Zoe S. Walters

Lymph node (LN) metastasis in adenocarcinomas of the upper gastrointestinal (GI) tract is a major determinant of patient outcomes following multimodal therapy and surgical resection. Despite its clinical significance, the biology enabling metastatic persistence within lymph nodes remains poorly understood, particularly given the immunologically active nature of the LN microenvironment. This review systematically evaluates the current literature investigating the metastatic LN microenvironment in upper GI adenocarcinomas. A comprehensive search of three databases identified 289 articles, of which 20 met inclusion criteria after screening. Synthesis of the available evidence highlighted findings across cell types within the metastatic LN microenvironment, although B cells and fibroblasts remain comparatively underexplored. Three principal themes emerged from the literature, supporting a hypothesis of dysfunctional anti-tumour immunity within metastatic LNs: macrophages and neutrophils demonstrated pro-tumoural phenotypes characterised by the expression of SPP1 and CXCR4 , respectively. Markers of T cell exhaustion were elevated within metastatic LNs although PD-1/PD-L1 expression was lowered, suggesting the use of non-classical immune checkpoint pathways. Current evidence indicates that macrophage- and neutrophil-mediated signalling, including SPP1/CD44 interactions and increased IL-8 expression, may contribute to an immunosuppressive metastatic niche. Collectively, this review consolidates the existing evidence surrounding the metastatic LN microenvironment in upper GI adenocarcinomas and identifies key biological and translational gaps requiring further investigation.

Rhabdomyosarcoma (RMS) is a predominantly paediatric cancer that is classified by the presence or absence of a PAX-FOXO1 fusion gene, which is associated with a worse prognosis. Previous classification was based on histology, alveolar RMS (ARMS) or embryonal RMS (ERMS). In other paediatric cancers, fusion gene status has been shown to associate with differences in the tumour microenvironment (TME). However, comprehensive understanding of the TME in RMS and how it may differ between subtypes is lacking. This systematic review aimed to identify differences in the TME between fusion-positive RMS and fusion-negative RMS, to better understand how the fusion gene drives malignancy. The Web of Science, MEDLINE (Ovid), and EMBASE (Ovid) were searched to identify relevant studies investigating the TME in RMS. A total of 17 studies met the inclusion criteria and were included in the review, but only three studies specified fusion status in their sample data. Nine studies investigated the extracellular matrix and stroma, and another nine investigated the immune microenvironment. Significant differences in CD163+ macrophages, matrix metalloproteinases and stromal platelet-derived growth factor receptors-α/β were observed between ARMS and ERMS. Regarding fusion status, there were differences in the prevalence of T cell dysfunction, NECTIN-3 expression, and genes related to PD-1 signalling and interferon (IFN) response. This review highlights a need for further research of the TME in each fusion subtype. This will improve our understanding of how the fusion gene drives malignancy and ultimately aids in the development of novel treatment strategies.

The tumor microenvironment (TME) comprises all non-tumor elements of cancer and strongly influences disease progression and phenotype. To understand tumor biology and accurately test new therapeutic strategies, representative models should contain both tumor cells and normal cells of the TME. Here we describe and characterize co-culture tumor-derived organoids and cancer-associated fibroblasts (CAFs), a major component of the TME, in matrix-embedded assembloid models of esophageal adenocarcinoma (EAC). We demonstrate that the assembloid models faithfully recapitulate the differentiation status of EAC and different CAF phenotypes found in the EAC patient TME. We evaluate cell phenotypes by combining tissue clearing techniques with wholemount immunofluorescence and histology, providing a practical framework for characterization of cancer assembloids.

Background: Rhabdomyosarcomas (RMS) are predominantly paediatric sarcomas thought to originate from muscle precursor cells due to impaired myogenic differentiation. Despite intensive treatment, 5-year survival for patients with advanced disease remains low (< 30%), highlighting a need for novel therapies to improve outcomes. Differentiation therapeutics are agents that induce differentiation of cancer cells from malignant to benign. The histone methyltransferase, Enhancer of Zeste Homolog 2 (EZH2) suppresses normal skeletal muscle differentiation and is highly expressed in RMS tumours. Results: We demonstrate combining inhibition of the epigenetic modulator EZH2 with the differentiating agent retinoic acid (RA) is more effective at reducing cell proliferation in RMS cell lines than single agents alone. In PAX3-FOXO1 positive RMS cells this is due to an RA-driven induction of the interferon pathway resulting in apoptosis. In fusion negative RMS, combination therapy led to an EZH2i-driven upregulation of myogenic signalling resulting in differentiation. In both subtypes, EZH2 is significantly associated with enrichment of trimethylated lysine 27 on histone 3 (H3K27me3) in genes that are downregulated in untreated RMS cells and upregulated with EZH2 inhibitor treatment. These results provide insight into the mechanism that drives the anti-cancer effect of the EZH2/RA single agent and combination treatment and indicate that the reduction of EZH2 activity combined with the induction of RA signalling represents a potential novel therapeutic strategy to treat both subtypes of RMS. Conclusions: The results of this study demonstrate the potential utility of combining EZH2 inhibitors with differentiation agents for the treatment of paediatric rhabdomyosarcomas. As EZH2 inhibitors are currently undergoing clinical trials for adult and paediatric solid tumours and retinoic acid differentiation agents are already in clinical use this presents a readily translatable potential therapeutic strategy. Moreover, as inhibition of EZH2 in the poor prognosis FPRMS subtype results in an inflammatory response, it is conceivable that this strategy may also synergise with immunotherapies for a more effective treatment in these patients.

Rhabdomyosarcomas (RMS) are predominantly pediatric sarcomas thought to originate from muscle precursor cells due to impaired myogenic differentiation. Despite intensive treatment, 5-year survival for patients with advanced disease remains low (Highlights EZH2 expression is upregulated fusion positive (FPRMS) and fusion negative (FNRMS) rhabdomyosarcomas EZH2 inhibition combined with retinoic acid treatment was investigated RMS cell models. Combination treatment reduced cell proliferation and tumor spheroid volume. Combination treatment in FPRMS resulted in apoptosis in FPRMS via interferon signaling. Conversely, combination treatment in fusion negative RMS resulted in myogenic differentiation.