Publications

The 30th Annual North American Cystic Fibrosis Conference, Orange County Convention Center, Orlando, Florida, October 27–29

<p>Ewing sarcoma is infiltrated by immunosuppressive macrophages and functionally impaired APCs. <b>A,</b> UMAP and dotplot of the myeloid compartment, showing conventional dendritic cells type 1 and 2 (cDC1, cDC2), undifferentiated macrophages (Mo), and differentiated macrophage populations (Mφ) and a selection of their marker genes. <b>B,</b> Macrophage-specific scores for proinflammatory and anti-inflammatory signatures as well as M1 and M2 signatures. <b>C,</b> Antigen presentation and costimulatory capacity score as constructed with genes shown in <a href="#SMF7" target="_blank">Supplementary Fig. S7D</a>. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001 versus Mo. <b>D,</b> Barplot showing percentage of costimulatory gene-expressing Mo, Mφ, and cDCs. Neuroblastoma (NBL; Kildisiute and colleagues) data included as comparative group. Mean percentages and error bars are plotted for individual samples. Samples with <5 cells per group were excluded. <i>*</i>, <i>P <</i> 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001. <b>E,</b> Violin plot depicting DC maturation score for Ewing sarcoma and NBL cDCs. Z-scores were calculated using a curated MSigDB gene set (“LINDSTEDT_DENDRITIC_CELL_MATURATION_B”). This gene set includes upregulated genes both at 8 and 48 hours in response to inflammatory stimuli. ***, <i>P</i> < 0.001. <b>F,</b> Violin plots showing the <i>STAT3</i> and <i>NFKB1</i> gene expression levels of Ewing sarcoma and NBL cDCs. ***, <i>P</i> < 0.001 with Bonferroni correction.</p>

<p>Table S4</p>

ABSTRACT Cancers rely on multiple, heterogeneous processes at different scales, pertaining to many biomedical fields. Therefore, understanding cancer is necessarily an interdisciplinary task that requires placing specialised experimental and clinical research into a broader conceptual, theoretical, and methodological framework. Without such a framework, oncology will collect piecemeal results, with scant dialogue between the different scientific communities studying cancer. We argue that one important way forward in service of a more successful dialogue is through greater integration of applied sciences (experimental and clinical) with conceptual and theoretical approaches, informed by philosophical methods. By way of illustration, we explore six central themes: ( i ) the role of mutations in cancer; ( ii ) the clonal evolution of cancer cells; ( iii ) the relationship between cancer and multicellularity; ( iv ) the tumour microenvironment; ( v ) the immune system; and ( vi ) stem cells. In each case, we examine open questions in the scientific literature through a philosophical methodology and show the benefit of such a synergy for the scientific and medical understanding of cancer.

Binucleated polyploid cells are common in many animal tissues, where they arise by endomitosis, a non-canonical cell cycle in which cells enter M phase but do not undergo cytokinesis. Different steps of cytokinesis have been shown to be inhibited during endomitosis M phase in rodents, but it is currently unknown how human cells undergo endomitosis. In this study, we use fetal-derived human hepatocyte organoids (Hep-Orgs) to investigate how human hepatocytes initiate and execute endomitosis. We find that cells in endomitosis M phase have normal mitotic timings, but lose membrane anchorage to the midbody during cytokinesis, which is associated with the loss of four cortical anchoring proteins, RacGAP1, Anillin, SEPT9, and citron kinase (CIT-K). Moreover, reduction of WNT activity increases the percentage of binucleated cells in Hep-Orgs, an effect that is dependent on the atypical E2F proteins, E2F7 and E2F8. Together, we have elucidated how hepatocytes undergo endomitosis in human Hep-Orgs, providing new insights into the mechanisms of endomitosis in mammals.

RNA life span at single-cell resolution RNA transcripts are an easily accessed representation of gene expression, but we lack a comprehensive view of the life span of RNA within the single cell. Battich et al. developed a method to sequence messenger RNA labeled with 5-ethynyl-uridine (EU) in single cells (scEU-seq), which allows estimation of RNA transcription and degradation rates. When examining intestinal organoid cells, scEU-seq data can be used to discern between transcription and degradation during development, indicating that this method can be applied to better understand the relationship between gene expression and RNA degradation during development. Science , this issue p. 1151

<p>Table S2</p>

Crypt base intestinal stem cells (ISCs) maintain the epithelial lining of the intestines, but their frequencies are reduced in experimental models of graft vs. host disease (GVHD). Paneth cells provide an epithelial contribution to the stem cell niche, but their frequencies are also reduced in GVHD mouse models and in patients with GVHD. Mechanisms regulating ISCs and their niche after damage remain poorly understood. Interleukin-33 (IL-33) is an immunomodulatory alarmin typically thought to target lymphocytes such as T cells and innate lymphoid cells. In experimental models of allogeneic bone marrow transplantation, IL-33 can promote or attenuate GVHD, depending on the cellular target and the timing of the exposure. A secreted isoform of the IL-33 receptor, termed soluble ST2 (sST2), acts as a negative regulator by blocking IL-33 from binding to the membrane-bound isoform that mediates intracellular signaling. sST2 also has diagnostic and prognostic utility, serving as a biomarker for GVHD severity. While IL-33 has well-described effects on lymphocytes populations, there is limited understanding of the role of IL-33 signaling within the intestinal mucosa after damage. Utilizing experimental radiation injury to model conditioning-related intestinal pathology, we investigated the role of the IL-33/ST2 axis in epithelial damage and regeneration. Wild-type (WT) mice increased IL-33 mRNA and protein expression in the small intestine within days after 10 Gy total body irradiation (TBI). Intestinal sST2 expression also increased quickly in WT mice after TBI, but not in IL-33 -/- mice, suggesting that IL-33 itself was a regulator of sST2. While the ileal mucosa appeared similar at baseline in wild-type and IL-33 -/- mice, the IL-33 -/- mice demonstrated more severe radiation injury 5 days after 10 Gy TBI, with more crypt loss, loss of ISCs and Paneth cells, and an attenuated regenerative response with smaller crypts post-TBI containing fewer Ki67 + cells. 3-D whole-mount confocal microscopy of ileal tissue from IL-33-GFP reporter mice indicated that the crypt epithelium did not express IL-33 at baseline, but Olfm4 + ISCs adjacent to Paneth cells began to express IL-33 following radiation injury (Fig 1). Anti-Thy1-mediated lymphocyte depletion prior to irradiation of WT and IL-33 -/- mice did not abrogate the increased radiation sensitivity of IL-33 -/- mice, indicating a role for IL-33 beyond immunomodulation and suggesting that IL-33 could have direct effects within the epithelium. Intestinal organoid cultures confirmed epithelial expression of IL-33, confirmed increased radiation sensitivity of IL-33 -/- epithelium, and identified impaired epidermal growth factor (EGF) expression in the absence of IL-33. Furthermore, treatment of intestinal organoids with IL-33 induced EGF mRNA and protein expression, both of which were abrogated by the p38 inhibitor SB202190. Treatment of purified Lysozyme-DsRed + Paneth cells indicated that IL-33 directly promoted their production of EGF. In vivo, TBI-induced EGF upregulation was absent in IL-33 -/- mice. To further investigate this newly identified epithelial IL-33/ST2/EGF pathway in the ISC compartment, RiboTag mice, which facilitate analysis of lineage-specific proteome-relevant gene expression with minimal tissue processing and artifact introduction, were crossed to Olfm4-Cre and Lysozyme-Cre transgenics. Lineage-specific ribosome targeting was confirmed by immunostains and qPCR, and this approach also confirmed ISC upregulation of IL-33 following radiation injury and Paneth cell upregulation of EGF and sST2. Finally, to investigate the importance of this radiation-induced EGF upregulation, WT mice were treated with the EGFR inhibitor gefitinib following irradiation, which resulted in more severe radiation-induced ISC loss, similar to IL-33 deficiency. Furthermore, epithelial regeneration and ISC recovery were augmented in IL-33 -/- mice after TBI by treatment with exogenous EGF (Fig 2). These findings reveal an unknown pathway of stem cell niche regulation and crypt regeneration whereby the Paneth cell niche responds dynamically upon injury by upregulating EGF and the stem cells orchestrate regeneration by regulating their niche with IL-33. This regenerative circuit also highlights the breadth of IL-33 activity beyond immunomodulation and the therapeutic potential of EGF administration for treatment of intestinal injury.