Exploration of the tissue architecture of IC and ID β <i>HLTF</i> KO pancreata.

(A) Two-dimensional batch-corrected t-distributed stochastic neighbor embedding (t-SNE) visualization of the UMI counts from the entire IC vs ID dataset. (B) Pancreata clusters 16 and 18 are shown in t-SNE space. (C) Pancreatic genes insulin 1 (Ins1) and insulin 2 (Ins2) illustrated differential gene expression in IC vs ID β Hltf KO mice in t-SNE plots of clusters 16 and 18. The unique expression of immune cell markers in IC vs ID tissue is elaborated in t-SNE plots of these clusters. Slamf6 and Il2rb were added to previously identified markers for NK cells (GzmA, Klrb1b). Five markers for B cells (Pax5, Blk, Fcmr, Fcrla, Tnfrasf9) and three markers for activation of innate immunity (Bpifb1, Serpinb3a, Defb36) were unique to cluster 18.</p

Immunolabeling with anti-γH2AX.

Extensive γH2AX labeling in HLTF-/-CDX ID Hltf KO mice from endogenous DNA damage (A) strongly contrasts with the absence of γH2AX labeling in HLTF+/+CDX ID Hltf KO mice (B). CDX tumors (T) as well as tumor cells in lymphatic intravascular niches (blue arrow heads) of the mouse TME are labeled. (C) γH2AX labeling in mouse testis during meiosis (positive control) occurs in all intermediate and type B spermatogonia (last mitotic cells) and in preleptotene through zygotene spermatocytes during normal spermatogenesis. Panels A-C, 40X magnification.</p

Statistics for spatial transcriptomics outcome for Visium_FFPE_Mouse_Pancreas in ID β <i>Hltf</i>-KO (Sample ID, A and B) and IC β <i>Hltf</i>-KO (Sample ID, C and D).

Statistics for spatial transcriptomics outcome for Visium_FFPE_Mouse_Pancreas in ID β Hltf-KO (Sample ID, A and B) and IC β Hltf-KO (Sample ID, C and D).</p

<i>t</i>-SNE projection of spots and a violin plot.

Principle component analysis (PCA) was run on the normalized filtered feature-barcoded matrix to reduce the number of feature dimensions prior to clustering. For gene expression after PCA, t-distributed neighbor embedding (t-SNE) was run to visualize spots in a 2-D space, followed by clustering to group spots with similar expression profiles. t-SNE colored by UMI counts per spot (A) and spots by clustering (B) are shown for the entire data set. Comparative expression (C) analysis revealed variability in the spatiotemporal distribution of HIF-1a expression across 16 clusters for the entire data set.</p

Catastrophic consequences of Hltf-deletion from pancreatic β cells during development.

Recruitment of NK cells expressing Prf1-GzmA—triggered by the Hmgb1-RAGE axis in response to impaired DNA-damage repair—perpetuated DNA damage and selective loss of pancreatic β cells. Created with Biorender.com.</p

Statistics for spatial transcriptomics outcome for Visium_FFPE_<i>HLTF</i>^-/-CDX in ID <i>Hltf</i> KO mice (A and B) and <i>HLTF</i>^<i>+/+</i>CDX in ID <i>Hltf</i> KO (C and D).

Statistics for spatial transcriptomics outcome for Visium_FFPE_HLTF-/-CDX in ID Hltf KO mice (A and B) and HLTF+/+CDX in ID Hltf KO (C and D).</p

Differential γH2Ax pan-staining and TUNEL assay.

Abundant γH2Ax in β cells from IC β Hltf KO mice (A) compared to minimal immunostaining in β cells from ID β Hltf KO mice (B). Two-types of γH2Ax pan-staining are evident. β cells from IC β Hltf KO mice have apoptotic rings and the β cells from ID β Hltf KO mice have limited pan-nuclear staining of the entire nucleus. Results from the terminal deoxynuceotidyl transferase dUTP nick-end labeling (TUNEL) assay (C), which detects β cell death-associated DNA fragmentation (3’-OH termini), indicates the amount of DNA damage is more than the targeted β cells can efficiently repair when the animals are IC. Cell-death in IC Hltf +/+ controls and ID β Hltf KO mice was negligible. A positive mouse testis control (D) was included because apoptosis is an important component of normal spermatogenesis.</p

Indicators of OXPHOS and glutathione biosynthesis in <i>Hltf</i>^<i>-/-</i>CDX model.

(A) Photomicrograph emphasizes gradient of positive immunostaining for MT-CYB in tumor cells of the Hltf-/-CDX including tumor cells in lymphatic intravascular niches (blue arrow heads) of the mouse TME. 10X magnification. (B) Violin plots from spatial transcriptomics depicts regional gene expression. Each violin plot is a composite of genes. The top plot is for the trio of genes in GSH synthesis, i.e., the rate-limiting enzyme glutamate cysteine ligase (GCL), which is composed of catalytic (GCLC) and modifier (GCLM) subunits, and GSH synthetase (GSS). The bottom plot shows gene expression for mitochondrial glutathione transporters, SLC25A10 and SLC25A39. (C) S-glutathionylation was visualized with a biotin switch assay and confocal imaging, which allowed for optimal detection of S-glutathionylated proteins over potential background signals. For data presentation, the maximal projection confocal images obtained from a z stack (500 nm slice) using 10x (upper) and 60x (lower) objectives (oil) are shown. Cells were visualized using Nikon T1-E microscope with A1 confocal and images were taken using NIS software. Merged images were obtained with transmitted light and excitation at 405 nm (DAPI, blue-violet) nuclei and 488 (streptavidin-labeled, green) S-glutathionylated proteins. Human tumor and mouse endothelial cells (yellow asterisk) harbor S-glutathionylated proteins. The mouse endothelium is in the lamina propria that subtends the mucosal epithelium.</p

NC3Rs ARRIVE guidelines checklist (fillable).

Helicase-like transcription factor (HLTF) also known as SMARCA3, protects genome integrity. A tumor suppressor, HLTF is expressed in tumor cells but not in the tumor microenvironment (TME) in early-stage colorectal cancer (CRC). With disease progression, there is high concordance between epigenetic silencing of HLTF in CRC cells and negligible HLTF expression in the TME. We developed a cell line-derived xenograft (CDX) model and show for the first time that HLTF-deletion in cancer cells and the TME results in metabolic reprogramming that mitigates oxidative stress in lymphatic intravascular metastatic niches. The two metabolic pathways that derive energy from glucose—glycolysis and oxidative phosphorylation (OXPHOS)—are variously utilized by cancer cells depending upon the TME. HIF-1α, a master regulator of glycolysis, was eliminated from a role in reprogramming metabolism to satisfy CDX energetic requirements by RNAseq and spatial transcriptomics. Variability in the gut microbiome, with a putative role in altered metabolism, was also eliminated. HLTF-deleted cancer cells recovered from DNA damage at a transcriptomic level induction of DNA repair and OXPHOS genes linked to an amoeboid-associated phenotype at the tumor border (confocal microscopy). HLTF-deleted cancer and endothelial cells of lymphatic (PDPN) intravascular niches in the TME shared a site-specific protein S-glutathionylation signature (2D DIGE, MALDI-TOF/TOF mass spectrometry) for three glycolytic enzymes (PGK1 Cys379/380, PGAM1 Cys55, ENOA1 Cys119) that diverted glycolysis in support of continued glutathione biosynthesis. The collective absence of HLTF/Hltf from tumor and TME achieved redox homeostasis throughout the CDX and promoted metastasis.</div

Sample quality control and RNAseq outcomes.

Epigenetic mechanisms are integral to pancreatic β cell function. Promoter hypermethylation of the helicase like-transcription factor (HLTF) gene—a component of the cellular DNA damage response that contributes to genome stability—has been implicated in age-associated changes in β cells. To study HLTF, we generated global and β cell-specific (β) Hltf knockout (KO) immune competent (IC) and immune deficient (ID) Rag2-/IL2- mice. IC global and β Hltf KO mice were neonatal lethal whereas ID global and β Hltf KO newborn mice had normal survival. This focused our investigation on the effects of Rag2 interruption with common gamma chain interruption on β cell function/survival. Three-way transcriptomic (RNAseq) analyses of whole pancreata from IC and ID newborn β Hltf KO and wild type (Hltf +/+) controls combined with spatially resolved transcriptomic analysis of formalin fixed paraffin embedded tissue, immunohistochemistry and laser scanning confocal microscopy showed DNA damage caused by β Hltf KO in IC mice upregulated the Hmgb1-Rage axis and a gene signature for innate immune cells. Perforin-delivered granzyme A (GzmA) activation of DNase, Nme1, showed damaged nuclear single-stranded DNA (γH2AX immunostaining). This caspase-independent method of cell death was supported by transcriptional downregulation of Serpinc1 gene that encodes a serine protease inhibitor of GzmA. Increased transcriptional availability of complement receptors C3ar1 and C5ar1 likely invited crosstalk with Hmgb1 to amplify inflammation. This study explores the complex dialog between β cells and immune cells during development. It has implications for the initiation of type I diabetes in utero when altered gene expression that compromises genome stability invokes a localized inflammatory response.</div