TGFß-induced EN1 promotes tumor budding of adenoid cystic carcinoma in patient-derived organoid model.

Adenoid cystic carcinoma (ACC) and basal cell adenoma (BCA) share many histological characteristics and often need a differential diagnosis in clinical pathology. Recently, we found homeobox protein engrailed-1 (EN1) was a potential diagnostic marker for ACC in an organoids library of salivary gland tumors (SGTs). Here we aim to confirm EN1 as a differential diagnostic marker for ACC, and further investigate the regulatory mechanism and biological function of EN1 in tumor progression. The transcriptional analysis, quantitative polymerase chain reaction, Western blot and immunohistochemistry staining were performed and revealed that EN1 was specifically and highly expressed in ACC, and accurately differentiated ACC from BCA. Furthermore, TGFß signaling pathway was found associated with ACC, and the regulation of EN1 through TGFß was detected in the human ACC cell lines and patient-derived organoids (PDOs). TGFß-induced EN1 was important in promoting tumor budding in the PDOs model. Interestingly, a high level of EN1 and TGFß1 in the budding tips was observed in ACC clinical samples, and the expression of EN1 and TGFß1 in ACC was significantly associated with the clinical stage. In summary, our study verified EN1 is a good diagnostic marker to differentiate ACC from BCA. TGFß-induced EN1 facilitates the tumor budding of ACC, which might be an important mechanism related to the malignant phenotype of ACC.

Changing smoking habits and the occurrence of lung cancer in Sweden-a population analysis.

BACKGROUND: The objective is to estimate the importance of the decrease of smoking habits in Sweden for the occurrence of lung cancer. METHODS: The change in smoking habits in the general population was retrieved from surveys and on taxation of sale of cigarettes. We used data from the Swedish Cancer Register on incidence of lung cancer between 1970 and 2021, stratified for sex, age and cell type, and compared the occurrence overtime in ages between 40 and 84 years. RESULTS: The sale of cigarettes peaked in 1980 to 1800 cigarettes per person and decreased to 600 per person in 2021. The change in incidence rates of squamous cell cancer and other cell types varied over time, sex, and age in a pattern that partly seems to be explained by change in the prevalence of daily smokers. The incidence of adenocarcinoma was similar in men and women 1970-2021 and increased, e.g. for women and men 75-79 years of age from around 20 cases in early 1970s to around 120 cases per 100 000 person-years in the 2020s. CONCLUSIONS: Our data indicate that the risk of lung cancer several years after smoking cessation is less favourable than previously studies have indicated. There is a similar increase in the incidence of adenocarcinoma in men and women which is hard to explain only with changing smoking habits. The change from non-filter to filter cigarettes in the 1960s-1970s may be a contributing factor.

Combined Transcriptomic and Protein Array Cytokine Profiling of Human Stem Cells from Dental Apical Papilla Modulated by Oral Bacteria.

Stem cells from the apical papilla (SCAP) are a promising resource for use in regenerative endodontic treatment (RET) that may be adversely affected by oral bacteria, which in turn can exert an effect on the success of RET. Our work aims to study the cytokine profile of SCAP upon exposure to oral bacteria and their supernatants-Fusobacterium nucleatum and Enterococcus faecalis-as well as to establish their effect on the osteogenic and immunogenic potentials of SCAP. Further, we target the presence of key proteins of the Wnt/ß-Catenin, TGF-ß, and NF-κB signaling pathways, which play a crucial role in adult osteogenic differentiation of mesenchymal stem cells, using the Western blot (WB) technique. The membrane-based sandwich immunoassay and transcriptomic analysis showed that, under the influence of F. nucleatum (both bacteria and supernatant), the production of pro-inflammatory cytokines IL-6, IL-8, and MCP-1 occurred, which was also confirmed at the mRNA level. Conversely, E. faecalis reduced the secretion of the aforementioned cytokines at both mRNA and protein levels. WB analysis showed that SCAP co-cultivation with E. faecalis led to a decrease in the level of the key proteins of the Wnt/ß-Catenin and NF-κB signaling pathways: ß-Catenin (p = 0.0068 *), LRP-5 (p = 0.0059 **), and LRP-6 (p = 0.0329 *), as well as NF-kB (p = 0.0034 **) and TRAF6 (p = 0.0285 *). These results suggest that oral bacteria can up- and downregulate the immune and inflammatory responses of SCAP, as well as influence the osteogenic potential of SCAP, which may negatively regulate the success of RET.

Osteoblast-derived factors promote metastatic potential in human prostate cancer cells, in part via non-canonical transforming growth factor ß (TGFß) signaling.

BACKGROUND: Transforming growth factor ß (TGFß) functions as a double-edged sword in prostate cancer tumorigenesis. In initial stages of the disease, TGFß acts as a growth inhibitor upon tumor cells, whereas it in later stages of disease rather promotes invasion and metastatic potential. One well-known cellular source of TGFß in the bone metastatic site is the bone-forming osteoblasts. Here we have studied the effects by osteoblast-derived factors on metastatic potential in several human prostate cancer cell lines. METHODS: Effects on metastatic potential in prostate cancer cells by osteoblast-derived factors were studied in vitro using several methods, including Transwell migration and evaluation of formation of pro-migratory protrusions. Confocal microscopy was used to evaluate possible changes in differentiation state in tumor cells by analysis of markers for epithelial-to-mesenchymal transition (EMT). The Matrigel-on-top 3D culture method was used for further assessment of metastatic characteristics in tumor cells by analysis of formation of filopodium-like protrusions (FLPs). RESULTS: Osteoblast-derived factors increased migration of PC-3U cells, an effect less prominent in cells overexpressing a mutated type I TGFß receptor (TßRI) preventing non-canonical TRAF6-dependent TGFß signaling. Osteoblast-derived factors also increased the formation of long protrusions and loss of cell-cell contacts in PC-3U cells, suggesting induction of a more aggressive phenotype. In addition, treatment with TGFß or osteoblast-derived factors of PC-3U cells in Matrigel-on-top 3D cultures promoted formation of FLPs, previously shown to be essential for metastatic establishment. CONCLUSIONS: These findings suggests that factors secreted from osteoblasts, including TGFß, can induce several cellular traits involved in metastatic potential of PC-3U cells, further strengthening the role for bone cells to promote metastatic tumor cell behavior.

Regulated intramembrane proteolysis of the TGFß type I receptor conveys oncogenic signals.

Cancer cells produce high levels of TGFß, a multipotent cytokine. Binding of TGFß to its cell surface receptors, the transmembrane serine/threonine kinases TßRII and TßRI, causes phosphorylation and activation of intracellular latent Smad transcription factors. Nuclear Smads act in concert with specific transcription factors to reprogram epithelial cells to become invasive mesenchymal cells. TGFß also propagates non-canonical signals, so it is crucial to have a better understanding of the underlying molecular mechanisms which favor this pathway. Here we highlight our recent discovery that TGFß promotes the proteolytic cleavage of TßRI in cancer cells, resulting in the liberation and nuclear translocation of its intracellular domain, acting as co-regulator to transcribe pro-invasive genes. This newly identified oncogenic TGFß pathway resembles the Notch signaling pathway. We discuss our findings in relation to Notch and provide a short overview of other growth factors that transduce signals via nuclear translocation of their cell surface receptors.

Human transforming growth factor ß type I receptor in complex with kinase inhibitor SB505124.

The crystal structure of the intracellular domain of transforming growth factor ß type I receptor (TßR1) in complex with the competitive inhibitor SB505124 is presented. The study provides insights into the structure and function of TßR1 in complex with SB505124, and as such offers molecular-level understanding of the inhibition of this critical signalling pathway. The potential of SB505124 as an avenue for therapy in cancer treatment is discussed on basis of the results.

Robust approach for production of the human oncology target Aurora kinase B in complex with its binding partner INCENP.

Protein kinases are key players in many eukaryotic signal transduction cascades and are as a result often linked to human disease. In humans, the mitotic protein kinase family of Aurora kinases consist of three members: Aurora A, B and C. All three members are involved in cell division with proposed implications in various human cancers. The human Aurora kinase B has in particular proven challenging to study with structural biology approaches, and this is mainly due to difficulties in producing the large quantities of active enzyme required for such studies. Here, we present a novel and E. coli-based production system that allows for production of milligram quantities of well-folded and active human Aurora B in complex with its binding partner INCENP. The complex is produced as a continuous polypeptide chain and the resulting fusion protein is cleaved with TEV protease to generate a stable and native heterodimer of the Aurora B:INCENP complex. The activity, stability and degree of phosphorylation of the protein complex was quantified by using a coupled ATPase assay, 31P NMR spectroscopy and mass spectrometry. The developed production system enables isotope labeling and we here report the first 1H-15N-HSQC of the human Aurora B:INCENP complex. Our developed production strategy paves the way for future structural and functional studies of Aurora B and can as such assist the development of novel anticancer drugs targeting this important mitotic protein kinase.

The TßRI promotes migration and metastasis through thrombospondin 1 and ITGAV in prostate cancer cells.

TGFß potently modifies the extracellular matrix (ECM), which is thought to favor tumor cell invasion. However, the mechanism whereby the cancer cells employ the ECM proteins to facilitate their motility is largely unknown. In this study we used RNA-seq and proteomic analysis to examine the proteins secreted by castration-resistant prostate cancer (CRPC) cells upon TGFß treatment and found that thrombospondin 1 (THBS1) was observed to be one of the predominant proteins. The CRISPR Cas9, or siRNA techniques was used to downregulate TGFß type I receptor (TßRI) to interfere with TGFß signaling in various cancer cells in vitro. The interaction of ECM proteins with the TßRI in the migratory prostate cancer cells in response to TGFß1 was demonstrated by several different techniques to reveal that THBS1 mediates cell migration by interacting with integrin subunit alpha V (ITGAV) and TßRI. Deletion of TßRI or THBS1 in cancer cells prevented their migration and invasion. THBS1 belongs to a group of tumorigenic ECM proteins induced via TGFß signaling in CRPC cells, and high expression of THBS1 in human prostate cancer tissues correlated with the degree of malignancy. TGFß-induced production of THBS1 through TßRI facilitates the invasion and metastasis of CRPC cells as shown in vivo xenograft animal experiments.

Polyubiquitination of transforming growth factor ß (TGFß)-associated kinase 1 mediates nuclear factor-κB activation in response to different inflammatory stimuli.

The transcription factor nuclear factor κB (NF-κB) plays a central role in regulating inflammation in response to several external signals. The TGFß-associated kinase 1 (TAK1) is an upstream regulator of NF-κB signaling. In TGFß-stimulated cells, TAK1 undergoes Lys-63-linked polyubiquitination at Lys-34 by TNF receptor-associated factor 6 and is thereby activated. The aim of this study was to investigate whether TAK1 polyubiquitination at Lys-34 is also essential for NF-κB activation via TNF receptor, IL-1 receptor and toll-like receptor 4. We observed that TAK1 polyubiquitination occurred at Lys-34 and required the E3 ubiquitin ligase TNF receptor-associated factor 6 after stimulation of cells with IL-1ß. Polyubiquitination of TAK1 also occurred at Lys-34 in cells stimulated by TNF-α and LPS, which activates TLR4, as well as in HepG2 and prostate cancer cells stimulated with TGFß, which in all cases resulted in NF-κB activation. Expression of a K34R-mutant TAK1 led to a reduced NF-κB activation, IL-6 promoter activity, and proinflammatory cytokine secretion by TNF-α-stimulated PC-3U cells. Similar results were obtained in the mouse macrophage cell line RAW264.7 after LPS treatment. In conclusion, polyubiquitination of TAK1 is correlated with activation of TAK1 and is essential for activation of NF-κB signaling downstream of several receptors.

Exploring the impact of oral bacteria remnants on stem cells from the Apical papilla: mineralization potential and inflammatory response.

Introduction: Bacterial persistence is considered one of the main causal factors for regenerative endodontic treatment (RET) failure in immature permanent teeth. This interference is claimed to be caused by the interaction of bacteria that reside in the root canal with the stem cells that are one of the essentials for RET. The aim of the study was to investigate whether prolonged exposure of stem cells from the apical papilla (SCAP) to bacterial remnants of Fusobacterium nucleatum, Actinomyces gerensceriae, Slackia exigua, Enterococcus faecalis, Peptostreptococcaceae yurii, commonly found in infected traumatized root canals, and the probiotic bacteria Lactobacillus gasseri and Limosilactobacillus reuteri, can alter SCAP's inflammatory response and mineralization potential. Methods: To assess the effect of bacterial remnants on SCAP, we used UV-C-inactivated bacteria (as cell wall-associated virulence factors) and bacterial DNA. Histochemical staining using Osteoimage Mineralization Assay and Alizarin Red analysis was performed to study SCAP mineralization, while inflammatory and osteo/odontogenic-related responses of SCAPs were assessed with Multiplex ELISA. Results: We showed that mineralization promotion was greater with UV C-inactivated bacteria compared to bacterial DNA. Immunofluorescence analysis detected that the early mineralization marker alkaline phosphatase (ALP) was increased by the level of E. coli lipopolysaccharide (LPS) positive control in the case of UV-C-inactivated bacteria; meanwhile, DNA treatment decreased the level of ALP compared to the positive control. SCAP's secretome assessed with Multiplex ELISA showed the upregulation of pro-inflammatory factors IL-6, IL-8, GM-CSF, IL-1b, neurotrophic factor BDNF, and angiogenic factor VEGF, induced by UV-C-killed bacteria. Discussion: The results suggest that long term stimulation (for 21 days) of SCAP with UV-C-inactivated bacteria stimulate their mineralization and inflammatory response, while DNA influence has no such effect, which opens up new ideas about the nature of RET failure.

Helicobacter pylori attachment-blocking antibodies protect against duodenal ulcer disease.

The majority of the world population carry the gastric pathogen Helicobacter pylori. Fortunately, most individuals experience only low-grade or no symptoms, but in many cases the chronic inflammatory infection develops into severe gastric disease, including duodenal ulcer disease and gastric cancer. Here we report on a protective mechanism where H. pylori attachment and accompanying chronic mucosal inflammation can be reduced by antibodies that are present in a vast majority of H. pylori carriers. These antibodies block binding of the H. pylori attachment protein BabA by mimicking BabA's binding to the ABO blood group glycans in the gastric mucosa. However, many individuals demonstrate low titers of BabA blocking antibodies, which is associated with an increased risk for duodenal ulceration, suggesting a role for these antibodies in preventing gastric disease.

Non-Smad signaling pathways.

Transforming growth factor-beta (TGFß) is a key regulator of cell fate during embryogenesis and has also emerged as a potent driver of the epithelial-mesenchymal transition during tumor progression. TGFß signals are transduced by transmembrane type I and type II serine/threonine kinase receptors (TßRI and TßRII, respectively). The activated TßR complex phosphorylates Smad2 and Smad3, converting them into transcriptional regulators that complex with Smad4. TGFß also uses non-Smad signaling pathways such as the p38 and Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways to convey its signals. Ubiquitin ligase tumor necrosis factor (TNF)-receptor-associated factor 6 (TRAF6) and TGFß-associated kinase 1 (TAK1) have recently been shown to be crucial for the activation of the p38 and JNK MAPK pathways. Other TGFß-induced non-Smad signaling pathways include the phosphoinositide 3-kinase-Akt-mTOR pathway, the small GTPases Rho, Rac, and Cdc42, and the Ras-Erk-MAPK pathway. Signals induced by TGFß are tightly regulated and specified by post-translational modifications of the signaling components, since they dictate the subcellular localization, activity, and duration of the signal. In this review, we discuss recent findings in the field of TGFß-induced responses by non-Smad signaling pathways.

The Synergistic Cooperation between TGF-ß and Hypoxia in Cancer and Fibrosis.

Transforming growth factor ß (TGF-ß) is a multifunctional cytokine regulating homeostasis and immune responses in adult animals and humans. Aberrant and overactive TGF-ß signaling promotes cancer initiation and fibrosis through epithelial-mesenchymal transition (EMT), as well as the invasion and metastatic growth of cancer cells. TGF-ß is a key factor that is active during hypoxic conditions in cancer and is thereby capable of contributing to angiogenesis in various types of cancer. Another potent role of TGF-ß is suppressing immune responses in cancer patients. The strong tumor-promoting effects of TGF-ß and its profibrotic effects make it a focus for the development of novel therapeutic strategies against cancer and fibrosis as well as an attractive drug target in combination with immune regulatory checkpoint inhibitors. TGF-ß belongs to a family of cytokines that exert their function through signaling via serine/threonine kinase transmembrane receptors to intracellular Smad proteins via the canonical pathway and in combination with co-regulators such as the adaptor protein and E3 ubiquitin ligases TNF receptor-associated factor 4 (TRAF4) and TNF receptor-associated factor 6 (TRAF6) to promote non-canonical pathways. Finally, the outcome of gene transcription initiated by TGF-ß is context-dependent and controlled by signals exerted by other growth factors such as EGF and Wnt. Here, we discuss the synergistic cooperation between TGF-ß and hypoxia in development, fibrosis and cancer.

The type II TGF-ß receptor phosphorylates Tyr182 in the type I receptor to activate downstream Src signaling.

Transforming growth factor-ß (TGF-ß) signaling has important roles during embryonic development and in tissue homeostasis. TGF-ß ligands exert cellular effects by binding to type I (TßRI) and type II (TßRII) receptors and inducing both SMAD-dependent and SMAD-independent intracellular signaling pathways, the latter of which includes the activation of the tyrosine kinase Src. We investigated the mechanism by which TGF-ß stimulation activates Src in human and mouse cells. Before TGF-ß stimulation, inactive Src was complexed with TßRII. Upon TGF-ß1 stimulation, TßRII associated with and phosphorylated TßRI at Tyr182. Binding of Src to TßRI involved the interaction of the Src SH2 domain with phosphorylated Tyr182 and the interaction of the Src SH3 domain with a proline-rich region in TßRI and led to the activation of Src kinase activity and Src autophosphorylation. TGF-ß1-induced Src activation required the kinase activities of TßRII and Src but not that of TßRI. Activated Src also phosphorylated TßRI on several tyrosine residues, which may stabilize the binding of Src to the receptor. Src activation was required for the ability of TGF-ß to induce fibronectin production and migration in human breast carcinoma cells and to induce α-smooth muscle actin and actin reorganization in mouse fibroblasts. Thus, TGF-ß induces Src activation by stimulating a direct interaction with TßRI that depends on tyrosine phosphorylation of TßRI by TßRII.

The ubiquitin-ligase TRAF6 and TGFß type I receptor form a complex with Aurora kinase B contributing to mitotic progression and cytokinesis in cancer cells.

BACKGROUND: Transforming growth factor ß (TGFß) is overexpressed in several advanced cancer types and promotes tumor progression. We have reported that the intracellular domain (ICD) of TGFß receptor (TßR) I is cleaved by proteolytic enzymes in cancer cells, and then translocated to the nucleus in a manner dependent on the endosomal adaptor proteins APPL1/2, driving an invasiveness program. How cancer cells evade TGFß-induced growth inhibition is unclear. METHODS: We performed microarray analysis to search for genes regulated by APPL1/2 proteins in castration-resistant prostate cancer (CRPC) cells. We investigated the role of TßRI and TRAF6 in mitosis in cancer cell lines cultured in 10% FBS in the absence of exogenous TGFß. The molecular mechanism of the ubiquitination of AURKB by TRAF6 in mitosis and the formation of AURKB-TßRI complex in cancer cell lines and tissue microarrays was also studied. FINDINGS: During mitosis and cytokinesis, AURKB-TßRI complexes formed in midbodies in CRPC and KELLY neuroblastoma cells. TRAF6 induced polyubiquitination of AURKB on K85 and K87, protruding on the surface of AURKB to facilitate its activation. AURKB-TßRI complexes in patient's tumor tissue sections correlated with the malignancy of prostate cancer. INTERPRETATION: The AURKB-TßRI complex may become a prognostic biomarker for patients with risk of developing aggressive PC. FUNDING: Swedish Medical Research Council (2019-01598, ML; 2015-02757 and 2020-01291, CHH), the Swedish Cancer Society (20 0964, ML), a regional agreement between Umeå University and Region Västerbotten (ALF; RV-939377, -967041, -970057, ML). The European Research Council (787472, CHH). KAW 2019.0345, and the Kempe Foundation SMK-1866; ML. National Microscopy Infrastructure (NMI VR-RFI 2016-00968).

Significance of PI3K signalling pathway in clear cell renal cell carcinoma in relation to VHL and HIF status.

Renal cell carcinoma (RCC) includes diverse tumour types characterised by various genetic abnormalities. The genetic changes, like mutations, deletions and epigenetic alterations, play a crucial role in the modification of signalling networks, tumour pathogenesis and prognosis. The most prevalent RCC type, clear cell RCC (ccRCC), is asymptomatic in the early stages and has a poorer prognosis compared with the papillary and the chromophobe types RCCs. Generally, ccRCC is refractory to chemotherapy and radiation therapy. Loss of von Hippel-Lindau (VHL) gene and upregulation of hypoxia-inducible factors (HIF), the signature of most sporadic ccRCC, promote multiple growth factors. Hence, VHL/HIF and a variety of pathways, including phosphatase and TEnsin homolog on chromosome 10/phosphatidylinositol-3-kinase (PI3K)/AKT, are closely connected and contribute to the ontogeny of ccRCC. In the recent decade, multiple targeting agents have been developed based on blocking major signalling pathways directly or indirectly involved in ccRCC tumour progression, metastasis, angiogenesis and survival. However, most of these drugs have limitations; either metastatic ccRCC develops resistance to these agents, or despite blocking receptors, tumour cells use alternate signalling pathways. This review compiles the state of knowledge about the PI3K/AKT signalling pathway confined to ccRCC and its cross-talks with VHL/HIF pathway.

Cytokine Secretion, Viability, and Real-Time Proliferation of Apical-Papilla Stem Cells Upon Exposure to Oral Bacteria.

The use of stem cells from the apical papilla (SCAPs) has been proposed as a means of promoting root maturation in permanent immature teeth, and plays a significant role in regenerative dental procedures. However, the role of SCAPs may be compromised by microenvironmental factors, such as hypoxic conditions and the presence of bacteria from infected dental root canals. We aim to investigate oral bacterial modulation of SCAP in terms of binding capacity using flow cytometry and imaging, real-time cell proliferation monitoring, and cytokine secretion (IL-6, IL-8, and TGF-ß isoforms) under anaerobic conditions. SCAPs were exposed to key species in dental root canal infection, namely Actinomyces gerensceriae, Slackia exigua, Fusobacterium nucleatum, and Enterococcus faecalis, as well as two probiotic strains, Lactobacillus gasseri strain B6 and Lactobacillus reuteri (DSM 17938). We found that A. gerensceriae, S. exigua, F. nucleatum, and E. faecalis, but not the Lactobacillus probiotic strains bind to SCAPs on anaerobic conditions. Enterococcus faecalis and F. nucleatum exhibited the strongest binding capacity, resulting in significantly reduced SCAP proliferation. Notably, F. nucleatum, but not E. faecalis, induce production of the proinflammatory chemokine IL-8 and IL-10 from SCAPs. Production of TGF-ß1 and TGF-ß2 by SCAPs was dependent on species, cell line, and time, but secretion of TGF-ß3 did not vary significantly over time. In conclusion, SCAP response is compromised when exposed to bacterial stimuli from infected dental root canals in anaerobic conditions. Thus, stem cell-mediated endodontic regenerative studies need to include microenvironmental conditions, such as the presence of microorganisms to promote further advantage in the field.

Smad7 Enhances TGF-ß-Induced Transcription of c-Jun and HDAC6 Promoting Invasion of Prostate Cancer Cells.

Transforming growth factor ß (TGF-ß) enhances migration and invasion of cancer cells, causing life-threatening metastasis. Smad7 expression is induced by TGF-ß to control TGF-ß signaling in a negative feedback manner. Here we report an additional function of Smad7, i.e., to enhance TGF-ß induction of c-Jun and HDAC6 via binding to their regulatory regions, promoting migration and invasion of prostate cancer cells. Lysine 102 in Smad7 is crucial for binding to specific consensus sites in c-Jun and HDAC6, even when endogenous Smad2, 3, and 4 were silenced by siRNA. A correlation between the mRNA expression of Smad7 and HDAC6, Smad7 and c-Jun, and c-Jun and HDAC6 was found in public databases from analyses of prostate cancer tissues. High expression of Smad7, HDAC6, and c-Jun correlated with poor prognosis for patients with prostate cancer. The knowledge that Smad7 can activate transcription of proinvasive genes leading to prostate cancer progression provides clinically relevant information.

Fluorophore-conjugated Helicobacter pylori recombinant membrane protein (HopQ) labels primary colon cancer and metastases in orthotopic mouse models by binding CEA-related cell adhesion molecules.

HopQ is an outer-membrane protein of Helicobacter pylori that binds to human carcinoembryonic antigen-related cell-adhesion molecules (CEACAMs) with high specificity. We aimed to investigate fluorescence targeting of CEACAM-expressing colorectal tumors in patient-derived orthotopic xenograft (PDOX) models with fluorescently labeled recombinant HopQ (rHopQ). Western blotting, flow cytometry and ELISA were performed to determine the efficiency of rHopQ binding to CEACAMs. rHopQ was conjugated to IR800DyeCW (rHopQ-IR800). Nude mice received orthotopic implantation of colon cancer tumors. Three weeks later, mice were administered 25 µg or 50 µg HopQ-IR800 and imaged 24 or 48 h later. Intravital images were analyzed for tumor-to-background ratio (TBR). Flow cytometry and ELISA demonstrated binding of HopQ to CEACAM1, 3 and 5. Dose-response intravital imaging in PDOX models demonstrated optimal results 48 h after administration of 50 µg rHopQ-IR800 (TBR = 3.576) in our protocol. Orthotopic models demonstrated clear tumor margins of primary tumors and small regional metastases with a mean TBR = 3.678 (SD ±â€¯1.027). rHopQ showed specific binding to various CEACAMs in PDOX models. rHopQ may be useful for CEACAM-positive tumor and metastasis detection for pre-surgical diagnosis, intra-operative imaging and fluorescence-guided surgery.