Two-photon Microscopy Research Articles

First hyperpolarizability of cellulose nanocrystals: an experimental and theoretical investigation.

Cellulose nanocrystals (CNCs) have attracted considerable interest due to their optical properties, though their nonlinear optical behavior remains largely unexplored. In this paper, we investigate the second-order nonlinear optical (SONLO) response of CNCs through both experimental and theoretical investigations. Hyper-Rayleigh scattering (HRS) experiments revealed values comparable to well-known nonlinear optical biomaterials, such as collagen, and on par with inorganic reference materials like KDP. The strong response in CNCs can be attributed to the well-ordered structure of the cellulose chains, which enhances the overall susceptibility of the nanoparticles. Quantum chemical modeling using density functional theory (DFT) was employed to simulate the molecular hyperpolarizability of CNCs. The study reduced the complex first hyperpolarizability tensor of the CNCs to two key components, βzzz and βzyy. An electrostatic model was applied to account for the CNCs' shape and dielectric properties, leading to strong agreement with the experimental data. Our findings highlight the potential of CNCs for optoelectronic applications and provide valuable insights for characterizing CNC-based mesomaterials through two-photon microscopy.

Advancements in organic fluorescent materials: unveiling the potential of peripheral group modification in dithienyl-diketopyrrolopyrrole derivatives for one- and two-photon bioimaging.

The quest for novel organic fluorescent materials capable of two-photon absorption (2PA) has intensified in recent years due to their promising applications in biological imaging. Two-photon fluorescence microscopy (2PFM) offers high spatial-temporal resolution, reduced photodamage, and deeper tissue penetration compared to conventional techniques. However, the development of bright two-photon molecular markers remains a challenge, necessitating compounds with high fluorescence quantum yield and 2PA cross-section (σ2PA). Strategies such as increasing π-conjugation have shown promise but are hindered by synthesis complexities and limited biocompatibility. Alternatively, incorporating electron-donating (ED) or electron-withdrawing (EW) peripheral groups in a main structure has emerged as a viable approach, leading to significant enhancements in σ2PA. This study highlights the advantages and challenges of these strategies, emphasizing the importance of exploring new organic compounds and evaluating the efficacy of peripheral groups for advanced two-photon bioimaging applications.

Cell Division Imaging of Tobacco BY-2 Cells in a 3D Volume by Two-Photon Spinning-Disk Confocal Microscopy.

Plant cells have a unique organization of microtubules during mitosis and cytokinesis. However, it is difficult to observe cell division in plant cells because of the distortion of images caused by thick samples. We addressed this issue by two-photon excitation coupled with spinning-disk confocal microscopy. Herein, we describe the method for observing microtubules and nuclei during cell division in tobacco BY-2 cells by two-photon spinning-disk confocal microscopy. The protocol includes the transformation of BY-2 cells with a plasmid for the expression of tubulin and histone markers, preparation of cells for microscopy, and operation of the two-photon spinning-disk microscope.

Molecular Design of a Diketopyrrolopyrrole Conjugated Oligoelectrolyte Capable of Imaging Intracellular Vesicle Membrane Dynamics.

Conjugated oligoelectrolytes (COEs) are lipid bilayer spanning optical reporters that hold promise for delineating spatiotemporal changes in subcellular compartments. However, their ability to probe a broader range of biological processes remains restricted due to the lack of environmentally-responsive chemical functionalities. Herein, the study reports a novel COE, namely COE-KP, for monitoring spatiotemporal changes in the endolysosomal vesicles. COE-KP features a central diketopyrrolopyrrole functional group in the optically active conjugated core that confers photophysical properties suitable for bioimaging, in particular responding to the presence of hydrogen bonding functionalities within the hydrophobic domain of the lipid bilayer. COE-KP can thus discriminate cells in different growth states through two-photon fluorescence lifetime imaging microscopy (FLIM) with excitation in the NIR-II range. These changes in lifetime most reasonably reflect the degree of water permeability through the membrane and are not linked to notable differences in membrane tension or solvent polarity. Furthermore, using stimulated emission depletion (STED) nanoscopy, it is possible to directly visualize membrane-bound COEs within cellular vesicles. These results illustrate further opportunities for applying COE-based reporters for super-resolution microscopy of organelle membranes, visualization of subcellular membrane morphologies, and imaging long-term changes in membrane properties.

Endoplasmic reticulum stress-related deficits in calcium clearance promote neuronal dysfunction that is prevented by SERCA2 gene augmentation.

Disruption of calcium (Ca2+) homeostasis in neurons is a hallmark of neurodegenerative diseases. Here, we investigate the mechanisms leading to Ca2+ dysregulation and ask whether altered Ca2+ dynamics impinge on neuronal stress and circuit dysfunction. Using two-photon microscopy, we show that ocular hypertension, a major risk factor in glaucoma, and optic nerve crush injury disrupt the capacity of retinal neurons to clear cytosolic Ca2+ leading to impaired light-evoked responses. Gene- and protein expression analysis reveal the loss of the sarco-endoplasmic reticulum (ER) Ca2+-ATPase2 pump (SERCA2/ATP2A2) in injured retinal neurons from mice and patients with primary open-angle glaucoma. Pharmacological activation or neuron-specific gene delivery of SERCA2 is sufficient to rescue single-cell Ca2+ dynamics and promote robust survival of damaged neurons. Furthermore, SERCA2 gene supplementation reduces ER stress, reestablishes circuit balance, and restores visual behaviors. Our findings reveal that enhancing the Ca2+ clearance capacity of vulnerable neurons alleviates organelle stress and promotes neurorecovery.

UNet-Att: a self-supervised denoising and recovery model for two-photon microscopic image

Two-photon microscopy is indispensable in cell and molecular biology for its high-resolution visualization of cellular and molecular dynamics. However, the inevitable low signal-to-noise conditions significantly degrade image quality, obscuring essential details and complicating morphological analysis. While existing denoising methods such as CNNs, Noise2Noise, and DeepCAD serve broad applications in imaging, they still have limitations in preserving texture structures and fine details in two-photon microscopic images affected by complex noise, particularly in sophisticated structures like neuronal synapses. To improve two-photon microscopy image denoising effectiveness, by experimenting on real two-photon microscopy images, we propose a novel deep learning framework, the UNet-Att model, which integrates a specifically tailored UNet++ architecture with attention mechanisms. Specifically, this approach consists of a sophisticated downsampling module for extracting hierarchical features at varied scales, and an innovative attention module that strategically emphasizes salient features during the integration process. The architecture is completed by an ingenious upsampling pathway that reconstructs the image with high fidelity, ensuring the retention of textural integrity. Additionally, the model supports end-to-end training, optimizing its denoising efficacy. The UNet-Att model proves to surpass mainstream algorithms in the dual objectives of denoising and preserving the textural intricacies of original images, which is evidenced by an increase of 9.42 dB in the high Peak Signal-to-Noise Ratio (PSNR) coupled with an improvement of 0.1131 in the Structural Similarity Index Measurement (SSIM). The ablation experiments reveal the effectiveness of each module. The associated Python packages and datasets of UNet-Att are freely available at https://github.com/ZjjDh/UNet-Att.

TIMP1 regulates ferroptosis in osteoblasts by inhibiting TFRC ubiquitination: an in vitro and in vivo study

BackgroundIn clinical practice, alterations in the internal environment of type 2 diabetes can significantly affect bone quality. While the increased risk of fractures among diabetic patients is well-established, the precise mechanisms by which hyperglycemia influences bone quality remain largely unclear.MethodsWestern blotting, immunohistochemistry (IHC), and micro-CT were used to examine ferroptosis-related protein expression and bone morphology changes in the bone tissues of type 2 diabetic mice. The CCK8 assay determined the optimal conditions for inducing ferroptosis in osteoblasts by high glucose and high fat (HGHF). Ferroptosis phenotypes in osteoblasts were analyzed using flow cytometry, Western blotting, and two-photon laser confocal microscopy. Transcriptomic sequencing of the control and HGHF groups, followed by bioinformatic analysis, identified and validated key genes. TIMP1 was knocked down in osteoblasts to assess its impact on ferroptosis, while TFRC expression was inhibited and activated to verify the role of TIMP1 in regulating ferroptosis through TFRC. The therapeutic effect of TIMP1 inhibition on osteoporosis was evaluated in a type 2 diabetic mouse model.ResultsThe expression of TIMP1 is increased in type 2 diabetic osteoporosis. In vitro, TIMP1 knockout inhibited ferroptosis in osteoblasts induced by high glucose and high fat (HGHF). However, overexpression of TFRC reversed the ferroptosis inhibition caused by TIMP1 knockout. Suppression of TIMP1 expression alleviated the progression of osteoporosis in type 2 diabetic mice. Mechanistic studies suggest that TIMP1 regulates HGHF-induced ferroptosis in osteoblasts through TFRC.ConclusionThis study demonstrates that TIMP1 expression is increased during type 2 diabetic osteoporosis and that TIMP1 promotes ferroptosis in osteoblasts by regulating TFRC. These findings suggest that TIMP1 is a promising novel therapeutic target for type 2 diabetic osteoporosis.

The sperm hook as a functional adaptation for migration and self-organized behavior.

In most murine species, spermatozoa exhibit a falciform apical hook at the head end. The function of the sperm hook is not yet clearly understood. In this study, we investigate the role of the sperm hook in the migration of spermatozoa through the female reproductive tract in Mus musculus (C57BL/6), using a deep tissue imaging custom-built two-photon microscope. Through live reproductive tract imaging, we found evidence indicating that the sperm hook aids in the attachment of spermatozoa to the epithelium and facilitates interactions between spermatozoa and the epithelium during migration in the uterus and oviduct. We also observed synchronized sperm beating, which resulted from the spontaneous unidirectional rearrangement of spermatozoa in the uterus. Based on live imaging of spermatozoa-epithelium interaction dynamics, we propose that the sperm hook plays a crucial role in successful migration through the female reproductive tract by providing anchor-like mechanical support and facilitating interactions between spermatozoa and the female reproductive tract in the house mouse.

Early and late place cells during postnatal development of the hippocampus

A proportion of hippocampal CA1 neurons function as place cells from the onset of navigation, which are referred to as early place cells. It is not clear whether this subset of neurons is predisposed to become place cells during early stages, or if all neurons have this potential. Here, we longitudinally imaged the activity of CA1 neurons in developing male rats during navigation with both one-photon and two-photon microscopy. Our results suggested that a largely consistent population of cells functioned as early place cells, demonstrating higher spatial coding abilities across environments and a tendency to form more synchronous cell assemblies. Early place cells were present in both deep and superficial layers of CA1. Cells in the deep layer exhibited greater synchrony than those in the superficial layer during early ages. These results support the theory that an initial cognitive map is primarily shaped by a predetermined set of hippocampal cells.

A compact module for video-rate image mosaics in two-photon microscopy

A compact module for video-rate image mosaics in two-photon microscopy

High-power, frequency-doubled all-polarization-maintaining fiber laser system at 925 nm.

We demonstrate a high-power 925-nm pulsed laser system based on a frequency-doubled, all-polarization-maintaining (PM) fiber laser source operating at 1.8 µm. The seed is a figure-9 mode-locked oscillator, which incorporates a nonlinear amplifying loop mirror. After power scaling and pulse compression, the 1.8-µm laser source can provide femtosecond pulses with a repetition rate of 31.3 MHz and an output power of 2.24 W. Through frequency doubling in a nonlinear crystal, the 925-nm laser delivers a pulse duration of 503 fs and an output power of 818 mW, which is the highest power provided by all-PM fiber laser systems at this wavelength, as far as we know. Furthermore, this 925-nm all-PM fiber laser is employed as the excitation light source for two-photon microscopy (TPM) and second-harmonic generation (SHG) microscopy.

A novel rhodopsin-based voltage indicator for simultaneous two-photon optical recording with GCaMP in vivo.

Genetically encoded voltage indicators (GEVIs) allow optical recording of membrane potential from targeted cells in vivo . However, red GEVIs that are compatible with two-photon microscopy and that can be multiplexed in vivo with green reporters like GCaMP, are currently lacking. To address this gap, we explored diverse rhodopsin proteins as GEVIs and engineered a novel GEVI, 2Photron, based on a rhodopsin from the green algae Klebsormidium nitens . 2Photron, combined with two photon ultrafast local volume excitation (ULoVE), enabled multiplexed readout of spiking and subthreshold voltage simultaneously with GCaMP calcium signals in visual cortical neurons of awake, behaving mice. These recordings revealed the cell-specific relationship of spiking and subthreshold voltage dynamics with GCaMP responses, highlighting the challenges of extracting underlying spike trains from calcium imaging.

Breaking Abbe's diffraction limit with harmonic deactivation microscopy.

Nonlinear optical microscopy provides elegant means for label-free imaging of biological samples and condensed matter systems. The widespread areas of application could even be increased if resolution was improved, which the famous Abbe diffraction limit now restrains. Super-resolution techniques can break the diffraction limit but most rely on fluorescent labeling. This makes them incompatible with (sub)femtosecond temporal resolution and applications that demand the absence of labeling. Here, we introduce harmonic deactivation microscopy (HADES) for breaking the diffraction limit in nonfluorescent samples. By controlling the harmonic generation process on the quantum level with a second donut-shaped pulse, we confine the third-harmonic generation to three times below the original focus size of a scanning microscope. We demonstrate that resolution improvement by deactivation is more efficient for higher harmonic orders and only limited by the maximum applicable deactivation-pulse fluence. This provides a route toward sub-100-nanometer resolution in a regular nonlinear microscope.

Reactive Stroma and Acinar Morphology in Prostate Cancer: Implications for Progression and Prognostic Assessment.

Prostate cancer (PC) remains a significant global health concern, with prognostic assessments largely reliant on the Gleason Classification System. While it has proven effective, subjectivity in interpretation persists, prompting the need for complementary approaches. Reactive stroma (RS) has emerged as a potential candidate for enhancing PC characterization, as it reflects intricate interactions among stromal, epithelial, and extracellular matrix components. To shed light on this, we conducted a comprehensive study. Two expert pathologists independently analyzed consecutive prostate biopsies (n = 120 patients), categorized into four groups based on Gleason scores. Four acinar patterns were described, denoted as A, B, C, and D. Our study uncovered a noteworthy presence of RS, predominantly within poorly differentiated tumors. Stromogenic tumors, characterized by high RS content, were particularly associated with Gleason scores of 4 + 3 and ≥ 8. Intriguingly, acinar patterns, including the distinctive B and D patterns, exhibited strong correlations with stromogenic tumors. Incorporating quantitative imaging techniques (Second Harmonic Generation and Two-Photon Excitation Fluorescence Microscopy), we examined collagen fiber density within the stroma. Our analysis revealed a direct relationship between RS intensity and collagen fiber counts, particularly prominent in patterns B and D. These findings suggest that the stromal reaction in PC is closely linked to acinar morphology and collagen deposition. Moreover, rudimentary microacini at the tumor periphery, associated with intense RS and patterns B and D, may signify an unfavorable prognosis. Our study highlights the potential of RS as an additional prognostic factor in PC. It underscores the intricate interplay between acinar patterns, RS intensity, and collagen fiber density, providing valuable insights for future prognostic assessments and therapeutic strategies. Further exploration of these relationships is essential for a comprehensive understanding of PC progression and management.

Imaging Brain Tissue with Quantum Light at Low Power.

Light-induced tissue damage is a crucial limitation for traditional microscopy of the living brain, underscoring the need for new techniques that minimize exposure of samples to light. Here, we tested the hypothesis that quantum light, i.e., entangled photons, could detect brain structures at a lower excitation energy. In a proof of principle, we show microscopic images of fixed brain tissue in the hippocampus area created by fluorescence selective excitation in the process of entangled two-photon absorption in a scanning microscope. Quantum-enhanced entangled two-photon microscopy (TPM) had brain imaging capabilities at an unprecedented low excitation intensity of ∼3.6 × 107 photons/s, orders of magnitude lower than the excitation level for the classical two-photon fluorescence image obtained in the same microscope. The extremely low light probe intensity demonstrated in entangled TPM is of critical importance in the investigation of neural activity to minimize heating and photobleaching during repetitive imaging. It may have important functional implications in optogenetic technology, removing unintended heating and accumulated photodamage effects. This technology also opens avenues in spatially resolved brain tissue investigations with quantum light, providing new capabilities in local spectroscopy.

Dynamic changes in the structure and function of brain mural cells around chronically implanted microelectrodes

Integration of neural interfaces with minimal tissue disruption in the brain is ideal to develop robust tools that can address essential neuroscience questions and combat neurological disorders. However, implantation of intracortical devices provokes severe tissue inflammation within the brain, which requires a high metabolic demand to support a complex series of cellular events mediating tissue degeneration and wound healing. Pericytes, peri-vascular cells involved in blood-brain barrier maintenance, vascular permeability, waste clearance, and angiogenesis, have recently been implicated as potential perpetuators of neurodegeneration in brain injury and disease. While the intimate relationship between pericytes and the cortical microvasculature have been explored in other disease states, their behavior following microelectrode implantation, which is responsible for direct blood vessel disruption and dysfunction, is currently unknown. Using two-photon microscopy we observed dynamic changes in the structure and function of pericytes during implantation of a microelectrode array over a 4-week implantation period. Pericytes respond to electrode insertion through transient increases in intracellular calcium and underlying constriction of capillary vessels. Within days following the initial insertion, we observed an influx of new, proliferating pericytes which contribute to new blood vessel formation. Additionally, we discovered a potentially novel population of reactive immune cells in close proximity to the electrode-tissue interface actively engaging in encapsulation of the microelectrode array. Finally, we determined that intracellular pericyte calcium can be modulated by intracortical microstimulation in an amplitude- and frequency-dependent manner. This study provides a new perspective on the complex biological sequelae occurring at the electrode-tissue interface and will foster new avenues of potential research consideration and lead to development of more advanced therapeutic interventions towards improving the biocompatibility of neural electrode technology.

Adult Neurogenesis: A Review of Current Perspectives and Implications for Neuroscience Research

Background: The study of new neuron formation in the adult brain has sparked controversy and ignited interest among scientists in recent times, these include its occurrence and location in the adult human brain, functional significance, variation in study methods, translation from animal model to human, and ethical challenges involving neural stem cell research. Aim: To provide a comprehensive understanding of adult neurogenesis, functional significance, and challenges and explore the latest advances in the study of adult neurogenesis. Methodology: An extensive and systematic search of electronic databases (Medline, Scopus, Web of Science) was conducted using keywords related to adult neurogenesis and techniques involved in its study. Results: The mechanism of adult neurogenesis was found to occur in specific brain regions such as the subgranular zone of the dentate gyrus and subventricular zone of the lateral ventricle. Adult neurogenesis is vital neural plasticity, providing a potential mechanism for the brain to adapt and reorganize in response to environmental cues and experiences. Cutting-edge research and sophisticated imaging techniques, such as two-photon microscopy, MRI, optogenetic, and stem-cell-based therapies have provided deeper insight into the study of adult neurogenesis. Conclusion: The study of neurogenesis is important for understanding nervous system development, physiology, pathology, and exploring neuroplasticity. Its advancement is challenged by some ethical concerns regarding embryonic, pluripotent stem cells, and the need for safe, and noninvasive study methods. Although recent breakthroughs in neuroimaging, microscopic techniques, and genetic tools are aiding real-time study of adult neurogenesis.

IMMU-28. LOW-INTENSITY PULSED ULTRASOUND COMBINED WITH POLY-ICLC AND INTERLEUKIN-2 UNLOCKS THE IMMUNE PRIVILEGE OF THE BRAIN BY PROMOTING EPITOPE SPREADING AND ACTIVATION OF CNS-SPECIFIC NAÏVE T CELLS

Abstract Immunotherapies targeting glioma, such as chimeric antigen receptor (CAR) T cell therapies, offer new therapeutic avenues; however, their application has yielded limited success in clinical trials so far. Due to the antigenic heterogeneity of gliomas, targeting a few or several antigens still results in recurrence with “antigen-loss” tumors, indicating the need to engage the endogenous immune system to eliminate these therapy-resistant tumor cells. However, natural anatomical barriers and an immunosuppressive environment allow brain tumors to evade detection by the endogenous immune system. Low-intensity pulsed ultrasound combined with microbubbles (LIPU/MB) has recently been shown to transiently open the blood-brain barrier (BBB), enabling penetration of cells and drugs into the CNS. Using intravital two-photon imaging, two-photon microscopy, multiparametric spectral flow cytometry, and bone marrow chimera mice, alongside blood samples from glioblastoma patients treated with LIPU/MB, we observed that LIPU/MB promotes migration of CNS antigen-presenting cells from the perivascular space into the periphery. To assess the immunological relevance of LIPU/MB-mediated CNS antigen presentation to the peripheral immune system, we have developed new transgenic mice (GFAP-minigene mice) with an intact BBB expressing four immunogenic peptides under the CNS-specific GFAP promoter. In GFAP-minigene mice, LIPU/MB combined with toll-like receptor 3 agonist poly-ICLC and Interleukin-2 promoted priming, activation, and homing of naïve CNS-specific T cells into the brain parenchyma. Cytotoxic T cell activity in the brain was accompanied by MHC-I and MHC-II epitope spreading, mirrored by the occurrence of endogenous T cells in the brain, deep cervical lymph nodes, and spleen harboring other CNS-(minigene) antigen-specific T cell receptors. Collectively, our findings suggest that LIPU/MB combined with poly-ICLC can overcome inherent limitations of immunotherapy by enhancing CNS epitope spreading and activation of naïve CNS-antigen-specific T cells within the context of an intact blood-brain barrier.

Senolysis potentiates endothelial progenitor cell adhesion to and integration into the brain vasculature

BackgroundOne of the most severe consequences of ageing is cognitive decline, which is associated with dysfunction of the brain microvasculature. Thus, repairing the brain vasculature could result in healthier brain function.MethodsTo better understand the potential beneficial effect of endothelial progenitor cells (EPCs) in vascular repair, we studied the adhesion and integration of EPCs using the early embryonic mouse aorta–gonad–mesonephros – MAgEC 10.5 endothelial cell line. The EPC interaction with brain microvasculature was monitored ex vivo and in vivo using epifluorescence, laser confocal and two-photon microscopy in healthy young and old animals. The effects of senolysis, EPC activation and ischaemia (two-vessel occlusion model) were analysed in BALB/c and FVB/Ant: TgCAG-yfp_sb #27 mice.ResultsMAgEC 10.5 cells rapidly adhered to brain microvasculature and some differentiated into mature endothelial cells (ECs). MAgEC 10.5-derived endothelial cells integrated into microvessels, established tight junctions and co-formed vessel lumens with pre-existing ECs within five days. Adhesion and integration were much weaker in aged mice, but were increased by depleting senescent cells using abt-263 or dasatinib plus quercetin. Furthermore, MAgEC 10.5 cell adhesion to and integration into brain vessels were increased by ischaemia and by pre-activating EPCs with TNFα.ConclusionsCombining progenitor cell therapy with senolytic therapy and the prior activation of EPCs are promising for improving EPC adhesion to and integration into the cerebral vasculature and could help rejuvenate the ageing brain.

EXTH-13. COMBINED ANTI-ANGIOGENIC TREATMENT TO INCREASE INTRATUMORAL PERSISTENCE OF CAR T-CELLS IN LUNG CANCER BRAIN METASTASES

Abstract BACKGROUND Metastatic lung cancer, especially when it spreads to the brain, shows a devastating prognosis. Immunotherapeutic approaches including CAR T-cells show promise in hematologic malignancies. However, the presence of immunosuppressive factors within the tumor microenvironment and physical barriers like elevated interstitial pressure due to dysfunctional tumor vessels are one of the main obstacles especially in solid tumors. Evaluation of the potential synergy between anti-angiogenetic drugs and CAR T-cells in this context are an ongoing investigation. METHODS Here, we set up an immunocompetent syngeneic orthotopic cerebral metastasis model in mice by combining a chronic cranial window with repetitive intracerebral two-photon laser scanning microscopy (TPLSM). This model allows in vivo characterization of fluorescent tumor cells and CAR T-cells on a single cell level over time. Intraparenchymal injection of red fluorescent EpCAM-transduced Lewis Lung carcinoma cells (EpCAM/tdtLL/2 cells) followed by injection of EpCAM-directed or undirected CAR T-cells into the adjacent brain tissue was performed. Additionally, aVEGF-A combined with aVEGF-R2 and the respective isotype control were injected intraperitoneally. RESULTS Compared to undirected CAR T-cells, mice receiving EpCAM-directed CAR T-cells showed higher intratumoral CAR T-cell densities after intraparenchymal injection. This finding was accompanied with reduced tumor growth and eventually translates into a survival benefit. However, intratumoral CAR T-cell numbers diminish over time indicating insufficient persistence inside of the tumor microenvironment. Additional anti-VEGF-A/VEGF-R2 treatment results in reduced tumor growth after EpCAM/GFPCAR T-cell and GFPT-cell treatment, respectively. Interestingly, in vivo imaging reveals higher intratumoral CAR T-cell numbers after anti-angiogenic treatment compared to isotype controls pointing towards enhanced CAR T-cell persistence resulting in a survival benefit. CONCLUSION Collectively, our findings indicate that locally injected CAR T-cells may safely induce relevant anti-tumor effects in brain metastases from lung cancer. Additional anti-angiogenic treatment provides a mechanism to enhance intratumoral CAR T-cell persistence and reduce tumor growth.