Tissue clearing and immunostaining to visualize the spatial organization of vasculature and tumor cells in mouse liver

The liver has a complex and hierarchical segmental organization of arteries, portal veins, hepatic veins and lymphatic vessels. In-depth imaging of liver vasculature and malignancies could improve knowledge on tumor micro-environment, local tumor growth, invasion, as well as metastasis. Non-invasive imaging techniques such as computed tomography (CT), magnetic resonance imaging (MRI) and positron-emission transmission (PET) are routine for clinical imaging, but show inadequate resolution at cellular and subcellular level. In recent years, tissue clearing – a technique rendering tissues optically transparent allowing enhanced microscopy imaging – has made great advances. While mainly used in the neurobiology field, recently more studies have used clearing techniques for imaging other organ systems as well as tumor tissues. In this study, our aim was to develop a reproducible tissue clearing and immunostaining model for visualizing intrahepatic blood microvasculature and tumor cells in murine colorectal liver metastases. CLARITY and 3DISCO/iDISCO+ are two established clearing methods that have been shown to be compatible with immunolabelling, most often in neurobiology research. In this study, CLARITY unfortunately resulted in damaged tissue integrity of the murine liver lobes and no specific immunostaining. Using the 3DISCO/iDISCO+ method, liver samples were successfully rendered optically transparent. After which, successful immunostaining of the intrahepatic microvasculature using panendothelial cell antigen MECA-32 and colorectal cancer cells using epithelial cell adhesion molecule (EpCAM) was established. This approach for tumor micro-environment tissue clearing would be especially valuable for allowing visualization of spatial heterogeneity and complex interactions of tumor cells and their environment in future studies

Modeling resistance of colorectal peritoneal metastases to immune checkpoint blockade in humanized mice

Background The immunogenic nature of metastatic colorectal cancer (CRC) with high microsatellite instability (MSI-H) underlies their responsiveness to immune checkpoint blockade (ICB). However, resistance to ICB is commonly observed, and is associated with the presence of peritoneal-metastases and ascites formation. The mechanisms underlying this site-specific benefit of ICB are unknown. Methods We created a novel model for spontaneous multiorgan metastasis in MSI-H CRC tumors by transplanting patient-derived organoids (PDO) into the cecum of humanized mice. Anti-programmed cell death protein-1 (PD-1) and anti-cytotoxic T-lymphocytes-Associated protein 4 (CTLA-4) ICB treatment effects were analyzed in relation to the immune context of primary tumors, liver metastases, and peritoneal metastases. Immune profiling was performed by immunohistochemistry, flow cytometry and single-cell RNA sequencing. The role of B cells was assessed by antibody-mediated depletion. Immunosuppressive cytokine levels (interleukin (IL)-10, transforming growth factor (TGF)b1, TGFb2, TGFb3) were determined in ascites and serum samples by ELISA. Results PDO-initiated primary tumors spontaneously metastasized to the liver and the peritoneum. Peritoneal-metastasis formation was accompanied by the accumulation of ascites. ICB completely cleared liver metastases and reduced primary tumor mass but had no effect on peritoneal metastases. This mimics clinical observations. After therapy discontinuation, primary tumor masses progressively decreased, but peritoneal metastases displayed unabated growth. Therapy efficacy correlated with the formation of tertiary lymphoid structures (TLS)-containing B cells and juxtaposed T cells-and with expression of an interferon-γsignature together with the B cell chemoattractant CXCL13. B cell depletion prevented liver-metastasis clearance by anti-CTLA-4 treatment. Peritoneal metastases were devoid of B cells and TLS, while the T cells in these lesions displayed a dysfunctional phenotype. Ascites samples from patients with cancer with peritoneal metastases and from the mouse model contained significantly higher levels of IL-10, TGFb1, TGFb2 and TGFb3 than serum samples. Conclusions By combining organoid and humanized mouse technologies, we present a novel model for spontaneous multiorgan metastasis by MSI-H CRC, in which the clinically observed organ site-dependent benefit of ICB is recapitulated. Moreover, we provide empirical evidence for a critical role for B cells in the generation of site-dependent antitumor immunity following anti-CTLA-4 treatment. High levels of immunosuppressive cytokines in ascites may underlie the observed resistance of peritoneal metastases to ICB

Detection of Experimental Colorectal Peritoneal Metastases by a Novel PDGFRβ-Targeting Nanobody

Peritoneal metastases in colorectal cancer (CRC) belong to Consensus Molecular Subtype 4 (CMS4) and are associated with poor prognosis. Conventional imaging modalities, such as Computed Tomography (CT) and Fluorodeoxyglucose-Positron Emission Tomography (FDG-PET), perform very poorly in the detection of peritoneal metastases. However, the stroma-rich nature of these lesions provides a basis for developing molecular imaging strategies. In this study, conducted from 2019 to 2021, we aimed to generate a Platelet-Derived Growth Factor Receptor beta (PDGFRB)-binding molecular imaging tracer for the detection of CMS4 CRC, including peritoneal metastases. The expression of PDGFRB mRNA discriminated CMS4 from CMS1-3 (AUROC = 0.86 (95% CI 0.85–0.88)) and was associated with poor relapse-free survival. PDGFRB mRNA and protein levels were very high in all human peritoneal metastases examined (n = 66). Therefore, we generated a PDGFRB-targeting llama nanobody (VHH1E12). Biotin-labelled VHH1E12 bound to immobilized human and mouse PDGFRB with high affinity (EC50 human PDGFRB = 7 nM; EC50 murine PDGFRB = 0.8 nM), and to PDGFRB-expressing HEK293 cells grown in vitro. A pharmacokinetic analysis of IRDye-800CW-conjugated VHH1E12 in mice showed that the plasma half-life was 6 min. IRDye-800CW-conjugated VHH1E12 specifically accumulated in experimentally induced colorectal cancer peritoneal metastases in mice. A tissue analysis subsequently demonstrated co-localization of the nanobody with PDGFRB expression in the tumour stroma. Our results demonstrate the potential value of PDGFRB-targeted molecular imaging as a novel strategy for the non-invasive detection of CMS4 CRC, in particular, peritoneal metastases

KIT promotes tumor stroma formation and counteracts tumor-suppressive TGFβ signaling in colorectal cancer

Abstract Expression profiling has identified four consensus molecular subtypes (CMS1-4) in colorectal cancer (CRC). The receptor tyrosine kinase KIT has been associated with the most aggressive subtype, CMS4. However, it is unclear whether, and how, KIT contributes to the aggressive features of CMS4 CRC. Here, we employed genome-editing technologies in patient-derived organoids (PDOs) to study KIT function in CRC in vitro and in vivo. CRISPR-Cas9-mediated deletion of the KIT gene caused a partial mesenchymal-to-epithelial phenotype switch and a strong reduction of intra-tumor stromal content. Vice versa, overexpression of KIT caused a partial epithelial-to-mesenchymal phenotype switch, a strong increase of intra-tumor stromal content, and high expression of TGFβ1. Surprisingly, the levels of phosphorylated SMAD2 were significantly lower in KIT-expressing versus KIT-deficient tumor cells. In vitro analyses showed that TGFβ signaling in PDOs limits their regenerative capacity. Overexpression of KIT prevented tumor-suppressive TGFβ signaling, while KIT deletion sensitized PDOs to TGFβ-mediated growth inhibition. Mechanistically, we found that KIT expression caused a strong reduction in the expression of SMAD2, a central mediator of canonical TGFβ signaling. We propose that KIT induces a pro-fibrotic tumor microenvironment by stimulating TGFβ expression, and protects the tumor cells from tumor-suppressive TGFβ signaling by inhibiting SMAD2 expression

Impact of Global Fxr Deficiency on Experimental Acute Pancreatitis and Genetic Variation in the FXR Locus in Human Acute Pancreatitis

BACKGROUND: Infectious complications often occur in acute pancreatitis, related to impaired intestinal barrier function, with prolonged disease course and even mortality as a result. The bile salt nuclear receptor farnesoid X receptor (FXR), which is expressed in the ileum, liver and other organs including the pancreas, exhibits anti-inflammatory effects by inhibiting NF-κB activation and is implicated in maintaining intestinal barrier integrity and preventing bacterial overgrowth and translocation. Here we explore, with the aid of complementary animal and human experiments, the potential role of FXR in acute pancreatitis. METHODS: Experimental acute pancreatitis was induced using the CCK-analogue cerulein in wild-type and Fxr-/- mice. Severity of acute pancreatitis was assessed using histology and a semi-quantitative scoring system. Ileal permeability was analyzed in vitro by Ussing chambers and an in vivo permeability assay. Gene expression of Fxr and Fxr target genes was studied by quantitative RT-PCR. Serum FGF19 levels were determined by ELISA in acute pancreatitis patients and healthy volunteers. A genetic association study in 387 acute pancreatitis patients and 853 controls was performed using 9 tagging single nucleotide polymorphisms (SNPs) covering the complete FXR gene and two additional functional SNPs. RESULTS: In wild-type mice with acute pancreatitis, ileal transepithelial resistance was reduced and ileal mRNA expression of Fxr target genes Fgf15, SHP, and IBABP was decreased. Nevertheless, Fxr-/- mice did not exhibit a more severe acute pancreatitis than wild-type mice. In patients with acute pancreatitis, FGF19 levels were lower than in controls. However, there were no associations of FXR SNPs or haplotypes with susceptibility to acute pancreatitis, or its course, outcome or etiology. CONCLUSION: We found no evidence for a major role of FXR in acute human or murine pancreatitis. The observed altered Fxr activity during the course of disease may be a secondary phenomenon

Impact of Global <i>Fxr</i> Deficiency on Experimental Acute Pancreatitis and Genetic Variation in the <i>FXR - Figure 2 </i> Locus in Human Acute Pancreatitis

<p>A - Representative pancreatic histology following induction of acute pancreatitis (H&E staining, 20x and 100x consecutive magnifications for each experimental group): wild-type control (a,b); wild-type acute pancreatitis (c,d); Fxr^-/- control (e,f); Fxr^-/- acute pancreatitis (g,h). B – Semi-quantitative composite pancreatitis severity score of histopathological examination of pancreas samples from wild-type and Fxr^-/- mice with and without acute pancreatitis. Absence of <i>Fxr</i> does not result in more severe acute pancreatitis. C – Ileal mRNA expression of <i>Fgf15</i> in wild-type and Fxr^-/- mice with and without early acute pancreatitis. <i>Fgf15</i> expression was decreased in wild-type mice with acute pancreatitis. Bars indicate mean and SEM.</p

FGF19 plasma levels in patients with predicted severe acute pancreatitis during continuous enteral nutrition.

<p>For comparison, FGF19 plasma levels of healthy controls in the fasting state and after a single bolus of fat are also shown. The postprandial levels represent the average of plasma FGF19 levels at 2, 3, 4 and 6 hours after fat ingestion. There appears to be a blunted FGF19 release in the pancreatitis group. Bars indicate mean and SD.</p

Impact of Global <i>Fxr</i> Deficiency on Experimental Acute Pancreatitis and Genetic Variation in the <i>FXR - Figure 1 </i> Locus in Human Acute Pancreatitis

<p>A – Representative pancreatic histology of wild-type mice from the control group (a, b) and mice with early (c, d) and late (e, f) acute pancreatitis (H&E staining, 20x and 100x magnifications consecutively). Control mice have normal pancreatic morphology, whereas mice of the early pancreatitis group exhibit edema, influx of neutrophils and necrosis. Mice of the late pancreatitis group have no edema or necrosis, but show influx of lymphocytes and fibroblasts. B – Transepithelial electrical resistance of the ileum measured by Ussing chamber experiments. The resistance of the ileum was lower in the early pancreatitis group in comparison to both controls and the late pancreatitis group. C – Ileal mRNA expression of <i>Fxr</i> and FXR target-genes <i>Asbt</i>, <i>Shp</i>, <i>Fgf15</i>, and <i>Ibabp</i>, and <i>Villin</i> in wild-type mice of the control group, and the early and late pancreatitis groups. Expression of <i>Fxr</i>, <i>Asbt</i> and <i>Villin</i> did not differ between experimental groups. Expression of the other Fxr target genes was lower in early acute pancreatitis, but not in late pancreatitis. D – Ileal mRNA expression of genes implicated in intestinal barrier function, <i>iNos</i>, <i>Ang1</i>, <i>Car12</i>, <i>IL18</i>. Expression of <i>Ang1</i> was lowered in the early pancreatitis group, the other genes remained similar in the different experimental conditions. E – Hepatic expression of <i>Fxr</i>, <i>Shp</i>, <i>iNos</i> and <i>Ang1</i>. Hepatic <i>Ang1</i> was increased in early pancreatitis, whereas the expression of the other genes was lowered in the early acute pancreatitis group. Expression levels were normalized to cyclophilin expression. Bars indicate means and SEM, * p<0.05, ** p<0.01, *** p<0.001.</p