Microdissected Tissue Research Articles

Technology Innovation for Discovering Renal Autoantibodies in Autoimmune Conditions

Autoimmune glomerulonephritis is a homogeneous area of renal pathology with clinical relevance in terms of its numerical impact and difficulties in its treatment. Systemic lupus erythematosus/lupus nephritis and membranous nephropathy are the two most frequent autoimmune conditions with clinical relevance. They are characterized by glomerular deposition of circulating autoantibodies that recognize glomerular antigens. Technologies for studying renal tissue and circulating antibodies have evolved over the years and have culminated with the direct analysis of antigen–antibody complexes in renal bioptic fragments. Initial studies utilized renal microdissection to obtain glomerular tissue. Obtaining immunoprecipitates after partial proteolysis of renal tissue is a recent evolution that eliminates the need for tissue microdissection. New technologies based on ‘super-resolution microscopy’ have added the possibility of a direct analysis of the interaction between circulating autoantibodies and their target antigens in glomeruli. Peptide and protein arrays represent the new frontier for identifying new autoantibodies in circulation. Peptide arrays consist of 7.5 million aligned peptides with 16 amino acids each, which cover the whole human proteome; protein arrays utilize, instead, a chip containing structured proteins, with 26.000 overall. An example of the application of the peptide array is the discovery in membranous nephropathy of many new circulating autoantibodies including formin-like-1, a protein of podosomes that is implicated in macrophage movements. Studies that utilize protein arrays are now in progress and will soon be published. The contribution of new technologies is expected to be relevant for extending our knowledge of the mechanisms involved in the pathogenesis of several autoimmune conditions. They may also add significant tools in clinical settings and modify the therapeutic handling of conditions that are not considered to be autoimmune.

Lymphoid Aggregates in Canine Cutaneous and Subcutaneous Sarcomas: Immunohistochemical and Gene Expression Evidence for Tertiary Lymphoid Structures.

Canine cutaneous/subcutaneous soft-tissue sarcomas (STS) are diversely derived mesenchymal neoplasms with a risk of recurrence and/or metastasis depending on the extent of surgical excision and histologic grade. Lymphoid aggregates (LAs) are often described in these tumours but not characterised. In humans, LA characterised as tertiary lymphoid structures (TLSs) improve the prognosis of many tumours, including sarcomas. We sought to determine if LA meeting a size criterion (> 700 cells) in canine sarcomas met the criteria of TLS and the overall prevalence of LA of any size. RNA expression in large LAs versus aggregate-adjacent sarcoma tissue (AAS) was measured in laser capture microdissected tissue and compared to curl-derived RNA from aggregate-free sarcomas and lymph nodes. CD3, CD20, MUM-1 and PNAd expressions were measured using immunohistochemistry. CD20 and CD3 mRNA were more highly expressed in LA versus AAS (13.8 fold, p = 0.0003 and 2.3 fold, p = 0.043). This was supported by the IHC findings. The large LAs were also enriched in chemokine RNA expression characteristic of TLS (CXCR5 5.8 fold, p < 00001, CCL19 3.68 fold, p = 0.0209, CCL21 6.87 fold, p = 0.0209 and CXCL13 2.68 fold, p = 0.0924). Plasma cells and high endothelial venules were identified in LA containing tumours but not in control tissue. Large LAs were present in 12% of tumours, and LA of any size in 30%. We conclude that large LAs in canine STS are consistent with TLS.

8302 A Novel Tool to Map Estrogen Responsiveness Within Estrogen Sensitive Hypothalamic Neurons in Mice

Abstract Disclosure: A.L. Cara: None. A.L. Hall: None. K.K. Soma: None. J. van Veen: None. S.M. Correa: None. Identification of hormone sensitive cells has been traditionally evaluated by hormone receptor expression. Receptor positive cells can be labeled via expression of mRNA transcript via in situ hybridization, presence of protein via immunohistology, or via conditionally-dependent Cre recombinase driven reporter expression. However, these methods used to label and manipulate cells only indicate which cells are capable of sensing hormones, and not necessarily which cells are responding to a hormonal signal in a specific physiologic or behavioral context. Furthermore, broad labeling of all receptor positive cells may not accurately reflect the heterogeneous nature of certain cell populations, particularly in the brain. The hypothalamus is a brain region with well characterized hormone receptor expression, including estrogen receptors, such as ERα (encoded by the Esr1 gene). Estrogens have a broad array of physiologic and behavioral functions which are mediated through hypothalamic nuclei, including thermoregulation, metabolism, and reproduction. Neuronal populations mediating these functions have been characterized, targeted, and manipulated based on ERα expression, but the dynamic response to estrogens in these highly heterogeneous populations has yet to be uncovered. We developed a custom reporter virus that fluorescently labels estrogen-sensitive and responsive cells. The virus contains a flip-excision switch (FLEX) to drive Cre-dependent expression of a FusionRed fluorescent reporter, eight tandem estrogen response elements (ERE), and a destabilized enhanced green fluorescent protein (EGFP) with a 2-hour half-life. The Cre-dependent expression of FusionRed identifies all Esr1-Cre cells at the time of transduction, while the ERE-EGFP identifies a subset of Esr1-Cre+ cells that are actively responding to estrogens via nuclear receptor signaling. We stereotactically injected the ERE-EGFP virus to distinct hypothalamic nuclei of Esr1-Cre female mice. Following transduction, mice were sacrificed during different stages of the estrous cycle. Spatial analysis of ERE-EGFP expression revealed dynamic changes across the estrous cycle, with responses differing based on hypothalamic nucleus. Reporter expression dynamics were compared to estrogen levels measured by LC-MS/MS in ovary, serum, and microdissected brain tissue. A separate group of mice were ovariectomized and given estradiol benzoate (EB) or vehicle control, and neurons were flow sorted for bulk RNA sequencing. We discovered transcriptomic differences in FusionRed+ cells with high or low ERE-EGFP expression, including upregulation of estrogen target genes (e.g., Pgr, encoding progesterone receptor) with EB treatment in ERE-EGFP+ cells. In summary, this new viral reporter reveals divergent responses to estrogens across subsets of estrogen sensitive neurons, brain regions, and hormonal states. Presentation: 6/2/2024

A modified dual preparatory method for improved isolation of nucleic acids from laser microdissected fresh-frozen human cancer tissue specimens.

A central theme in cancer research is to increase our understanding of the cancer tissue microenvironment, which is comprised of a complex and spatially heterogeneous ecosystem of malignant and non-malignant cells, both of which actively contribute to an intervening extracellular matrix. Laser microdissection (LMD) enables histology selective harvest of cellular subpopulations from the tissue microenvironment for their independent molecular investigation, such as by high-throughput DNA and RNA sequencing. Although enabling, LMD often requires a labor-intensive investment to harvest enough cells to achieve the necessary DNA and/or RNA input requirements for conventional next-generation sequencing workflows. To increase efficiencies, we sought to use a commonplace dual preparatory (DP) procedure to isolate DNA and RNA from the same LMD harvested tissue samples. While the yield of DNA from the DP protocol was satisfactory, the RNA yield from the LMD harvested tissue samples was significantly poorer compared to a dedicated RNA preparation procedure. We determined that this low yield of RNA was due to incomplete partitioning of RNA in this widely used DP protocol. Here, we describe a modified DP protocol that more equally partitions nucleic acids and results in significantly improved RNA yields from LMD-harvested cells.

Resolving multi-image spatial lipidomic responses to inhaled toxicants by machine learning.

Regional responses to inhaled toxicants are essential to understand the pathogenesis of lung disease under exposure to air pollution. We evaluated the effect of combined allergen sensitization and ozone exposure on eliciting spatial differences in lipid distribution in the mouse lung that may contribute to ozone-induced exacerbations in asthma. Lung lobes from male and female BALB/c mice were cryosectioned and acquired by high resolution mass spectrometry imaging (MSI). Processed MSI peak annotations were validated by LC-MS/MS data from scraped tissue slides and microdissected lung tissue. Images were normalized and segmented into clusters. Interestingly, segmented clusters overlapped with stained serial tissue sections, enabling statistical analysis across biological replicates for morphologically relevant lung regions. Spatially distinct lipids had higher overall degree of unsaturated fatty acids in distal lung regions compared to proximal regions. Furthermore, the airway and alveolar epithelium exhibited significantly decreased sphingolipid and glycerophospholipid abundance in females, but not in males. We demonstrate the potential role of lipid saturation in healthy lung function and highlight sex differences in regional lung lipid distribution following ozone exposure. Our study provides a framework for future MSI experiments capable of relative quantification across biological replicates and expansion to multiple sample types, including human tissue.

A multistep computational approach reveals a neuro-mesenchymal cell population in the embryonic hematopoietic stem cell niche.

The first hematopoietic stem and progenitor cells (HSPCs) emerge in the Aorta-Gonad-Mesonephros (AGM) region of the mid-gestation mouse embryo. However, the precise nature of their supportive mesenchymal microenvironment remains largely unexplored. Here, we profiled transcriptomes of laser micro-dissected aortic tissues at three developmental stages and individual AGM cells. Computational analyses allowed the identification of several cell subpopulations within the E11.5 AGM mesenchyme, with the presence of a yet unidentified subpopulation characterized by the dual expression of genes implicated in adhesive or neuronal functions. We confirmed the identity of this cell subset as a neuro-mesenchymal population, through morphological and lineage tracing assays. Loss of function in the zebrafish confirmed that Decorin, a characteristic extracellular matrix component of the neuro-mesenchyme, is essential for HSPC development. We further demonstrated that this cell population is not merely derived from the neural crest, and hence, is a bona fide novel subpopulation of the AGM mesenchyme.

Abstract PR007: Molecular subtyping of lung adenocarcinoma premalignant lesions identifies features associated with aggressive disease

Abstract Lung cancer is the leading cause of cancer-related death. The five year survival rate had remained stagnant for decades, but more effective screening protocols using low-dose computed tomographic screening for high-risk individuals, along with progress in treatment for advanced-stage lung cancer with targeted therapy and immunotherapy, have helped decrease lung cancer mortality. However, improved approaches to lung cancer prevention and early detection are still urgently needed. Adenocarcinoma (LUAD) is the most common form of lung cancer. Following surgical resection of pre-invasive adenocarcinoma in situ, the five-year survival rate approaches 100%. This emphasizes the need for improved understanding of characteristics of aggressive premalignant lesions that would benefit from early interventions. In contrast to the well-characterized histopathologic sequence of lung squamous cell carcinoma, atypical adenomatous hyperplasia (AAH) and adenocarcinoma in situ (AIS) are the only known precursors in the sequence of LUAD pathogenesis. The features of AAH and AIS that lead to invasive LUAD are poorly characterized. The relationship of AIS and AAH to the histologically diverse subtypes of LUAD, ranging from the typically slow growing lepidic to aggressive solid subtypes, are also unclear. We hypothesized that transcriptomic changes in subsets of premalignant lesions are linked to distinct genomic and clinicopathologic features of malignant disease. To investigate this hypothesis, we performed exome sequencing and bulk RNA sequencing of laser capture microdissected tissue from tumor margins that included premalignant lesions, invasive tumor, and adjacent normal tissues. With our analysis, we discovered de novo subtypes of LUAD premalignant lesions based on gene expression. One of these subtypes had gene expression alterations similar to aggressive invasive LUAD solid tumors. This molecular adenomatous premalignant lesion subtype was also associated with an increased accumulation of cancer driver mutations. This subtype was further characterized by altered expression of immune-related pathways. Molecular signatures measured in adenomatous PML may thus enhance our understanding of pathway dysregulation and mutational heterogeneity occurring during LUAD carcinogenesis, and implicate immunotherapeutic strategies to prevent their progression to cancer. Citation Format: Kelley Anderson, Linh Tran, Kostyantyn Krysan, William Wallace, Gregory Fishbein, Emily Green, Gang Liu, Hanqiao Liu, Erin Kane, Sarah Mazzilli, Steven Dubinett, Avrum Spira, Marc Lenburg, Jennifer Beane. Molecular subtyping of lung adenocarcinoma premalignant lesions identifies features associated with aggressive disease [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr PR007.

Abstract C059: Hub gene analysis identifies CAD as a potential mediator for drug resistance in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a complex disease pathobiology and poor treatment options, emphasizing the need for novel therapeutic targets. Hub genes have high connectivity (i.e., exceptionally many interaction partners), for example, certain proteases with numerous interaction partners. Thereby, hub genes are often central biological regulators and dysregulated proteolysis is indeed a key trait of malignant tissues. Here, we aimed to identify novel protease-related hub genes with potential involvement in pancreatic cancer. We applied an integrative computational approach to compartment-specific multiOMIC profiles of laser-capture micro-dissected human PDAC tissues (n = 32) and queried associations of identified candidate genes with clinical outcomes in early (n = 186) and advanced PDAC patients (n = 253). This identified 20 protease-related genes with ≥ 10 physically verified interaction partners in pancreatic cancer. Among these, the pyrimidine biosynthesis regulator gene CAD emerged as a potentially druggable target in pancreatic cancer. CAD expression was nearly restricted to the malignant PDAC epithelium. Furthermore, higher CAD expression was associated with significantly worse overall survival in patients with advanced but not resectable disease, suggesting a role in treatment response rather than disease progression. Moreover, patients with higher CAD expression specifically demonstrated poor response to mFFX but not Gemcitabine-nab-Paclitaxel treatment, which is in line with previous reports on CAD expression being protective against 5-Fluouracil, a component of FFX. Moving forward, we will interrogate the role of CAD in PDAC tissue biology and scrutinize its involvement in FFX resistance, using patient-derived organoids. Citation Format: Foram Vyas, Barbara Grünwald, Kazeera Aliar, Gun Ho Jang, Grainne O'Kane, Thomas Kislinger, Steven Gallinger, Benjamin Haibe-Kains, Rama Khokha. Hub gene analysis identifies CAD as a potential mediator for drug resistance in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C059.

A Fast-Tracking Sample Preparation Protocol for Proteomics of Formalin-Fixed Paraffin-Embedded Tumor Tissues.

Archived tumor specimens are routinely preserved by formalin fixation and paraffin embedding. Despite the conventional wisdom that proteomics might be ineffective due to the cross-linking and pre-analytical variables, these samples have utility for both discovery and targeted proteomics. Building on this capability, proteomics approaches can be used to maximize our understanding of cancer biology and clinical relevance by studying preserved tumor tissues annotated with the patients' medical histories. Proteomics of formalin-fixed paraffin-embedded (FFPE) tissues also integrates with histological evaluation and molecular pathology strategies, so that additional collection of research biopsies or resected tumor aliquots is not needed. The acquisition of data from the same tumor sample also overcomes concerns about biological variation between samples due to intratumoral heterogeneity. However, the protein extraction and proteomics sample preparation from FFPE samples can be onerous, particularly for small (i.e., limited or precious) samples. Therefore, we provide a protocol for a recently introduced kit-based EasyPep method with benchmarking against a modified version of the well-established filter-aided sample preparation strategy using laser-capture microdissected lung adenocarcinoma tissues from a genetically engineered mouse model. This model system allows control over the tumor preparation and pre-analytical variables while also supporting the development of methods for spatial proteomics to examine intratumoral heterogeneity. Data are posted in ProteomeXchange (PXD045879).

Neuronal MeCP2 in the dentate gyrus regulates mossy fiber sprouting of mice with temporal lobe epilepsy

Sprouting of mossy fibers, one of the most consistent findings in tissue from patients with mesial temporal lobe epilepsy, exhibits several uncommon axonal growth features and has been considered a paradigmatic example of circuit plasticity that occurs in the adult brain. Clarifying the mechanisms responsible may provide new insight into epileptogenesis as well as axon misguidance in the central nervous system. Methyl-CpG-binding protein 2 (MeCP2) binds to methylated genomic DNA to regulate a range of physiological functions implicated in neuronal development and adult synaptic plasticity. However, exploring the potential role of MeCP2 in the documented misguidance of axons in the dentate gyrus has not yet been attempted. In this study, a status epilepticus-induced decrease of neuronal MeCP2 was observed in the dentate gyrus (DG). An essential regulatory role of MeCP2 in the development of functional mossy fiber sprouting (MFS) was confirmed through stereotaxic injection of a recombinant adeno-associated virus (AAV) to up- or down-regulate MeCP2 in the dentate neurons. Chromatin immunoprecipitation sequencing (ChIP-seq) was performed to identify the binding profile of native MeCP2 using micro-dissected dentate tissues. In both dentate tissues and HT22 cell lines, we demonstrated that MeCP2 could act as a transcription repressor on miR-682 with the involvement of the DNA methylation mechanism. Further, we found that miR-682 could bind to mRNA of phosphatase and tensin homolog (PTEN) in a sequence specific manner, thus leading to the suppression of PTEN and excessive activation of mTOR. This study therefore presents a novel epigenetic mechanism by identifying MeCP2/miR-682/PTEN/mTOR as an essential signal pathway in regulating the formation of MFS in the temporal lobe epileptic (TLE) mice. Significance statementUnderstanding the mechanisms that regulate axon guidance is important for a better comprehension of neural disorders. Sprouting of mossy fibers, one of the most consistent findings in patients with mesial temporal lobe epilepsy, has been considered a paradigmatic example of circuit plasticity in the adult brain. Although abnormal regulation of DNA methylation has been observed in both experimental rodents and humans with epilepsy, the potential role of DNA methylation in this well-documented example of sprouting of dentate axon remains elusive. This study demonstrates an essential role of methyl-CpG-binding protein 2 in the formation of mossy fiber sprouting. The underlying signal pathway has been also identified. The data hence provide new insight into epileptogenesis as well as axon misguidance in the central nervous system.

Construction of multiplexed transcriptome NGS libraries of microdissected tissue samples based on combinational DNA barcode microbeads.

The combination of single-cell RNA sequencing and microdissection techniques that preserves positional information has become a major tool for spatial transcriptome analyses. However, high costs and time requirements, especially for experiments at the single cell scale, make it challenging for this approach to meet the demand for increased throughput. Therefore, we proposed combinational DNA barcode (CDB)-seq as a medium-throughput, multiplexed approach combining Smart-3SEQ and CDB magnetic microbeads for transcriptome analyses of microdissected tissue samples. We conducted a comprehensive comparison of conditions for CDB microbead preparation and related factors and then applied CDB-seq to RNA extracts, fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) mouse brain tissue samples. CDB-seq transcriptomic profiles of tens of microdissected samples could be obtained in a simple, cost-effective way, providing a promising method for future spatial transcriptomics.

NanoLC-MS/MS protein analysis on laser-microdissected wheat endosperm tissues: A comparison between aleurone, sub-aleurone and inner endosperm

Wheat kernel proteins are not homogeneously distributed throughout the endosperm. The goal of this study was to investigate the relative differences in protein composition between the aleurone, sub-aleurone and inner endosperm. Using laser microdissection followed by nanoLC-MS/MS, an innovative method combining high spatial specificity and analytical selectivity in sample-limited situations, 780 proteins were detected and classified by function. A higher proportion of gluten proteins was detected in the sub-aleurone than inner endosperm. Composition-wise, gluten from the sub-aleurone is relatively more enriched in ω-gliadins but impoverished in LMW-GS and γ-gliadins. While a basic set of albumins and globulins was detected in all three microdissected endosperm tissues, specific proteins, like puroindoline B, displayed a gradient. This study provides indications that both histological origin and relative positioning of the tissues drive the protein distribution. Knowledge of this protein distribution offers significant opportunities for the wheat manufacturing industry. Data available via ProteomeXchange, identifier PXD038743.

FRI278 Kisspeptin Neuron Projections And Expression Patterns Are Unaltered In Prenatally Androgenized Female Mice Modeling PCOS

Abstract Disclosure: M. Prescott: None. Y. Watanabe: None. A. Ruddenklau: None. L. Fisher: None. C.L. Jasoni: None. R.E. Campbell: Consulting Fee; Self; Bayer, Inc. Polycystic ovary syndrome (PCOS) is associated with impaired gonadal steroid hormone feedback and a hyperactive central reproductive axis, implicating the brain in PCOS pathophysiology. Excess prenatal androgen (PNA) exposure is linked with the development of PCOS, and preclinical evidence suggests that PNA can program altered connections within the gonadotropin-releasing hormone (GnRH) neuronal network. Kisspeptin neurons are critical upstream regulators of GnRH neurons, relaying hormone feedback and potently activating GnRH secretion, but their role in PCOS remains incompletely understood. This study tested the hypothesis that prenatal androgen excess programmes altered neuroanatomical connections between kisspeptin neurons and GnRH neurons and modified neuropeptide and steroid hormone receptor expression in kisspeptin neurons. To investigate this, we labelled, imaged and assessed kisspeptin fibers, GnRH neurons, and gonadal steroid hormone receptors in adult female transgenic kisspeptin reporter mice (Kiss1(Cre-GFP)Coll; ROSA26tdTomato) exposed prenatally to di-hydrotestosterone or an oil vehicle (VEH) on gestational days 16-18 (n=7-10/group). PNA mice were acyclic and all tissue was collected in diestrus. Fixed coronal brain sections were immunofluorescently labelled for GnRH and kisspeptin reporter tdTomato and imaged on a confocal microscope to assess close fiber contacts to the soma and proximal dendrite of GnRH neurons. Neither the proportion of GnRH neurons with kisspeptin fiber contacts (VEH: 86.3±3.4%, PNA: 87.3±4.3%), nor the mean number of kisspeptin fiber close appositions per GnRH neuron (VEH: 6.1±0.6, PNA: 5.7±0.8) was different between groups. Kisspeptin neuron number and kisspeptin neuron co-expression with estrogen and progesterone receptors was evaluated in immunolabelled sections throughout the rostral periventricular nucleus of the 3rd ventricle (RP3V) and the arcuate nucleus (ARN). PNA had no significant impact on kisspeptin neuron number or the proportion of neurons with gonadal hormone co-expression in either population. Expression of neuropeptides was quantified by RT-qPCR in micro-dissected RP3V and ARN tissue from a separate cohort of adult PNA and VEH mice (n=5-8/group). PNA did not affect Kiss1, Tac2 or Pdyn expression in the ARN or Kiss1 expression in the RP3V. These data indicate that PNA does not modify anatomical connections between RP3V kisspeptin neurons and GnRH neurons, nor does it have a robust impact on steroid hormone responsiveness or neuropeptide expression of kisspeptin neurons. While the neuroanatomical relationship between ARN kisspeptin neurons and the GnRH neuron distal dendron in PNA mice remains to be determined, these data suggest that PNA-mediated neuroendocrine dysfunction is not due to programmed changes in the neuroanatomical projections or gonadal hormone sensitivity of kisspeptin neurons. Presentation: Friday, June 16, 2023

Bitter taste receptor (TAS2R) 46 in human skeletal muscle: expression and activity.

Bitter taste receptors are involved not only in taste perception but in various physiological functions as their anatomical location is not restricted to the gustatory system. We previously demonstrated expression and activity of the subtype hTAS2R46 in human airway smooth muscle and broncho-epithelial cells, and here we show its expression and functionality in human skeletal muscle cells. Three different cellular models were used: micro-dissected human skeletal tissues, human myoblasts/myotubes and human skeletal muscle cells differentiated from urine stem cells of healthy donors. We used qPCR, immunohistochemistry and immunofluorescence analysis to evaluate gene and protein hTAS2R46 expression. In order to explore receptor activity, cells were incubated with the specific bitter ligands absinthin and 3ß-hydroxydihydrocostunolide, and calcium oscillation and relaxation were evaluated by calcium imaging and collagen assay, respectively, after a cholinergic stimulus. We show, for the first time, experimentally the presence and functionality of a type 2 bitter receptor in human skeletal muscle cells. Given the tendentially protective role of the bitter receptors starting from the oral cavity and following also in the other ectopic sites, and given its expression already at the myoblast level, we hypothesize that the bitter receptor can play an important role in the development, maintenance and in the protection of muscle tissue functions.

P53 loss of heterozygosity (LOH) in formalin-fixed paraffin-embedded leiomyosarcoma (LMS): a novel report.

The occurrence of p53 loss of heterozygosity (LOH) is a common genetic event in malignancy. LOH occurs when a heterozygous locus loses one of its two parental alleles, becoming homozygous at that locus, by either copy number loss (CNL-LOH) or by becoming copy number neutral (CNN-LOH). A role for CNL-LOH (cnLOH) has been postulated in cancer aetiology. Loss of heterozygosity (LOH) results in irreversible genetic loss. LOH was determined in DNA extracted from formalin-fixed paraffin-embedded (FFPE) leiomyosarcoma (LMS) specimens in a retrospective study from 30 patients, to assess the prognostic significance of LOH. The findings were analysed and their validity assessed. LOH was an adverse prognostic factor in LMS. Prospective uniform standardisation of formalin-fixation techniques is required. DNA was extracted from 169 formalin-fixed paraffin blocks of 30 patients with LMS, following extensive tissue microdissection. Genomic DNA was amplified using the polymerase chain reaction (PCR) technique. Fluorescence-based microsatellite PCR was used to detect and quantitate heterozygosity loss. LOH was detected at gene locus 17p13 in 16 LMS (Four grade 2 and 12 grade 3 LMS). LOH was not detected in 14 LMS cases (one grade 1, five grade 2 and eight grade 3 LMS). LOH was associated with shorter patient survival. The results reported herein endorse the value of utilizing FFPE DNA in identifying LOH as a prognostic factor in LMS. The results have implications for tumour biobanking and precision medicine in patients with sarcomas.

Deciphering Stromal Changes between Metastatic and Non-metastatic Canine Mammary Carcinomas

Cancer-associated stroma (CAS) is widely recognized to influence development and progression of epithelial tumours including breast cancer. Canine mammary tumours (CMTs) such as simple canine mammary carcinomas represent valuable models for human breast cancer also with respect to stromal reprogramming. However, it remains unclear whether and how CAS changes in metastatic tumours compared to non-metastatic ones. To characterize stromal changes between metastatic and non-metastatic CMTs and identify potential drivers of tumour progression, we analysed CAS and matched normal stroma from 16 non-metastatic and 15 metastatic CMTs by RNA-sequencing of microdissected FFPE tissue. We identified 1438 differentially regulated genes between CAS and normal stroma, supporting previous results demonstrating stromal reprogramming in CMTs to be comparable with CAS in human breast cancer and validating deregulation of pathways and genes associated with CAS. Using primary human fibroblasts activated by treatment with TGFβ, we demonstrate some of the strongest expression changes to be conserved in fibroblasts across species. Furthermore, we identify 132 differentially expressed genes between CAS from metastatic and non-metastatic tumours, with strong changes in pathways including chemotaxis, regulation of apoptosis, immune response and TGFβ signalling and validate deregulation of several targets using RT-qPCR. Finally, we identify specific upregulation of COL6A5, F5, GALNT3, CIT and MMP11 in metastatic CAS, suggesting high stromal expression of these targets to be linked to malignancy and metastasis of CMTs. In summary, our data present a resource supporting further research into stromal changes of the mammary gland in relation to metastasis with implications for both canine and human mammary cancer.

A High-Throughput Workflow for FFPE Tissue Proteomics.

Laser capture microdissection (LCM) has become an indispensable tool for mass spectrometry-based proteomic analysis of specific regions obtained from formalin-fixed paraffin-embedded (FFPE) tissue samples in both clinical and research settings. Low protein yields from LCM samples along with laborious sample processing steps present challenges for proteomic analysis without sacrificing protein and peptide recovery. Automation of sample preparation workflows is still under development, especially for samples such as laser-capture microdissected tissues. Here, we present a simplified and rapid workflow using adaptive focused acoustics (AFA) technology for sample processing for high-throughput FFPE-based proteomics. We evaluated three different workflows: standard extraction method followed by overnight trypsin digestion, AFA-assisted extraction and overnight trypsin digestion, and AFA-assisted extraction simultaneously performed with trypsin digestion. The use of AFA-based ultrasonication enables automated sample processing for high-throughput proteomic analysis of LCM-FFPE tissues in 96-well and 384-well formats. Further, accelerated trypsin digestion combined with AFA dramatically reduced the overall processing times. LC-MS/MS analysis revealed a slightly higher number of protein and peptide identifications in AFA accelerated workflows compared to standard and AFA overnight workflows. Further, we did not observe any difference in the proportion of peptides identified with missed cleavages or deamidated peptides across the three different workflows. Overall, our results demonstrate that the workflow described in this study enables rapid and high-throughput sample processing with greatly reduced sample handling, which is amenable to automation.

Transcriptome Analysis Identifies Oncogenic Tissue Remodeling during Progression from Common Nevi to Early Melanoma

Early detection and treatment of melanoma, the most aggressive skin cancer, improves the median 5-year survival rate of patients from 25% to 99%. Melanoma development involves a stepwise process during which genetic changes drive histological alterations within nevi and surrounding tissue. Here, we perform a comprehensive analysis of publicly available gene expression datasets of melanoma, common or congenital nevi (CN), and dysplastic nevi (DN), and assess molecular & genetic pathways leading to early melanoma. Our results demonstrate several pathways reflective of ongoing local structural tissue remodeling activity likely involved during the transition from benign to early-stage melanoma. These processes include the gene expression of cancer associated fibroblasts (CAF), collagens, extracellular matrix (ECM) and integrins, which assist early melanoma development and the immune surveillance which plays a substantial role at this early stage. Further, we show genes upregulated in dysplastic nevi (DN) are also overexpressed in melanoma tissue, supporting the notion DN may serve as a transitional phase towards oncogenesis. Lastly, we reveal CN collected from healthy individuals exhibit different gene signatures compared to histologically benign nevi tissue located adjacent to melanoma (adjacent nevi, AN). We show the expression profile of micro-dissected AN tissue is more similar to melanoma compared to CN, revealing the melanoma influence on this annexed tissue.

Single-nucleus RNA sequencing of pre-malignant liver reveals disease-associated hepatocyte state with HCC prognostic potential

Current approaches to staging chronic liver diseases have limited utility for predicting liver cancer risk. Here, we employed single-nucleus RNA sequencing (snRNA-seq) to characterize the cellular microenvironment of healthy and pre-malignant livers using two distinct mouse models. Downstream analyses unraveled a previously uncharacterized disease-associated hepatocyte (daHep) transcriptional state. These cells were absent in healthy livers but increasingly prevalent as chronic liver disease progressed. Copy number variation (CNV) analysis of microdissected tissue demonstrated that daHep-enriched regions are riddled with structural variants, suggesting these cells represent a pre-malignant intermediary. Integrated analysis of three recent human snRNA-seq datasets confirmed the presence of a similar phenotype in human chronic liver disease and further supported its enhanced mutational burden. Importantly, we show that high daHep levels precede carcinogenesis and predict a higher risk of hepatocellular carcinoma development. These findings may change the way chronic liver disease patients are staged, surveilled, and risk stratified.

Abstract 4320: A novel digitally directed tissue microdissection platform integrates digital pathology with molecular analysis: Case study in metastatic colorectal cancer

Abstract Artificial intelligence (AI) based digital pathology has been getting rapid adoption over the last 2 to 3 years These AI based digital pathology models can analyze samples at high resolution and scale Retaining that resolution at scale through molecular analysis is key to the discovery of novel biomarkers We have developed a technology that can integrate AI-supported digital pathology (DP) algorithms to identify informative tissue regions for selective microdissection to improve molecular analysis of FFPE material The system is capable of extracting DNA, RNA or proteins with cellular (10 micron) resolution from FFPE tissue sections. The samples are ready for use in off-the-shelf molecular analysis workflows (qPCR, Next Gen Sequencing, etc) The system decreases the tumor cellularity requirements for clinical analysis by extracting only the area enriched for informative biomarkers To demonstrate the capability of the system we used an AI based digital pathology model that was developed to identify multiple cellular phenotypes from a H&amp;E stained colorectal cancer FFPE tissue sections We used the digital pathology output to guide the microdissection of RNA from tumor buds, carcinoma, immature stroma and inflammatory stroma in a paired designed RNA seq experiment The data showed that the system is capable of enriching the tumor-bud tissue content from&amp;lt;1% in the tissue to ~50% purity in the crude lysate Bioinformatic analysis of the RNAseq signatures showed that the tumor buds had a distinct RNAseq signature relative to the other three cell types and that the gene expression pattern was consistent with EMT pathways. The methodology is scalable, robust, and simpler than current microdissection techniques. This technology can be used to develop novel tests by integrating with AI algorithms, targeting tissue enriched for predictive biomarkers, and enabling low tumor cellularity samples to be analyzed with high precision. Citation Format: John H. Butler, Bidhan Chaudhuri, Katie Konigsfeld, Rish Pai. A novel digitally directed tissue microdissection platform integrates digital pathology with molecular analysis: Case study in metastatic colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4320.