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Eagle view multiplex scanning: unveiling cellular interactions in tumor explants

Multiplex fluorescent immunohistochemistry (mF-IHC) stands as a powerful method that enables researchers to gain better insights into the complex interactions between cells in biological samples. In essence, this approach uses multiple fluorescent markers simultaneously to label various structures within a sample. By employing this multidimensional technique, researchers can visualize the spatial relationships between different targets, resulting in a more comprehensive understanding of cellular interactions.
This spatial context is crucial in cancer research, as it enables the study of tumor microenvironments and the assessment of treatment effectiveness.

Dr. Meggy Suarez-Carmona, a researcher at the Helmholtz Institute for Translational Oncology in Germany is using mF-IHC to advance cancer treatment research. Currently completing her postdoctoral project in the group of Prof. Niels Halama, Dr. Suarez-Carmona is investigating resistance mechanisms to immunotherapy in solid tumors, with a focus on ovarian cancer. In her research, Dr. Suarez-Carmona employs the NanoZoomer^Ⓡ S60 Digital slide scanner (Hamamatsu Photonics) to acquire whole slide images of small tumor explants stained using the mF-IHC technique. This method helps gather a vast amount of information on how tumors interact with immune cells following different treatments, all from just one small sample. This gives valuable insights into potential therapeutic strategies.

During her experiments, Dr. Suarez-Carmona (left) receives support from Alicia Höflich (right), the technician responsible for conducting the stains. Together, they have developed a staining protocol that targets five different markers on the same sample. The glass slide with a section of tumor is stained with fluorescent dyes and then automatically scanned using Hamamatsu’s NanoZoomer S60 Digital slide scanner. Dr. Suarez-Carmona explains that this method enables her to address multiple questions using just one slide, such as: “Is the tumor infiltrated by numerous T cells?”, “Are T cells forming aggregates, suggesting their activation? and “What is the proportion of cytotoxic T cells relative to the overall T cell population (CD8-to-CD3 ratio)?” Additionally, a preliminary understanding of immunosuppression is gained through the density of scavenger macrophages. These markers not only provide prognosis information but also indicate whether a response to immunotherapy is occurring.

The NanoZoomer S60 Digital slide scanner provides Dr. Suarez-Carmona with an eagle’s view of the localization and distribution of T cells, and tumor-associated macrophages within and around the tumor core. Notably, one of the most advantageous features of the NanoZoomer S60 Digital slide scanner lies in its flexibility. Designed to provide up to six different filter sets, the NanoZoomer S60 Digital slide scanner offers a wide range of fluorophores for their applications. In Dr. Suarez-Carmona’s case, her NanoZoomer S60 Digital slide scanner is equipped with six different filter sets, allowing her to effectively scan the multiplex IHC.

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“Thanks to the scanner’s speed and flexibility, we can quickly scan the entire layer of tumor explants labeled with multiple markers.”

Dr. Suarez-Carmona

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Figure 1: Overview of the mF-IHC staining for immune and cancer cells on a cryosection of epithelial ovarian cancer. Cy3-EpCAM stains epithelial (cancer) cells. Cy2-CD3 stains pan T cells. Cy7-CD8 stains cytotoxic effector T cells. Cy5-CD163 stains scavenger tumor-associated macrophages. Images are acquired at the 40× magnification. The white square delineates the area illustrated in Figure 2.

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Figure 2: Micro-images illustrating an mF-IHC for immune and cancer cell markers on a section of epithelial ovarian cancer. Cy3-EpCAM stains epithelial (cancer) cells. Cy2-CD3 stains pan T cells. Cy7-CD8 stains cytotoxic effector T cells. Cy5-CD163 stains scavenger tumor-associated macrophages. Images are acquired at the 40× magnification.

IHC protocol for multiplex stain of five markers on paraformaldehyde-fixed tissue

Provided by Dr. Suarez-Carmona, please find below an example of her IHC protocol for multiplex stain of five markers on paraformaldehyde-fixed tissue.

For this purpose, we used 6 µm-thick frozen tissue sections from a tumor, in this example, a specimen from an epithelial ovarian carcinoma.
The sections were fixed in paraformaldehyde 4 % for 20 minutes, then thoroughly washed in PBS, prior permeabilization with triton X100 0.1 % for 5 minutes.
The tissue sections were incubated with anti-CD3 antibody for 1 hour at room temperature, washed, and then incubated overnight with all other antibodies at 4 °C (see Table 1 for a list of antibodies).
The tissue was counterstained with DAPI for 20 minutes and mounted with ProLong Gold Antifade Mountant (Invitrogen). To ensure no cross-detection of a stain in another filter, the fluorophores were organized so that no two spectrally-adjacent colors could generate a colocalized stain (i.e. CD3 and CD8 were not detected by two consecutive filters).
Finally, the whole slide images were acquired with the Hamamatsu NanoZoomer S60 Digital slide scanner at 40× magnification.

Table 1: Reagents and references

Reagent	Reference	Antibody concentration
Anti-CD3 [UCHT1] Alexa Fluor 488	300415 (BioLegend)	0.5 µg/ml
Anti-EpCAM [VU1D9] Alexa Fluor 555	5488S (Cell Signaling)	0.15 µg/ml
Anti-CD163 [GHI/61] PerCP	333626 (BioLegend)	4 µg/ml
Anti-CD8 [SK1] APC-Cy7	348793 (BD Biosciences)	0.5 µg/ml
DAPI	D9542-1mg (Sigma-Aldrich)	2 µg/ml

Multiplex fluorescent immunohistochemistry stands as a pivotal tool in cancer research, exemplified by Dr. Meggy Suarez-Carmona's work on deciphering mechanisms of resistance to immunotherapy in solid tumors. Overcoming challenges posed by limited samples, mF-IHC enables researchers to extract detailed spatially resolved information from one single biopsy or a tumor explant. Utilizing the NanoZoomer S60 Digital slide scanner, Dr. Suarez-Carmona gains an eagle's view of cellular interactions, addressing multiple questions simultaneously. This transformative approach not only enhances our understanding of cancer biology and immunology but also holds promise for more effective therapeutic interventions in solid tumors.

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Meggy Suarez-Carmona, PhD, is a researcher at the Helmholtz Institute for Translational Oncology (HI-TRON Mainz), a Helmholtz Institute of the DKFZ, Mainz, Germany. Dr Suarez-Carmona graduated in biomedical sciences at the University of Liège (Belgium) and works at the German Cancer Research Centre (DKFZ) in Germany, where she endeavors to decipher the mechanisms of resistance to chemotherapy and immunotherapy. Dr Suarez-Carmona specializes in the use of human tissue explant models as personalized, clinically predictive tools for target discovery and drug development in tumor immunology and immunotherapy, with a strong focus on macrophage repolarization drugs.

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Marta Böhle, PhD, is an Application Engineer at Hamamatsu, specializing in both research and clinical whole slide imaging using NanoZoomer^® scanners. With a PhD in Neuroscience and extensive experience in diverse imaging techniques, Marta is passionate about imaging and dedicated to supporting customers in their projects.

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