grown as orthotopic tumors in athymic nude mice and PAF1 knockdown was induced by administration of doxycycline in drinking water. Tumor growth and metastasis were monitored via IVIS imaging. CSCs were isolated from pancreatic cancer cell populations using flow cytometry and characterized by tumor sphere formation, tumor formation in nude mice, and expression of CSC markers. Isolated CSCs were depleted of PAF1 using the CRISPR/Cas9 system. PAF1-regulated genes in CSCs were identified via RNA-seq and PCR array analyses of cells with PAF1 knockdown. Proteins that interact with PAF1 in CSCs were identified by immunoprecipitations and mass spectrometry. We performed chromatin immunoprecipitation sequencing of CSCs to confirm the binding of the PAF1 sub-complex to target genes. RESULTS: Pancreatic cancer cells depleted of PAF1 formed smaller and fewer tumor spheres in culture and orthotopic tumors and metastases in mice. Isolated CSCs depleted of PAF1 downregulated markers of self-renewal (NANOG, SOX9, and ß-CATENIN), of CSCs (CD44v6, and ALDH1), and the metastasis-associated gene signature, compared to CSCs without knockdown of PAF1. The role of PAF1 in CSC maintenance was independent of its RNA polymerase II-associated factor 1 complex component identity. We identified DDX3 and PHF5A as proteins that interact with PAF1 in CSCs and demonstrated that the PAF1-PHF5A-DDX3 sub-complex bound to the promoter region of Nanog, whose product regulates genes that control stemness. Levels of the PAF1-DDX3 and PAF1-PHF5A were increased and co-localized in human pancreatic tumor specimens, human pancreatic tumor-derived organoids, and organoids derived from tumors of KPC mice, compared with controls. Binding of DDX3 and PAF1 to the Nanog promoter, and the self-renewal capacity of CSCs, were decreased in cells incubated with the DDX3 inhibitor RK-33. CSCs depleted of PAF1 downregulated genes that regulate stem cell features (Flot2, Taz, Epcam, Erbb2, Foxp1, Abcc5, Ddr1, Muc1, Pecam1, Notch3, Aldh1a3, Foxa2, Plat, and Lif). CONCLUSIONS: In pancreatic CSCs, PAF1 interacts with DDX3 and PHF5A to regulate expression of NANOG and other genes that regulate stemness. Knockdown of PAF1 reduces the ability of orthotopic pancreatic tumors to develop and progress in mice and their numbers of CSCs. Strategies to target the PAF1-PHF5A-DDX3 complex might be developed to slow or inhibit progression of pancreatic cancer.

MicroRNA regulation of K-Ras in pancreatic cancer and opportunities for therapeutic intervention.

The Ras family of GTPases is involved in cell proliferation, cell survival, and angiogenesis. It is upregulated in several cancers, including pancreatic cancer (PC) and leads to uncontrolled growth and aggressiveness. PC is well known to be a lethal disease with poor prognosis, plagued by limited therapeutic modalities. MicroRNAs (miRNAs), which are short non-coding RNA molecules, have recently emerged as regulators of signaling networks and have shown potential to target pathway components for therapeutic use in several malignancies. K-Ras mutations are widespread in PC cases (90%), with mutations detectable as early as pancreatic intraepithelial neoplasias and in later metastatic stages alike; therefore, these mutations in K-Ras are obvious drivers and potential targets for PC therapy. Several K-Ras targeting miRNAs have lately been discovered, and many of them have shown promise in combating pancreatic tumor growth in vitro and in mouse models. However, the field of miRNA therapy is still in its infancy, and miRNA mimics or anti-miRNA oligonucleotides that target Ras pathway have thus far not been evaluated in PC patients. In this review, we summarize the role of several miRNAs that regulate oncogenic K-Ras signaling in PC, with their prospective roles as therapeutic agents for targeting K-Ras pathway.

Correction: Global analysis of human glycosyltransferases reveals novel targets for pancreatic cancer pathogenesis.

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

Global analysis of human glycosyltransferases reveals novel targets for pancreatic cancer pathogenesis.

BACKGROUND: Several reports have shown the role of glycosylation in pancreatic cancer (PC), but a global systematic screening of specific glycosyltransferases (glycoTs) in its progression remains unknown. METHODS: We demonstrate a rigorous top-down approach using TCGA-based RNA-Seq analysis, multi-step validation using RT-qPCR, immunoblots and immunohistochemistry. We identified six unique glycoTs (B3GNT3, B4GALNT3, FUT3, FUT6, GCNT3 and MGAT3) in PC pathogenesis and studied their function using CRISPR/Cas9-based KD systems. RESULTS: Serial metastatic in vitro models using T3M4 and HPAF/CD18, generated in house, exhibited decreases in B3GNT3, FUT3 and GCNT3 expression on increasing metastatic potential. Immunohistochemistry identified clinical significance for GCNT3, B4GALNT3 and MGAT3 in PC. Furthermore, the effects of B3GNT3, FUT3, GCNT3 and MGAT3 were shown on proliferation, migration, EMT and stem cell markers in CD18 cell line. Talniflumate, GCNT3 inhibitor, reduced colony formation and migration in T3M4 and CD18 cells. Moreover, we found that loss of GCNT3 suppresses PC progression and metastasis by downregulating cell cycle genes and ß-catenin/MUC4 axis. For GCNT3, proteomics revealed downregulation of MUC5AC, MUC1, MUC5B including many other proteins. CONCLUSIONS: Collectively, we demonstrate a critical role of O- and N-linked glycoTs in PC progression and delineate the mechanism encompassing the role of GCNT3 in PC.

Novel therapies hijack the blood-brain barrier to eradicate glioblastoma cancer stem cells.

Glioblastoma (GBM) is amongst the most aggressive brain tumors with a dismal prognosis. Despite significant advances in the current multimodality therapy including surgery, postoperative radiotherapy (RT) and temozolomide (TMZ)-based concomitant and adjuvant chemotherapy (CT), tumor recurrence is nearly universal with poor patient outcomes. These limitations are in part due to poor drug penetration through the blood-brain barrier (BBB) and resistance to CT and RT by a small population of cancer cells recognized as tumor-initiating cells or cancer stem cells (CSCs). Though CT and RT kill the bulk of the tumor cells, they fail to affect CSCs, resulting in their enrichment and their development into more refractory tumors. Therefore, identifying the mechanisms of resistance and developing therapies that specifically target CSCs can improve response, prevent the development of refractory tumors and increase overall survival of GBM patients. Small molecule inhibitors that can breach the BBB and selectively target CSCs are emerging. In this review, we have summarized the recent advancements in understanding the GBM CSC-specific signaling pathways, the CSC-tumor microenvironment niche that contributes to CT and RT resistance and the use of novel combination therapies of small molecule inhibitors that may be used in conjunction with TMZ-based chemoradiation for effective management of GBM.

Disruption of C1galt1 Gene Promotes Development and Metastasis of Pancreatic Adenocarcinomas in Mice.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinomas (PDACs) produce higher levels of truncated O-glycan structures (such as Tn and sTn) than normal pancreata. Dysregulated activity of core 1 synthase glycoprotein-N-acetylgalactosamine 3-ß-galactosyltransferase 1 (C1GALT1) leads to increased expression of these truncated O-glycans. We investigated whether and how truncated O-glycans contributes to the development and progression of PDAC using mice with disruption of C1galt1. METHODS: We crossed C1galt1 floxed mice (C1galt1loxP/loxP) with KrasG12D/+; Trp53R172H/+; Pdx1-Cre (KPC) mice to create KPCC mice. Growth and progression of pancreatic tumors were compared between KPC and KPCC mice; pancreatic tissues were collected and analyzed by immunohistochemistry; immunofluorescence; and Sirius red, alcian blue, and lectin staining. We used the CRISPR/Cas9 system to disrupt C1GALT1 in human PDAC cells (T3M4 and CD18/HPAF) and levels of O-glycans were analyzed by lectin blotting, mass spectrometry, and lectin pulldown assay. Orthotopic studies and RNA sequencing analyses were performed with control and C1GALT1 knockout PDAC cells. C1GALT1 expression was analyzed in well-differentiated (n = 36) and poorly differentiated (n = 23) PDAC samples by immunohistochemistry. RESULTS: KPCC mice had significantly shorter survival times (median 102 days) than KPC mice (median 200 days) and developed early pancreatic intraepithelial neoplasias at 3 weeks, PDAC at 5 weeks, and metastasis at 10 weeks compared with KPC mice. Pancreatic tumors that developed in KPCC mice were more aggressive (more invasive and metastases) than those in KPC mice, had a decreased amount of stroma, and had increased production of Tn. Poorly differentiated PDAC specimens had significantly lower levels of C1GALT1 than well-differentiated PDACs. Human PDAC cells with knockout of C1GALT1 had aberrant glycosylation of MUC16 compared with control cells and increased expression of genes that regulate tumorigenesis and metastasis. CONCLUSIONS: In studies of KPC mice with disruption of C1galt1, we found that loss of C1galt1 promotes development of aggressive PDACs and increased metastasis. Knockout of C1galt1 leads to increased tumorigenicity and truncation of O-glycosylation on MUC16, which could contribute to increased aggressiveness.

Cigarette Smoke Induces Stem Cell Features of Pancreatic Cancer Cells via PAF1.

BACKGROUND & AIMS: Cigarette smoking is a major risk factor for pancreatic cancer. Aggressive pancreatic tumors contain cancer cells with stem cell features. We investigated whether cigarette smoke induces stem cell features in pancreatic cancer cells. METHODS: KrasG12D; Pdx1-Cre mice were exposed to cigarette smoke or clean air (controls) for up to 20 weeks; pancreata were collected and analyzed by histology, quantitative reverse transcription polymerase chain reaction, and confocal immunofluorescence microscopy. HPNE and Capan1 cells were exposed to cigarette smoke extract (CSE), nicotine and nicotine-derived carcinogens (NNN or NNK), or clean air (controls) for 80 days and evaluated for stem cell markers and features using flow cytometry-based autofluorescence, sphere formation, and immunoblot assays. Proteins were knocked down in cells with small interfering RNAs. We performed RNA sequencing analyses of CSE-exposed cells. We used chromatin immunoprecipitation assays to confirm the binding of FOS-like 1, AP-1 transcription factor subunit (FOSL1) to RNA polymerase II-associated factor (PAF1) promoter. We obtained pancreatic ductal adenocarcinoma (PDAC) and matched nontumor tissues (n = 15) and performed immunohistochemical analyses. RESULTS: Chronic exposure of HPNE and Capan1 cells to CSE caused them to increase markers of stem cells, including autofluorescence and sphere formation, compared with control cells. These cells increased expression of ABCG2, SOX9, and PAF1, via cholinergic receptor nicotinic alpha 7 subunit (CHRNA7) signaling to mitogen-activated protein kinase 1 and FOSL1. CSE-exposed pancreatic cells with knockdown of PAF1 did not show stem cell features. Exposure of cells to NNN and NNK led to increased expression of CHRNA7, FOSL1, and PAF1 along with stem cell features. Pancreata from KrasG12D; Pdx1-Cre mice exposed to cigarette smoke had increased levels of PAF1 mRNA and protein, compared with control mice, as well as increased expression of SOX9. Levels of PAF1 and FOSL1 were increased in PDAC tissues, especially those from smokers, compared with nontumor pancreatic tissue. CSE exposure increased expression of PHD-finger protein 5A, a pluripotent transcription factor and its interaction with PAF1. CONCLUSIONS: Exposure to cigarette smoke activates stem cell features of pancreatic cells, via CHRNA7 signaling and FOSL1 activation of PAF1 expression. Levels of PAF1 are increased in pancreatic tumors of humans and mice with chronic cigarette smoke exposure.

Concise Review: Current Status of Three-Dimensional Organoids as Preclinical Models.

Three-dimensional (3D) cultures use the property of some cells to self-organize in matrices and generate structures that can be programmed to represent an organ or a pathology. Organoid cultures are the 3D cultivation of source tissue (ranging from cells to tissue fragments) in a support matrix and specialized media that nearly resembles the physiological environment. Depending on the source tissue, growth factors, and inhibitors provided, organoids can be programmed to recapitulate the biology of a system and progression of pathology. Organoids are genetically stable, and genetically amenable, making them very suitable tools to study tissue homeostasis and cancer. In this Review, we focus on providing recent technical advances from published literature to efficiently use organoids as a tool for disease modeling and therapeutics. Also, we discuss stem cell biology principles used to generate multiple organoids and their characteristics, with a brief description of methodology. A major theme of this review is to expand organoid applications to the study disease progression and drug response in different cancers. We also discuss shortcomings, limitations, and advantages of developed 3D cultures, with the rationale behind the methodology. Stem Cells 2018;36:1329-1340.

Single stage treatment of complex aortic pathology (mega-aortic syndrome) using frozen elephant trunk technique.

This is a case report of single-stage total thoracic aortic repair by the frozen elephant trunk technique for a 75-yearold female with a complex aortic pathology of mega-aorta extending from the ascending aorta to the descending aorta. We used a Thora flex™ Hybrid device, a frozen elephant trunk device with four branched grafts and the distal stent graft. The distal stent graft was inserted into the downstream descending aorta via an aortic arch and positioned 15 cm beyond the left subclavian artery after total arch replacement had been performed using a four-branch graft. The postoperative course was unremarkable with no complications. A post procedural computed tomography scan demonstrated complete exclusion of the descending thoracic aneurysm without endoleak. Therefore, fixing the whole mega-aorta in a single stage using the frozen elephant trunk was effective and safe.

Analysis of sulfates on low molecular weight heparin using mass spectrometry: structural characterization of enoxaparin.

INTRODUCTION: Structural characterization of low molecular weight heparin (LMWH) is critical to meet biosimilarity standards. In this context, the review focuses on structural analysis of labile sulfates attached to the side-groups of LMWH using mass spectrometry. A comprehensive review of this topic will help readers to identify key strategies for tackling the problem related to sulfate loss. At the same time, various mass spectrometry techniques are presented to facilitate compositional analysis of LMWH, mainly enoxaparin. Areas covered: This review summarizes findings on mass spectrometry application for LMWH, including modulation of sulfates, using enzymology and sample preparation approaches. Furthermore, popular open-source software packages for automated spectral data interpretation are also discussed. Successful use of LC/MS can decipher structural composition for LMWH and help evaluate their sameness or biosimilarity with the innovator molecule. Overall, the literature has been searched using PubMed by typing various search queries such as 'enoxaparin', 'mass spectrometry', 'low molecular weight heparin', 'structural characterization', etc. Expert commentary: This section highlights clinically relevant areas that need improvement to achieve satisfactory commercialization of LMWHs. It also primarily emphasizes the advancements in instrumentation related to mass spectrometry, and discusses building automated software for data interpretation and analysis.

Novel role of O-glycosyltransferases GALNT3 and B3GNT3 in the self-renewal of pancreatic cancer stem cells.

BACKGROUND: Glycosylation plays a critical role in the aggressiveness of pancreatic cancer (PC). Emerging evidences indicate significant involvement of cancer stem cells (CSCs) in PC aggressiveness. However, the importance of glycosylation in pancreatic cancer stem cells (PCSCs) is yet to be addressed. Hence, we evaluated the potential role of glycosylation in maintenance of stemness of PCSCs. METHODS: Effect of glycosylation specific inhibitors on growth and PCSCs of PC cells was assessed by MTT assay and Side Population (SP) analysis. Isolated PCSCs/SP were characterized using molecular and functional assays. Expression of tumor-associated carbohydrate antigens (TACAs) was analyzed in PCSCs by western blotting. Effect of tunicamycin on PCSCs was analyzed by tumorsphere, clonogenicity, migration assay and immunoblotting for CSCs markers. The differential expression of glycogenes in PCSCs compared to non-CSCs were determined by RT-qPCR, immunoblotting and immunofluorescence. Co-expression of GALNT3 and B3GNT3 with CD44v6 was assessed in progression stages of KrasG12D; Pdx-1-Cre (KC) and KrasG12D; p53R172H; Pdx-1-Cre (KPC) tumors by immunofluorescence. Transient and CRISPR/Cas9 silencing of GALNT3 and B3GNT3 was performed to examine their effect on CSCs maintenance. RESULTS: Inhibition of glycosylation decreased growth and CSCs/SP in PC cells. PCSCs overexpressed CSC markers (CD44v6, ESA, SOX2, SOX9 and ABCG2), exhibited global expressional variation of TACAs and showed higher self-renewal potential. Specifically, N-glycosylation inhibition, significantly decreased tumorsphere formation, migration, and clonogenicity of PCSCs, as well as hypo-glycosylated CD44v6 and ESA. Of note, glycosyltransferases (GFs), GALNT3 and B3GNT3, were significantly overexpressed in PCSCs and co-expressed with CD44v6 at advanced PDAC stages in KC and KPC tumors. Further, GALNT3 and B3GNT3 knockdown led to a decrease in the expression of cell surface markers (CD44v6 and ESA) and self-renewal markers (SOX2 and OCT3/4) in PCSCs. Interestingly, CD44v6 was modified with sialyl Lewis a in PCSCs. Finally, CRISPR/Cas9-mediated GALNT3 KO significantly decreased self-renewal, clonogenicity, and migratory capacity in PCSCs. CONCLUSIONS: Taken together, for the first time, our study showed the importance of glycosylation in mediating growth, stemness, and maintenance of PCSCs. These results indicate that elevated GALNT3 and B3GNT3 expression in PCSCs regulate stemness through modulating CSC markers.