Resistance of HNSCC cell models to pan-FGFR inhibition depends on the EMT phenotype associating with clinical outcome.

BACKGROUND: Focal adhesion signaling involving receptor tyrosine kinases (RTK) and integrins co-controls cancer cell survival and therapy resistance. However, co-dependencies between these receptors and therapeutically exploitable vulnerabilities remain largely elusive in HPV-negative head and neck squamous cell carcinoma (HNSCC). METHODS: The cytotoxic and radiochemosensitizing potential of targeting 10 RTK and ß1 integrin was determined in up to 20 3D matrix-grown HNSCC cell models followed by drug screening and patient-derived organoid validation. RNA sequencing and protein-based biochemical assays were performed for molecular characterization. Bioinformatically identified transcriptomic signatures were applied to patient cohorts. RESULTS: Fibroblast growth factor receptor (FGFR 1-4) targeting exhibited the strongest cytotoxic and radiosensitizing effects as monotherapy and combined with ß1 integrin inhibition, exceeding the efficacy of the other RTK studied. Pharmacological pan-FGFR inhibition elicited responses ranging from cytotoxicity/radiochemosensitization to resistance/radiation protection. RNA sequence analysis revealed a mesenchymal-to-epithelial transition (MET) in sensitive cell models, whereas resistant cell models exhibited a partial epithelial-to-mesenchymal transition (EMT). Accordingly, inhibition of EMT-associated kinases such as EGFR caused reduced adaptive resistance and enhanced (radio)sensitization to FGFR inhibition cell model- and organoid-dependently. Transferring the EMT-associated transcriptomic profiles to HNSCC patient cohorts not only demonstrated their prognostic value but also provided a conclusive validation of the presence of EGFR-related vulnerabilities that can be strategically exploited for therapeutic interventions. CONCLUSIONS: This study demonstrates that pan-FGFR inhibition elicits a beneficial radiochemosensitizing and a detrimental radioprotective potential in HNSCC cell models. Adaptive EMT-associated resistance appears to be of clinical importance, and we provide effective molecular approaches to exploit this therapeutically.

A transcriptomic pan-cancer signature for survival prognostication and prediction of immunotherapy response based on endothelial senescence.

BACKGROUND: The microvascular endothelium inherently controls nutrient delivery, oxygen supply, and immune surveillance of malignant tumors, thus representing both biological prerequisite and therapeutic vulnerability in cancer. Recently, cellular senescence emerged as a fundamental characteristic of solid malignancies. In particular, tumor endothelial cells have been reported to acquire a senescence-associated secretory phenotype, which is characterized by a pro-inflammatory transcriptional program, eventually promoting tumor growth and formation of distant metastases. We therefore hypothesize that senescence of tumor endothelial cells (TEC) represents a promising target for survival prognostication and prediction of immunotherapy efficacy in precision oncology. METHODS: Published single-cell RNA sequencing datasets of different cancer entities were analyzed for cell-specific senescence, before generating a pan-cancer endothelial senescence-related transcriptomic signature termed EC.SENESCENCE.SIG. Utilizing this signature, machine learning algorithms were employed to construct survival prognostication and immunotherapy response prediction models. Machine learning-based feature selection algorithms were applied to select key genes as prognostic biomarkers. RESULTS: Our analyses in published transcriptomic datasets indicate that in a variety of cancers, endothelial cells exhibit the highest cellular senescence as compared to tumor cells or other cells in the vascular compartment of malignant tumors. Based on these findings, we developed a TEC-associated, senescence-related transcriptomic signature (EC.SENESCENCE.SIG) that positively correlates with pro-tumorigenic signaling, tumor-promoting dysbalance of immune cell responses, and impaired patient survival across multiple cancer entities. Combining clinical patient data with a risk score computed from EC.SENESCENCE.SIG, a nomogram model was constructed that enhanced the accuracy of clinical survival prognostication. Towards clinical application, we identified three genes as pan-cancer biomarkers for survival probability estimation. As therapeutic perspective, a machine learning model constructed on EC.SENESCENCE.SIG provided superior pan-cancer prediction for immunotherapy response than previously published transcriptomic models. CONCLUSIONS: We here established a pan-cancer transcriptomic signature for survival prognostication and prediction of immunotherapy response based on endothelial senescence.

5'-Ectonucleotidase CD73/NT5E supports EGFR-mediated invasion of HPV-negative head and neck carcinoma cells.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) of malignant cells is a driving force of disease progression in human papillomavirus-negative (HPV-negative) head and neck squamous cell carcinomas (HNSCC). Sustained hyper-activation of epidermal growth factor receptor (EGFR) induces an invasion-promoting subtype of EMT (EGFR-EMT) characterized by a gene signature ("'EGFR-EMT_Signature'") comprising 5´-ectonucleotidase CD73. Generally, CD73 promotes immune evasion via adenosine (ADO) formation and associates with EMT and metastases. However, CD73 regulation through EGFR signaling remains under-explored and targeting options are amiss. METHODS: CD73 functions in EGFR-mediated tumor cell dissemination were addressed in 2D and 3D cellular models of migration and invasion. The novel antagonizing antibody 22E6 and therapeutic antibody Cetuximab served as inhibitors of CD73 and EGFR, respectively, in combinatorial treatment. Specificity for CD73 and its role as effector or regulator of EGFR-EMT were assessed upon CD73 knock-down and over-expression. CD73 correlation to tumor budding was studied in an in-house primary HNSCC cohort. Expression correlations, and prognostic and predictive values were analyzed using machine learning-based algorithms and Kaplan-Meier survival curves in single cell and bulk RNA sequencing datasets. RESULTS: CD73/NT5E is induced by the EGF/EGFR-EMT-axis and blocked by Cetuximab and MEK inhibitor. Inhibition of CD73 with the novel antagonizing antibody 22E6 specifically repressed EGFR-dependent migration and invasion of HNSCC cells in 2D. Cetuximab and 22E6 alone reduced local invasion in a 3D-model. Interestingly, combining inefficient low-dose concentrations of Cetuximab and 22E6 revealed highly potent in invasion inhibition, substantially reducing the functional IC50 of Cetuximab regarding local invasion. A role for CD73 as an effector of EGFR-EMT in local invasion was further supported by knock-down and over-expression experiments in vitro and by high expression in malignant cells budding from primary tumors. CD73 expression correlated with EGFR pathway activity, EMT, and partial EMT (p-EMT) in malignant single HNSCC cells and in large patient cohorts. Contrary to published data, CD73 was not a prognostic marker of overall survival (OS) in the TCGA-HNSCC cohort when patients were stratified for HPV-status. However, CD73 prognosticated OS of oral cavity carcinomas. Furthermore, CD73 expression levels correlated with response to Cetuximab in HPV-negative advanced, metastasized HNSCC patients. CONCLUSIONS: In sum, CD73 is an effector of EGF/EGFR-mediated local invasion and a potential therapeutic target and candidate predictive marker for advanced HPV-negative HNSCC.

A transcriptomic map of EGFR-induced epithelial-to-mesenchymal transition identifies prognostic and therapeutic targets for head and neck cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) is both a driver oncogene and a therapeutic target in advanced head and neck squamous cell carcinoma (HNSCC). However, response to EGFR treatment is inconsistent and lacks markers for treatment prediction. This study investigated EGFR-induced epithelial-to-mesenchymal transition (EMT) as a central parameter in tumor progression and identified novel prognostic and therapeutic targets, and a candidate predictive marker for EGFR therapy response. METHODS: Transcriptomic profiles were analyzed by RNA sequencing (RNA-seq) following EGFR-mediated EMT in responsive human HNSCC cell lines. Exclusive genes were extracted via differentially expressed genes (DEGs) and a risk score was determined through forward feature selection and Cox regression models in HNSCC cohorts. Functional characterization of selected prognostic genes was conducted in 2D and 3D cellular models, and findings were validated by immunohistochemistry in primary HNSCC. RESULTS: An EGFR-mediated EMT gene signature composed of n = 171 genes was identified in responsive cell lines and transferred to the TCGA-HNSCC cohort. A 5-gene risk score comprising DDIT4, FADD, ITGB4, NCEH1, and TIMP1 prognosticated overall survival (OS) in TCGA and was confirmed in independent HNSCC cohorts. The EGFR-mediated EMT signature was distinct from EMT hallmark and partial EMT (pEMT) meta-programs with a differing enrichment pattern in single malignant cells. Molecular characterization showed that ITGB4 was upregulated in primary tumors and metastases compared to normal mucosa and correlated with EGFR/MAPK activity in tumor bulk and single malignant cells. Preferential localization of ITGB4 together with its ligand laminin 5 at tumor-stroma interfaces correlated with increased tumor budding in primary HNSCC tissue sections. In vitro, ITGB4 knock-down reduced EGFR-mediated migration and invasion and ITGB4-antagonizing antibody ASC8 impaired 2D and 3D invasion. Furthermore, a logistic regression model defined ITGB4 as a predictive marker of progression-free survival in response to Cetuximab in recurrent metastatic HNSCC patients. CONCLUSIONS: EGFR-mediated EMT conveyed through MAPK activation contributes to HNSCC progression upon induction of migration and invasion. A 5-gene risk score based on a novel EGFR-mediated EMT signature prognosticated survival of HNSCC patients and determined ITGB4 as potential therapeutic and predictive target in patients with strong EGFR-mediated EMT.

Liquid BIOpsy for MiNimal RESidual DiSease Detection in Head and Neck Squamous Cell Carcinoma (LIONESS)-a personalised circulating tumour DNA analysis in head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) remain a substantial burden to global health. Cell-free circulating tumour DNA (ctDNA) is an emerging biomarker but has not been studied sufficiently in HNSCC. METHODS: We conducted a single-centre prospective cohort study to investigate ctDNA in patients with p16-negative HNSCC who received curative-intent primary surgical treatment. Whole-exome sequencing was performed on formalin-fixed paraffin-embedded (FFPE) tumour tissue. We utilised RaDaRTM, a highly sensitive personalised assay using deep sequencing for tumour-specific variants, to analyse serial pre- and post-operative plasma samples for evidence of minimal residual disease and recurrence. RESULTS: In 17 patients analysed, personalised panels were designed to detect 34 to 52 somatic variants. Data show ctDNA detection in baseline samples taken prior to surgery in 17 of 17 patients. In post-surgery samples, ctDNA could be detected at levels as low as 0.0006% variant allele frequency. In all cases with clinical recurrence to date, ctDNA was detected prior to progression, with lead times ranging from 108 to 253 days. CONCLUSIONS: This study illustrates the potential of ctDNA as a biomarker for detecting minimal residual disease and recurrence in HNSCC and demonstrates the feasibility of personalised ctDNA assays for the detection of disease prior to clinical recurrence.

Expression and function of epithelial cell adhesion molecule EpCAM: where are we after 40 years?

EpCAM (epithelial cell adhesion molecule) was discovered four decades ago as a tumor antigen on colorectal carcinomas. Owing to its frequent and high expression on carcinomas and their metastases, EpCAM serves as a prognostic marker, a therapeutic target, and an anchor molecule on circulating and disseminated tumor cells (CTCs/DTCs), which are considered the major source for metastatic cancer cells. Today, EpCAM is reckoned as a multi-functional transmembrane protein involved in the regulation of cell adhesion, proliferation, migration, stemness, and epithelial-to-mesenchymal transition (EMT) of carcinoma cells. To fulfill these functions, EpCAM is instrumental in intra- and intercellular signaling as a full-length molecule and following regulated intramembrane proteolysis, generating functionally active extra- and intracellular fragments. Intact EpCAM and its proteolytic fragments interact with claudins, CD44, E-cadherin, epidermal growth factor receptor (EGFR), and intracellular signaling components of the WNT and Ras/Raf pathways, respectively. This plethora of functions contributes to shaping intratumor heterogeneity and partial EMT, which are major determinants of the clinical outcome of carcinoma patients. EpCAM represents a marker for the epithelial status of primary and systemic tumor cells and emerges as a measure for the metastatic capacity of CTCs. Consequentially, EpCAM has reclaimed potential as a prognostic marker and target on primary and systemic tumor cells.

EpCAM ectodomain EpEX is a ligand of EGFR that counteracts EGF-mediated epithelial-mesenchymal transition through modulation of phospho-ERK1/2 in head and neck cancers.

Head and neck squamous cell carcinomas (HNSCCs) are characterized by outstanding molecular heterogeneity that results in severe therapy resistance and poor clinical outcome. Inter- and intratumoral heterogeneity in epithelial-mesenchymal transition (EMT) was recently revealed as a major parameter of poor clinical outcome. Here, we addressed the expression and function of the therapeutic target epidermal growth factor receptor (EGFR) and of the major determinant of epithelial differentiation epithelial cell adhesion molecule (EpCAM) in clinical samples and in vitro models of HNSCCs. We describe improved survival of EGFRlow/EpCAMhigh HNSCC patients (n = 180) and provide a molecular basis for the observed disparities in clinical outcome. EGF/EGFR have concentration-dependent dual capacities as inducers of proliferation and EMT through differential activation of the central molecular switch phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) and EMT transcription factors (EMT-TFs) Snail, zinc finger E-box-binding homeobox 1 (Zeb1), and Slug. Furthermore, soluble ectodomain of EpCAM (EpEX) was identified as a ligand of EGFR that activates pERK1/2 and phosphorylated AKT (pAKT) and induces EGFR-dependent proliferation but represses EGF-mediated EMT, Snail, Zeb1, and Slug activation and cell migration. EMT repression by EpEX is realized through competitive modulation of pERK1/2 activation strength and inhibition of EMT-TFs, which is reflected in levels of pERK1/2 and its target Slug in clinical samples. Accordingly, high expression of pERK1/2 and/or Slug predicted poor outcome of HNSCCs. Hence, EpEX is a ligand of EGFR that induces proliferation but counteracts EMT mediated by the EGF/EGFR/pERK1/2 axis. Therefore, the emerging EGFR/EpCAM molecular cross talk represents a promising target to improve patient-tailored adjuvant treatment of HNSCCs.

Safety and Efficacy of Blue Light Laser Treatment in Hereditary Hemorrhagic Telangiectasia.

BACKGROUND AND OBJECTIVES: Hereditary hemorrhagic telangiectasia (HHT) is a hereditary condition that is associated with arteriovenous malformations. A common site for these malformations is the nasal mucosa, which is associated with severe epistaxis and debilitation for affected patients. We evaluated the efficacy and safety of blue light laser technology in treating these endonasal manifestations in a retrospective chart analysis. Additionally, we compared blue light laser technology to bipolar coagulation in an animal model. STUDY DESIGN/MATERIALS AND METHODS: We performed a retrospective chart analysis of all patients that were diagnosed with HHT and received endonasal blue light laser treatment between 10/2017 and 04/2019. In addition, we performed bipolar or blue light laser coagulation of all macroscopically visible vessels on thyroid gland lobes (n = 4) from Dunkin-Hartley Guinea Pigs. Hematoxylin-eosin (HE) staining was then used to visualize depth and area of coagulation surrounding these vessels. RESULTS: One hundred and fifty-one treatments in 23 patients were analyzed. Under regular blue light laser treatment, quality of life (QOL), indicated on a visual analog scale from 1 to 10, gradually increased significantly from 5.6 ± 0.5 (before the first treatment) to 7.5 ± 0.9 (after the second treatment). Following this, QOL remained steady throughout additional treatments. Adverse effects were not recorded. HE staining showed that coagulation depth (162 ± 56 vs. 586 ± 192 µm) and area (74 ± 35 vs. 1015 ± 449 µm2 ) were significantly lower after laser treatment. CONCLUSION: Blue light laser therapy is safe and efficient in treating HHT. Damage to the surrounding tissue is significantly lower compared with bipolar coagulation. © 2020 The Authors. Lasers in Surgery and Medicine published by Wiley Periodicals LLC.

Membrane-associated epithelial cell adhesion molecule is slowly cleaved by γ-secretase prior to efficient proteasomal degradation of its intracellular domain.

Regulated intramembrane proteolysis (RIP) is a key mechanism for activating transmembrane proteins such as epithelial cell adhesion molecule (EpCAM) for cellular signaling and degradation. EpCAM is highly expressed in carcinomas and progenitor and embryonic stem cells and is involved in the regulation of cell adhesion, proliferation, and differentiation. Strictly sequential cleavage of EpCAM through RIP involves initial shedding of the extracellular domain by α-secretase (ADAM) and ß-secretase (BACE) sheddases, generating a membrane-tethered C-terminal fragment EpCTF. Subsequently, the rate-limiting γ-secretase complex catalyzes intramembrane cleavage of EpCTF, generating an extracellular EpCAM-Aß-like fragment and an intracellular EpICD fragment involved in nuclear signaling. Here, we have combined biochemical approaches with live-cell imaging of fluorescent protein tags to investigate the kinetics of γ-secretase-mediated intramembrane cleavage of EpCTF. We demonstrate that γ-secretase-mediated proteolysis of exogenously and endogenously expressed EpCTF is a slow process with a 50% protein turnover in cells ranging from 45 min to 5.5 h. The slow cleavage was dictated by γ-secretase activity and not by EpCTF species, as indicated by cross-species swapping experiments. Furthermore, both human and murine EpICDs generated from EpCTF by γ-secretase were degraded efficiently (94-99%) by the proteasome. Hence, proteolytic cleavage of EpCTF is a comparably slow process, and EpICD generation does not appear to be suited for rapidly transducing extracellular cues into nuclear signaling, but appears to provide steady signals that can be further controlled through efficient proteasomal degradation. Our approach provides an unbiased bioassay to investigate proteolytic processing of EpCTF in single living cells.

A high-content screen for small-molecule regulators of epithelial cell-adhesion molecule (EpCAM) cleavage yields a robust inhibitor.

Epithelial cell-adhesion molecule (EpCAM) is a transmembrane protein that regulates cell cycle progression and differentiation and is overexpressed in many carcinomas. The EpCAM-induced mitogenic cascade is activated via regulated intramembrane proteolysis (RIP) of EpCAM by ADAM and γ-secretases, generating the signaling-active intracellular domain EpICD. Because of its expression pattern and molecular function, EpCAM is a valuable target in prognostic and therapeutic approaches for various carcinomas. So far, several immunotherapeutic strategies have targeted the extracellular domain of EpCAM. However, targeting the intracellular signaling cascade of EpCAM holds promise for specifically interfering with EpCAM's proliferation-stimulating signaling cascade. Here, using a yellow fluorescence protein-tagged version of the C-terminal fragment of EpCAM, we established a high-content screening (HCS) of a small-molecule compound library (n = 27,280) and characterized validated hits that target EpCAM signaling. In total, 128 potential inhibitors were initially identified, of which one compound with robust inhibitory effects on RIP of EpCAM was analyzed in greater detail. In summary, our study demonstrates that the development of an HCS for small-molecule inhibitors of the EpCAM signaling pathway is feasible. We propose that this approach may also be useful for identifying chemical compounds targeting other disorders involving membrane cleavage-dependent signaling pathways.

Cleavage and cell adhesion properties of human epithelial cell adhesion molecule (HEPCAM).

Human epithelial cell adhesion molecule (HEPCAM) is a tumor-associated antigen frequently expressed in carcinomas, which promotes proliferation after regulated intramembrane proteolysis. Here, we describe extracellular shedding of HEPCAM at two α-sites through a disintegrin and metalloprotease (ADAM) and at one ß-site through BACE1. Transmembrane cleavage by γ-secretase occurs at three γ-sites to generate extracellular Aß-like fragments and at two ϵ-sites to release human EPCAM intracellular domain HEPICD, which is efficiently degraded by the proteasome. Mapping of cleavage sites onto three-dimensional structures of HEPEX cis-dimer predicted conditional availability of α- and ß-sites. Endocytosis of HEPCAM warrants acidification in cytoplasmic vesicles to dissociate protein cis-dimers required for cleavage by BACE1 at low pH values. Intramembrane cleavage sites are accessible and not part of the structurally important transmembrane helix dimer crossing region. Surprisingly, neither chemical inhibition of cleavage nor cellular knock-out of HEPCAM using CRISPR-Cas9 technology impacted the adhesion of carcinoma cell lines. Hence, a direct function of HEPCAM as an adhesion molecule in carcinoma cells is not supported and appears to be questionable.

EpCAM and the biology of hepatic stem/progenitor cells.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein, which is frequently and highly expressed on carcinomas, tumor-initiating cells, selected tissue progenitors, and embryonic and adult stem cells. During liver development, EpCAM demonstrates a dynamic expression, since it can be detected in fetal liver, including cells of the parenchyma, whereas mature hepatocytes are devoid of EpCAM. Liver regeneration is associated with a population of EpCAM-positive cells within ductular reactions, which gradually lose the expression of EpCAM along with maturation into hepatocytes. EpCAM can be switched on and off through a wide panel of strategies to fine-tune EpCAM-dependent functional and differentiative traits. EpCAM-associated functions relate to cell-cell adhesion, proliferation, maintenance of a pluripotent state, regulation of differentiation, migration, and invasion. These functions can be conferred by the full-length protein and/or EpCAM-derived fragments, which are generated upon regulated intramembrane proteolysis. Control by EpCAM therefore not only depends on the presence of full-length EpCAM at cellular membranes but also on varying rates of the formation of EpCAM-derived fragments that have their own regulatory properties and on changes in the association of EpCAM with interaction partners. Thus spatiotemporal localization of EpCAM in immature liver progenitors, transit-amplifying cells, and mature liver cells will decisively impact the regulation of EpCAM functions and might be one of the triggers that contributes to the adaptive processes in stem/progenitor cell lineages. This review will summarize EpCAM-related molecular events and how they relate to hepatobiliary differentiation and regeneration.

TKTL1 expression in human malign and benign cell lines.

BACKGROUND: Overexpression of transketolase-like 1 protein TKTL1 in cancer cells has been reported to correlate with enhanced glycolysis and lactic acid production. Furthermore, enhanced TKTL1 expression was put into context with resistance to chemotherapy and ionizing radiation. Here, a panel of human malign and benign cells, which cover a broad range of chemotherapy and radiation resistance as well as reliance on glucose metabolism, was analyzed in vitro for TKTL1 expression. METHODS: 17 malign and three benign cell lines were characterized according to their expression of TKTL1 on the protein level with three commercially available anti-TKTL1 antibodies utilizing immunohistochemistry and Western blot, as well as on mRNA level with three published primer pairs for RT-qPCR. Furthermore, sensitivities to paclitaxel, cisplatin and ionizing radiation were assessed in cell survival assays. Glucose consumption and lactate production were quantified as surrogates for the "Warburg effect". RESULTS: Considerable amounts of tktl1 mRNA and TKTL1 protein were detected only upon stable transfection of the human embryonic kidney cell line HEK293 with an expression plasmid for human TKTL1. Beyond that, weak expression of endogenous tktl1 mRNA was measured in the cell lines JAR and U251. Western blot analysis of JAR and U251 cells did not detect TKTL1 at the expected size of 65 kDa with all three antibodies specific for TKTL1 protein and immunohistochemical staining was observed with antibody JFC12T10 only. All other cell lines tested here revealed expression of tktl1 mRNA below detection limits and were negative for TKTL1 protein. However, in all cell lines including TKTL1-negative HEK293-control cells, antibody JFC12T10 detected multiple proteins with different molecular weights. Importantly, JAR and U251 did neither demonstrate an outstanding production of lactic acid nor increased resistance against chemotherapeutics or to ionizing radiation, respectively. CONCLUSION: Using RT-qPCR and three different antibodies we observed only exceptional occurrence of TKTL1 in a panel of malignant human cell lines in vitro. The presence of TKTL1 was unrelated to either the rate of glucose consumption/lactic acid production or resistance against chemo- and radiotherapy.

Reducing tumor growth and angiogenesis using a triple therapy measured with Contrast-enhanced ultrasound (CEUS).

BACKGROUND: To evaluate the in vivo response by detecting the anti-angiogenic and invasion-inhibiting effects of a triple-combination-therapy in an experimental-small-animal-squamous-cell-carcinoma-model using the "flash-replenishment" (FR) method to assess tissue hemodynamics via contrast-enhanced-ultrasound (CEUS). METHODS: Human hypopharynx-carcinoma-cells were subcutaneously injected into the left flank of 22-female-athymic-nude-rats. After seven days of subcutaneous tumor growth, FR-measurements were performed on each rat. Treatment-group and control-group were treated every day for a period of one week, with the treatment-group receiving solvents containing a triple therapy of Upamostat®, Celecoxib® and Ilomastat® and the control-group solvents only. On day seven, follow-up measurements were performed using the same measurement protocol to assess the effects of the triple therapy. VueBox® was used to quantify the kinetic parameters and additional immunohistochemistry analyses were performed for comparison with and validation of the CEUS results against established methods (Proliferation/Ki-67, vascularization/CD31, apoptosis/caspase3). RESULTS: Compared to the control-group, the treatment-group that received the triple-therapy resulted in a reduction of tumor growth by 48.6% in size. Likewise, the immunohistochemistry results showed significant decreases in tumor proliferation and vascularization in the treatment-group in comparison to the control-group of 26%(p ≤ 0.05) and 32.2%(p ≤ 0.05) respectively. Correspondingly, between the baseline and follow-up measurements, the therapy-group was associated with a significant(p ≤ 0.01) decrease in the relative-Blood-Volume(rBV) in both the whole tumor(wt) and hypervascular tumor(ht) areas (p ≤ 0.01), while the control-group was associated with a significant (p ≤ 0.01) increase of the rBV in the wt area and a non-significant increase (p ≤ 0.16) in the ht area. The mean-transit-time (mTT) of the wt and the ht areas showed a significant increase (p ≤ 0.01) in the follow-up measurements in the therapy group. CONCLUSION: The triple-therapy is feasible and effective in reducing both tumor growth and vascularization. In particular, compared with the placebo-group, the triple-therapy-group resulted in a reduction in tumor growth of 48.6% in size when assessed by CEUS and a significant reduction in the number of vessels in the tumor of 32% as assessed by immunohistochemistry. As the immunohistochemistry supports the CEUS findings, CEUS using the "flash replenishment"(FR) method appears to provide a useful assessment of the anti-angiogenic and invasion-inhibiting effects of a triple combination therapy.

Dynamic EpCAM expression on circulating and disseminating tumor cells: causes and consequences.

Formation of metastasis is the most important and lethal step in cancer progression. Circulating and disseminated cancer cells (CTCs/DTCs) in blood and bone marrow are considered as potential metastases-inducing cells. Their detection and characterization has, therefore, become a field of major interest in translational and clinical research in oncology. The main strategy to detect these cells relies thus far on the epithelial characteristics of carcinoma cells and epithelial cell adhesion molecule (EpCAM) represents the most commonly used epithelial marker to capture CTCs/DTCs. Recent data, however, demonstrated a dynamic expression of EpCAM associated with a loss during epithelial-to-mesenchymal transition. The present review summarizes the potential mechanisms and reasons for a dynamic expression of EpCAM.

Quantification of oxygen consumption in head and neck cancer using fluorescent sensor foil technology.

Introduction: Head and neck squamous cell carcinoma (HNSCC) patients suffer from frequent local recurrences that negatively impact on prognosis. Hence, distinguishing tumor and normal tissue is of clinical importance as it may improve the detection of residual tumor tissue in surgical resection margins and during imaging-based surgery planning. Differences in O2 consumption (OC) can be used to this aim, as they provide options for improved surgical, image-guided approaches. Methods: In the present study, the potential of a fluorescent sensor foil-based technology to quantify OC in HNSCC was evaluated in an in vitro 3D model and in situ in patients. Results: In vitro measurements of OC using hypopharyngeal and esophageal cell lines allowed a specific detection of tumor cell spheroids embedded together with cancer-associated fibroblasts in type I collagen extracellular matrix down to a diameter of 440 µm. Pre-surgery in situ measurements were conducted with a handheld recording device and sensor foils with an oxygen permeable membrane and immobilized O2-reactive fluorescent dyes. Lateral tongue carcinoma and carcinoma of the floor of the mouth were chosen for analysis owing to their facilitated accessibility. OC was evaluated over a time span of 60 seconds and was significantly higher in tumor tissue compared to healthy mucosa in the vicinity of the tumor. Discussion: Hence, OC quantification using fluorescent sensor foil-based technology is a relevant parameter for the differentiation of tumor tissue of the head and neck region and may support surgery planning.

Proteomic Profiling of Serum Extracellular Vesicles Identifies Diagnostic Signatures and Therapeutic Targets in Breast Cancer.

Analysis of extracellular vesicles (EVs) is a promising noninvasive liquid biopsy approach for breast cancer (BC) detection, prognosis, and therapeutic monitoring. A comprehensive understanding of the characteristics and proteomic composition of BC-specific EVs from human samples is required to realize the potential of this strategy. In this study, we applied a mass spectrometry-based, data-independent acquisition (DIA) proteomic approach to characterize human serum EVs derived from patients with BC (n = 126) and healthy donors (HDs, n = 70) in a discovery cohort and validated the findings in five independent cohorts. Examination of the EV proteomes enabled construction of specific EV protein classifiers for diagnosing BC and distinguishing patients with metastatic disease. Of note, TALDO1 was found to be an EV biomarker of distant metastasis of BC. In vitro and in vivo analysis confirmed the role of TALDO1 in stimulating BC invasion and metastasis. Finally, high-throughput molecular docking and virtual screening of a library consisting of 271,380 small molecules identified a potent TALDO1 allosteric inhibitor, AO-022, which could inhibit BC migration in vitro and tumor progression in vivo. Together, this work elucidates the proteomic alterations in the serum EVs of BC patients to guide development of improved diagnosis, monitoring, and treatment strategies.

TSPAN8+ myofibroblastic cancer-associated fibroblasts promote chemoresistance in patients with breast cancer.

Cancer-associated fibroblasts (CAFs) are abundant stromal cells in the tumor microenvironment that promote cancer progression and relapse. However, the heterogeneity and regulatory roles of CAFs underlying chemoresistance remain largely unclear. Here, we performed a single-cell analysis using high-dimensional flow cytometry analysis and identified a distinct senescence-like tetraspanin-8 (TSPAN8)+ myofibroblastic CAF (myCAF) subset, which is correlated with therapeutic resistance and poor survival in multiple cohorts of patients with breast cancer (BC). TSPAN8+ myCAFs potentiate the stemness of the surrounding BC cells through secretion of senescence-associated secretory phenotype (SASP)-related factors IL-6 and IL-8 to counteract chemotherapy. NAD-dependent protein deacetylase sirtuin 6 (SIRT6) reduction was responsible for the senescence-like phenotype and tumor-promoting role of TSPAN8+ myCAFs. Mechanistically, TSPAN8 promoted the phosphorylation of ubiquitin E3 ligase retinoblastoma binding protein 6 (RBBP6) at Ser772 by recruiting MAPK11, thereby inducing SIRT6 protein destruction. In turn, SIRT6 down-regulation up-regulated GLS1 and PYCR1, which caused TSPAN8+ myCAFs to secrete aspartate and proline, and therefore proved a nutritional niche to support BC outgrowth. By demonstrating that TSPAN8+SIRT6low myCAFs were tightly associated with unfavorable disease outcomes, we proposed that the combined regimen of anti-TSPAN8 antibody and SIRT6 activator MDL-800 is a promising approach to overcome chemoresistance. These findings highlight that senescence contributes to CAF heterogeneity and chemoresistance and suggest that targeting TSPAN8+ myCAFs is a promising approach to circumvent chemoresistance.

Metabolic pathway-based subtypes associate glycan biosynthesis and treatment response in head and neck cancer.

Head and Neck Squamous Cell Carcinoma (HNSCC) is a heterogeneous malignancy that remains a significant challenge in clinical management due to frequent treatment failures and pronounced therapy resistance. While metabolic dysregulation appears to be a critical factor in this scenario, comprehensive analyses of the metabolic HNSCC landscape and its impact on clinical outcomes are lacking. This study utilized transcriptomic data from four independent clinical cohorts to investigate metabolic heterogeneity in HNSCC and define metabolic pathway-based subtypes (MPS). In HPV-negative HNSCCs, MPS1 and MPS2 were identified, while MPS3 was enriched in HPV-positive cases. MPS classification was associated with clinical outcome post adjuvant radio(chemo)therapy, with MPS1 consistently exhibiting the highest risk of therapeutic failure. MPS1 was uniquely characterized by upregulation of glycan (particularly chondroitin/dermatan sulfate) metabolism genes. Immunohistochemistry and pilot mass spectrometry imaging analyses confirmed this at metabolite level. The histological context and single-cell RNA sequencing data identified the malignant cells as key contributors. Globally, MPS1 was distinguished by a unique transcriptomic landscape associated with increased disease aggressiveness, featuring motifs related to epithelial-mesenchymal transition, immune signaling, cancer stemness, tumor microenvironment assembly, and oncogenic signaling. This translated into a distinct histological appearance marked by extensive extracellular matrix remodeling, abundant spindle-shaped cancer-associated fibroblasts, and intimately intertwined populations of malignant and stromal cells. Proof-of-concept data from orthotopic xenotransplants replicated the MPS phenotypes on the histological and transcriptome levels. In summary, this study introduces a metabolic pathway-based classification of HNSCC, pinpointing glycan metabolism-enriched MPS1 as the most challenging subgroup that necessitates alternative therapeutic strategies.

CD19-independent instruction of murine marginal zone B-cell development by constitutive Notch2 signaling.

B cell-specific gene ablation of Notch2 results in the loss of the marginal zone (MZ) B-cell lineage. To analyze the effects of constitutive Notch2 signaling in B cells, we have generated a transgenic mouse strain that allows the conditional expression of a constitutively active, intracellular form of Notch2 (Notch2IC). Expression of Notch2IC at the earliest developmental stages of the B-cell lineage completely abolished B-cell generation and led to the development of ectopic T cells in the bone marrow (BM), showing that Notch2IC is acting redundantly with Notch1IC in driving ectopic T-cell differentiation. In B cells clearly committed to the B-cell lineage induction of Notch2IC drove all cells toward the MZ B-cell compartment at the expense of follicular B cells. Notch2IC-expressing B cells reflected the phenotype of wild-type MZ B cells for their localization in the MZ, the expression of characteristic surface markers, their enhanced proliferation after stimulation, and increased basal activity of Akt, Erk, and Jnk. Notch2IC-driven MZ B-cell generation in the spleen was achieved even in the absence of CD19. Our results implicate that a constitutive Notch2 signal in transitional type 1 B cells is sufficient to drive MZ B-cell differentiation.