Francis Jacob

Ovarian cancer metastasis to the human omentum disrupts organ homeostasis and induces fundamental tissue reprogramming

High-resolution imaging mass cytometry to map subcellular structures

Imaging mass cytometry (IMC) is a powerful multiplexed imaging technology used to investigate cell phenotypes and spatial organization of tissue in health and disease. The spatial resolution of IMC is presently at 1 µm, enabling the resolution of single cells and large subcellular compartments but not submicrometer intracellular structures. Here we report a method to improve the resolution of IMC so that it approaches that of light microscopy. High-resolution IMC (HR-IMC) uses an oversampling approach coupled with point-spread function-based deconvolution to achieve a resolution below 350 nm. We demonstrate the performance of HR-IMC in resolving subcellular structures, such as nuclear foci and mitochondrial networks previously undetectable with IMC, and applied it to visualize chemotherapy-induced perturbation of patient-derived ovarian cancer cells. HR-IMC extends highly multiplex IMC analyses into the subcellular regime, enabling analysis of cell biological features and characteristics of disease.

De novo detection of somatic variants in high-quality long-read single-cell RNA sequencing data

In cancer, genetic and transcriptomic variations generate clonal heterogeneity, leading to treatment resistance. Long-read single-cell RNA sequencing (LR scRNA-seq) has the potential to detect genetic and transcriptomic variations simultaneously. Here, we present LongSom, a computational workflow leveraging high-quality LR scRNA-seq data to call de novo somatic single-nucleotide variants (SNVs), including in mitochondria (mtSNVs), copy number alterations (CNAs), and gene fusions, to reconstruct the tumor clonal heterogeneity. Before somatic variant calling, LongSom reannotates marker gene-based cell types using cell mutational profiles. LongSom distinguishes somatic SNVs from noise and germline polymorphisms by applying an extensive set of hard filters and statistical tests. Applying LongSom to human ovarian cancer samples, we detected clinically relevant somatic SNVs that were validated against matched DNA samples. Leveraging somatic SNVs and fusions, LongSom found subclones with different predicted treatment outcomes. In summary, LongSom enables de novo variant detection without the need for normal samples, facilitating the study of cancer evolution, clonal heterogeneity, and treatment resistance.

Diagnosing and staging epithelial ovarian cancer by serum glycoproteomic profiling

There is a need for diagnostic tests for screening, triaging and staging of epithelial ovarian cancer (EOC). Glycoproteomics of blood samples has shown promise for biomarker discovery. We applied glycoproteomics to serum of people with EOC or benign pelvic masses and healthy controls. A total of 653 analytes were quantified and assessed in multivariable models, which were tested in an independent cohort. Additionally, we analyzed glycosylation patterns in serum markers and in tissues. We identified a biomarker panel that distinguished benign lesions from EOC with sensitivity and specificity of 83.5% and 90.1% in the training set, and of 86.7 and 86.7% in the test set, respectively. ROC analysis demonstrated strong performance across a range of cutoffs. Fucosylated multi-antennary glycopeptide markers were higher in late-stage than in early-stage EOC. A comparable pattern was found in late-stage EOC tissues. Blood glycopeptide biomarkers have the potential to distinguish benign from malignant pelvic masses, and early- from late-stage EOC. Glycosylation of circulating and tumor tissue proteins may be related. This study supports the hypothesis that blood glycoproteomic profiling can be used for EOC diagnosis and staging and it warrants further clinical evaluation.

Detection of isoforms and genomic alterations by high-throughput full-length single-cell RNA sequencing in ovarian cancer

Abstract Understanding the complex background of cancer requires genotype-phenotype information in single-cell resolution. Here, we perform long-read single-cell RNA sequencing (scRNA-seq) on clinical samples from three ovarian cancer patients presenting with omental metastasis and increase the PacBio sequencing depth to 12,000 reads per cell. Our approach captures 152,000 isoforms, of which over 52,000 were not previously reported. Isoform-level analysis accounting for non-coding isoforms reveals 20% overestimation of protein-coding gene expression on average. We also detect cell type-specific isoform and poly-adenylation site usage in tumor and mesothelial cells, and find that mesothelial cells transition into cancer-associated fibroblasts in the metastasis, partly through the TGF-β/miR-29/Collagen axis. Furthermore, we identify gene fusions, including an experimentally validated IGF2BP2::TESPA1 fusion, which is misclassified as high TESPA1 expression in matched short-read data, and call mutations confirmed by targeted NGS cancer gene panel results. With these findings, we envision long-read scRNA-seq to become increasingly relevant in oncology and personalized medicine.

A 3D multi-cellular tissue model of the human omentum to study the formation of ovarian cancer metastasis

Reliable and predictive experimental models are urgently needed to study metastatic mechanisms of ovarian cancer cells in the omentum. Although models for ovarian cancer cell adhesion and invasion were previously investigated, the lack of certain omental cell types, which influence the metastatic behavior of cancer cells, limits the application of these tissue models. Here, we describe a 3D multi-cellular human omentum tissue model, which considers the spatial arrangement of five omental cell types. Reproducible tissue models were fabricated combining permeable cell culture inserts and bioprinting technology to mimic metastatic processes of immortalized and patient-derived ovarian cancer cells. The implementation of an endothelial barrier further allowed studying the interaction between cancer and endothelial cells during hematogenous dissemination and the impact of chemotherapeutic drugs. This proof-of-concept study may serve as a platform for patient-specific investigations in personalized oncology in the future.

High‐grade serous peritoneal cancer follows a high stromal response signature and shows worse outcome than ovarian cancer

In the era of personalized medicine, where transition from organ‐based to individualized genetic diagnosis takes place, the tailoring of treatment in cancer becomes increasingly important. This is particularly true for high‐grade, advanced FIGO stage serous adenocarcinomas of the ovary (OC), fallopian tube (TC), and peritoneum (PC), which are currently all treated identically. We analyzed three independent patient cohorts using histopathologically classified diagnosis and various molecular approaches (transcriptomics, immunohistochemistry, next‐generation sequencing, fluorescent and chromogenic in situ hybridization). Using multivariate Cox regression model, we found that PC is more aggressive compared with advanced‐stage OC independent of residual disease as shown by an earlier relapse‐free survival in two large cohorts (HR: 2.63, CI: 1.59–4.37, P < 0.001, and HR: 1.66, CI: 1.04–2.63, P < 0.033). In line with these findings, transcriptomic data revealed differentially expressed gene signatures identifying PC as high stromal response tumors. The third independent cohort (n = 4054) showed a distinction between these cancer types for markers suggested to be predictive for chemotherapy drug response. Our findings add additional evidence that ovarian and peritoneal cancers are epidemiologically and molecularly distinct diseases. Moreover, our data also suggest consideration of the tumor‐sampling site for future diagnosis and treatment decisions.

Collagen-rich omentum is a premetastatic niche for integrin α2-mediated peritoneal metastasis

The extracellular matrix (ECM) plays critical roles in tumor progression and metastasis. However, the contribution of ECM proteins to early metastatic onset in the peritoneal cavity remains unexplored. Here, we suggest a new route of metastasis through the interaction of integrin alpha 2 (ITGA2) with collagens enriched in the tumor coinciding with poor outcome in patients with ovarian cancer. Using multiple gene-edited cell lines and patient-derived samples, we demonstrate that ITGA2 triggers cancer cell adhesion to collagen, promotes cell migration, anoikis resistance, mesothelial clearance, and peritoneal metastasis in vitro and in vivo. Mechanistically, phosphoproteomics identify an ITGA2-dependent phosphorylation of focal adhesion kinase and mitogen-activated protein kinase pathway leading to enhanced oncogenic properties. Consequently, specific inhibition of ITGA2-mediated cancer cell-collagen interaction or targeting focal adhesion signaling may present an opportunity for therapeutic intervention of metastatic spread in ovarian cancer.

Glycosphingolipids are mediators of cancer plasticity through independent signaling pathways

The molecular repertoire promoting cancer cell plasticity is not fully elucidated. Here, we propose that glycosphingolipids (GSLs), specifically the globo and ganglio series, correlate and promote the transition between epithelial and mesenchymal cells. The epithelial character of ovarian cancer remains stable throughout disease progression, and spatial glycosphingolipidomics reveals elevated globosides in the tumor compartment compared with the ganglioside-rich stroma. CRISPR-Cas9 knockin mediated truncation of endogenous E-cadherin induces epithelial-to-mesenchymal transition (EMT) and decreases globosides. The transcriptomics analysis identifies the ganglioside-synthesizing enzyme ST8SIA1 to be consistently elevated in mesenchymal-like samples, predicting poor outcome. Subsequent deletion of ST8SIA1 induces epithelial cell features through mTOR

Phosphoproteomics Reveals L1CAM-Associated Signaling Networks in High-Grade Serous Ovarian Carcinoma: Implications for Radioresistance and Tumorigenesis

Quantitative phosphoproteomics enables the comprehensive analysis of signaling pathways driven by overexpressed cancer receptors, revealing the molecular mechanisms that underpin tumor progression and therapy resistance. The glycoprotein L1 cell adhesion molecule (L1CAM) is overexpressed in high-grade serous ovarian carcinoma (HGSOC) and plays a crucial role in carcinogenesis by regulating cancer stem cell properties. Here, CRISPR–Cas9-mediated knockout of L1CAM in ovarian cancer OVCAR8 and OVCAR4 cells significantly impaired anchor-independent growth in soft agar assays and reduced clonogenic survival following external beam irradiation. In vivo, L1CAM knockout decreased cancer stem cell frequency and significantly decreased tumorigenicity. To uncover L1CAM-regulated signaling networks, we employed quantitative phosphoproteomics and proteomics. Bioinformatics analyses and validation studies revealed L1CAM-associated pathways that contribute to radioresistance through DNA repair processes and mammalian target or rapamycin complex 1 (mTORC1)-mediated signaling. In conclusion, our study established a link between L1CAM-dependent tumorigenesis and radioresistance, both hallmarks of cancer stemness, with phosphorylation of key proteins involved in DNA damage response. This study further emphasizes the value of quantitative phosphoproteomics in cancer research, showcasing its ability to enhance understanding of cancer progression and therapy resistance.

P-cadherin overexpression is associated with early transformation of the Fallopian tube epithelium and aggressiveness of tubo-ovarian high-grade serous carcinoma

Abstract Tubo-ovarian high-grade serous carcinoma (HGSC) with proficient homologous recombination (HR) DNA repair (HRP) accounts for approximately 50% of cases and is associated with platinum-resistance and poor prognosis. We hypothesize that the acquisition of hybrid phenotypes displaying both epithelial and mesenchymal (E/M) features may be involved in the malignant transformation and tumour dissemination in this subgroup. Therefore, we analysed, by digital pathology, the expression and prognostic significance of 3 classic cadherins (E-cadherin, epithelial marker; N-cadherin, mesenchymal marker; and P-cadherin, candidate marker of hybrid E/M) in 577 formalin-fixed paraffin-embedded human samples representing the putative stepwise serous carcinogenesis in the Fallopian tube epithelium (FTE). We observed a non-canonical N-to-P-cadherin switch along the carcinogenic progression, with a statistically significant overexpression of P-cadherin in pre-malignant and malignant samples, compared to the control FTE. Interestingly, this overexpression was most pronounced in precursor lesions and HGSC cells from malignant ascites. Tumours with high P-cadherin expression were significantly associated with worse overall survival, especially in the subgroup without BRCA1/2 mutations. Transient P-cadherin knock-down resulted in in vitro significant reduction of functional hybrid E/M hallmarks, namely decreased anoikis resistance, reduced collective migration and invasion in a representative platinum-resistant HRP cell line. Taken together, our results suggest that P-cadherin overexpression is an early event in the serous carcinogenesis and may be involved in hybrid E/M activation in HRP-HGSC, further supporting this adhesion molecule as a promising biomarker for this poor prognostic subgroup.

Protocol for quantifying drug sensitivity in 3D patient-derived ovarian cancer models

Three-dimensional (3D) ex vivo cultures allow the study of cancer progression and drug resistance mechanisms. Here, we present a protocol for measuring on-target drug sensitivity in a scaffold-free 3D culture system through quantification of apoptotic tumor cells. We provide detailed steps for sample processing, immunofluorescence staining, semi-high-throughput confocal imaging, and imaged-based quantification of 3D cultures. This protocol is versatile and can be applied in principle to any patient-derived material.

Tertiary lymphoid structures and B cells determine clinically relevant T cell phenotypes in ovarian cancer

AbstractIntratumoral tertiary lymphoid structures (TLSs) have been associated with improved outcome in various cohorts of patients with cancer, reflecting their contribution to the development of tumor-targeting immunity. Here, we demonstrate that high-grade serous ovarian carcinoma (HGSOC) contains distinct immune aggregates with varying degrees of organization and maturation. Specifically, mature TLSs (mTLS) as forming only in 16% of HGSOCs with relatively elevated tumor mutational burden (TMB) are associated with an increased intratumoral density of CD8+ effector T (TEFF) cells and TIM3+PD1+, hence poorly immune checkpoint inhibitor (ICI)-sensitive, CD8+ T cells. Conversely, CD8+ T cells from immunologically hot tumors like non-small cell lung carcinoma (NSCLC) are enriched in ICI-responsive TCF1+ PD1+ T cells. Spatial B-cell profiling identifies patterns of in situ maturation and differentiation associated with mTLSs. Moreover, B-cell depletion promotes signs of a dysfunctional CD8+ T cell compartment among tumor-infiltrating lymphocytes from freshly isolated HGSOC and NSCLC biopsies. Taken together, our data demonstrate that – at odds with NSCLC – HGSOC is associated with a low density of follicular helper T cells and thus develops a limited number of mTLS that might be insufficient to preserve a ICI-sensitive TCF1+PD1+ CD8+ T cell phenotype. These findings point to key quantitative and qualitative differences between mTLSs in ICI-responsive vs ICI-irresponsive neoplasms that may guide the development of alternative immunotherapies for patients with HGSOC.