Optical Barcoding Research Articles

Overcoming fixation and permeabilization challenges in flow cytometry by optical barcoding and multi-pass acquisition.

The fixation and permeabilization of cells are essential for labeling intracellular biomarkers in flow cytometry. However, these chemical treatments often alter fragile targets, such as cell surface and fluorescent proteins (FPs), and can destroy chemically-sensitive fluorescent labels. This reduces measurement accuracy and introduces compromises into sample workflows, leading to losses in data quality. Here, we demonstrate a novel multi-pass flow cytometry approach to address this long-standing problem. Our technique utilizes individual cell barcoding with laser particles, enabling sequential analysis of the same cells with single-cell resolution maintained. Chemically-fragile protein markers and their fluorochrome conjugates are measured prior to destructive sample processing and adjoined to subsequent measurements of intracellular markers after fixation and permeabilization. We demonstrate the effectiveness of our technique in accurately measuring intracellular FPs and methanol-sensitive antigens and fluorophores, along with various surface and intracellular markers. This approach significantly enhances assay flexibility, enabling accurate and comprehensive cellular analysis without the constraints of conventional one-time measurement flow cytometry. This innovation paves new avenues in flow cytometry for a wide range of applications in immuno-oncology, stem cell research, and cell biology.

Overcoming fixation and permeabilization challenges in flow cytometry by optical barcoding and multi-pass acquisition.

The fixation and permeabilization of cells are essential for labeling intracellular biomarkers in flow cytometry. However, these chemical treatments often alter fragile targets, such as cell surface and fluorescent proteins, and can destroy chemically-sensitive fluorescent labels. This reduces measurement accuracy and introduces compromises into sample workflows, leading to losses in data quality. Here, we demonstrate a novel multi-pass flow cytometry approach to address this long-standing problem. Our technique utilizes individual cell barcoding with laser particles, enabling sequential analysis of the same cells with single-cell resolution maintained. Chemically-fragile protein markers and their fluorochrome conjugates are measured prior to destructive sample processing and adjoined to subsequent measurements of intracellular markers after fixation and permeabilization. We demonstrate the effectiveness of our technique in accurately measuring intracellular fluorescent proteins and methanol-sensitive antigens and fluorophores, along with various surface and intracellular markers. This approach significantly enhances assay flexibility, enabling accurate and comprehensive cell analysis without the constraints of conventional one-time measurement flow cytometry. This innovation paves new avenues in flow cytometry for a wide range of applications in immuno-oncology, stem cell research, and cell biology.

Abstract 1210: High-throughput time-resolved single-cell analysis of T-cell activation

Abstract How immune cells respond to stimuli is fundamental in developing adoptive cell therapies, vaccines, and other immunotherapies. T cells dynamically undergo activation, exhaustion, expansion, phenotype switching, and cell killing. Flow cytometry is the gold standard method for characterizing these at high throughput, typically by comparing T cell populations before and at different timepoints after treatment. This approach is limited to population-level analyses and overlooks any heterogeneity of individual T cell responses over time, which may contribute to variability in response. Here, we describe the development of a novel flow cytometry-based method (Time-lapse flowTM) to characterize immune cell responses over time at the single cell level. To measure the same cells repeatedly on a flow cytometer, we employed a new optical barcoding technology called laser particles (LPs) that provide individual cells with a unique spectral signature that can be tracked over the course of multiple measurements. Using a custom flow cytometer with fluorescence and LP detection, we performed a multi-day assay to capture how T cell marker expression changes over time after CD3/CD28 activation. Specifically, we used releasable antibodies to quantify PD1, CD69 and CD25 at each time-point and correlated these expression kinetics with cell function at the end of the assay by measuring cytokine secretion (IFNg, IL2, TNFa) after PMA/ionomycin stimulation. We found significant cellular heterogeneity in kinetic profiles of PD1, CD69 and CD25 expression, with different dominant ‘kinetic modes’ present for each marker. For example, while a large proportion of PD1 expression was transient (positive on day 1, but not on days 2 and 3), nearly all cells that express CD69 on day 1 retained their expression on day 2. We also found that T cells with late expression of PD1 had significantly more cytokine secretion compared to cells that were initially positive for PD1. These results were replicated with at least 3 replicate samples for multiple donors. Differences in kinetic phenotypes were not explained by conventional phenotyping with CD4, CD8, CD45RA and CCR7. To date, immune cell phenotyping with flow cytometry involves classifying cell types through their marker expression profiles at a single time-point. Here, we introduce a novel approach for acquiring time-resolved single-cell data with a flow cytometer and applied it to study T cell activation. We found novel kinetic phenotypes of T cells defined by their marker expression over time, which also correlated to their functional potential (cytokine secretion). These kinetic phenotypes could not be identified with conventional flow cytometry. Our approach could have significant implications for immunotherapy development, particularly in identifying the most suitable cell populations to use, understanding mechanisms of exhaustion and persistence, and elucidating the basis of donor variability. Citation Format: Sheldon J. Kwok, Sarah Forward, Emane Rose Assita, Trevor Brown, Marissa Fahlberg, Pratip Chattopadhyay. High-throughput time-resolved single-cell analysis of T-cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1210.

Abstract LB_A04: Quantitative digital spatial protein profiling of non-small cell lung cancer brain metastasis

Abstract Metastatic brain tumor is the most common form of adult central nervous system (CNS) tumor, and the majority of the cerebral metastasis originate from lung cancer. Along with recent combination strategies applying immune checkpoint inhibitor, evaluating tumor microenvironment of the brain metastasis (BM) needs to be unveiled. In this study, we tried to disclose immune context information of BM of non-small cell lung cancer (NSCLC) by using highly multiplex NanoString digital spatial profiling (DSP) method. Seven cases of NSCLC and metastatic brain tumor were evaluated. To detect proteins at quantitative and high multiplex with spatial resolution, NanoString optical barcoding technology was used to digitally profile protein expression in FFPE samples. Immuno-oncology protein targets, including immune cell markers and checkpoint proteins are included. DSP identified differentially expressed proteins between primary NSCLC and BM. Proteins associated with immune regulation, evasion, and adaptive immunity, such as IDO-1, PD1, PDL1, STAT3, PTEN, and CD44 revealed decreased expression in patients with BM (P<0.01). The expression of pS6 was significantly increased in BM (P<0.01), which is associated with activation induced T-cell death. This study demonstrated differentially expressed proteins in BM associated with immune regulation and evasion via NanoString DSP platform. This study highlighted the localized expression of immune-related proteins of BM in patients with NSCLC. Moreover, our results presented the possibility of using digitally quantified and spatially allocated novel method in BM eliminating the subjectivity and variability associated with the immunohistochemistry techniques. Citation Format: Tae Jung Kim. Quantitative digital spatial protein profiling of non-small cell lung cancer brain metastasis [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A04.

Particle ID: A Multiplexed Hydrogel Bead Platform for Biomedical Applications.

We present a new platform based on hydrogel beads for multiplex analysis that can be fabricated, barcoded, and functionalized in a single step using a simple microfluidic assembly and a photo-cross-linking process. The beads are generated in a two-phase flow fluidic system and photo-cross-linking of the polymer in the aqueous phase by C,H insertion cross-linking (CHic). The size and shape of the hydrogel particles can be controlled over a wide range by fluidic parameters. During the fabrication of the beads, they are barcoded by using physical and optical barcoding strategies. Magnetic beads and fluorescent particles, which allow identification of the production batch number, are added simultaneously as desired, resulting in complex, multifunctional beads in a one-step reaction. As an example of biofunctionalization, Borrelia antigens were immobilized on the beads. Serum samples that originated from infected and non-infected patients could be clearly distinguished, and the sensitivity was as good as or even better than ELISA, the state of the art in clinical diagnostics. The ease of the one-step production process and the wide range of barcoding parameters offer strong advantages for multiplexed analytics in the life sciences and medical diagnostics.

Ultrasmall InGa(As)P Dielectric and Plasmonic Nanolasers.

Nanolasers have great potential for both on-chip light sources and optical barcoding particles. We demonstrate ultrasmall InGaP and InGaAsP disk lasers with diameters down to 360 nm (198 nm in height) in the red spectral range. Optically pumped, room-temperature, single-mode lasing was achieved from both disk-on-pillar and isolated particles. When isolated disks were placed on gold, plasmon polariton lasing was obtained with Purcell-enhanced stimulated emission. UV lithography and plasma ashing enabled wafer-scale fabrication of nanodisks with an intended random size variation. Silica-coated nanodisk particles generated stable subnanometer spectra from within biological cells across an 80 nm bandwidth from 635 to 715 nm.

Vacuum pressure imaging solution based on fiber-optic Fabry-Pérot interferometer and optical barcode

Slight variations in vacuum can have a huge impact on industrial, medical, and scientific applications. For sub-atmospheric pressure measurements, conventional demodulation methods for fiber-optic sensors based on the wavelength tracking or power interrogation of the single resonant peak encounter difficulties in the direct vacuum level detection, in addition to cost and sensitivity issues which are one of the key obstacles for further applications of sensing probes. Here we demonstrate a total solution based on a fiber-based Fabry-Pérot interferometer (FPI) and an optical barcode based on Fréchet distance. The vacuum sensing fiber tip based on FPI prepared using the fixed-length cutting and fiber-end grinding techniques address the need for low-cost, high-sensitivity sensing for potential applications. Optical barcoding technique provides direct vacuum information by monitoring the overall behavior of the spectral resonance peaks. This work lays the foundation for the development of a low-cost, compact vacuum pressure sensing solution with direct vacuum reading.

Microcavity- and Microlaser-Based Optical Barcoding: A Review of Encoding Techniques and Applications.

Optical microbarcodes have recently received a great deal of interest because of their suitability for a wide range of applications, such as multiplexed assays, cell tagging and tracking, anticounterfeiting, and product labeling. Spectral barcodes are especially promising because they are robust and have a simple readout. In addition, microcavity- and microlaser-based barcodes have very narrow spectra and therefore have the potential to generate millions of unique barcodes. This review begins with a discussion of the different types of barcodes and then focuses specifically on microcavity-based barcodes. While almost any kind of optical microcavity can be used for barcoding, currently whispering-gallery microcavities (in the form of spheres and disks), nanowire lasers, Fabry-Pérot lasers, random lasers, and distributed feedback lasers are the most frequently employed for this purpose. In microcavity-based barcodes, the information is encoded in various ways in the properties of the emitted light, most frequently in the spectrum. The barcode is dependent on the properties of the microcavity, such as the size, shape, and the gain materials. Various applications of these barcodes, including cell tracking, anticounterfeiting, and product labeling are described. Finally, the future prospects for microcavity- and microlaser-based barcodes are discussed.

Lymphatic Invasion of Plakoglobin-Dependent Tumor Cell Clusters Drives Formation of Polyclonal Lung Metastases in Colon Cancer

Patients with colon cancer with liver metastases may be cured with surgery, but the presence of additional lung metastases often precludes curative treatment. Little is known about the processes driving lung metastasis. This study aimed to elucidate the mechanisms governing lung vs liver metastasis formation. Patient-derived organoid (PDO) cultures were established from colon tumors with distinct patterns of metastasis. Mouse models recapitulating metastatic organotropism were created by implanting PDOs into the cecum wall. Optical barcoding was applied to trace the origin and clonal composition of liver and lung metastases. RNA sequencing and immunohistochemistry were used to identify candidate determinants of metastatic organotropism. Genetic, pharmacologic, invitro, and invivo modeling strategies identified essential steps in lung metastasis formation. Validation was performed by analyzing patient-derived tissues. Cecum transplantation of 3 distinct PDOs yielded models with distinct metastatic organotropism: liver only, lung only, and liver and lung. Liver metastases were seeded by single cells derived from select clones. Lung metastases were seeded by polyclonal clusters of tumor cells entering the lymphatic vasculature with very limited clonal selection. Lung-specific metastasis was associated with high expression of desmosome markers, including plakoglobin. Plakoglobin deletion abrogated tumor cell cluster formation, lymphatic invasion, and lung metastasis formation. Pharmacologic inhibition of lymphangiogenesis attenuated lung metastasis formation. Primary human colon, rectum, esophagus, and stomach tumors with lung metastases had a higher N-stage and more plakoglobin-expressing intra-lymphatic tumor cell clusters than those without lung metastases. Lung and liver metastasis formation are fundamentally distinct processes with different evolutionary bottlenecks, seeding entities, and anatomic routing. Polyclonal lung metastases originate from plakoglobin-dependent tumor cell clusters entering the lymphatic vasculature at the primary tumor site.

Pursuing excitonic energy transfer with programmable DNA-based optical breadboards.

DNA nanotechnology has now enabled the self-assembly of almost any prescribed 3-dimensional nanoscale structure in large numbers and with high fidelity. These structures are also amenable to site-specific modification with a variety of small molecules ranging from drugs to reporter dyes. Beyond obvious application in biotechnology, such DNA structures are being pursued as programmable nanoscale optical breadboards where multiple different/identical fluorophores can be positioned with sub-nanometer resolution in a manner designed to allow them to engage in multistep excitonic energy-transfer (ET) via Förster resonance energy transfer (FRET) or other related processes. Not only is the ability to create such complex optical structures unique, more importantly, the ability to rapidly redesign and prototype almost all structural and optical analogues in a massively parallel format allows for deep insight into the underlying photophysical processes. Dynamic DNA structures further provide the unparalleled capability to reconfigure a DNA scaffold on the fly in situ and thus switch between ET pathways within a given assembly, actively change its properties, and even repeatedly toggle between two states such as on/off. Here, we review progress in developing these composite materials for potential applications that include artificial light harvesting, smart sensors, nanoactuators, optical barcoding, bioprobes, cryptography, computing, charge conversion, and theranostics to even new forms of optical data storage. Along with an introduction into the DNA scaffolding itself, the diverse fluorophores utilized in these structures, their incorporation chemistry, and the photophysical processes they are designed to exploit, we highlight the evolution of DNA architectures implemented in the pursuit of increased transfer efficiency and the key lessons about ET learned from each iteration. We also focus on recent and growing efforts to exploit DNA as a scaffold for assembling molecular dye aggregates that host delocalized excitons as a test bed for creating excitonic circuits and accessing other quantum-like optical phenomena. We conclude with an outlook on what is still required to transition these materials from a research pursuit to application specific prototypes and beyond.

Enabling spectral barcoding of SERS nanotags using gold nanostars

Gold nanostars have shown the great potential in design of SERS nanotags for optical spectral barcoding technology.

Modeling Colorectal Cancer Progression Reveals Niche-Dependent Clonal Selection.

Simple SummaryColorectal cancer (CRC) starts as a localized tumor and becomes a systemic disease with fatal consequences. However, clonal dynamics during progression are not well understood. Here, we present various techniques to model the different stages of CRC progression. Using genetically engineered mouse models (GEMMs) or organoid transplantation, localized tumors as well as liver metastases with characteristic intra-tumor heterogeneity were generated. The optical barcoding of transplanted organoids revealed niche-dependent clonal selection, implying that distinct niche factors control clonal outgrowth.Colorectal cancer (CRC) is among the deadliest cancers worldwide, with metastasis being the main cause of patient mortality. During CRC progression the complex tumor ecosystem changes in its composition at virtually every stage. However, clonal dynamics and associated niche-dependencies at these stages are unknown. Hence, it is of importance to utilize models that faithfully recapitulate human CRC to define its clonal dynamics. We used an optical barcoding approach in mouse-derived organoids (MDOs) that revealed niche-dependent clonal selection. Our findings highlight that clonal selection is controlled by a site-specific niche, which critically contributes to cancer heterogeneity and has implications for therapeutic intervention.

Mastering the use of cellular barcoding to explore cancer heterogeneity

Tumours are often composed of a multitude of malignant clones that are genomically unique, and only a few of them may have the ability to escape cancer therapy and grow as symptomatic lesions. As a result, tumours with a large degree of genomic diversity have a higher chance of leading to patient death. However, clonal fate can be driven by non-genomic features. In this context, new technologies are emerging not only to track the spatiotemporal fate of individual cells and their progeny but also to study their molecular features using various omics analysis. In particular, the recent development of cellular barcoding facilitates the labelling of tens to millions of cancer clones and enables the identification of the complex mechanisms associated with clonal fate in different microenvironments and in response to therapy. In this Review, we highlight the recent discoveries made using lentiviral-based cellular barcoding techniques, namely genetic and optical barcoding. We also emphasize the strengths and limitations of each of these technologies and discuss some of the key concepts that must be taken into consideration when one is designing barcoding experiments. Finally, we suggest new directions to further improve the use of these technologies in cancer research.

Optical barcoding using polarisation sensitive plasmonic biosensors for the detection of self-assembled monolayers

Periodic subwavelength apertures have the ability to passively detect variations in the dielectric properties of the local sample environment through modification of the plasmon resonances associated with these structures. The resulting resonance peak can effectively provide a ‘fingerprint’ indicative of the dielectric properties of the medium within the near-surface region. Here we report on the use of bimodal silver-based plasmonic colour filters for molecular sensing. Firstly, by exploring the optical output of these devices as a function of the incident polarisation for a range of different analytes of known refractive index, we were able to both maximise and quantify their sensitivity. We then apply this concept to the real-time monitoring of the formation of self-assembled monolayers based on detection of the optical output using a spectrometer. This highlights the potential for bimodal plasmonic devices to be able to dynamically monitor variations in the local environment down to the level of single molecules without the need for specific functionalisation or labelling. Advantages of using this technique include the ability for these devices to be miniaturised and to dynamically tailor their optical output permitting the analysis of very small sample volumes and maximise their dynamic range for a specific analyte.

Longitudinal Monitoring of Intra-Tumoural Heterogeneity Using Optical Barcoding of Patient-Derived Colorectal Tumour Models.

Simple SummaryColorectal cancer (CRC) is the second most common cancer worldwide. Despite improvements in the clinical management of CRC, outcomes for those with metastatic disease remain extremely poor. One reason for this is tumour heterogeneity, which refers to the observation that each cell within complex tumour cell populations displays different genetic features and biological behaviours. Such tumour heterogeneity is known to impact treatment efficacy and promote tumour recurrence. Here, we present a multi-colour barcoding methodology that allows for different lineages of colorectal cancer cells to be identified and monitored, thus allowing for tumour heterogeneity to be quantified in real-time. We show that discrete cell lineages can be quantified by both fluorescence microscopy and flow cytometry. Using this approach, we show that the cell culture models that are traditionally used in cancer research display limited heterogeneity, whereas patient-derived organoids—which are generated from fresh tumour resections—more faithfully represent the heterogeneity observed in cancer patients.Geno- and phenotypic heterogeneity amongst cancer cell subpopulations are established drivers of treatment resistance and tumour recurrence. However, due to the technical difficulty associated with studying such intra-tumoural heterogeneity, this phenomenon is seldom interrogated in conventional cell culture models. Here, we employ a fluorescent lineage technique termed “optical barcoding” (OBC) to perform simultaneous longitudinal tracking of spatio-temporal fate in 64 patient-derived colorectal cancer subclones. To do so, patient-derived cancer cell lines and organoids were labelled with discrete combinations of reporter constructs, stably integrated into the genome and thus passed on from the founder cell to all its clonal descendants. This strategy enables the longitudinal monitoring of individual cell lineages based upon their unique optical barcodes. By designing a novel panel of six fluorescent proteins, the maximum theoretical subpopulation resolution of 64 discriminable subpopulations was achieved, greatly improving throughput compared with previous studies. We demonstrate that all subpopulations can be purified from complex clonal mixtures via flow cytometry, permitting the downstream isolation and analysis of any lineages of interest. Moreover, we outline an optimized imaging protocol that can be used to image optical barcodes in real-time, allowing for clonal dynamics to be resolved in live cells. In contrast with the limited intra-tumour heterogeneity observed in conventional 2D cell lines, the OBC technique was successfully used to quantify dynamic clonal expansions and contractions in 3D patient-derived organoids, which were previously demonstrated to better recapitulate the heterogeneity of their parental tumour material. In summary, we present OBC as a user-friendly, inexpensive, and high-throughput technique for monitoring intra-tumoural heterogeneity in in vitro cell culture models.

Imaging-based optical barcoding for relative humidity sensing based on meta-tip

Abstract In a wide range of applications such as healthcare treatment, environmental monitoring, food processing and storage, and semiconductor chip manufacturing, relative humidity (RH) sensing is required. However, traditional fiber-optic humidity sensors face the challenges of miniaturization and indirectly obtaining humidity values. Here, we propose and demonstrate an optical barcode technique by cooperating with RH meta-tip, which can predict the humidity values directly. Such RH meta-tip is composed of fiber-optic sensor based on surface plasmon resonance (SPR) effect and graphene oxide film as humidity sensitizer. While SPR sensor is composed of multimode fiber (MMF) integrated with metallic metasurface. Dynamic time warping (DTW) algorithm is used to obtain the warp path distance (WPD) sequence between the measured reflection spectrum and the spectra of the precalibrated database. The distance sequence is transformed into a pseudo-color barcode, and the humidity value is corresponded to the lowest distance, which can be read by human eyes. The RH measurement depends on the collective changes of the reflection spectrum rather than tracking a single specific resonance peak/dip. This work can open up new doors to the development of a humidity sensor with direct RH recognition by human eyes.

The site of breast cancer metastases dictates their clonal composition and reversible transcriptomic profile.

Intratumoral heterogeneity is a driver of breast cancer progression, but the nature of the clonal interactive network involved in this process remains unclear. Here, we optimized the use of optical barcoding to visualize and characterize 31 cancer subclones in vivo. By mapping the clonal composition of thousands of metastases in two clinically relevant sites, the lungs and liver, we found that metastases were highly polyclonal in lungs but not in the liver. Furthermore, the transcriptome of the subclones varied according to their metastatic niche. We also identified a reversible niche-driven signature that was conserved in lung and liver metastases collected during patient autopsies. Among this signature, we found that the tumor necrosis factor-α pathway was up-regulated in lung compared to liver metastases, and inhibition of this pathway affected metastasis diversity. These results highlight that the cellular and molecular heterogeneity observed in metastases is largely dictated by the tumor microenvironment.

Lanthanide-Based Molecular Cluster-Aggregates: Optical Barcoding and White-Light Emission with Nanosized {Ln20 } Compounds.

Counterfeit goods represent a major problem to companies, governments, and customers, affecting the global economy. In order to protect the authenticity of products and documents, optical anti-counterfeit technologies have widely been employed via the use of discrete molecular species, extended metal-organic frameworks (MOFs), and nanoparticles. Herein, for the first time we demonstrate the potential use of molecular cluster-aggregates (MCA) as optical barcodes via composition and energy transfer control. The tuneable optical properties for the [Ln20 (chp)30 (CO3 )12 (NO3 )6 (H2 O)6 ], where chp- =deprotonated 6-chloro-2-pyridinol, allow the fine control of the emission colour output, resulting in high-security level optical labelling with a precise read-out. Moreover, a unique tri-doped composition of GdIII , TbIII , and EuIII led to MCAs with white-light emission. The presented methodology is a unique approach to probe the effect of composition control on the luminescent properties of nanosized molecular material.

Multiplexed optical barcoding of cells via photochemical programming of bioorthogonal host-guest recognition.

Modern chemical and biological studies are undergoing a paradigm shift, where understanding the fate of individual cells, in an apparently homogeneous population, is becoming increasingly important. This has inculcated a growing demand for developing strategies that label individual cells with unique fluorescent signatures or barcodes so that their spatiotemporal trajectories can be mapped in real time. Among various approaches, light-regulated methods employing photocaged fluorophores have received particular attention, owing to their fine spatiotemporal control over labelling. However, their multiplexed use to barcode large numbers of cells for interrogating cellular libraries or complex tissues remains inherently challenging, due to the lack of multiple spectrally distinct photoactivated states in the currently available photocaged fluorophores. We report here an alternative multiplexable strategy based on optically controlled host–guest recognition in the cucurbit[7]uril (CB[7]) system that provides spatial control over the positioning of fluorophores to generate distinct barcodes in ‘user-defined’ cells. Using a combination of three spectrally distinct CB[7]-conjugated fluorophores and by sequentially performing cycles of photoactivation and fluorophore encoding, we demonstrate 10-color barcoding in microtubule-targeted fixed cells as well as 7-color barcoding in cell surface glycan targeted live MCF7 cells.

HGG-16. EXOSOME-MEDIATED INTER-CLONAL INTERACTIONS IN PEDIATRIC GBM AND DIPG

Pediatric Glioblastoma (pGBM) and Diffuse Intrinsic Pontine Glioma (DIPG) are highly heterogeneous brain tumors which we demonstrated are comprised by distinct sub-clones interacting in a functional network. Exosomes are known to mediate the crosstalk between tumor and its microenvironment. Based on this, we aimed to investigate the role of exosomes in mediating pGBM and DIPG inter-clonal communication. By using optical barcoding for single cell-tracking, we generated two bulk multicolor patient derived-cell lines (one DIPG H3.3K27M and one pGBM histone WT) from which we obtained two and five single cell-derived clones respectively. The sub-clones demonstrated significantly phenotypic differences in terms of morphology, growth, adhesion, migration and invasion properties. In particular, co-culture experiments, with the two most different clones for both cell-lines, confirmed the cell-cell interaction key role in driving their more aggressive phenotype. Furthermore, we found that pGBM and DIPG sub-clones release exosomes which are actively and differentially up-taken by individual clones. Analysis of the exosomal microRNAs showed a different profile between the two selected clones in each cell-line. In particular, we found a pool of five upregulated microRNAs in 1C5 clone (DIPG cell-line) strongly associated to Wnt-signaling and PI3K-AKT pathway. Similarly, a pool of five upregulated microRNAs for 5E2 clone (pGBM cell-line) were found associated with focal adhesion and PI3K-AKT pathway. Our study may provide novel therapeutic strategies by interfering with the exosome-mediated inter-clonal communication in pGBM and DIPG.