Mapping the complexity and diversity of tertiary lymphoid structures in primary and peritoneal metastatic gastric cancer.

BACKGROUND: Tertiary lymphoid structures (TLSs) are thought to stimulate antitumor immunity and positively impact prognosis and response to immune checkpoint blockade. In gastric cancers (GCs), however, TLSs are predominantly found in GC with poor prognosis and limited treatment response. We, therefore, hypothesize that immune cell composition and function of TLS depends on tumor location and the tumor immune environment. METHODS: Spatial transcriptomics and immunohistochemistry were used to characterize the phenotype of CD45+ immune cells inside and outside of TLS using archival resection specimens from GC primary tumors and peritoneal metastases. RESULTS: We identified significant intrapatient and interpatient diversity of the cellular composition and maturation status of TLS in GC. Tumor location (primary vs metastatic site) accounted for the majority of differences in TLS maturity, as TLS in peritoneal metastases were predominantly immature. This was associated with higher levels of tumor-infiltrating macrophages and Tregs and less plasma cells compared with tumors with mature TLS. Furthermore, mature TLSs were characterized by overexpression of antitumor immune pathways such as B cell-related pathways, MHC class II antigen presentation while immature TLS were associated with protumor pathways, including T cell exhaustion and enhancement of DNA repair pathways in the corresponding cancer. CONCLUSION: The observation that GC-derived peritoneal metastases often contain immature TLS which are associated with immune suppressive regulatory tumor-infiltrating leucocytes, is in keeping with the lack of response to immune checkpoint blockade and the poor prognostic features of peritoneal metastatic GC, which needs to be taken into account when optimizing immunomodulatory strategies for metastatic GC.

Phenotypic immune characterization of gastric and esophageal adenocarcinomas reveals profound immune suppression in esophageal tumor locations.

Background: Tumors in the distal esophagus (EAC), gastro-esophageal junction including cardia (GEJAC), and stomach (GAC) develop in close proximity and show strong similarities on a molecular and cellular level. However, recent clinical data showed that the effectiveness of chemo-immunotherapy is limited to a subset of GEAC patients and that EACs and GEJACs generally benefit less from checkpoint inhibition compared to GACs. As the composition of the tumor immune microenvironment drives response to (immuno)therapy we here performed a detailed immune analysis of a large series of GEACs to facilitate the development of a more individualized immunomodulatory strategy. Methods: Extensive immunophenotyping was performed by 14-color flow cytometry in a prospective study to detail the immune composition of untreated gastro-esophageal cancers (n=104) using fresh tumor biopsies of 35 EACs, 38 GEJACs and 31 GACs. The immune cell composition of GEACs was characterized and correlated with clinicopathologic features such as tumor location, MSI and HER2 status. The spatial immune architecture of a subset of tumors (n=30) was evaluated using multiplex immunohistochemistry (mIHC) which allowed us to determine the tumor infiltration status of CD3+, CD8+, FoxP3+, CD163+ and Ki67+ cells. Results: Immunophenotyping revealed that the tumor immune microenvironment of GEACs is heterogeneous and that immune suppressive cell populations such as monocytic myeloid-derived suppressor cells (mMDSC) are more abundant in EACs compared to GACs (p<0.001). In contrast, GACs indicated a proinflammatory microenvironment with elevated frequencies of proliferating (Ki67+) CD4 Th cells (p<0.001), Ki67+ CD8 T cells (p=0.002), and CD8 effector memory-T cells (p=0.024). Differences between EACs and GACs were confirmed by mIHC analyses showing lower densities of tumor- and stroma-infiltrating Ki67+ CD8 T cells in EAC compared to GAC (both p=0.021). Discussions: This comprehensive immune phenotype study of a large series of untreated GEACs, identified that tumors with an esophageal tumor location have more immune suppressive features compared to tumors in the gastro-esophageal junction or stomach which might explain the location-specific responses to checkpoint inhibitors in this disease. These findings provide an important rationale for stratification according to tumor location in clinical studies and the development of location-dependent immunomodulatory treatment approaches.

Taxonomic Profiling of Fecal and Intestinal Microbiota in the African Turquoise Killifish Nothobranchius furzeri.

Killifish are emerging as a new laboratory system in which to study a range of questions, from the genetic basis of embryo dormancy to life history trait evolution, age-dependent neurodegeneration, and the connection between microbial community structure and biology of aging. Over the past decade, advances in high-throughput sequencing have helped uncover the vast diversity of microbial communities present in environmental samples and on host epithelia. Here, we describe an optimized protocol to study the taxonomic composition of intestinal and fecal microbiota in laboratory-raised as well as natural killifish populations and provide comprehensive step-by-step instructions for tissue sampling, high-throughput genomic DNA extraction, and the generation of 16S V3V4 rRNA and 16S V4 rRNA gene libraries.

Sequencing the Immunoglobulin Heavy-Chain Locus (IgH) in Turquoise Killifish (Nothobranchius furzeri).

B cells undergo a process of somatic recombination leading to the synthesis of a staggering diversity of transmembrane and secreted antibodies. This process dates back to the evolution of jawed vertebrates and is pivotal to the origin of lymphocyte-based adaptive immune responses. Here, we optimized a sequencing-based protocol to characterize B-cell-specific IgH transcripts from bulk RNA in the African turquoise killifish, an emerging model organism characterized by naturally short life span and by a vast range of age-dependent dysfunctions.

The role of the gut microbiome during host ageing.

Gut microbial communities participate in key aspects of host biology, including development, nutrient absorption, immunity and disease. During host ageing, intestinal microbes undergo dramatic changes in composition and function and can shift from commensal to pathogenic. However, whether they play a causal role in host ageing and life span has remained an open question for a long time. Recent work in model organisms has revealed for the first time that gut microbes can modulate ageing, opening new questions and opportunities to uncover novel ageing-modulating mechanisms and to design anti-ageing interventions by targeting the gut microbiota.

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri).

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish - the turquoise killifish (Nothobranchius furzeri) - has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test - in a short time - experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span - ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.