FULL PAPER www.advtherap.com Deep Tissue Translocation of Graphene Oxide Sheets in Human Glioblastoma 3D Spheroids and an Orthotopic Xenograft Model Irene de Lázaro, Paul Sharp, Cansu Gurcan, Ahmet Ceylan, Maria Stylianou, Thomas Kisby, Yingxian Chen, Sandra Vranic, Katharine Barr, Hadiseh Taheri, Asuman Ozen, Cyrill Bussy, Acelya Yilmazer,* and Kostas Kostarelos* 1. Introduction Its anatomical localization, a highly heterogeneous and drug-resistant tumor cell population and a “cold” immune microenvironment, all challenge the treat- Glioblastoma multiforme, which equates ment of glioblastoma. Nanoscale drug delivery systems, including graphene ox- to grade IV glioma, is the most frequent form of cancer of the central nervous sys- ide (GO) flakes, may circumvent some of these issues bypassing biological bar- tem and one of the most aggressive. The riers, delivering multiple cargoes to impact several pathways simultaneously, or current standard of care entails surgical targeting the immune compartment. Here, the interactions of GO flakes with in resection followed by radiotherapy, plus vitro (U-87 MG three-dimensional spheroids, without stromal or immune com- concomitant and maintenance chemother- partments) and in vivo (U-87 MG orthotopic xenograft) models of glioblastoma apy with temozolomide.[1] However, despite these interventions, the median overall sur- are investigated. In vitro, GO flakes translocated deeply into the spheroids with vival (OS) after diagnosis does not surpass little internalization in tumor cells. In vivo, intracranially administered GO also the 18-month threshold. Furthermore, an- show extensive distribution throughout the tumor and demonstrate no impact tiangiogenic therapy with the anti vascular on tumor growth and progression for the duration of the study. Internalization endothelial growth factor (VEGF) antibody within tumor cells is also scarce, with the majority of flakes preferentially bevacizumab has only shown modest ben- taken up by microglia/macrophages. The results indicate that GO flakes could efit in recurrent glioblastoma and does not significantly extend OS in newly diagnosed offer deep and homogenous distribution throughout glioblastoma tumors and patients.[2] a means to target their myeloid compartment. Further studies are warranted Several challenges contribute to the dif- to investigate the mechanisms of GO flakes transport within the tumor mass ficulty in treating glioblastoma, which have and their capacity to deliver bioactive cargoes but, ultimately, this information been extensively reviewed by others.[3] First, could inform the development of immunotherapies against glioblastoma. the anatomical localization of the tumor into one of the body’s most vital organs not only sets hurdles to its complete surgical Dr. I. de Lázaro, Dr. P. Sharp, M. Stylianou, T. Kisby, Y. Chen, Dr. S. Vranic, Dr. I. de Lázaro Dr. K. Barr, Dr. C. Bussy, Prof. K. Kostarelos Wyss Institute for Biologically Inspired Engineering at Harvard University Nanomedicine Lab, National Graphene Institute and Faculty of Biology, 3 Blackfan Circle Boston, MA 02115, USA Medicine and Health C. Gurcan, H. Taheri, Dr. A. Yilmazer University of Manchester Department of Biomedical Engineering, Faculty of Engineering AV Hill Building, Manchester M13 9PT, UK Ankara University E-mail: kostas.kostarelos@manchester.ac.uk Ankara 06830, Turkey Dr. I. de Lázaro E-mail: ayilmazer@ankara.edu.tr John A Paulson School of Engineering and Applied Sciences C. Gurcan, Dr. A. Yilmazer Harvard University Stem Cell Institute 58 Oxford Street Cambridge, MA 02138, USA Ankara University Ankara 06520, Turkey Dr. A. Ceylan, Prof. A. Ozen The ORCID identification number(s) for the author(s) of this article Department of Histology Embryology, Faculty of Veterinary Medicine can be found under https://doi.org/10.1002/adtp.202000109 Ankara University © 2020 The Authors. Published by Wiley-VCH GmbH. This is an open Ankara 06110, Turkey access article under the terms of the Creative Commons Attribution Prof. K. Kostarelos License, which permits use, distribution and reproduction in any Catalan Institute of Nanoscience and Nanotechnology (ICN2) medium, provided the original work is properly cited. UAB Campus Bellaterra DOI: 10.1002/adtp.202000109 Barcelona 08193, Spain Adv. Therap. 2021, 4, 2000109 2000109 (1 of 15) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com resection but is also shielded by the blood–brain barrier (BBB). in vivo (orthotopic xenograft) models of the disease. We report The BBB complicates access of systemically administered thera- extensive translocation of the material throughout the tumor peutics to the brain, and the search for strategies that bypass this mass and preferential internalization by the macrophage com- barrier is a very active field in drug delivery research.[4] Addition- partment in vivo, which may offer an opportunity to utilize GO ally, the reported heterogeneity in the molecular signature and as delivery vector targeting this cell population in glioblastoma. mutation load of glioblastoma cells[5] brings in a major compli- cation in the search for efficient drugs, as it is unlikely that tar- geting a single pathway will achieve significant therapeutic out- 2. Results comes. In fact, no significant progress has been made in the de- velopment of new chemotherapies for glioblastoma since the ap- Throughout this work, we used endotoxin-free GO flakes of aver- proval of temozolomide in 2005.[6] Finally, the immune microen- age lateral dimensions of a few hundred nm prepared by a mod- vironment in glioblastoma also appears to be a main culprit of the ified Hummers’ method.[18] A summary of the physicochemical poor prognosis of the disease. Glioblastomas are “cold” tumors characterization of the GO flakes utilized in our study, which have with a paucity of T cells, especially CD8+ cytotoxic T cells, and been described in a previous publication,[19] is provided in Table a predominant myeloid compartment (about 12% of the tumor S1 in the Supporting Information. These GO sheet suspensions mass and 30% of all infiltrating cells) that shows an immunosup- have been shown to be noncytotoxic in a number of cell lines pressive M2-like phenotype.[7] Glioblastoma cells also contribute at the range of concentrations studied here.[15,20] We additionally to the immunosuppressive microenvironment by secreting and confirmed the absence of cytotoxicity induced by the material in expressing immunosuppressive cytokines and surface proteins, Uppsala 87 Malignant Glioma (U-87 MG) cells specifically (Fig- respectively.[7a] ure S1, Supporting Information). The development of nanoscale delivery vectors may offer opportunities to circumvent some of these challenges. For example, nanoparticles can be engineered to efficiently cross the 2.1. GO Induces U-87 MG Spheroid Growth without Cell BBB and they can also protect their cargo from degradation in Proliferation the blood or extracellular space prior to reaching its target.[8] A single nanocarrier can also accommodate and deliver various To investigate the interactions of GO with glioblastoma cells, we therapeutic agents,[9] which is of interest in order to target mul- first exposed 3D spheroids composed of human U-87 MG glioma tiple pathways simultaneously, and the intrinsic properties of cells to 10 or 100 µg mL−1 of GO flakes, dispersed in the cell cul- some nanomaterials can also be exploited for multimodal ther- ture medium, and measured spheroid growth for 12 d. We used apy. An example is the induction of hyperthermia concomitant spheroids because they recapitulate the 3D in vivo architecture with temozolomide delivery.[10] In addition, nanoparticles can be of the tumor and its cell–cell interactions more closely than 2D decorated with targeting ligands to deliver their cargo to specific monolayers.[21] Spheroids were prepared from U-87 MG single cell populations, including glioma cells.[11] Overall, nanoscale cell suspensions and left to form for 3 d prior to GO exposure. GO formulations could improve the treatment of glioblastoma and induced a dose-dependent increase in spheroid diameter (Figure several clinical trials are underway (see Glaser et al.[12] ), but 1a). From day 2 after exposure, statistically significant differences only one nanoparticle formulation has so far received regulatory in spheroid size were observed, and those became larger over approval for this indication. Nanotherm® , a magnetic iron oxide time (Figure 1b, p-values are shown in Table S2, Supporting In- nanoparticle formulation developed to induce hyperthermia, was formation). We used light sheet fluorescence microscopy (LSFM) approved by the European medicines agency (EMA) in 2011.[13] 7 d after treatment to confirm that the exposure to GO induced Graphene oxide (GO), a relatively recent addition to the an increase in tumor spheroid volume (X/Y/Z directions) and not nanomaterial toolbox, offers several advantages over other just in a 2D plane (data not shown). However, several observa- nanocarriers. First, its two-dimensional (2D) structure in the tions suggested that the increase in size was not a result of cell form of oxidized graphene sheets provides a large surface-to- division. First, we enzymatically dissociated individual U-87 MG volume ratio that offers increased loading capacity compared to spheroids into single-cell suspensions, 10 d after GO exposure, other nanoparticles. The presence of multiple oxygen functional- and counted the number of cells with a hemocytometer. No sig- ities grants high dispersibility in aqueous-based biological fluids nificant differences were found in the cell number per spheroid and also allows covalent and noncovalent conjugation possibili- among treatment groups (Figure 1c). Next, we sought to obtain ties with chemically diverse small molecules and biologicals.[14] more precise information on potential differences in the rate of GO is also internalized by phagocytic and nonphagocytic cells cell division, as errors could be introduced due to cell loss or in- with very low cytotoxicity.[14c,15] Overall, GO represents the complete dissociation when generating a single-cell suspension. most widely studied 2D material as a delivery vector in vitro We performed immunohistochemistry (IHC) to detect Ki67, a and in preclinical models in vivo,[14b,14c,15,16] even though its marker of cell proliferation, in cryosections obtained from U-87 interactions with relevant models of glioblastoma remain poorly MG spheroids 7 d after exposure (Figure 1d) and quantified the described.[17] Here, we aimed to take a first step toward the abundance of Ki67+ cells (Figure 1e). A small but significant de- investigation of GO as a potential delivery vector for the treat- crease in the number of proliferating Ki67+ cells was observed ment of glioblastoma. We characterized the interactions of thin, in GO-treated spheroids, which was also dose dependent (Fig- biomedical-grade GO flakes (i.e., endotoxin-free and highly ure 1e). This observation was also supported by the levels of Ki67 dispersible in biological fluids) of controlled dimensions (lateral mRNA measured by real-time quantitative reverse transcription size < 1µm, thickness 1–2 nm) with in vitro (3D spheroids) and polymerase chain reaction (RT-qPCR), which were significantly Adv. Therap. 2021, 4, 2000109 2000109 (2 of 15) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com Figure 1. Exposure to GO induces U-87 MG spheroid volume growth, in a dose-dependent manner, without cell proliferation. U-87 MG spheroids were exposed to 0, 10, and 100 µg mL−1 GO in the cell culture medium. a) Images of spheroids 0, 1, 7, and 12 d after exposure (scale bars represent 500 µm). Adv. Therap. 2021, 4, 2000109 2000109 (3 of 15) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com downregulated with the GO treatment 12 d after exposure (Fig- shows brightfield images of tissue sections utilized for Raman ure 1f). Overall, these results confirmed that the exposure of U-87 mapping, as well as the individual maps of different regions of MG spheroids to GO flakes in vitro correlated with a significant each spheroid that have been combined in Figure 3b. Further de- increase in spheroid volume but a decrease in tumor cell prolif- tails of Raman data acquisition and analysis are provided in Fig- eration. ure S2 and Table S3 in the Supporting Information. We also performed transmission electron microscopy (TEM) 2.2. GO Flakes Translocate Deeply into U-87 MG Spheroids but in nearby sections, which provided much higher resolution than Remain Primarily within the Extracellular Compartment LSFM or Raman mapping, to confirm the subcellular localiza- tion of GO flakes (Figure 3c). The material accumulated predomi- We next set out to investigate the distribution of GO flakes nantly in the extracellular space, with minimal intracellular local- within the spheroid 3D structure. The optical properties of ization, although we were able to identify various cells in which a GO allow label-free detection of the material by fluorescence small amount of GO was internalized in vacuole-like structures microscopy.[20] LSFM of whole mount spheroids exposed to GO (blue arrows). We also observed a small number of cells prepar- for 24 h showed penetration of the material for several layers of ing to uptake the material (green arrows). To further interrogate cells. Figure 2 shows optical sections taken at different depths the scarce cellular uptake of GO sheets in the spheroids, we ex- from the front to the back of the spheroid. Next, ultrathin sec- posed U-87 MG cells grown in 2D monolayers to the same con- tions of U-87 MG spheroids were obtained 7 d after GO exposure, centrations of the material (Figure S3, Supporting Information). for further investigation. Toluidine B staining analyzed by bright- The fluorescence signal of GO was barely observed in the intra- field microscopy demonstrated the increased accumulation of cellular space by confocal microscopy throughout the culture, as extracellular matrix (ECM) in GO-treated spheroids, in a dose- opposed to previous studies using different cell types exposed to dependent fashion (gray, unstained areas, compared to purple- the same concentrations of the same material.[15,20] This further stained ones, Figure 3a), which quite likely accounted for the in- confirmed the very limited degree of cellular uptake and internal- crease in spheroid volume reported in our study. These data fur- ization obtained within the 3D spheroids. ther supported our previous observation that the GO-induced in- Transport of GO from the periphery to the core of the crease in spheroid volume was not linked to an increase in cell spheroids could be ascribed to either a dynamic process medi- proliferation. ated by cell-to-cell transfer (i.e., transcytosis-like)[22] or a passive We next performed Raman mapping of neighboring spheroid mechanism via diffusion in the extra-cellular space, in between sections to localize the distribution of GO flakes (Figure 3b). adjacent cells. Considering the scarce evidence of cellular inter- This technique, and the use of sections, allowed us to investi- nalization found here, the latter is the most plausible mecha- gate the presence of GO at the core of the spheroids with more nism. In addition, TEM images also evidenced an increase in the confidence. No Raman fingerprints specific to GO were found abundance of collagen-like fibers within the extracellular space in the control nontreated spheroids. In contrast, when spheroids of GO-treated spheroids (indicated with red arrows in Figure 3c). were exposed to either 10 or 100 µg mL−1 of GO flakes, clear This aligned well with our observation of an increased presence Raman spectra specific to GO fingerprints, including D and G of ECM upon GO treatment using toluidine blue staining. Over- bands at about 1352 and 1594 cm−1 , respectively, could be iden- all, these results confirmed that GO is barely internalized by U-87 tified within the sections (shown in green in the maps, Fig- MG cells. Instead, it translocated deeply into 3D spheroids, most ure 3b). More interestingly, when increasing the concentration likely through the extracellular space, and could be responsible from 10 to 100 µg mL−1 , the mapping revealed that a larger sur- for triggering the increased secretion of ECM components by the face of the spheroids was positive for GO fingerprints, which cells within the spheroids. confirms again that the accumulation of GO flakes in spheroids is a dose-dependent phenomenon. In addition, GO positive re- 2.3. GO Has Minimal Impact on the Transcriptomic and gions were identified not only at the rim but also at the core of Proteomic Signatures of U-87 MG Spheroids the spheroid. This supported our LSFM observations that GO translocated deeply into the spheroids. Poor penetration into 3D We then examined whether the exposure to GO induced changes tissues is indeed a limitation suffered by a plethora of nanopar- in gene and protein expression in U-87 MG cells. Previous stud- ticles that perform well when tested in 2D monolayers, but fail ies using other cell lines have demonstrated that GO can have to translocate sufficiently and evenly into 3D structures resem- significant impact in gene expression, even in transcripts that are bling solid tumors.[21a] Figure S2 in the Supporting Information usually utilized as reference or housekeeping genes.[16b,23] For b) Spheroid growth curve. Data show mean diameter ± SD, n = 12. *p < 0.05, **p < 0.01, and p < 0.001 indicate statistically significant differences between untreated and [GO] = 10 µg mL−1 groups. 𝛿𝛿𝛿p < 0.001 indicates statistically significant differences between untreated and [GO] = 100 µg mL−1 groups. ɸɸp < 0.01 and ɸɸɸp < 0.001 indicate statistically significant differences between [GO] = 10 µg mL−1 and [GO] = 100 µg mL−1 groups. Statistics were assessed by one-way ANOVA and Tukey’s post hoc test or by Welch ANOVA and Games-Howell post hoc test, depending on the homogeneity of variance, and a summary is presented in Table S2 in the Supporting Information. c) Cell counting upon spheroid dissociation (10 d after treatment) shows no statistically significant (n.s.) differences in the number of cells among treatment groups, assessed by one-way ANOVA and Tukey’s post hoc test, n = 3. Data are presented as mean ± SD. d) Ki67 was detected in spheroid sections fixed 7 d after treatment by IHC and e) Ki67+ cells were quantified in four random fields per condition; *p < 0.05 and **p < 0.01 indicate statistically significant differences, assessed by one-way ANOVA and Tukey’s post hoc test (n = 4). Data are shown as mean ± SD. f) Ki67 mRNA levels were assessed by real-time RT-qPCR on day 12 after exposure. *p < 0.05 and **p < 0.01 indicate statistically significant differences, assessed by one-way ANOVA and Tukey’s post hoc test, (n = 3). Data are shown as mean fold change ± propagated error. Adv. Therap. 2021, 4, 2000109 2000109 (4 of 15) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com Figure 2. GO penetrates in U-87 MG spheroids but remains in the extracellular space. Images of spheroids one day after exposure to 0, 10, and 100 µg mL−1 GO, acquired using Lightsheet Z.1 fluorescence microscope. Frontal, upper quarter, and middle sections of the spheroids are shown. DAPI staining is shown in blue and GO autofluorescence signal is shown in magenta. Scale bars indicate 100 µm. this reason, we first determined the stability of the expression of TIMP2 (fold change = 1.55) and LAMB1 (1.34) were upregulated ten different candidate reference genes upon exposure of U-87 24 h after exposure (Table S5, Supporting Information). On MG spheroids to different doses of GO, and selected the combi- day 7, we only confirmed the upregulation of MMP9 (1.68) nation of GAPDH and MAPK1 as the most stable normalization and IDH1 (1.34) and the downregulation of NES (0.54). 12 d factor for further gene expression studies, according to the after exposure, only LAMB1 was slightly downregulated (fold Normfinder algorithm (Figure S4, Supporting Information). We change = 0.80) compared to untreated controls. The highest then performed real-time RT-qPCR to investigate the mRNA lev- dose of GO (100 µg mL−1 ) induced changes in a slightly larger els of several genes involved in critical pathways in glioblastoma number of transcripts, but fold changes were also small (all (Figure S5, Supporting Information). A total of 87 transcripts fold changes < 2.6, Table S6, Supporting Information). Most involved in proliferation (Figure S5a, Supporting Information), differentially expressed genes were involved in ECM deposition apoptosis (Figure S5b, Supporting Information), invasion and or remodeling and cell–cell adhesions. However, we did not find migration (Figure S5c, Supporting Information), integrins in- a clear pattern among the transcripts under study, since some volved in cell migration (Figure S5d, Supporting Information), of them were down- and others upregulated after GO treatment proteases involved in ECM remodeling (Figure S5e, Supporting (Figure S5 and Tables S5 and S6, Supporting Information). Information), genes involved in ECM deposition (Figure S5f, Prompted by the very few changes observed in the genes Supporting Information), angiogenesis and hypoxia (Figure S5g, interrogated, which we selected based on their relevance to Supporting Information), and other markers of glioblastoma glioblastoma progression, we decided to perform a broader, (Figure S5h, Supporting Information) were investigated. Table unbiased analysis of changes at the protein expression level S4 in the Supporting Information includes information related (Figure 4). For that, we performed liquid Chromatography with to all such genes and the primer pairs utilized in this study. tandem mass spectrometry (LC-MS/MS) upon exposure of P-values for genes that showed statistically significant changes U-87 MG spheroids to the highest dose of GO (100 µg mL−1 ). in mRNA expression are given in Table S5 in the Supporting Principal component analysis (PCA) demonstrated that the Information (10 µg mL−1 dose) and Table S6 in the Support- samples clustered according to experimental conditions, namely ing Information (100 µg mL−1 ). Changes in gene expression GO treatment or no treatment (Figure 4a). Investigation of were very small. At the lowest GO dose (10 µg mL−1 ), only differentially expressed proteins confirmed that only 37 out Adv. Therap. 2021, 4, 2000109 2000109 (5 of 15) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com Figure 3. GO distributes primarily within the extracellular space of U-87 MG spheroids and correlates with increased deposition of ECM. Spheroid sections were fixed 7 d after exposure to 0, 10, or 100 µg mL−1 GO. a) Toluidine blue staining of U-87 MG spheroid sections shows increased accumulation of ECM (gray areas) with increasing doses of GO. Scale bars indicate 100 µm. b) Chemical mapping by Raman spectroscopy imaging confirms dose- dependent distribution of GO flakes which is not limited to the rim of the spheroid but reaches the core, scale bars indicate 100 µm. c) GO flakes are clearly visible in the extracellular space of U-87 MG spheroids in TEM images, but intracellularly located GO is scarce. Red arrows indicate collagen fibers. Green arrows indicate cells preparing to phagocytize GO flakes. Blue arrows indicate vacuoles containing GO. Scale bars represent 1 or 5 µm as indicated in the images. of 1698 proteins were differentially expressed compared to differentially expressed proteins retrieved terms related to endo- untreated controls (Figure 4b and Tables S7 and S8, Supporting cytosis, phagocytosis, and vesicle transport as the most enriched Information). These results further supported our observation biological process terms (Figure 4c). Cellular compartment gene that the changes in the molecular signatures of U-87 MG cells ontology terms also included terms related to endocytic and upon exposure to GO were very small. Gene ontology analysis of cytoplasmic vesicles, as well as collagen containing-extracellular Adv. Therap. 2021, 4, 2000109 2000109 (6 of 15) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com Figure 4. Exposure to GO flakes induces minor changes in U-87 MG protein expression. U-87 MG spheroids treated for 7 d with the highest dose of GO (100 µg mL−1 ), or left untreated, were processed for proteomics analysis (n = 3). a) PCA with all detected proteins indicated that the samples clustered according to treatment. b) Volcano plot shows differentially expressed proteins between the groups, obtained with the DEP package of Bioconductor. Fold changes were calculated normalizing to the untreated group. The Clusterprofiler package (Bioconductor) was implemented to perform Gene Ontology. Dot plots show c) biological process, d) cellular compartment, and e) molecular function Gene Ontology terms. matrix and extracellular matrix, that scored as the 3rd and 4th recapitulating the in vivo microenvironment. In particular, the most enriched terms, respectively (Figure 4d). Finally, molecular model utilized here did not account for the presence of the function terms included several related to peptidase activity stromal, vascular, and immune compartments in glioblastoma, (Figure 4e). among other factors. For this reason, we set out to determine Overall, these analyses demonstrated that the exposure of U- the fate and effects of GO sheets following direct intratumoral 87 MG spheroids to GO only induced minor changes in the ex- injection in an orthotopic xenograft model of glioblastoma. pression of a small number of genes and proteins, most of them U-87 MG cells were implanted intracranially in athymic nude likely related to the attempts of the cells to uptake the material mice and tumors were left to grow for 16 d after inoculation. At and to the increased generation of ECM. This contrasts with the this point, mice received a second intracranial administration observations found in other cell types, in which changes in gene of either 1 µL of dextrose (vehicle control), 0.1 mg mL−1 GO, and protein expression were much more remarkable.[16b,23] How- or 1 mg mL−1 GO. Magnetic resonance imaging (MRI) of the ever, this could be linked to the much lower internalization of the tumor volume over time demonstrated no significant differences material in U-87 MG cells. in the evolution of tumor growth across treatment groups, for the duration of the study, contrary to our observations in vitro (Figure 5a,b). In addition, no differences were observed in 2.4. Stereotactic, Intratumorally Administered GO Sheets the survival of the mice for the duration of the study. Tumor Distribute Extensively throughout the Tumor Volume and Are volume was also measured from the reconstruction of H&E Internalized by Resident Microglia/Macrophages in vivo stained brain tissue sections, which confirmed the absence of differences in tumor size between groups (Figure 5c). These While 3D in vitro tumor models better represent the in vivo results suggested that intratumoral administration of GO did not scenario than their 2D counterparts, they are still far from fully significantly impact glioblastoma progression in the timeframe Adv. Therap. 2021, 4, 2000109 2000109 (7 of 15) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com Figure 5. GO administered intratumorally distributes extensively in a U-87 MG orthotopic xenograft and does not induce tumor growth or ECM deposi- tion. Athymic nude mice were implanted with 1 × 105 U-87 MG cells, in 1 µL PBS, into the right striatum and intracranially administered with 1µL of 5% Adv. Therap. 2021, 4, 2000109 2000109 (8 of 15) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com of our study (28 d after tumor cell inoculation). In addition, 3. Discussion histological investigation of the brains 12 d after intratumoral delivery of GO showed that the material had distributed exten- This study described the interactions of GO flakes with two mod- sively throughout the entire volume of the tumor but remained els of glioblastoma (in vitro and in vivo) as an initial step toward largely confined to its margins (Figure 5d). The extraordinary investigation of this material as a delivery platform technology distribution of GO throughout the tumor, in spite of the small for the treatment of glioblastoma. In vitro studies with 3D U- volume that was administered (1 µL), has important implica- 87 MG spheroids helped us determine the interactions and ef- tions in the potential of GO as a delivery vector for glioblastoma fects of GO in glioblastoma tumor cells alone (in the absence of therapies. an immune component). We have demonstrated that GO sheets Since we did not observe any significant increases in tu- transported and localized at the extracellular space throughout mor volume in vivo following GO treatment, contrary to our the spheroid volume, and that the material was scarcely internal- observations in spheroids in vitro, we decided to investigate ized by this tumor cell type (Figure 3c and Figure S4, Supporting the deposition of ECM in the tissue. We did not observe dif- Information) with no cytotoxicity at the dose-range studied (Fig- ferences in the deposition of collagen I in the tumor when ure S1, Supporting Information). Our cell internalization results GO was administered (Figure 5e–g). The lack of increased are in agreement with another previous report indicating strong ECM deposition is in marked contrast to the results of in vitro interactions of GO flakes with the plasma membrane, but very spheroids, so we next examined the subcellular distribution of poor uptake in U-87 MG and other glioma-like cells (at least three the material in vivo (Figure 6). We stained brain tumor sections times lower than other carbon nanomaterials).[17a] The lack of cy- from U-87 MG glioma mice administered with GO for IBA1+ totoxicity of GO in glioma cells has also been reported by others, cells, which are primarily macrophages and microglia. We used who also demonstrated its lack of genotoxicity.[17b] More studies phase contrast light microscopy to identify GO (black particles) are warranted in order to reveal the mechanisms of GO sheet in- and fluorescence microscopy to detect IBA1+ cells (Figure 6a). teraction with glioblastoma and other cancer cell types both in The data showed that >60% of GO particles identified were monolayer and 3D conformation. internalized by IBA1+ cells (Figure 6b). Because some glioma We did observe an increase in the deposition of ECM upon cells, including U-87 MG, can upregulate IBA1,[24] we also exposure of U-87 MG spheroids to GO in vitro (Figure 3a,c and costained brain tissue sections with an antibody against hu- Figure S2, Supporting Information). Indeed, many of the few man mitochondria (Mit) that allowed us to identify whether changes observed at the gene and protein levels were related to IBA1+ cells have mouse (macrophages/microglia) or otherwise ECM deposition and remodeling, according to gene ontology human (U-87 MG) origin (Figure 6c). Approximately 20% of (Figure 4). Because such processes are frequently utilized by GO particles colocalized with IBA1+ /Mit+ double positive cells cancer cells, including glioma cells, to migrate and infiltrate (i.e., originated from the U-87 MG xenograft, Figure 6d). This the tissues,[25] this could potentially be a source of concern on result was expected since we observed some, albeit low, inter- the safety of the material in its interaction with glioblastoma. nalization of GO flakes in glioblastoma cells in vitro. Overall, However, we confirmed that there were no alterations in ECM these results demonstrated that intratumoral administration deposition when the material was administered in vivo (Fig- of GO results in a predominant internalization of the nano- ure 5d). This discrepancy could be due to the predominant material by macrophages and microglia infiltrating the tumor, sequestration of GO sheets by macrophages and microglia in and that GO penetrates readily throughout the tumor with the orthotopic model (Figure 6). The increased deposition of no significant impact on tumor growth or progression. Given ECM in GO treated spheroids is likely responsible for their the crucial role that macrophages and microglia play in the dose-dependent increased volume, which we confirmed was not establishment of the immunosuppressive microenvironment in caused by higher levels of tumor cell proliferation. Again, these glioblastoma,[7b] these observations warrant the further investi- changes in tumor size were only observed in vitro and, overall, gation of GO as a vector system in the context of glioblastoma these findings highlight the inadequacy of oversimplified in vitro immunomodulation. models (even if in 3D) to recapitulate the in vivo scenario and dextrose, 0.1 mg mL−1 of GO, or 1 mg mL−1 GO 17–21 d later (n = 5). For longitudinal assessment of tumor growth, tumor bearing mice underwent MRI (n = 2 for dextrose, n = 3 for GO groups). a) Representative T2 -weighted gradient echo MRI images for a vehicle (5% dextrose) control mouse showing intracranial tumor growth. Baseline tumor volumes were measured 14 d post tumor inoculation (p.i.) to demarcate the center of the tumor for subsequent intratumoral administration of dextrose or GO. MRI was repeated on days 21 and 28 d p.i., to determine the effect of GO on tumor growth. Scale bar, 4 mm. b) MRI longitudinal assessment of tumor volume in dextrose and GO groups. Data are displayed as mean tumor volume ± standard error (SE). No statistical significance was found between the groups, assessed by one-way ANOVA. c) Tumor volume calculated from H&E stained sec- tions 28 d p.i. from dextrose (n = 5) and GO treated mice (1.0 or 0.1 mg mL−1 , n = 5 for each group). Data are displayed as mean tumor volume ± SD and each dot represents an individual tumor. No statistical significance was found between GO treated mice and vehicle controls (one-way ANOVA). d) GO distribution in intracranial tumors from two mice, 12 d after direct intratumoral injection of 1 mg mL−1 GO (1 µL). The images of frozen brains undergoing cryosectioning clearly show diffusion of the material throughout the tumor. H&E staining of the tissue indicates that GO remains confined within the tumor boundaries. e) Collagen I (green) and DAPI (blue) staining performed in brain sections collected 12 d after GO administration. Scale bar, 20 µm. f) Quantification of collagen I staining per field of view. Each dot represents the average number of collagen I positive pixels in three ran- domly selected fields of view within the brain tumor area, four tissue sections per animal, n = 5 animals per group. P-values indicated on the graph were assessed using a Mann–Whitney U test. g) Quantification of collagen I staining per field of view. Each dot represents the number of collagen I positive pixels in a randomly selected field of view within the brain tumor area. Three random fields of view per section, four tissue sections per animal, n = 5 animals per group. P-values indicated on the graph were assessed using a Mann–Whitney U test. Adv. Therap. 2021, 4, 2000109 2000109 (9 of 15) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com Figure 6. Intratumorally injected GO is predominantly internalized by IBA1+ macrophages and microglia. a) Representative bright-field (phase-contrast) and immunofluorescence images of U-87 MG xenografts collected 12 d after dextrose or 1 mg mL−1 GO administration (n = 5) show predominant localization of GO (black) in IBA1+ cells (red). Scale bar, 20 µm. b) Quantification of the percentage of GO (black particles) in IBA1+ cells. Each dot represents the average of quantified GO particles in two fields of view from three separate frozen brain tumor sections per animal, n = 6 per group. c) Representative bright-field (phase-contrast) and immunofluorescence images of IBA1 and human mitochondria (Mit) staining. d) Quantification of the percentage of GO (black particles) in IBA1+ /Mit+ cells (human origin). Each dot represents the average of quantified GO particles in two fields of view from three separate frozen brain tumor sections per animal, n = 5 per group. confirm that the results obtained in such systems should be in- flakes by IBA1+ macrophages and microglia in the orthotopic terpreted with caution. Indeed, there is an urgent need for more xenograft model (Figure 6). These cell types acquire a M2-like physiologically relevant ex vivo models that not only recapitulate phenotype within the tumor which is partially responsible for the the 3D architecture of the tissue, but also integrate stromal and immunosuppressive microenvironment that contributes to the immune compartments, perfusable vasculature and that mimic poor prognosis of glioblastoma.[7b] Due to its detrimental role the chemical and mechanical properties of the in vivo ECM.[26] in this and other so-called “cold tumors”, there is an increasing One of the most interesting findings from this study was number of strategies aiming to either deplete or reprogram the preferential uptake of intratumorally administered GO the macrophage compartment toward a M1-like “tumoricidal” Adv. Therap. 2021, 4, 2000109 2000109 (10 of 15) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com or “proinflammatory” phenotype.[27] Different approaches have 4. Conclusion explored a plethora of pathways including macrophage de- pletion with small molecule inhibitors or antibodies against We have shown here that GO flakes of controlled lateral dimen- the colony-stimulating factor 1 (CSF-1) receptor[28] and M2 to sions (<1 µm) distribute extensively throughout the tumor mass, M1 macrophage re-education via small drug toll-like receptor are scarcely uptaken into glioblastoma cells, and are preferen- (TLR) agonists,[29] cytokines[30] or microRNAs,[31] among others. tially internalized by macrophages and microglia. These findings We envision that preferential internalization of GO flakes in suggest that GO could be utilized as a platform for the devel- IBA1+ cells may offer an opportunity to utilize this material as a opment of immunomodulation strategies against glioblastoma nanocarrier that delivers the necessary cues for these and similar based on targeting the macrophage and microglia compartment. strategies. The great versatility that GO offers to incorporate, Studies that thoroughly investigate the impact of GO uptake on covalently or noncovalently, and deliver very chemically diverse the macrophage phenotype and the capacity of these vectors to payloads will likely also be an advantage toward these strategies. efficiently deliver immunomodulatory cues should follow. In addition, reports have also pointed at an intrinsic capacity of GO flakes to induce M1 to M2 macrophage polarization, 5. Experimental Section although the mechanisms are still not fully understood.[32] Indeed, the interactions of GO and other graphene derived Reagents and Chemicals: Cell culture reagents and chemicals were pur- materials with the immune system are being thoroughly re- chased from Sigma-Aldrich (Merck, UK) unless otherwise stated. GO: Biological-grade GO was produced at the University of Manch- searched, incorporating concepts of “immunity-by-design” that ester following a modified Hummers’ method and size separation by aim to provide next generation immune-targeted therapeutics centrifugation as previously reported.[18] GO flakes of average lateral through rational material design.[33] Overall, strategies targeting dimensions of few hundred nm and one to three carbon atom layers in the macrophage compartment will likely synergize with other thickness were selected for this study based on their relevance to biomed- immunotherapy, chemotherapy, and radiotherapy interven- ical applications. Sheets of such dimensions have been termed “small tions against glioblastoma, and thus combination studies are GO flakes” in previous studies[18] to differentiate them from counterparts with larger or shorter lateral dimensions. Table S1 in the Supporting Infor- warranted. Beyond immunotherapy, recent studies have also mation summarizes the main physicochemical properties of the specific shown that macrophages that uptake drug-loaded nanoparticles batch of material utilized in this study, which was fully characterized in a can act as local depots for extended release of the therapeutic previous study.[19] The material was confirmed endotoxin-free. payload.[34] Cell Culture: U-87 MG human glioblastoma cell line was obtained To our knowledge, the intratumoral distribution of GO sheets from the American Type Culture Collection (ATCC) and authenticated at throughout the human orthotopic glioblastoma xenograft mass the University of Manchester. Cells were maintained in Dulbecco’s Modi- observed in this work has not been previously reported. Other fied Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, UK) and 1% antibiotics (penicillin groups have pursued the intravenous administration of modi- and streptomycin, PenStrep). For in vivo studies, U-87 MGcells were used fied GO, functionalized either with transferrin or with iron ox- below passage 10. ide nanoparticles, in an allogeneic rat glioma model.[35] How- 3D Spheroid Formation: U-87 MG cells grown as monolay- ever, none of their studies has determined the translocation of ers were detached into a single-cell suspension with trypsin/ GO into the brain, let alone its distribution and internalization ethylenediaminetetraacetic acid (EDTA). 10 000 cells were seeded in by different cell types populating the tissue. Our studies demon- each well of a 96-well plate, previously coated with 1% agar, in 200 µL complete cell culture medium (composition above). Spheroids were left strated consistently the capacity of GO sheets to diffuse exten- to form for 3 d before the exposure to GO flakes. sively throughout the entire tumor volume, both in vitro (Fig- U-87 MG Exposure to GO Flakes: For studies with U-87 MG spheroids, ures 2 and 3) and in vivo (Figure 5). In the latter, uptake by mi- cell culture medium was removed on day 3 after spheroid seeding and gratory macrophages and microglia may have also contributed replaced with cell culture medium containing 10 or 100 µg mL−1 of GO to the distribution of the material. This extensive translocation flakes, without FBS. U-87 MG cells grown in monolayers were also ex- constitutes a unique feature among different nanoparticles and posed to GO flakes at 10 or 100 µg mL−1 concentrations without FBS. Af- ter 4 h of incubation, GO-containing medium replaced by fresh complete adds further support to the potential of GO as a delivery vec- medium. tor in direct, intratumoral therapeutic strategies against glioblas- Monitoring of U-87 MG Spheroid Growth: The diameter of U-87 MG toma. Indeed, even small drug molecules can suffer in terms of spheroids exposed to GO was measured on the day of exposure and 2, distal spread and transport within the brain because the ECM 4, 6, 8,10, and 12 d after treatment. Images were taken with an inverted is very densely packed with charged proteins that can bind the light microscope (Primovert, Zeiss), focusing on the equatorial plane. 12 molecules and inhibit their penetration into the tissue.[36] There- spheroids were monitored per condition. fore, it is of great importance to engineer delivery vectors that Cell Counting from Dissociated Spheroids: Spheroids treated with 0, 10, or 100 µg mL−1 of GO flakes were dissociated into single cell suspen- achieve uniform drug/gene delivery across the tumor to treat ag- sions 10 d after the exposure. In brief, n = 8 spheroids per condition were gressive gliomas and potentially other brain disorders. We rec- centrifuged and treated with 0.01% trypsin/EDTA solution. Following en- ognize that intracranial administration as utilized in this study zymatic treatment, single cells were resuspended in complete media and is not the first choice in the clinic. However, stereotactic brain cells were counted on a hemocytometer. surgery has been used for decades and is justified in such se- Annexin V/propidium iodide (PI) Staining and Flow Cytometry: U-87 vere and life-threatening scenario.[37] Other alternatives to con- MG cells seeded in P12-well plates (Costar, Corning) were treated with GO flakes (10 and 100 µg mL−1 , 0.5 mL per well), as described above, for sider are the administration of GO nanoparticles at the time of 24 h. After treatment, cells were detached using 0.05% trypsin–EDTA for surgery, in the case of resectable tumors, or further functional- 10 min, followed by the addition of FBS (30 µL per well, Gibco, Thermo ization of the flakes to incorporate molecules that facilitate BBB Fisher Scientific, UK), and collected in 1.5 mL tubes. Cells were centrifuged translocation. for 5 min at 1500 rpm and resuspended in 1X diluted Annexin-binding Adv. Therap. 2021, 4, 2000109 2000109 (11 of 15) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com buffer (200 µL per tube, v13246, Thermo Fisher Scientific, UK). 1 µL per by two specific D and G bands at about 1352 and 1594 cm−1 , respectively, tube of Annexin V (A13201, Thermo Fisher Scientific, UK) was added and and a less prominent 2D band at about 2707 cm−1 . This characteristic incubated with the cells for 20 min in the dark. Samples were stored on spectrum can be used as fingerprints to reveal the presence of GO in any ice, 1 µL per tube of propidium iodide (P4864-10ML) was added 30 s matrix, including sections of spheroids embedded in resin. Herein, Raman before the analysis by FACSVerse flow cytometry using the fluorescein mapping of the GO location within spheroid sections was performed us- isothiocyanate (FITC, for Annexin V) and phycoerythrin (PE, for propidium ing a HORIBA XploRA PLUS Raman microscope working with a 638 nm iodide) channels. Excitation/emission wavelength: Annexin V = 495/519, excitation wavelength. Raman mapping was performed using a 50x ob- propidium iodide = 493/636. jective by collecting a Raman spectrum at each single pixel of the region Cryosectioning of 3D Spheroids: Spheroids were collected and frozen of interest, using a 638 nm laser operating at 25% of power, through a in Cryomatrix embedding resin (Thermo Fisher Scientific, UK). The frozen 300 µm hole and 100 µm slit, and an acquisition time of 1 s for each spec- spheroid blocks were sectioned at 10 µm thickness using a Leica CM1510- trum. A step size of 2 µm was used between each collected spectrum. 3 cryostat (Leica Biosystems, Germany). Cryosections were placed on poly- Each Raman map was about 15–20 h of data collection; the size of each l-lysine microscope slides (Thermo Fisher Scientific, UK) and preserved at map was adapted to the spheroid to be mapped. Due to the increasing −20 °C until use. size of spheroids with the applied GO concentration, Raman maps of non- Immunohistochemical Staining (IHC): Spheroid cryosections were treated spheroids were hence made of a single map; while Raman maps fixed in pure acetone for 10 min and rehydrated in phosphate buffered of 10 and 100 µg mL−1 GO treated spheroids were made of two to three saline (PBS) with 0.3% triton (PBS-T) for 15 min. Endogenous peroxidase and three to four maps, respectively (Figure S2, Supporting Information). activity was blocked by quenching with H2 O2 (3% in methanol) for Consecutive maps were slightly overlapping in order to allow stitching 30 min. Sections were incubated with 10% normal goat serum for 30 min and reconstruction of the final map using common map features at the to prevent nonspecific binding of antibodies. Subsequently, the sections edges. were incubated overnight with primary antibodies at 4 °C. After three Raman Image Analysis: A convolution[38] -based fuzzy template washings in PBS, the biotinylated secondary antibody (Histostain-Plus matching algorithm was used to identify the representative peaks for GO IHC Kit, Invitrogen, USA) was added and incubated for 30 min at RT. Later, at shifts of approximately 1352 and 1594 cm−1 based on a template of sections were treated with enzyme-conjugated streptavidin (Histostain- a typical GO fingerprint. Convolution computes the “overlap” between Plus IHC Kit, Invitrogen, USA) for 30 min at RT and then visualized two functions and is a standard technique in signals processing and im- with 3,3-diaminobenzidine tetrahydrochloride (Invitrogen, USA). Finally, age analysis. It is used for template matching in popular tools such as sections were counterstained with Gill’s hematoxylin, dehydrated, and OpenCV.[39] This algorithm was implemented in Python 3.7.4 and makes mounted in Entellan (Merck, Germany). Images were acquired with a extensive use of the SciPy signal library. First, data containing undefined microscope digital camera (DFC450, Leica, Germany) integrated into a values were excluded, and the rest were smoothed by a median filter, which light microscope (DM2500, Leica, Germany). Antibody information is removed most cosmic ray spikes. To reduce the computational load for the provided in Table S9 in the Supporting Information. convolution step, spectra were filtered based on the means of shifts inside Light Sheet Fluorescence Microscopy (LSFM) of Wholemount U-87 MG and outside the D and G bands region. Then, the individual spectra were Spheroids: U-87 MG spheroids were exposed to GO in agar-coated 96 clipped to the size of the template, such that they included the location of well-plates as described above and fixed for whole mount imaging 24 h the characteristic GO peaks. This further reduced the computational load after treatment. In brief, spheroids were transferred to 1.5 mL Eppendorf of the convolution step and avoided false positives due to coincidentally tubes, washed three times with PBS (10 min each), and later fixed in similar peaks at other shifts. The clipped spectra were normalized. Finally, 4% paraformaldehyde (PFA), 0.5% triton (2.5 h at 4 °C). After a wash the smoothed, filtered, clipped, and normalized spectra were convolved in PBS for 10 min, the samples were incubated with 6 × 10−6 m 4′,6- with the template. If the value of the maximum of the convolution was diamidino-2-phenylindole (DAPI) at RT for 40 min. Finally, the spheroids greater than a predefined threshold, then the “scipy.signal.find_peaks” were washed twice in PBS (10 min each) and mounted in 1% low melting function was used to confirm the presence of the representative GO peaks. agarose in MilliQ water, in capillary tubes. For that purpose, agarose was The code used is available at https://github.com/kebarr/raman_analysis. warmed up to 60 °C and then left to cool down to 37 °C to avoid damaging The algorithm was validated by testing it using samples treated with dif- the spheroids. LSFM was performed with a Lightsheet Z.1 microscope ferent concentrations of GO, and the results are summarized in Table S3 (Zeiss). DAPI was excited with the 405 nm laser line and its emission was in the Supporting Information. It can be seen that very few spectra were detected using a bandpass filter (420–470 nm). GO was excited with the identified as containing GO in the case of the control (untreated) samples, 561 nm laser line of the microscope and its fluorescence was recorded and that the number of GO positive sites as a proportion of total sites in in 620–690 emission range. In order to remove any autofluorescence those treated with 100 µg mL−1 GO was, roughly, an order of magnitude background, the microscope was manipulated to incorporate a longpass greater than in the samples treated with 10 µg mL−1 GO. filter (Thorlabs, cutoff 850 nm). Gene Expression: The spheroids were removed from 96-well plates and TEM and Toluidine Blue Staining of Ultrathin Spheroid Sections: lysed by vortexing in lysis buffer from the Purelink RNA Mini Kit (Invit- Spheroids were fixed in 2.5% glutaraldehyde and 4% PFA buffered with rogen). RNA extraction was performed following the manufacturer’s in- 0.1 m sodium cacodylate buffer (pH 7.4) at RT for 2 h and overnight at structions. cDNA was synthesized from 0.5 µg of RNA template using 4 °C. They were then washed with 0.1 m sodium cacodylate buffer and the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) postfixed in 2% osmium tetroxide (OsO4 ). Following several washes with following the manufacturer’s protocol. High throughput RT-qPCR was 0.1 m sodium cacodylate buffer, the spheroids were stained en-bloc with then performed using Fluidigm Biomark HD with a 96.96 dynamic ar- 2% uranyl acetate and dehydrated in a graded ethanol series. This was ray format. Data was normalized to the geometric mean of two refer- followed by specimen embedding in Araldite M mixtures and was per- ence genes (GAPDH and MAPK1) following the Livak method (2−ΔΔCt ). formed according to standard procedures. The Araldite M blocks were GAPDH and MAPK1 were identified as the most stable housekeeping then trimmed, semi-thin sectioned (150–200 nm), and stained with tolui- genes implementing the Normfinder algorithm[40] (Figure S2, Support- dine blue (to aid with the orientation and visualization of samples). Sub- ing Information). n = 3 biological replicates (each including 24 U-87 MG sequently, thin sections were cut and transferred onto copper mesh grids. spheroids to obtain sufficient RNA) were analyzed in duplicates. Data are The grids were stained in 2% uranyl acetate in glass distilled water for shown as fold change ± error (Figure 1f) or in heatmaps showing the mean 15 min. The grids were then secondarily stained with lead citrate for 5 min fold change (Figure S5, Supporting Information). Error was propagated √ and washed with distilled water. Imaging was performed at 120 kV on a following the formula: Error (a + b) = Error(a)2 + Error(b)2 . Statistical Jeol TEM, (JEM1400). Raman Mapping of Spheroid Sections: Semi-thin spheroid sections analysis was performed using ΔCt values. A list of the genes included in prepared as described above were used for Raman spectroscopy imaging the study and the primers used is provided in Table S4 in the Supporting in which GO can be identified by a typical Raman spectrum characterized Information. Adv. Therap. 2021, 4, 2000109 2000109 (12 of 15) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com Mass Spectroscopy: Spheroids (15 per group) were collected and cen- moved and postfixed overnight at 4 °C and later placed in sucrose solu- trifuged at 200 g for 10 min. Pellets were resuspended in lysis buffer tion (30% in PBS) until loss of buoyancy. The brains were snap frozen in (7 m urea, 2 m thiourea, 4% 3-((3-cholamidopropyl) dimethylammonio)-1- precooled isopentane (−50 °C) and coronal sections (20 µm thick) were propanesulfonate (CHAPS), and 20 × 10−3 m Tris-HCl, pH 8.8) to isolate taken using a cryostat (Leica CM3050, Leica Biosystems, Germany) that proteins. Bicinchoninic Acid (BCA) assay (Pierce, Thermo Fisher Scien- incorporated the entire tumor. Sections were either stained with hema- tific, UK) was used to quantify total protein concentration. The profiling toxylin and eosin (H&E) using standard protocols or immunofluorescence of proteins was performed using nano Acquity ultra performance liquid staining. chromatography coupled with LC-MS/MS system on Synapt instrument For the latter, frozen brain sections were fixed in 4% PFA. After fixation, (Waters). For each sample, a total of 500 ng tryptic protein digest was blocking buffer composed of 5% normal donkey serum, 1% bovine serum used. The analysis was performed at the Acibadem Labmed Laboratories albumin, and 0.2% triton in PBS was incubated with the brain sections for (Turkey). The data were first processed with ProteinLynx Global Server v. 1.5 h at RT. Sections were then incubated with primary antibodies at 4 °C 2.5, IdentityE software and Progenesis v4 (Waters). Differential expression overnight. After washing the primary antibody (three times with PBS, 5 min analysis and subsequent plotting was then performed in R (version 3.6.1) each), secondary antibodies were added to the sections for 1 h. Sections following the Differential Enrichment analysis of Proteomics data (DEP) were finally washed three times with PBS (5 min each) and mounted in package in Bioconductor: https://bioconductor.org/packages/devel/bioc/ Prolong Gold medium with DAPI. For antibody information, see Table S9 vignettes/DEP/inst/doc/DEP.html. From this analysis, a list of statistically in the Supporting Information. significant, differentially expressed proteins is obtained. The Uniprot IDs Quantification of Collagen I in Brain Tumor Sections: Collagen I was de- of these proteins were converted into Entrez IDs, then Gene Ontology tected by immunofluorescence performed as described above. The per- analysis was performed using ClusterProfiler. The list of differentially ex- cent of collagen I positive pixels was analyzed using Image J software pressed proteins is included in Table S7 (upregulated) and Table S8 (down- (NIH, Bethesda, USA), as follows. Individual images (field of view at regulated) in the Supporting Information. 20x magnification) were saved as eight-bit images. A threshold (Thresh- Animals: Fifteen female athymic nude-Foxn1nu mice were purchased old = 6) was set on the collagen I channel in comparison with the original from Envigo (UK), housed in individually ventilated cages in groups of 5, image. The images of collage I staining (red pixels) and the background kept in a 12/12 h light/dark cycle with environmental enrichment and ad (black pixels) were transformed into binary images where the Collagen I libitum access to food and water. All procedures were carried out in accor- staining was in black and the rest, white color. A table was generated us- dance with the Animal (Scientific Procedures) Act 1986 (UK), approved by ing the “List” function in the Histogram window, showing the number of the University of Manchester Ethical Review Committee and under a UK black (“0” row) and white (“255” row) pixels. The percentage of collagen I Home Office Project License 70/7763. positive pixels was calculated as [black pixels/(black + white pixels) *100]. U-87 MG Orthotopic Xenograft: Fifteen female athymic nude mice (8– Three random fields of view were analyzed in each of four brain tumor 9 weeks old) were anaesthetized using isoflurane (3% induction and 1–2% tissue sections per mouse (n = 5). maintenance in medical oxygen, at a rate of 1.5 L min−1 ) and placed in a Quantification of GO and IBA1+ Cells in Brain Tumor Sections: Im- rodent stereotactic frame. After the incision site was sterilized, a 0.7 mm munofluorescence for IBA1+ cells and human mitochondria (Mit) was bore hole was drilled above the right striatum at the following co-ordinates: performed as described above. Images were captured as z-stacks using 0.0 mm anterior and 2.5 mm lateral from bregma. A 10 µL Hamilton sy- an inverted SP8 laser scanning confocal microscope. The fields were se- ringe (701 RN) fitted with a 26-gauge needle was advanced to 3 mm below lected using the bright field channel to detect black particles that indicate the cortical surface and withdrawn by 0.4 to 2.6 mm. 1 µL PBS containing the presence of GO. Images were analyzed in FIJI (Image J software). Black 1 × 105 U-87 MGcells was then injected into the intracranial pocket at a particles identified as GO were drawn individually in the images obtained rate of 0.2 µL min−1 . After injection, the needle was kept in place for 3 min from GO injected brains and saved in region of interest (ROI) manager. before being slowly extracted to limit cell build-up in the needle track. No black particles were found in dextrose injected brains. Markings from Intratumoral Administration of GO: 17–21 d after tumor inoculation, GO particles were overlapped with the sum of the images of IBA1 staining mice bearing U-87 MG gliomas (n = 15) were assigned to three exper- in which black particles were in focus. The total number of GO particles imental groups (n = 5 each) for intratumoral delivery of: 5% dextrose, and the number of GO particles that colocalized with IBA1+ cells per field 1.0 mg mL−1 GO in 5% dextrose or 0.1 mg mL−1 GO in 5% dextrose, in of view were counted and the percentage was calculated. The same quan- 1 µL total volume. Mice were anaesthetized and positioned in a stereotac- tification was used to calculate the number of GO particles in IBA1+ /Mit+ tic frame and a 33-gauge needle was advanced through the original bore double positive cells. hole to a depth of 2.6 mm below the cortical surface. The solution was Statistical Analysis: Statistical analysis was performed with GraphPad delivered at a rate of 0.2 µL min−1 . Prism (version 8) and IBM SPSS Statistics (version 25) software. A non- MRI Acquisition and Analysis: MRI was performed 2, 3, and 4 weeks af- parametric test (Mann–Whitney) was used to analyze data that were not ter tumor inoculation (5% dextrose control, n = 2; GO 1 mg mL−1 , n = 3; normally distributed. When data were normally distributed, Levene’s test GO 0.1 mg mL−1 , n = 3). MRI was conducted using a 7 Tesla magnet was first implemented to confirm the homogeneity of variance. When connected to a Bruker Advance III console (Bruker Biospin Ltd, UK). Mice the variance was homogeneous, statistically significant differences among were placed in a custom-built Perspex magnet capsule with a cylindrical groups were analyzed by one-way analysis of variance (ANOVA) and surface coil and imaged under isoflurane anesthesia (3% induction and 1– Tukey’s post hoc test. When the variance was not homogeneous, statisti- 2% maintenance in medical oxygen at a rate of 1.5 L min−1 ). Respiratory cally significant differences among groups were analyzed by Welch ANOVA rate was monitored throughout imaging and anesthetic level controlled and Games-Howell post hoc test. The number of replicates and specific based on respiratory parameters (60–90 bpm). After localizing imaging test implemented for each study are indicated in each figure legend and on three orthogonal axes, the whole brain was imaged using T2 weighted the relevant Experimental sections. Data are presented as mean ± stan- MRI: Rapid Acquisition with Relaxation Enhancement (RARE) pulse se- dard deviation (SD), unless otherwise specified in the figure legend. quence, repetition time = 2200 ms, echo time 33 ms, RARE factor 8, field of view (FOV) 30 mm*30 mm matrix size 512*512, 17 contiguous slices, Supporting Information thickness 0.8 mm, averages = 5. Tumor volumes were determined using ImageJ software (National Institutes of Health, NIH, Bethesda, USA). Tu- Supporting Information is available from the Wiley Online Library or from mor area was delineated for each coronal slice and the total tumor volume the author. generated from the acquired measurements. Tissue Processing and Immunohistochemistry: Four weeks after tumor implantation, U-87 MGtumor-bearing mice (n = 15) were anaesthetized Acknowledgements with 4% isoflurane and culled by transcardial perfusion with heparinized K.K., P.S., S.V., and M.S. would like to acknowledge the United Kingdom PBS (10 units mL−1 ), followed by 4% PFA in PBS. The brains were re- Research and Innovation (UKRI) Engineering and Physical Sciences Adv. Therap. 2021, 4, 2000109 2000109 (13 of 15) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com Research Council (EPSRC) for funding most of this work under the 2D- S. A. 2013, 110, 4009; b) A. P. Patel, I. Tirosh, J. J. Trombetta, A. K. Health Programme Grant (EP/P00119X/1). K.K. and I.d.L. would also like Shalek, S. M. Gillespie, H. Wakimoto, D. P. Cahill, B. V. Nahed, W. T. to acknowledge the UKRI (EPSRC) for an International Centre-to-Centre Curry, R. L. Martuza, D. N. Louis, O. Rozenblatt-Rosen, M. L. Suva, grant (EP/S030719/1) between the University of Manchester and Harvard A. Regev, B. E. Bernstein, Science 2014, 344, 1396. University that funded part of this work. T.K. would like to thank the UKRI [6] J. D. Bernstock, J. H. Mooney, A. Ilyas, G. Chagoya, D. Estevez- (EPSRC and Medical Research Council, MRC) Center for Doctoral Train- Ordonez, A. Ibrahim, I. Nakano, J Neurosurg. 2019, 1. Epub ahead ing (CDT) in Regenerative Medicine (EP/L014904/1) for a funded PhD of print. studentship. The authors would like to thank Dr. Marta Quintanilla and [7] a) E. K. Nduom, M. Weller, A. B. Heimberger, Neurooncology 2015, Dr. David Spiller for help with LSFM. The equipment at the University 17, vii9; b) D. Hambardzumyan, D. H. Gutmann, H. Kettenmann, of Manchester Bioimaging and Genomic Technologies facilities used in Nat. Neurosci. 2016, 19, 20. this study were purchased with grants from the Biotechnology and Biolog- [8] a) L. B. Thomsen, T. Linemann, S. Birkelund, G. A. Tarp, T. Moos, ical Sciences Research Council (BBSRC), Wellcome Trust, and the Univer- Materials (Basel) 2019, 12; b) C. Englert, A. K. Trutzschler, M. Raasch, sity of Manchester Strategic Fund. A.Y., C.G., and H.T. would like to thank the project “G-IMMUNOMICS” funded under the Joint Transnational Call T. Bus, P. Borchers, A. S. Mosig, A. Traeger, U. S. Schubert, J. Con- (JTC) Graphene 2015. A.Y. is thankful to the Turkish Academy of Sciences trolled Release 2016, 241, 1; c) C. He, P. Cai, J. Li, T. Zhang, L. Lin, A. (TUBA) for the financial support. Z. Abbasi, J. T. Henderson, A. M. Rauth, X. Y. Wu, J. Controlled Release 2017, 246, 98. [9] C. Lei, Y. Cui, L. Zheng, P. K. Chow, C. H. Wang, Biomaterials 2013, Conflict of Interest 34, 7483. [10] A. Marino, A. Camponovo, A. Degl’Innocenti, M. Bartolucci, C. The authors declare no conflict of interest. Tapeinos, C. Martinelli, D. De Pasquale, F. Santoro, V. Mollo, S. Arai, M. Suzuki, Y. Harada, A. Petretto, G. Ciofani, Nanoscale 2019, 11, 21227. Author Contributions [11] P. Saalik, P. Lingasamy, K. Toome, I. Mastandrea, L. Rousso-Noori, A. Tobi, L. Simon-Gracia, H. Hunt, P. Paiste, V. R. Kotamraju, G. Bergers, I.d.L., A.Y., and K.K. initiated, designed, and coordinated the study. I.d.L. T. Asser, T. Ratsep, E. Ruoslahti, R. Bjerkvig, D. Friedmann-Morvinski, contributed to experiments (spheroid growth, LSFM) and data analysis T. Teesalu, J. Controlled Release 2019, 308, 109. (gene expression, proteomics), prepared the figures and wrote the paper [12] T. Glaser, I. Han, L. Wu, X. Zeng, Front. Pharmacol. 2017, 8, 166. with contributions and feedback from coauthors. P.S. performed in vivo [13] K. Maier-Hauff, F. Ulrich, D. Nestler, H. Niehoff, P. Wust, B. Thiesen, studies, including cell and nanomaterial administration, MRI imaging, tis- H. Orawa, V. Budach, A. Jordan, J. Neurooncol. 2011, 103, 317. sue processing, staining, and imaging. 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