Combined Radiation and Anti-CTLA4 Induces a CD8+ Immune Response in an Orthotopic Mouse Model of Breast Cancer Which is Detectable with CD8 ImmunoPET at an Early Timepoint after Treatment

Abstract

<h3>Purpose/Objective(s)</h3> The purpose of this study is to characterize the CD8+ T cell response to radiation treatment (RT) combined with anti-CTLA4 in an orthotopic mouse model of breast cancer and to determine whether CD8 immuno-position emission tomography (immunoPET) can be used to image this response. Combinatorial therapy is an active area of investigation for potentiating CD8+ T cell responses and improving tumor response rates. CD8 immunoPET is being explored in early clinical trials but the optimal timing for imaging after RT + immunotherapy is unknown, and the feasibility of CD8 immunoPET after RT in a preclinical orthotopic model has not been demonstrated. <h3>Materials/Methods</h3> 4T1 tumors were implanted in a mammary fat pad of female BALB/c mice and treated conformally with 8Gy x3 followed by anti-CTLA4 (200ug x3). Tumors were harvested prior, during and after treatment at multiple timepoints for analysis of CD8 staining by immunohistochemistry (IHC). CD8+ cell density was quantified using Visiopharm software; comparisons were made by one-way ANOVA of log transformed data, with p values adjusted for multiple comparisons. For imaging studies, a Zr-89 radiolabeled CD8-specific cys-diabody was injected the final day of treatment and static images were acquired after 24 hours with a PET/CT scanner. Activity (% injected dose/gram, %ID/g) was confirmed with ex vivo biodistribution studies; unpaired t test was used to compare tumor activity from treated vs untreated mice. In an exploratory analysis, linear regression was performed for 7 tumor samples with paired IHC and imaging data. <h3>Results</h3> Tumors treated with RT + anti-CTLA4 demonstrated increased CD8+ cell density by IHC 24 hours post-treatment compared to untreated tumors (mean cell count/mm² 1106.0 vs 57.3, p<0.001). At this timepoint, tumors and tissues enriched for CD8+ cells (including lymph nodes, thymus, and spleen) were visualized with CD8 immunoPET in all mice. %ID/g was increased for treated tumors, n = 3, compared to untreated tumors, n = 4 (mean %ID/g 3.97 vs 2.15, p=0.039), consistent with greater CD8+ T cell infiltration into treated tumors. Repeat imaging with a second cohort of mice confirmed a statistically significant increase in %ID/g for treated tumors. Linear regression of paired IHC/imaging samples showed a trend toward correlation of higher CD8+ density with higher %ID/g (r² = 0.53). <h3>Conclusion</h3> RT + anti-CTLA4 induces a CD8+ immune response 24 hours after completion of treatment in an orthotopic mouse model of breast cancer which is detectable by CD8 immunoPET, correlating with increased CD8+ cell infiltration by IHC. This model will facilitate further studies that are underway to characterize longitudinal immune responses after RT. These preclinical data could inform use of CD8 immunoPET as an early biomarker after RT in future clinical studies.