Mitophagy in Intestinal Epithelial Cells Triggers Adaptive Immunity during Tumorigenesis.

In colorectal cancer patients, a high density of cytotoxic CD8+ T cells in tumors is associated with better prognosis. Using a Stat3 loss-of-function approach in two wnt/ß-catenin-dependent autochthonous models of sporadic intestinal tumorigenesis, we unravel a complex intracellular process in intestinal epithelial cells (IECs) that controls the induction of a CD8+ T cell based adaptive immune response. Elevated mitophagy in IECs causes iron(II)-accumulation in epithelial lysosomes, in turn, triggering lysosomal membrane permeabilization. Subsequent release of proteases into the cytoplasm augments MHC class I presentation and activation of CD8+ T cells via cross-dressing of dendritic cells. Thus, our findings highlight a so-far-unrecognized link between mitochondrial function, lysosomal integrity, and MHC class I presentation in IECs and suggest that therapies triggering mitophagy or inducing LMP in IECs may prove successful in shifting the balance toward anti-tumor immunity in colorectal cancer.

Expansion of T memory stem cells with superior anti-tumor immunity by Urolithin A-induced mitophagy.

T memory stem cells (TSCM) display increased self-renewal and prolonged survival capabilities, thus preventing T cell exhaustion and promoting effective anti-tumor T cell responses. TSCM cells can be expanded by Urolithin A (UA), which is produced by the commensal gut microbiome from foods rich in ellagitannins and is known to improve mitochondrial health. Oral UA administration to tumor-bearing mice conferred strong anti-tumor CD8+ T cell immunity, whereas ex vivo UA pre-treated T cells displayed improved anti-tumor function upon adoptive cell transfer. UA-induced TSCM formation depended on Pink1-mediated mitophagy triggering cytosolic release of the mitochondrial phosphatase Pgam5. Cytosolic Pgam5 dephosphorylated ß-catenin, which drove Wnt signaling and compensatory mitochondrial biogenesis. Collectively, we unravel a critical signaling pathway linking mitophagy to TSCM formation and suggest that the well-tolerated metabolic compound UA represents an attractive option to improve immune therapy.

Intestinal tumorigenesis initiated by dedifferentiation and acquisition of stem-cell-like properties.

Cell-type plasticity within a tumor has recently been suggested to cause a bidirectional conversion between tumor-initiating stem cells and nonstem cells triggered by an inflammatory stroma. NF-κB represents a key transcription factor within the inflammatory tumor microenvironment. However, NF-κB's function in tumor-initiating cells has not been examined yet. Using a genetic model of intestinal epithelial cell (IEC)-restricted constitutive Wnt-activation, which comprises the most common event in the initiation of colon cancer, we demonstrate that NF-κB modulates Wnt signaling and show that IEC-specific ablation of RelA/p65 retards crypt stem cell expansion. In contrast, elevated NF-κB signaling enhances Wnt activation and induces dedifferentiation of nonstem cells that acquire tumor-initiating capacity. Thus, our data support the concept of bidirectional conversion and highlight the importance of inflammatory signaling for dedifferentiation and generation of tumor-initiating cells in vivo.

High-fat-diet-mediated dysbiosis promotes intestinal carcinogenesis independently of obesity.

Several features common to a Western lifestyle, including obesity and low levels of physical activity, are known risk factors for gastrointestinal cancers. There is substantial evidence suggesting that diet markedly affects the composition of the intestinal microbiota. Moreover, there is now unequivocal evidence linking dysbiosis to cancer development. However, the mechanisms by which high-fat diet (HFD)-mediated changes in the microbial community affect the severity of tumorigenesis in the gut remain to be determined. Here we demonstrate that an HFD promotes tumour progression in the small intestine of genetically susceptible, K-ras(G12Dint), mice independently of obesity. HFD consumption, in conjunction with K-ras mutation, mediated a shift in the composition of the gut microbiota, and this shift was associated with a decrease in Paneth-cell-mediated antimicrobial host defence that compromised dendritic cell recruitment and MHC class II molecule presentation in the gut-associated lymphoid tissues. When butyrate was administered to HFD-fed K-ras(G12Dint) mice, dendritic cell recruitment in the gut-associated lymphoid tissues was normalized, and tumour progression was attenuated. Importantly, deficiency in MYD88, a signalling adaptor for pattern recognition receptors and Toll-like receptors, blocked tumour progression. The transfer of faecal samples from HFD-fed mice with intestinal tumours to healthy adult K-ras(G12Dint) mice was sufficient to transmit disease in the absence of an HFD. Furthermore, treatment with antibiotics completely blocked HFD-induced tumour progression, suggesting that distinct shifts in the microbiota have a pivotal role in aggravating disease. Collectively, these data underscore the importance of the reciprocal interaction between host and environmental factors in selecting a microbiota that favours carcinogenesis, and they suggest that tumorigenesis is transmissible among genetically predisposed individuals.

[The model transferability of AI in digital pathology : Potential and reality]. / Die Modelltransferierbarkeit von KI in der digitalen Pathologie : Potenzial und Realität.

OBJECTIVE: Artificial intelligence (AI) holds the potential to make significant advancements in pathology. However, its actual implementation and certification for practical use are currently limited, often due to challenges related to model transferability. In this context, we investigate the factors influencing transferability and present methods aimed at enhancing the utilization of AI algorithms in pathology. MATERIALS AND METHODS: Various convolutional neural networks (CNNs) and vision transformers (ViTs) were trained using datasets from two institutions, along with the publicly available TCGA-MIBC dataset. These networks conducted predictions in urothelial tissue and intrahepatic cholangiocarcinoma (iCCA). The objective was to illustrate the impact of stain normalization, the influence of various artifacts during both training and testing, as well as the effects of the NoisyEnsemble method. RESULTS: We were able to demonstrate that stain normalization of slides from different institutions has a significant positive effect on the inter-institutional transferability of CNNs and ViTs (respectively +13% and +10%). In addition, ViTs usually achieve a higher accuracy in the external test (here +1.5%). Similarly, we showcased how artifacts in test data can negatively affect CNN predictions and how incorporating these artifacts during training leads to improvements. Lastly, NoisyEnsembles of CNNs (better than ViTs) were shown to enhance transferability across different tissues and research questions (+7% Bladder, +15% iCCA). DISCUSSION: It is crucial to be aware of the transferability challenge: achieving good performance during development does not necessarily translate to good performance in real-world applications. The inclusion of existing methods to enhance transferability, such as stain normalization and NoisyEnsemble, and their ongoing refinement, is of importance.

[Challenges of automation in quantitative evaluation of liver biopsies : Automatic quantification of liver steatosis]. / Herausforderungen der Automation bei der quantitativen Auswertung von Leberbiopsien : Automatische Quantifizierung von Leberverfettung.

BACKGROUND: Metabolic dysfunction-associated steatotic liver disease (MASLD), or non-alcoholic fatty liver disease (NAFLD), is a common disease that is diagnosed through manual evaluation of liver biopsies, an assessment that is subject to high interobserver variability (IBV). IBV can be reduced using automated methods. OBJECTIVES: Many existing computer-based methods do not accurately reflect what pathologists evaluate in practice. The goal is to demonstrate how these differences impact the prediction of hepatic steatosis. Additionally, IBV complicates algorithm validation. MATERIALS AND METHODS: Forty tissue sections were analyzed to detect steatosis, nuclei, and fibrosis. Data generated from automated image processing were used to predict steatosis grades. To investigate IBV, 18 liver biopsies were evaluated by multiple observers. RESULTS: Area-based approaches yielded more strongly correlated results than nucleus-based methods (⌀ Spearman rho [ρ] = 0.92 vs. 0.79). The inclusion of information regarding tissue composition reduced the average absolute error for both area- and nucleus-based predictions by 0.5% and 2.2%, respectively. Our final area-based algorithm, incorporating tissue structure information, achieved a high accuracy (80%) and strong correlation (⌀ Spearman ρ = 0.94) with manual evaluation. CONCLUSION: The automatic and deterministic evaluation of steatosis can be improved by integrating information about tissue composition and can serve to reduce the influence of IBV.

[Fluorescence confocal microscopy-complete digitization of pathology]. / Fluoreszenzbasierte Konfokalmikroskopie ­ vollständige Digitalisierung der Pathologie.

BACKGROUND: Fluorescence-based confocal microscopy (FCM) can be used to create virtual H&E sections in real time. So far, FCM has been used in dermato-, uro-, and gynecopathology. FCM allows the creation of a completely digitized frozen section, which could potentially replace conventional frozen sections in the future. OBJECTIVE: The aim of the current work is to implement FCM technology as a component of fully digitized processes in the pathological workflow. For this purpose, the current use of FCM in liver transplant pathology will be extended to other disciplines such as urology and otorhinolaryngology. MATERIALS AND METHODS: The FCM technique continues to be used prospectively on native tissue samples from potential donor livers. Conventional frozen sections are used comparatively to virtual FCM scans. RESULTS: The data show a nearly perfect agreement for the detection of cholangitis, fibrosis, and malignancy, and a high level of agreement for, e.g., macrovesicular steatosis, inflammation, steatohepatitis, and necrosis between virtual FCM scans and conventional routine diagnostic frozen sections. CONCLUSION: Since the availability of time- and cost-intensive frozen section diagnostics in the context of transplant pathology in continuous operation (24/7) is now only established at very few university centers in Germany due to an increasing shortage of specialists, the use of FCM could be an important building block in the current process leading towards a fully digitized pathology workflow and should thus be extended to various disciplines.

Colorectal Cancer Organoid-Stroma Biobank Allows Subtype-Specific Assessment of Individualized Therapy Responses.

In colorectal cancers, the tumor microenvironment plays a key role in prognosis and therapy efficacy. Patient-derived tumor organoids (PDTO) show enormous potential for preclinical testing; however, cultured tumor cells lose important characteristics, including the consensus molecular subtypes (CMS). To better reflect the cellular heterogeneity, we established the colorectal cancer organoid-stroma biobank of matched PDTOs and cancer-associated fibroblasts (CAF) from 30 patients. Context-specific phenotyping showed that xenotransplantation or coculture with CAFs improves the transcriptomic fidelity and instructs subtype-specific stromal gene expression. Furthermore, functional profiling in coculture exposed CMS4-specific therapeutic resistance to gefitinib and SN-38 and prognostic expression signatures. Chemogenomic library screening identified patient- and therapy-dependent mechanisms of stromal resistance including MET as a common target. Our results demonstrate that colorectal cancer phenotypes are encrypted in the cancer epithelium in a plastic fashion that strongly depends on the context. Consequently, CAFs are essential for a faithful representation of molecular subtypes and therapy responses ex vivo. SIGNIFICANCE: Systematic characterization of the organoid-stroma biobank provides a resource for context dependency in colorectal cancer. We demonstrate a colorectal cancer subtype memory of PDTOs that is independent of specific driver mutations. Our data underscore the importance of functional profiling in cocultures for improved preclinical testing and identification of stromal resistance mechanisms. This article is featured in Selected Articles from This Issue, p. 2109.

How to learn with intentional mistakes: NoisyEnsembles to overcome poor tissue quality for deep learning in computational pathology.

There is a lot of recent interest in the field of computational pathology, as many algorithms are introduced to detect, for example, cancer lesions or molecular features. However, there is a large gap between artificial intelligence (AI) technology and practice, since only a small fraction of the applications is used in routine diagnostics. The main problems are the transferability of convolutional neural network (CNN) models to data from other sources and the identification of uncertain predictions. The role of tissue quality itself is also largely unknown. Here, we demonstrated that samples of the TCGA ovarian cancer (TCGA-OV) dataset from different tissue sources have different quality characteristics and that CNN performance is linked to this property. CNNs performed best on high-quality data. Quality control tools were partially able to identify low-quality tiles, but their use did not increase the performance of the trained CNNs. Furthermore, we trained NoisyEnsembles by introducing label noise during training. These NoisyEnsembles could improve CNN performance for low-quality, unknown datasets. Moreover, the performance increases as the ensemble become more consistent, suggesting that incorrect predictions could be discarded efficiently to avoid wrong diagnostic decisions.

Inflammatory fibroblasts mediate resistance to neoadjuvant therapy in rectal cancer.

Standard cancer therapy targets tumor cells without considering possible damage on the tumor microenvironment that could impair therapy response. In rectal cancer patients we find that inflammatory cancer-associated fibroblasts (iCAFs) are associated with poor chemoradiotherapy response. Employing a murine rectal cancer model or patient-derived tumor organoids and primary stroma cells, we show that, upon irradiation, interleukin-1α (IL-1α) not only polarizes cancer-associated fibroblasts toward the inflammatory phenotype but also triggers oxidative DNA damage, thereby predisposing iCAFs to p53-mediated therapy-induced senescence, which in turn results in chemoradiotherapy resistance and disease progression. Consistently, IL-1 inhibition, prevention of iCAFs senescence, or senolytic therapy sensitizes mice to irradiation, while lower IL-1 receptor antagonist serum levels in rectal patients correlate with poor prognosis. Collectively, we unravel a critical role for iCAFs in rectal cancer therapy resistance and identify IL-1 signaling as an attractive target for stroma-repolarization and prevention of cancer-associated fibroblasts senescence.

AKT-dependent NOTCH3 activation drives tumor progression in a model of mesenchymal colorectal cancer.

Recently, a transcriptome-based consensus molecular subtype (CMS) classification of colorectal cancer (CRC) has been established, which may ultimately help to individualize CRC therapy. However, the lack of animal models that faithfully recapitulate the different molecular subtypes impedes adequate preclinical testing of stratified therapeutic concepts. Here, we demonstrate that constitutive AKT activation in intestinal epithelial cells markedly enhances tumor invasion and metastasis in Trp53ΔIEC mice (Trp53ΔIECAktE17K) upon challenge with the carcinogen azoxymethane. Gene-expression profiling indicates that Trp53ΔIECAktE17K tumors resemble the human mesenchymal colorectal cancer subtype (CMS4), which is characterized by the poorest survival rate among the four CMSs. Trp53ΔIECAktE17K tumor cells are characterized by Notch3 up-regulation, and treatment of Trp53ΔIECAktE17K mice with a NOTCH3-inhibiting antibody reduces invasion and metastasis. In CRC patients, NOTCH3 expression correlates positively with tumor grading and the presence of lymph node as well as distant metastases and is specifically up-regulated in CMS4 tumors. Therefore, we suggest NOTCH3 as a putative target for advanced CMS4 CRC patients.

Loss of Stat6 affects chromatin condensation in intestinal epithelial cells causing diverse outcome in murine models of inflammation-associated and sporadic colon carcinogenesis.

While great advances have been achieved regarding the genetic basis of colorectal cancer, the complex role of cell-cell communication and cytokine-induced signaling during its pathogenesis remains less understood. Signal transducer and activator of transcription 6 (Stat6) is the main transcription factor of interleukin-4 (IL-4) signaling and its participation in the development of various tumor types has been already reported. Here we aimed to examine the contribution of Stat6 in intestinal epithelial cells (IEC) in mouse models of intestinal carcinogenesis. Wild-type (WT), Stat6 knockout (Stat6-/-), and intestinal epithelial cell-specific IL-4Rα knockout (Il-4rαΔIEC) mice were subjected to colitis-associated (AOM/DSS) and colitis-independent (sporadic) carcinogenesis. IEC proliferation, apoptosis and RNA expression were evaluated by immunohistochemical, immunoblot, and RT-PCR analysis. We found that Stat6-/- mice developed more tumors in the colitis-associated carcinogenesis model. This was accompanied by a more pronounced inflammatory response during colitis and an elevated Stat3-dependent proliferation of IEC. Increased sensitivity to DSS-induced colitis was caused by elevated cell death in response to the initial carcinogen exposure as Stat6 deficiency led to increased chromatin compaction affecting DNA damage response in IEC upon treatment with alkylating agents independently of IL-4Rα engagement. Thus, loss of Stat6 caused more severe colitis and increased tumor load, however loss-of-initiated Stat6-/- IEC prevented tumor formation in the absence of overt inflammation. Our data unravel unexpected IL-4-independent functions of Stat6 in chromatin compaction in intestinal epithelial cells ultimately providing both tumor suppressive as well as tumor promoting effects in different models of intestinal tumorigenesis.

IKKß acts as a tumor suppressor in cancer-associated fibroblasts during intestinal tumorigenesis.

Cancer-associated fibroblasts (CAFs) comprise one of the most important cell types in the tumor microenvironment. A proinflammatory NF-κB gene signature in CAFs has been suggested to promote tumorigenesis in models of pancreatic and mammary skin cancer. Using an autochthonous model of colitis-associated cancer (CAC) and sporadic cancer, we now provide evidence for a tumor-suppressive function of IKKß/NF-κB in CAFs. Fibroblast-restricted deletion of Ikkß stimulates intestinal epithelial cell proliferation, suppresses tumor cell death, enhances accumulation of CD4(+)Foxp3(+) regulatory T cells, and induces angiogenesis, ultimately promoting colonic tumor growth. In Ikkß-deficient fibroblasts, transcription of negative regulators of TGFß signaling, including Smad7 and Smurf1, is impaired, causing up-regulation of a TGFß gene signature and elevated hepatocyte growth factor (HGF) secretion. Overexpression of Smad7 in Ikkß-deficient fibroblasts prevents HGF secretion, and pharmacological inhibition of Met during the CAC model confirms that enhanced tumor promotion is dependent on HGF-Met signaling in mucosa of Ikkß-mutant animals. Collectively, these results highlight an unexpected tumor suppressive function of IKKß/NF-κB in CAFs linked to HGF release and raise potential concerns about the use of IKK inhibitors in colorectal cancer patients.

IL-6R/STAT3/miR-34a feedback loop promotes EMT-mediated colorectal cancer invasion and metastasis.

Members of the miR-34 family are induced by the tumor suppressor p53 and are known to inhibit epithelial-to-mesenchymal transition (EMT) and therefore presumably suppress the early phases of metastasis. Here, we determined that exposure of human colorectal cancer (CRC) cells to the cytokine IL-6 activates the oncogenic STAT3 transcription factor, which directly represses the MIR34A gene via a conserved STAT3-binding site in the first intron. Repression of MIR34A was required for IL-6-induced EMT and invasion. Furthermore, we identified the IL-6 receptor (IL-6R), which mediates IL-6-dependent STAT3 activation, as a conserved, direct miR-34a target. The resulting IL-6R/STAT3/miR-34a feedback loop was present in primary colorectal tumors as well as CRC, breast, and prostate cancer cell lines and associated with a mesenchymal phenotype. An active IL-6R/STAT3/miR-34a loop was necessary for EMT, invasion, and metastasis of CRC cell lines and was associated with nodal and distant metastasis in CRC patient samples. p53 activation in CRC cells interfered with IL-6-induced invasion and migration via miR-34a-dependent downregulation of IL6R expression. In Mir34a-deficient mice, colitis-associated intestinal tumors displayed upregulation of p-STAT3, IL-6R, and SNAIL and progressed to invasive carcinomas, which was not observed in WT animals. Collectively, our data indicate that p53-dependent expression of miR-34a suppresses tumor progression by inhibiting a IL-6R/STAT3/miR-34a feedback loop.

Loss of p53 in enterocytes generates an inflammatory microenvironment enabling invasion and lymph node metastasis of carcinogen-induced colorectal tumors.

Loss of p53 is considered to allow progression of colorectal tumors from the adenoma to the carcinoma stage. Using mice with an intestinal epithelial cell (IEC)-specific p53 deletion, we demonstrate that loss of p53 alone is insufficient to initiate intestinal tumorigenesis but markedly enhances carcinogen-induced tumor incidence and leads to invasive cancer and lymph node metastasis. Whereas p53 controls DNA damage and IEC survival during the initiation stage, loss of p53 during tumor progression is associated with increased intestinal permeability, causing formation of an NF-κB-dependent inflammatory microenvironment and the induction of epithelial-mesenchymal transition. Thus, we propose a p53-controlled tumor-suppressive function that is independent of its well-established role in cell-cycle regulation, apoptosis, and senescence.

Interleukin-11 is the dominant IL-6 family cytokine during gastrointestinal tumorigenesis and can be targeted therapeutically.

Among the cytokines linked to inflammation-associated cancer, interleukin (IL)-6 drives many of the cancer "hallmarks" through downstream activation of the gp130/STAT3 signaling pathway. However, we show that the related cytokine IL-11 has a stronger correlation with elevated STAT3 activation in human gastrointestinal cancers. Using genetic mouse models, we reveal that IL-11 has a more prominent role compared to IL-6 during the progression of sporadic and inflammation-associated colon and gastric cancers. Accordingly, in these models and in human tumor cell line xenograft models, pharmacologic inhibition of IL-11 signaling alleviated STAT3 activation, suppressed tumor cell proliferation, and reduced the invasive capacity and growth of tumors. Our results identify IL-11 signaling as a potential therapeutic target for the treatment of gastrointestinal cancers.