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Detection of tumor targeting by in vivo imaging (fluorescent intensity, FLI)of CEA TCB formats having bivalent or monovalent binding to CEA and directly labeled with Alexa647.

Supplementary Methods and Figure Legends.

Purpose: CEA TCB (RG7802, RO6958688) is a novel T-cell bispecific antibody, engaging CD3ϵ upon binding to carcinoembryonic antigen (CEA) on tumor cells. Containing an engineered Fc region, conferring an extended blood half-life while preventing side effects due to activation of innate effector cells, CEA TCB potently induces tumor lysis in mouse tumors. Here we aimed to characterize the pharmacokinetic profile, the biodistribution, and the mode of action of CEA TCB by combining in vitro and in vivo fluorescence imaging readouts.Experimental Design: CEA-expressing tumor cells (LS174T) and human peripheral blood mononuclear cells (PBMC) were cocultured in vitro or cografted into immunocompromised mice. Fluorescence reflectance imaging and intravital 2-photon (2P) microscopy were employed to analyze in vivo tumor targeting while in vitro confocal and intravital time-lapse imaging were used to assess the mode of action of CEA TCB.Results: Fluorescence reflectance imaging revealed increased ratios of extravascular to vascular fluorescence signals in tumors after treatment with CEA TCB compared with control antibody, suggesting specific targeting, which was confirmed by intravital microscopy. Confocal and intravital 2P microscopy showed CEA TCB to accelerate T-cell–dependent tumor cell lysis by inducing a local increase of effector to tumor cell ratios and stable crosslinking of multiple T cells to individual tumor cells.Conclusions: Using optical imaging, we demonstrate specific tumor targeting and characterize the mode of CEA TCB–mediated target cell lysis in a mouse tumor model, which supports further clinical evaluation of CEA TCB. Clin Cancer Res; 22(17); 4417–27. ©2016 AACR.See related commentary by Teijeira et al., p. 4277

Pharmacokinetic profiles of CEA TCB in mice, additional information on CD3 signaling, T cell activation, tumor cell lysis and cytokine release upon CEA TCB and untargeted control TCB treatment.

In vitro live cell confocal imaging of LS174T RFP cells co-cultured with CFSE-labeled T-cells (E:T = 3:1) in the presence of CEA or a control TCB, respectively.

Purpose: CEA TCB is a novel IgG-based T-cell bispecific (TCB) antibody for the treatment of CEA-expressing solid tumors currently in phase I clinical trials (NCT02324257). Its format incorporates bivalent binding to CEA, a head-to-tail fusion of CEA- and CD3e-binding Fab domains and an engineered Fc region with completely abolished binding to FcγRs and C1q. The study provides novel mechanistic insights into the activity and mode of action of CEA TCB.Experimental Design: CEA TCB activity was characterized on 110 cell lines in vitro and in xenograft tumor models in vivo using NOG mice engrafted with human peripheral blood mononuclear cells.Results: Simultaneous binding of CEA TCB to tumor and T cells leads to formation of immunologic synapses, T-cell activation, secretion of cytotoxic granules, and tumor cell lysis. CEA TCB activity strongly correlates with CEA expression, with higher potency observed in highly CEA-expressing tumor cells and a threshold of approximately 10,000 CEA-binding sites/cell, which allows distinguishing between high- and low-CEA–expressing tumor and primary epithelial cells, respectively. Genetic factors do not affect CEA TCB activity confirming that CEA expression level is the strongest predictor of CEA TCB activity. In vivo, CEA TCB induces regression of CEA-expressing xenograft tumors with variable amounts of immune cell infiltrate, leads to increased frequency of activated T cells, and converts PD-L1 negative into PD-L1–positive tumors.Conclusions: CEA TCB is a novel generation TCB displaying potent antitumor activity; it is efficacious in poorly infiltrated tumors where it increases T-cell infiltration and generates a highly inflamed tumor microenvironment. Clin Cancer Res; 22(13); 3286–97. ©2016 AACR.

Representative videos of intravital two-photon (2P) microscopy of tumors treated with CEA TCB. Tumor cells red, T cells green.

Supplementary Figure S1. CEA TCB leads stimulates the expression of CD25, a T-cell activation marker, on both T-helper (CD4+) and cytotoxic T-cells (CD8+) after incubation with LS174T tumor cells. Supplementary Figure S2. CEA TCB induces efficient MKN45 tumor cell lysis and T-cell activation in the presence of these tumor cells. Supplementary Figure S3. Both CD4+ and CD8+ T-cells contribute to CEA-TCB mediated killing. Supplementary Figure S4. Ex vivo analysis of in vivo tumor targeting. Supplementary Figure S5. Analysis of T-cell migratory persistence index.

2P time-lapse acquisition of LS174T / human PBMC cografts on day 4 after implantation (baseline), 24 h after control and 24 h after CEA TCB treatment. LS174T RFP tumor cells (red), CFSE labeled T-cells (green). Scale bar indicates 50 μm.

Representative videos of intravital two-photon (2P) microscopy of tumors treated with untargeted TCB. Tumor cells red, T cells green.

List, Source and CEA binding sites of Cell Lines used in the study

Assessment of binding of CEA TCB to human and cynomolgus monkey CEA- and CD3-expressing cells, internalization and ADCC, CDC activity.

Ex vivo analysis of the CEA TCB (magenta) mediated cross-linking of CFSE labeled T-cells (green) to tumor cells. 3D reconstructions of confocal z-stacks acquired on tumor sections obtained from huPBMC (CFSE labeled T-cells)/LS174T RFP co-grafts, 24 h after intravenous A647 labeled CEA TCB injections are shown. Bar, 10 μm. Cell nuclei (DAPI) are shown in blue.

Supplemental Material and Methods describing CEA TCB production and purification process, determination of affinity and avidity, genetic characterization of cell lines, determination of CEA binding sites, assessment of T cell proliferation and activation, Jurkat NFAT assay, confocal microscopy, FRET, SDPK, in vivo efficacy and imaging studies. Supplemental Video Legends are also within the file.

Extracellular vesicles (EVs), nanovesicles released by cells to effectively exchange biological information, are gaining interest as drug delivery system. Yet, analogously to liposomes, they show short blood circulation times and accumulation in the liver and the spleen. For tissue specific delivery, EV surfaces will thus have to be functionalized. We present a novel platform for flexible modification of EVs with target-specific ligands based on the avidin-biotin system. Genetic engineering of donor cells with a glycosylphosphatidylinositol-anchored avidin (GPI-Av) construct allows the isolation of EVs displaying avidin on their surface, functionalized with any biotinylated ligand. For proof of concept, GPI-Av EVs were modified with i) a biotinylated antibody or ii) de novo designed and synthesized biotinylated ligands binding carbonic anhydrase IX (CAIX), a membrane associated enzyme overexpressed in cancer. Functionalized EVs showed specific binding and uptake by CAIX-expressing cells, demonstrating the power of the system to prepare EVs for cell-specific drug delivery.

Purpose: CEA TCB (RG7802, RO6958688) is a novel T-cell bispecific antibody, engaging CD3ϵ upon binding to carcinoembryonic antigen (CEA) on tumor cells. Containing an engineered Fc region, conferring an extended blood half-life while preventing side effects due to activation of innate effector cells, CEA TCB potently induces tumor lysis in mouse tumors. Here we aimed to characterize the pharmacokinetic profile, the biodistribution, and the mode of action of CEA TCB by combining in vitro and in vivo fluorescence imaging readouts. Experimental Design: CEA-expressing tumor cells (LS174T) and human peripheral blood mononuclear cells (PBMC) were cocultured in vitro or cografted into immunocompromised mice. Fluorescence reflectance imaging and intravital 2-photon (2P) microscopy were employed to analyze in vivo tumor targeting while in vitro confocal and intravital time-lapse imaging were used to assess the mode of action of CEA TCB. Results: Fluorescence reflectance imaging revealed increased ratios of extravascular to vascular fluorescence signals in tumors after treatment with CEA TCB compared with control antibody, suggesting specific targeting, which was confirmed by intravital microscopy. Confocal and intravital 2P microscopy showed CEA TCB to accelerate T-cell–dependent tumor cell lysis by inducing a local increase of effector to tumor cell ratios and stable crosslinking of multiple T cells to individual tumor cells. Conclusions: Using optical imaging, we demonstrate specific tumor targeting and characterize the mode of CEA TCB–mediated target cell lysis in a mouse tumor model, which supports further clinical evaluation of CEA TCB. Clin Cancer Res; 22(17); 4417–27. ©2016 AACR. See related commentary by Teijeira et al., p. 4277

Zusammenfassung80% der Schüler berichten über Kopfschmerzen im Laufe von 90 Tagen. Eine frühe Erkrankung an häufigen Kopfschmerzen bedingt eine höhere Wahrscheinlichkeit auch später im Erwachsenenalter an Kopfschmerzen zu leiden. Es ist deshalb sinnvoll, über Möglichkeiten einer Prävention von Kopfschmerzen im Schulalter nachzudenken. In der vorliegenden Übersicht werden die Ergebnisse von Studien, die die Umsetzbarkeit von meist psychologischen Interventionen wie Entspannungsübungen, Verhaltenstherapie oder Psychoedukation über Kopfschmerzen untersuchten, vorgestellt. Meist relativ kleine Studien weisen darauf hin, dass internetbasierte Verfahren oder Programme, die vorwiegend auf Selbststudium basieren, zu einer signifikanten Besserung führen. Vor diesem Hintergrund führten wir eine Studie zu dem Einfluss einer einstündigen Psychoedukation über Risikofaktoren, den Zusammenhang zu Muskelspannung der peri - kraniellen Muskulatur mit Demonstration von Dehnübungen und Wachheit gegenüber Stressoren durch. Diese bei 12 bis 19 Jahre alten Schülern durchgeführte Studie konnte zeigen, dass niedrigschwellige Intervention zur signifikanten Reduktion von Kopfschmerzen in der Gruppe führte, ein auch noch nach 7 Monaten nachweisbarer Effekt. Der Ansatz, über eine Gesundheitsaufklärung in der Schule, eine nachhaltige Besserung der Kopfschmerzen zu erreichen, wird mit unseren aktuellen Daten unterstützt.

Aim The aim of the present analysis is to confirm or refute the association of neck pain to migraine or tension-type headache and to assess whether this association is independent of other risk factors for headache. Methods Secondary school students were invited to complete a questionnaire on headache and lifestyle factors in a cross-sectional study. Neck pain was assessed via (a) a screening question concerning neck pain and (b) denoting affected areas in schematic drawings of the human body. Results Absolute increment in prevalence of headache with pain in the shoulder-neck region was between 7.5% and 9.6%. Gender, grade, stress and lifestyle factors were assessed as potential confounding factors. Nearly all factors were associated with shoulder-neck pain and most with headache. After adjustment for confounders, the association of neck pain with headache was almost completely confined to migraine (OR 2.39; 95% CI 1.48–3.85) and migraine + tension-type headache (OR 2.12; 95% CI 1.50–2.99), whereas the association with isolated tension-type headache was negligible (OR 1.22, 95% CI 0.87–1.69). Conclusion Neck pain is associated with migraine but not with tension-type headache. A possible link between migraine and neck pain may be the cervico-trigeminal convergence of neck and meningeal sensory afferents or a disturbed descending inhibition in migraine.

Contains fulltext : 170562.pdf (Publisher’s version ) (Closed access) Cancer metastases arise from a multi-step process that requires metastasizing tumor cells to adapt to signaling input from varying tissue environments [1]. As an early metastatic event, cancer cell dissemination occurs through different migration programs, including multicellular, collective, and single-cell mesenchymal or amoeboid migration [2-4]. Migration modes can interconvert based on changes in cell adhesion, cytoskeletal mechanotransduction [5], and/or proteolysis [6], most likely under the control of transcriptional programs such as the epithelial-to-mesenchymal transition (EMT) [7, 8]. However, how plasticity of tumor cell migration and EMT is spatiotemporally controlled and connected upon challenge by the tumor microenvironment remains unclear. Using 3D cultures of collectively invading breast and head and neck cancer spheroids, here we identify hypoxia, a hallmark of solid tumors [9], as an inducer of the collective-to-amoeboid transition (CAT), promoting the dissemination of amoeboid-moving single cells from collective invasion strands. Hypoxia-induced amoeboid detachment was driven by hypoxia-inducible factor 1 (HIF-1), followed the downregulation of E-cadherin, and produced heterogeneous cell subsets whose phenotype and migration were dependent ( approximately 30%) or independent ( approximately 70%) of Twist-mediated EMT. EMT-like and EMT-independent amoeboid cell subsets showed stable amoeboid movement over hours as well as leukocyte-like traits, including rounded morphology, matrix metalloproteinase (MMP)-independent migration, and nuclear deformation. Cancer cells undergoing pharmacological stabilization of HIFs retained their constitutive ability for early metastatic seeding in an experimental model of lung metastasis, indicating that hypoxia-induced CAT enhances cell release rather than early organ colonization. Induced by metabolic challenge, amoeboid movement may thus constitute a common endpoint of both EMT-dependent and EMT-independent cancer dissemination programs. 9 p.

The complexity of the tumor microenvironment is difficult to mimic in vitro, particularly regarding tumor–host interactions. To enable better assessment of cancer immunotherapy agents in vitro, we developed a three-dimensional (3D) heterotypic spheroid model composed of tumor cells, fibroblasts, and immune cells. Drug targeting, efficient stimulation of immune cell infiltration, and specific elimination of tumor or fibroblast spheroid areas were demonstrated following treatment with a novel immunocytokine (interleukin-2 variant; IgG-IL2v) and tumor- or fibroblast-targeted T cell bispecific antibody (TCB). Following treatment with IgG-IL2v, activation of T cells, NK cells, and NKT cells was demonstrated by increased expression of the activation marker CD69 and enhanced cytokine secretion. The combination of TCBs with IgG-IL2v molecules was more effective than monotherapy, as shown by enhanced effects on immune cell infiltration; activation; increased cytokine secretion; and faster, more efficient elimination of targeted cells. This study demonstrates that the 3D heterotypic spheroid model provides a novel and versatile tool for in vitro evaluation of cancer immunotherapy agents and allows for assessment of additional aspects of the activity of cancer immunotherapy agents, including analysis of immune cell infiltration and drug targeting., Cancer Immunology, Immunotherapy, 66 (1), ISSN:0340-7004, ISSN:1432-0851

Background The identified preventable risk factors for primary headache in adolescents are smoking; consumption of coffee or alcoholic mixed drinks; physical inactivity; muscle pain in the head, neck, or shoulder region; and chronic stress. Objective To investigate the interrelation of headache with other health complaints and the specificity of the above-mentioned risk factors for headache in adolescents. Methods A total of 1,260 students (grades 10 and 11) filled in questionnaires on headache, dietary, and lifestyle factors. The type of headache and health complaints such as dizziness, abdominal pain, musculoskeletal pains, symptoms of possible fatigue syndrome, and psychic complaints were assessed. Results Isolated headache was found in 18% of the headache sufferers; most frequently isolated tension-type headache (78.2%). Only among adolescents with a combination of headache (mainly migraine) and other health complaints, significant associations for almost all analyzed risk factors were found. The strength of the associations with the considered risk factors was very similar in all three analyzed strata except for considerably lower odds ratios for isolated headache. Conclusion All analyzed risk factors are nonspecific for headache in adolescents because they also increase the risk for other health complaints. Interventions, therefore, should consider a holistic approach focusing not only on headache but also on a broader spectrum of health complaints.

Introduction Several risk factors for headache have been identified, some of which are potentially amenable to interventions. The potential effect of such interventions can be predicted by the population-attributable risk fraction (PARF). We assessed PARFs of the the following risk factors: neck muscle pain, chronic stress, alcohol consumption, smoking, coffee consumption, and physical inactivity. We studied the maximal possible effect achievable by avoidance of these risk factors. Methods Two approaches to estimate PARFs are compared, which assess their cumulative and individual impact of risk factors by age: the Levin formula and the average attributable fraction. Results The overall impact for removal of all six risk factors amounts to 19.7% for the average attributable fraction. Neck tension and consumption of alcohol ranked as the strongest population-attributable risk factor for any headache. The potential impact for migraine was considerably higher (43.8%). With increasing age, the overall impact of risk factors on headache increases by 18.9%. Conclusion Based on the estimations of the most appropriate approach, up to 20% of headaches in general and up to 43% of migraine in adolescents might be preventable by removing risk factors amenable to intervention, with increasing proportions by age.

Purpose: CEA TCB is a novel IgG-based T-cell bispecific (TCB) antibody for the treatment of CEA-expressing solid tumors currently in phase I clinical trials (NCT02324257). Its format incorporates bivalent binding to CEA, a head-to-tail fusion of CEA- and CD3e-binding Fab domains and an engineered Fc region with completely abolished binding to FcγRs and C1q. The study provides novel mechanistic insights into the activity and mode of action of CEA TCB. Experimental Design: CEA TCB activity was characterized on 110 cell lines in vitro and in xenograft tumor models in vivo using NOG mice engrafted with human peripheral blood mononuclear cells. Results: Simultaneous binding of CEA TCB to tumor and T cells leads to formation of immunologic synapses, T-cell activation, secretion of cytotoxic granules, and tumor cell lysis. CEA TCB activity strongly correlates with CEA expression, with higher potency observed in highly CEA-expressing tumor cells and a threshold of approximately 10,000 CEA-binding sites/cell, which allows distinguishing between high- and low-CEA–expressing tumor and primary epithelial cells, respectively. Genetic factors do not affect CEA TCB activity confirming that CEA expression level is the strongest predictor of CEA TCB activity. In vivo, CEA TCB induces regression of CEA-expressing xenograft tumors with variable amounts of immune cell infiltrate, leads to increased frequency of activated T cells, and converts PD-L1 negative into PD-L1–positive tumors. Conclusions: CEA TCB is a novel generation TCB displaying potent antitumor activity; it is efficacious in poorly infiltrated tumors where it increases T-cell infiltration and generates a highly inflamed tumor microenvironment. Clin Cancer Res; 22(13); 3286–97. ©2016 AACR.

MRI is a method of choice for assessing anatomical structures or angiogenesis-related parameters noninvasively during tumor progression. Typically, tumor tissue displays a high degree of heterogeneity that can be evaluated using pattern analysis (PA), which comprises shape and texture analysis. This work aims at implementing PA methods to study angiogenesis in a murine tumor model and testing its sensitivity with regard to detecting changes elicited by administration of a drug. Twelve balb/c-nude mice were injected subcutaneously with 10(6) C51 cells (colon carcinoma). A first group (N = 6) of animals was treated with dimethyloxalylglycine, a drug known to stabilize hypoxia-inducible-factor-α, which among other functions, is involved in angiogenesis. The second group (N = 6) was treated with saline. MRI experiments assessing tumor blood volume and permeability-maps (K(trans) ) were performed immediately before and 6 days after drug treatment. Data have been analyzed using standard histogram analysis and PA. Standard histogram analysis did not reveal any difference between the two groups, neither before nor after the treatment. In contrast, PA revealed significant differences between drug and placebo treated mice in the texture of the TBV and K(trans) maps after drug treatment, but not with regard to tumors shapes. The results indicated that in view of the heterogeneity of tumor tissue, standard histogram analysis appears insensitive in picking-up differences in response to treatment, while PA appears to be particularly sensitive to changes in texture.

Summary What is already known about this subject Gestational weight gain is a modifiable risk factor for childhood overweight. What this study adds Exceeding the recommended gestational weight gain increases the risk for childhood overweight by about 30%. Interventions to avoid excessive weight gain are needed. Background Overweight and obesity in children are thought to be related to prenatal priming. Gestational weight gain (GWG) might be a potential modifiable risk factor for childhood overweight. Objective To quantify the associations of inadequate and excessive GWG with childhood overweight. Methods The electronic literature search in six databases was complemented by a hand search for relevant articles. Papers were selected and quality was assessed. The effect estimates of the individual studies were pooled using a random-effects model. Results The literature search yielded 1492 results in total, of which seven observational studies met the inclusion criteria. The studies had medium to good quality. The pooled estimate for the association between excessive GWG and childhood overweight yielded an odds ratio (OR) of 1.38 (95% confidence interval [CI]: 1.21–1.57). The association between inadequate GWG and childhood overweight yielded an OR of 0.91 (95% CI: 0.85–0.98). Conclusion These data provide evidence for at least a 21% risk for childhood overweight related to excessive GWG. Therefore, further efforts to design appropriate interventions against excessive GWG may appear warranted.

Item does not contain fulltext With the emergence of multimodal imaging strategies, genetically encoded reporters that can be flexibly combined with any imaging modality become highly attractive. Here we describe the use of glycosylphosphatidylinositol (GPI)-anchored avidin, an avidin moiety targeted to the extracellular side of cell membranes via a GPI anchor, as a reporter for in vivo imaging. Being present on the outside of cells, avidin can be visualized with any type of biotinylated imaging agent, without the requirement that the probe be membrane-permeable. We used the avidin-GPI system to monitor the activity of hypoxia-inducible factors (HIFs)-oxygen-sensing transcription factors, which play a major role in regulating cancer progression-in a mouse tumor allograft model. METHODS: Mouse C51 cells were stably transfected with pH3SVG, a reporter construct driving the expression of avidin-GPI from an HIF-sensitive promoter. The transfected cells were subcutaneously implanted into BALB/c nude mice. At 10 d after tumor inoculation, mice received an intravenous injection of either alexa-594-biocytin or (67)Ga-DOTA-biotin, and tumor HIF activity was imaged using fluorescence reflectance imaging or SPECT. RESULTS: In vitro cell experiments demonstrated the functionality and HIF-dependent regulation of the avidin-GPI reporter construct. In vivo, avidin-GPI was targeted specifically in allograft tumors with biotinylated imaging probes using both fluorescence imaging and SPECT. Analysis of the reporter expression pattern on ex vivo tumor tissue sections indicated a good overlap, with areas of hypoxia. CONCLUSION: We have demonstrated the utility of avidin-GPI as a reporter for multimodal in vivo imaging using both a fluorescence and a SPECT approach to assess intracellular oxygen signaling in a mouse tumor model. 01 maart 2011

European journal of nuclear medicine and molecular imaging 42(9), 1430-1438 (2015). doi:10.1007/s00259-015-3084-8, Published by Springer-Verl., Heidelberg [u.a.]

Introduction: The hypoxia-inducible factor (HIF)-1 pathway can stimulate tumor cell migration and metastasis. Furthermore, hypoxic tumors are associated with a poor prognosis. Besides the HIF-1 pathway, the unfolded protein response (UPR) is also induced by hypoxic conditions. The PKR-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-arm of the UPR induces expression of lysosomal-associated membrane protein 3 (LAMP3), a factor that has been linked to metastasis and poor prognosis in solid tumors. In this study the role of UPR-induced LAMP3 in hypoxia-mediated migration of breast cancer cells was examined. Methods: A number of in vitro metastasis models were used to study the migration and invasion of MDA-MB-231 breast cancer cells under hypoxic conditions. PERK, ATF4 and their downstream factor LAMP3 were knocked down to examine their role in cell migration. In addition, multicellular tumor spheroids were used to study the involvement of the tumor microenvironment in invasion. Results: Using transwell assays, migration of different breast cancer cell lines was assessed. A direct correlation was found between cell migration and baseline LAMP3 expression. Furthermore, moderate hypoxia (1% O-2) was found to be optimal in stimulating migration of MDA-MB-231 cells. siRNA mediated knockdown of PERK, ATF4 and LAMP3 reduced migration of cells under these conditions. Using gap closure assays, similar results were found. In a three-dimensional invasion assay into collagen, LAMP3 knockdown cells showed a diminished capacity to invade compared to control cells when collectively grown in multicellular spheroids. Conclusions: Thus, the PERK/ATF4/LAMP3-arm of the UPR is an additional pathway mediating hypoxia-induced breast cancer cell migration.

Tumor hypoxia, by elevating hypoxia-inducible factors (HIFs), is an established inducer of epithelial-to-mesenchymal transition (EMT) and subsequent cancer cell invasion and metastasis. However, how hypoxia impacts on tumor cell invasion programs and translates into modes of dissemination of moving tumor cells remains unknown. Using breast cancer (BC) and head and neck cancer (HNC) cells which efficiently invade from multicellular spheroids into 3D fibrillar collagen, we addressed which signaling programs that control cell-cell adhesions, cell-matrix interactions and cytoskeletal dynamics and which modes of tumor cell migration are induced by hypoxia. While both cancer models primarily invaded collectively under normoxic conditions, severe hypoxia (0.2% oxygen) or pharmacological stabilization of HIF-1 using the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG) induced EMT-like detachment and migration of single cells into collagen, exhibiting a 2- and 4-fold increase of single BC and HNC cells, respectively. Besides infrequent spindle-shaped movement, hypoxia or DMOG predominantly induced the transition from collective to amoeboid single-cell migration, with blebby amoeboid migration as the predominant migration mode (4- and 3-fold increase for BC and HNC, respectively) next to filopodial amoeboid migration. Hypoxia-induced amoeboid migration is characterized by variably fast and persistent migration, reaching up to several 100 μm/day, mediated by actin-rich filopodial or blebby protrusions at the leading edge. Criteria for amoeboid movement included (i) actin-containing blebs that interacted with collagen structures in a polarized fashion, (ii) weak dependence on matrix-metalloproteinase-mediated collagen remodeling, (iii) low dependence on focalized integrin-mediated matrix adhesions, and (iv) high dependence on RhoA-mediated actomyosin contraction for efficient bleb formation and migration speed. Interference with ROCK signaling using Y-27632 and myosin using blebbistatin significantly inhibited hypoxia-induced amoeboid dissemination and bleb formation. Thus, hypoxia induces plasticity of cancer invasion modes, with efficient blebby amoeboid dissemination as primary outcome. While blebby amoeboid motion is known as a rudimentary migration mode in embryonic stem cells, here we observe blebby amoeboid migration as a simple (low MMP- and integrin dependent) but efficient salvage migration mode of cancer cells under severe hypoxic stress. Citation Format: Veronika te Boekhorst, Liying Jiang, Steffi Lehmann, Alba Zuidema, Peter Friedl. Hypoxia-induced amoeboid cancer cell migration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5062.

Tumor hypoxia, by elevating hypoxia-inducible factors (HIFs), is an established inducer of epithelial-to-mesenchymal transition (EMT) and subsequent cancer cell invasion and metastasis. Using epithelial carcinoma spheroids in 3D fibrillar collagen, we characterized the invasion patterns as well as cellular and molecular mechanisms of hypoxia-induced cancer cell migration modes. While epithelial cancer cells show collective invasion under normoxic conditions, hypoxia or pharmacological stabilization of HIF-1 using the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG) induced EMT-like detachment and migration of single cells. Besides mesenchymal movement, most epithelial cells converted to amoeboid migration with characteristic actin-rich filopodal or distinctive blebby protrusions towards the direction of migration. Whereas mesenchymal migrating cells moved with low velocities in a directionally persistent manner, amoeboid migrating cells generated a broad spectrum of low to high velocities, but with less persistent invasion paths. Sub-cellular molecular analysis showed typical amoeboid features particularly in hypoxic blebby single cell migration, including low to lacking collagen degradation along the migration path and insensitivity to broad-spectrum matrix metalloproteinase (MMP)-inhibitor GM6001, non-focalized cortical actin cytoskeleton within blebs that interacted with collagen structures and low-beta1 integrin expression with lack of integrin focalization. Thus, tumor hypoxia induces a diversity of single-cancer cell invasion modes, including blebby amoeboid migration, thereby enhancing predominantly MMP- and integrin-independent amoeboid dissemination in parallel to EMT induction. Citation Format: Steffi Lehmann, Veronika A M te Boekhorst, Julia Odenthal, Liying Jiang, Sjoerd van Helvert, Peter Friedl. Hypoxia-induced transition from collective to amoeboid single cell dissemination in epithelial cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4141. doi:10.1158/1538-7445.AM2015-4141

Background and purpose Headache is a common health problem in adolescents. There are a number of risk factors for headache in adolescents that are amenable to intervention. The aim of the study was to assess the effectiveness of a low-level headache prevention programme in the classroom setting to prevent these risk factors. Methods In all, 1674 students in 8th–10th grade at 12 grammar schools in greater Munich, Germany, were cluster randomized into intervention and control groups. A standardized 60-min prevention lesson focusing on preventable risk factors for headache (physical inactivity, coffee consumption, alcohol consumption and smoking) and providing instructions on stress management and neck and shoulder muscle relaxation exercises was given in a classroom setting. Seven months later, students were reassessed. The main outcome parameter was headache cessation. Logistic regression models with random effects for cluster and adjustment for baseline risk factors were calculated. Results Nine hundred students (intervention group N = 450, control group N = 450) with headache at baseline and complete data for headache and confounders were included in the analysis. Headache cessation was observed in 9.78% of the control group compared with 16.22% in the intervention group (number needed to treat = 16). Accounting for cluster effects and confounders, the probability of headache cessation in the intervention group was 1.77 (95% confidence interval = [1.08; 2.90]) higher than in the control group. The effect was most pronounced in adolescents with tension-type headache: odds ratio = 2.11 (95% confidence interval = [1.15; 3.80]). Conclusion Our study demonstrates the effectiveness of a one-time, classroom-based headache prevention programme.

IL-2 therapy can result in durable responses in a proportion of cancer patients, but the treatment is associated with significant toxicity. To improve the liabilities associated with IL-2 therapy, we recently developed a novel monomeric tumor-targeted immunocytokine (CEA-IL2v), which completely lacks binding to CD25 (IL-2Rα) but retains binding to IL-2Rβγ. When tested in classical 2D tumor/lymphocyte co-culture systems, CEA-IL2v induces concentration-dependent proliferation and activation of NK, CD4 and CD8 T cells, but differently from IL-2, does not lead to preferential activation of Tregs and induces lower levels Fas-induced apoptosis. To further explore the properties of CEA-IL2v, including the assessment of antibody targeting and stimulation of immune-cell activation and infiltration, we developed a novel 3D heterotypic microtissues model consisting of tumor cells, fibroblasts and lymphocytes. To address the penetration and targeting, heterotypic 3D microtissues consisting of CEA-expressing human tumor cells and fibroblasts were incubated with tumor-targeted IL-2v (CEA-IL2v), stroma-targeted IL-2v (FAP-IL2v), and the corresponding untargeted control (DP47-IL2v). CEA-IL2v successfully penetrated into microtissues and specifically bound to CEA-positive tumor cells, FAP-IL2v was specifically retained by FAP-expressing fibroblasts whereas the DP47-IL2v was not detected in any of the components. The activity of the immunocytokines was further evaluated by addition of human lymphocytes to 3D microtissues. CEA-IL2v and DP47-IL2v activated different lymphocyte subsets, stimulated their infiltration and selectively promoted the elimination of tumor cells as shown by (a) reduction of microtissue size, (b) increase of LDH release, (c) elimination of CEA-positive tumor cells, (d) increase of lymphocytes infiltrating into the microtissues and (e) increase of cytokine and chemokine levels secreted in the supernatants, including MIP-1β, IL-6 and IFN-γ. Taken together, the heterotypic tumor/fibroblast/lymphocyte 3D microtissue model presented here offers the possibility to monitor antibody penetration and targeting to tumor and stroma components, to study the interaction of tumor cells with immune cells in a system that more closely resembles the in vivo tumor microenvironment and thus provides an in vitro platform for optimal immunotherapy development. The study also points out that the tumor-targeted CEA-IL2v immunocytokine is an elegant tool for expansion and activation of immune effector cells, which may successfully be applied for cancer immunotherapy. Citation Format: Inja Waldhauer, Laura Morra, Steffi Lehmann, Irina Agarkova, Philine Zumstein, Jens M. Kelm, Pablo Umana, Christian Klein, Marina Bacac. Development of 3D microtissue models to study the activity of novel tumor-targeted immunotherapeutics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-264. doi:10.1158/1538-7445.AM2013-LB-264