Abstract

Multiplex Immunofluorescence for Evaluation of NK Cell Therapy in a Murine Model of Glioblastoma Multiforme

Submitted and written by Samantha Hicks, Texas A&M University

Hypothesis: We hypothesized that multiplex immunofluorescence could be utilized to evaluate NK cell infiltration in both tumor and adjacent brain tissue of immunodeficient NSG mice engrafted with glioblastoma multiforme (GBM) tumor cells. 

Significance: Presently, most adoptive cell transfer (ACT) therapies are inconsistently effective in solid tumors and are solely labeled for hematogenous neoplasia. One emerging ACT therapy utilizes NK cells, an immune cell that has a natural predilection for targeting and destroying tumor cells. NK cell therapy is showing significant promise for the treatment of solid tumors such as GBM, the most common primary brain tumor in human adults. There still exists a considerable need for improved immunotherapies for solid tumors and the development of novel ACT therapies with improved efficacy and toxicity profiles.

Methods: We evaluated genetically engineered NK cell therapy for persistence and expansion in the tumor microenvironment and its effects on normal brain and GBM tumor tissue. In this study, NSG mice were cerebrally implanted with GSC-20 patient-derived GBM cells transduced to express firefly luciferase and genetically engineered or non-transduced NK cells with one cohort given only tumor cells. Mice were monitored with in vivo bioluminescent imaging of tumor cells. Mice were then euthanized, and the engrafted tumors and brain tissue were collected, formalin-fixed, paraffin-embedded, and stained using H&E and Akoya Biosciences Opal 7-color kit for multiplex immunofluorescence (mIF). Antibodies for granzyme B, caspase 3, Ki-67, Iba-1, luciferase, and GFAP were utilized and evaluated. The mIF highlighted microglia, GBM cells, NK cells, astrocytes, apoptotic cells, and proliferating cells. Whole slides were scanned using an Aperio AT2 and Leica Versa 8. We used Halo v3.6.4 to quantify phenotypes of interest and determine spatial relationships of microglia and NK cells, which synergistically work to produce an inflammatory response in the brain. 

Conclusion: NK cells demonstrated extensive tumor infiltration and local brain inflammation. NK cell therapy shows strong promise for treating solid tumors and mIF may be a useful tool for evaluating NK cell persistence and proliferation in tumors and the tumor microenvironment.