Cancer cell invasion through physical barriers in the extracellular matrix (ECM) requires a complex synergy of traction force against the ECM, mechanosensitive feedback, and subsequent cytoskeletal rearrangement. PDMS microchannels were used to investigate the transition from mesenchymal to amoeboid invasion in cancer cells. Migration was faster in narrow 3 μm-wide channels than in wider 10 μm channels, even in the absence of cell-binding ECM proteins. Cells permeating narrow channels exhibited blebbing and had smooth leading edge profiles, suggesting an ECM-induced transition from mesenchymal invasion to amoeboid invasion. Live cell labeling revealed a mechanosensing period in which the cell attempts mesenchymal-based migration, reorganizes its cytoskeleton, and proceeds using an amoeboid phenotype. Rho/ROCK (amoeboid) and Rac (mesenchymal) pathway inhibition revealed that amoeboid invasion through confined environments relies on both pathways in a time- and ECM-dependent manner. This demonstrates that cancer cells can dynamically modify their invasion programming to navigate physically-confining matrix conditions.

NK cells play an important role in tumor immunosurveillance. Their reactivity is governed by various activating and inhibitory surface receptors, which include several members of the TNF/TNF receptor family. For more than 50 years, it has been recognized that tumor immunosurveillance and in particular NK cell antitumor reactivity is largely influenced by platelets, but the underlying mechanisms remain to be fully elucidated. Here we report that upon activation, which reportedly occurs following interaction with cancer cells, platelets upregulate the TNF family member RANKL. Comparative analysis of the expression of RANK among different NK cell subsets and RANKL on platelets in cancer patients and healthy volunteers revealed a distinct malignant phenotype, and platelet-derived RANKL was found to inhibit the activity of normal NK cells against cancer cells. Notably, NK cell antitumor reactivity could be partially restored by application of denosumab, a RANKL-neutralizing antibody approved for treatment of benign and malignant osteolysis. Together, our data not only unravel a novel mechanism of tumor immune evasion mediated by platelets, but they also provide a functional explanation for the clinical observation that denosumab, beyond protecting from bone loss, may prolong disease-free survival in patients with solid tumors.

Owing to their key role in several diseases including cancer, activating and inhibitory immune checkpoint molecules are increasingly exploited as targets for immunotherapy. Recently, we demonstrated that platelets, which largely influence tumor progression and immune evasion, functionally express the ligand of the checkpoint molecule GITR. This immunoreceptor modulates effector functions of T cells and NK cells with its function varying dependent on cellular context and activation state. Here, we provide a comparative analysis of platelet-derived GITRL (pGITRL) in breast cancer patients and healthy volunteers. The levels of pGITRL were found to be higher on platelets derived from cancer patients and appeared to be specifically regulated during tumor progression as exemplified by several clinical parameters including tumor stage/grade, the occurrence of metastases and tumor proliferation (Ki67) index. In addition, we report that pGITRL is upregulated during platelet maturation and particularly induced upon exposure to tumor-derived soluble factors. Our data indicate that platelets modulate the GITR/GITRL immune checkpoint in the context of malignant disease and provide a rationale to further study the GITR/GITRL axis for exploitation for immunotherapeutic intervention in cancer patients.

Although treatment options of acute myeloid leukemia (AML) have improved over the recent years, prognosis remains poor. Better understanding of the molecular mechanisms influencing and predicting treatment efficacy may improve disease control and outcome. Here we studied the expression, prognostic relevance and functional role of the tumor necrosis factor receptor (TNFR) family member Receptor Activator of Nuclear Factor (NF)-κB (RANK) in AML. We conducted an experimental ex vivo study using leukemic cells of 54 AML patients. Substantial surface expression of RANK was detected on primary AML cells in 35% of the analyzed patients. We further found that RANK signaling induced the release of cytokines acting as growth and survival factors for the leukemic cells and mediated resistance of AML cells to treatment with doxorubicin and cytarabine, the most commonly used cytostatic compounds in AML treatment. In line, RANK expression correlated with a dismal disease course as revealed by reduced overall survival. Together, our results show that RANK plays a yet unrecognized role in AML pathophysiology and resistance to treatment, and identify RANK as “functional” prognostic marker in AML. Therapeutic modulation of RANK holds promise to improve treatment response in AML patients.

Background The activating immunoreceptor NKG2D is expressed on cytotoxic lymphocytes (CTL) like NK cells, CD8⁺ cytotoxic T cells and γδ T cells. It potently stimulates CTL effector functions upon recognition of its ligands (NKG2DL) that are generally absent on healthy tissue, but induced by cellular stress including malignant transformation. Recent studies demonstrated that – somewhat surprisingly - also the NKG2D receptor can be expressed on tumor cells of various origins and may affect cellular functions of the malignant cells like tumor growth and metastasis. Here we report on the role of NKG2D expression in acute myeloid leukemia (AML). Methods Expression of NKG2D on the cell surface of patient AML cells and its correlation with differentiation/maturation as well as stemness characteristics depending on NKG2D expression were studied. Furthermore, the functional role of NKG2D in AML cells on signaling and stem cell properties was investigated. Results Primary leukemic cells of AML patients expressed substantial levels of NKG2D on the cell surface in approximately 40% of 156 investigated patients. Within the patient cohort, expression of NKG2D was associated with more differentiated subtypes of AML according to the FAB classification. Notably, within the leukemic cell population of individual patients, NKG2D expression significantly correlated with CD34 negativity. In line, after sorting bulk AML cells of patients according to NKG2D expression, only the subpopulation lacking NKG2D expression showed stemness characteristics like the ability to give rise to colonies in colony forming unit assays. Exposure of NKG2D-positive AML cells to agonistic NKG2D antibody caused release of the cytokines IL-6, IL-8, IL-10 and TNF, which act as growth and survival factors for AML cells and play an important role in disease pathophysiology. NKG2D stimulation further increased engraftment of primary AML cells in NSG mice. Signaling via NKG2D was found to enhance the metabolic activity/viability of AML cells and protected the leukemic cells from treatment with the cytostatic compounds cytarabine and doxorubicin that are routinely used for AML treatment. Conclusions Taken together, our data implicate that the NKG2D/NKG2DL system, beyond its involvement in immune surveillance, may directly affect AML pathophysiology. Ongoing investigations aim to unravel the NKG2D-mediated signaling pathways, consequences of autocrine/paracrine NKG2D/NKG2DL interaction in AML cells as well as the association of NKG2D negativity with known stemness/differentiation markers and clinical disease course. Ethics Approval The study was approved by IRB (ethics committee of the Faculty of Medicine of the Eberhard Karls Universitaet Tuebingen and of the University Hospital Tuebingen) and was conducted in accordance with the Declaration of Helsinki; reference number 13/2007V. Consent Human material was collected after obtaining written informed consent of patients/participants.