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.  

CASES/ ABSTRACTS

Submitted by Amelia Collins, University of Georgia

Evaluating host pathogen interactions of Bordetella bronchiseptica and Bordetella pertussis with respiratory epithelium utilizing an Air-Liquid-Interface system

The genus Bordetella contains many common respiratory disease-causing pathogen species in both humans and animals. Bordetella pertussis (Bp) and Bordetella bronchiseptica (Bb) cause Whooping cough in humans and Kennel cough in dogs, respectively. Bp and Bb reside in the respiratory tract and tightly adhere to epithelial cells during infection. Previous in vitro model systems used to evaluate the host-pathogen interactions occurring at this critical interface have not use polarized epithelial cells that simulate the human natural respiratory tract. This study utilizes an Air-Liquid-Interface culture system (ALI) using human broncho-tracheal (NHBE) cell cultures replete with cilia, goblet cells, mucus, and sol layers to evaluate how these pathogens interact with their niche: the ciliated epithelium that lines the mammalian airways. This study utilizes a highly virulent circulating strain of Bp (D420) and two isogenic mutant strains lacking key virulence factors (btrS and pertussis toxin) to evaluate tight junction integrity via trans-epithelial electrical resistance, bacterial growth on the apical surface, migration through the epithelium to the basal media, bacterial attachment & localization, and pro-inflammatory cytokine responses to infection. The data generated from these studies will reveal the roles of specific Bordetella virulence factors in various measurable aspects of their interactions with ciliated respiratory epithelia.

CASES/ ABSTRACTS

Submitted by Samantha Hicks, Texas A&M University

CASES/ ABSTRACTS

Submitted by Regan Smith, University of Georgia

Case Report

Submitted by Olivia Obringer, Michigan State University

My personal pet, Merlin, an 8mo old MN DSH presented to MSU VMC for inability to urinate. He was adopted by myself 29 days prior and had no history of urinary issues. His bladder was firm and painful upon palpation. An IV catheter was placed, and Merlin was sedated and unblocked. This procedure was uneventful, and urinary catheter positioning was successful. A urinalysis was submitted and was positive for crystals and negative for bacteria and casts. An abdominal radiograph of the bladder revealed 2-3 mildly radiolucent stones in the bladder. Ultrasound of the bladder confirmed the stones (see image below).

A cystotomy was performed and the stones were removed and submitted to the Minnesota Urolith Center. Merlin recovered well from surgery and began a diet of Hill’s c/d. 

A few weeks later, the results of the stone submission confirmed Merlin had ammonium urate stones. Because of this result, bile acids were run, and the results were as followed:

Bile Acids Fasting (Enzyme cycling): 8.6H [Range: 0.5-7.9] umol/L

Bile Acid 2 Hour (Enzyme cycling): 88.5 H [Range: 1.9-11.3] umol/L

Two weeks later, Merlin was sedated for a CT with angiogram and bloodwork. His bloodwork results revealed:

Urea Nitrogen: 7 L [19-36] mg/dL
Total Bili 0.1 [0.1-0.3] mg/dL
Direct Bili 0.0 [0.0-0.1] mg/dL
Indirect Bili 0.1 [0.1-0.3] mg/dL
ALP 104 H [13-48] U/L
ALT 71 [25-76] U/L
AST 22 [14-36] U/L

His CT report revealed a “single extrahepatic splenophrenic portosystemic shunt”.

Merlin never had any neurological signs (ataxia, seizures, dysphoria) and only ever had a history of chronic diarrhea. Due to the blood’s diversion of the liver, Merlin accumulated ammonium in his body which promoted the stone formation in his bladder.

Merlin had a good prognosis with surgery, and that option was pursued. An ameroid constrictor was placed around the shunt, and he did well during surgery and post-operatively. He was discharged the day after. Unfortunately, Merlin developed post-attenuation neurological signs (PANS) a few hours after returning home. He was disoriented, ataxic, and experiencing central blindness. Despite proactive treatment, Merlin continued to decline and developed cluster seizures. He was humanely euthanized.

I will miss him dearly.