Charakterisierung muriner und humaner Tumoren mit besonderem Fokus auf die Immunzellinfiltration mittels innovativer bildgebender Verfahren

Characterisation of murine and human tumours with a special focus on immune cell infiltration using innovative imaging techniques

by Andreas Falk Kremer
Doctoral thesis
Date of Examination:2024-09-04
Date of issue:2024-08-23
Advisor:Dr. Jeannine Mißbach-Güntner
Referee:Prof. Dr. Frauke Alves
Referee:Prof. Dr. Philipp Ströbel
Persistent Address: http://dx.doi.org/10.53846/goediss-10631

 

 

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Abstract

English

3D imaging of the immune cell status in a complex tumour volume represents an important step towards understanding the anti-tumour immune response and a decisive predictor of the efficacy of immune checkpoint inhibition. The gold standard for this issue is histology. However, due to the limitation of the sample size and the random selection of a small tumour area, this only allows an imprecise statement to be made about the 3D distribution of immune cells in a larger volume. A promising alternative here is light sheet microscopy, which allows both tomographic fluorescence images to be taken with cellular resolution and a 3D reconstruction of an entire tumour sample to be created. Two challenges were observed for light sheet microscopy, firstly to achieve a sufficient increase in transparency of tumour samples through clearing and secondly to achieve complete immunolabelling using fluorophore-coupled antibodies (NIRF probes). Three different protocols were tested for application to tumour tissue as part of the work. The protocol used here for aqueous clearing with Clarity, which is established on brain tissue, led to tissue damage in colon carcinoma, the increase in transparency was not sufficient for larger sample pieces of 0.5 cm3 and the NIRF probes could only specifically stain marginal areas of the human colon carcinoma samples. In the subsequent immunohistochemistry (IHC), EGFR and CD31 could no longer be stained and Ki-67 could only be stained very weakly compared to the control. The second method used was solvent-based clearing with benzyl alcohol benzyl benzoate (BABB). This led to a reduction in the size of the tissue, but provided the best result in terms of clearing, as it led to a very effective increase in transparency in all the tissues examined. Further experiments showed that BABB had a negative effect on the intensity of the NIRF probes. This clearing method was therefore particularly suitable for autofluorescence imaging without subsequent NIRF probe staining. Following solvent-based clearing, the target structures EGFR, CD31 and Ki-67 could be immunohistochemically stained. The third method, so-called Klarus clearing, represents an effective option for clearing low-collagen tumours, as demonstrated in this work on human colon carcinoma. Klarus clearing works with thiodiethanol, a non-toxic agent. This third method was able to ensure both a sufficient increase in transparency for light sheet microscopy and the detection of specific CD8-positive cytotoxic T cells. Furthermore, additional immunohistochemical staining could be carried out subsequently. Finally, the 3D distribution of cytotoxic T cells in collagen-poor colon carcinoma tissue could be visualised under the light sheet microscope using the clearing method developed in this work and adapted to tumour tissue.
Keywords: clearing