Myeloid-Derived Suppressor Cells: Therapeutic Target for Gastrointestinal Cancers.

Gastrointestinal cancers represent one of the more challenging cancers to treat. Current strategies to cure and control gastrointestinal (GI) cancers like surgery, radiation, chemotherapy, and immunotherapy have met with limited success, and research has turned towards further characterizing the tumor microenvironment to develop novel therapeutics. Myeloid-derived suppressor cells (MDSCs) have emerged as crucial drivers of pathogenesis and progression within the tumor microenvironment in GI malignancies. Many MDSCs clinical targets have been defined in preclinical models, that potentially play an integral role in blocking recruitment and expansion, promoting MDSC differentiation into mature myeloid cells, depleting existing MDSCs, altering MDSC metabolic pathways, and directly inhibiting MDSC function. This review article analyzes the role of MDSCs in GI cancers as viable therapeutic targets for gastrointestinal malignancies and reviews the existing clinical trial landscape of recently completed and ongoing clinical studies testing novel therapeutics in GI cancers.

Spatial profiling reveals tissue-specific neuro-immune interactions in gastroenteropancreatic neuroendocrine tumors.

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are heterogeneous malignancies that arise from complex cellular interactions within the tissue microenvironment. Here, we sought to decipher tumor-derived signals from the surrounding microenvironment by applying digital spatial profiling (DSP) to hormone-secreting and non-functional GEP-NETs. By combining this approach with in vitro studies of human-derived organoids, we demonstrated the convergence of cell autonomous immune and pro-inflammatory proteins that suggests their role in neuroendocrine differentiation and tumorigenesis. DSP was used to evaluate the expression of 40 neural- and immune-related proteins in surgically resected duodenal and pancreatic NETs (n = 20) primarily consisting of gastrinomas (18/20). A total of 279 regions of interest were examined between tumors, adjacent normal and abnormal-appearing epithelium, and the surrounding stroma. The results were stratified by tissue type and multiple endocrine neoplasia I (MEN1) status, whereas protein expression was validated by immunohistochemistry (IHC). A tumor immune cell autonomous inflammatory signature was further evaluated by IHC and RNAscope, while functional pro-inflammatory signaling was confirmed using patient-derived duodenal organoids. Gastrin-secreting and non-functional pancreatic NETs showed a higher abundance of immune cell markers and immune infiltrate compared with duodenal gastrinomas. Compared with non-MEN1 tumors, MEN1 gastrinomas and preneoplastic lesions showed strong immune exclusion and upregulated expression of neuropathological proteins. Despite a paucity of immune cells, duodenal gastrinomas expressed the pro-inflammatory and pro-neural factor IL-17B. Treatment of human duodenal organoids with IL-17B activated NF-κB and STAT3 signaling and induced the expression of neuroendocrine markers. In conclusion, multiplexed spatial protein analysis identified tissue-specific neuro-immune signatures in GEP-NETs. Duodenal gastrinomas are characterized by an immunologically cold microenvironment that permits cellular reprogramming and neoplastic transformation of the preneoplastic epithelium. Moreover, duodenal gastrinomas cell autonomously express immune and pro-inflammatory factors, including tumor-derived IL-17B, that stimulate the neuroendocrine phenotype. © 2024 The Pathological Society of Great Britain and Ireland.

Combined flat-field and frequency filter approach to correcting artifacts of multichannel two-photon microscopy.

Significance: Multiphoton microscopy (MPM) is a useful biomedical imaging tool for its ability to probe labeled and unlabeled depth-resolved tissue biomarkers at high resolution. Automated MPM tile scanning allows for whole-slide image acquisition but can suffer from tile-stitching artifacts that prevent accurate quantitative data analysis. Aim: We have investigated postprocessing artifact correction methods using ImageJ macros and custom Python code. Quantitative and qualitative comparisons of these methods were made using whole-slide MPM autofluorescence and second-harmonic generation images of human duodenal tissue. Approach: Image quality after artifact removal is assessed by evaluating the processed image and its unprocessed counterpart using the root mean square error, structural similarity index, and image histogram measurements. Results: Consideration of both quantitative and qualitative results suggest that a combination of a custom flat-field-based correction and frequency filtering processing step provide improved artifact correction when compared with each method used independently to correct for tiling artifacts of tile-scan MPM images. Conclusions: While some image artifacts remain with these methods, further optimization of these processing steps may result in computational-efficient methods for removing these artifacts that are ubiquitous in large-scale MPM imaging. Removal of these artifacts with retention of the original image information would facilitate the use of this imaging modality in both research and clinical settings, where it is highly useful in collecting detailed morphologic and optical properties of tissue.

Zfp281 and Zfp148 control CD4+ T cell thymic development and TH2 functions.

How CD4+ lineage gene expression is initiated in differentiating thymocytes remains poorly understood. Here, we show that the paralog transcription factors Zfp281 and Zfp148 control both this process and cytokine expression by T helper cell type 2 (TH2) effector cells. Genetic, single-cell, and spatial transcriptomic analyses showed that these factors promote the intrathymic CD4+ T cell differentiation of class II major histocompatibility complex (MHC II)-restricted thymocytes, including expression of the CD4+ lineage-committing factor Thpok. In peripheral T cells, Zfp281 and Zfp148 promoted chromatin opening at and expression of TH2 cytokine genes but not of the TH2 lineage-determining transcription factor Gata3. We found that Zfp281 interacts with Gata3 and is recruited to Gata3 genomic binding sites at loci encoding Thpok and TH2 cytokines. Thus, Zfp148 and Zfp281 collaborate with Gata3 to promote CD4+ T cell development and TH2 cell responses.

Design, fabrication, and preclinical testing of a miniaturized, multispectral, chip-on-tip, imaging probe for intraluminal fluorescence imaging of the gastrointestinal tract.

Gastrointestinal cancers continue to account for a disproportionately large percentage of annual cancer deaths in the US. Advancements in miniature imaging technology combined with a need for precise and thorough tumor detection in gastrointestinal cancer screenings fuel the demand for new, small-scale, and low-cost methods of localization and margin identification with improved accuracy. Here, we report the development of a miniaturized, chip-on-tip, multispectral, fluorescence imaging probe designed to port through a gastroscope working channel with the aim of detecting cancerous lesions in point-of-care endoscopy of the gastrointestinal lumen. Preclinical testing has confirmed fluorescence sensitivity and supports that this miniature probe can locate structures of interest via detection of fluorescence emission from exogenous contrast agents. This work demonstrates the design and preliminary performance evaluation of a miniaturized, single-use, chip-on-tip fluorescence imaging system, capable of detecting multiple fluorochromes, and devised for deployment via the accessory channel of a standard gastroscope.

The bHLH transcription factor ASCL1 promotes differentiation of endocrine cells in the stomach and is regulated by Notch signaling.

Notch signaling regulates gastrointestinal stem cell proliferation and differentiation yet Notch-regulated transcriptional effectors of gastric epithelial cell differentiation are poorly understood. Here we tested the role of the bHLH transcription factor Achaete-Scute homolog 1 (ASCL1) in gastric epithelial cell differentiation, and its regulation by Notch. Newborn Ascl1 null mice showed a loss of expression of markers of neurogenin-3-dependent enteroendocrine cells, with normal expression of enterochromaffin-like cells, mucous cells, chief cells, and parietal cells. In adult mice, Ascl1 gene expression was observed in the stomach, but not the intestine, with higher expression in antral than corpus epithelium. Lineage tracing in Ascl1-CreERT2; Rosa26-LSL-tdTomato mice revealed single, scattered ASCL1+ cells in the gastric epithelium, demonstrating expression in antral gastrin- and serotonin-producing endocrine cells. ASCL1-expressing endocrine cells persisted for several weeks posttamoxifen labeling with a half-life of approximately 2 months. Lineage tracing in Gastrin-CreERT2 mice demonstrated a similar lifespan for gastrin-producing cells, confirming that gastric endocrine cells are long-lived. Finally, treatment of Ascl1-CreERT2; Rosa26-LSL-tdTomato mice with the pan-Notch inhibitor dibenzazepine increased the number of lineage-labeled cells in the gastric antrum, suggesting that Notch signaling normally inhibits Ascl1 expression. Notch regulation of Ascl1 was also demonstrated in a genetic mouse model of Notch activation, as well as Notch-manipulated antral organoid cultures, thus suggesting that ASCL1 is a key downstream Notch pathway effector promoting endocrine cell differentiation in the gastric epithelium.NEW & NOTEWORTHY Although Notch signaling is known to regulate cellular differentiation in the stomach, downstream effectors are poorly described. Here we demonstrate that the bHLH transcription factor ASCL1 is expressed in endocrine cells in the stomach and is required for formation of neurogenin-3-dependent enteroendocrine cells but not enterochromaffin-like cells. We also demonstrate that Ascl1 expression is inhibited by Notch signaling, suggesting that ASCL1 is a Notch-regulated transcriptional effector directing enteroendocrine cell fate in the mouse stomach.

Wide field-of-view fluorescence imaging for organ-level lineage tracing of rare intestinal stem cell populations.

Significance: Lineage tracing using fluorescent reporters is a common tool for monitoring the expression of genes and transcription factors in stem cell populations and their progeny. The zinc-binding protein 89 (ZBP-89/Zfp148 mouse gene) is a transcription factor that plays a role in gastrointestinal (GI) stem cell maintenance and cellular differentiation and has been linked to the progression of colon cancer. While lineage tracing is a useful tool, it is commonly performed with high-magnification microscopy on a small field of view within tissue sections, thereby limiting the ability to resolve reporter expression at the organ level. Furthermore, this technique requires extensive tissue processing, which is time consuming and requires euthanizing the animal. Further knowledge could be elucidated by measuring the expression of fluorescent reporters across entire organs with minimal tissue processing. Aim: We present the application of wide-field fluorescence imaging for whole-organ lineage tracing of an inducible Zfp148-tdTomato-expressing transgenic mouse line to assess the expression of ZBP-89/Zfp148 in the GI tract. Approach: We measured tdTomato fluorescence in ex vivo organs at time points between 24 h and 6 months post-induction. Fluctuations in tdTomato expression were validated by fluorescence microscopy of tissue sections. Results: Quantification of the wide field-of-view images showed a statistically significant increase in fluorescent signal across the GI tract between transgenic mice and littermate controls. The results also showed a gradient of decreasing reporter expression from proximal to distal intestine, suggesting a higher abundance of ZBP-89 expressing stem cells, or higher expression of ZBP-89 within the stem cells, in the proximal intestine. Conclusions: We demonstrate that wide-field fluorescence imaging is a valuable tool for monitoring whole-organ expression of fluorescent reporters. This technique could potentially be applied in vivo for longitudinal assessment of a single animal, further enhancing our ability to resolve rare stem cell lineages spatially and temporally.

Clinically Defined Mutations in MEN1 Alter Its Tumor-suppressive Function Through Increased Menin Turnover.

Loss of the tumor suppressor protein menin is a critical event underlying the formation of neuroendocrine tumors (NET) in hormone-expressing tissues including gastrinomas. While aberrant expression of menin impairs its tumor suppression, few studies explore the structure-function relationship of clinical multiple endocrine neoplasia, type 1 (MEN1) mutations in the absence of a complete LOH at both loci. Here, we determined whether clinical MEN1 mutations render nuclear menin unstable and lead to its functional inactivation. We studied the structural and functional implications of two clinical MEN1 mutations (R516fs, E235K) and a third variant (A541T) recently identified in 10 patients with gastroenteropancreatic (GEP)-NETs. We evaluated the subcellular localization and half-lives of the mutants and variant in Men1-null mouse embryo fibroblast cells and in hormone-expressing human gastric adenocarcinoma and NET cell lines. Loss of menin function was assessed by cell proliferation and gastrin gene expression assays. Finally, we evaluated the effect of the small-molecule compound MI-503 on stabilizing nuclear menin expression and function in vitro and in a previously reported mouse model of gastric NET development. Both the R516fs and E235K mutants exhibited severe defects in total and subcellular expression of menin, and this was consistent with reduced half-lives of these mutants. Mutated menin proteins exhibited loss of function in suppressing tumor cell proliferation and gastrin expression. Treatment with MI-503 rescued nuclear menin expression and attenuated hypergastrinemia and gastric hyperplasia in NET-bearing mice. Clinically defined MEN1 mutations and a germline variant confer pathogenicity by destabilizing nuclear menin expression. Significance: We examined the function of somatic and germline mutations and a variant of MEN1 sequenced from gastroenteropancreatic NETs. We report that these mutations and variant promote tumor cell growth and gastrin expression by rendering menin protein unstable and prone to increased degradation. We demonstrate that the menin-MLL (mixed lineage leukemia) inhibitor MI-503 restores menin protein expression and function in vitro and in vivo, suggesting a potential novel therapeutic approach to target MEN1 GEP-NETs.

Schlafen4+-MDSC in Helicobacter-induced gastric metaplasia reveals role for GTPases.

Introduction: MDSCs express SCHLAFEN 4 (SLFN4) in Helicobacter-infected stomachs coincident with spasmolytic polypeptide-expressing metaplasia (SPEM), a precursor of gastric cancer. We aimed to characterize SLFN4+ cell identity and the role of Slfn4 in these cells. Methods: Single-cell RNA sequencing was performed on immune cells sorted from PBMCs and stomachs prepared from uninfected and 6-month H. felis-infected mice. Knockdown of Slfn4 by siRNA or PDE5/6 inhibition by sildenafil were performed in vitro. Intracellular ATP/GTP levels and GTPase activity of immunoprecipitated Slfn4 complexes were measured using the GTPase-Glo assay kit. The intracellular level of ROS was quantified by the DCF-DA fluorescent staining, and apoptosis was determined by cleaved Caspase-3 and Annexin V expression. Gli1CreERT2 x Slfn4 fl/fl mice were generated and infected with H. felis. Sildenafil was administered twice over 2 weeks by gavaging H. felis infected mice ~4 months after inoculation once SPEM had developed. Results: Slfn4 was highly induced in both monocytic and granulocytic MDSCs from infected stomachs. Both Slfn4 +-MDSC populations exhibited strong transcriptional signatures for type-I interferon responsive GTPases and exhibited T cell suppressor function. SLFN4-containing protein complexes immunoprecipitated from myeloid cell cultures treated with IFNa exhibited GTPase activity. Knocking down Slfn4 or PDE5/6 inhibition with sildenafil blocked IFNa induction of GTP, SLFN4 and NOS2. Moreover, IFNa induction of Slfn +-MDSC function was inhibited by inducing their reactive oxygen species (ROS) production and apoptosis through protein kinase G activation. Accordingly, in vivo disruption of Slfn4 in Gli1CreERT2 x Slfn4 fl/fl mice or pharmacologic inhibition by sildenafil after Helicobacter infection also suppressed SLFN4 and NOS2, reversed T cell suppression and mitigated SPEM development. Conclusion: Taken together, SLFN4 regulates the activity of the GTPase pathway in MDSCs and precludes these cells from succumbing to the massive ROS generation when they acquire MDSC function.

High-fat diet plus HNF1A variant promotes polyps by activating ß-catenin in early-onset colorectal cancer.

The incidence of early-onset colorectal cancer (EO-CRC) is rising and is poorly understood. Lifestyle factors and altered genetic background possibly contribute. Here, we performed targeted exon sequencing of archived leukocyte DNA from 158 EO-CRC participants, which identified a missense mutation at p.A98V within the proximal DNA binding domain of Hepatic Nuclear Factor 1 α (HNF1AA98V, rs1800574). The HNF1AA98V exhibited reduced DNA binding. To test function, the HNF1A variant was introduced into the mouse genome by CRISPR/Cas9, and the mice were placed on either a high-fat diet (HFD) or high-sugar diet (HSD). Only 1% of the HNF1A mutant mice developed polyps on normal chow; however, 19% and 3% developed polyps on the HFD and HSD, respectively. RNA-Seq revealed an increase in metabolic, immune, lipid biogenesis genes, and Wnt/ß-catenin signaling components in the HNF1A mutant relative to the WT mice. Mouse polyps and colon cancers from participants carrying the HNF1AA98V variant exhibited reduced CDX2 and elevated ß-catenin proteins. We further demonstrated decreased occupancy of HNF1AA98V at the Cdx2 locus and reduced Cdx2 promoter activity compared with WT HNF1A. Collectively, our study shows that the HNF1AA98V variant plus a HFD promotes the formation of colonic polyps by activating ß-catenin via decreasing Cdx2 expression.

MENIN-mediated regulation of gastrin gene expression and its role in gastrinoma development.

The Multiple Endocrine Neoplasia I (MEN1) locus encodes the protein MENIN, which functions as a tumor suppressor protein in neuroendocrine tissues. Gastrinomas are neuroendocrine neoplasms that overproduce the hormone gastrin and can arise sporadically or as part of the MEN1 syndrome, in which mutations in the MEN1 gene lead to loss or inactivation of MENIN protein. Gastrin is a peptide hormone that is primarily synthesized in the gastric antrum and stimulates the secretion of histamine from enterochromaffin-like (ECL) cells and subsequently acid from parietal cells in the gastric corpus. In addition, gastrin exerts a mitogenic function primarily on ECL cells and progenitor cells in the gastric isthmus. Current studies seek to understand how MEN1 mutations generate a mutant MENIN protein that abrogates its tumor suppressor function. Mutations in the MEN1 gene are broadly distributed throughout its nine protein-coding exons, making it difficult to correlate protein structure with its function. Although disruption of the Men1 locus in mice causes functional neuroendocrine tumors in the pituitary and pancreas, gastrinomas do not develop in these transgenic animal models. Prior studies of human gastrinomas suggest that tissue-specific microenvironmental cues in the submucosal foregut may contribute to tumorigenesis by reprogramming of epithelial cells toward the neuroendocrine phenotype. Accordingly, recent studies suggest that neural crest-derived cells are also sensitive to reprogramming when MEN1 is deleted or mutated. Thus, the goal of this report is to review our current understanding of how MENIN modulates gastrin gene expression while highlighting its role in the prevention/suppression of neuroendocrine cell transformation.

The next big questions in cancer research.

Our understanding of tumorigenesis and cancer progression as well as clinical therapies for different cancer types have evolved dramatically in recent years. However, even with this progress, there are big challenges for scientists and oncologists to tackle, ranging from unpacking the molecular and cellular mechanisms involved to therapeutics and biomarker development to quality of life in the aftermath of therapy. In this article, we asked researchers to comment on the questions that they think are important to address in the coming years.

Helicobacter pylori treatment knowledge, access and barriers: A cross-sectional study.

BACKGROUND: Helicobacter pylori (Hp) is among the most common bacterial infections in the world and one of the most common infectious agents linked to malignancy, gastric cancer (GC). Within the US there is high disparity in the rates of Hp infection and associated diseases. Hp infection is treatable, and knowledge may influence screening and treatment seeking behaviors. MATERIALS AND METHODS: In this cross-sectional study of 1042 respondents recruited from the Online Amazon MTurk platform, we sought to assess baseline knowledge of Hp and to gain insight into barriers related to Hp care. RESULTS: Just over half (52.3%) reported some prior knowledge of Hp with 11.7% (n = 122) reporting being treated for Hp themselves and 21.4% reporting family members diagnosed with Hp. Of respondents reporting prior treatment, 95 (78%) reported GI upset and 27 (21%) reported not completing medications. Specific to Hp and GC, 70% indicated that a belief that the treatment was worse than the symptoms would affect their willingness to seek care, while 81% indicated knowing Hp can cause GC would affect their treatment decisions and knowing their gastric symptoms were caused by Hp would affect their willingness to receive care. CONCLUSIONS: Knowledge of Hp in this US sample of online respondents is low and self-reported difficulties with treatment compliance is high. Increasing awareness of this infection and addressing the challenges to treatment compliance could potentially reduce rates of Hp antibiotic resistance and progression to GC or other complications of Hp infection.

Combined multiphoton microscopy and somatostatin receptor type 2 imaging of pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (PNETs) are a rare but increasingly more prevalent cancer with heterogeneous clinical and pathological presentation. Surgery is the preferred treatment for all hormone-expressing PNETs and any PNET greater than 2 cm, but difficulties arise when tumors are multifocal, metastatic, or small in size due to lack of effective surgical localization. Existing techniques such as intraoperative ultrasound provide poor contrast and resolution, resulting in low sensitivity for such tumors. Somatostatin receptor type 2 (SSTR2) is commonly overexpressed in PNETs and presents an avenue for targeted tumor localization. SSTR2 is often used for pre-operative imaging and therapeutic treatment, with recent studies demonstrating that somatostatin receptor imaging (SRI) can be applied in radioguided surgery to aid in removal of metastatic lymph nodes and achieving negative surgical margins. However not all PNETs express SSTR2, indicating labeled SRI could benefit from using a supplemental label-free technique such as multiphoton microscopy (MPM), which has proven useful in improving the accuracy of diagnosing more common exocrine pancreatic cancers. Our work tests the suitability of combined SRI and MPM for localizing PNETs by imaging and comparing samples of PNETs and normal pancreatic tissue. Specimens were labeled with a novel SSTR2-targeted contrast agent and imaged using fluorescence microscopy, and subsequently imaged using MPM to collect four autofluorescent channels and second harmonic generation. Our results show that a combination of both SRI and MPM provides enhanced contrast and sensitivity for localizing diseased tissue, suggesting that this approach could be a valuable clinical tool for surgical localization and treatment of PNETs.

Tissue- and cell-specific properties of enterochromaffin cells affect the fate of tumorigenesis toward nonendocrine adenocarcinoma of the small intestine.

Small intestinal neuroendocrine tumors (SI-NETs) are serotonin-secreting well-differentiated neuroendocrine tumors of putative enterochromaffin (EC) cell origin. However, EC cell-derived tumorigenesis remains poorly understood. Here, we examined whether the gain of Myc and the loss of RB1 and Trp53 function in EC cells result in SI-NET using tryptophan hydroxylase 1 (TPH1) Cre-ERT2-driven RB1fl Trp53fl MycLSL (RPM) mice. TPH1-Cre-induced gain of Myc and loss of RB1 and Trp53 function resulted in endocrine or neuronal tumors in pancreas, lung, enteric neurons, and brain. Lineage tracing indicated that the cellular origin for these tumors was TPH1-expressing neuroendocrine, neuronal, or their precursor cells in these organs. However, despite that TPH1 is most highly expressed in EC cells of the small intestine, we observed no incidence of EC cell tumors. Instead, the tumor of epithelial cell origin in the intestine was exclusively nonendocrine adenocarcinoma, suggesting dedifferentiation of EC cells into intestinal stem cells (ISCs) as a cellular mechanism. Furthermore, ex vivo organoid studies indicated that loss of functions of Rb1 and Trp53 accelerated dedifferentiation of EC cells that were susceptible to apoptosis with expression of activated MycT58A, suggesting that the rare dedifferentiating cells escaping cell death went on to develop adenocarcinomas. Lineage tracing demonstrated that EC cells in the small intestine were short-lived compared with neuroendocrine or neuronal cells in other organs. In contrast, EC cell-derived ISCs were long-lasting and actively cycling and thus susceptible to transformation. These results suggest that tissue- and cell-specific properties of EC cells such as rapid cell turnover and homeostatic dedifferentiation, affect the fate and rate of tumorigenesis induced by genetic alterations and provide important insights into EC cell-derived tumorigenesis.NEW & NOTEWORTHY Small intestinal neuroendocrine tumors are of putative enterochromaffin (EC) cell origin and are the most common malignancy in the small intestine, followed by adenocarcinoma. However, the tumorigenesis of these tumor types remains poorly understood. The present lineage tracing studies showed that tissue- and cell-specific properties of EC cells such as rapid cell turnover and homeostatic dedifferentiation affect the fate and rate of tumorigenesis induced by genetic alterations toward a rare occurrence of adenocarcinoma.

Quantitative characterization of duodenal gastrinoma autofluorescence using multiphoton microscopy.

BACKGROUND: Duodenal gastrinomas (DGASTs) are neuroendocrine tumors that develop in the submucosa of the duodenum and produce the hormone gastrin. Surgical resection of DGASTs is complicated by the small size of these tumors and the tendency for them to develop diffusely in the duodenum. Endoscopic mucosal resection of DGASTs is an increasingly popular method for treating this disease due to its low complication rate but suffers from poor rates of pathologically negative margins. Multiphoton microscopy can capture high-resolution images of biological tissue with contrast generated from endogenous fluorescence (autofluorescence [AF]) through two-photon excited fluorescence (2PEF). Second harmonic generation is another popular method of generating image contrast with multiphoton microscopy (MPM) and is a light-scattering phenomenon that occurs predominantly from structures such as collagen in biological samples. Some molecules that contribute to AF change in abundance from processes related to the cancer disease process (e.g., metabolic changes, oxidative stress, and angiogenesis). STUDY DESIGN/MATERIALS AND METHODS: MPM was used to image 12 separate patient samples of formalin-fixed and paraffin-embedded duodenal gastrinoma slides with a second-harmonic generation (SHG) channel and four 2PEF channels. The excitation and emission profiles of each 2PEF channel were tuned to capture signal dominated by distinct fluorophores with well-characterized fluorescent spectra and known connections to the physiologic changes that arise in cancerous tissue. RESULTS: We found that there was a significant difference in the relative abundance of signal generated in the 2PEF channels for regions of DGASTs in comparison to the neighboring tissues of the duodenum. Data generated from texture feature extraction of the MPM images were used in linear discriminant analysis models to create classifiers for tumor versus all other tissue types before and after principal component analysis (PCA). PCA improved the classifier accuracy and reduced the number of features required to achieve maximum accuracy. The linear discriminant classifier after PCA distinguished between tumor and other tissue types with an accuracy of 90.6%-93.8%. CONCLUSIONS: These results suggest that multiphoton microscopy 2PEF and SHG imaging is a promising label-free method for discriminating between DGASTs and normal duodenal tissue which has implications for future applications of in vivo assessment of resection margins with endoscopic MPM.

Live-cell imaging in human colonic monolayers reveals ERK waves limit the stem cell compartment to maintain epithelial homeostasis.

The establishment and maintenance of different cellular compartments in tissues is a universal requirement across all metazoans. Maintaining the correct ratio of cell types in time and space allows tissues to form patterned compartments and perform complex functions. Patterning is especially evident in the human colon, where tissue homeostasis is maintained by stem cells in crypt structures that balance proliferation and differentiation. Here, we developed a human 2D patient derived organoid screening platform to study tissue patterning and kinase pathway dynamics in single cells. Using this system, we discovered that waves of ERK signaling induced by apoptotic cells play a critical role in maintaining tissue patterning and homeostasis. If ERK is activated acutely across all cells instead of in wave-like patterns, then tissue patterning and stem cells are lost. Conversely, if ERK activity is inhibited, then stem cells become unrestricted and expand dramatically. This work demonstrates that the colonic epithelium requires coordinated ERK signaling dynamics to maintain patterning and tissue homeostasis. Our work reveals how ERK can antagonize stem cells while supporting cell replacement and the function of the gut.

GFAP-directed Inactivation of Men1 Exploits Glial Cell Plasticity in Favor of Neuroendocrine Reprogramming.

BACKGROUND & AIMS: Efforts to characterize the signaling mechanisms that underlie gastroenteropancreatic neoplasms (GEP-NENs) are precluded by a lack of comprehensive models that recapitulate pathogenesis. Investigation into a potential cell-of-origin for gastrin-secreting NENs revealed a non-cell autonomous role for loss of menin in neuroendocrine cell specification, resulting in an induction of gastrin in enteric glia. Here, we investigated the hypothesis that cell autonomous Men1 inactivation in glial fibrillary acidic protein (GFAP)-expressing cells induced neuroendocrine differentiation and tumorigenesis. METHODS: Transgenic GFAPΔMen1 mice were generated by conditional GFAP-directed Men1 deletion in GFAP-expressing cells. Cre specificity was confirmed using a tdTomato reporter. GFAPΔMen1 mice were evaluated for GEP-NEN development and neuroendocrine cell hyperplasia. Small interfering RNA-mediated Men1 silencing in a rat enteric glial cell line was performed in parallel. RESULTS: GFAPΔMen1 mice developed pancreatic NENs, in addition to pituitary prolactinomas that phenocopied the human MEN1 syndrome. GFAPΔMen1 mice exhibited gastric neuroendocrine hyperplasia that coincided with a significant loss of GFAP expression. Men1 deletion induced loss of glial-restricted progenitor lineage markers and an increase in neuroendocrine genes, suggesting a reprogramming of GFAP+ cells. Deleting Kif3a, a mediator of Hedgehog signaling, in GFAP-expressing cells attenuated neuroendocrine hyperplasia by restricting the neuroendocrine cell fate. Similar results in the pancreas were observed when Sox10 was used to delete Men1. CONCLUSIONS: GFAP-directed Men1 inactivation exploits glial cell plasticity in favor of neuroendocrine differentiation.