MACS® Imaging and Microscopy Days

MACS® Imaging and Microscopy Days

September 20–21, 2022 | Miltenyi Biotec, Bergisch Gladbach

Understand nature‘s complexity through omni-dimensional microscopy

We are looking forward to breaking down the recent complex findings from experts in our face-to-face event, the MACS Imaging and Microscopy Days at Miltenyi Biotec headquarters. With leading scientists from the fields of spatial biology and 3D imaging, come join us in sharing recent findings and achievements with the latest microscopy and spatial biology technologies.

Miltenyi Biotec
Friedrich-Ebert-Straße 68 
51429 Bergisch Gladbach 
Germany
September 20, 2022 | 2:00 p.m.–7:00 p.m.
September 21, 2022 | 9:00 a.m.–2:00 p.m.

MACS Imaging and Microscopy Days

September 20, 2022 | 2:00 p.m.–7:00 p.m.

September 21, 2022 | 9:00 a.m.–2:00 p.m.

Agenda

DAY 1

September 20, 2022

 

1:00 p.m.

Miltenyi Biotec company tour for invited speakers
Host: Dr. Stefan Eulitz, Customer Liaison Manager, Miltenyi Biotec

2:00 p.m.–
2:45 p.m.

Introduction into Miltenyi Biotec's workflow solutions for imaging and microscopy
Speaker: Dr. Stefan Eulitz, Customer Liaison Manager, Miltenyi Biotec

 

SPATIAL BIOLOGY SESSION

2:45 p.m.–
3:25 p.m.

Multimodal imaging to decode immunological landscapes in infections and cancer
Speaker: Prof. Daniel Engel, Institute of Experimental Immunology and Imaging, University Hospital Essen, Germany

3:25 p.m.–
3:40 p.m.

Coffee break

3:40 p.m.–
4:20 p.m.

Visualizing cancer – From functional phenotyping to precision immunotherapy
Speaker: Dr. Christian Seitz and Sophia Scheuermann, University Children’s Hospital Tübingen, Germany

4:20 p.m.–
5:00 p.m.

MACSima Imaging Platform – deciphering the cellular composition and novel biomarker in health and disease
Speaker: Dr. Andreas Bosio, Head of Molecular Technologies & Stem Cell Therapy, Miltenyi Biotec

5:00 p.m.–
5:15 p.m.

Coffee break

5:15 p.m.–
5:35 p.m.

Rare components of the tumor microenvironment – detection of native human dendritic cell subsets
Speaker: Dr. Rebekka Wehner, Institute of Immunology, Medical Faculty Carl Gustav Carus  Technical University, Dresden, Germany

5:35 p.m.–
6:05 p.m.

Resolving the spatial architecture of the liver at single-cell resolution
Speaker: Birthe Haest, VIB Center for Inflammation Research (IRC), Ghent, Belgium

6:05 p.m.–
6:45 p.m.

Human myeloid cells in tissue: from inflammation to cancer
Speaker: Dr. Egle Kvedaraite, Department of Pathology and Cancer Diagnostics Karolinska University Hospital, Sweden

7:00 p.m - late

Dinner

 

DAY 2

 

September 21, 2022

 

LARGE SCALE IMAGING SESSION

9:00 a.m.–
9:40 a.m.

Endocrine cell renewal: making possible the 3rd dimension!
Speaker: Prof. Clara Álvarez, Centre for Research in Molecular Medicine and Chronic Diseases (CIMUS), Spain

9:40 a.m.–
10:20 a.m.

3D imaging to elucidate the neurobiology of cancer
Speaker: Dr. Fanny Mann, Institut de Biologie du Développement de Marseille, France

10:20 a.m.–10:35 a.m.

Coffee break

10:35 a.m.–11:15 a.m.

Imaging whole mouse bodies and human organs on the Ultramicroscope Blaze™
Speaker: Moritz Negwer, Helmholtz Zentrum Munich, Germany

11:15 a.m.–11:55 a.m.

Probing vascular development, function and pathofunction with the ultramicroscope
Speaker: Prof. Friedemann Kiefer, European Institute for Molecular Imaging, Münster, Germany

11:55 a.m.–12:30 p.m.

Coffee break and lunch

12:30 p.m.–
1:10 p.m.

A multi-modal imaging workflow for monitoring CAR T cell therapy against solid tumors from whole-body to single-cell level
Speaker: Dr. Rita Pfeifer, Research and Development, Miltenyi Biotec

1:10 p.m.–
1:50 p.m.

Immune cell dynamics within normal liver and hepatocellular carcinoma
Speaker: Dr. Federica Moalli, Division of Immunology, Transplantation and Infectious Diseases. Experimental Imaging Centre. IRCCS San Raffaele Scientific Institute

1:50 p.m.

Closing words

Abstracts and speaker information

Introduction into Miltenyi Biotec's workflow solutions for imaging and microscopy

Dr. Stefan Eulitz, Customer Liaison Manager, Miltenyi Biotec

Abstract
Cells are the building blocks of life and nature. They can be extremely versatile and specifically adapted to their environment. Therefore, they exist in many shapes, states, and types. Unveiling the function and interaction of cells provides the key to understanding nature’s complexity. However, the interplay of different cell types at a certain location under specific environmental conditions can only be analyzed in the overall context. 

To start deciphering these complex processes and interdependencies you need advanced techniques that can provide you with the necessary insight. This is the reason why Miltenyi Biotec continuously develops cutting-edge imaging technologies that allow you to observe dynamic processes in living animals, analyze whole animals and organs in 3D, and perform the world's deepest characterization of cells. We are happy to take you with us on a journey to the possibilities of imaging technologies and towards understanding nature's complexity.

Speaker information
Dr. Stefan Eulitz is Global Customer Liaison Manager at Miltenyi Biotec. With more than 10 years' experience of cyclic imaging in industry and academia, Stefan combines bench and business knowledge. During his Ph.D. at the Rheinische-Friedrich-Wilhelms-Universität Bonn, Germany, and as consultant at the French company Alcimed, he gained deep knowledge of various imaging technologies as well as drug target and biomarker discovery. Stefan joined Miltenyi Biotec in 2015 to support the development of the MACSima™ Imaging Platform as Deputy Development Program Leader and currently supports researchers in the adaption of Miltenyi Biotec’s imaging technologies from basic research and development to commercial research strategies. 

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Multimodal imaging to decode immunological landscapes in infections and cancer

Prof. Daniel Engel, Institute of Experimental Immunology and Imaging, University Hospital Essen, Germany

Abstract
The Department of Immunodynamics investigates immunological mechanisms that underlie diseases such as persistent infections and cancer. We employ state of the art approaches, such as imaging mass spectrometry, bioinformatics, and microscopy to understand the mechanisms that spatially regulate leukocyte migration and function. We develop interdisciplinary co-registration algorithms that combine multimodal imaging approaches, such as immunohistochemistry (IHC), matrix-assisted laser desorption/ionization mass spectrometry imaging, (MALDI MSI) and liquid chromatography with tandem mass spectrometry (LC-MS/MS). Our overall aim is to decode immunological landscapes to develop novel therapeutic approaches to treat diseases. 

Speaker information
Prof. Engel and the department of immunodynamics investigates immunological mechanisms that underlie diseases such as persistent infections and cancer. By employing state of the art approaches, such as imaging mass spectrometry, bioinformatics, and multiplex microscopy they look to understand the mechanisms that spatially regulate leukocyte migration and function. The department develops interdisciplinary co-registration algorithms that combine multimodal imaging approaches from multiplex microscopy and imaging mass spectrometry to decode immunological landscapes and develop novel therapeutic approaches to treat diseases.

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Visualizing cancer – From functional phenotyping to precision immunotherapy

Dr. Christian Seitz and Sophia Scheuermann, University Children’s Hospital Tübingen, Germany

Abstract
What are the underlaying mechanisms that lead to the limited effects of immune therapies, in specific, chimeric antigen receptor (CAR) technologies, in solid tumors and how can we identify new therapeutic approaches to overcome those hurdles? By using the MACSimaTM platform, we try to answer these questions for different solid cancers such as pediatric sarcomas and neuroblastomas, as well as for major adult cancer subtypes like lung and liver cancer. 

We started our experiments doing feasibility studies with reference tissue, different tumor entities and defined comprehensive antibody panels with more than 100 markers. This enabled us to identify a variety of stromal components, immune cells, heterogeneous tumor tissue, and clinically relevant and druggable target antigens, like immune checkpoints. On the one hand, we implemented the ultra-high content imaging workflow into our pre-clinical research to study the interaction between CAR-T cells, the tumor, and its tumor microenvironment (TME) i.e. in cell culture experiments and in vivo studies, whereas the goal is the characterization of CAR-T cells in a reproducible and coherent way. On the other hand, the MACSimaTM is used as an “on-the-fly” diagnostics tool in Tübingen, which complements classical pathology, offers a deeper and previously impossible understanding of tumor samples, and has the potential to improve precision immunotherapy.

Speaker information
Christian Seitz, M.D., is pediatrician at the University Children’s Hospital Tübingen, Germany. He studied medicine at the universities in Ulm and Frankfurt, Germany, as well as at the National Cancer Institute in Bethesda, MD, USA. He received his doctorate, Dr. med., from the Goethe University Frankfurt, Germany. In 2014, he started his clinical training in paediatrics under Rupert Handgretinger at the University Children’s Hospital Tübingen, focusing on stem cell transplantation and cellular therapies. His scientific work is focused on the development and clinical translation of modular chimeric antigen receptor (CAR) technologies and the interaction of CAR T cells in the tumor microenvironment.

Sophia Scheuermann, M.Sc. in Biochemistry, is a PhD candidate in the Advanced Cellular Therapies (ACT) Lab of Dr. Christian Seitz at the University Children's Hospital Tübingen, Germany. She studied life sciences and biochemistry at the Universities of Konstanz and Tübingen. She extended her knowledge about protein biochemistry, mass spectrometry, and immunology as a visiting researcher at the University of British Columbia, Vancouver, Canada. She finished her studies in 2020 in the lab of Dr. Christian Seitz with her master's thesis about "Strategies to improve the performance of CAR-T cells by targeted integration". In her current scientific work, she is focusing on the improvement of cellular and immune therapies for solid tumor settings. In specific, she is interested in the investigations of the interplay between chimeric antigen receptor (CAR) therapy and the tumor microenvironment (TME).

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MACSima™  Imaging Platform – deciphering the cellular composition and novel biomarker in health and disease

Dr. Andreas Bosio, Head of Molecular Technologies & Stem Cell Therapy, Miltenyi Biotec

Abstract
Understanding a cells nature and identity by measuring the expression of a huge number of proteins in the context of its neighbour cells opens new insights into the biology of healthy and diseased tissues. We have used MICS and light-sheet microscopy to address a broad range of diverse scientific questions, two of which will be discussed in this  talk. First, we analyzed human glioblastoma, ovarian, and pancreatic carcinoma, and 16 healthy tissues identifying EPCAM/THY1 as a potential target for CAR T cell therapy for ovarian carcinoma. Using an adapter CAR T cell approach, we show selective killing of cells only if both markers are expressed. Secondly, as part of a human developmental cell atlas initiative we are working on the discovery of novel biomarkers for cell types in the developing gonad to better understand the cellular mechanisms underlying sex determination. Here we show first results outlining protein expression, cell types, and architecture of the developing gonad.

Speaker information
Andreas Bosio received a degree in chemistry and a PhD in biochemistry at the University of Cologne, Germany. He was CSO of MEMOREC Stoffel GmbH before joining Miltenyi Biotec in 2003 where he is now heading the R&D department for Molecular Technologies and Stem Cell Therapy.   

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Rare components of the tumor microenvironment – detection of native human dendritic cell subsets

Dr. Rebekka Wehner, Institute of Immunology, Medical Faculty Carl Gustav Carus  Technical University, Dresden, Germany

Abstract
As professional antigen-presenting cells, dendritic cells (DC) play an essential role in the activation and maintenance of antitumor immune responses. They stimulate NK cells, bridge innate and adaptive immune systems, and facilitate T-cell priming. Therefore, they are associated with improved clinical outcomes in various cancers. 

Nevertheless, DCs show high plasticity and can develop a tumor-induced tolerogenic phenotype. These DCs promote the development of an immunosuppressive milieu and are associated with poor patient survival. Multiparameter fluorescence microscopy imaging provides the opportunity to dissect the role of DCs and their relationship to other cell types in the tumor microenvironment. This talk presents our work in the DC research field since quantification and spatial organization of native human dendritic cell subsets may support the identification of pathways as well as mechanisms which promote cancer elimination and therapy response.

Speaker Information
Rebekka Wehner graduated in biology and did her PhD at the Institute of Immunology at the Faculty of Medicine, TU Dresden, Germany. Here, she started her research on native human dendritic cells (DC) and 6-sulfo LacNAc(slan)-positive monocytes, a subset of non-classical monocytes (formally termed MDC8+DC, slanDC) in the context of anti-tumor immune responses and therapies. Her current research focus comprises the analysis of the immune architecture of different cancers using multiplex fluorescence microscopy. Since 2016, she coordinates the NCT/UCC Immune Monitoring Unit Dresden, which offers an accompanying immune monitoring program during clinical studies. She promotes the characterization of immune cells in the blood, bone marrow, or tissues of tumor patients via multiparametric flow cytometry and immunohistochemistry.

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Resolving the spatial architecture of the liver at single-cell resolution

Birthe Haest, VIB Center for Inflammation Research (IRC), Ghent, Belgium

Abstract
The liver is the largest solid organ in our body, yet it remains incompletely characterized. Single-cell sequencing technologies allow the analysis of the cellular compositions of organs, yet often lack one of the most essential aspects of biology: the spatial context of cells. To fully understand how tissues function and cells communicate, we need not only a precise idea of gene and protein expression per cell type, but also detailed information on their position in relation to other cells and structures that make up the organ.  To generate a spatial proteogenomic atlas of the healthy human and murine liver we have combined single-cell CITE-seq, single-nuclei sequencing, 100-plex spatial transcriptomics and 100-plex spatial proteomics (MACSima™). This has enabled the identification of validated strategies to reliably discriminate, purify, and localize all hepatic cell subsets. Moreover, the proteogenomic analysis enabled us to identify the respective cellular niches of three distinct hepatic macrophage subtypes. This further allowed the microenvironmental circuits driving the unique transcriptomic identities of these distinct macrophages to be identified, including an evolutionarily conserved cell-cell circuit between Stellate cells and Kupffer cells that is crucial for Kupffer cell development.

Speaker Information
Birthe Haest is a PhD student in the laboratory of Myeloid Cell Biology in Tissue Homeostasis and Regeneration of Prof Martin Guilliams at Flanders Institute for Biotechnology (VIB)-UGent Center for Inflammation Research. In 2019, she obtained her Master degree in Biochemistry and Biotechnology at Ghent University. For her master’s thesis she went to Maynooth, Ireland, to study macrophage activation. Currently, she is investigating the cell-cell interactions that drive liver regeneration.

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Human myeloid cells in tissue: from inflammation to cancer

Dr. Egle Kvedaraite, Department of Pathology and Cancer Diagnostics Karolinska University Hospital, Sweden

Abstract
Myeloid cells are a part of innate immunity, playing a major role in orchestrating innate and adaptive immune responses in the tissue microenvironment. While work performed in experiment model systems has significantly increased our knowledge of fundamental myeloid cell functions, studies in well-designed clinical cohorts applying high-dimensional analytic pipelines, including high-dimensional microscopy, are important for understanding immune cell functions in human health and disease, such as inflammatory conditions and cancer. The specific functional responses of myeloid cells will be discussed in two different clinical contexts: inflammatory bowel disease (IBD), a chronic disorder of the gastrointestinal tract and Langerhans cell histiocytosis (LCH), an inflammatory myeloid neoplasia that may engage multiple tissue sites.

Speaker Information
Egle Kvedaraite completed her MD in 2016 and PhD in 2022, as a part of highly competitive Physician Scientist Training Program at Karolinska Institutet, Sweden. Currently, Dr Kvedaraite is a licensed physician and continues to combine her clinical training in clinical pathology and cancer diagnostics with focus on neuropathology at Karolinska University Hospital with research at the Department of Medical Biochemistry and Biophysics, Karolinska Institutet. The focus of Egle’s PhD was human myeloid cells in cancer, inflammation and infection, with research being performed at the Center for Infectious Medicine (CIM), Karolinska Institutet. CIM was funded in 2002 as one of six national “Strategic Research Centers” in Sweden and has recently been ranked as #1 center for translational medicine at Karolinska Institutet. In addition, Egle has performed research studying myeloid cells at the Singapore Immunology Network, Agency for Science, and Technology and Research (ASTAR). Egle’s interests lie within the areas of human immunology, myeloid cells, and stromal-immune cell interactions in tissue environment.

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Endocrine cell renewal: making possible the 3rd dimension!

Prof. Clara Álvarez, Centre for Research in Molecular Medicine and Chronic Diseases, Spain

Abstract
The emergence of new microscopy technologies such as light-sheet fluorescence microscopy (LSFM) proposes 3D characterization of organs, from a volumetric point of view to a detailed cellular level, preventing the need for sectioning. Practically, almost all protocols published for LSFM are standardized for brain. Thus, small endocrine glands with high epithelial cellular density, and a large amount of stored hormones, arise as an important challenge. 

Our objective with LSFM is the study of two of those endocrine organs, the pituitary and the thyroid gland. The pituitary gland contains five different types of endocrine cells organized in tight acini secreting six different hormones. Currently, not much is known about the pituitary cell renewal during the different key life steps of longitudinal growth, puberty, pregnancy, or metabolic adaptation. This is important since pituitary adenomas are responsible for 15% of head tumors, giving both anatomical compression and excess hormone secretion symptoms, like in acromegaly/gigantism. Likewise, negative differentiation or slow renewal originate disease through reduced hormone secretion, like in dwarfism. We discovered the pituitary stem cell niche conserved in rodents (mouse, rat) and human pituitary. Two sources of new cells are proposed for pituitary cell renewal, proliferation of already differentiated endocrine cells, and recruitment from the pituitary stem cell niche. We aim to study the contribution of each system through LSFM. The thyroid gland is organized in follicles, hollow structures containing colloid, a material composed of thyroglobulin, a heavily glycosylated protein with covalent iodination in tyrosine residues. We are developing a LSFM protocol to be able to localize major proteins of the functional thyroid phenotype, like the sodium/iodine transporter NIS. Our long-term role is to apply LSFM standardized protocols to human thyroid tissues present in our collection of surgery surpluses, in order to improve diagnostic of proliferative benign disease and thyroid cancers. A collection of directly labeled antibodies is being tested.

Speaker information
A medical doctor and professor of physiology and endocrinology at the University of Santiago de Compostela (USC), Prof. Clara Alvarez heads the lab of Neoplasia and Endocrine Differentiation at the Centre for Research in Molecular Medicine and Chronic Disease (CIMUS). 
Prof. Alvarez’s background focused on endocrinology and clinical research, in addition to holding a surgical background with special emphasis on studies related to the thyroid and pituitary gland. Most of these studies have been centered on physiological mechanisms involved in cancer cell proliferation and normal cellular turnover, in addition to their implications for the development of different pathological processes. Additionally, during the last 15 years, Prof. Alvarez has acted as a PI working in well-defined endocrinology cellular models (human primary cultures from surgical surplus of patients’ surgeries, mouse, and rat in vivo models of endocrine diseases such as hypothyroidism and primary cultures of endocrine stem cells) and has been published in journals such as EMBO J, Clin Can Res, Oncogene, Annals Oncol, J Clin Endocrinol Metab, Endocrine Rel Cancer or Endocrinology. Additional group collaborations working in a range of related research from hypothalamic regulation of energy balance to the recent role in the European consortium for human nano-encapsulated insulin for oral administration, have led to publications in the aforementioned journals and others like Cell Metab, Nature Comm, J Control Release, Trends Mol Med, Mol Met or FASEB J. Additionally, such research activity of the group has aroused the interest of a large number of audiences having given lectures by invitation, among others, at ITC (worldwide), ECE and ENEA (European), ENDO (American). 

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3D imaging to elucidate the neurobiology of cancer

Dr. Fanny Mann, The Marseille Developmental Biology Institute, France

Abstract
Peripheral nerve projections are found in all tissues and organs of the body, even in malignant tumors regardless of their site of emergence and spread. A growing body of work indicates that cancer cells and cells of the nervous system engage in a reciprocal dialogue with beneficial, but sometimes detrimental, consequences for disease progression. Nerve infiltration in the tumor microenvironment has therefore recently emerged as a prognostic biomarker and a potential therapeutic target for cancer treatment. 

This presentation will focus on a three-dimensional imaging method to analyze the organization of neural networks in cleared tumor samples. We will illustrate how this approach, applied to a mouse model of pancreatic ductal adenocarcinoma, has allowed us to demonstrate subtype-specific remodeling of neurons during disease development.

Speaker Information
Fanny Mann is a CNRS research director at the Institute of Developmental Biology of Marseille (IBDM) in France. She obtained her PhD in neuroscience at the University of Lyon 1 in 1999, then followed with a post-doctoral fellowship at the University of Cambridge in the UK. Back in France, she initiated an independent line of research on the molecular mechanisms of neural circuit development. Her work was awarded the CNRS bronze medal in 2010. Recently, the research of her group has been oriented towards the mechanisms of neuroplasticity induced by tumor development and their consequences on cancer progression.

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Imaging whole mouse bodies and human organs on the UltraMicroscope Blaze™ 

Moritz Negwer, Institute of Tissue Engineering and Regenerative Medicine (iTERM), Helmholtz  Munich, Germany

Abstract
In the Ertürk laboratory at the Helmholtz Munich, we combine large-scale clearing with light-sheet imaging and AI-supported data analysis. Our DISCO-family of clearing techniques enables us to reliably clear large samples, up to the size of an adult mouse in which we can immunolabel cells with nanobodies. This enables us to track cancer cells down to the single-cell metastasis level and neurons including their axon bundles throughout the entire mouse body, in an unbiased way. For example, our approach led us to discover - previously unknown - channels between the skull bone marrow and the dura mater, which allow immune cells from the bone to shuttle towards the brain and participate in neuroinflammatory responses. Using this approach, we can also make complex, three-dimensional networks such as the vasculature of the brain. In all  those cases, we generate our data with a fleet of UltraMicroscope Blazes custom-fitted with mouse-sized sample chambers and large-scale stages. In order to make sense of the terabytes of data we produce with each scan, we build sophisticated analysis pipelines built on deep learning that allow for a truly unbiased analysis. Lastly, we do not limit ourselves to the mouse, and have developed the solvent-based SHANEL clearing for labelling and clearing donated human organs, such as kidneys, lung, heart, and brain. 

Speaker Information
Moritz Negwer studied Neurobiology at Radboud University in Nijmegen, the Netherlands, with an internship at UC Irvine in California. He completed his PhD at the Donders Institute and Radboudumc in the laboratories of Nael Nadif Kasri and Dirk Schubert in 2021, studying the influence of the epigenetic modulator Ehmt1 on the development of GABAergic inhibitory neurons in the mouse brain. During this PhD he discovered the magic of tissue clearing and spent the next three years optimizing a mouse-brain clearing and analysis pipeline using a Miltenyi Biotec Ultramicroscope II. He found that once the imaging is done and the data is analyzed, brain-wide cell distributions are an excellent hypothesis-generating tool. His twin experience in light-sheet microscopy and data analysis prepared him well for his current position as Staff Scientist at the Ertürk laboratory at the Helmholtz Zentrum in Munich, Germany. There, he trains new lab members in whole-mouse body clearing, sets up collaboration projects, and keeps the fleet of Miltenyi Biotec UltraMicroscope Blazes scanning and the analysis pipelines running. He is currently working on projects ranging from cancer to neuroscience, and is broadly interested in pushing the technological limit of tissue clearing and light-sheet imaging.  

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Probing vascular development, function and pathofunction with the UltraMicroscope

Prof. Friedemann Kiefer, European Institute for Molecular Imaging, Münster, Germany

Abstract
At the European Institute for Molecular Imaging / Multiscale imaging center (EIMI/MIC at the University of Münster), we investigate morphogenetic processes that shape and maintain the vascular systems, with a special emphasis on the development und function of lymphatic vessels. Functional specialization of lymphatics vessel beds is only recently being recognized. 

It becomes increasingly clear that the tissue-specific functions of lymphatic vessels result not only from specialized lymphatic endothelial cells but also to a large degree from their specific spatial arrangement forming characteristic vessel beds. A comprehensive analysis of specific vessel beds requires volume imaging. More recently, we have turned to physiological and pathophysiological processes during adult life, in particular inflammation, where the interplay of blood and lymphatic vessels, tissue oxygenation and leukocytes is crucial for the maintenance of organ function.

Speaker information
Prof. Dr. Friedemann Kiefer is the current Professor for Intravital Molecular Imaging at the European Institute for Molecular Imaging (EIMI) located at the Multiscale Imaging Center (MIC) in Münster, Germany. He acts as deputy spokesperson and coordinator of the Integrated Research Training Group “Multiscale Imaging”, at the Collaborative Research Centre 1450 "Multiscale Imaging of organ-specific inflammation”. He is also an appointed member of the Research Foundation – Flanders expert panel Bio1: Molecular and Cellular Biology, and on the editorial board of several biomedical journals. He received his Diploma in Biochemistry and Physiological Chemistry at the University of Tübingen, performed his dissertation at the Research Institute of Molecular Pathology, Vienna and obtained a Habilitation in Cell Biology, from the Faculty of Medicine, Philipps-Universität Marburg.

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A multi-modal imaging workflow for monitoring CAR T cell therapy against solid tumors from whole-body to single-cell level

Dr. Rita Pfeifer, Research and Development, Miltenyi Biotec

Abstract
As CAR T cell therapy continues to become a mainstay pillar in cancer treatment, there is a growing need to understand the function and interaction of the therapeutic cells in their native environment in order to further improve this treatment modality. Imaging platforms with spatiotemporal information can provide a profound insight into therapeutic ongoings during different stages of therapy in this way accelerating the establishment of successful therapies. 

Thus, in this study, we aimed to retrieve multi-scale information on therapeutic effects following CAR T cell therapy using various imaging approaches. In vivo and ex vivo CAR T cell tracking was conducted using a subcutaneous pancreatic adenocarcinoma xenograft model. Longitudinal whole-body in vivo BLT imaging displayed differential biodistribution of therapeutic and mock CAR T cells and a tumor antigen-dependent accumulation of the immune cells at lesion site. Notably, T cell homing and accumulation within the tumor did not correlate with therapeutic efficacy throughout the study suggesting a complex interplay between the CAR T and tumor cells. Subsequent high-resolution profiling of tumors by LSFM revealed that the majority of tumor-infiltrating T cells was located within the tumor periphery in a heterogeneous, island-like distribution and only a small fraction was able to reach the tumor core. An in-depth characterization of the intratumoral T cells by means of MICS imaging revealed phenotypic differences between CAR and mock T cells. Taken together, the herein established imaging workflow provides new insights for the development of novel, more efficacious CAR T cell immunotherapies.

Speaker Information
Dr. Rita Pfeifer studied Biochemistry at the University of Tübingen, Germany, followed by a position as Research Fellow at the Children’s Hospital Los Angeles, USA. Thereafter, she returned to Germany to start her Ph.D. at the University of Tübingen and Miltenyi Biotec. Since 2018, she continues her work with Miltenyi Biotec as Team Coordinator Research and Development, focusing on in vivo and ex vivo tracking and the development of CAR T cell therapies against solid tumors

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Immune cell dynamics within normal liver and hepatocellular carcinoma

Dr. Federica Moalli, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy / Experimental Imaging Centre, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Abstract
T cells are actively scanning pMHC-presenting cells in lymphoid organs and nonlymphoid tissues with divergent topologies and confinement. In the liver, antigen-specific effector CD8+ T cells play a critical role in controlling hepatic infections, such as the one caused by hepatitis B virus (HBV), and liver tumours, such as hepatocellular carcinoma (HCC). We published recent results where we coupled advanced dynamic imaging with dedicated mouse models of HBV pathogenesis to show that circulating effector CD8+ T cells aimed at viral clearance initially arrest in liver sinusoids by preferentially docking onto platelets that have previously adhered to liver sinusoids. 

Upon detachment from platelets, effector CD8+ T cells crawl within the sinusoids irrespective of bloodstream direction, and probe underlying hepatocytes for the presence of antigen by extending filopodia-like protrusions through the sinusoidal fenestrae. Effector CD8+ T cells recognize hepatocellular antigen and perform effector functions (i.e., IFN-γ production and hepatocyte killing) while still in the intravascular space. We recently demonstrated that the effector functions of CD8+ T cells in the liver are negatively regulated by group 1 innate lymphoid cells, by limiting local IL-2 concentration.

We are now working on the identification of the determinants of hepatic effector CD8+ T cell trafficking, antigen recognition and effector functions during HCC. 
Thus, we established a murine model of HCC in which the tumorigenic hepatocytes express a nominal antigen (TAg) and a fluorescent protein to follow the transformed hepatocytes. After TAg-specific effector CD8+ T cell (TE) adoptive transfer in tumor-bearing mice, some respond to the TE treatment, yet we established a therapeutic threshold of 10 mm3 lesion volume dividing lesions between responders (volume <10 mm3) and non-responders (>10 mm3). Our goal is to address the determinants that confer a therapeutic activity to the TAg-specific TE in responders and the ones that dampen the activity in the non-responders HCC lesions.
 
A better understanding of how adaptive immunity mediates pathogen clearance and tumor elimination may lead to improved vaccination and treatment strategies for immunotherapy of infectious diseases and cancer.

Speaker information
With a master degree in medical biotechnology from Milan University, Dr. Moalli followed her studies with a PhD in Immunology at Vita Salute University, San Raffaele Scientific Institute. From 2005-2010, Dr. Moalli worked in the laboratory of Dr. Cecilia Garlanda, under the supervision of Prof. Alberto Mantovani, Humanitas Clinical Research Center, Milan, working on the definition of the in vivo and in vitro functions of the long Pentraxin 3 (PTX3), a component of the humoral arm of innate immunity, in innate responses to pathogens and in inflammatory responses. From 2011-2016,  Dr. Moalli moved to Bern (Switzerland) as a postdoctoral researcher in the laboratory of Prof. Jens V. Stein, Theodor Kocher Institute, University of Bern, to study the molecular mediators at the basis of T cell migration and APC interaction, using intra vital two-photon microscopy, OPT (optical projection tomography) system and LSFM (laser sheet fluorescent microscopy) system. In 2017, she moved to Milan (Italy) as a Senior Postdoctoral Researcher in the laboratory of Prof. Matteo Iannacone, San Raffaele Scientific Institute, Milan. Her focus lies in studying the complex dynamics of immune system in carcinogenesis, with a particular focus on the spatiotemporal dynamics of CD8+ T cells within liver tumors.

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Recordings from the MACS Imaging and Microscopy Day 2021

Our solutions for imaging and microscopy