Search

Revealing the 3D composition of intact tissue specimens is essential for understanding cell and organ biology in health and disease. State-of-the-art 3D microscopy techniques aim to capture tissue volumes on an ever-increasing scale, while also retaining sufficient resolution for single-cell analysis. Furthermore, spatial profiling through multi-marker imaging is fast developing, providing more context and better distinction between cell types. Following these lines of technological advance, we here present a protocol based on FUnGI (fructose, urea and glycerol clearing solution for imaging) optical clearing of tissue before multispectral large-scale single-cell resolution 3D (mLSR-3D) imaging, which implements 'on-the-fly' linear unmixing of up to eight fluorophores during a single acquisition. Our protocol removes the need for repetitive illumination, thereby allowing larger volumes to be scanned with better image quality in less time, also reducing photo-bleaching and file size. To aid in the design of multiplex antibody panels, we provide a fast and manageable intensity equalization assay with automated analysis to design a combination of markers with balanced intensities suitable for mLSR-3D. We demonstrate effective mLSR-3D imaging of various tissues, including patient-derived organoids and xenografted tumors, and, furthermore, describe an optimized workflow for mLSR-3D imaging of formalin-fixed paraffin-embedded samples. Finally, we provide essential steps for 3D image data processing, including shading correction that does not require pre-acquired shading references and 3D inhomogeneity correction to correct fluorescence artefacts often afflicting 3D datasets. Together, this provides a one-week protocol for eight-fluorescent-marker 3D visualization and exploration of intact tissue of various origins at single-cell resolution.Multispectral large-scale single-cell resolution 3D imaging allows up to eight fluorophores to be captured in a single acquisition. This protocol enables the visualization and exploration of large intact tissue volumes.

Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a ‘super engager’ behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies.

Despite advances in three-dimensional (3D) imaging, it remains challenging to profile all the cells within a large 3D tissue, including the morphology and organization of the many cell types present. Here, we introduce eight-color, multispectral, large-scale single-cell resolution 3D (mLSR-3D) imaging and image analysis software for the parallelized, deep learning–based segmentation of large numbers of single cells in tissues, called segmentation analysis by parallelization of 3D datasets (STAPL-3D). Applying the method to pediatric Wilms tumor, we extract molecular, spatial and morphological features of millions of cells and reconstruct the tumor’s spatio-phenotypic patterning. In situ population profiling and pseudotime ordering reveals a highly disorganized spatial pattern in Wilms tumor compared to healthy fetal kidney, yet cellular profiles closely resembling human fetal kidney cells could be observed. In addition, we identify previously unreported tumor-specific populations, uniquely characterized by their spatial embedding or morphological attributes. Our results demonstrate the use of combining mLSR-3D and STAPL-3D to generate a comprehensive cellular map of human tumors. An imaging software for large-scale microscopy data shows how cells are organized in tissues.

Our understanding of the cellular composition and architecture of cancer has primarily advanced using 2D models and thin slice samples. This has granted spatial information on fundamental cancer biology and treatment response. However, tissues contain a variety of interconnected cells with different functional states and shapes, and this complex organization is impossible to capture in a single plane. Furthermore, tumours have been shown to be highly heterogenous, requiring large‐scale spatial analysis to reliably profile their cellular and structural composition. Volumetric imaging permits the visualization of intact biological samples, thereby revealing the spatio‐phenotypic and dynamic traits of cancer. This review focuses on new insights into cancer biology uniquely brought to light by 3D imaging and concomitant progress in cancer modelling and quantitative analysis. 3D imaging has the potential to generate broad knowledge advance from major mechanisms of tumour progression to new strategies for cancer treatment and patient diagnosis. We discuss the expected future contributions of the newest imaging trends towards these goals and the challenges faced for reaching their full application in cancer research.

Revealing the 3D composition of intact tissue specimens is essential for understanding cell and organ biology in health and disease. State-of-the-art 3D microscopy techniques aim to capture tissue volumes on an ever-increasing scale, while also retaining sufficient resolution for single-cell analysis. Furthermore, spatial profiling through multi-marker imaging is fast developing, providing more context and better distinction between cell types. Following these lines of technological advance, we here present a protocol based on FUnGI (fructose, urea and glycerol clearing solution for imaging) optical clearing of tissue before multispectral large-scale single-cell resolution 3D (mLSR-3D) imaging, which implements 'on-the-fly' linear unmixing of up to eight fluorophores during a single acquisition. Our protocol removes the need for repetitive illumination, thereby allowing larger volumes to be scanned with better image quality in less time, also reducing photo-bleaching and file size. To aid in the design of multiplex antibody panels, we provide a fast and manageable intensity equalization assay with automated analysis to design a combination of markers with balanced intensities suitable for mLSR-3D. We demonstrate effective mLSR-3D imaging of various tissues, including patient-derived organoids and xenografted tumors, and, furthermore, describe an optimized workflow for mLSR-3D imaging of formalin-fixed paraffin-embedded samples. Finally, we provide essential steps for 3D image data processing, including shading correction that does not require pre-acquired shading references and 3D inhomogeneity correction to correct fluorescence artefacts often afflicting 3D datasets. Together, this provides a one-week protocol for eight-fluorescent-marker 3D visualization and exploration of intact tissue of various origins at single-cell resolution.

In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.

Despite advances in three-dimensional (3D) imaging, it remains challenging to profile all the cells within a large 3D tissue, including the morphology and organization of the many cell types present. Here, we introduce eight-color, multispectral, large-scale single-cell resolution 3D (mLSR-3D) imaging and image analysis software for the parallelized, deep learning-based segmentation of large numbers of single cells in tissues, called segmentation analysis by parallelization of 3D datasets (STAPL-3D). Applying the method to pediatric Wilms tumor, we extract molecular, spatial and morphological features of millions of cells and reconstruct the tumor's spatio-phenotypic patterning. In situ population profiling and pseudotime ordering reveals a highly disorganized spatial pattern in Wilms tumor compared to healthy fetal kidney, yet cellular profiles closely resembling human fetal kidney cells could be observed. In addition, we identify previously unreported tumor-specific populations, uniquely characterized by their spatial embedding or morphological attributes. Our results demonstrate the use of combining mLSR-3D and STAPL-3D to generate a comprehensive cellular map of human tumors.

Surgical procedures such as tumor resection and biopsy are still the gold standard for diagnosis and (determination of) treatment of solid tumors, and are prognostically beneficial for patients. However, growing evidence suggests that even a minor surgical trauma can influence several (patho) physiological processes that might promote postoperative metastatic spread and tumor recurrence. Local effects include tumor seeding and a wound healing response that can promote tumor cell migration, proliferation, differentiation, extracellular matrix remodeling, angiogenesis and extravasation. In addition, local and systemic immunosuppression impairs antitumor immunity and contributes to tumor cell survival. Surgical manipulation of the tumor can result in cancer cell release into the circulation, thus increasing the chance of tumor cell dissemination. To prevent these undesired effects of surgical interventions, therapeutic strategies targeting immune response exacerbation or alteration have been proposed. This review summarizes the current literature regarding these local, systemic and secondary site effects of surgical interventions on tumor progression and dissemination, and discusses studies that aimed to identify potential therapeutic approaches to prevent these effects in order to further increase the clinical benefit from surgical procedures.

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.

In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.

Although biopsies and tumor resection are prognostically beneficial for glioblastomas (GBM), potential negative effects have also been suggested. Here, using retrospective study of patients and intravital imaging of mice, we identify some of these negative aspects, including stimulation of proliferation and migration of non-resected tumor cells, and provide a strategy to prevent these adverse effects. By repeated high-resolution intravital microscopy, we show that biopsy-like injury in GBM induces migration and proliferation of tumor cells through chemokine (C-C motif) ligand 2 (CCL-2)-dependent recruitment of macrophages. Blocking macrophage recruitment or administrating dexamethasone, a commonly used glucocorticoid to prevent brain edema in GBM patients, suppressed the observed inflammatory response and subsequent tumor growth upon biopsy both in mice and in multifocal GBM patients. Taken together, our study suggests that inhibiting CCL-2-dependent recruitment of macrophages may further increase the clinical benefits from surgical and biopsy procedures.

Introduction: Glioblastoma (GBM) belongs to the most aggressive cancers. Main pathophysiological feature of GBM is fast and widespread invasion of the brain parenchyma by a subpopulation of progenitor tumour cells, rendering localized therapies ineffective. In addition, brain tumour initiating cells (BTIC) from GBM may repopulate the tumour site after initially effective treatments. Thus, the invasive progenitor tumour cell fraction is a strategic prime target for early and sustained anti-invasion therapies. To date, the dependency on and interaction with the microenvironment of progenitor cells and their adaptive development from the tumour cell fraction has not been thoroughly investigated. Methods: With organotypic brain slice cultures (OBSC), single cell RNA expression analysis and bioinformatics workup, we established a novel integrated approach to investigate the expression profile of invasion leading cells. A micromanipulator adapter was developed to isolate different three subpopulations within human GBM progenitor cells: leader, follower, and stationary, from the OBSCs. In comparison, we use long-term intravital imaging to analyse the behaviour and interaction of the three cellular fractions with each other and the microenvironment in vivo. Imaging through a cranial window in the brain of a living mouse enables us to visualize single tumour cells. Using a photoswitchable vector expressed in BTICs we specifically label and isolate leader, follower, and stationary cells separately to compare leader cell behaviour in vivo, in situ, and in the patient situation. Findings: The in situ OBSC model is a valuable tool to analyse migration and invasion in conditions simulating normal brain tissue including some microenvironmental influences. This allowed us to monitor human BTIC cell invasion in real time. Invasive cells emerge from the initial cell population, driving invasion into the surrounding tissue. Those leader cells differ in morphology and behaviour from stationary cells, which remain at the implantation site. A subsequent microarray analysis of single cells revealed that leader cells have a markedly distinct expression pattern in comparison to follower and stationary cells. This leader cell-specific expression signature is currently verified in fresh patient tumour biopsies, other BTICs analysed in the OBSC as model, and in the cranial window model. Updated results will be presented at the WFNOS meeting. Outlook: We showed that invasive leader cells have a specific functional and molecular profile. A thorough characterization of these cells in respect to their interaction with and response to the microenvironment that may be involved in the process of transforming cells into invasive leader cells is ongoing. We hypothesize that a deep characterization of these events will shed light on the chronology of one of the gliomagenesis milestones, namely invasion.

Multipotent human adipose tissue mesenchymal stromal cells (hAMSCs) are promising therapy vehicles with tumor-homing capacity that can be easily modified to deliver cytotoxicity activating systems in the proximity of tumors. In a previous work, we observed that hAMSCs are very effective delivering cytotoxicity to glioma tumors. However, these results were difficult to reconcile with the relatively few hAMSCs surviving implantation. We use a bioluminescence imaging (BLI) platform to analyze the behavior of bioluminescent hAMSCs expressing HSV-tTK in a U87 glioma model and gain insight into the therapeutic mechanisms. Tumor-implanted hAMSCs express the endothelial marker PECAM1(CD31), integrate in tumor vessels and associate with CD133-expressing glioma stem cells (GSC). Inhibition of endothelial lineage differentiation in hAMSCs by Notch1 shRNA had no effect on their tumor homing and growth-promoting capacity but abolished the association of hAMSCs with tumor vessels and CD133+ tumor cells and significantly reduced their tumor-killing capacity. The current strategy allowed the study of tumor/stroma interactions, showed that tumor promotion and tumor-killing capacities of hAMSCs are based on different mechanisms. Our data strongly suggest that the therapeutic effectiveness of hAMSCs results from their association with special tumor vascular structures that also contain GSCs.

In vivo testing is a mandatory last step in scaffold development. Agile longitudinal noninvasive real-time monitoring of stem cell behavior in biomaterials implanted in live animals should facilitate the development of scaffolds for tissue engineering. We report on a noninvasive bioluminescence imaging (BLI) procedure for simultaneous monitoring of changes in the expression of multiple genes to evaluate scaffold performance in vivo. Adipose tissue-derived stromal mensenchymal cells were dually labeled with Renilla red fluorescent protein and firefly green fluorescent protein chimeric reporters regulated by cytomegalovirus and tissue-specific promoters, respectively. Labeled cells were induced to differentiate in vitro and in vivo, by seeding in demineralized bone matrices (DBMs) and monitored by BLI. Imaging results were validated by RT-polymerase chain reaction and histological procedures. The proposed approach improves molecular imaging and measurement of changes in gene expression of cells implanted in live animals. This procedure, applicable to the simultaneous analysis of multiple genes from cells seeded in DBMs, should facilitate engineering of scaffolds for tissue repair.

Human mesenchymal stromal cells, whether from the bone marrow or adipose tissue (hASCs), are promising cell therapy agents. However, generation of abundant cells for therapy remains to be a challenge, due to the need of lengthy expansion and the risk of accumulating genomic defects during the process. We show that hASCs can be easily induced to a reversible fast-proliferating phenotype (FP-ASCs) that allows rapid generation of a clinically useful quantity of cells in

10 páginas, 4 figuras, 1 tabla.-- et al., Hypothyroidism is the most common hormonal disease in adults, which is frequently accompanied by learning and memory impairments and emotional disorders. However, the deleterious effects of thyroid hormones deficiency on emotional memory are poorly understood and often underestimated. To evaluate the consequences of hypothyroidism on emotional learning and memory, we have performed a classical Pavlovian fear conditioning paradigm in euthyroid and adult-thyroidectomized Wistar rats. In this experimental model, learning acquisition was not impaired, fear memory was enhanced, memory extinction was delayed and spontaneous recovery of fear memory was exacerbated in hypothyroid rats. The potentiation of emotional memory under hypothyroidism was associated with an increase of corticosterone release after fear conditioning and with higher expression of glucocorticoid and mineralocorticoid receptors in the lateral and basolateral nuclei of the amygdala, nuclei that are critically involved in the circuitry of fear memory. Our results demonstrate for the first time that adult-onset hypothyroidism potentiates fear memory and also increases vulnerability to develop emotional memories. Furthermore, our findings suggest that enhanced corticosterone signaling in the amygdala is involved in the pathophysiological mechanisms of fear memory potentiation. Therefore, we recommend evaluating whether inappropriate regulation of fear in patients with post-traumatic stress and other mental disorders is associated with abnormal levels of thyroid hormones, especially those patients refractory to treatment., This work was supported by grants BFU2007-62979 and BFU 2010-16498 (A.G.-F) and SAF2009-09129 (C.V.) from the Spanish Ministry of Science and Innovation. I.F.-L. was recipient of a fellowship and A.M.-P. of a contract both from the Ministry of Science and Innovation, Spain.

A biopsy is for most types of cancer the golden standard for a definitive cancer diagnosis, as it provides the most accurate analysis of tissues. However taking biopsies creates a wound thereby potentially changing the composition and architecture of the tissue. In non-pathological situations, wounded tissue ‘repairs’ itself by a cascade of cellular events, including the induction of an inflammatory response that promotes cell proliferation and migration of surrounding cells to close the wound. Proliferation and migration are two malignant processes that promote tumor progression and metastatic disease. Thus it is vital to identify the potential effect of biopsy on the behavior of cells that remain in the tumor. By making use of repeated high-resolution intravital microscopy (IVM) we studied how the behavior of tumor cells locally changes upon biopsy. Our results showed that biopsy-like injury triggers a wound-healing response observed as increased tumor cell migration and proliferation. 24 hours after biopsy-like injury there was a 1.75 fold increase in migratory cells and a 1.52 fold increase in proliferating cells compared to the control group that did not received an injury. IVM showed that the observed effects were mediated by a CCL-2- dependent inflammation response. Interestingly, immune cell ablation by dexamethasone, a glucocorticoid commonly used to prevent inflammation resulted in decreased tumor cell motility and proliferation and suppressed the observed wound healing response. This result opens new therapeutic possibilities to prevent biopsy undesired effects on tumor cell behavior. Taken together, our data show that biopsy induces a local wound-healing response in the tumor through an immune system mediated mechanism and illustrate that IVM is a powerful technique to study local and temporal changes in cell behavior that cannot be assessed by other techniques (e.g. immunohistochemistry) that only provide a snapshot of a dynamic tissue. Furthermore this tool can be useful to determine local and dynamic responses of (tumor) cells to other more aggressive clinical interventions, such as surgical resection or chemotherapeutic treatment. Citation Format: Maria Alieva, Burcin Colak, Carla Boquetale, Jacco Van Rheenen. Intravital microscopy reveals biopsy induced local wound-healing response. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A84.

Genetically modified adipose tissue derived mesenchymal stromal cells (hAMSCs) with tumor homing capacity have been proposed for localized therapy of chemo- and radiotherapy resistant glioblastomas. We demonstrate an effective procedure to optimize glioblastoma therapy based on the use of genetically modified hAMSCs and in vivo non invasive monitoring of tumor and therapeutic cells. Glioblastoma U87 cells expressing Photinus pyralis luciferase (Pluc) were implanted in combination with hAMSCs expressing a trifunctional Renilla reniformis luciferase-red fluorescent protein-thymidine kinase reporter in the brains of SCID mice that were subsequently treated with ganciclovir (GCV). The resulting optimized therapy was effective and monitoring of tumor cells by bioluminescence imaging (BLI) showed that after 49 days GCV treatment reduced significantly the hAMSC treated tumors; by a factor of 10(4) relative to controls. Using a Pluc reporter regulated by an endothelial specific promoter and in vivo BLI to image hAMSC differentiation we gained insight on the therapeutic mechanism. Implanted hAMSCs homed to tumor vessels, where they differentiated to endothelial cells. We propose that the tumor killing efficiency of genetically modified hAMSCs results from their association with the tumor vascular system and should be useful vehicles to deliver localized therapy to glioblastoma surgical borders following tumor resection.