SuperCUT, an unsupervised multimodal image registration with deep learning for biomedical microscopy.

Numerous imaging techniques are available for observing and interrogating biological samples, and several of them can be used consecutively to enable correlative analysis of different image modalities with varying resolutions and the inclusion of structural or molecular information. Achieving accurate registration of multimodal images is essential for the correlative analysis process, but it remains a challenging computer vision task with no widely accepted solution. Moreover, supervised registration methods require annotated data produced by experts, which is limited. To address this challenge, we propose a general unsupervised pipeline for multimodal image registration using deep learning. We provide a comprehensive evaluation of the proposed pipeline versus the current state-of-the-art image registration and style transfer methods on four types of biological problems utilizing different microscopy modalities. We found that style transfer of modality domains paired with fully unsupervised training leads to comparable image registration accuracy to supervised methods and, most importantly, does not require human intervention.

Genomic alterations and diagnosis of renal cancer.

The application of molecular profiling has made substantial impact on the classification of urogenital tumors. Therefore, the 2022 World Health Organization incorporated the concept of molecularly defined renal tumor entities into its classification, including succinate dehydrogenase-deficient renal cell carcinoma (RCC), FH-deficient RCC, TFE3-rearranged RCC, TFEB-altered RCC, ALK-rearranged RCC, ELOC-mutated RCC, and renal medullary RCC, which are characterized by SMARCB1-deficiency. This review aims to provide an overview of the most important molecular alterations in renal cancer, with a specific focus on the diagnostic value of characteristic genomic aberrations, their chromosomal localization, and associations with renal tumor subtypes. It may not yet be the time to completely shift to a molecular RCC classification, but undoubtedly, the application of molecular profiling will enhance the accuracy of renal cancer diagnosis, and ultimately guide personalized treatment strategies for patients.

Ex Vivo Drug Testing in Patient-derived Papillary Renal Cancer Cells Reveals EGFR and the BCL2 Family as Therapeutic Targets.

BACKGROUND: Immune checkpoint inhibitors and antiangiogenic agents are used for first-line treatment of advanced papillary renal cell carcinoma (pRCC) but pRCC response rates to these therapies are low. OBJECTIVE: To generate and characterise a functional ex vivo model to identify novel treatment options in advanced pRCC. DESIGN, SETTING, AND PARTICIPANTS: We established patient-derived cell cultures (PDCs) from seven pRCC samples from patients and characterised them via genomic analysis and drug profiling. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Comprehensive molecular characterisation in terms of copy number analysis and whole-exome sequencing confirmed the concordance of pRCC PDCs with the original tumours. We evaluated their sensitivity to novel drugs by generating drug scores for each PDC. RESULTS AND LIMITATIONS: PDCs confirmed pRCC-specific copy number variations such as gains in chromosomes 7, 16, and 17. Whole-exome sequencing revealed that PDCs retained mutations in pRCC-specific driver genes. We performed drug screening with 526 novel and oncological compounds. Whereas exposure to conventional drugs showed low efficacy, the results highlighted EGFR and BCL2 family inhibition as the most effective targets in our pRCC PDCs. CONCLUSIONS: High-throughput drug testing on newly established pRCC PDCs revealed that inhibition of EGFR and BCL2 family members could be a therapeutic strategy in pRCC. PATIENT SUMMARY: We used a new approach to generate patient-derived cells from a specific type of kidney cancer. We showed that these cells have the same genetic background as the original tumour and can be used as models to study novel treatment options for this type of kidney cancer.

RNAi Screening Uncovers a Synthetic Sick Interaction between CtIP and the BARD1 Tumor Suppressor.

Human CtIP is best known for its role in DNA end resection to initiate DNA double-strand break repair by homologous recombination. Recently, CtIP has also been shown to protect reversed replication forks from nucleolytic degradation upon DNA replication stress. However, still little is known about the DNA damage response (DDR) networks that preserve genome integrity and sustain cell survival in the context of CtIP insufficiency. Here, to reveal such potential buffering relationships, we screened a DDR siRNA library in CtIP-deficient cells to identify candidate genes that induce synthetic sickness/lethality (SSL). Our analyses unveil a negative genetic interaction between CtIP and BARD1, the heterodimeric binding partner of BRCA1. We found that simultaneous disruption of CtIP and BARD1 triggers enhanced apoptosis due to persistent replication stress-induced DNA lesions giving rise to chromosomal abnormalities. Moreover, we observed that the genetic interaction between CtIP and BARD1 occurs independently of the BRCA1-BARD1 complex formation and might be, therefore, therapeutical relevant for the treatment of BRCA-defective tumors.

Tracing Clonal Dynamics Reveals that Two- and Three-dimensional Patient-derived Cell Models Capture Tumor Heterogeneity of Clear Cell Renal Cell Carcinoma.

BACKGROUND: Extensive DNA sequencing has led to an unprecedented view of the diversity of individual genomes and their evolution among patients with clear cell renal cell carcinoma (ccRCC). OBJECTIVE: To understand subclonal architecture and dynamics of patient-derived two-dimensional (2D) and three-dimensional (3D) ccRCC models in vitro, in order to determine whether they mirror ccRCC inter- and intratumor heterogeneity. DESIGN, SETTING, AND PARTICIPANTS: We have established a comprehensive platform of living renal cancer cell models from ccRCC surgical specimens. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We confirmed the concordance of 2D and 3D patient-derived cell (PDC) models with the original tumor tissue in terms of histology, biomarker expression, cancer driver mutations, and copy number alterations. We addressed inter- and intrapatient heterogeneity by analyzing clonal dynamics during serial passaging. RESULTS AND LIMITATIONS: In-depth genetic characterization verified the presence of heterogeneous cell populations, and revealed a high degree of similarity between subclonal compositions of monolayer and organoid cell cultures and the corresponding parental ccRCCs. Clonal dynamics were evident during serial passaging of cells in vitro, suggesting that PDC cultures can offer insights into evolutionary potential and treatment susceptibility of ccRCC subclones in vivo. Proof-of-concept drug profiling using selected ccRCC-targeted therapy agents highlighted patient-specific vulnerabilities in PDC models that could not be anticipated by interrogating commercially available cell lines. CONCLUSIONS: We demonstrate that PDC models mirror inter- and intratumor heterogeneity of ccRCC in vitro. Based on our findings, we envision that the use of these models will advance our understanding of the trajectories that cause genetic diversity and their consequences for treatment on an individual level. PATIENT SUMMARY: In this study, we developed two- and three-dimensional patient-derived models from clear cell renal cell carcinoma (ccRCC) as "mini-tumors in a dish." We show that these cell models retain important features of the human ccRCCs such as the profound tumor heterogeneity, thus highlighting their importance for cancer research and precision medicine.

Liquid Biopsies in Renal Cell Carcinoma-Recent Advances and Promising New Technologies for the Early Detection of Metastatic Disease.

Clear cell renal cell carcinoma (ccRCC) displays a highly varying clinical progression, from slow growing localized tumors to very aggressive metastatic disease (mRCC). Almost a third of all patients with ccRCC show metastatic dissemination at presentation while another third develop metastasis during the course of the disease. Survival rates of mRCC patients remain low despite the development of novel targeted treatment regimens. Biomarkers indicating disease progression could help to define its aggressive potential and thus guide patient management. However, molecular markers that can reliably assess metastatic dissemination and disease recurrence in ccRCC have not been recommended for clinical practice to date. Liquid biopsies could provide an attractive and non-invasive method to determine the risk of recurrence or metastatic dissemination during follow-up and thus assist the search for surveillance biomarkers in ccRCC tumors. A wide spectrum of circulating molecules have already shown considerable potential for ccRCC diagnosis and prognostication. In this review, we outline state of the art of the key circulating analytes such as cfDNA, cfRNA, proteins, and exosomes that may serve as biomarkers for the longitudinal monitoring of ccRCC progression to metastasis. Moreover, we address some of the prevailing limitations in the past approaches and present promising adoptable technologies that could help to pursue the implementation of liquid biopsies as a prognostic tool for mRCC.

Cancer Sample Biobanking at the Next Level: Combining Tissue With Living Cell Repositories to Promote Precision Medicine.

Biorespositories of formalin-fixed and paraffin-embedded (FFPE) or fresh frozen human tissues from malignant diseases generated as integral part of the diagnostic workup in many pathology departments have been pivotal resources for translational cancer studies. However, such tissue biobanks have traditionally contained only non-viable specimens and thus cannot enable functional assays for the discovery and validation of therapeutic targets or the assessment of drug responses and resistance to treatment. To overcome these limitations, we have developed a next-generation comprehensive biobanking platform that includes the generation of patient-derived in vitro cell models from colorectal, pancreatic and kidney cancers among others. As such patient-derived cell (PDC) models retain important features of the original human tumors, they have emerged as relevant tools for more dynamic clinical and experimental analyses of cancer. Here, we describe details of the complex processes of acquisition and processing of patient-derived samples, propagation, annotation, characterization and distribution of resulting cell models and emphasize the requirements of quality assurance, organizational considerations and investment into resources. Taken together, we show how clinical tissue collections can be taken to the next level thus promising major new opportunities for understanding and treating cancer in the context of precision medicine.

CtIP-Mediated Fork Protection Synergizes with BRCA1 to Suppress Genomic Instability upon DNA Replication Stress.

Protecting stalled DNA replication forks from degradation by promiscuous nucleases is essential to prevent genomic instability, a major driving force of tumorigenesis. Several proteins commonly associated with the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) have been implicated in the stabilization of stalled forks. Human CtIP, in conjunction with the MRE11 nuclease complex, plays an important role in HR by promoting DSB resection. Here, we report an unanticipated function for CtIP in protecting reversed forks from degradation. Unlike BRCA proteins, which defend nascent DNA strands from nucleolytic attack by MRE11, we find that CtIP protects perturbed forks from erroneous over-resection by DNA2. Finally, we uncover functionally synergistic effects between CtIP and BRCA1 in mitigating replication-stress-induced genomic instability. Collectively, our findings reveal a DSB-resection- and MRE11-independent role for CtIP in preserving fork integrity that contributes to the survival of BRCA1-deficient cells.

Scavenger receptor BI promotes cytoplasmic accumulation of lipoproteins in clear-cell renal cell carcinoma.

Clear-cell renal cell carcinomas (ccRCCs) are characterized by inactivation of the von Hippel-Lindau (VHL) gene and intracellular lipid accumulation by unknown pathomechanisms. The immunochemical analysis of 356 RCCs revealed high abundance of apoA-I and apoB, as well as scavenger receptor BI (SR-BI) in the ccRCC subtype. Given the characteristic loss of VHL function in ccRCC, we used VHL-defective and VHL-proficient cells to study the potential influence of VHL on lipoprotein uptake. VHL-defective patient-derived ccRCC cells and cell lines (786O and RCC4) showed enhanced uptake as well as less resecretion and degradation of radio-iodinated HDL and LDL (125I-HDL and 125I-LDL, respectively) compared with the VHL-proficient cells. The ccRCC cells showed enhanced vascular endothelial growth factor (VEGF) and SR-BI expression compared with normal kidney epithelial cells. Uptake of 125I-HDL and 125I-LDL by patient-derived normal kidney epithelial cells as well as the VHL-reexpressing ccRCC cell lines, 786-O-VHL and RCC4-O-VHL cells, was strongly enhanced by VEGF treatment. The knockdown of the VEGF coreceptor, neuropilin-1 (NRP1), as well as blocking of SR-BI significantly reduced the uptake of lipoproteins into ccRCC cells in vitro. LDL stimulated proliferation of 786-O cells more potently than 786-O-VHL cells in a NRP1- and SR-BI-dependent manner. In conclusion, enhanced lipoprotein uptake due to increased activities of VEGF/NRP1 and SR-BI promotes lipid accumulation and proliferation of VHL-defective ccRCC cells.

FANCD2 and CtIP cooperate to repair DNA interstrand crosslinks.

The resolution of DNA interstrand crosslinks (ICLs) requires a complex interplay between several processes of DNA metabolism, including the Fanconi anemia (FA) pathway and homologous recombination (HR). FANCD2 monoubiquitination and CtIP-dependent DNA-end resection represent key events in FA and HR activation, respectively, but very little is known about their functional relationship. Here, we show that CtIP physically interacts with both FANCD2 and ubiquitin and that monoubiquitinated FANCD2 tethers CtIP to damaged chromatin, which helps channel DNA double-strand breaks generated during ICL processing into the HR pathway. Consequently, CtIP mutants defective in FANCD2 binding fail to associate with damaged chromatin, which leads to increased levels of nonhomologous end-joining activity and ICL hypersensitivity. Interestingly, we also observe that CtIP depletion aggravates the genomic instability in FANCD2-deficient cells. Thus, our data indicate that FANCD2 primes CtIP-dependent resection during HR after ICL induction but that CtIP helps prevent illegitimate recombination in FA cells.

Targeting DNA double-strand break signalling and repair: recent advances in cancer therapy.

Genomic instability, a hallmark of almost all human cancers, drives both carcinogenesis and resistance to therapeutic interventions. Pivotal to the ability of a cell to maintain genome integrity are mechanisms that signal and repair deoxyribonucleic acid (DNA) double-strand breaks (DSBs), one of the most deleterious lesions induced by ionising radiation and various DNA-damaging chemicals. On the other hand, many current therapeutic regimens that effectively kill cancer cells are based on the induction of excessive DSBs. However, these drugs often lack selectivity for tumour cells, which results in severe side effects for the patients, thus compromising their therapeutic potential. Therefore, the development of novel tumour-specific treatment strategies is required. Unlike normal cells, however, cancer cells are often characterised by abnormalities in the DNA damage response including defects in cell cycle checkpoints and/or DNA repair, rendering them particularly sensitive to the induction of DSBs. Therefore, new anticancer agents designed to exploit these vulnerabilities are becoming promising drugs for enhancing the specificity and efficacy of future cancer therapies. Here, we summarise the latest preclinical and clinical developments in cancer therapy based on the current knowledge of DSB signalling and repair, with a special focus on the combination of small molecule inhibitors with synthetic lethality approaches.

Functional connection between histone acetyltransferase Gcn5p and methyltransferase Hmt1p.

Histone acetylation and methylation are linked to a variety of nuclear activities, most notably transcriptional regulation. Both synergistic and antagonistic relationships between these two modifications have been reported in different systems. Here we show that the budding yeast histone H4 arginine 3 (R3) methyltransferase Hmt1p binds acetylated histones H3 and H4, and importantly, that acetylated H4 is a significantly better methylation substrate for Hmt1p. Kinetic studies show that acetylation at any of the four acetylatable lysine residues of histone H4 results in more efficient methylation. Among the four, K8 acetylation imposes the strongest effect on reducing K(M), consistent with the observed acetylation-stimulated interaction. In vivo, hmt1Delta cells rescue the transcriptional defect caused by GCN5 deletion, indicating that one of the functions of Gcn5p is to neutralize the negative effect of Hmt1p. Mutating either K8 or R3 to alanine causes similar growth defects in selective histone and gcn5 mutant background, suggesting that these two residues function in the same pathway for optimal vegetative growth. Together, these results reveal a functional connection between histone acetylation, methylation, and two of the responsible enzymes, Gcn5p and Hmt1p.