Multicellular Spheroids Research Articles

Bright and Photostable TADF‐Emitting Zirconium(IV) Pyridinedipyrrolide Complexes: Efficient Dyes for Decay Time‐Based Temperature Sensing and Imaging

AbstractLuminescence thermometry represents a technique of choice for measurements in small objects and imaging of temperature distribution. However, most state‐of‐the‐art luminescent probes are limited in spectral characteristics, brightness, photostability, and sensitivity. Molecular thermometers of the new generation utilizing air and moisture‐stable zirconium(IV) pyridinedipyrrolide complexes can address all these limitations. The dyes emit pure thermally activated delayed fluorescence without any prompt fluorescence and show a unique combination of attractive features: a) visible light excitation and emission in the orange/red region, b) high luminescence brightness (quantum yields ≈0.5 in toluene and 0.8–1.0 in polystyrene matrix), c) excellent photostability, d) suitability for two‐photon excitation and e) mono‐exponential decay on the order of tens to hundreds of microseconds with strongly temperature‐dependent lifetimes (between −2.5 and −2.9% K−1 in polystyrene at 25 °C). Immobilization in gas‐blocking polymers yields sensing materials for self‐referenced decay time read‐out that are manufactured in two common formats: planar optodes and water‐dispersible nanoparticles. Positively charged nanoparticles are demonstrated to be suitable for nanothermometry in live cells and multicellular spheroids. Negatively charged nanoparticles represent advanced analytical tools for imaging temperature gradients in samples of small volumes such as microfluidic devices.

BODIPY-Based Photothermal Agents with Excellent Phototoxic Indices for Cancer Treatment.

Here, we report six novel, easily accessible BODIPY-based agents for cancer treatment. In contrast to established photodynamic therapy (PDT) agents, these BODIPY-based compounds show additional photothermal activity and their cytotoxicity is not dependent on the generation of reactive oxygen species (ROS). The agents show high photocytotoxicity upon irradiation with light and low dark toxicity in different cancer cell lines in 2D culture as well as in 3D multicellular tumor spheroids (MCTSs). The ratio of dark to light toxicity (phototoxic index, PI) of these agents reaches striking values exceeding 830,000 after irradiation with energetically low doses of light at 630 nm. The oxygen-dependent mechanism of action (MOA) of established photosensitizers (PSs) hampers effective clinical deployment of these agents. Under hypoxic conditions (0.2% O2), which are known to limit the efficiency of conventional PSs in solid tumors, photocytotoxicity was induced at the same concentration levels, indicating an oxygen-independent photothermal MOA. With a PI exceeding 360,000 under hypoxic conditions, both PI values are the highest reported to date. We anticipate that small molecule agents with a photothermal MOA, such as the BODIPY-based compounds reported in this work, may overcome this barrier and provide a new avenue to cancer therapy.

Yield stress: A tipping point of cell unjamming.

Tissue mechanics is impacted not only by the properties of the composite cells, but also by the behavior of collective cells, such as cell unjamming and extended differential adhesion. In former studies, we used multicellular spheroids (MS) of healthy epithelial cells (jammed solid) and cancerous mesenchymal cells (unjammed fluid) as our model system to investigate the tissue mechanics of cell clusters. We realized even in a simple cell cluster system, the mechanical behavior differed between core/shell region, which we concluded due to the different domination of nuclei (bulk) and cortical rim formed by cell cytoskeleton (tissue surface tension). As we also observed in cancer spheroids, nuclei seem to dominate the whole mechanics of MTS (no reaction to chemical perturbation of the cell cytoskeleton). By specifically probing the nucleus region of a single cell under AFM, and combining its nucleus morphology aside as an indicator, we could measure how much yield stress a cell will need to become motile (unjammed). Based on our preliminary results, we see a different linear growth of the Young's modulus with elongated nuclei of healthy epithelial cells (high-slope) and cancerous mesenchymal cells (low-slope). Knowing the different behavior of such cell types under the constriction studies (mesenchymal cells do not pass through the confinement whereas epithelial cells do), the nuclei domination of the mesenchymal cells is unassailable. Therefore, to establish the connection between the nucleus-based yield stress to its motility are strongly required. To do so, we are able to derive an unjamming-based marker that detects the ability of cells to move in static histological images, which will be a big step to understand the role of the cancer cell unjamming in the metastatic cascade.

Identification of mesenchymal stem cells based on the passaging effect using machine learning approaches.

For diseases that lack efficient therapeutic approaches, stem cell therapy is an emerging alternative treatment, wherein cell quality assessment is an essential step before cell transplantation into patients. Cell quality is generally evaluated using approaches, such as immunohistochemistry and flow cytometry, which involve complex laboratory routines. In this milieu, several studies have demonstrated the feasibility of applying image processing and analysis to assist cell quality assessment more efficiently. We developed an image-based analytical tool using cell tracking, cytometric analyses, and gating with time-lapse microscopy to investigate the migratory behavior of human mesenchymal stem cells (hMSCs) cultured on chitosan membranes. Using this tool, systematic studies were carried out to inspect variations in cell quality by extracting two morphological and seven migratory features of cells of interest. We found that chitosan membrane-induced multi-cellular spheroids move at a higher speed and in a more consistent direction than single cells. The hMSCs showed a tendency to aggregate with strong 3-D spheroid formability and increased mobility during maturation; however, both these tendencies decreased with degeneration. Furthermore, we automated the cell quality assessment procedure by utilizing machine learning. We selected 570 hMSCs, 390 adipose-derived adult stem cells (hADSCs) and 180 human placenta-derived multipotent cells (hPDMCs), with complete movement trajectories from 25 videos, at three different passages. The differentiation accuracy between hADSCs and hPDMCs in the mature passage was 96.3%, which was higher than that in the early (82.6%) and late (80.5%) passages, whereas the accuracy of passage identification was 62.8% for hADSCs and 70.6% for hPDMCs. The limited accuracy of passage identification may be owing to the insufficient number of morphological features. This study shows the potential of applying machine learning in monitoring the quality of stem cells during in vitro culture.

3D co-culture of macrophages and fibroblasts in a sessile drop array for unveiling the role of macrophages in skin wound-healing

Three-dimensional (3D) heterotypic multicellular spheroid models play important roles in researches of the proliferation and remodeling phases in wound healing. This study aimed to develop a sessile drop array to cultivate 3D spheroids and simulate wound healing stage in vitro using NIH-3T3 fibroblasts and M2-type macrophages. By the aid of the offset of surface tension and gravity, the sessile drop array is able to transfer cell suspensions to spheroids via the superhydrophobic surface of each microwell. Meanwhile, each microwell has a cylinder hole at its bottom that provides adequate oxygen to the spheroid. It demonstrated that the NIH-3T3 fibroblast spheroid and the 3T3 fibroblast/M2-type macrophage heterotypic multicellular spheroid can form and maintain physiological activities within nine days. In order to further investigate the structure without destroying the entire spheroid, we reconstructed its 3D morphology using transparent processing technology and the Z-stack function of confocal microscopy. Additionally, a nano antibody-based 3D immunostaining assay was used to analyze the proliferation and differentiation characteristics of these cells. It found that M2-type macrophages were capable of promoting the differentiation of 3T3 fibroblast spheroid. In this study, a novel, inexpensive platform was constructed for developing spheroids, as well as a 3D immunofluorescence method for investigating the macrophage-associated wound healing microenvironment.

A method for reproducible high-resolution imaging of 3D cancer cell spheroids.

Multicellular tumour cell spheroids embedded within three-dimensional (3D) hydrogels or extracellular matrices (ECM) are widely used as models to study cancer growth and invasion. Standard methods to embed spheroids in 3D matrices result in random placement in space which limits the use of inverted fluorescence microscopy techniques, and thus the resolution that can be achieved to image molecular detail within the intact spheroid. Here, we leverage UV photolithography to microfabricate PDMS (polydimethylsiloxane) stamps that allow for generation of high-content, reproducible well-like structures in multiple different imaging chambers. Addition of multicellular tumour spheroids into stamped collagen structures allows for precise positioning of spheroids in 3D space for reproducible high-/super-resolution imaging. Embedded spheroids can be imaged live or fixed and are amenable to immunostaining, allowing for greater flexibility of experimental approaches. We describe the use of these spheroid imaging chambers to analyse cell invasion, cell-ECM interaction, ECM alignment, force-dependent intracellular protein dynamics and extension of fine actin-based protrusions with a variety of commonly used inverted microscope platforms. This method enables reproducible, high-/super-resolution live imaging of multiple tumour spheroids, that can be potentially extended to visualise organoids and other more complex 3D in vitro systems.

Multicellular Quadruple Colorectal Cancer Spheroids as an In Vitro Tool for Antiangiogenic Potential Evaluation of Nanoparticles

AbstractThe encapsulation of bevacizumab (BVZ), angiogenesis inhibitor antibody, into nanocarriers is explored aiming at enhancing its efficacy in colorectal cancer (CRC) treatment while eliminating potential side effects. Still, the translation of such nanomedicines into clinics is not straightforward owing to their preclinical screening in simplistic models, that do not mimic the complexity and heterogeneity of the CRC microenvironment. Herein, the development of a multicellular spheroid of CRC as an advanced preclinical model of CRC capable of screening the antiangiogenic potential of novel nanomedicines is proposed. For that, a quadruple co‐culture is established through the combination of HCT116 cells, human pulmonary microvascular endothelial cell (HPMEC), fibroblasts, and macrophages. It is demonstrated that the developed model displays intrinsic CRC features, such as the organization of cells, expression of tumor microenvironment, extracellular matrix, and the formation of a necrotic core. Moreover, the model is shown to be composed mostly of HCT116 (93%), followed by hypoxia‐inducible factor (3%), HPMEC (3%), and macrophages (1%). Gellan gum/chitosan nanoparticles encapsulating BVZ exhibit superior antiangiogenic properties when compared to free BVZ. Overall, the developed nanoparticles can further be explored as a promising approach in CRC treatment.

Comparative Analysis of Dasatinib Effect between 2D and 3D Tumor Cell Cultures

Three-dimensional cell culture methods are able to confer new predictive relevance to in vitro tumor models. In particular, the 3D multicellular tumor spheroids model is considered to better resemble tumor complexity associated with drug resistance compared to the 2D monolayer model. Recent advances in 3D printing techniques and suitable biomaterials have offered new promises in developing 3D tissue cultures at increased reproducibility and with high-throughput characteristics. In our study, we compared the sensitivity to dasatinib treatment in two different cancer cell lines, prostate cancer cells DU145 and glioblastoma cells U87, cultured in the 3D spheroids model and in the 3D bioprinting model. DU145 and U87 cells were able to proliferate in 3D alginate/gelatin bioprinted structures for two weeks, forming spheroid aggregates. The treatment with dasatinib demonstrated that bioprinted cells were considerably more resistant to drug toxicity than corresponding cells cultured in monolayer, in a way that was comparable to behavior observed in the 3D spheroids model. Recovery and analysis of cells from 3D bioprinted structures led us to hypothesize that dasatinib resistance was dependent on a scarce penetrance of the drug, a phenomenon commonly reported also in spheroids. In conclusion, the 3D bioprinted model utilizing alginate/gelatin hydrogel was demonstrated to be a suitable model in drug screening when spheroid growth is required, offering advantages in feasibility, reproducibility, and scalability compared to the classical 3D spheroids model.

Highly efficient fabrication of functional hepatocyte spheroids by a magnetic system for the rescue of acute liver failure

Engineering hepatocytes as multicellular cell spheroids can improve their viability after implantation in vivo for effective rescue of the devastating acute liver failure (ALF). However, there is still a lack of straightforward methods for efficient generation of functional hepatocyte spheroids. In this study, a magnetic system, consisting of magnetic microwell arrays and magnet blocks, is developed to realize magnetically controlled 3D cell capture and spatial confinement-mediated cell aggregation. The cell spheroids with smaller size show superior hepatic functions than the larger-sized counterparts. Notably, the intrinsic magnetism of magnetic microwell arrays can regulate superoxide anions in hepatocyte spheroids and herein promote various biological processes, including antioxidation, hepatocyte-related functions, and pro-angiogenic potential. Ectopic implantation of the functional cell spheroids in ALF-challenged mice significantly prolongs the animal survival, ameliorates inflammation, and promotes liver regeneration. Hence, application of the magnetic system for generation of functionally enhanced hepatocyte spheroids holds great potential for clinical translation in the future.

Geometric analysis enables biological insight from complex non-identifiable models using simple surrogates.

An enduring challenge in computational biology is to balance data quality and quantity with model complexity. Tools such as identifiability analysis and information criterion have been developed to harmonise this juxtaposition, yet cannot always resolve the mismatch between available data and the granularity required in mathematical models to answer important biological questions. Often, it is only simple phenomenological models, such as the logistic and Gompertz growth models, that are identifiable from standard experimental measurements. To draw insights from complex, non-identifiable models that incorporate key biological mechanisms of interest, we study the geometry of a map in parameter space from the complex model to a simple, identifiable, surrogate model. By studying how non-identifiable parameters in the complex model quantitatively relate to identifiable parameters in surrogate, we introduce and exploit a layer of interpretation between the set of non-identifiable parameters and the goodness-of-fit metric or likelihood studied in typical identifiability analysis. We demonstrate our approach by analysing a hierarchy of mathematical models for multicellular tumour spheroid growth experiments. Typical data from tumour spheroid experiments are limited and noisy, and corresponding mathematical models are very often made arbitrarily complex. Our geometric approach is able to predict non-identifiabilities, classify non-identifiable parameter spaces into identifiable parameter combinations that relate to features in the data characterised by parameters in a surrogate model, and overall provide additional biological insight from complex non-identifiable models.

Phototoxicity of temoporfin-loaded cyclodextrin nanosponges in stroma-rich three-dimensional models of head and neck cancer

Photodynamic therapy is a multistage treatment, in which cancerous and precancerous cells are destroyed by light activation of a drug (photosensitizer). For a long time, high cellular uptake of the photosensitizer was an important indication of efficient PDT, while the role of photosensitizer penetration was unexplored. Recently, we have demonstrated that nanosponges based on hypercrosslinked β-cyclodextrin polymer (β-CDp) can increase drug penetration at the cost of their cellular uptake in multicellular spheroids, paving the way for studying the impact of penetration on PDT response.In the present work, we used β-CDp nanosponges to deliver temoporfin to the depth of stroma-rich head and neck cancer multicellular spheroids and then assess PDT response. Encapsulation of temoporfin in β-CDp nanosponges resulted in increased penetration and more uniform distribution of temoporfin in spheroids, however, was also associated with a two-fold reduction of cellular uptake compared to the free drug. Nevertheless, we demonstrated that β-CDp nanosponges possess similar PDT efficiency as the free drug in stroma-rich head and neck cancer multicellular spheroids. Overall, this study suggests that β-CDp nanosponges are a strong candidate for in vivo studies as they have fewer “off-target” effects while providing a similar therapeutic response.

Switching the Mode of Cell Death between Apoptosis and Autophagy by Histone Deacetylase 6 Inhibition Levels.

Inhibition of histone deacetylase (HDAC) has been demonstrated to be an effective strategy for cancer treatment. In this work, we have developed a new agent Ir-VPA, which exhibits the cell death mode switching between apoptosis and autophagy due to the distinct level of HDAC6 inhibition. Ir-VPA indicates the best anticancer activity to HeLa cells, and could be hydrolyzed due to the high expression of the esterase in HeLa cells. Ir-VPA could accumulate in nuclei, induce severe DNA damages and cell cycle arrest at G2/M phase. The anticancer mechanism of Ir-VPA to HeLa cells was dependent on the HDAC6 inhibitory performance, as the caspase dependent apoptosis at low concentration (IC50 ) and autophagy with the autophagy flux blockage at high concentration (2×IC50 ). This is resulted from the distinct inhibitory levels of HDAC6, as moderate/complete inhibition at the concentration of IC50 /2×IC50 .In the presence of autophagic inhibitor chloroquine, the apoptotic population elevated from 32.7 % to 61.7 %, indicating that Ir-VPA could activate apoptotic process through the autophagolysosome fusion inhibition. Ir-VPA also exhibits excellent antiproliferative behavior to 3D HeLa multicellular tumor spheroids (MCTSs). This work not only provided a new HDAC6 inhibitor and novel anticancer mechanism for the effective treatment of cervical cancer, but also demonstrated the strategy to conjugate the metal fragment with active organic drug to enhance the anticancer performance.

Extracellular vesicle‑mediated miR‑126‑3p transfer contributes to inter‑cellular communication in the liver tumor microenvironment.

Extracellular vesicles (EVs) and their contents are gaining recognition as important mediators of intercellular communication through the transfer of bioactive molecules, such as non‑coding RNA. The present study comprehensively assessed the microRNA (miRNA/miR) content within EVs released from HepG2 liver cancer (LC) cells and LX2 hepatic stellate cells (HSCs) and determined the contribution of EV miRNA to intercellular communication. Using both transwell and spheroid co‑cultures of LC cells and HSCs, miR‑126‑3p within EV was established as a mediator of HSC to LC cell communication that influenced tumor cell migration and invasion, as well as the growth of multicellular LC/HSC spheroids. Manipulation of miR‑126‑3p either by enforced expression using pre‑miR‑126‑3p or by inhibition using antimiR‑126‑3p did not alter tumor cell viability, proliferation or sensitivity to either sorafenib or regorafenib. By contrast, enforced expression of miR‑126‑3p decreased tumor‑cell migration. Knockdown of miR‑126‑3p in tumor cells increased disintegrin and metalloproteinase domain‑containing protein 9 (ADAM9) expression and in HSCs increased collagen‑1A1 accumulation with an increase in compactness of multicellular spheroids. Within LC/HSC spheroids, ADAM9 and vascular endothelial growth factor expression was increased by silencing of miR‑126‑3p but diminished with the restoration of miR‑126‑3p. These studies implicate miR‑126‑3p in functional effects on migration, invasion and spheroid growth of tumor cells in the presence of HSCs, and thereby demonstrate functional EV‑RNA‑based intercellular signaling between HSCs and LC cells that is directly relevant to tumor‑cell behavior.

Microfluidically-generated Encapsulated Spheroids (μ-GELS): An All-Aqueous Droplet Microfluidics Platform for Multicellular Spheroids Generation.

Spheroids are three-dimensional clusters of cells that serve as in vitro tumor models to recapitulate in vivo morphology. A limitation of many existing on-chip platforms for spheroid formation is the use of cytotoxic organic solvents as the continuous phase in droplet generation processes. All-aqueous methods do not contain cytotoxic organic solvents but have so far been unable to achieve complete hydrogel gelation on chip. Here, we describe an enhanced droplet microfluidic platform that achieves on-chip gelation of all-aqueous hydrogel multicellular spheroids (MCSs). Specifically, we generate dextran-alginate droplets containing MCF-7 breast cancer cells, surrounded by polyethylene glycol, at a flow-focusing junction. Droplets then travel to a second flow-focusing junction where they interact with calcium chloride and gel on chip to form hydrogel MCSs. On-chip gelation of the MCSs is possible here because of an embedded capillary at the second junction that delays the droplet gelation, which prevents channel clogging problems that would otherwise exist. In drug-free experiments, we demonstrate that MCSs remain viable for 6 days. We also confirm the applicability of this system for cancer drug testing by observing that dose-dependent cell death is achievable using doxorubicin.

Stiffness-Transformable Nanoplatforms Responsive to the Tumor Microenvironment for Enhanced Tumor Therapeutic Efficacy.

Herein, we report, for the first time, a unique stiffness-transformable manganese oxide hybridized mesoporous organosilica nanoplatform (MMON) for enhancing tumor therapeutic efficacy. The prepared MMONs had a quasi-spherical morphology and were completely transformed into soft bowl-like nanocapsules in the simulated tumor microenvironment through the breakage of Mn-O bonds, which decreased their Young's modulus from 165.7 to 84.5 MPa. Due to their unique stiffness transformation properties, the MMONs had reduced macrophage internalization, improved tumor cell uptake, and enhanced penetration of multicellular spheroids. In addition, in vivo experiments showed that the MMONs displayed a 3.79- and 2.90-fold decrease in non-specific liver distribution and a 2.87- and 1.83-fold increase in tumor accumulation compared to their soft and stiff counterparts, respectively. Furthermore, chlorin e6 (Ce6) modified MMONs had significantly improved photodynamic therapeutic effect.

Stiffness‐Transformable Nanoplatforms Responsive to the Tumor Microenvironment for Enhanced Tumor Therapeutic Efficacy

AbstractHerein, we report, for the first time, a unique stiffness‐transformable manganese oxide hybridized mesoporous organosilica nanoplatform (MMON) for enhancing tumor therapeutic efficacy. The prepared MMONs had a quasi‐spherical morphology and were completely transformed into soft bowl‐like nanocapsules in the simulated tumor microenvironment through the breakage of Mn−O bonds, which decreased their Young's modulus from 165.7 to 84.5 MPa. Due to their unique stiffness transformation properties, the MMONs had reduced macrophage internalization, improved tumor cell uptake, and enhanced penetration of multicellular spheroids. In addition, in vivo experiments showed that the MMONs displayed a 3.79‐ and 2.90‐fold decrease in non‐specific liver distribution and a 2.87‐ and 1.83‐fold increase in tumor accumulation compared to their soft and stiff counterparts, respectively. Furthermore, chlorin e6 (Ce6) modified MMONs had significantly improved photodynamic therapeutic effect.

Long-Term Simulation of Microgravity Induces Changes in Gene Expression in Breast Cancer Cells.

Microgravity changes the gene expression pattern in various cell types. This study focuses on the breast cancer cell lines MCF-7 (less invasive) and MDA-MB-231 (triple-negative, highly invasive). The cells were cultured for 14 days under simulated microgravity (s-µg) conditions using a random positioning machine (RPM). We investigated cytoskeletal and extracellular matrix (ECM) factors as well as focal adhesion (FA) and the transmembrane proteins involved in different cellular signaling pathways (MAPK, PAM and VEGF). The mRNA expressions of 24 genes of interest (TUBB, ACTB, COL1A1, COL4A5, LAMA3, ITGB1, CD44, VEGF, FLK1, EGFR, SRC, FAK1, RAF1, AKT1, ERK1, MAPK14, MAP2K1, MTOR, RICTOR, VCL, PXN, CDKN1, CTNNA1 and CTNNB1) were determined by quantitative real-time PCR (qPCR) and studied using STRING interaction analysis. Histochemical staining was carried out to investigate the morphology of the adherent cells (ADs) and the multicellular spheroids (MCSs) after RPM exposure. To better understand this experimental model in the context of breast cancer patients, a weighted gene co-expression network analysis (WGCNA) was conducted to obtain the expression profiles of 35 breast cell lines from the HMS LINCS Database. The qPCR-verified genes were searched in the mammalian phenotype database and the human genome-wide association studies (GWAS) Catalog. The results demonstrated the positive association between the real metastatic microtumor environment and MCSs with respect to the extracellular matrix, cytoskeleton, morphology, different cellular signaling pathway key proteins and several other components. In summary, the microgravity-engineered three-dimensional MCS model can be utilized to study breast cancer cell behavior and to assess the therapeutic efficacies of drugs against breast cancer in the future.

PDLLA length on anti-breast cancer efficacy of acid-responsive self-assembling mPEG-PDLLA‒docetaxel conjugates

Engineering small-molecule drugs into nanoparticulate formulations provides an unprecedented opportunity to improve the performance of traditional chemo drugs, but suffers from poor compatibility between drugs and nanocarriers. Stimuli-responsive mPEG-PDLLA–drug conjugate-based nanomedicines can facilitate the exploitation of beneficial properties of the carrier and enable the practical fabrication of highly efficacious self-assembled nanomedicines. However, the influence of hydrophobic length on the performance of this type of nanomedicine is little known. Here we synthesized two acid-sensitive ketal-linked mPEG-PDLLA–docetaxel prodrugs with different lengths of PDLLA, and engineered them into self-assembled sub-20 nm micellar nanomedicines for breast cancer chemotherapy. We found that the nanomedicine consisting of a mPEG-PDLLA–docetaxel prodrug with the shorter length of PDLLA stood out due to its potent cytotoxicity, deep penetration into multicellular spheroids, and improved in vivo anticancer performance. Additionally, our prodrug-based nanomedicines outperformed the generic formulation of commercial Nanoxel in terms of safety profile, tolerated doses, and tumor suppression. Our findings indicate that the hydrophobic content of a polymeric prodrug nanomedicine plays an important role in the performance of the nanomedicine, and should be instructive for developing polymeric prodrug-based nanomedicines with clinical translational potential.

Observe without disturbing: tracer particles sense local stresses in cell collectives without affecting the cancer cell dynamics.

Measurements of local stresses on the cancer cells (CCs), inferred by embedding inert compressible tracer particles (TPs) in a growing multicellular spheroid (MCS), show that pressure decreases monotonically as the distance from the core of the MCS increases. How faithfully do the TPs report the local stresses in the CCs is an important question because pressure buildup in the MCS is dynamically generated due to CC division, which implies that the CC dynamics should be minimally altered by the TPs. Here using theory and simulations, we show that although the TP dynamics is unusual, exhibiting sub-diffusive behavior on times less than the CC division times and hyper-diffusive dynamics in the long-time limit, they do not affect the long-time CC dynamics. The CC pressure profile within the MCS, which decays from a high value at the core to the periphery, is almost identical with and without the TPs. That the TPs have a small effect on the local stresses in the MCS implies that they are reasonale reporters of the CC microenvironment.

Evaluating nanoparticle localisation in glioblastoma multicellular tumour spheroids by surface enhanced Raman scattering.

Glioblastoma multiforme (GBM) is a particularly aggressive and high-grade brain cancer, with poor prognosis and life expectancy, in urgent need of novel therapies. These severe outcomes are compounded by the difficulty in distinguishing between cancerous and non-cancerous tissues using conventional imaging techniques. Metallic nanoparticles (NPs) are advantageous due to their diverse optical and physical properties, such as their targeting and imaging potential. In this work, the uptake, distribution, and location of silica coated gold nanoparticles (AuNP-SHINs) within multicellular tumour spheroids (MTS) derived from U87-MG glioblastoma cells was investigated by surface enhanced Raman scattering (SERS) optical mapping. MTS are three-dimensional in vitro tumour mimics that represent a tumour in vivo much more closely than that of a two-dimensional cell culture. By using AuNP-SHIN nanotags, it is possible to readily functionalise the inner gold surface with a Raman reporter, and the outer silica surface with an antibody for tumour specific targeting. The nanotags were designed to target the biomarker tenascin-C overexpressed in U87-MG glioblastoma cells. Immunochemistry indicated that tenascin-C was upregulated within the core of the MTS, however limitations such as NP size, quiescence, and hypoxia, restricted the penetration of the nanotags to the core and they remained in the outer proliferating cells of the spheroids. Previous examples of MTS studies using SERS demonstrated the incubation of NPs on a 2D monolayer of cells, with the subsequent formation of the MTS from these pre-incubated cells. Here, we focus on the localisation of the NPs after incubation into pre-formed MTS to establish a better understanding of targeting and NP uptake. Therefore, this work highlights the importance for the investigation and translation of NP uptake into these 3D in vitro models.