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210 New approaches to understanding the tumor microenvironment: in situ detection of immune checkpoint interactions in cancer patient tissue samples
  1. Jonas Vennberg,
  2. Desirée Edén,
  3. Sara Bodbin,
  4. Ka IAu Ieong,
  5. Lena Beckman,
  6. Daniel Ekman and
  7. Agata Zieba-Wicher
  1. Navinci Diagnostics, Uppsala, Uppsala, Sweden
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Receptors PD-1 and LAG3, and their respective ligands PD-L1 and MHCII are immune checkpoint (IC) interactions that lead to inhibitory signals in the T cell and promote immune exhaustion to avoid autoimmunity. Both these pathways, however, can be hijacked as immune escape routes by cancer cells in the tumor microenvironment (TME). Hence their blockade is an attractive and widely applied immunotherapeutic approach. However, the efficacy of immune checkpoint inhibitors (ICIs) has been limited, even for patients positive for diagnostic biomarkers. Patient stratification is traditionally based on immunohistochemical (IHC) stainings for single IC proteins such as PD-L1 which often prove inconclusive. Patient outcomes may have better correlation to high levels IC interactions which are indicative of pathway activation rather than to single protein expression.

Methods Formalin-fixed, paraffin-embedded tissue (FFPE) sections as well as tissue microarrays (TMAs) were stained using specific pairs of antibodies against either PD1 and PD-L1, or LAG3 and MHCII in order to examine the activation of these immunosuppressive pathways in cancer tissues. To ensure that interactions and not simply an overlap of fluorescent signal was detected, we employed the Naveni™ proximity ligation method. This technology detects proteins in situ, including in FFPE tissue, located within an interaction range (<40 nm) by means of antibodies conjugated to oligonucleotides that create amplified fluorescent or chromogenic signal only if the proximity requirement is fulfilled.

Results PD1/PD-L1 and LAG3/MHCII interactions as assessed by proximity ligation technology were observed in healthy tonsil tissue. The two interactions were generally overlapping and predominantly observed within germinal centers of the tonsil, known as the sites of communication between antigen-presenting cells and T cells. The PD1/PD-L1 interaction was further assessed and found to stain positively in different TMAs, including tumor tissues from patients with non-small cell lung cancer (NSCLC), malignant melanoma, colon cancer and pancreatic cancer. Moreover, the PD1/PD-L1 interaction was also found in draining lymph nodes of patients with NSCLC and malignant melanoma.

Conclusions Interactions such as PD1/PD-L1 and LAG3/MHCII that can be visualized in cancer patient tissues using the proximity ligation assay are promising targets with a more biologically relevant profile than traditional IHC. Understanding the dynamics of ICIs within the TME can enhance the development of effective strategies to overcome immune escape mechanisms employed by cancer cells. Further research is needed to validate the utility of ICIs as biomarkers and explore their therapeutic potential, advancing cancer immunotherapy and improving patient outcomes.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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