Tumor microenvironment phenotyping and imaging

The tumor microenvironment (TME) is inherently complex. Due to its highly variable cell composition and the high number of proteins and structures involved in tumor formation, an in-depth analysis of tumor tissues requires the examination of a plethora of parameters.  

Novel imaging technologies like ultra-high content imaging or 3D visualization by light sheet microscopy allow researchers to dive deep into limited sample material, laying the foundation for understanding TME complexity. 

Ultra high content imaging using MICS (MACSima Imaging Cyclic Staining) technology on the MACSima Imaging Platform allows for automated fluorescence microscopic analysis of hundreds of markers on a single sample. MICS technology can be applied to decipher complex questions in TME research, like target or biomarker identification, protein characterization, TIL phenotyping, or cancer classification as shown in the exemplary data below.

Identification of CAR target candidates in the TME of a pancreatic adenocarcinoma

Expression of a multitude of CAR target candidates within the primary pancreatic cancer tissues from patients was assessed using the MACSima Imaging Platform. The full story is published in Nature Communications (Schäfer, D. et al. 2021). Licensed under a Creative Commons Attribution 4.0 International License

CAR target candidate identification in pancreatic adenocarcinoma

Talk
Early identification of target candidates for pancreatic adenocarcinoma immunotherapy

In this symposium, recorded at the virtual EACR 2021, you get insights into how Miltenyi Biotec’s cancer research solutions support the discovery of novel target candidates to develop innovative therapeutic
approaches.

Biomarker screening in pancreatic ductal adenocarcinoma

MICS technology allows for cyclic staining and analysis of hundreds of potential marker candidates on one tissue section. The data below show exemplary immunofluorescence images for different markers of interest analyzed in an automated 97-marker imaging run.

Biomarker screening in pancreatic ductal adenocarcinoma
Inter- and intratumor heterogeneity of epithelial MUC1 expression in ovarian carcinomas

Evaluation of the specificity and tissue distribution of cancer associated proteins in ovarian carcinoma

The automated MACSima Imaging Platform enables assessment of multiple samples at a time. Here, the expression of MUC1 was analyzed in patient-derived ovarian carcinoma tissues. Multiparameter expression analysis of MUC1 and markers indicative of cell lineages and proliferation status, i.e., CD326 (epithelial), Ki-67 (proliferation), CD31 (endothelial), CD90 (stromal), and CD45 (differentiated hematopoietic cells) are shown.

Classification of primary glioblastoma tumors

Classification of primary glioblastoma tumors

Classification of different patient-derived primary glioblastoma tumors based on the expression of PDGFRα, p53, synaptophysin, CD44, nestin, podoplanin, GFAP, and EGFR based on a classification scheme published by Motomura, K. et al. 2012.

Identification of different cell populations in a colon carcinoma sample 

Characterization of tumor-infiltrating leukocytes (TILs) in human colon 

In this experiment, 75 different markers were detected on a colon carcinoma section. Images were acquired with the MACSima Imaging System and data were analyzed using the powerful and intuitive Qi Tissue Image Analysis Software.

Based on the intensity profile of the cells, several distinct cell populations could be identified. Using this information, it was possible to identify the populations that could infiltrate the tumor region.

MACSima Imaging System

Video
Powerful and automated: Ultra high content imaging with the MACSima™ Imaging Platform 

Learn how the MACSima Imaging System can stain hundreds of markers on a single sample, and stain multiple samples at a time, all in an entirely automated fashion. See more than ever before: Unlock new insights and advance your research further, faster!

Light sheet microscopy uses a laser light sheet that is perpendicular to the axis of observation to excite a single plane of a labeled sample. Illumination of only a thin layer of the z-axis results in high-quality 3D imaging of entire organs with only low and strictly localized photodamage and bleaching effects. Moving the sample along the z-axis through the light sheet produces a stack of images that can be combined to visualize large biological samples in 3D with high resolution (Mohr, H. et al. 2021 and Taranda, J. and Turcan, S. 2021).

The fully automated UltraMicroscope Blaze light sheet microscope in combination with tissue clearing enables 3D visualization of heterogeneous tumor physiology. It allows for quantification of multiple tumor parameters and CAR T cell infiltration at a cellular and even subcellular level for multiple samples and can be applied for:

  • Section-free 3D histological analysis.
  • Visualization, quantification, and evaluation of cellular therapies within the TME.
  • Visualization and quantification of single disseminated cancer cells in whole animal models.
  • Drug target identification for cancer treatments in a whole mouse body or entire organs.

Video
Visualization of infiltrating CAR T cells in a pancreatic carcinoma xenograft 

The video shows a xenograft of a human pancreatic carcinoma cell line with infiltrating CAR T cells. Sample preparation involved whole-mount immunolabeling followed by ethyl cinnamate (ECi) clearing. The 3D data of the tumor were created with the UltraMicroscope II light sheet microscope. CAR T cells were labeled using the REA844 clone conjugated with Vio® 667 (violet), detecting human CD271 (LNGFR). Vasculature was stained by rhodamine-lectin (orange). Remaining GFP expression of carcinoma cells is shown in green.

3D visualization of thorax metastases targeted and missed by a drug candidate

Visualization of metastases in a mouse thorax

3D visualization of a mouse thorax with the UltraMicroscope Blaze detects metastases targeted by a drug candidate (white) versus metastases missed by the drug candidate (magenta).

Reference list
Publications using UltraMicroscope light sheet imaging systems in cancer research

Video
Imaging single cancer cells disseminated in whole mouse bodies

Source: Chenchen Pan et al. Deep learning reveals cancer metastasis and therapeutic antibody targeting in the entire body, P1661–1676.E19. 
Copyright (2019), with permission from Elsevier.

UltraMicroscope Blaze
  • Hahn, A. (2020) The preclinical amyloid sensitive composite to determine subtle cognitive differences in preclinical Alzheimer's disease. Sci. Rep. 10(1): 13583.
  • Mohr, H. et al. (2021) Mutation of the cell cycle regulator p27kip1 drives pseudohypoxic pheochromocytoma development. Cancers 13(1): 126.
  • Motomura, K. et al. (2012) Immunohistochemical analysis-based proteomic subclassification of newly diagnosed glioblastomas. Cancer Sci. 103(10):1871–9.
  • Schäfer, D. et al. (2021) Identification of CD318, TSPAN8 and CD66c as target candidates for CAR T cell based immunotherapy of pancreatic adenocarcinoma. Nat. Commun. 12(1): 1453.
  • Taranda, J. and Turcan, S. (2021) 3D whole-brain imaging approaches to study brain tumors. Cancers 13(8): 1897.

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