Biology subjects

Morkel, M.

Publications and source records attributed to Morkel, M..

13 recordsLinked to original sources

bioRxiv2026

Tumor-versus-non-tumor T cell enrichment supported by single-cell and spatial phenotyping enables antigen-agnostic prioritization of candidate tumor-reactive TCRs

Introduction: Tumor-reactive T cell receptors (TCRs) are promising candidates for the manufacturing of safe and effective adoptive TCR-T cell therapies, but their efficient identification remains a major challenge. We previously established an antigen-agnostic TCR-repertoire analysis workflow that prioritizes candidate TCRs based on intratumoral clonal expansion and high tumor-to-non-tumor (T/N) frequency ratios. Here, we investigated whether T/N-selected clonotypes show independent transcriptional and spatial features consistent with tumor association. Methods: Bulk and single-cell TCR repertoire sequencing of paired tumor and adjacent non-tumor tissues was used to identify clonotypes with high T/N ratios across 15 cancer cases, comprising six non-small cell lung cancers, five pancreatic cancers, two breast cancers, and two colorectal cancers. Candidate clonotypes were further characterized by paired single-cell TCR and gene expression profiling and, in two PDAC cases, by Xenium spatial transcriptomics to assess their cellular phenotype, spatial localization, and proximity to malignant cells. Results: High T/N ratios consistently marked T cell clonotypes with features of tumor reactivity across the studied solid tumor entities. The analysis included Cluster-A, a lead T cell cluster comprising highly similar TCRs shared among 14 HLA-A*02:01-positive patients in the present cohort. Cluster-A clonotypes were consistently enriched in tumor relative to matched non-tumor tissue, and previous functional studies demonstrated tumor recognition by Cluster-A members. In a focused analysis of a pancreatic cancer case, Cluster-A CD8 T cells were selectively detected in tumor tissue, preferentially localized near malignant epithelial cells, and displayed an antigen-experienced effector-memory phenotype with prominent expression of granzymes A and K (GZMA/GZMK). Importantly, in another PDAC case that lacked Cluster-A clonotypes, these features extended to independent clonotypes, revealing a continuous tumor-association axis in which increasing T/N ratios were associated with progressively closer localization to malignant cells and increased GZMA/GZMK expression. Thus, repertoire enrichment, spatial tumor proximity, and effector-associated transcriptional states converged across independent tumor-enriched clonotypes. Outlook: These findings support the T/N ratio as a robust primary criterion for identifying candidate tumor-reactive TCRs. Integration of single-cell and spatial transcriptomics provides supporting biological evidence of tumor association and a framework for prioritizing therapeutic TCR candidates when functional validation is limited.

immunology↗

bioRxiv2026

Identification of non-covalent inhibitors for the atypical peroxiredoxin PRDX5 as a therapeutic strategy in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive asbestos-linked cancer with limited therapeutic options and a dismal 5-year survival rate of [~]5%. While aberrant production of reactive oxygen and nitrogen species (ROS/RNS) is a hallmark of MPM, targeted approaches to exploit these redox vulnerabilities remain scarce. Here, using the MOSAIC multimodal cancer patient atlas, we identify Peroxiredoxin 5 (PRDX5) as being significantly upregulated in the epithelioid subtype of MPM. We show that MPM cells exhibit enhanced resistance to nitrosative and oxidative stress compared to healthy mesothelial cells, a phenotype correlated with basal PRDX5 expression. Next, utilising a machine learning guided discovery pipeline, we identified three putative allosteric pockets in PRDX5 and conducted a virtual screen of 3.6 million compounds. High-throughput biochemical validation of 452 candidates yielded 36 non-covalent hits, including sub-micromolar inhibitors. These findings establish PRDX5 as a novel, subtype specific therapeutic target in MPM and provide a chemical framework for the development of next-generation redox-modulating oncology treatments.

cancer biology↗

bioRxiv2024

Reporter-based screening identifies RAS-RAF stabilizing mutations as drivers of resistance to broad-spectrum RAS inhibition in colorectal cancer

Therapy-induced acquired resistance limits the clinical effectiveness of mutation-specific RAS inhibitors in colorectal cancer. It is unknown whether broad-spectrum active-state RAS inhibitors meet similar limitations. Here, we identify and categorize mechanisms of resistance to the broad-spectrum active-state RAS inhibitor RMC-7977 in colorectal cancer cell lines. We found that KRAS-mutant colorectal cancer cell lines are universally sensitive to RMC-7977, inhibiting the RAS-RAF-MEK-ERK axis, halting proliferation and in some cases inducing apoptosis. To monitor KRAS downstream effector pathway activity, we developed a compartment-specific dual-color ERK activity reporter. RMC-7977 treatment reduced reporter activity. However, long-term dose escalation with RMC-7977 revealed multiple patterns of reporter reactivation in emerging resistant cell populations that correlated with phosphorylation states of compartment-specific ERK targets. Cells sorted for high, low, or cytoplasmic reporter activity exhibited distinct patterns of genomic mutations, phospho-protein, and transcriptional activities. Notably, all resistant subpopulations showed dynamic ERK regulation in the presence of the RAS inhibitor, unlike the parental sensitive cell lines. High levels of RAS downstream activities were observed in cells characterized by a KRAS Y71H resistance mutation. In contrast, RAS inhibitor-resistant populations with low, or cytoplasmic ERK reporter reactivation displayed different genetic alterations, among them RAF1 S257L and S259P mutations. Colorectal cancer cells resistant to RMC-7977 and harboring the RAF1 mutation specifically exhibited synergistic sensitivity to concurrent RAS and RAF inhibition. Our findings endorse reporter-assisted screening together with single-cell analyses as a powerful approach for dissecting the complex landscape of therapy resistance. The strategy offers opportunities to develop clinically relevant combinatorial treatments to counteract emergence of resistant cancer cells.

cancer biology↗

bioRxiv2024

High-confidence calling of normal epithelial cells allows identification of a novel stem-like cell state in the colorectal cancer microenvironment

Single-cell analyses can be confounded by assigning unrelated groups of cells to common developmental trajectories. For instance, cancer cells and admixed normal epithelial cells could potentially adopt similar cell states thus complicating analyses of their developmental potential. Here, we develop and benchmark CCISM (for Cancer Cell Identification using Somatic Mutations) to exploit genomic single nucleotide variants for the disambiguation of cancer cells from genomically normal non-cancer epithelial cells in single-cell data. In colorectal cancer datasets, we find that our method and others based on gene expression or allelic imbalances identify overlapping sets of cancer versus normal epithelial cells, depending on molecular characteristics of individual cancers. Further, we define consensus cell identities of normal and cancer epithelial cells with higher transcriptome cluster homogeneity than those derived using existing tools. Using the consensus identities, we identify significant shifts of cell state distributions in genomically normal epithelial cells developing in the cancer microenvironment, with immature states increased at the expense of terminal differentiation throughout the colon, and a novel stem-like cell state arising in the left colon. Trajectory analyses show that the new cell state extends the pseudo-time range of normal colon stem-like cells in a cancer context. We identify cancer-associated fibroblasts as sources of WNT and BMP ligands potentially contributing to increased plasticity of stem cells in the cancer microenvironment. Our analyses advocate careful interpretation of cell heterogeneity and plasticity in the cancer context and the consideration of genomic information in addition to gene expression data when possible. Novelty and ImpactSingle-cell analyses have become standard to assess cell heterogeneity and developmental hierarchies in cancer tissues. However, these datasets are complex and contain cancer and non-cancer lineage cells. Here, we develop and systematically benchmark tools to distinguish between cancer and non-cancer single-cell transcriptomes, based on gene expression or different levels of genomic information. We provide strategies to combine results of different tools into consensus calls tailored to the biology and genetic characteristics of the individual cancer.

cancer biology↗

bioRxiv2023

Robust detection of clinically relevant features in single-cell RNA profiles of patient-matched fresh and formalin-fixed paraffin-embedded (FFPE) lung cancer tissue

Single-cell transcriptional profiling reveals cell heterogeneity and clinically relevant traits in intra-operatively collected patient-derived tissue. However, the established approach to perform such analyses on freshly collected tissue constitutes an important limitation since it requires prospective collection and immediate processing. Therefore, the ability to perform single-cell RNA sequencing from archived tissues would be very beneficial in a clinical setting. Here, we benchmark single-cell gene expression profiles from patient-matched fresh, cryopreserved and FFPE cancer tissue. We find that fresh tissue and FFPE routine blocks can be employed for the robust detection of clinically relevant traits on the single-cell level. Specifically, single-cell maps of fresh patient tissues and corresponding FFPE tissue blocks could be integrated into common low-dimensional representations, and cell subtype clusters showed highly correlated transcriptional strengths of signaling pathways, Hallmark and clinically useful signatures, despite some variability in expression of individual genes due to technological differences. FFPE tissue blocks revealed higher cell diversity compared to fresh tissue. In contrast, single-cell profiling of cryopreserved tissue was prone to artifacts in the clinical setting. Our analysis suggests that single-cell RNA sequencing from FFPE tissues is comparable to and can replace analyses from fresh tissue. This highlights the potential of single-cell profiling in the analysis of retrospectively and prospectively collected archival pathology cohorts and dramatically increases the applicability in translational projects.

pathology↗

bioRxiv2022

Oncogenic signalling is coupled to colorectal cancer cell differentiation state

Colorectal cancer progression is intrinsically linked to stepwise deregulation of the intestinal differentiation trajectory. In this process, sequential mutations of APC/Wnt, KRAS, TP53 and SMAD4 stepwisely enable an oncogenic signalling network. Here, we developed a novel mass cytometry antibody panel to analyse colorectal cancer cell differentiation and signalling in human isogenic colorectal cancer organoids and in patient-derived cultures. We define a differentiation axis following EphrinB2 abundance in all tumour progression states from normal to cancer. We show that during colorectal cancer progression, oncogenes decrease dependence on external factors and shape distribution of cells along the differentiation axis. In this regard, subsequent mutations can have stem cell-promoting or restricting effects. Individual nodes of the signalling network remain coupled to the differentiation state, regardless of the presence of oncogenic signals. Our work underscores the key role of cell plasticity as a hallmark of cancer that is gradually unlocked during colorectal cancer progression.

cancer biology↗

bioRxiv2021

The single-cell transcriptional landscape of lung carcinoid tumors

Lung carcinoid tumors, also referred to as pulmonary neuroendocrine tumors or lung carcinoids, are rare neoplasms of the lung with a more favorable prognosis than other subtypes of lung cancer. Still, some patients suffer from relapsed disease and metastatic spread while no consensus treatment exists for metastasized carcinoids. Several recent single-cell studies have provided detailed insights into the cellular heterogeneity of more common lung cancers, such as adeno- and squamous cell carcinoma. However, the characteristics of lung carcinoids on the single-cell level are yet completely unknown. To study the cellular composition and single-cell gene expression profiles in lung carcinoids, we applied single-cell RNA sequencing to three lung carcinoid tumor samples and normal lung tissue. The single-cell transcriptomes of carcinoid tumor cells reflected intertumoral heterogeneity associated with clinicopathological features, such as tumor necrosis and proliferation index. The immune microenvironment was specifically enriched in noninflammatory monocyte-derived myeloid cells. Tumor-associated endothelial cells were characterized by distinct gene expression profiles. A spectrum of vascular smooth muscle cells and pericytes predominated the stromal microenvironment. We found a small proportion of myofibroblasts exhibiting features reminiscent of cancer-associated fibroblasts. Stromal and immune cells exhibited potential paracrine interactions which may shape the microenvironment via NOTCH, VEGF, TGF{beta} and JAK/STAT signaling. Moreover, single-cell gene signatures of pericytes and myofibroblasts demonstrated prognostic value in bulk gene expression data. Here, we provide first comprehensive insights into the cellular composition and single-cell gene expression profiles in lung carcinoids, demonstrating the non-inflammatory and vessel-rich nature of their tumor microenvironment, and outlining relevant intercellular interactions which could serve as future therapeutic targets.

cancer biology↗

bioRxiv2021

Post-entry, spike-dependent replication advantage of B.1.1.7 and B.1.617.2 over B.1 SARS-CoV-2 in an ACE2-deficient human lung cell line

Epidemiological data demonstrate that SARS-CoV-2 variants of concern (VOC) B.1.1.7 and B.1.617.2 are more transmissible and infections are associated with a higher mortality than non-VOC virus infections. Phenotypic properties underlying their enhanced spread in the human population remain unknown. B.1.1.7 virus isolates displayed inferior or equivalent spread in most cell lines and primary cells compared to an ancestral B.1 SARS-CoV-2, and were outcompeted by the latter. Lower infectivity and delayed entry kinetics of B.1.1.7 viruses were accompanied by inefficient proteolytic processing of spike. B.1.1.7 viruses failed to escape from neutralizing antibodies, but slightly dampened induction of innate immunity. The bronchial cell line NCI-H1299 supported 24- and 595-fold increased growth of B.1.1.7 and B.1.617.2 viruses, respectively, in the absence of detectable ACE2 expression and in a spike-determined fashion. Superior spread in NCI-H1299 cells suggests that VOCs employ a distinct set of cellular cofactors that may be unavailable in standard cell lines.

microbiology↗

bioRxiv2020

Single-cell RNA sequencing reveals distinct tumor microenvironmental patterns in lung adenocarcinoma

Recent developments in immuno-oncology demonstrate that not only cancer cells, but also features of the tumor microenvironment guide precision medicine. Still, the relationship between tumor and microenvironment remains poorly understood. To overcome this limitation and identify clinically relevant microenvironmental and cancer features, we applied single-cell RNA sequencing to lung adenocarcinomas. While the highly heterogeneous carcinoma cell transcriptomes reflected histological grade and activity of relevant oncogenic pathways, our analysis revealed two distinct microenvironmental patterns. We identified a prognostically unfavorable group of tumors with a microenvironment composed of cancer-associated myofibroblasts, exhausted CD8+ T cells, proinflammatory monocyte-derived macrophages and plasmacytoid dendritic cells (CEP2 pattern) and a prognostically favorable group characterized by myeloid dendritic cells, anti-inflammatory monocyte-derived macrophages, normal-like myofibroblasts, NK cells and conventional T cells (MAN2C pattern). Our results show that single-cell gene expression profiling allows to identify patient subgroups based on the tumor microenvironment beyond cancer cell-centric profiling.

cancer biology↗

bioRxiv2020

An Integrative Genetic, Epigenetic and Proteomic Characterization of Pancreatic Neuroendocrine Neoplasms (PanNENs) defines Distinct Molecular Features of α- and β-cell like Subgroups

Pancreatic Neuroendocrine Carcinomas (PanNECs) are high-grade, poorly-differentiated tumors grouped together with Pancreatic Neuroendocrine Tumors (PanNETs) and placed within the Pancreatic Neuroendocrine Neoplasms (PanNENs) WHO tumor classification. Despite recent studies suggesting the endocrine origin of low-grade PanNETs, high-grade PanNEC origin remains unknown. DNA methylation analysis using the Illumina 850K beadchip array was conducted on 57 PanNEN samples, including 14 PanNECs. Distinct methylation profiles separated PanNEN samples into two major groups, clearly distinguishing high-grade PanNECs from other PanNETs including high-grade NETG3. DNA mutations, copy number changes and Immunohistochemistry of pancreatic cell-type markers PDX1, ARX and SOX9 were utilized to further characterize PanNECs and their hierarchical cell of origin in the pancreas. Phylo-epigenetic and cell-type signature features using methylation data from normal alpha, beta, acinar and ductal adult cells indicate an exocrine cell of origin for PanNECs, thus separating them in cell lineage from other PanNENs of endocrine origin. Our study provides a robust and clinically relevant method relying on methylation profiles to clearly distinguish PanNECs from PanNETG3s to improve patient stratification and treatment.

cancer biology↗

bioRxiv2020

A census of cell types and paracrine interactions in colorectal cancer

In colorectal cancer, oncogenic mutations transform a hierarchically organized and homeostatic epithelium into invasive cancer tissue lacking visible organization. We sought to define colorectal cancer cell types and signals controlling their development. More than 30,000 epithelial single cell transcriptomes of tumors and matched non-cancerous tissues of twelve colorectal cancer patients were clustered into six patient-overarching groups defined by differential activities of oncogenic signaling pathways such as mitogen-activated protein kinase and oncogenic traits such as replication stress. RNA metabolic labeling and assessment of RNA velocity in patient-derived organoids revealed developmental trajectories of colorectal cancer cells organized along a mitogen-activated protein kinase activity gradient. This was in contrast to normal colon organoid cells developing along graded Wnt activity. Experimental targeting of EGFR-BRAF-MEK in cancer organoids affected signaling and gene expression contingent on predictive KRAS/BRAF mutations and induced cell plasticity overriding default developmental trajectories, providing a basis for non-genetic resistance to targeted therapies.

cancer biology↗

bioRxiv2018

Cell type-dependent differential activation of ERK by oncogenic KRAS or BRAF in the mouse intestinal epithelium

Mutations activating the KRAS GTPase or the BRAF kinase are frequent in colorectal cancer and are thought to constitutively activate the terminal mitogen-activated protein kinase, ERK. Using mass cytometry, we found graded phosphorylation of ERK anti-correlated with cell differentiation in patient-derived colorectal cancer organoids, independent of KRAS mutational status. Reporter, single cell transcriptome and mass cytometry analyses showed that transgenic KRASG12V activated ERK in a cell type-specific pattern in mouse intestinal organoids. In contrast, transgenic BRAFV600E triggered high ERK activity and downstream gene expression in all intestinal cell types, followed by epithelial disorganisation. Quantitative network modelling from perturbation data revealed that activation of ERK is shaped by cell type-specific MEK to ERK feed forward and negative feedback signalling. We identified dual-specificity phosphatases as candidate modulators of ERK activity between intestinal cell types. Furthermore, we found that oncogenic KRAS, together with {beta}-Catenin, favoured expansion of crypt cells with high ERK activity. Our experiments highlight key differences between ERK activity elicited by the BRAF or KRAS oncogenes in colorectal cancer and find unexpected heterogeneity in a signalling pathway with fundamental relevance for cancer therapy.

cell biology↗

bioRxiv2018

Non-canonical HIF-1 stabilization is essential for intestinal tumorigenesis

The hypoxia-inducible transcription factor HIF-1 is appreciated as a promising target for cancer therapy. However, conditional deletion of HIF-1 and HIF-1 target genes in cells of the tumor microenvironment can result in accelerated tumor growth, calling for a detailed characterization of the cellular context to fully comprehend HIF-1s role in tumorigenesis. We dissected cell type-specific functions of HIF-1 for intestinal tumorigenesis by lineage-restricted deletion of the Hif1a locus. Intestinal epithelial cell-specific Hif1a loss reduced activation of wnt/{beta}-catenin, tumor-specific metabolism and inflammation, significantly inhibiting tumor growth. Deletion of Hif1a in myeloid cells reduced the expression of fibroblast-activating factors in tumor-associated macrophages resulting in decreased abundance of tumor-associated fibroblasts and robustly reduced tumor formation. Interestingly, hypoxia was detectable only sparsely and without spatial association with nuclear HIF-1 in intestinal adenomas, pointing towards a functional importance of hypoxia-independent, i.e. non-canonical HIF-1 stabilization that has not been previously appreciated. This adds a further layer of complexity to the regulation of HIF-1 and suggests that hypoxia and HIF-1 stabilization can be uncoupled in cancer. Collectively, our data show that HIF-1 is a pivotal pro-tumorigenic factor for intestinal tumor formation, controlling key oncogenic programs in both the epithelial tumor compartment and the tumor microenvironment.

cancer biology↗