Mapping the breast tumor microenvironment: proximity analysis reveals spatial relationships between macrophage subtypes and metastasis-initiating cancer cells.

Metastasis is responsible for the majority of cancer-related fatalities. We previously identified specific cancer cell populations responsible for metastatic events which are cytokeratin-14 (CK14) and E-cadherin positive in luminal tumors, and E-cadherin and vimentin positive in triple-negative tumors. Since cancer cells evolve within a complex ecosystem comprised of immune cells and stromal cells, we sought to decipher the spatial interactions of these aggressive cancer cell populations within the tumor microenvironment (TME). We used imaging mass cytometry to detect 36 proteins in tumor microarrays containing paired primary and metastatic lesions from luminal or triple-negative breast cancers (TNBC), resulting in a dataset of 1,477,337 annotated cells. Focusing on metastasis-initiating cell populations, we observed close proximity to specific fibroblast and macrophage subtypes, a relationship maintained between primary and metastatic tumors. Notably, high CK14 in luminal cancer cells and high vimentin in TNBC cells correlated with close proximity to specific macrophage subtypes (CD163intCD206intPDL1intHLA-DR+ or PDL1highARG1high). Our in-depth spatial analysis demonstrates that metastasis-initiating cancer cells consistently colocalizes with distinct cell populations within the TME, suggesting a role for these cell-cell interactions in promoting metastasis.

PanIN and CAF transitions in pancreatic carcinogenesis revealed with spatial data integration.

This study introduces a new imaging, spatial transcriptomics (ST), and single-cell RNA-sequencing integration pipeline to characterize neoplastic cell state transitions during tumorigenesis. We applied a semi-supervised analysis pipeline to examine premalignant pancreatic intraepithelial neoplasias (PanINs) that can develop into pancreatic ductal adenocarcinoma (PDAC). Their strict diagnosis on formalin-fixed and paraffin-embedded (FFPE) samples limited the single-cell characterization of human PanINs within their microenvironment. We leverage whole transcriptome FFPE ST to enable the study of a rare cohort of matched low-grade (LG) and high-grade (HG) PanIN lesions to track progression and map cellular phenotypes relative to single-cell PDAC datasets. We demonstrate that cancer-associated fibroblasts (CAFs), including antigen-presenting CAFs, are located close to PanINs. We further observed a transition from CAF-related inflammatory signaling to cellular proliferation during PanIN progression. We validate these findings with single-cell high-dimensional imaging proteomics and transcriptomics technologies. Altogether, our semi-supervised learning framework for spatial multi-omics has broad applicability across cancer types to decipher the spatiotemporal dynamics of carcinogenesis.

The postnatal injection of AAV9-FOXG1 rescues corpus callosum agenesis and other brain deficits in the mouse model of FOXG1 syndrome.

Heterozygous mutations in the FOXG1 gene manifest as FOXG1 syndrome, a severe neurodevelopmental disorder characterized by structural brain anomalies, including agenesis of the corpus callosum, hippocampal reduction, and myelination delays. Despite the well-defined genetic basis of FOXG1 syndrome, therapeutic interventions targeting the underlying cause of the disorder are nonexistent. In this study, we explore the therapeutic potential of adeno-associated virus 9 (AAV9)-mediated delivery of the FOXG1 gene. Remarkably, intracerebroventricular injection of AAV9-FOXG1 to Foxg1 heterozygous mouse model at the postnatal stage rescues a wide range of brain pathologies. This includes the amelioration of corpus callosum deficiencies, the restoration of dentate gyrus morphology in the hippocampus, the normalization of oligodendrocyte lineage cell numbers, and the rectification of myelination anomalies. Our findings highlight the efficacy of AAV9-based gene therapy as a viable treatment strategy for FOXG1 syndrome and potentially other neurodevelopmental disorders with similar brain malformations, asserting its therapeutic relevance in postnatal stages.

Multi-omic profiling of intraductal papillary neoplasms of the pancreas reveals distinct expression patterns and potential markers of progression.

In order to advance our understanding of precancers in the pancreas, 69 pancreatic intraductal papillary neoplasms (IPNs), including 64 intraductal papillary mucinous neoplasms (IPMNs) and 5 intraductal oncocytic papillary neoplasms (IOPNs), 32 pancreatic cyst fluid samples, 104 invasive pancreatic ductal adenocarcinomas (PDACs), 43 normal adjacent tissues (NATs), and 76 macro-dissected normal pancreatic ducts (NDs) were analyzed by mass spectrometry. A total of 10,246 proteins and 22,284 glycopeptides were identified in all tissue samples, and 756 proteins with more than 1.5-fold increase in abundance in IPMNs relative to NDs were identified, 45% of which were also identified in cyst fluids. The over-expression of selected proteins was validated by immunolabeling. Proteins and glycoproteins overexpressed in IPMNs included those involved in glycan biosynthesis and the immune system. In addition, multiomics clustering identified two subtypes of IPMNs. This study provides a foundation for understanding tumor progression and targets for earlier detection and therapies. Significance: This multilevel characterization of intraductal papillary neoplasms of the pancreas provides a foundation for understanding the changes in protein and glycoprotein expression during the progression from normal duct to intraductal papillary neoplasm, and to invasive pancreatic carcinoma, providing a foundation for informed approaches to earlier detection and treatment.

Hepatocytes coordinate immune evasion in cancer via release of serum amyloid A proteins.

T cell infiltration into tumors is a favorable prognostic feature, but most solid tumors lack productive T cell responses. Mechanisms that coordinate T cell exclusion are incompletely understood. Here we identify hepatocyte activation via interleukin-6/STAT3 and secretion of serum amyloid A (SAA) proteins 1 and 2 as important regulators of T cell surveillance of extrahepatic tumors. Loss of STAT3 in hepatocytes or SAA remodeled the tumor microenvironment with infiltration by CD8+ T cells, while interleukin-6 overexpression in hepatocytes and SAA signaling via Toll-like receptor 2 reduced the number of intratumoral dendritic cells and, in doing so, inhibited T cell tumor infiltration. Genetic ablation of SAA enhanced survival after tumor resection in a T cell-dependent manner. Likewise, in individuals with pancreatic ductal adenocarcinoma, long-term survivors after surgery demonstrated lower serum SAA levels than short-term survivors. Taken together, these data define a fundamental link between liver and tumor immunobiology wherein hepatocytes govern productive T cell surveillance in cancer.

Transfer Learning Reveals Cancer-Associated Fibroblasts Are Associated with Epithelial-Mesenchymal Transition and Inflammation in Cancer Cells in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by an immunosuppressive tumor microenvironment enriched with cancer-associated fibroblasts (CAF). This study used a convergence approach to identify tumor cell and CAF interactions through the integration of single-cell data from human tumors with human organoid coculture experiments. Analysis of a comprehensive atlas of PDAC single-cell RNA sequencing data indicated that CAF density is associated with increased inflammation and epithelial-mesenchymal transition (EMT) in epithelial cells. Transfer learning using transcriptional data from patient-derived organoid and CAF cocultures provided in silico validation of CAF induction of inflammatory and EMT epithelial cell states. Further experimental validation in cocultures demonstrated integrin beta 1 (ITGB1) and vascular endothelial factor A (VEGFA) interactions with neuropilin-1 mediating CAF-epithelial cell cross-talk. Together, this study introduces transfer learning from human single-cell data to organoid coculture analyses for experimental validation of discoveries of cell-cell cross-talk and identifies fibroblast-mediated regulation of EMT and inflammation. SIGNIFICANCE: Adaptation of transfer learning to relate human single-cell RNA sequencing data to organoid-CAF cocultures facilitates discovery of human pancreatic cancer intercellular interactions and uncovers cross-talk between CAFs and tumor cells through VEGFA and ITGB1.

Feasibility of using a depth camera or pressure mat for visual feedback balance training with functional electrical stimulation.

Individuals with incomplete spinal-cord injury/disease are at an increased risk of falling due to their impaired ability to maintain balance. Our research group has developed a closed-loop visual-feedback balance training (VFBT) system coupled with functional electrical stimulation (FES) for rehabilitation of standing balance (FES + VFBT system); however, clinical usage of this system is limited by the use of force plates, which are expensive and not easily accessible. This study aimed to investigate the feasibility of a more affordable and accessible sensor such as a depth camera or pressure mat in place of the force plate. Ten able-bodied participants (7 males, 3 females) performed three sets of four different standing balance exercises using the FES + VFBT system with the force plate. A depth camera and pressure mat collected centre of mass and centre of pressure data passively, respectively. The depth camera showed higher Pearson's correlation (r > 98) and lower root mean squared error (RMSE < 10 mm) than the pressure mat (r > 0.82; RMSE < 4.5 mm) when compared with the force plate overall. Stimulation based on the depth camera showed lower RMSE than that based on the pressure mat relative to the FES + VFBT system. The depth camera shows potential as a replacement sensor to the force plate for providing feedback to the FES + VFBT system.

Multimodal immune phenotyping reveals microbial-T cell interactions that shape pancreatic cancer.

Microbes are an integral component of the tumor microenvironment. However, determinants of microbial presence remain ill-defined. Here, using spatial-profiling technologies, we show that bacterial and immune cell heterogeneity are spatially coupled. Mouse models of pancreatic cancer recapitulate the immune-microbial spatial coupling seen in humans. Distinct intra-tumoral niches are defined by T cells, with T cell-enriched and T cell-poor regions displaying unique bacterial communities that are associated with immunologically active and quiescent phenotypes, respectively, but are independent of the gut microbiome. Depletion of intra-tumoral bacteria slows tumor growth in T cell-poor tumors and alters the phenotype and presence of myeloid and B cells in T cell-enriched tumors but does not affect T cell infiltration. In contrast, T cell depletion disrupts the immunological state of tumors and reduces intra-tumoral bacteria. Our results establish a coupling between microbes and T cells in cancer wherein spatially defined immune-microbial communities differentially influence tumor biology.

AI-Based Geometry Recognition for Evaluating the Feasibility of Intensified Reaction and Separation Systems.

Reactive distillation (RD) provides notable advantages over conventional processes, regarding reduced energy requirements and CO2 emissions. However, as the benefits of RD may not be universally applicable, a comprehensive feasibility assessment is necessary. This study introduced an automated feasibility evaluation procedure for an RD column using an AI-based region recognition approach, reducing the reliance on expert knowledge and heuristics in graphical methods. Through k-means clustering-based image segmentation, topological information on the reaction and separation reachable region was extracted from ternary diagram landscapes. Subsequently, the extracted information was integrated into tray-by-tray calculations to automate the evaluation. This geometric calculation procedure was applied to assess the feasibility of RD columns with different types of reactions. The feasibility results were obtained within seconds, demonstrating the efficiency of the proposed approach. Furthermore, case studies validated the feasibility of the evaluation results for three practical examples using rigorous simulations, confirming its reliability and applicability.

Immune landscape of tertiary lymphoid structures in hepatocellular carcinoma (HCC) treated with neoadjuvant immune checkpoint blockade.

Neoadjuvant immunotherapy is thought to produce long-term remissions through induction of antitumor immune responses before removal of the primary tumor. Tertiary lymphoid structures (TLS), germinal center-like structures that can arise within tumors, may contribute to the establishment of immunological memory in this setting, but understanding of their role remains limited. Here, we investigated the contribution of TLS to antitumor immunity in hepatocellular carcinoma (HCC) treated with neoadjuvant immunotherapy. We found that neoadjuvant immunotherapy induced the formation of TLS, which were associated with superior pathologic response, improved relapse free survival, and expansion of the intratumoral T and B cell repertoire. While TLS in viable tumor displayed a highly active mature morphology, in areas of tumor regression we identified an involuted TLS morphology, which was characterized by dispersion of the B cell follicle and persistence of a T cell zone enriched for ongoing antigen presentation and T cell-mature dendritic cell interactions. Involuted TLS showed increased expression of T cell memory markers and expansion of CD8+ cytotoxic and tissue resident memory clonotypes. Collectively, these data reveal the circumstances of TLS dissolution and suggest a functional role for late-stage TLS as sites of T cell memory formation after elimination of viable tumor.

Three-dimensional analysis of ductular reactions and their correlation with liver regeneration and fibrosis.

The liver has multiple regeneration modes, including hepatocellular hypertrophy and self-renewal of hepatocytes. When hepatocyte proliferation is impaired, hepatic progenitor cells may proliferate through ductular reaction (DR), differentiate into hepatocytes, and contribute to fibrosis. However, the three-dimensional spatial relationship between DR and regenerating hepatocytes and dynamic changes in DR associated with fibrosis remain poorly understood. Here, we performed three-dimensional (3D) imaging of cleared 42 liver explants with chronic and acute liver diseases and 4 normal livers to visualize DR. In chronic hepatic liver diseases, such as viral hepatitis, steatohepatitis, autoimmune hepatitis, and cryptogenic cirrhosis, the total length and number of branches of DR showed a significant positive correlation. We studied the spatial relationship between DR and GS-expressing cells using glutamine synthetase (GS) and cytokeratin 19 (CK19) as markers of liver regeneration and DR, respectively. The percentage of CK19-positive cells that co-expressed GS was less than 10% in chronic liver diseases. In contrast, nearly one-third of CK19-positive cells co-expressed GS in acute liver diseases, and chronic cholestatic liver diseases, including primary biliary cholangitis and primary sclerosing cholangitis, showed no co-expression. We also found that DR was longer and had more branching in livers with progressive fibrosis compared to those with regressive fibrosis. Our results suggest that DR displays varying degrees of spatial complexity and contribution to liver regeneration. DR may serve as hepatobiliary junctions that maintain continuity between hepatocytes and bile ducts rather than hepatocyte regeneration in chronic liver diseases.

Systematic review of challenging issues in pathology of intraductal papillary mucinous neoplasms.

BACKGROUND: Intraductal papillary mucinous neoplasms (IPMNs) are a cystic precursor to pancreatic cancer. IPMNs deemed clinically to be at high-risk for malignant progression are frequently treated with surgical resection, and pathological examination of the pancreatectomy specimen is a key component of the clinical care of IPMN patients. METHODS: Systematic literature reviews were conducted around eight topics of clinical relevance in the examination of pathological specimens in patients undergoing resection of IPMN. RESULTS: This review provides updated perspectives on morphological subtyping of IPMNs, classification of intraductal oncocytic papillary neoplasms, nomenclature for high-grade dysplasia, assessment of T stage, distinction of carcinoma associated or concomitant with IPMN, role of molecular assessment of IPMN tissue, role of intraoperative assessment by frozen section, and preoperative evaluation of cyst fluid cytology. CONCLUSIONS: This analysis provides the foundation for data-driven approaches to several challenging issues in the pathology of IPMNs.

Treatment Response in First-Line Metastatic Pancreatic Ductal Adenocarcinoma Is Stratified By a Composite Index of Tumor Proliferation and CD8 T-Cell Infiltration.

PURPOSE: Determinants of treatment outcomes to chemotherapy-based regimens in metastatic pancreatic ductal adenocarcinoma (PDA) remain ill-defined. Our aim was to examine tissue-based correlates of treatment response and resistance using matched baseline and on-treatment biopsies collected from patients with PDA treated in the first-line metastatic setting. EXPERIMENTAL DESIGN: Patients with treatment-naïve metastatic PDA were enrolled in a Phase II trial (NCT02077881) investigating gemcitabine plus nab-paclitaxel in combination with indoximod, an orally administered small-molecule inhibitor of the IDO pathway. Baseline and on-treatment biopsies (week 8) of metastatic lesions (88% liver) were collected from a cohort of responders (N = 8) and non-responders (N = 8) based on RECIST v1.1 and examined by multiplex IHC and mRNA sequencing. RESULTS: Treatment altered the transcriptional profile of metastatic lesions with a decrease in tumor cell proliferation independent of treatment response. The antiproliferative response was seen in both basal and classical PDA subtypes. PDA subtype was not associated with survival outcomes; instead, genes involved in immune activation distinguished responders from non-responders. Tumor response was associated with an increase in CD3+ and CD8+ T-cell infiltrates into metastatic lesions. A composite of decreased tumor proliferation in response to treatment and increased CD8 T-cell infiltration in metastatic lesions identified responders and associated with a favorable survival outcome. CONCLUSIONS: Our findings suggest that inhibiting cancer cell proliferation alone in PDA is insufficient to produce tumor responses and support a role for tumor-extrinsic mechanisms, such as CD8+ T cells, which combine with the cancer cell proliferation index to define treatment outcomes.

Identifying priorities for balance interventions through a participatory co-design approach with end-users.

BACKGROUND: Most individuals living with spinal cord injuries/diseases (SCI/D) or stroke experience at least one fall each year; hence, the development of interventions and technologies that target balance control is needed. The purpose of this study was to identify and explore the priorities for balance-focused interventions and technologies from the perspectives of end-users to assist with the design of an intervention that combines functional electrical stimulation (FES) with visual feedback training for standing balance. METHODS: Two individuals with SCI/D, one individual with stroke, two physical therapists (PT) and one hospital administrator were recruited. Participants attended three focus group meetings that followed a participatory co-design approach. A semi-structured interview guide, developed from the FAME (Feasibility, Appropriateness, Meaningfulness, Effectiveness, Economic Evidence) framework, was used to lead the discussion, querying participants' experiences with balance deficits and interventions, and FES. Meetings were audio-recorded and transcribed verbatim. An iterative and reflexive inductive thematic analysis was applied to the transcripts by three researchers. RESULTS: Four themes were identified: (1) Balance is meaningful for daily life and rehabilitation. Participants acknowledged various factors influencing balance control and how balance deficits interfered with participation in activities. End-users stressed the importance of continuing to work on one's balance after discharge from hospital-based rehabilitation. (2) Desired characteristics of balance interventions. Participants explained that balance interventions should be tailored to an individual's unique needs and goals, relevant to their lives, balance their safety and risk, and be engaging. (3) Prior experiences with FES to inform future therapeutic use. Participants with stroke or SCI/D described initial apprehension with FES, but experienced numerous benefits that motivated them to continue with FES. Challenges with FES were mentioned, including wires, cost, and time of set up. (4) Potential role of FES in balance interventions. Participants felt that FES would complement balance interventions; however, they had not experienced this combination of therapies previously. CONCLUSIONS: End-users described how their experiences with balance deficits, rehabilitation, and FES informed their priorities for balance interventions. The findings inform the design and implementation of future balance interventions for individuals with SCI/D or stroke, including an intervention involving FES and visual feedback training.

Associations of Dietary Intakes of Total and Specific Types of Fat with Blood Lipid Levels in the Filipino Women's Diet and Health Study (FiLWHEL).

Background: Limited evidence exists on the association between dietary fat intake and lipid profiles in Southeast Asian populations. Objectives: We aimed to examine the cross-sectional associations of dietary intake of total and specific types of fat with dyslipidemia in Filipino immigrant women in Korea. Methods: We included 406 Filipino women married to Korean in the Filipino Women's Diet and Health Study (FiLWHEL). Dietary fat intake was assessed using 24-hour recalls. Impaired blood lipid profiles were defined as high total cholesterol (TC) (≥200 mg/dL), high triglyceride (TG) (≥150 mg/dL), high LDL Cholesterol (LDL-C) (≥ 130 mg/dL), or low HDL cholesterol (HDL-C) (<50 mg/dL). The genomic DNA samples were genotyped using DNA chip. The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using multivariate logistic regression. Results: Substituting carbohydrates with dietary saturated fat (SFA) intake was associated with increased prevalence of dyslipidemia; ORs (95% CIs) for subsequent tertiles compared to the first tertile were 2.28 (1.19-4.35), and 2.88 (1.29-6.39) (P for trend = 0.02). When we examined individual markers, ORs (95% CIs, P for trend) comparing the third to the first tertile were 3.62 (1.53-8.55, 0.01) for high TC, 1.46 (0.42-5.10, 0.72) for high TG, 4.00 (1.48-10.79, 0.02) for high LDL-C, and 0.69 (0.30-1.59, 0.36) for low HDL-C. When we examined the interaction by LDL-C-related polymorphisms, the association with dyslipidemia was more pronounced among participants with CC alleles than among those with T alleles of rs6102059 (P for interaction = 0.01). Conclusions: High dietary SFA intake was significantly associated with a high prevalence of dyslipidemia in Filipino women in Korea. Further prospective cohort studies are warranted to determine risk factors for CVD in Southeast Asian populations.

Screening for pancreatic cancer has the potential to save lives, but is it practical?

INTRODUCTION: Most patients with pancreatic cancer present with advanced stage, incurable disease. However, patients with high-grade precancerous lesions and many patients with low-stage disease can be cured with surgery, suggesting that early detection has the potential to improve survival. While serum CA19.9 has been a long-standing biomarker used for pancreatic cancer disease monitoring, its low sensitivity and poor specificity have driven investigators to hunt for better diagnostic markers. AREAS COVERED: This review will cover recent advances in genetics, proteomics, imaging, and artificial intelligence, which offer opportunities for the early detection of curable pancreatic neoplasms. EXPERT OPINION: From exosomes, to circulating tumor DNA, to subtle changes on imaging, we know much more now about the biology and clinical manifestations of early pancreatic neoplasia than we did just five years ago. The overriding challenge, however, remains the development of a practical approach to screen for a relatively rare, but deadly, disease that is often treated with complex surgery. It is our hope that future advances will bring us closer to an effective and financially sound approach for the early detection of pancreatic cancer and its precursors.

Stepping responses for reactive balance for individuals with incomplete spinal cord injury.

Incomplete spinal cord injury (iSCI) causes impairment of reactive balance control, leading to higher fall risk. In our previous work, we found that individuals with iSCI were more likely to exhibit multiple-step response during the lean-and-release (LR) test, where the participant leaned forward while a tether supported 8-12% of the body weight and received a sudden release, inducing reactive steps. Here we investigated the foot placement of people with iSCI during the LR test using margin-of-stability (MOS). Twenty-one individuals with iSCI (age: 56.1 ± 16.1 years old; mass: 72.5 ± 19.0 kg; height: 166 ± 12 cm), and fifteen age- and sex-matched able-bodied (AB) individuals (age: 56.1 ± 12.9 years old; mass: 57.4 ± 10.9 kg; height: 164 ± 8 cm) participated in the study. The participants performed ten trials of the LR test and also completed clinical assessment of balance and strengths, including the Mini-Balance Evaluations Systems Test, the Community Balance and Mobility Scale, gait speed, and lower extremity manual muscle testing. MOS was significantly smaller during multiple-step responses than during single-step responses for both individuals with iSCI and AB counterparts. Using binary logistic regression and receiver operating characteristic analyses, we demonstrated that MOS can distinguish single- and multiple-step responses. In addition, individuals with iSCI demonstrated significantly larger intra-subject variability of MOS compared to AB individuals at first foot contact. Further, we found that MOS correlated with clinical measures of balance including one for reactive balance. We conclude that individuals with iSCI were less likely to demonstrate foot placement with sufficiently large MOS, which may increase the tendency to exhibit multiple-step responses.

Mechanisms of antigen-induced reversal of CNS inflammation in experimental demyelinating disease.

Autoimmune central nervous system (CNS) demyelinating diseases are a major public health burden and poorly controlled by current immunosuppressants. More precise immunotherapies with higher efficacy and fewer side effects are sought. We investigated the effectiveness and mechanism of an injectable myelin-based antigenic polyprotein MMPt (myelin oligodendrocyte glycoprotein, myelin basic protein and proteolipid protein, truncated). We find that it suppresses mouse experimental autoimmune encephalomyelitis without major side effects. MMPt induces rapid apoptosis of the encephalitogenic T cells and suppresses inflammation in the affected CNS. Intravital microscopy shows that MMPt is taken up by perivascular F4/80+ cells but not conventional antigen-presenting dendritic cells, B cells, or microglia. MMPt-stimulated F4/80+ cells induce reactive T cell immobilization and apoptosis in situ, resulting in reduced infiltration of inflammatory cells and chemokine production. Our study reveals alternative mechanisms that explain how cognate antigen suppresses CNS inflammation and may be applicable for effectively and safely treating demyelinating diseases.