Tissue Profile Research Articles

Metabolism and spatial transcription resolved heterogeneity of glutamine metabolism in cervical carcinoma

BackgroundReprogramming of cellular metabolism is a pivotal mechanism employed by tumor cells to facilitate cell growth, proliferation, and differentiation, thereby propelling the progression of cancer. A comprehensive analysis of the transcriptional and metabolic landscape of cervical squamous cell carcinoma (CSCC) at high resolution could greatly enhance the precision of management and therapeutic strategies for this malignancy.MethodsThe Air-flow-assisted Desorption Electrospray Ionization Mass Spectro-metric Imaging (AFADESI-MSI) and Spatial Transcriptomics techniques (ST) were employed to investigate the metabolic and transcription profiles of CSCC and normal tissues. For clinical validation, the expression of ASCT2(Ala, Ser, Cys transporter 2) was assessed using immune histochemistry in 122 cases of cervical cancer and 30 cases of cervicitis.ResultsThe AFADESI-MSI findings have revealed metabolic differences among different CSCC patients. Among them, the metabolic pathways of glutamine show more significant differences. After in situ detection of metabolites, the intensity of glutamate is observed to be significantly higher in cancerous tissue compared to normal tissue, but the intensity is not uniform. To elucidate the potential factors underlying alterations in glutamine metabolism across tissues, we employ ST to quantify mRNA levels. This analysis unveils significant perturbations in glutamine metabolism accompanied by extensive heterogeneity within cervical cancer tissues. After conducting a comprehensive analysis, it has been revealed that the differential expression of ASCT2(encoded by SLC1A5) in distinct regions of cervical cancer tissues plays a pivotal role in inducing heterogeneity in glutamine metabolism. Furthermore, the higher the expression level of ASCT2, the higher the intensity of glutamate is in the region. Further verification, it is found that the expression of ASCT2 protein in CSCC tissues is significantly higher than that in normal tissues (105/122, 86.07%).ConclusionsThis finding suggests that the variation in glutamine metabolism is not uniform throughout the tumor. The differential expression of ASCT2 in different regions of cervical cancer tissues seems to play a key role in causing this heterogeneity. This research has opened up new avenues for exploring the glutamine metabolic characteristics of CSCC which is essential for developing more effective targeted therapies.

Elevation of Granulocyte Colony Stimulating Factor in Human AMD Donor RPE-Choroid.

Choroidal inflammation, complement deposition, and accumulation of C-reactive protein (CRP) are involved in age-related macular degeneration (AMD) pathology. The pro-inflammatory signals that regulate immune cell recruitment in the choroid of patients with AMD remain to be determined. We performed cytokine profiling of human AMD and age-matched control donor tissue to identify inflammatory molecules upregulated in AMD tissue. Protein was isolated from 25 AMD and 21 control donor RPE/choroid macular punches. Total protein was quantified, and 50 µg assayed for expression of 40 cytokines using an inflammation array. We validated the elevated expression of granulocyte colony stimulating factor (G-CSF) protein by ELISA in a second cohort of 22 control and 26 AMD donors. To identify an AMD associated stressor responsible for upregulating G-CSF we assayed for changes in G-CSF protein secretion in RPE/choroid organ cultures treated with the monomeric (m)CRP, an inflammatory protein elevated in AMD. Using a multiplex array, we identified elevated G-CSF protein in the choroid of AMD donors compared to age-matched non-AMD controls. Differential expression of G-CSF was confirmed via ELISA in an independent cohort of samples (P = 0.01). The mCRP, which is deposited in AMD choroids, increased G-CSF protein secretion in RPE/choroid organ cultures. Single nuclei RNA sequencing identified choroidal endothelial cells and fibroblasts as the primary cell types responsible for increased G-CSF secretion in response to mCRP. The G-CSF receptor is expressed primarily by choroidal macrophages and dendritic cells and anti-G-CSFR colocalizes with anti-CD45 and anti-CD68 in human donor choroid tissue. Elevated G-CSF expression in AMD donor tissue as a result of increased levels of mCRP may be involved in immune cell recruitment in AMD contributing to inflammatory stress in the choroid.

Differential activity of MAPK signalling defines fibroblast subtypes in pancreatic cancer

Fibroblast heterogeneity is increasingly recognised across cancer conditions. Given their important contribution to disease progression, mapping fibroblasts’ heterogeneity is critical to devise effective anti-cancer therapies. Cancer-associated fibroblasts (CAFs) represent the most abundant cell population in pancreatic ductal adenocarcinoma (PDAC). Whether CAF phenotypes are differently specified by PDAC cell lineages remains to be elucidated. Here, we reveal an important role for the MAPK signalling pathway in defining PDAC CAF phenotypes. We show that epithelial MAPK activity promotes the myofibroblastic differentiation of CAFs by sustaining the expression and secretion of TGF-β1. We integrate single-cell profiling of post-perturbation transcriptional responses from mouse models with cellular and spatial profiles of human tissues to define a MAPKhigh CAF (mapCAF) phenotype. We show that this phenotype associates with basal-like tumour cells and reduced frequency of CD8+ T cells. In addition to elevated MAPK activity, this mapCAF phenotype is characterized by TGF-β signalling, hypoxia responsive signatures, and immunoregulatory gene programs. Furthermore, the mapCAF signature is enriched in myofibroblastic CAFs from various cancer conditions and correlates with reduced response to immune checkpoint inhibition in melanoma. Altogether, our data expand our knowledge on CAF phenotype heterogeneity and reveal a potential strategy for targeting myofibroblastic CAFs in vivo.

Revealing key proteins in comparison of tumor and para-tumor tissues in stage I esophageal squamous cell carcinoma: A combined gene expression clustering and protein interaction network analysis

Esophageal Squamous Cell Carcinoma (ESCC) which is diagnosed in advanced stages and metastasis to the vital organs, remained the sixth fatal malignancy among other types of cancer-related death. Considering the lack of specific clinical symptoms in the early development of the disease, finding suitable biomarkers and tissue-specific alterations for effective screening of ESCC is targeted in this project. Gene expression profiles of tumor tissue and para-tumors of stage I ESCC patients were retrieved from the Gene Expression Omnibus (GEO) database. The significant tissue-specific differentially expressed genes (DEGs) were identified and studied based on fold change distribution and the functional role of genes in the action map. A total number of 11,483 significant DEGs discriminated the tumor tissue from para-tumor tissue. Clustering analysis of significant DEGs led to identifying 220 DEGs as the final significant genes. Protein interaction network and action map analysis of the final gene list showed matrix metallopeptidase-9 (MMP9) as the critical gene related to the development of ESCC diseases in stage I. Significant expression change of MMP9 in esophageal carcinoma was validated in UALCAN (The University of Alabama at Birmingham Cancer Data Analysis Portal). This study highlighted the pivotal role of MMP9 in combination with SPP1, MMP13, and IL18 as a possible biomarkers panel in studies of tumor invasion and prognosis for stage I ESCC disease.

Novel biomarker in hepatocellular carcinoma: Stearoyl-CoA desaturase 1

BackgroundIn recent years, more and more studies have shown that reprogramming lipid metabolism plays an important role in the occurrence and development of hepatocellular carcinoma (HCC). However, there is a lack of systematic exploration of fatty acid (FA) profiles in HCC. AimsThis study aims to systematically investigate the FA profile in HCC and assess the diagnostic potential of stearoyl-CoA desaturase 1 (SCD1) as a biomarker for HCC. MethodsThe FA profile in HCC tissues was detected by gas chromatography mass spectrometry. Abnormal FA metabolism was analyzed by qRT-PCR, Western blot. Immunohistochemical and bioinformatics analysis were used to analyze SCD1 expression and function. Receiver operating characteristic curves were used to analyze the diagnostic efficacy of SCD1, and the relationship between SCD1 and immune infiltration in HCC was analyzed by the biological information method. ResultsFAs were found to accumulate in the HCC samples, and abnormal FA metabolism in HCC related to the upregulation of the expression and activity of SCD1. The combination of SCD1 and alpha-fetoprotein produced a greater area under the receiver operating characteristic curve (0.925, P < 0.001) than SCD1 or alpha-fetoprotein alone. It also showed better sensitivity (77.5 %). Besides, high SCD1 expression was found to be related to immune infiltration in HCC. ConclusionSCD1 can serve as a reliable biomarker for HCC diagnosis.

Novel placental biomarker shows predictive potential for spontaneous preterm labor.

Human parturition involve many events among mother, fetus, and placenta, and the initiation of these events is the consequence of activation of a series of endocrine and immune responses. Multiple underlying pathways account for the cascade of events that culminate in spontaneous preterm labor. In this study, we aimed to characterize these signaling pathways of placental origin at molecular levels. We used single-cell RNA-sequencing (sc-RNA-seq) analysis to probe transcriptional heterogeneity in human placenta delivered at preterm or term and then used RNA in situ hybridization (RNA-ISH) assay on formalin-fixed paraffin-embedded placental tissues to validate these results. By using sc-RNA-seq on villous cytotrophoblast isolated from a preterm placenta, we found that signaling pathways implicated in initiation of term or preterm labor including ferroptosis, kisspeptin, and senescence were constitutively activated in distinct cellular clusters of these trophoblastic stem cells. RNA-ISH-based spatial gene expression profiling in formalin-fixed paraffin-embedded tissues revealed that Pregnancy Specific beta-1-Glycoprotein 4 (PSG4), a potent molecular driver for cellular aging, was significantly increased in preterm placentas (N=30) compared to their full-term counterparts (N=9). Furthermore, PSG4 mRNA signals were predominantly detected in the villous syncytiotrophoblast and the discontinuous monolayer of cytotrophoblast as well as the intervillous space where maternal blood circulates. Our study provides strong support for PSG4 overexpression serving as a biomarker for pregnant women at risk for preterm delivery, which can allow for development of timely and clinical preventive strategies.

Proteomic Analysis of Tissue Proteins Related to Lateral Lymph Node Metastasis in Papillary Thyroid Microcarcinoma.

Patients with lateral lymph node metastasis (LLNM) may experience higher locoregional recurrence rates and poorer prognoses compared to those without LLNM, highlighting the need for effective preoperative stratification to reliably assess risk LLNM. In this study, we collected PTMC samples from Peking Union Medical College Hospital and employed data-independent acquisition mass spectrometry proteomics technique to identify protein profiles in PTMC tissues with and without LLNM. Pseudo temporal analysis and single sample gene set enrichment analysis were conducted in combination with The Cancer Genome Atlas Thyroid Carcinoma for functional coordination analysis and the construction of a prediction model based on random forest. Non-negative matrix factorization (NMF) clustering was utilized to classify molecular subtypes of PTMC. Our findings revealed that the differential activation of pathways such as MAPK and PI3K was critical in enhancing the lateral lymph node metastatic potential of PTMC. We successfully screened biomarkers via machine learning and public databases, creating an effective prediction model for metastasis. Additionally, we explored the mechanism of metastasis-associated PTMC subtypes via NMF clustering. These insights into LLNM mechanisms in PTMC may contribute to future biomarker screening and the identification of therapeutic targets.

Metabolomic profile of cerebral tissue after acoustically-mediated blood-brain barrier opening in a healthy rat model: a focus on the contralateral side.

Microbubble (MB)-assisted ultrasound (US) is an innovative modality for the non-invasive, targeted, and efficient delivery of therapeutic molecules into the brain. Previously, we reported the first metabolomic signature of blood-brain barrier opening (BBBO) induced by MB-assisted US. In the present study, the neurometabolic consequences of acoustically-mediated BBBO on cerebral tissue were investigated using multimodal metabolomics approaches. Sinusoid US waves (1 MHz, peak negative pressure 0.6 MPa, burst length 10 ms, total treatment time 30 s, MB bolus dose 0.7 × 105 MBs/g) were applied on the rats' right striatum (ipsilateral side). Brain was collected and both striata were then dissected 3 h, 2 days, and 1 week after BBBO. After tissue preparation, the samples were analyzed using nuclear magnetic resonance spectrometry (NMRS) and high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS). Our findings showed a slight disruption of metabolic pathways in contralateral striata of animals. Analyses of metabolic pathways indicated changes in amino acid metabolisms. In addition, tryptophan derivate dosages revealed the perturbation of a central metabolite of the kynurenine pathway (i.e., 3-hydroxy-kynurenine). In conclusion, the acoustically-mediated BBBO of the ipsilateral cerebral hemisphere induced significant change in metabolism of contralateral one.

Differential expression of keratin and keratin associated proteins are linked with hair loss condition in spontaneously mutated inbred mice

Hair loss condition is heritable and is influenced by multifactorial inheritance. In the present study, spontaneously mutated mice showed hair loss phenotype with defect in the first cycle of hair follicle formation leading to cyclic alopecia. These mutant mice follow autosomal recessive inheritance pattern. The transcriptomic profile and differential gene expression analysis of skin tissues by RNA-sequencing at different stages of hair cycle formation was performed. The genes with significant differential genes expression levels in each stage of hair cycle formation were identified and most of these genes were shown to be associated with keratinization process and hair follicle formation. Transcriptome profiling followed by QPCR validation revealed that mRNA levels of Krt16, Alox15, Fetub (upregulated) and Msx2 (downregulated) were significantly differentially expressed in mutant skin tissues during late anagen and catagen stages. Krt6b mRNA and protein levels were significantly higher in the mutant mice during all stages of first hair cycle formation. The present study provides basis for understanding the differential gene expression of hair-related genes, including keratinization-associated proteins and its relevance. These mutant mice can serve as a model for studying hair loss condition that can be further used in the identification, evaluation and treatment strategies for alopecia condition.

Exploring differential miRNA expression profiles in muscular and visceral adipose tissue of patients with severe obesity.

This study aims to investigate the differential miRNA expression profile between the visceral white adipose tissue and the skeletal muscle of people with obesity undergoing bariatric surgery. Skeletal muscle and visceral adipose tissue samples of 10 controls and 38 people with obesity (50% also with type 2 diabetes) undergoing bariatric surgery were collected. miRNA expression profiles were analyzed using Next-Generation Sequencing and subsequently validated using RT-PCR. Approximately 69% of miRNAs showed similar expression in both tissues, however, 55 miRNAs were preferentially expressed in visceral adipose tissue and 53 in skeletal muscle. miR-122b-5p was uniquely identified in skeletal muscle, while miR-1-3p and miR-206 were upregulated in skeletal muscle. Conversely, miR-224-5p and miR-335-3p exhibited upregulation in visceral adipose tissue. Notably, distinctions related to the presence of type 2 diabetes were observed solely in the expression of miR-1-3p and miR-206 in visceral adipose tissue. This is the first study unveiling distinct miRNA expression profiles in paired samples of visceral adipose tissue and skeletal muscle in humans. The identification of obesity-specific miRNAs in these tissues opens up promising avenues for research into potential biomarkers for obesity diagnosis and treatment.

Towards routine proteome profiling of FFPE tissue: insights from a 1,220-case pan-cancer study.

Proteome profiling of formalin-fixed paraffin-embedded (FFPE) specimens has gained traction for the analysis of cancer tissue for the discovery of molecular biomarkers. However, reports so far focused on single cancer entities, comprised relatively few cases and did not assess the long-term performance of experimental workflows. In this study, we analyze 1220 tumors from six cancer entities processed over the course of three years. Key findings include the need for a new normalization method ensuring equal and reproducible sample loading for LC-MS/MS analysis across cohorts, showing that tumors can, on average, be profiled to a depth of >4000 proteins and discovering that current software fails to process such large ion mobility-based online fractionated datasets. We report the first comprehensive pan-cancer proteome expression resource for FFPE material comprising 11,000 proteins which is of immediate utility to the scientific community, and can be explored via a web resource. It enables a range of analyses including quantitative comparisons of proteins between patientsand cohorts, the discovery of protein fingerprints representing the tissue of origin or proteins enriched in certain cancer entities.

High expression of BBOX1 in paracancerous tissue is associated with poor prognosis in hepatocellular carcinoma patients

The molecular profile of paracancerous tissue has been reported to be prognostic for recurrence in patients with different cancers. This study investigated the clinical significance of the protein expression of gamma-butyrobetaine hydroxylase 1 (BBOX1) in tumor and paracancerous tissues of hepatocellular carcinoma (HCC) patients. This study assessed the role of BBOX1 mRNA in tumor and paracancerous tissues in HCC via bioinformatics analysis. The protein levels of BBOX1 in paired tumor and paracancerous tissues from 83 HCC patients were determined by immunohistochemistry. The associations among BBOX1 protein levels, clinicopathological characteristics and the survival probability of HCC patients were also analyzed. The results revealed that BBOX1 mRNA expression was significantly lower in HCC tissues than in noncancerous tissues. HCC patients with low BBOX1 mRNA expression had a significantly poorer overall survival than those with high BBOX1 expression did. Immunohistochemical analysis of BBOX1 protein expression further confirmed that its levels were markedly lower in HCC tissues than in paracancerous tissues. High BBOX1 expression in paracancerous tissues correlated with poor overall survival and disease-free survival in HCC patients. These findings suggest that the paracancerous BBOX1 expression might be a credible indicator for overall survival or disease-free survival in HCC patients.

Abstract PR008: Cross-talk between metastatic cells and host systems: Neutrophil metabolic adaptation, immune profiling, and systemic metabolic shifts in tumor progression and cachexia

Abstract Oral squamous cell carcinoma (OSCC) is a prevalent type of head and neck cancer with a high mortality rate, partly due to late diagnosis often accompanied by metastasis. Cachexia, a complex syndrome marked by severe weight loss and muscle wasting, frequently accompanies advanced cancers including OSCC. Notably, 50-70% of OSCC patients exhibit cachexia at diagnosis, which persists post-treatment, hinting at a metastatic contribution to this syndrome. One significant challenge in studying cancer cachexia is the lack of well-characterized pre-clinical models that accurately reflect human disease. Traditional mouse models often involve subcutaneously transplanted cells, which fail to mimic the natural tumor-host interactions and metastatic progression seen in patients. These models do not typically exhibit metastasis and differ significantly from the genetic and molecular landscape of human cancers. To address these limitations, we developed a syngeneic mouse model by orthotopically transplanting MOC2 murine OSCC cells into the tongues of B6J mice, thereby closely mimicking the tumor microenvironment and metastatic behavior of human OSCC. By enhancing metastasis with a palm-enriched high-fat diet, we observed progressive tumor growth, metastasis, and body weight loss, independent of food intake reduction, indicating metabolic dysregulation rather than anorexia. Comprehensive analyses, including single-cell RNA sequencing (scRNAseq) of immune cells and transcriptomic and metabolomic profiling of tumor and host tissues, identified significant gene expression changes associated with metastasis and cachexia. Enhanced metastasis correlated with lipid metabolism changes in lung metastases, suggesting that metastatic cells exploit lipids for growth. Further, immune profiling revealed neutrophils' metabolic adaptation in metastasis, with implications for both tumor progression and cachexia. Targeting key metabolic and inflammatory pathways in tumor and immune cells holds potential for mitigating metastasis and cachexia. Early-stage serum metabolomics indicated systemic metabolic shifts, emphasizing the need to understand the interplay between tumor metastasis and host metabolism. These findings underscore the complex mechanisms underlying OSCC-induced cachexia, and the need for new targeted therapeutic strategies to disrupt this lethal synergy between cancer progression and metabolic deterioration of the host. Citation Format: Blanca Majem, Josefina Martin, Liudmila Shevkova, Claudia Bigas, Gloria Pascual, Salvador Aznar-Benitah. Cross-talk between metastatic cells and host systems: Neutrophil metabolic adaptation, immune profiling, and systemic metabolic shifts in tumor progression and cachexia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR008.

In-Depth Proteome Profiling of the Hippocampus of LDLR Knockout Mice Reveals Alternation in Synaptic Signaling Pathway.

The low-density lipoprotein receptor (LDLR) is a major apolipoprotein receptor that regulates cholesterol homeostasis. LDLR deficiency is associated with cognitive impairment by the induction of synaptopathy in the hippocampus. Despite the close relationship between LDLR and neurodegenerative disorders, proteomics research for protein profiling in the LDLR knockout (KO) model remains insufficient. Therefore, understanding LDLR KO-mediated differential protein expression within the hippocampus is crucial for elucidating a role of LDLR in neurodegenerative disorders. In this study, we conducted first-time proteomic profiling of hippocampus tissue from LDLR KO mice using tandem mass tag (TMT)-based MS analysis. LDLR deficiency induces changes in proteins associated with the transport of diverse molecules, and activity of kinase and catalyst within the hippocampus. Additionally, significant alterations in the expression of components in the major synaptic pathways were found. Furthermore, these synaptic effects were verified using a data-independent acquisition (DIA)-based proteomic method. Our data will serve as a valuable resource for further studies to discover the molecular function of LDLR in neurodegenerative disorders.

Global metabolomic profiling of tumor tissue and paired serum samples to identify biomarkers for response to neoadjuvant FOLFIRINOX treatment of human pancreatic cancer.

Neoadjuvant chemotherapy (NAT) is increasingly used for the treatment of non-metastatic pancreatic ductal adenocarcinoma (PDAC) and is established as a standard of care for borderline resectable and locally advanced PDAC. However, full exploitation of its clinical benefits is limited by the lack of biomarkers that assess treatment response. To address this unmet need, global metabolomic profiling was performed on tumor tissue and paired serum samples from patients with treatment-naïve (TN; n = 18) and neoadjuvant leucovorin calcium (folinic acid), fluorouracil, irinotecan hydrochloride and oxaliplatin (FOLFIRINOX)-treated (NAT; n = 17) PDAC using liquid chromatography mass spectrometry. Differentially abundant metabolites (DAMs) in TN versus NAT groups were identified and their correlation with various clinical parameters was assessed. Metabolomics profiling identified 40 tissue and five serum DAMs in TN versus NAT PDAC. In general, DAMs associated with amino acid andnucleotide metabolism were lower in NAT compared to TN. Four DAMs-3-hydroxybutyric acid (BHB), 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), glycochenodeoxycholate and citrulline-were common to both tissue and serum and showed a similar pattern of differential abundance in both groups. A strong positive correlation was observed between serum carbohydrate 19-9 antigen (CA 19-9) and tissue carnitines (C12, C18, C18:2) and N8-acetylspermidine. The reduction in CA 19-9 following NAT correlated negatively with serum deoxycholate levels, and the latter correlated positively with survival. This study revealed neoadjuvant-chemotherapy-induced changes in metabolic pathways in PDAC, mainly amino acid and nucleotide metabolism, and these correlated with reduced CA 19-9 following neoadjuvant FOLFIRINOX treatment.

Levilactobacillus brevis 47f: Bioadaptation to Low Doses of Xenobiotics in Aquaculture.

Agricultural and industrial activities are increasing pollution of water bodies with low doses of xenobiotics that have detrimental effects on aquaculture. The aim of this work was to determine the possibility of using Levilactobacillus brevis 47f culture in fish aquaculture under the influence of low doses of xenobiotics as an adaptogen. An increase in the survival of Danio rerio individuals exposed to the xenobiotic bisphenol A solution and fed with the L. brevis 47f was shown compared to control groups and, at the same time, the cytokine profile in the intestinal tissues of Danio rerio was also investigated. Analysis of differential gene expression of the L. brevis 47f grown under the action of high concentrations of bisphenol A showed changes in mRNA levels of a number of genes, including genes of various transport proteins, genes involved in fatty acid synthesis, genes of transcriptional regulators, genes of the arabinose operon, and the oppA gene. The identification of L. brevis 47f proteins from polyacrylamide gel by mass spectrometry revealed L-arabinose isomerase, Clp chaperone subunit, ATP synthase subunits, pentose phosphate pathway and glycolysis enzyme proteins, which are likely part of the L. brevis 47f strain's anti-stress response, but probably do not affect its adaptogenic activity toward Danio rerio.

Proteomic profiling of oral squamous cell carcinoma tissues reveals altered immune-related proteins: implications for personalized therapy

ABSTRACT Objectives Oral squamous cell carcinoma poses a substantial global health challenge marked by rising mortality rate. Recently, immunotherapy has shown promising results in cancer management by enhancing immune response. Thus, identifying additional immune-related markers is critical for advancing immunotherapy treatments. Methods Data-independent acquisition (DIA) mass spectrometry approach was used to explore differentially expressed immune-related proteins in oral cancer tissues compared to adjacent non-cancerous tissues. Functional significance was identified through Gene Ontology, pathway, and network analysis. Gene expression of identified proteins was validated using transcriptomic data. Results DIA analysis identified 29,459 precursors corresponding to 3429 proteins. Among these, 1060 proteins were differentially expressed, with 166 being immune-related. Differentially regulated proteins were involved in innate immune response, mitochondrial ATP synthesis, and neutrophil degranulation. Pathway analysis of immune-related proteins showed perturbation in anti-tumor immunity-related pathways such as interferon signaling, TCR signaling, PD-1 signaling, and antigen processing and presentation. Significance of these pathways was further reinforced by the strong interactions identified in the protein–protein interaction network analysis. Additionally, gene expression analysis showed similar mRNA expression patterns for key proteins involved in altered pathways, including ISG15, IFIT1/3, HLA-A/C and OAS2/3. Conclusions Further validation of these proteins could establish them as potential targets for personalized therapy.

Abstract PR010: High purity CTC RNA sequencing identifies poor prognosis lineage states in castrate resistant prostate cancer

Abstract Background: Metastatic prostate cancer is an androgen receptor (AR) driven disease for which multiple AR targeted therapies are FDA approved, leading to improvements in patient outcomes. However, development of treatment resistance remains universal, driven by AR alterations as well as lineage state transitions that bypass AR signaling and culminate in neuroendocrine prostate cancer (NEPC) with poor prognosis. Liquid biopsies could enable longitudinal molecular analysis to identify such lineage transitions, but bulk RNA sequencing of circulating tumor cells (CTCs) has been limited by the challenge of isolating sufficiently pure samples for global signaling pathway analysis. We developed a novel approach to CTC purification that results in purity comparable to tissue biopsies and performed the first large-scale CTC RNA-seq study in a multi-institutional cohort of patients with metastatic prostate cancer. Using this approach, we identified multiple prostate cancer lineage states associated with prognosis. Methods: 273 blood samples were collected from 117 patients with histologically confirmed metastatic prostate cancer treated at the University of Wisconsin Carbone Cancer Center, William S. Middleton Memorial Veterans Hospital, UC San Diego Moores Cancer Center and Dana Farber Cancer Institute. CTCs were isolated with our automated microfluidic technology integrating negative and positive selection for CTC enrichment. CTCs were captured immunomagnetically followed by RNA isolation on chip and RNA sequencing. A modification of the ESTIMATE algorithm was used to infer sample tumor purity, and CIBERSORTx was used to infer immune content. Overall survival (OS) was defined as date of death or last contact relative to first CTC sample collection. Results: 146 samples from 70 patients met our &amp;gt;50% purity threshold for gene expression pathway analysis. Single sample pathway analysis identified four CTC transcriptional phenotypes: luminal A (high AR signaling, intermediate proliferation), luminal B (high AR signaling, high proliferation), low proliferation, and neuroendocrine. Compared to patients with low CTC burden/low tumor purity (median OS NR, n=63), patients with luminal A (n=21) and low proliferation (n=12) phenotypes had similar survival, while patients with luminal B (n=18) and NE (n=3) had markedly shorter survival (LumB: median OS 6 months, HR 9.1, log rank p&amp;lt;0.0001, NE: median OS 3.7 months, HR 11.8, log rank p=0.0019). Conclusion: We report the largest CTC RNA sequencing cohort of patients with metastatic prostate cancer and demonstrate that CTC RNA-seq identifies prostate cancer lineage states mirroring those described in tissue profiling. In addition to an NE phenotype concordant with tissue histology and associated with markedly inferior overall survival, we identified a more common luminal B CTC subtype defined by persistent AR signaling and high proliferation and associated with poor prognosis comparable to NEPC. Citation Format: Marina N Sharifi, Jamie M Sperger, Amy K Taylor, Katharine E Tippins, Shannon R Reese, Viridiana Carreno, Katherine R Kaufmann, Alex H Chang, Luke A Nunamaker, Charlotte Linebarger, Kyle T Helzer, Matthew Bootsma, Grace C Blitzer, John Floberg, David Kosoff, Rana R McKay, Xiao X Wei, Shuang G Zhao, Joshua M Lang. High purity CTC RNA sequencing identifies poor prognosis lineage states in castrate resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR010.

Abstract B011: Circulating tumor DNA profiling in colorectal cancer to detect guideline-based targeted mutations at a Quebec health care center

Abstract Liquid biopsy is an emerging tool in oncology, providing minimally invasive detection of tumor- specific DNA in body fluids, known as circulating tumor DNA (ctDNA). Colorectal cancer (CRC) has shown a high rate of shedding of ctDNA, making it a promising prognostic and possibly predictive biomarker in the personalized management of patients with CRC. Therefore, the half-life of ctDNA in circulation is estimated to be between 16 minutes and several hours, indicating a need for rapid processing. This study aimed to explore the concordance between the mutational profiles in tumor tissue and ctDNA from plasma obtained using EDTA tubes as well as the correlation between ctDNA and disease stage, to evaluate the potential of implementing liquid biopsy testing in CRC cases, following biobank standards. In this retrospective cohort, 58 patients with CRC stage T1 to T4 were enrolled at the Research Institute McGill University Health Centre (RI-MUHC). Tumor tissue was obtained through surgical resection or biopsies of formalin-fixed paraffin-embedded tissue, and blood samples were drawn pre-surgery using EDTA tubes. Cell-free DNA (cfDNA) was extracted from 1-2mL plasma samples using QIAamp Circulating Nucleic Acids. Next-Generation Sequencing using a 52-gene (AmpliSeq for Illumina Focus Panel) was performed to identify the tumor mutations and subsequently droplet digital PCR (ddPCR) was performed to identify the plasma mutations. Among the 58 patients, the mean age was 62 years (range 29-86), and 29 were women. Forty- one patients had left-sided tumor. At least 1 tumor-specific mutation was detected in all tissue tumor samples. Overall, the most frequently identified mutations in this cohort were: BRAF V600E (22.4%), KRAS G12D (12.1%), KRAS G12V and KRAS G13D (8.6%). Cell-free DNA was obtained from 32 patients and showed a mean concentration of 975 ng/mL (range 400- 1,995) for stage T1, 584 ng/mL (range 228-1,300) for stage T2, 1,127 ng/mL (range 249-4,160) for stage T3 and 2,083 ng/mL (range 860-3,680) for stage T4. ddPCR revealed mutation concordance between ctDNA and matched tumor tissue of 28.1%. When tumors were stratified by stage, the concordance was 0% (n=0/3) in patients with T1 tumors, 16.7% (n=1/6) in patients with T2 tumors, 25% (n=5/20) in patients with T3 tumors and 100% (3/3) in patients with T4 tumors. This study showed that ctDNA detection of mutations has excellent concordance in stage T4 tumors, highlighting the potential of ctDNA in advanced stage disease. However, in T1-3 tumor stages the concordance was low, showing the importance of preanalytical steps such as the use of cell-free DNA preservative tubes for sensitive and accurate detection of ctDNA in patients with earlier stage disease. Citation Format: Monyse de Nobrega, Kyle Dickinson, Saba Alsaddah, Alexandra Bartolomucci, Tadhg Ferrier, Anne Mahalia Olivier Mahalia Olivier, Mina Farag, Gertuda Evaristo, Sophie Camilleri-Broet, Andrea Gomez, Thupten Tsering, Lawrence Lee, Pierre Olivier Fiset, Julia V. Burnier. Circulating tumor DNA profiling in colorectal cancer to detect guideline-based targeted mutations at a Quebec health care center [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B011.

Abstract PR014: Robust disease monitoring using CSF liquid biopsies collected from children undergoing cellular therapy for malignant central nervous system tumors

Abstract Background: Central nervous system (CNS) tumors remain the leading cause of cancer-related mortality in children, necessitating more effective treatment options. Chimeric Antigen Receptor T-cells (CAR-T) have emerged as a promising strategy to treat CNS tumors, with B7H3 (CD276) representing an attractive target due to its high expression across a broad range of entities. B7H3-CAR-T therapy is currently being evaluated in a St. Jude Children’s Research Hospital Phase I clinical trial (NCT05835687) for the treatment of relapse/refractory pediatric CNS tumors. Monitoring disease progression after CAR-T infusion by magnetic resonance imaging (MRI) remains challenging, emphasizing the need for more sensitive biomarkers of treatment response. Detection of circulating tumor DNA (ctDNA) in cerebrospinal fluid (CSF) liquid biopsies holds tremendous potential for minimal residual disease (MRD) monitoring in the clinical setting. Here, we leverage CSF liquid biopsies for longitudinal disease profiling in pediatric CNS tumor patients receiving B7H3-CAR-T immunotherapy. Methods: CSF samples were collected from Ommaya reservoirs of 9 pediatric patients with relapse/refractory CNS tumors undergoing B7H3-CAR-T therapy. CSF was sampled at study enrollment, and weekly intervals pre- and post-infusion. Cell-free DNA (cfDNA) was extracted from 115 longitudinally collected CSF samples and subjected to enzymatic methylation sequencing (EM-seq). Implementation of a custom computational workflow enabled detection of tumor-derived copy number variations (CNVs), estimation of malignant vs. non-malignant fractions, and molecular classification of tumor entity. Results: Tumor-derived CNVs were detected in 7/9 (78%) CSF samples collected at enrollment, prior to immunotherapy. Liquid biopsy-inferred CNV profiles and entity classifications were highly concordant with matched tumor tissue profiles. Chromosomal alterations that were restricted to CSF profiles indicated clonal evolution. Increased ctDNA burden was repeatedly observed after B7H3-CAR-T infusions, suggesting subclinical antitumor activity. Transient subclonal alterations emerging in response to treatment were also identified in a subset of patients. Conclusions: EM-seq is a feasible method for serial tracking of ctDNA burden in patients receiving cellular immunotherapy. CSF liquid biopsies capture the global landscape of tumor heterogeneity and genetic alterations that arise during therapy, providing a sensitive platform for detecting clinical response and disclosing potential mechanisms of treatment resistance. Citation Format: Anna Kostecka, Kyle S Smith, Katie Han, Hong Lin, Deanna Langfitt, Tara Walhart, Stephen Gottschalk, Giedre Krenciute, Christopher DeRenzo, Kelsey Bertrand, Paul A Northcott. Robust disease monitoring using CSF liquid biopsies collected from children undergoing cellular therapy for malignant central nervous system tumors [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR014.