Publications

<div>Abstract<p>Previous studies have described that tumor organoids can capture the diversity of defined human carcinoma types. Here, we describe conditions for long-term culture of human mucosal organoids. Using this protocol, a panel of 31 head and neck squamous cell carcinoma (HNSCC)–derived organoid lines was established. This panel recapitulates genetic and molecular characteristics previously described for HNSCC. Organoids retain their tumorigenic potential upon xenotransplantation. We observe differential responses to a panel of drugs including cisplatin, carboplatin, cetuximab, and radiotherapy <i>in vitro</i>. Additionally, drug screens reveal selective sensitivity to targeted drugs that are not normally used in the treatment of patients with HNSCC. These observations may inspire a personalized approach to the management of HNSCC and expand the repertoire of HNSCC drugs.</p>Significance:<p>This work describes the culture of organoids derived from HNSCC and corresponding normal epithelium. These tumoroids recapitulate the disease genetically, histologically, and functionally. <i>In vitro</i> drug screening of tumoroids reveals responses to therapies both currently used in the treatment of HNSCC and those not (yet) used in clinical practice.</p><p><i>See related commentary by Hill and D'Andrea, p. 828</i>.</p><p><i>This article is highlighted in the In This Issue feature, p. 813</i></p></div>

Significance Barrett’s esophagus (BE), the premalignant condition of esophageal adenocarcinoma, is categorized into different stages which correlate with the risk of developing carcinoma. We performed single-cell DNA-sequencing experiments with fresh biopsies, which revealed the appearance of a specific T > C and T > G mutational signature, known as COSMIC signature SBS17, in BE cells that are chromosomally unstable. The SBS17-specific mutations were, however, not detected in chromosomally stable BE cells. Additionally, we performed single-cell RNA sequencing experiments which identified seven genes that facilitate the distinction between different BE stages on histological sections.

In the Netherlands, occupational physicians can diagnose burnout when employees report sick. Their guideline distinguishes between stress complaints lasting < 3 months, adaptation disorder with complaints lasting 3-6 months and burnout with complaints lasting > 6 months at moment of diagnosis. The Four-Dimensional Symptom Questionnaire (4DSQ) is recommended to assess and monitor. The new Burnout Assessment Tool (BAT) might be an alternative. Research questions were: 1) What are the sickness absence duration, 4DSQ and BAT-scores of absentees diagnosed with stress complaints, adaptation disorder and burnout?; 2) Is the BAT a better predictor of sickness absence duration than the 4DSQ? From 2020-2023, data was collected among all 3,462 employees who were absent due to stress complaints, adaptation disorder or burnout at one large occupational health and safety service via registry data and an employee questionnaire. Results show that two-thirds of those with stress complaints, half of them with adaptation disorder and one third with burnout were absent for less than half a year. 4DSQ and BAT scores differed significantly between the three groups (p<.05). The BAT explained 0.8% of variance in absence duration (p< .001) when controlled for covariates, which explained 1.6% of variance (p< .001). The explanation by BAT was 1.9% within the group with stress complaints (p = 0.002), .4% within the group with adaptation disorder (p=.022) and absent within the group with burnout. Comparable results were found for the 4DSQ. The differences in absence duration between the three groups aligned with the Dutch guidelines. The BAT nor the 4DSQ were clinically relevant predictors of duration of absence for stress-related diagnoses among absentees in the Netherlands. To reduce productivity loss, it seems better to report sick when still in the stage of stress complaints than to wait until a full burnout has developed. The BAT and 4DSQ might help risk assessment in the working population. Key messages • The sickness absence duration of Dutch employees diagnosed with burnout is much longer than that of those with adaptation disorder or stress complaints. • Both the instrument recommended in the guideline and the new instrument have no clinical relevance in predicting sickness absence duration of stress-related diagnoses.

Abstract T cell therapies have shown great promise, especially for the treatment of hematological malignancies, but this success has not been translatable for most solid tumors. Therefore, efforts have been put towards improving their efficacy, including optimization of the subset composition of the T cell product and development of strategies to overcome immunosuppression by the tumor microenvironment. Here, we combined advanced immune-organoid co-cultures and live 3D imaging to understand how different T cell subsets and immune cells in the tumor microenvironment can affect T cell therapy efficacy. We found that CD4+ and CD8+ subsets of therapeutic T cells cooperate to boost anti-tumor function, while tumor-associated macrophages (TAMs) have an opposing effect and reduce tumor-targeting by engineered T cells. Through our dynamic imaging-readout (Dekkers, Alieva Nature Biotech. 2022), we observed that a high level of interactions are involved in these modulating events, indicating direct crosstalk between the different cell types. To further investigate the impact of these interactions, we developed a 3D interactome mapping tool which enables us to profile all occurring cell-cell interactions in our 3D imaging datasets and determine their functional outcome. With this tool we identified a specific interaction between CD4+ and CD8+ T cells, where a subset of CD4+ T cells acquired a potent helper function over time by first engaging with tumor organoids and subsequently inducing CD8+ T cell target localization via direct contact. Multi-omics integration with single cell transcriptomic data allowed us to study the underlying molecular pathways involved in this interaction, leading to the identification of a crucial binding partner involved in this cooperative tumor-targeting behavior. Using the same strategy, we are currently investigating how interactions between TAMs and therapeutic T cells modulate their tumor-targeting activity. Together, these data highlight how cellular interactions can play an important role in engineered T cell function and we are currently exploring strategies to target these axes of immune cell communication to maximize therapy efficacy. Citation Format: Amber K.L. Wezenaar, Celina Honhoff, Helena van Eck, Elena Jiménez Curiel, Annelisa M Cornel, Femke C.A. Ringnalda, Tineke Aarts-Riemens, Jürgen Kuball, Zsolt Sebestyen, Hans Clevers, Stefan Nierkens, Johanna F. Dekkers, Ellen J. Wehrens, Maria Alieva, Anne C. Rios. Communication is key: Understanding the modulating effect of immune cell crosstalk on T cell therapy function [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B003.

<p>Cell-cell interaction output T/NK vs. myeloid cells</p>

<p>Differential gene enrichment from CRISPR-Cas9 genome-wide screens</p>

The small GTPases H, K, and NRAS are molecular switches that are indispensable for proper regulation of cellular proliferation and growth. Mutations in this family of proteins are associated with cancer and result in aberrant activation of signaling processes caused by a deregulated recruitment of downstream effector proteins. In this study, we engineered novel variants of the Ras-binding domain (RBD) of the kinase CRAF. These variants bound with high affinity to the effector binding site of active Ras. Structural characterization showed how the newly identified mutations cooperate to enhance affinity to the effector binding site compared to RBDwt. The engineered RBD variants closely mimic the interaction mode of naturally occurring Ras effectors and as dominant negative affinity reagent block their activation. Experiments with cancer cells showed that expression of these RBD variants inhibits Ras signaling leading to a reduced growth and inductions of apoptosis. Using the optimized RBD variants, we stratified patient-derived colorectal cancer organoids according to Ras dependency, which showed that the presence of Ras mutations was insufficient to predict sensitivity to Ras inhibition.

Studies of work and family issues have used predominately between-subject cross-sectional designs. While some researchers have called for more longitudinal studies, others have suggested that a more strategic way forward for the field is the use of daily diary studies (Frone, Russell, & Cooper, 1992). This study explores different types of interference between work and non-work domains, using a diary study methodology. This study consisted of a two-week diary study among 12 Irish employees. Analysis involved a two-step procedure involving both content analysis and interpretative phenomenological analysis (IPA). Results indicated that while individuals reported strain-based and time-based interference, evidence was also found of interference concerning persistent and intrusive thoughts. Despite some limitations to the study, this paper demonstrates that diary studies represent an opportunity for researchers to explore work and non-work experiences in a sensitive and ecologically valid way.

Abstract Pseudomonas aeruginosa is an opportunistic pathogen that causes major healthcare concerns due to its virulence and high intrinsic resistance to antimicrobial agents. Therefore, new treatments are highly needed. An interesting approach is to target quorum sensing (QS). QS regulates the production of a wide variety of virulence factors and biofilm formation in P. aeruginosa . This study describes the identification of paecilomycone as inhibitor of QS in both C. violaceum and P. aeruginosa . Paecilomycone strongly inhibited the production of virulence factors, including various phenazines, and biofilm formation. In search of the working mechanism, we found that paecilomycone inhibited the production of 4-hydroxy-2-heptylquinoline (HHQ) and 3,4- dihydroxy-2-heptylquinoline (PQS), but not 2’-aminoacetophenone (2-AA). We suggest that paecilomycone affects QS in P. aeruginosa by targeting the PqsBC complex and alternative targets, or alters processes that influence the enzymatic activity of the PqsBC complex. The toxicity of paecilomycone towards eukaryotic cells and organisms was low, making it an interesting lead for further clinical research. Importance Antibiotics are becoming less effective against bacterial infections due to the evolution of resistance among bacteria. Pseudomonas aeruginosa is a Gram-negative pathogen that causes major healthcare concerns and is difficult to treat due to its high intrinsic resistance to antimicrobial agents. Therefore, new targets are needed and an interesting approach is to target quorum sensing (QS). QS is the communication system in bacteria that regulates multiple pathways including the production of virulence factors and biofilm formation, which leads to high toxicity in the host and low sensitivity to antibiotics, respectively. We found a compound, named paecilomycone, which inhibited biofilm formation and the production of various virulence factors in P. aeruginosa. The toxicity of paecilomycone towards eukaryotic cells and organisms was low, making it an interesting lead for further clinical research.

Abstract Various species of intestinal microbiota have been associated with the development of colorectal cancer (CRC), yet a direct role of bacteria in the occurrence of oncogenic mutations has not been established. Escherichia coli can carry the pathogenicity island pks within their genomes, which encodes a set of enzymes that synthesize colibactin. This compound can alkylate nascent DNA on adenine residues, and it has been shown to induce double-strand breaks in cultured cells. We hypothesized that pks+ E. coli are carcinogenic through the mutagenic action of colibactin. To test this, we exposed human intestinal organoids for 5 months to genotoxic pks+ Escherichia coli by repeated luminal injection. Whole-genome sequencing (WGS) of clonal organoids before and after exposure revealed a distinct and reproducible mutational signature, which was completely absent in organoids that were injected with isogenic pks-deficient bacteria. This signature encompasses a single base substitution trinucleotide signature (SBS-pks) with a marked transcriptional strand bias in line with damage on adenine residues, which was accompanied by a specific indel signature (ID-pks). Moreover, a distinct preference for the wider sequence context was detected, fitting a structural model of colibactin bound to DNA. This mutational signature was detected in three cohorts containing more than 6,000 human cancer genomes, and contribution of this signature could be detected in a subset of CRC genomes and more rarely in other cancer types. Finally, we identified driver genes harboring the colibactin target motif and could attribute driver mutations from CRC genomes to the signature. Our study describes a highly specific mutational cancer signature that reflects mutagenic activity of pks+ bacteria in the intestinal lumen, which can induce oncogenic mutations and could contribute to cancer initiation. This abstract is also being presented as Poster A02. Citation Format: Jens Puschhof, Cayetano Pleguezuelos-Manzano, Axel Rosendahl Huber, Ruben van Bostel, Hans Clevers. A mutational signature in human colorectal cancer induced by genotoxic pks+ E. coli [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr PR02.

Objective Serrated polyps (hyperplastic polyps, sessile or traditional serrated adenomas), which can arise in a sporadic or polyposis setting, predispose to colorectal cancer (CRC), especially those with microsatellite instability (MSI) due to MLH1 promoter methylation (MLH1 me+ ). We investigate genetic alterations in the serrated polyposis pathway. Design We used a combination of exome sequencing and target gene Sanger sequencing to study serrated polyposis families, sporadic serrated polyps and CRCs, with validation by analysis of The Cancer Genome Atlas (TCGA) cohort, followed by organoid-based functional studies. Results In one out of four serrated polyposis families, we identified a germline RNF43 mutation that displayed autosomal dominant cosegregation with the serrated polyposis phenotype, along with second-hit inactivation through loss of heterozygosity or somatic mutations in all serrated polyps (16), adenomas (5) and cancer (1) examined, as well as coincidental BRAF mutation in 62.5% of the serrated polyps. Concurrently, somatic RNF43 mutations were identified in 34% of sporadic sessile/traditional serrated adenomas, but 0% of hyperplastic polyps (p=0.013). Lastly, in MSI CRCs, we found significantly more frequent RNF43 mutations in the MLH1 me+ (85%) versus MLH1 me− (33.3%) group (p<0.001). These findings were validated in the TCGA MSI CRCs (p=0.005), which further delineated a significant differential involvement of three Wnt pathway genes between these two groups (RNF43 in MLH1 me+ ; APC and CTNNB1 in MLH1 me− ); and identified significant co-occurrence of BRAF and RNF43 mutations in the MSI (p<0.001), microsatellite stable (MSS) (p=0.002) and MLH1 me+ MSI CRCs (p=0.042). Functionally, organoid culture of serrated adenoma or mouse colon with CRISPR-induced RNF43 mutations had reduced dependency on R-spondin1. Conclusions These results illustrate the importance of RNF43, along with BRAF mutation in the serrated neoplasia pathway (both the sporadic and familial forms), inform genetic diagnosis protocol and raise therapeutic opportunities through Wnt inhibition in different stages of evolution of serrated polyps.

No abstract is provided for this article.

The original version of this Article contained an error in the Discussion section.Additionally, the Reference, which is listed below as Reference 44, was omitted.As a result, "Most importantly, a cross-comparison of these cell types between organs has not previously been given." now reads: "Last year, by comparing the transcriptome of human epithelial cells in nasal, bronchial, and epididymal samples, Paranjapye et al. described conservation of ionocytes between these tissues 44 .However, a complementing crosscomparison of these cell types between other organs has not previously been given."