Targeting Gαi2 in neutrophils protects from myocardial ischemia reperfusion injury.

Neutrophils are not only involved in immune defense against infection but also contribute to the exacerbation of tissue damage after ischemia and reperfusion. We have previously shown that genetic ablation of regulatory Gαi proteins in mice has both protective and deleterious effects on myocardial ischemia reperfusion injury (mIRI), depending on which isoform is deleted. To deepen and analyze these findings in more detail the contribution of Gαi2 proteins in resident cardiac vs circulating blood cells for mIRI was first studied in bone marrow chimeras. In fact, the absence of Gαi2 in all blood cells reduced the extent of mIRI (22,9% infarct size of area at risk (AAR) Gnai2-/- → wt vs 44.0% wt → wt; p < 0.001) whereas the absence of Gαi2 in non-hematopoietic cells increased the infarct damage (66.5% wt → Gnai2-/- vs 44.0% wt → wt; p < 0.001). Previously we have reported the impact of platelet Gαi2 for mIRI. Here, we show that infarct size was substantially reduced when Gαi2 signaling was either genetically ablated in neutrophils/macrophages using LysM-driven Cre recombinase (AAR: 17.9% Gnai2fl/fl LysM-Cre+/tg vs 42.0% Gnai2fl/fl; p < 0.01) or selectively blocked with specific antibodies directed against Gαi2 (AAR: 19.0% (anti-Gαi2) vs 49.0% (IgG); p < 0.001). In addition, the number of platelet-neutrophil complexes (PNCs) in the infarcted area were reduced in both, genetically modified (PNCs: 18 (Gnai2fl/fl; LysM-Cre+/tg) vs 31 (Gnai2fl/fl); p < 0.001) and in anti-Gαi2 antibody-treated (PNCs: 9 (anti-Gαi2) vs 33 (IgG); p < 0.001) mice. Of note, significant infarct-limiting effects were achieved with a single anti-Gαi2 antibody challenge immediately prior to vessel reperfusion without affecting bleeding time, heart rate or cellular distribution of neutrophils. Finally, anti-Gαi2 antibody treatment also inhibited transendothelial migration of human neutrophils (25,885 (IgG) vs 13,225 (anti-Gαi2) neutrophils; p < 0.001), collectively suggesting that a therapeutic concept of functional Gαi2 inhibition during thrombolysis and reperfusion in patients with myocardial infarction should be further considered.

mitoBKCa is functionally expressed in murine and human breast cancer cells and potentially contributes to metabolic reprogramming.

Alterations in the function of K+ channels such as the voltage- and Ca2+-activated K+ channel of large conductance (BKCa) reportedly promote breast cancer (BC) development and progression. Underlying molecular mechanisms remain, however, elusive. Here, we provide electrophysiological evidence for a BKCa splice variant localized to the inner mitochondrial membrane of murine and human BC cells (mitoBKCa). Through a combination of genetic knockdown and knockout along with a cell permeable BKCa channel blocker, we show that mitoBKCa modulates overall cellular and mitochondrial energy production, and mediates the metabolic rewiring referred to as the 'Warburg effect', thereby promoting BC cell proliferation in the presence and absence of oxygen. Additionally, we detect mitoBKCa and BKCa transcripts in low or high abundance, respectively, in clinical BC specimens. Together, our results emphasize, that targeting mitoBKCa could represent a treatment strategy for selected BC patients in future.

Pharmacological targeting of the mitochondrial calcium-dependent potassium channel KCa3.1 triggers cell death and reduces tumor growth and metastasis in vivo.

Ion channels are non-conventional, druggable oncological targets. The intermediate-conductance calcium-dependent potassium channel (KCa3.1) is highly expressed in the plasma membrane and in the inner mitochondrial membrane (mitoKCa3.1) of various cancer cell lines. The role mitoKCa3.1 plays in cancer cells is still undefined. Here we report the synthesis and characterization of two mitochondria-targeted novel derivatives of a high-affinity KCa3.1 antagonist, TRAM-34, which retain the ability to block channel activity. The effects of these drugs were tested in melanoma, pancreatic ductal adenocarcinoma and breast cancer lines, as well as in vivo in two orthotopic models. We show that the mitochondria-targeted TRAM-34 derivatives induce release of mitochondrial reactive oxygen species, rapid depolarization of the mitochondrial membrane, fragmentation of the mitochondrial network. They trigger cancer cell death with an EC50 in the µM range, depending on channel expression. In contrast, inhibition of the plasma membrane KCa3.1 by membrane-impermeant Maurotoxin is without effect, indicating a specific role of mitoKCa3.1 in determining cell fate. At sub-lethal concentrations, pharmacological targeting of mitoKCa3.1 significantly reduced cancer cell migration by enhancing production of mitochondrial reactive oxygen species and nuclear factor-κB (NF-κB) activation, and by downregulating expression of Bcl-2 Nineteen kD-Interacting Protein (BNIP-3) and of Rho GTPase CDC-42. This signaling cascade finally leads to cytoskeletal reorganization and impaired migration. Overexpression of BNIP-3 or pharmacological modulation of NF-κB and CDC-42 prevented the migration-reducing effect of mitoTRAM-34. In orthotopic models of melanoma and pancreatic ductal adenocarcinoma, the tumors at sacrifice were 60% smaller in treated versus untreated animals. Metastasis of melanoma cells to lymph nodes was also drastically reduced. No signs of toxicity were observed. In summary, our results identify mitochondrial KCa3.1 as an unexpected player in cancer cell migration and show that its pharmacological targeting is efficient against both tumor growth and metastatic spread in vivo.

IKCa channels control breast cancer metabolism including AMPK-driven autophagy.

Ca2+-activated K+ channels of intermediate conductance (IK) are frequently overexpressed in breast cancer (BC) cells, while IK channel depletion reduces BC cell proliferation and tumorigenesis. This raises the question, of whether and mechanistically how IK activity interferes with the metabolic activity and energy consumption rates, which are fundamental for rapidly growing cells. Using BC cells obtained from MMTV-PyMT tumor-bearing mice, we show that both, glycolysis and mitochondrial ATP-production are reduced in cells derived from IK-deficient breast tumors. Loss of IK altered the sub-/cellular K+- and Ca2+- homeostasis and mitochondrial membrane potential, ultimately resulting in reduced ATP-production and metabolic activity. Consequently, we find that BC cells lacking IK upregulate AMP-activated protein kinase activity to induce autophagy compensating the glycolytic and mitochondrial energy shortage. Our results emphasize that IK by modulating cellular Ca2+- and K+-dynamics contributes to the remodeling of metabolic pathways in cancer. Thus, targeting IK channel might disturb the metabolic activity of BC cells and reduce malignancy.

Influence of the external breast prosthesis on the postural control of women who underwent mastectomy: Cross-sectional study.

Women after mastectomy may decide to either have a breast reconstruction or use an external breast prosthesis. AIM: The aim of the presented research was to evaluate the influence of external breast prosthesis on postural stability in women after mastectomy. METHODS AND PROCEDURES: In the study 52 women after unilateral mastectomy took part. The study consisted of 4 parts: 1) anthropometric measurements; 2) measurements of upper limb circumference; 3) assessment of weight-bearing (WB); and 4) posturographic tests (PT). OUTCOMES AND RESULTS: Differences in the arm circumferences on the amputated (A) and non-amputated (NA) sides did not confirm the occurrence of lymphedema in limb on amputated side. The results of the WB between the A and NA body sides in both tested conditions, i.e., with open and closed eyes, showed no significant differences between the test with and without an external prosthesis. No statistically differences have been observed between posturometric parameters with and without breast prosthesis during both PT. In comparing the posturometric parameters between the PT with open and closed eyes, the sway path of the center of pressure was statistically significantly longer when eyes were closed in both conditions, i.e., with and without breast prosthesis. CONCLUSION AND IMPLICATIONS: The finding show that 1) external breast prosthesis does not have a significant influence on the symmetry of loading on the A and NA body sides and on the postural stability of women after unilateral mastectomy and 2) exclusion visual control during PT increases postural instability in women after unilateral mastectomy.

Monitoring extracellular ion and metabolite dynamics with recombinant nanobody-fused biosensors.

Ion and analyte changes in the tumor microenvironment (TME) alter the metabolic activity of cancer cells, promote tumor cell growth, and impair anti-tumor immunity. Consequently, accurate determination and visualization of extracellular changes of analytes in real time is desired. In this study, we genetically combined FRET-based biosensors with nanobodies (Nbs) to specifically visualize and monitor extracellular changes in K+, pH, and glucose on cell surfaces. We demonstrated that these Nb-fused biosensors quantitatively visualized K+ alterations on cancer and non-cancer cell lines and primary neurons. By implementing a HER2-specific Nb, we generated functional K+ and pH sensors, which specifically stained HER2-positive breast cancer cells. Based on the successful development of several Nb-fused biosensor combinations, we anticipate that this approach can be readily extended to other biosensors and will open new opportunities for the study of extracellular analytes in advanced experimental settings.

Cysteine-Rich LIM-Only Protein 4 (CRP4) Promotes Atherogenesis in the ApoE-/- Mouse Model.

Vascular smooth muscle cells (VSMCs) can switch from their contractile state to a synthetic phenotype resulting in high migratory and proliferative capacity and driving atherosclerotic lesion formation. The cysteine-rich LIM-only protein 4 (CRP4) reportedly modulates VSM-like transcriptional signatures, which are perturbed in VSMCs undergoing phenotypic switching. Thus, we hypothesized that CRP4 contributes to adverse VSMC behaviours and thereby to atherogenesis in vivo. The atherogenic properties of CRP4 were investigated in plaque-prone apolipoprotein E (ApoE) and CRP4 double-knockout (dKO) as well as ApoE-deficient CRP4 wildtype mice. dKO mice exhibited lower plaque numbers and lesion areas as well as a reduced content of α-smooth muscle actin positive cells in the lesion area, while lesion-associated cell proliferation was elevated in vessels lacking CRP4. Reduced plaque volumes in dKO correlated with significantly less intra-plaque oxidized low-density lipoprotein (oxLDL), presumably due to upregulation of the antioxidant factor peroxiredoxin-4 (PRDX4). This study identifies CRP4 as a novel pro-atherogenic factor that facilitates plaque oxLDL deposition and identifies the invasion of atherosclerotic lesions by VSMCs as important determinants of plaque vulnerability. Thus, targeting of VSMC CRP4 should be considered in plaque-stabilizing pharmacological strategies.

Physiological and Pathophysiological Roles of Metabolic Pathways for NET Formation and Other Neutrophil Functions.

Neutrophils are the most numerous cells in the leukocyte population and essential for innate immunity. To limit their effector functions, neutrophils are able to modulate glycolysis and other cellular metabolic pathways. These metabolic pathways are essential not only for energy usage, but also for specialized effector actions, such as the production of reactive oxygen species (ROS), chemotaxis, phagocytosis, degranulation, and the formation of neutrophil extracellular traps (NETs). It has been demonstrated that activated viable neutrophils can produce NETs, which consists of a DNA scaffold able to bind granule proteins and microorganisms. The formation of NETs requires the availability of increased amounts of adenosine triphosphate (ATP) as it is an active cellular and therefore energy-dependent process. In this article, we discuss the glycolytic and other metabolic routes in association with neutrophil functions focusing on their role for building up NETs in the extracellular space. A better understanding of the requirements of metabolic pathways for neutrophil functions may lead to the discovery of molecular targets suitable to develop novel anti-infectious and/or anti-inflammatory drugs.

Assessing K+ ions and K+ channel functions in cancer cell metabolism using fluorescent biosensors.

Cancer represents a leading cause of death worldwide. Hence, a better understanding of the molecular mechanisms causing and propelling the disease is of utmost importance. Several cancer entities are associated with altered K+ channel expression which is frequently decisive for malignancy and disease outcome. The impact of such oncogenic K+ channels on cell patho-/physiology and homeostasis and their roles in different subcellular compartments is, however, far from being understood. A refined method to simultaneously investigate metabolic and ionic signaling events on the level of individual cells and their organelles represent genetically encoded fluorescent biosensors, that allow a high-resolution investigation of compartmentalized metabolite or ion dynamics in a non-invasive manner. This feature of these probes makes them versatile tools to visualize and understand subcellular consequences of aberrant K+ channel expression and activity in K+ channel related cancer research.

Subunits of BK channels promote breast cancer development and modulate responses to endocrine treatment in preclinical models.

BACKGROUND AND PURPOSE: Pore-forming α subunits of the voltage- and Ca2+ -activated K+ channel with large conductance (BKα) promote malignant phenotypes of breast tumour cells. Auxiliary subunits such as the leucine-rich repeat containing 26 (LRRC26) protein, also termed BKγ1, may be required to permit activation of BK currents at a depolarized resting membrane potential that frequently occur in non-excitable tumour cells. EXPERIMENTAL APPROACH: Anti-tumour effects of BKα loss were investigated in breast tumour-bearing MMTV-PyMT transgenic BKα knockout (KO) mice, primary MMTV-PyMT cell cultures, and in a syngeneic transplantation model of breast cancer derived from these cells. The therapeutic relevance of BK channels in the context of endocrine treatment was assessed in human breast cancer cell lines expressing either low (MCF-7) or high (MDA-MB-453) levels of BKα and BKγ1, as well as in BKα-negative MDA-MB-157. KEY RESULTS: BKα promoted breast cancer onset and overall survival in preclinical models. Conversely, lack of BKα and/or knockdown of BKγ1 attenuated proliferation of murine and human breast cancer cells in vitro. At low concentrations, tamoxifen and its major active metabolites stimulated proliferation of BKα/γ1-positive breast cancer cells, independent of the genomic signalling controlled by the oestrogen receptor. Finally, tamoxifen increased the relative survival time of BKα KO but not of wild-type tumour cell recipient mice. CONCLUSION AND IMPLICATIONS: Breast cancer initiation, progression, and tamoxifen sensitivity depend on functional BK channels thereby providing a rationale for the future exploration of the oncogenic actions of BK channels in clinical outcomes with anti-oestrogen therapy. LINKED ARTICLES: This article is part of a themed issue on New avenues in cancer prevention and treatment (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.12/issuetoc.

Investigating the K+ sensitivity of cellular metabolism by extracellular flux analysis.

We have recently demonstrated that the activity of hexokinase 2 is dependent on the intracellular potassium ion (K+) concentration ([K+]). To analyze the K+ dependency of the cell metabolism in cell populations, we used an extracellular flux analyzer to assess oxygen consumption and acidification rates as well-established measures of oxidative- and glycolytic metabolic activities. This protocol describes in detail how a potential K+ sensitivity of the cell metabolism can be elucidated by extracellular flux analysis. For complete details on the use and execution of this protocol, please refer to Bischof et al. (2021).

Rise of cGMP by partial phosphodiesterase-3A degradation enhances cardioprotection during hypoxia.

3',5'-cyclic guanosine monophosphate (cGMP) is a druggable second messenger regulating cell growth and survival in a plethora of cells and disease states, many of which are associated with hypoxia. For example, in myocardial infarction and heart failure (HF), clinical use of cGMP-elevating drugs improves disease outcomes. Although they protect mice from ischemia/reperfusion (I/R) injury, the exact mechanism how cardiac cGMP signaling is regulated in response to hypoxia is still largely unknown. By monitoring real-time cGMP dynamics in murine and human cardiomyocytes using in vitro and in vivo models of hypoxia/reoxygenation (H/R) and I/R injury combined with biochemical methods, we show that hypoxia causes rapid but partial degradation of cGMP-hydrolyzing phosphodiesterase-3A (PDE3A) protein via the autophagosomal-lysosomal pathway. While increasing cGMP in hypoxia prevents cell death, partially reduced PDE3A does not change the pro-apoptotic second messenger 3',5'-cyclic adenosine monophosphate (cAMP). However, it leads to significantly enhanced protective effects of clinically relevant activators of nitric oxide-sensitive guanylyl cyclase (NO-GC). Collectively, our mouse and human data unravel a new mechanism by which cardiac cGMP improves hypoxia-associated disease conditions.

Unveiling the K+-sensitivity of cell metabolism using genetically encoded, FRET-based K+, glucose, and ATP biosensors.

Investigating dynamic changes of mitochondrial ATP and cytosolic glucose levels of single living cells over time by genetically encoded biosensors provides an informative readout of their metabolic activities. Here, we describe how to monitor the metabolic K+-sensitivity of HEK293 cells exploiting ATP-, glucose-, and K+ probes. Fluorescence live-cell imaging of these Förster resonance energy transfer-based biosensors over time in response to gramicidin, an ionophoric peptide, indicated an absolute dependency of cellular ATP homeostasis on high intracellular K+ levels. For complete information on the generation and use of this protocol please refer to Bischof et al. (2021).

Cyclic GMP-Dependent Regulation of Vascular Tone and Blood Pressure Involves Cysteine-Rich LIM-Only Protein 4 (CRP4).

The cysteine-rich LIM-only protein 4 (CRP4), a LIM-domain and zinc finger containing adapter protein, has been implicated as a downstream effector of the second messenger 3',5'-cyclic guanosine monophosphate (cGMP) pathway in multiple cell types, including vascular smooth muscle cells (VSMCs). VSMCs and nitric oxide (NO)-induced cGMP signaling through cGMP-dependent protein kinase type I (cGKI) play fundamental roles in the physiological regulation of vascular tone and arterial blood pressure (BP). However, it remains unclear whether the vasorelaxant actions attributed to the NO/cGMP axis require CRP4. This study uses mice with a targeted deletion of the CRP4 gene (CRP4 KO) to elucidate whether cGMP-elevating agents, which are well known for their vasorelaxant properties, affect vessel tone, and thus, BP through CRP4. Cinaciguat, a NO- and heme-independent activator of the NO-sensitive (soluble) guanylyl cyclase (NO-GC) and NO-releasing agents, relaxed both CRP4-proficient and -deficient aortic ring segments pre-contracted with prostaglandin F2α. However, the magnitude of relaxation was slightly, but significantly, increased in vessels lacking CRP4. Accordingly, CRP4 KO mice presented with hypotonia at baseline, as well as a greater drop in systolic BP in response to the acute administration of cinaciguat, sodium nitroprusside, and carbachol. Mechanistically, loss of CRP4 in VSMCs reduced the Ca2+-sensitivity of the contractile apparatus, possibly involving regulatory proteins, such as myosin phosphatase targeting subunit 1 (MYPT1) and the regulatory light chain of myosin (RLC). In conclusion, the present findings confirm that the adapter protein CRP4 interacts with the NO-GC/cGMP/cGKI pathway in the vasculature. CRP4 seems to be part of a negative feedback loop that eventually fine-tunes the NO-GC/cGMP axis in VSMCs to increase myofilament Ca2+ desensitization and thereby the maximal vasorelaxant effects attained by (selected) cGMP-elevating agents.

Potassium Channels in Cancer.

Neoplastic transformation is reportedly associated with alterations of the potassium transport across plasma and intracellular membranes. These alterations have been identified as crucial elements of the tumourigenic reprogramming of cells. Potassium channels may contribute to cancer initiation, malignant progression and therapy resistance of tumour cells. The book chapter focusses on (oncogenic) potassium channels frequently upregulated in different tumour entities, upstream and downstream signalling of these channels, their contribution to the maintenance of cancer stemness and the formation of an immunosuppressive tumour microenvironment. In addition, their role in adaptation to tumour hypoxia, metabolic reprogramming, as well as tumour spreading and metastasis is discussed. Finally, we discuss how (oncogenic) potassium channels may confer treatment resistance of tumours against radiation and chemotherapy and thus might be harnessed for new therapy strategies, for instance, by repurposing approved drugs known to target potassium channels.

Potassium ions promote hexokinase-II dependent glycolysis.

High expression levels of mitochondria-associated hexokinase-II (HKII) represent a hallmark of metabolically highly active cells such as fast proliferating cancer cells. Typically, the enzyme provides a crucial metabolic switch towards aerobic glycolysis. By imaging metabolic activities on the single-cell level with genetically encoded fluorescent biosensors, we here demonstrate that HKII activity requires intracellular K+. The K+ dependency of glycolysis in cells expressing HKII was confirmed in cell populations using extracellular flux analysis and nuclear magnetic resonance-based metabolomics. Reductions of intracellular K+ by gramicidin acutely disrupted HKII-dependent glycolysis and triggered energy stress pathways, while K+ re-addition promptly restored glycolysis-dependent adenosine-5'-triphosphate generation. Moreover, expression and activation of KV1.3, a voltage-gated K+ channel, lowered cellular K+ content and the glycolytic activity of HEK293 cells. Our findings unveil K+ as an essential cofactor of HKII and provide a mechanistic link between activities of distinct K+ channels and cell metabolism.

CRIP1 expression in monocytes related to hypertension.

Hypertension is a complex and multifactorial disorder caused by lifestyle and environmental factors, inflammation and disease-related genetic factors and is a risk factor for stroke, ischemic heart disease and renal failure. Although circulating monocytes and tissue macrophages contribute to the pathogenesis of hypertension, the underlying mechanisms are poorly understood. Cysteine rich protein 1 (CRIP1) is highly expressed in immune cells, and CRIP1 mRNA expression in monocytes associates with blood pressure (BP) and is up-regulated by proinflammatory modulation suggesting a link between CRIP1 and BP regulation through the immune system. To address this functional link, we studied CRIP1 expression in immune cells in relation to BP using a human cohort study and hypertensive mouse models. CRIP1 expression in splenic monocytes/macrophages and in circulating monocytes was significantly affected by angiotensin II (Ang II) in a BP-elevating dose (2 mg/kg/day). In the human cohort study, monocytic CRIP1 expression levels were associated with elevated BP, whereas upon differentiation of monocytes to macrophages this association along with the CRIP1 expression level was diminished. In conclusion, CRIP1-positive circulating and splenic monocytes seem to play an important role in hypertension related inflammatory processes through endogenous hormones such as Ang II. These findings suggest that CRIP1 may affect the interaction between the immune system, in particular monocytes, and the pathogenesis of hypertension.

Functional Coupling of Slack Channels and P2X3 Receptors Contributes to Neuropathic Pain Processing.

The sodium-activated potassium channel Slack (KNa1.1, Slo2.2, or Kcnt1) is highly expressed in populations of sensory neurons, where it mediates the sodium-activated potassium current (IKNa) and modulates neuronal activity. Previous studies suggest that Slack is involved in the processing of neuropathic pain. However, mechanisms underlying the regulation of Slack activity in this context are poorly understood. Using whole-cell patch-clamp recordings we found that Slack-mediated IKNa in sensory neurons of mice is reduced after peripheral nerve injury, thereby contributing to neuropathic pain hypersensitivity. Interestingly, Slack is closely associated with ATP-sensitive P2X3 receptors in a population of sensory neurons. In vitro experiments revealed that Slack-mediated IKNa may be bidirectionally modulated in response to P2X3 activation. Moreover, mice lacking Slack show altered nocifensive responses to P2X3 stimulation. Our study identifies P2X3/Slack signaling as a mechanism contributing to hypersensitivity after peripheral nerve injury and proposes a potential novel strategy for treatment of neuropathic pain.

KCa3.1 Channels Confer Radioresistance to Breast Cancer Cells.

KCa3.1 K+ channels reportedly contribute to the proliferation of breast tumor cells and may serve pro-tumor functions in the microenvironment. The putative interaction of KCa3.1 with major anti-cancer treatment strategies, which are based on cytotoxic drugs or radiotherapy, remains largely unexplored. We employed KCa3.1-proficient and -deficient breast cancer cells derived from breast cancer-prone MMTV-PyMT mice, pharmacological KCa3.1 inhibition, and a syngeneic orthotopic mouse model to study the relevance of functional KCa3.1 for therapy response. The KCa3.1 status of MMTV-PyMT cells did not determine tumor cell proliferation after treatment with different concentrations of docetaxel, doxorubicin, 5-fluorouracil, or cyclophosphamide. KCa3.1 activation by ionizing radiation (IR) in breast tumor cells in vitro, however, enhanced radioresistance, probably via an involvement of the channel in IR-stimulated Ca2+ signals and DNA repair pathways. Consistently, KCa3.1 knockout increased survival time of wildtype mice upon syngeneic orthotopic transplantation of MMTV-PyMT tumors followed by fractionated radiotherapy. Combined, our results imply that KCa3.1 confers resistance to radio- but not to chemotherapy in the MMTV-PyMT breast cancer model. Since KCa3.1 is druggable, KCa3.1 targeting concomitant to radiotherapy seems to be a promising strategy to radiosensitize breast tumors.

From in vitro to ex vivo: subcellular localization and uptake of graphene quantum dots into solid tumors.

Among various nanoparticles tested for pharmacological applications over the recent years, graphene quantum dots (GQDs) seem to be promising candidates for the construction of drug delivery systems due to their superior biophysical and biochemical properties. The subcellular fate of incorporated nanomaterial is decisive for transporting pharmaceuticals into target cells. Therefore a detailed characterization of the uptake of GQDs into different breast cancer models was performed. The demonstrated accumulation inside the endolysosomal system might be the reason for the particles' low toxicity, but has to be overcome for cytosolic or nuclear drug delivery. Furthermore, the penetration of GQDs into precision-cut mammary tumor slices was studied. These constitute a far closer to reality model system than monoclonal cell lines. The constant uptake into the depth of the tissue slices underlines the systems' potential for drug delivery into solid tumors.