3D imaging for driving cancer discovery.

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.

Endothelial differentiation of adipose tissue-derived mesenchymal stromal cells in glioma tumors: implications for cell-based therapy.

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.

BEHAV3D: a 3D live imaging platform for comprehensive analysis of engineered T cell behavior and tumor response.

Modeling immuno-oncology by using patient-derived material and immune cell co-cultures can advance our understanding of immune cell tumor targeting in a patient-specific manner, offering leads to improve cellular immunotherapy. However, fully exploiting these living cultures requires analysis of the dynamic cellular features modeled, for which protocols are currently limited. Here, we describe the application of BEHAV3D, a platform that implements multi-color live 3D imaging and computational tools for: (i) analyzing tumor death dynamics at both single-organoid or cell and population levels, (ii) classifying T cell behavior and (iii) producing data-informed 3D images and videos for visual inspection and further insight into obtained results. Together, this enables a refined assessment of how solid and liquid tumors respond to cellular immunotherapy, critically capturing both inter- and intratumoral heterogeneity in treatment response. In addition, BEHAV3D uncovers T cell behavior involved in tumor targeting, offering insight into their mode of action. Our pipeline thereby has strong implications for comparing, prioritizing and improving immunotherapy products by highlighting the behavioral differences between individual tumor donors, distinct T cell therapy concepts or subpopulations. The protocol describes critical wet lab steps, including co-culture preparations and fast 3D imaging with live cell dyes, a segmentation-based image processing tool to track individual organoids, tumor and immune cells and an analytical pipeline for behavioral profiling. This 1-week protocol, accessible to users with basic cell culture, imaging and programming expertise, can easily be adapted to any type of co-culture to visualize and exploit cell behavior, having far-reaching implications for the immuno-oncology field and beyond.

A multispectral 3D live organoid imaging platform to screen probes for fluorescence guided surgery.

Achieving complete tumor resection is challenging and can be improved by real-time fluorescence-guided surgery with molecular-targeted probes. However, pre-clinical identification and validation of probes presents a lengthy process that is traditionally performed in animal models and further hampered by inter- and intra-tumoral heterogeneity in target expression. To screen multiple probes at patient scale, we developed a multispectral real-time 3D imaging platform that implements organoid technology to effectively model patient tumor heterogeneity and, importantly, healthy human tissue binding.

Bridging live-cell imaging and next-generation cancer treatment.

By providing spatial, molecular and morphological data over time, live-cell imaging can provide a deeper understanding of the cellular and signalling events that determine cancer response to treatment. Understanding this dynamic response has the potential to enhance clinical outcome by identifying biomarkers or actionable targets to improve therapeutic efficacy. Here, we review recent applications of live-cell imaging for uncovering both tumour heterogeneity in treatment response and the mode of action of cancer-targeting drugs. Given the increasing uses of T cell therapies, we discuss the unique opportunity of time-lapse imaging for capturing the interactivity and motility of immunotherapies. Although traditionally limited in the number of molecular features captured, novel developments in multidimensional imaging and multi-omics data integration offer strategies to connect single-cell dynamics to molecular phenotypes. We review the effect of these recent technological advances on our understanding of the cellular dynamics of tumour targeting and discuss their implication for next-generation precision medicine.

Uncovering the mode of action of engineered T cells in patient cancer organoids.

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.

Neutrophils Promote Glioblastoma Tumor Cell Migration after Biopsy.

Glioblastoma is diagnosed by biopsy or, if clinically feasible, tumor resection. However, emerging evidence suggests that this surgical intervention may increase the risk of tumor cell spread. It has been hypothesized that the damage to the tumor leads to infiltration of immune cells that consequently form an environment that favors tumor cell motility. In mouse glioma models, it was previously found that biopsy induced migration of tumor cells in vivo and that recruitment of monocytes from the blood was involved in this effect. However, the role of neutrophils in this process is still unclear. Here, we study the contribution of neutrophils on the pro-migratory effect of surgical interventions in glioma. Using repetitive intravital microscopy, in vivo migration of glioma tumor cells before and after biopsy was compared in mice systemically depleted of neutrophils. Interestingly, macrophages/microglia were almost completely absent from neutrophil-depleted tumors, indicating that neutrophils may be indirectly involved in biopsy-induced migration of glioma tumor cells through the recruitment of macrophages to the tumor. To further investigate whether neutrophils have the potential to also directly promote glioblastoma tumor cell migration, we performed in vitro migration assays using human neutrophils. Indeed, wound-healing of human primary glioblastoma tumor cell lines was promoted by human neutrophils. The pro-migratory effects of human neutrophils on glioblastoma tumor cells could also be recapitulated in transwell migration assays, indicating that soluble factor(s) are involved. We therefore provide evidence for both an indirect and direct involvement of neutrophils in tumor spread following biopsy of glioblastoma tumors.

Multispectral confocal 3D imaging of intact healthy and tumor tissue using mLSR-3D.

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.

Revealing the spatio-phenotypic patterning of cells in healthy and tumor tissues with mLSR-3D and STAPL-3D.

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.

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy.

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.

Intravital imaging of glioma border morphology reveals distinctive cellular dynamics and contribution to tumor cell invasion.

The pathogenesis of glioblastoma (GBM) is characterized by highly invasive behavior allowing dissemination and progression. A conclusive image of the invasive process is not available. The aim of this work was to study invasion dynamics in GBM using an innovative in vivo imaging approach. Primary brain tumor initiating cell lines from IDH-wild type GBM stably expressing H2B-Dendra2 were implanted orthotopically in the brains of SCID mice. Using high-resolution time-lapse intravital imaging, tumor cell migration in the tumor core, border and invasive front was recorded. Tumor cell dynamics at different border configurations were analyzed and multivariate linear modelling of tumor cell spreading was performed. We found tumor border configurations, recapitulating human tumor border morphologies. Not only tumor borders but also the tumor core was composed of highly dynamic cells, with no clear correlation to the ability to spread into the brain. Two types of border configurations contributed to tumor cell spreading through distinct invasion patterns: an invasive margin that executes slow but directed invasion, and a diffuse infiltration margin with fast but less directed movement. By providing a more detailed view on glioma invasion patterns, our study may improve accuracy of prognosis and serve as a basis for personalized therapeutic approaches.

High-resolution 3D imaging of fixed and cleared organoids.

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.

Potential impact of invasive surgical procedures on primary tumor growth and metastasis.

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.

Preventing inflammation inhibits biopsy-mediated changes in tumor cell behavior.

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.

Imaging windows for long-term intravital imaging: General overview and technical insights.

Intravital microscopy is increasingly used to visualize and quantitate dynamic biological processes at the (sub)cellular level in live animals. By visualizing tissues through imaging windows, individual cells (e.g., cancer, host, or stem cells) can be tracked and studied over a time-span of days to months. Several imaging windows have been developed to access tissues including the brain, superficial fascia, mammary glands, liver, kidney, pancreas, and small intestine among others. Here, we review the development of imaging windows and compare the most commonly used long-term imaging windows for cancer biology: the cranial imaging window, the dorsal skin fold chamber, the mammary imaging window, and the abdominal imaging window. Moreover, we provide technical details, considerations, and trouble-shooting tips on the surgical procedures and microscopy setups for each imaging window and explain different strategies to assure imaging of the same area over multiple imaging sessions. This review aims to be a useful resource for establishing the long-term intravital imaging procedure.

Fast-proliferating adipose tissue mesenchymal-stromal-like cells for therapy.

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 <2 weeks of culture. Expanded FP-ASCs retain their finite expansion capacity and pluripotent properties. Despite the high proliferation rate, FP-ASCs show genomic stability by array-comparative genomic hybridization, and did not generate tumors when implanted for a long time in an SCID mouse model. Comparative analysis of gene expression patterns revealed a set of genes that can be used to characterize FP-ASCs and distinguish them from hASCs. As potential candidate therapeutic agents, FP-ASCs displayed high vasculogenic capacity in Matrigel assays. Moreover, application of hASCs and FP-ASCs in a fibrin scaffold over a myocardium infarct model in SCID mice showed that both cell types can differentiate to endothelial and myocardium lineages, although FP-ASCs were more potent angiogenesis inducers than hASCs, at promoting myocardium revascularization.

Calcium phosphate glass improves angiogenesis capacity of poly(lactic acid) scaffolds and stimulates differentiation of adipose tissue-derived mesenchymal stromal cells to the endothelial lineage.

The angiogenic capacity of a new biomaterial composite of poly(lactic acid) and calcium phosphate glass (PLA/CaP) was analyzed by noninvasive bioluminescence imaging (BLI) and histological procedures. Human adipose tissue-derived mesenchymal stromal cells expressing cytomegalovirus (CMV) promoter regulated Photinus pyralis luciferase (hAMSC-PLuc) grew up to 30 times the initial cell load, in vitro, when seeded in PLA/CaP scaffolds, but suffered an initial growth crisis followed by recovery when the scaffolds were subcutaneously implanted in SCID mice. To analyze changes in gene expression, hAMSC-PLuc cells were double labeled with a CMV promoter regulated Renilla reniformis luciferase and a Photinus pyralis luciferase reporter regulated by either the PECAM promoter or a hypoxia response element (HRE) artificial promoter and seeded in PLA/CaP and PLA scaffolds implanted in SCID mice. Analysis by BLI showed that hAMSCs in scaffolds were induced to differentiate to the endothelial lineage and did this faster in PLA/CaP than in PLA scaffolds. Endothelial differentiation correlated with a decrease in the activity of HRE regulated luciferase expression, indicative of a reduction of hypoxia. Histological analysis showed that PLA/CaP scaffolds were colonized by a functional host vascular system. Moreover, colonization by isolectin B(4) positive host cells was more effective in PLA/CaP than in PLA scaffolds, corroborating BLI results.

Bioluminescence imaging of cardiomyogenic and vascular differentiation of cardiac and subcutaneous adipose tissue-derived progenitor cells in fibrin patches in a myocardium infarct model.

BACKGROUND: Adipose tissue-derived progenitor cells (ATDPCs) isolated from human cardiac adipose tissue are useful for cardiac regeneration in rodent models. These cells do not express cardiac troponin I (cTnI) and only express low levels of PECAM-1 when cultured under standard conditions. The purpose of the present study was to evaluate changes in cTnI and PECAM-1 gene expression in cardiac ATDPCs following their delivery through a fibrin patch to a murine model of myocardial infarction using a non-invasive bioluminescence imaging procedure. METHODS AND RESULTS: Cardiac and subcutaneous ATDPCs were doubly transduced with lentiviral vectors for the expression of chimerical bioluminescent-fluorescent reporters driven by constitutively active and tissue-specific promoters (cardiac and endothelial for cTnI and PECAM-1, respectively). Labeled cells mixed with fibrin were applied as a 3-D fibrin patch over the infarcted tissue. Both cell types exhibited de novo expression of cTnI, though the levels were remarkably higher in cardiac ATDPCs. Endothelial differentiation was similar in both ATDPCs, though cardiac cells induced vascularization more effectively. The imaging results were corroborated by standard techniques, validating the use of bioluminescence imaging for in vivo analysis of tissue repair strategies. Accordingly, ATDPC treatment translated into detectable functional and morphological improvements in heart function. CONCLUSIONS: Both ATDPCs differentiate to the endothelial lineage at a similar level, cardiac ATDPCs differentiated more readily to the cardiomyogenic lineage than subcutaneous ATDPCs. Non-invasive bioluminescence imaging was a useful tool for real time monitoring of gene expression changes in implanted ATDPCs that could facilitate the development of procedures for tissue repair.

In vivo bioluminescence imaging of cell differentiation in biomaterials: a platform for scaffold development.

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.

Glioblastoma therapy with cytotoxic mesenchymal stromal cells optimized by bioluminescence imaging of tumor and therapeutic cell response.

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.