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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

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