Key regulator PNPLA8 drives phospholipid reprogramming induced proliferation and migration in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and leads to the poorest patient outcomes despite surgery and chemotherapy treatment. Exploring new molecular mechanisms of TNBC that could lead to the development of novel molecular targets are critically important for improving therapeutic options for treating TNBC. METHODS: We sought to identify novel therapeutic targets in TNBC by combining genomic and functional studies with lipidomic analysis, which included mechanistic studies to elucidate the pathways that tie lipid profile to critical cancer cell properties. Our studies were performed in a large panel of human breast cancer cell lines and patient samples. RESULTS: Comprehensive lipid profiling revealed that phospholipid metabolism is reprogrammed in TNBC cells. We discovered that patatin-like phospholipase domain-containing lipase 8 (PNPLA8) is overexpressed in TNBC cell lines and tissues from breast cancer patients. Silencing of PNPLA8 disrupted phospholipid metabolic reprogramming in TNBC, particularly affecting the levels of phosphatidylglycerol (PG), phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and glycerophosphocholine (GPC). We showed that PNPLA8 is essential in regulating cell viability, migration and antioxidation in TNBC cells and promoted arachidonic acid and eicosanoid production, which in turn activated PI3K/Akt/Gsk3ß and MAPK signaling. CONCLUSIONS: Our study highlights PNPLA8 as key regulator of phospholipid metabolic reprogramming and malignant phenotypes in TNBC, which could be further developed as a novel molecular treatment target.

Lipidomic Phenotyping Of Human Small Intestinal Organoids Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

In the past decade, interest in organoids for biomedical research has surged, resulting in a higher demand for advanced imaging techniques. Traditional specimen embedding methods pose challenges, such as analyte delocalization and histological assessment. Here, we present an optimized sample preparation approach utilizing an Epredia M-1 cellulose-based embedding matrix, which preserves the structural integrity of fragile small intestinal organoids (SIOs). Additionally, background interference (delocalization of analytes, nonspecific (histological) staining, matrix ion clusters) was minimized, and we demonstrate the compatibility with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). With our approach, we can conduct label-free lipid imaging at the single-cell level, thereby yielding insights into the spatial distribution of lipids in both positive and negative ion modes. Moreover, M-1 embedding allows for an improved coregistration with histological and immunohistochemical (IHC) stainings, including MALDI-IHC, facilitating combined untargeted and targeted spatial information. Applying this approach, we successfully phenotyped crypt-like (CL) and villus-like (VL) SIOs, revealing that PE 36:2 [M - H]- (m/z 742.5) and PI 38:4 [M - H]- (m/z 885.5) display higher abundance in CL organoids, whereas PI 36:1 [M - H]- (m/z 863.6) was more prevalent in VL organoids. Our findings demonstrate the utility of M-1 embedding for advancing organoid research and unraveling intricate biological processes within these in vitro models.

MET Signaling Mediates Intestinal Crypt-Villus Development, Regeneration, and Adenoma Formation and Is Promoted by Stem Cell CD44 Isoforms.

BACKGROUND & AIMS: Resistance of metastatic human colorectal cancer cells to drugs that block epidermal growth factor (EGF) receptor signaling could be caused by aberrant activity of other receptor tyrosine kinases, activating overlapping signaling pathways. One of these receptor tyrosine kinases could be MET, the receptor for hepatocyte growth factor (HGF). We investigated how MET signaling, and its interaction with CD44 (a putative MET coreceptor regulated by Wnt signaling and highly expressed by intestinal stem cells [ISCs] and adenomas) affects intestinal homeostasis, regeneration, and adenoma formation in mini-gut organoids and mice. METHODS: We established organoid cultures from ISCs stimulated with HGF or EGF and assessed intestinal differentiation by immunohistochemistry. Mice with total epithelial disruption of MET (AhCre/Metfl/fl/LacZ) or ISC-specific disruption of MET (Lgr5Creert2/Metfl/fl/LacZ) and control mice (AhCre/Met+/+/LacZ, Lgr5Creert2/Met+/+/LacZ) were exposed to 10 Gy total body irradiation; intestinal tissues were collected, and homeostasis and regeneration were assessed by immunohistochemistry. We investigated adenoma organoid expansion stimulated by HGF or EGF using adenomas derived from Lgr5Creert2/Metfl/fl/Apcfl/fl and Lgr5Creert2/Met+/+/Apcfl/fl mice. The same mice were evaluated for adenoma prevalence and size. We also quantified adenomas in AhCre/Metfl/fl/Apcfl/+ mice compared with AhCre/Met+/+/Apcfl/+ control mice. We studied expansion of organoids generated from crypts and adenomas, stimulated by HGF or EGF, that were derived from mice expressing different CD44 splice variants (Cd44+/+, Cd44-/-, Cd44s/s, or Cd44v4-10/v4-10 mice). RESULTS: Crypts incubated with EGF or HGF expanded into self-organizing mini-guts with similar levels of efficacy and contained all differentiated cell lineages. MET-deficient mice did not have defects in intestinal homeostasis. Total body irradiation reduced numbers of proliferating crypts in AhCre/Metfl/fl/LacZ mice. Lgr5Creert2/Metfl/fl/LacZ mice had impaired regeneration of MET-deficient ISCs. Adenoma organoids stimulated with EGF or HGF expanded to almost twice the size of nonstimulated organoids. MET-deficient adenoma organoids did not respond to HGF stimulation, but did respond to EGF. ISC-specific disruption of Met (Lgr5Creert2/Metfl/fl/Apcfl/fl mice) caused a twofold increase in apoptosis in microadenomas, resulting in an approximately 50% reduction of microadenoma numbers and significantly reduced average adenoma size. Total epithelial disruption of Met (AhCre/Metfl/fl/Apcfl/+ mice) resulted in an approximate 50% reduction in (micro)adenoma numbers. Intestinal crypts from Cd44-/- mice did not expand to the same extent as crypts from Cd44+/+ mice on stimulation with HGF, but had the same response to EGF. The negative effect on HGF-mediated growth was overcome by expression of CD44v4-10, but not by CD44s. Similarly, HGF-mediated expansion of adenoma organoids required CD44v4-10. CONCLUSIONS: In studies of intestinal organoid cultures and mice with inducible deletion of MET, we found HGF receptor signaling to regulate intestinal homeostasis and regeneration, as well as adenoma formation. These activities of MET are promoted by the stem cell CD44 isoform CD44v4-10. Our findings provide rationale for targeting signaling via MET and CD44 during anti-EGF receptor therapy of patients with colorectal cancer or in patients resistant to EGF receptor inhibitors.

Intestinal Fatty Acid Binding Protein as a Predictor of Early Mesenteric Injury Preceding Clinical Presentation: A Case Report.

Introduction: Diagnosing non-occlusive mesenteric ischaemia (NOMI) in patients is complicated, due to poor signs and symptoms and non-specific laboratory tests, leading to a high mortality rate. This case study presents the rare case of a patient who developed mesenteric ischaemia after an emergency thoracic endovascular aneurysm repair (TEVAR) for a type B aortic dissection (TBAD) and peri-operative cardiogenic shock. Study outcomes revealed that intestinal fatty acid binding protein (I-FABP) identified early mucosal damage two days before the clinical presentation. Report: A 43 year old male patient was admitted to the emergency department with an acute TBAD and a dissection of the superior mesenteric artery (SMA), for which TEVAR was performed with additional stent placement in the SMA. Peri-operatively, the patient went into cardiogenic shock with a sustained period of hypotension. Post-operatively, the plasma I-FABP levels were measured prospectively, revealing an initial increase on post-operative day five (551.1 pg/mL), which continued beyond day six (610.3 pg/mL). On post-operative day seven, the patient developed a fever and demonstrated signs of peritonitis and bowel perforation. He underwent an emergency laparotomy, followed by an ileocaecal resection (<100 cm) with a transverse ileostomy. Pathological analysis confirmed the diagnosis of mesenteric ischaemia. Discussion: The diagnosis of NOMI in critically ill patients is often complicated, and the currently available diagnostic markers lack the specificity and sensitivity to detect early intestinal injury. This case report highlights that elevated I-FABP in plasma levels may indicate the presence of early mesenteric injury. Further research needs to be conducted before I-FABP can be applied in daily practice.

Diagnostic potential of plasma biomarkers and exhaled volatile organic compounds in predicting the different stages of acute mesenteric ischaemia: protocol for a multicentre prospective observational study (TACTIC study).

INTRODUCTION: Acute mesenteric ischaemia (AMI) is a life-threatening condition with short-term mortality of up to 80%. The diagnosis of AMI has remained troublesome due to the non-specific clinical presentation, symptoms and laboratory findings. Early unambiguous diagnosis of AMI is critical to prevent progression from reversible to irreversible transmural intestinal damage, thereby decreasing morbidity and improving survival. The present study aims to validate a panel of plasma biomarkers and investigate volatile organic compound (VOC) profiles in exhaled air as a tool to timely and accurately diagnose AMI. METHODS AND ANALYSIS: In this international multicentre prospective observational study, 120 patients (>18 years of age) will be recruited with clinical suspicion of AMI. Clinical suspicion is based on: (1) clinical manifestation, (2) physical examination, (3) laboratory measurements and (4) the physician's consideration to perform a CT scan. The patient's characteristics, repetitive blood samples and exhaled air will be prospectively collected. Plasma levels of mucosal damage markers intestinal fatty acid-binding protein and villin-1, as well as transmural damage marker smooth muscle protein 22-alpha, will be assessed by ELISA. Analysis of VOCs in exhaled air will be performed by gas chromatography time-of-flight mass spectrometry. Diagnosis of AMI will be based on CT, endovascular and surgical reports, clinical findings, and (if applicable) verified by histopathological examination. ETHICS AND DISSEMINATION: The study protocol was approved by the Medical Research Ethics Committee (METC) of Maastricht University Medical Centre+ and Maastricht University (METC azM/UM), the Netherlands (METC19-010) and the Ethics Committee Research UZ/KU Leuven, Belgium (S63500). Executive boards and local METCs of other Dutch participating centres Gelre Ziekenhuizen (Apeldoorn), Medisch Spectrum Twente (Enschede), and University Medical Centre Groningen have granted permission to carry out this study. Study results will be disseminated via open-access peer-reviewed scientific journals and national/international conferences. TRIAL REGISTRATION NUMBER: NCT05194527.

Proteomics analysis of human intestinal organoids during hypoxia and reoxygenation as a model to study ischemia-reperfusion injury.

Intestinal ischemia-reperfusion (IR) injury is associated with high mortality rates, which have not improved in the past decades despite advanced insight in its pathophysiology using in vivo animal and human models. The inability to translate previous findings to effective therapies emphasizes the need for a physiologically relevant in vitro model to thoroughly investigate mechanisms of IR-induced epithelial injury and test potential therapies. In this study, we demonstrate the use of human small intestinal organoids to model IR injury by exposing organoids to hypoxia and reoxygenation (HR). A mass-spectrometry-based proteomics approach was applied to characterize organoid differentiation and decipher protein dynamics and molecular mechanisms of IR injury in crypt-like and villus-like human intestinal organoids. We showed successful separation of organoids exhibiting a crypt-like proliferative phenotype, and organoids exhibiting a villus-like phenotype, enriched for enterocytes and goblet cells. Functional enrichment analysis of significantly changing proteins during HR revealed that processes related to mitochondrial metabolism and organization, other metabolic processes, and the immune response were altered in both organoid phenotypes. Changes in protein metabolism, as well as mitophagy pathway and protection against oxidative stress were more pronounced in crypt-like organoids, whereas cellular stress and cell death associated protein changes were more pronounced in villus-like organoids. Profile analysis highlighted several interesting proteins showing a consistent temporal profile during HR in organoids from different origin, such as NDRG1, SDF4 or DMBT1. This study demonstrates that the HR response in human intestinal organoids recapitulates properties of the in vivo IR response. Our findings provide a framework for further investigations to elucidate underlying mechanisms of IR injury in crypt and/or villus separately, and a model to test therapeutics to prevent IR injury.