15th European Molecular Imaging Meeting
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Immuno-Oncology Imaging

Session chair: Erik Aarntzen (Nijmegen, Netherlands); Bettina Weigelin (Tuebingen, Germany)
 
Shortcut: PW02
Date: Wednesday, 25 August, 2021, 7:15 p.m. - 9:00 p.m.
Session type: Poster

Contents

Click at talk title to open the abstract

120

Deciphering cellular interactions at a single-cell resolution for developing efficient and targeted cell-based immunotherapies

Robert Weingarten1, Maryam Rezaei1, Bianca Altvater2, Sareetha Kailayangiri2, Kathrin Kirchhoff1, Franziska S. Bockeloh1, Hans Kleine-Brüggeney1, Sebastian Bühren1

1 evorion biotechnologies GmbH, Münster, Germany
2 University Children’s Hospital Münster, Department of Pediatric Hematology and Oncology, Münster, Germany

Introduction

In the tumor microenvironment (TME), cancer cells directly interact with stroma and infiltrating immune cells. These interactions play a decisive role in tumor outgrowth and the control of tumor progression. A deep understanding of the crosstalk (juxtacrine or paracrine) between cancer cells and immune cells within the heterogeneous TME is crucial for developing efficient cell-based immunotherapies and improving cancer diagnostic procedures.

Methods

To investigate cellular communication, we developed a unique microfluidic chip-based technology that enables the co-culturing of different types of cells in hydrogel beads. Further, we can array these cell-laden beads on a chip and perform live-cell interaction analysis in a physiologically relevant 3D microenvironment at single-cell resolution. Our functional phenomics approach has a unique advantage that allows researchers to perform temporal cellular interactions and link the functional phenotype directly to the underlying molecular parameters including secreted molecules, cellular proteins, enzymatic activities & mRNAs. The whole approach is highly parallelized, providing a comprehensive analysis of thousands of cellular interactions and underlying molecular profiles at the same time.

Results/Discussion

We performed a multiparametric analysis of cellular interactions between endothelial cells (HUVEC) and breast cancer cells (MCF7) cells by applying our technology [1]. Live-cell 3D co-culture imaging showed a time-independent uptake of extracellular vesicles of apoptotic HUVECs cells by MCF7 cells [2]. In addition, we also investigated the interaction between CAR-T cells and cancer cell populations at single-cell resolution. This study showed heterogeneities in interactions of CAR-T cells and cancer cells in a 3D microenvironment. We observed three phenotypic categories, killing, serial killing, and no killing phenotypes of CAR-T cells when interacting with cancer cells. We were able to detect svFv secretion from genetically modified T-cells acting as micro-pharmacies in a highly parallelized approach.

Conclusions

A deep characterization of the cellular communication between cancer cells and immune cells within the heterogeneous tumor microenvironment by our technology will revolutionize the development of immunotherapies against solid tumors. We are currently developing the technology to enable the linking of these functional phenotypes with the transcriptomic multi-omics analysis by retrieving the beads from the chip.

Disclosure

I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation.

References
[1] Kleine-Brüggeney H, van Vliet LD, Mulas C, Gielen F, Agley CC, Silva JCR, Smith A, Chalut K, Hollfelder F 2019,‘Long-Term Perfusion Culture of Monoclonal Embryonic Stem Cells in 3D Hydrogel Beads for Continuous Optical Analysis of Differentiation’, Small., Feb;15(5):e1804576. doi: 10.1002/smll.201804576
[2] Rezaei M, Martins Cavaco AC, Stehling M, Nottebaum A, Brockhaus K, Caliandro MF, Schelhaas S, Schmalbein F, Vestweber D, Eble JA. 2020, ‘Extracellular Vesicle Transfer from Endothelial Cells Drives VE-Cadherin Expression in Breast Cancer Cells, Thereby Causing Heterotypic Cell Contacts’, Cancers (Basel). 2020 Aug 1;12(8):2138. doi: 10.3390/cancers12082138
Keywords: Cellular interactions, tumor microenvironment, single-cell imaging, 3D in-vitro co-culture
121

99mTc-Timanocept Spect Imaging As A Potential Non-Invasive Method To Quantify CD206+ Tumor-Associated M2-like Macrophages

Alexanne Bouchard1, 3, Hugo Sikner1, 3, Mathieu Moreau2, Emeric Limagne3, Pierre-Simon Bellaye3, 1, Evelyne Kohli1, Bertrand Collin2, 3

1 Université de Bourgogne Franche comté, UMR / INSERM / Ub / Agrosup / 1231, Dijon, France
2 Université de Bourgogne Franche Comté, UMR / Ub / CNRS / 6302, Dijon, France
3 Centre anticancéreux Georges François Leclerc, Dijon, France

Introduction

In oncology, modulation of tumor microenvironment (TME) and its follow-up by molecular imaging is crucial because of its central role in cancer. In the TME, M2-like macrophages have been associated with cancer aggressiveness, therapy resistance and poor prognosis. The aim of our study was to assess if M2-like macrophages (i) could be tracked in vivo into the TME with SPECT imaging and (ii) could be modulated by the inhibition of Gp96, an ER chaperone involved in inflammatory processes that we have previously shown to be expressed at the cell membrane of human M2 macrophages [1].

Methods

Tissues from mouse triple negative breast cancer (TNBC, 4T1 cell line) and colon cancer (CC, CT26 cell line) were analyzed by immunohistochemistry (IHC) to detect the presence of Gp96 and CD206 (a marker of M2 macrophages) into the TME. Specific CD206 in vivo imaging on 4T1- and CT26-tumor bearing mice receiving or not a specific inhibitor of Gp96 (PU-WS13) was performed with 99mTc-Tilmanocept SPECT (i.v injection, 15MBq/mouse). Images were performed at 1h, 4h and 24h post-injection. Ex vivo gamma counting of tumors was performed after the last imaging. Radioactivity content measured with gamma counting or on images was expressed as percentage of the injected dose per gram of tissue (% ID/g). For 4T1 tumors, tumor growth and collagen content were also assessed.

Results/Discussion

IHC experiments demonstrated an overexpression of Gp96 in tumor cells as well as the presence of M2-like macrophages expressing both CD206 and Gp96 in 4T1 tumors while CT26 tumors only showed an upregulation of Gp96. These results are confirmed with in vivo SPECT imaging. 99mTc-Tilmanocept tumor uptake was significantly higher in 4T1- compared to CT26-tumor bearing mice (2.53 %ID/g and 1.08 %ID/g respectively, p=0.009). Based on these results, the 4T1 model was chosen to study the impact of Gp96 inhibition. Interestingly, PU-WS13 induced a significant decrease in 99mTc-Tilmanocept tumor uptake compared to untreated mice (1.12 %ID/g and 0.78 %ID/g respectively, p=0.0011) as well as a lower number of CD206+ M2-like macrophages compared to untreated mice. These results correlated with a reduced tumor growth and collagen content in 4T1 tumors.

Conclusions

99mTc-Tilmanocept SPECT imaging might represent an innovative non-invasive strategy to quantify CD206+ tumor- associated macrophages as a biomarker relevant for prognosis, therapeutic prediction and/or monitoring of solid tumors. The potential effects of PU-WS13 on the modulation of M2-like macrophages are currently investigated.

Disclosure

I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of the presentation

References
[1] Chaumonnot Killian, Masson Sophie, Sikner Hugo, Bouchard Alexanne, Baverel Valentin, Bellaye Pierre-Simon, Collin Bertrand, Garrido Carmen, Evelyne Kohli, 2021, ‘The HSP GRP94 interacts with macrophage intracellular complement C3 and impacts M2 profile during ER stress’, Cell death Dis, 2021 Jan 22;12(1):114.
Keywords: M2-like macrophages, Tilmanocept, Spect imaging, Tumor Microenvironment, Gp96
122

Interaction of PDAC tumor organoids with CD68-EGFP+ cells upon nanovaccine treatment

Nathalia Ferreira1, Andrea Markus1, Daniele Ferrari1, Sana Savedipour2, Luis Cruz2, Frauke Alves1, 3, Fernanda Ramos-Gomes1

1 Max Planck Institute for Experimental Medicine, Göttingen, Germany
2 Leiden University Medical Center, Department of Radiology, Leiden, Netherlands
3 University Medical Center Göttingen, , Institute of Diagnostic and Interventional Radiology, Clinic for Hematology and Medical Oncology, Göttingen, Germany

Introduction

Pancreatic adenocarcinoma (PDAC) is projected to significantly increase and become the second cause of cancer-related deaths before 20301. Immunotherapy has revolutionized the treatment of cancer, however, it has failed to show benefits in PDAC treatment2. Ultimately, new immunotherapeutic approaches will require overcoming the immune suppression present in PDAC. Using a peptide-based nanovaccine to enhance the immune response, in this work we used a live imaging method to monitor the interaction of PDAC tumor organoids with immune cells obtained from the blood of nanovaccine-treated mice.

Methods

Murine KPC pancreatic tumor organoids were established as previously described3. To evaluate the reaction of CD68macrophages/monocytes towards KPC tumor organoids upon nanovaccine injection, nanovaccine or saline solution was subcutaneously administered in CD68-EGFP+ transgenic mice. After 24 h, peripheral blood was collected and viable CD68-EGFP+ cells were sorted using the Aria II FACS scanner (BD FACSAria™). Nuclear staining of organoids was achieved by overnight incubation of medium containing Draq5 (Biolegend). CD68-EGFR+ sorted cells were co-cultured with the KPC organoids, and live-cell imaging was performed using a Zeiss LSM880 confocal laser scanning microscope (Carl Zeiss Microscopy GmbH). The experiments were performed under live-cell environmental conditions (37oC/5% CO2).

Results/Discussion

A significant increase of CD68-EGFP+ cells was measured in peripheral blood 24 h after nanovaccine treatment in comparison to saline-treated mice, suggesting a direct enhancement of these cells in the blood of vaccinated mice (Fig. 1). Live confocal imaging showed a higher CD68-EGFP+ cells interaction with KPC organoids (Fig. 2). Within 16 h of co-culture of CD68-EGFP+ cells from vaccinated animals with KPC organoids, a significant amount of these cells infiltrated the organoids (Fig. 2, upper panel). In comparison, the number of infiltrating CD68-EGFP+ cells in KPC organoids was substantially lower with CD68-EGFP+ cells obtained from the blood of control animals (Fig. 2, lower panel). These results show that the nanovaccine increased CD68-EGFP+ cells circulation in the blood and that these cells interact with PDAC tumor organoids. M1/M2- like phenotype profiling of CD68-EGFP+ cells and their interaction with other immune cells is under investigation.

Conclusions

We demonstrated the capacity of a novel peptide-based nanovaccine to enhance CD68+ cells in the blood and the capability of these cells to actively target PDAC tumor organoids. In the future, this live-cell imaging approach will be applied to investigate the interplay of CD68+ cells with other immune cells, especially T-cells, and optimize this nanovaccine approach.

Acknowledgement

Special acknowledgment to Bärbel Heidrich for technical assistance. This project has received financial support from the European Union´s Horizon 2020 research and innovation program under the Marie Sklodowska- Curie grant agreement nº 861190.

Disclosure

We have no financial interest or relationship to disclose regarding the subject matter of this presentation.

References
[1] Kenner, B. J. et al. Early Detection of Pancreatic Cancer - A Defined Future Using Lessons from Other Cancers: A White Paper. Pancreas (2016)
[2] Feng, M. et al. PD-1/PD-L1 and immunotherapy for pancreatic cancer. Cancer Letters (2017).
[3] Baker, L. A. & Tuveson, D. A. Generation and culture of tumor and metastatic organoids from murine models of pancreatic ductal adenocarcinoma. Methods in Molecular Biology (2019)
Enhanced CD68-EGFP+ circulating cells in the blood upon nanovaccine treatment

The graphs show that 24h after nanovaccine treatment, the percentage of circulating CD68-EGFP+ cells in the peripheral blood is significantly increased in mice that received the nanovaccine compared to those that received saline only. N= 3 per group, test performed: Unpaired t-test, **p < 0.05.

CD68-EGFP cell interaction with KPC tumor organoids
Representative confocal 3D images (left) showing the interaction of CD68-EGFP+ cells (green) from saline- (lower panel) or nanovaccine-treated (upper panel) mice with KPC organoids (nuclear dye, red). Orthogonal views (right) show a close view of the proximity between CD68-EGFP+ cells (green) and KPC organoids. Scale bar represents 50 μm.
Keywords: PDAC, Immunotherapy, Organoids, Nanovaccine
123

The development of a NIR-fluorescent tracer for fluorescence molecular endoscopy and ex-vivo imaging for the assessment of treatment with immune-checkpoint inhibition therapy

Wouter T. R. Hooghiemstra1, 2, Matthijs D. Linssen1, 2, Janny J. van Zanden2, Annelies Jorritsma-Smit2, Wouter B. Nagengast1, Marjolijn N. Lub-de Hooge2

1 University Medical Center Groningen, Department of Gastroenterology and Hepatology, Groningen, Netherlands
2 University Medical Center Groningen, Department of Clinical Pharmacy and Pharmacology, Groningen, Netherlands

Introduction

Therapy with checkpoint inhibitors fills a gap in the treatment for specific cancers, that do not show optimal response to standard-of-care treatment. To limit patient exposure to unnecessary side effects and reduce costs, predicting which patient benefits from this treatment is necessary. Uptake of 89Zr-atezolizumab was shown to be a strong predictor to therapy response1. The aim of this study is to use an antibody targeting PD-L1 labelled with fluorescent dye as an imaging agent to predict response and a PD-1 targeting tracer to assess therapy response in esophageal cancer patients.

Methods

Commercially available anti PD-L1 antibodies Atezolizumab, Durvalumab and the anti PD-1 antibody Nivolumab were labeled with IRDye 800CW, IRDye 680LT NHS-esters (LI-COR, Lincoln,NE). Additionally, atezolizumab was also labeled with ZW800-1 dye (Curadel, Natick MA). Both after labelling and after purification the tracers were analyzed on SE-HPLC to assess protein integrity, label efficiency (LE) and concentrations of the protein and free dye. One combination of antibody and dye were chosen for each target to further optimize the labelling process and transfer to GMP production. Labelling optimization was done by assessing the optimal dye:protein labelling ratio and formulation buffer for long-time storage.

Results/Discussion

We successfully conjugated durvalumab and nivolumab to all of the available dyes, in contrast we were unable to successfully conjugate atezolizumab to any of the dyes. For durvalumab we chose to move forward in combination with the 680LT dye, to have possibilities of imaging on multiple wavelengths in the future. Optimization of the labeling process yielded an optimal dye:protein ratio of 2:1 and storage in a phosphate buffer with pH 7.0. For this labelling we achieved a label efficiency of about 88% with a 90% protein yield, 0% protein aggregates and <0,5% unconjugated dye.

Since the results of optimization are sufficient, we are now moving forward with the tech transfer to prepare for GMP production of the tracer for clinical use. As the 680LT dye is not yet used in a clinical setting, we are also preparing to do a toxicity study with the new tracer and with unconjugated dye.

Conclusions

We were able to successfully conjugate the antibodies durvalumab and nivolumab to IRDye 800CW and 680LT, in contrast we were unable to conjugate atezolizumab to any of the fluorescent dyes except ZW800-1. We chose to continue with durvalumab and IRDye 680LT, of which the labeling optimization and tech transfer are finished and a toxicity study is in preparation. The labeling process of nivolumab still needs to be optimized.

Acknowledgement

This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No 831514 (Immune-Image). The JU receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA

Disclosure

I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation.

References
[1] Bensch, F, van der Veen, EL 2018, '89Zr-atezolizumab imaging as a non-invasive approach to assess clinical response to PD-L1 blockade in cancer', Nature Medicine, 26-NOV-2018, 1852-1858
Keywords: molecular imaging, fluorescent tracer, fluorescence molecular imaging, durvalumab, immune checkpoint imaging
124

3d visualization of human tumors with special focus on T-cell infiltration by light sheet microscopy

Andreas F. Kremer1, Diana Pinkert-Leetsch1, 2, Frauke Alves1, 2, 3, Fernanda Ramos-Gomes2, Jeannine Missbach-Güntner1

1 University Medical Center Goettingen, Institute of Diagnostic and Interventional Radiology, Goettingen, Germany
2 Max Planck Institute for Experimental Medicine, Translational Molecular Imaging, Goettingen, Germany
3 University Medical Center Goettingen, Clinic for Hematology and Medical Oncology, Goettingen, Germany

Introduction

In the context of cancer (ca) immunotherapy, major successes have been achieved in recent years with immune checkpoint inhibitors. This is especially true for tumors infiltrated by immune effector cells. However, immunohistochemical (IHC) staining methods provide insufficient two dimensional (2d) information about the distribution of immune cells within the tumor, Therefore, the aim of this study was to apply a new clearing/staining protocol for imaging of human tumor samples in 3d using a novel light sheet fluorescence microscope (LSM) in order to assess the cytotoxic T-cell (CTL) status.

Methods

Human tumor specimen (0.5cm3) of breast and colon were analyzed by LSM (LaVision Biotec, a Miltenyi Biotec Company). To validate the findings, individual tissue sections were histologically analyzed to determine morphology and CTL status prior to application of the clearing, using H&E, MTS, anti-(α)CD4 and αCD8 stainings. Tumor samples were treated with the here newly developed Klarus clearing method with thiodiethanol (TDE). CTLs within the 3d tumor samples were stained with the αCD8-fluorophore antibody. The LSM scans were performed with different filter sets to enable not only the detection of the CD8 protein but also autofluorescence signals in 3d. Subsequently, the samples were embedded in paraffin, sectioned and the results were validated by 2-photon microscopy (2-PM) and IHC.

Results/Discussion

Examination of colon-ca by LSM revealed a homogenous distribution of fluorescence signals in the rim of the 3d tumor specimen, indicative for a CD8+CTL specific staining. In a higher magnification, the membrane localization of antibody, specific for CD8+ cells, was clearly detectable. CTL fluorescent signals were visualized in the 3d context of colon crypts, blood vessels and accumulation of tumor cells visualized by autofluorescence signals. Subsequent 2-PM analysis confirmed the presence of CD8+CTLs. 2d IHC showed a homogeneous CTL distribution all over the tumor, revealing a reduced penetration depth of the αCD8-fluorophore antibody into the tissue sample in the LSM approach. Histology of mammary-ca revealed a strong desmoplastic reaction, causing a high refractive index and scattering artifacts, impeding high-resolution LSM. In colon adenoma, no specific fluorescence was detected due to the preserved epithelial barrier, hindering the antibody to penetrate into the tissue.

Conclusions

Depending on tissue composition, different 3d distribution patterns (homogeneous, perivascular, focal) of CTLs were defined by LSM. Moreover, autofluorescence of vessels, epithelia and stroma facilitates the co-registration of fluorescent CTLs to morphological structures. Therefore, LSM is a promising method for the evaluation of CTL distribution in whole tissue samples, to study CTL infiltration in the context of complex tumor heterogeneity.

Acknowledgement

We thank Bettina Jeep, Julia Schirmer and Sabine Wolfgramm for excellent technical assistance in animal work, staining procedures, embedding and histological staining.

Disclosure

I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation.

References
[1] Fridman et al. 2012): Fridman, W. H.; Pagès, F.; Sautès-Fridman, C.; Galon, J.: The immune contexture in human tumours: impact on clinical outcome. In: Nature reviews. Cancer 12 (2012) 4, S. 298 - 306.
[2] Melero I, Rouzaut A, Motz GT, Coukos G (2014): T-cell and NK-cell infiltration into solid tumors: a key limiting factor for efficacious cancer immunotherapy. Cancer Discov 4, 522–526
[3] Pagès F, Galon J, Dieu-Nosjean M-C, Tartour E, Sautès-Fridman C, Fridman W-H (2010): Immune infiltration in human tumors: a prognostic factor that should not be ignored. Oncogene 29, 1093–1102
Richardson DS, Lichtman JW (2015): Clarifying Tissue Clearing. Cell 162, 246–257
Keywords: mammary carcinoma, colon carcinoma, T-cell infiltration, light sheet microscopy
127

Ultrahigh-content imaging helps to identify CAR target candidates against pancreatic adenocarcinoma

Daniel Schäfer1, 2, 3, Stefan Tomiuk1, Laura N. Küster1, Wa'el Al Rawashdeh1, Janina Henze1, 2, 3, German Tischler-Höhle1, David J. Agorku1, Janina Brauner1, Cathrin Linnartz1, Dominik Lock1, Andrew Kaiser1, Christoph Herbel1, Dominik Eckardt1, Melina Lamorte6, Dorothee Lenhard6, Julia Schüler6, Philipp Ströbel4, Jeannine Missbach-Güntner3, Diana Pinkert-Leetsch3, 5, Frauke Alves2, 3, 5, Andreas Bosio1, Olaf Hardt1

1 Miltenyi Biotec B.V. & Co. KG, R&D, Bergisch Gladbach, Germany
2 University Medical Center Göttingen, Clinic for Hematology and Medical Oncology, Göttingen, Germany
3 University Medical Center Göttingen, Institute for Diagnostic and Interventional Radiology, Göttingen, Germany
4 University Medical Center Göttingen, Institute for Pathology, Göttingen, Germany
5 Max Planck Institute for Experimental Medicine, Translational Molecular Imaging, Göttingen, Germany
6 Charles River Discovery Research Services GmbH, Freiburg, Germany

Introduction

Chimeric antigen receptor (CAR) T cells have become a new pillar of cancer therapy. They proved outstanding efficacies in leukemic patients, formerly believed to be beyond treatment. Their remarkable success in the context of liquid tumors could not yet be translated to the field of solid malignancies. One major issue remains the restricted availability of safe and tumor-specific antigens. Here, we show how a newly developed cyclic immunofluorescence microscopy platform (MACSima) enables the observation of dozens of markers on the same tissue and helps to identify novel CAR T cell targets.

Methods

We screened around 400 surface antigens on 17 patient derived xenografts (PDX) using flow cytometry. Expressed target candidates were prioritized based on healthy tissue expression using RNAseq, mass spectrometry and antibody databases. The novel MACSima platform was used to verify the expression of target candidates on human pancreatic tumors and dissecting the different cell linages within. 32 CAR constructs were designed specific for suitable targets and evaluated in vitro using cytokine release, marker expression and killing as read-outs. Most promising constructs were challenged in vivo. Here, MACSima helped to investigate differences between treatment groups and escape mechanisms. Finally, a healthy tissue multiarray was analyzed, to confirm low expression of the chosen targets.

Results/Discussion

We identified a set of 50 surface antigens on the PDXs in the initial flow screening. RNAseq, mass spectrometry and antibody databases were used to prioritize the target candidates and the MACSima platform helped to reveal, that CLA, CD66c, CD318 and TSPAN8 were the targets with the highest tumor specificity and safety profile. While for CD66c, CD318 and TSPAN8 most efficient CAR constructs were determined in vitro, no effective CAR T cells specific for CLA could be generated, due to self-antigenicity. These CARs were also efficient in vivo and MACSima was used to show that low efficacies are not caused by target downregulation, but are rather caused by intrinsic CAR function. MACSima unravelled also the restricted healthy tissue expression of CD66c, CD318 and TSPAN8, making them interesting targets for CAR treatment. Overall, the newly developed cyclic immunofluorescence platform proved to be a powerful and versatile tool, when it comes to multiplexing on the very same tissue section.

Conclusions

This study offers a glimpse at the new possibilities opened by the introduction of the MACSima imaging platform. It was used to bypass a major roadblock prohibiting effective cellular immunotherapy: a lack of suitable tumor-specific antigens. Often either the amount of tissue or stainable surface markers restricts a holistic understanding of tissue expression. The MACSima platform is a convenient solution to both.

Acknowledgement

We want to thank Michail Knaul and Jutta Kollet for their help and advice regarding bioinformatical matters and Sandy Reiß, Lara Minnerup, Vera Dittmer, Abigail J. Deloria, Sabine Wolfgramm, Niels Werchau, and Simon Lennartz for excellent technical assistance.

Disclosure

I or one of my co-authors have the following financial interest or relationship(s) to disclose regarding the subject matter of this presentation:
D.S., L.N.K., S.T., G.T.H., D.A., W.A.R., J.H., J.B., C.L., D. Lock, A.K., C.H., D.E., A.B., and O.H. are employees of Miltenyi Biotec B.V. & Co. KG. M.L., D. Lenhard, and J.S. are employees of Charles River Discovery Research Services GmbH. All other authors declare no competing interests. There are patent applications pending related to this work.

References
[1] Siegel, R. L., Miller, K. D. & Jemal, A. Cancer statistics, 2018. CA Cancer J. Clin. 68, 7–30 (2018).
[2] Kleeff, J. et al. Pancreatic cancer. Nat. Rev. Dis. Prim. 2, 16022 (2016).
[3] Akce, M., Zaidi, M. Y., Waller, E. K., El-Rayes, B. F. & Lesinski, G. B. The potential of CAR T cell therapy in pancreatic cancer. Front. Immunol. 9, 2166 (2018).
Expression and localization of several target candidates on a human PDAC tissue
Selection of 15 cyclic immunofluorescence images of a representative human PDAC tissue to evaluate co-expression of the marker of interest (MOI) with cytokeratin-positive tumor cells.
Immunofluorescence image of a human PDAC tissue comparing eight markers
This is an eight marker overlay of a human PDAC tissue, which was stained for 97 markers in a single cyclic immunolfuorescence run using the MACSima platform. In the center a tertiary lymphoid structure is visible containing B cells, T cells and several myeloid cells. In its perimeter several cancerous ducts can be observed.
Keywords: CAR T cell, PDAC, pancreatic cancer, microscopy, multiplexing