Nonlinear progression across the occult transition establishes cancer lethality.

Cancer screening is based upon a linear model of growth and invasion. Yet, early dissemination during the lengthy pre-diagnostic phase suggests that nonlinearity in growth can also occur. Therefore, we quantitatively traced the invisible and visible phases of tumorigenesis in the mammary gland for more than two-thousand tumors. Dynamic mathematical models of the invisible phase revealed an occult checkpoint resulting in nonlinear progression of transformed field cells. We found that expansile fields have increased dwell time at the occult checkpoint resulting in a large reservoir of image detectable precursors prior to invasion. In contrast, slowly proliferating lesions disseminate early and then transition rapidly through an occult checkpoint in a process we term nascent lethality. Our data illustrate how nonlinear growth across an occult checkpoint can account for a paradoxical increase in early-stage cancer detection without a dramatic reduction in metastatic burden. Highlights: Growth during the invisible phase of tumorigenesis is a nonlinear processField size and field growth rate are uncoupled from metastatic potentialOccult transition rates vary by genotypeNascent lethal lesions are currently undetectable.

Overcoming Xenoantigen Immunity to Enable Cellular Tracking and Gene Regulation with Immune-competent "NoGlow" Mice.

The ability to temporally regulate gene expression and track labeled cells makes animal models powerful biomedical tools. However, sudden expression of xenobiotic genes [e.g., GFP, luciferase (Luc), or rtTA3] can trigger inadvertent immunity that suppresses foreign protein expression or results in complete rejection of transplanted cells. Germline exposure to foreign antigens somewhat addresses these challenges; however, native fluorescence and bioluminescence abrogates the utility of reporter proteins and highly spatiotemporally restricted expression can lead to suboptimal xenoantigen tolerance. To overcome these unwanted immune responses and enable reliable cell tracking/gene regulation, we developed a novel mouse model that selectively expresses antigen-intact but nonfunctional forms of GFP and Luc, as well as rtTA3, after CRE-mediated recombination. Using tissue-specific CREs, we observed model and sex-based differences in immune tolerance to the encoded xenoantigens, illustrating the obstacles of tolerizing animals to foreign genes and validating the utility of these "NoGlow" mice to dissect mechanisms of central and peripheral tolerance. Critically, tissue unrestricted NoGlow mice possess no detectable background fluorescence or luminescence and exhibit limited adaptive immunity against encoded transgenic xenoantigens after vaccination. Moreover, we demonstrate that NoGlow mice allow tracking and tetracycline-inducible gene regulation of triple-transgenic cells expressing GFP/Luc/rtTA3, in contrast to transgene-negative immune-competent mice that eliminate these cells or prohibit metastatic seeding. Notably, this model enables de novo metastasis from orthotopically implanted, triple-transgenic tumor cells, despite high xenoantigen expression. Altogether, the NoGlow model provides a critical resource for in vivo studies across disciplines, including oncology, developmental biology, infectious disease, autoimmunity, and transplantation. SIGNIFICANCE: Multitolerant NoGlow mice enable tracking and gene manipulation of transplanted tumor cells without immune-mediated rejection, thus providing a platform to investigate novel mechanisms of adaptive immunity related to metastasis, immunotherapy, and tolerance.

Gut mucosal cells transfer α-synuclein to the vagus nerve.

Epidemiological and histopathological findings have raised the possibility that misfolded α-synuclein protein might spread from the gut to the brain and increase the risk of Parkinson's disease. Although past experimental studies in mouse models have relied on gut injections of exogenous recombinant α-synuclein fibrils to study gut-to-brain α-synuclein transfer, the possible origins of misfolded α-synuclein within the gut have remained elusive. We recently demonstrated that sensory cells of intestinal mucosa express α-synuclein. Here, we employed mouse intestinal organoids expressing human α-synuclein to observe the transfer of α-synuclein protein from epithelial cells in organoids to cocultured nodose neurons devoid of α-synuclein. In mice expressing human α-synuclein, but no mouse α-synuclein, α-synuclein fibril-templating activity emerged in α-synuclein-seeded fibril aggregation assays in intestine, vagus nerve, and dorsal motor nucleus. In newly engineered transgenic mice that restrict pathological human α-synuclein expression to intestinal epithelial cells, α-synuclein fibril-templating activity transfered to the vagus nerve and dorsal motor nucleus. Subdiaphragmatic vagotomy prior to induction of α-synuclein expression in intestinal epithelial cells effectively protected the hindbrain from emergence of α-synuclein fibril-templating activity. Overall, these findings highlight a potential non-neuronal source of fibrillar α-synuclein protein that might arise in gut mucosal cells.

Gut mucosal cells transfer α-synuclein to the vagus nerve.

Epidemiological and histopathological findings have raised the possibility that misfolded α-synuclein protein might spread from the gut to the brain and increase the risk of Parkinson's disease (PD). While past experimental studies in mouse models have relied on gut injections of exogenous recombinant α-synuclein fibrils to study gut to brain α-synuclein transfer, the possible origins of misfolded α-synuclein within the gut have remained elusive. We recently demonstrated that sensory cells of the gut mucosa express α-synuclein. In this study, we employed mouse intestinal organoids expressing human α-synuclein to observe the transfer of α-synuclein protein from gut epithelial cells in organoids co-cultured with vagal nodose neurons that are otherwise devoid of α-synuclein expression. In intact mice that express pathological human α-synuclein, but no mouse α-synuclein, α-synuclein fibril templating activity emerges in α-synuclein seeded fibril aggregation assays in tissues from the gut, vagus nerve, and dorsal motor nucleus. In newly engineered transgenic mice that restrict pathological human α-synuclein expression to intestinal epithelial cells, α-synuclein fibril-templating activity transfers to the vagus nerve and to the dorsal motor nucleus. Subdiaphragmatic vagotomy prior to the induction of α-synuclein expression in the gut epithelial cells effectively protects the hindbrain from the emergence of α-synuclein fibril templating activity. Overall, these findings highlight a novel potential non-neuronal source of fibrillar α-synuclein protein that might arise in gut mucosal cells.

Membrane structure and internalization dynamics of human Flower isoforms hFWE3 and hFWE4 indicate a conserved endocytic role for hFWE4.

Human Flower (hFWE) isoforms hFWE1-4 are putative transmembrane (TM) proteins that reportedly mediate fitness comparisons during cell competition through extracellular display of their C-terminal tails. Isoform topology, subcellular localization, and duration of plasma membrane presentation are essential to this function. However, disagreement persists regarding the structure of orthologous fly and mouse FWEs, and experimental evidence for hFWE isoform subcellular localization or membrane structure is lacking. Here, we used AlphaFold2 and subsequent molecular dynamics-based structural predictions to construct epitope-tagged hFWE3 and hFWE4, the most abundant human isoforms, for experimental determination of their structure and internalization dynamics. We demonstrate that hFWE3 resides in the membrane of the endoplasmic reticulum (ER), while hFWE4 partially colocalizes with Rab4-, Rab5-, and Rab11-positive vesicles as well as with the plasma membrane. An array of imaging techniques revealed that hFWE4 positions both N- and C-terminal tails and a loop between second and third TM segments within the cytosol, while small (4-12aa) loops between the first and second and the third and fourth TM segments are either exposed to the extracellular space or within the lumen of cytoplasmic vesicles. Similarly, we found hFWE3 positions both N- and C-terminal tails in the cytosol, while a short loop between TM domains extends into the ER lumen. Finally, we demonstrate that hFWE4 exists only transiently at the cell surface and is rapidly internalized in an AP-2- and dynamin-1-dependent manner. Collectively, these data are consistent with a conserved role for hFWE4 in endocytic processes.

A LGR5 reporter pig model closely resembles human intestine for improved study of stem cells in disease.

Intestinal epithelial stem cells (ISCs) are responsible for intestinal epithelial barrier renewal; thereby, ISCs play a critical role in intestinal pathophysiology research. While transgenic ISC reporter mice are available, advanced translational studies lack a large animal model. This study validates ISC isolation in a new porcine Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5) reporter line and demonstrates the use of these pigs as a novel colorectal cancer (CRC) model. We applied histology, immunofluorescence, fluorescence-activated cell sorting, flow cytometry, gene expression quantification, and 3D organoid cultures to whole tissue and single cells from the duodenum, jejunum, ileum, and colon of LGR5-H2B-GFP and wild-type pigs. Ileum and colon LGR5-H2B-GFP, healthy human, and murine biopsies were compared by mRNA fluorescent in situ hybridization (FISH). To model CRC, adenomatous polyposis coli (APC) mutation was induced by CRISPR/Cas9 editing in porcine LGR5-H2B-GFP colonoids. Crypt-base, green fluorescent protein (GFP) expressing cells co-localized with ISC biomarkers. LGR5-H2B-GFPhi cells had significantly higher LGR5 expression (p < .01) and enteroid forming efficiency (p < .0001) compared with LGR5-H2B-GFPmed/lo/neg cells. Using FISH, similar LGR5, OLFM4, HOPX, LYZ, and SOX9 expression was identified between human and LGR5-H2B-GFP pig crypt-base cells. LGR5-H2B-GFP/APCnull colonoids had cystic growth in WNT/R-spondin-depleted media and significantly upregulated WNT/ß-catenin target gene expression (p < .05). LGR5+ ISCs are reproducibly isolated in LGR5-H2B-GFP pigs and used to model CRC in an organoid platform. The known anatomical and physiologic similarities between pig and human, and those shown by crypt-base FISH, underscore the significance of this novel LGR5-H2B-GFP pig to translational ISC research.

Modeling collective cell behavior in cancer: Perspectives from an interdisciplinary conversation.

Collective cell behavior contributes to all stages of cancer progression. Understanding how collective behavior emerges through cell-cell interactions and decision-making will advance our understanding of cancer biology and provide new therapeutic approaches. Here, we summarize an interdisciplinary discussion on multicellular behavior in cancer, draw lessons from other scientific disciplines, and identify future directions.

HSP90-Specific nIR Probe Identifies Aggressive Prostate Cancers: Translation from Preclinical Models to a Human Phase I Study.

A noninvasive test to discriminate indolent prostate cancers from lethal ones would focus treatment where necessary while reducing overtreatment. We exploited the known activity of heat shock protein 90 (Hsp90) as a chaperone critical for the function of numerous oncogenic drivers, including the androgen receptor and its variants, to detect aggressive prostate cancer. We linked a near-infrared fluorescing molecule to an HSP90 binding drug and demonstrated that this probe (designated HS196) was highly sensitive and specific for detecting implanted prostate cancer cell lines with greater uptake by more aggressive subtypes. In a phase I human study, systemically administered HS196 could be detected in malignant nodules within prostatectomy specimens. Single-cell RNA sequencing identified uptake of HS196 by malignant prostate epithelium from the peripheral zone (AMACR+ERG+EPCAM+ cells), including SYP+ neuroendocrine cells that are associated with therapeutic resistance and metastatic progression. A theranostic version of this molecule is under clinical testing.

Veterinary Management of Harderian Gland Tumors in Cancer Rainbow (crainbow) HER2-Positive Mice.

A Cancer Rainbow mouse line that expresses 3 fluorescently labeled isoforms of the tumor-driver gene HER2 (HER2BOW) was developed recently for the study of tumorigenesis in the mammary gland. The expression of 1 of the 3 HER2 isoforms in HER2BOW mice is induced through the Cre/lox system. However, in addition to developing palpable mammary tumors, HER2BOW mice developed orbital tumors, specifically of the Harderian gland. Mice were euthanized, and histopathologic examination of the Harderian gland tumors was performed. Tumors were characterized by adenomatous hyperplasia to multinodular adenomas of the Harderian gland. Fluorescent imaging of the Harderian gland tissue confirmed the expression of HER2 in the tumors. Here we discuss monitoring and palliative approaches to allow attainment of humane experimental endpoints of mammary tumor growth in this mouse line. We describe a range of interventions, including close monitoring, topical palliative care, and surgical bilateral enucleation. Based on our data and previous reports in the literature, the overexpression of HER2 in Harderian gland tissue and subsequent tumor formation likely was driven by MMTV-Cre expression in the Harderian gland.

Measuring Nonapoptotic Caspase Activity with a Transgenic Reporter in Mice.

The protease caspase-3 is a key mediator of apoptotic programmed cell death. But weak or transient caspase activity can contribute to neuronal differentiation, axonal pathfinding, and synaptic long-term depression. Despite the importance of sublethal, or nonapoptotic, caspase activity in neurodevelopment and neural plasticity, there has been no simple method for mapping and quantifying nonapoptotic caspase activity (NACA) in rodent brains. We therefore generated a transgenic mouse expressing a highly sensitive and specific fluorescent reporter of caspase activity, with peak signal localized to the nucleus. As a proof of concept, we first obtained evidence that NACA influences neurophysiology in an amygdalar circuit. Then focusing on the amygdala, we were able to quantify a sex-specific persistent elevation in caspase activity in females after restraint stress. This simple in vivo caspase activity reporter will facilitate systems-level studies of apoptotic and nonapoptotic phenomena in behavioral and pathologic models.

HER2 Isoforms Uniquely Program Intratumor Heterogeneity and Predetermine Breast Cancer Trajectories During the Occult Tumorigenic Phase.

HER2-positive breast cancers are among the most heterogeneous breast cancer subtypes. The early amplification of HER2 and its known oncogenic isoforms provide a plausible mechanism in which distinct programs of tumor heterogeneity could be traced to the initial oncogenic event. Here a Cancer rainbow mouse simultaneously expressing fluorescently barcoded wildtype (WTHER2), exon-16 null (d16HER2), and N-terminally truncated (p95HER2) HER2 isoforms is used to trace tumorigenesis from initiation to invasion. Tumorigenesis was visualized using whole-gland fluorescent lineage tracing and single-cell molecular pathology. We demonstrate that within weeks of expression, morphologic aberrations were already present and unique to each HER2 isoform. Although WTHER2 cells were abundant throughout the mammary ducts, detectable lesions were exceptionally rare. In contrast, d16HER2 and p95HER2 induced rapid tumor development. d16HER2 incited homogenous and proliferative luminal-like lesions which infrequently progressed to invasive phenotypes whereas p95HER2 lesions were heterogenous and invasive at the smallest detectable stage. Distinct cancer trajectories were observed for d16HER2 and p95HER2 tumors as evidenced by oncogene-dependent changes in epithelial specification and the tumor microenvironment. These data provide direct experimental evidence that intratumor heterogeneity programs begin very early and well in advance of screen or clinically detectable breast cancer. IMPLICATIONS: Although all HER2 breast cancers are treated equally, we show a mechanism by which clinically undetected HER2 isoforms program heterogenous cancer phenotypes through biased epithelial specification and adaptations within the tumor microenvironment.

Noncanonical scaffolding of Gαi and ß-arrestin by G protein-coupled receptors.

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) are common drug targets and canonically couple to specific Gα protein subtypes and ß-arrestin adaptor proteins. G protein-mediated signaling and ß-arrestin-mediated signaling have been considered separable. We show here that GPCRs promote a direct interaction between Gαi protein subtype family members and ß-arrestins regardless of their canonical Gα protein subtype coupling. Gαi:ß-arrestin complexes bound extracellular signal-regulated kinase (ERK), and their disruption impaired both ERK activation and cell migration, which is consistent with ß-arrestins requiring a functional interaction with Gαi for certain signaling events. These results introduce a GPCR signaling mechanism distinct from canonical G protein activation in which GPCRs cause the formation of Gαi:ß-arrestin signaling complexes.

Stimulation of Oncogene-Specific Tumor-Infiltrating T Cells through Combined Vaccine and αPD-1 Enable Sustained Antitumor Responses against Established HER2 Breast Cancer.

PURPOSE: Despite promising advances in breast cancer immunotherapy, augmenting T-cell infiltration has remained a significant challenge. Although neither individual vaccines nor immune checkpoint blockade (ICB) have had broad success as monotherapies, we hypothesized that targeted vaccination against an oncogenic driver in combination with ICB could direct and enable antitumor immunity in advanced cancers. EXPERIMENTAL DESIGN: Our models of HER2+ breast cancer exhibit molecular signatures that are reflective of advanced human HER2+ breast cancer, with a small numbers of neoepitopes and elevated immunosuppressive markers. Using these, we vaccinated against the oncogenic HER2Δ16 isoform, a nondriver tumor-associated gene (GFP), and specific neoepitopes. We further tested the effect of vaccination or anti-PD-1, alone and in combination. RESULTS: We found that only vaccination targeting HER2Δ16, a driver of oncogenicity and HER2-therapeutic resistance, could elicit significant antitumor responses, while vaccines targeting a nondriver tumor-specific antigen or tumor neoepitopes did not. Vaccine-induced HER2-specific CD8+ T cells were essential for responses, which were more effective early in tumor development. Long-term tumor control of advanced cancers occurred only when HER2Δ16 vaccination was combined with αPD-1. Single-cell RNA sequencing of tumor-infiltrating T cells revealed that while vaccination expanded CD8 T cells, only the combination of vaccine with αPD-1 induced functional gene expression signatures in those CD8 T cells. Furthermore, we show that expanded clones are HER2-reactive, conclusively demonstrating the efficacy of this vaccination strategy in targeting HER2. CONCLUSIONS: Combining oncogenic driver targeted vaccines with selective ICB offers a rational paradigm for precision immunotherapy, which we are clinically evaluating in a phase II trial (NCT03632941).

Heat shock protein 90-targeted photodynamic therapy enables treatment of subcutaneous and visceral tumors.

Photodynamic therapy (PDT) ablates malignancies by applying focused near-infrared (nIR) light onto a lesion of interest after systemic administration of a photosensitizer (PS); however, the accumulation of existing PS is not tumor-exclusive. We developed a tumor-localizing strategy for PDT, exploiting the high expression of heat shock protein 90 (Hsp90) in cancer cells to retain high concentrations of PS by tethering a small molecule Hsp90 inhibitor to a PS (verteporfin, VP) to create an Hsp90-targeted PS (HS201). HS201 accumulates to a greater extent than VP in breast cancer cells both in vitro and in vivo, resulting in increased treatment efficacy of HS201-PDT in various human breast cancer xenografts regardless of molecular and clinical subtypes. The therapeutic index achieved with Hsp90-targeted PDT would permit treatment not only of localized tumors, but also more diffusely infiltrating processes such as inflammatory breast cancer.

A cancer rainbow mouse for visualizing the functional genomics of oncogenic clonal expansion.

Field cancerization is a premalignant process marked by clones of oncogenic mutations spreading through the epithelium. The timescales of intestinal field cancerization can be variable and the mechanisms driving the rapid spread of oncogenic clones are unknown. Here we use a Cancer rainbow (Crainbow) modelling system for fluorescently barcoding somatic mutations and directly visualizing the clonal expansion and spread of oncogenes. Crainbow shows that mutations of ß-catenin (Ctnnb1) within the intestinal stem cell results in widespread expansion of oncogenes during perinatal development but not in adults. In contrast, mutations that extrinsically disrupt the stem cell microenvironment can spread in adult intestine without delay. We observe the rapid spread of premalignant clones in Crainbow mice expressing oncogenic Rspondin-3 (RSPO3), which occurs by increasing crypt fission and inhibiting crypt fixation. Crainbow modelling provides insight into how somatic mutations rapidly spread and a plausible mechanism for predetermining the intratumor heterogeneity found in colon cancers.

Slow-release delivery enhances the pharmacological properties of oral 5-hydroxytryptophan: mouse proof-of-concept.

5-hydroxytryptophan (5-HTP) has shown therapeutic promise in a range of human CNS disorders. But native 5-HTP immediate release (IR) is poorly druggable, as rapid absorption causes rapid onset of adverse events, and rapid elimination causes fluctuating exposure. Recently, we reported that 5-HTP delivered as slow-release (SR) in mice augmented the brain pro-serotonergic effect of selective serotonin reuptake inhibitors (SSRIs), without the usual adverse events associated with 5-HTP IR. However, our previous study entailed translational limitations, in terms of route, dose, and duration. Here we modeled oral 5-HTP SR in mice by administering 5-HTP via the food. We modeled oral SSRI treatment via fluoxetine in the water, in a regimen recapitulating clinical pharmacokinetics and pharmacodynamics. 5-HTP SR produced plasma 5-HTP levels well within the range enhancing brain 5-HT function in humans. 5-HTP SR robustly increased brain 5-HT synthesis and levels. When administered with an SSRI, 5-HTP SR enhanced 5-HT-sensitive behaviors and neurotrophic mRNA expression. 5-HTP SR's pro-serotonergic effects were stronger in mice with endogenous brain 5-HT deficiency. In a comprehensive screen, 5-HTP SR was devoid of overt toxicological effects. The present preclinical data, appreciated in the context of published 5-HTP clinical data, suggest that 5-HTP SR could represent a new therapeutic approach to the plethora of CNS disorders potentially treatable with a pro-serotonergic drug. 5-HTP SR might in particular be therapeutically relevant when brain 5-HT deficiency is pathogenic and as an adjunctive augmentation therapy to SSRI therapy.

Vaccine-Induced Memory CD8+ T Cells Provide Clinical Benefit in HER2 Expressing Breast Cancer: A Mouse to Human Translational Study.

PURPOSE: Immune-based therapy for metastatic breast cancer has had limited success, particularly in molecular subtypes with low somatic mutations rates. Strategies to augment T-cell infiltration of tumors include vaccines targeting established oncogenic drivers such as the genomic amplification of HER2. We constructed a vaccine based on a novel alphaviral vector encoding a portion of HER2 (VRP-HER2). PATIENTS AND METHODS: In preclinical studies, mice were immunized with VRP-HER2 before or after implantation of hHER2+ tumor cells and HER2-specific immune responses and antitumor function were evaluated. We tested VRP-HER2 in a phase I clinical trial where subjects with advanced HER2-overexpressing malignancies in cohort 1 received VRP-HER2 every 2 weeks for a total of 3 doses. In cohort 2, subjects received the same schedule concurrently with a HER2-targeted therapy. RESULTS: Vaccination in preclinical models with VRP-HER2 induced HER2-specific T cells and antibodies while inhibiting tumor growth. VRP-HER2 was well tolerated in patients and vaccination induced HER2-specific T cells and antibodies. Although a phase I study, there was 1 partial response and 2 patients with continued stable disease. Median OS was 50.2 months in cohort 1 (n = 4) and 32.7 months in cohort 2 (n = 18). Perforin expression by memory CD8 T cells post-vaccination significantly correlated with improved PFS. CONCLUSIONS: VRP-HER2 increased HER2-specific memory CD8 T cells and had antitumor effects in preclinical and clinical studies. The expansion of HER2-specific memory CD8 T cells in vaccinated patients was significantly correlated with increased PFS. Subsequent studies will seek to enhance T-cell activity by combining with anti-PD-1.

hCALCRL mutation causes autosomal recessive nonimmune hydrops fetalis with lymphatic dysplasia.

We report the first case of nonimmune hydrops fetalis (NIHF) associated with a recessive, in-frame deletion of V205 in the G protein-coupled receptor, Calcitonin Receptor-Like Receptor (hCALCRL). Homozygosity results in fetal demise from hydrops fetalis, while heterozygosity in females is associated with spontaneous miscarriage and subfertility. Using molecular dynamic modeling and in vitro biochemical assays, we show that the hCLR(V205del) mutant results in misfolding of the first extracellular loop, reducing association with its requisite receptor chaperone, receptor activity modifying protein (RAMP), translocation to the plasma membrane and signaling. Using three independent genetic mouse models we establish that the adrenomedullin-CLR-RAMP2 axis is both necessary and sufficient for driving lymphatic vascular proliferation. Genetic ablation of either lymphatic endothelial Calcrl or nonendothelial Ramp2 leads to severe NIHF with embryonic demise and placental pathologies, similar to that observed in humans. Our results highlight a novel candidate gene for human congenital NIHF and provide structure-function insights of this signaling axis for human physiology.

Inhibiting clathrin-mediated endocytosis of the leucine-rich G protein-coupled receptor-5 diminishes cell fitness.

The leucine-rich G protein-coupled receptor-5 (LGR5) is expressed in adult tissue stem cells of many epithelia, and its overexpression is negatively correlated with cancer prognosis. LGR5 potentiates WNT/ß-catenin signaling through its unique constitutive internalization property that clears negative regulators of the WNT-receptor complex from the membrane. However, both the mechanism and physiological relevance of LGR5 internalization are unclear. Therefore, a natural product library was screened to discover LGR5 internalization inhibitors and gain mechanistic insight into LGR5 internalization. The plant lignan justicidin B blocked the constitutive internalization of LGR5. Justicidin B is structurally similar to more potent vacuolar-type H+-ATPase inhibitors, which all inhibited LGR5 internalization by blocking clathrin-mediated endocytosis. We then tested the physiological relevance of LGR5 internalization blockade in vivo A LGR5-rainbow (LBOW) mouse line was engineered to express three different LGR5 isoforms along with unique fluorescent protein lineage reporters in the same mouse. In this manner, the effects of each isoform on cell fate can be simultaneously assessed through simple fluorescent imaging for each lineage reporter. LBOW mice express three different forms of LGR5, a wild-type form that constitutively internalizes and two mutant forms whose internalization properties have been compromised by genetic perturbations within the carboxyl-terminal tail. LBOW was activated in the intestinal epithelium, and a year-long lineage-tracing course revealed that genetic blockade of LGR5 internalization diminished cell fitness. Together these data provide proof-of-concept genetic evidence that blocking the clathrin-mediated endocytosis of LGR5 could be used to pharmacologically control cell behavior.