Preclinical Characterization of Cell-Based IL12 Immunotherapy Against Murine Acute Lymphoblastic Leukemia Using Intravital Imaging

Acute lymphoblastic leukemia (ALL) is a disease of the bone marrow (BM) microenvironment, in which ALL-stroma interactions, signaling molecules, and low oxygen tension all contribute to leukemia development and maintenance. The cytokine interleukin 12 (IL12) is a key primer of the initial specific T-cell immune response for resisting cancer progression, however its potential as an anti-leukemia immunotherapeutic agent has been largely under-examined. One potential reason for this oversight is the inability of current experimental systems to recapitulate the complex BM microenvironmental system ex vivo or obtain the resolution necessary in vivo to study the precise mechanism of effect for such proposed immunotherapies. Recently, our lab has developed a novel femur window chamber (FWC) intravital imaging mouse model capable of long-term spatiotemporal assessment of leukemias within the in vivo femoral BM at cellular resolution. We used this technique to characterize a new cell-based IL12 immunotherapy, in which patients are injected with their own blast cells engineered to express IL12. This approach has shown significant potential in preliminary preclinical trials, although its mechanism of effect is still unknown. Our study sought to establish the precise interaction between the modified IL12-expressing blast cells with the BM ALL population and immune system to determine a) how long post-ALL engraftment IL12 therapy continue to be effective, b) the optimal treatment administration route, and c) whether repeated infusions will yield added benefit.

The murine lymphoblastic cell line, 70Z/3, was transfected with either a constitutively active GFP marker (70Z/3-GFP) or a lentivirus expressing mCherry and IL12 (70Z/3-IL12-mCherry). The FWC was installed by shaving the femoral corticalis to reveal the BM cavity and securing a cover glass on top. Mice were then intravenously (IV) injected with 3x10⁶ 70Z/3-GFP cells 2 weeks post-surgery. Fluorescent cellular activity within the BM was monitored two times a week for four weeks via confocal microscopy (Zeiss LSM710). 3x10⁶ 70Z/3-IL12-mCherry cells were then injected into mice IV, intraperitoneally (IP), or intrafemorally (IF) at each experimental time point following initial inoculation. Leukemic burden was quantified as the percent total GFP signal within the BM space while immune cell activity was monitored using injected fluorescently-conjugated antibodies and ex vivo flow cytometry. Nonfluorescent leukemic and healthy mice were also imaged as controls.

Intravital imaging revealed that IV injected 70Z/3-GFP cells first enter the BM, on average, 10 days post-inoculation and achieve 90% infiltration within 3 weeks. Based on this timeline, we evaluated the effect of IL12 treatment administered 1, 2, or 3 weeks post-inoculation, as well as at the time of inoculation itself (co-injection). Mice treated with a single administration of 70Z/3-IL12-mCherry cells at 1 week showed a 26% reduction (p<0.05) in overall leukemia burden at the experimental endpoint, while mice treated at 3 weeks did not have a statistically significantly reduction in burden compared to untreated controls. We also observed a significant decline in BM immune cell populations, particularly macrophages, associated with increasing ALL percentage. We hypothesize that IL12 therapy is only effective below a specific threshold of disease, which corresponds to a minimum effective immune population still remaining in the BM. Current efforts are now underway to characterize this threshold and test the ability of repeated infusions to maintain immune cells within this effective range for the entirety of the treatment course. Interestingly, 70Z/3-IL12-mCherry cells co-injected with 70Z/3-GFP cells prevented ALL establishment in the BM altogether. Additionally, while IP injection has been the sole treatment administration technique used in previous IL12 studies, our results indicate that IV administration is the more effective option; not only do IV injected cells reach the BM faster and with greater quantity as compared to all other techniques tested, but it is also the more clinically relevant modality.

Our study not only demonstrates the ability of cell-based IL12 therapy to elicit a therapeutic response in ALL-bearing mice, it also reveals important insights into the biology of the immunotherapy, which can be applied to further augment the treatment's effect.