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Intravital Imaging of Bright Cyan-Fluorescent AML Model Reveals Impact of CXCR4 Inhibitor on AML and Immune Cell Dynamics

INTRODUCTION: CXCR4 chemokine receptor inhibitors such as BL-8040 (BioLineRx) have been investigated by us and others as possible anti-leukemic drugs due to their ability to "mobilize" leukemia cells out of the BM and into the circulation, where they are more sensitive to chemotherapy. However, the exact mode of cell relocation remains unclear. CXCR4/CXCL12 signaling pathway also participates in BM homing of immune cells, including both central memory T cells and immunosuppressive CD4+FoxP3+ T-regulatory cells (T-reg). Therefore, CXCR4 inhibition has the potential to either counteract or enhance the process of AML immune surveillance. Therefore, we sought to develop a syngeneic AML model for intravital 2-photon microscopy (TPM) compatible with existing immune reporter mouse strains, which typically occupy the green, yellow and red fluorescence channels.
 HYPOTHESIS: CXCR4 inhibition decreases AML and T cell BM cellularity by increasing the rate of intravascular cell entry and/or decreasing the rate of circulating cell homing back to BM.
 MODEL: The cyan-colored fluorescent protein mTurquoise2 was lentivirally introduced into C57BL6-origin AML cells containing the MLL, ENL-FLT3, ITD, and p53-/- mutations, termed AML1-mTurq2. Syngeneic FoxP3-GFP/CD11c-YFP/hCD2-DsRed reporter mice were generated by inter-breeding of the corresponding strains, respectively highlighting T-reg, myeloid antigen presenting cells, and all T cells. After intravenous infusion of 1E5 AML1-mTurq2 cells, 1-2% blasts appeared in peripheral blood on day 9, increasing to 70% on day 15-20 when animals had to be euthanized.
 TREATMENT: Mice with >1% blasts were given BL-8040 I.P. in two daily 400 µg doses followed by imaging 24 h later, or intravenously during imaging 10 µg and 50 µg one hour later.
 ANALYSIS: Disease progression was characterized by blood flow cytometry, symptom scoring and thick-mount organ tissue fluorescence microscopy. Intravital TPM of the calvarial bone marrow (BM) was performed through intact bone under general anesthesia. By interline multiplexing dual femtosecond lasers with four-sensor detection for 8 distinct channels, mTurquoise2 and SHG were recorded by the same sensor at, respectively, 860 and 990 nm excitation, along with GFP, YFP, DsRed and dextran-TRITC (blood tracer). AML and T cell subsets were 3-D tracked using Imaris software.
 RESULTS: AML1-mTurq2 cells stably and uniformly expressed bright cyan fluorescence, suitable for intravital TPM with low incident laser powers and fast imaging rates in deep tissue locations. In C57BL6 mice, sparse AML cell clusters were found in BM perivascular spaces on day 1 after cell infusion. AML cells were slowly motile (~4 um/min) and highly proliferative, gradually filling BM spaces and emerging in other organs. T cells and CD11c dendritic cells were present in leukemic BM, and the vasculature appeared largely intact and well perfused. T cells interacted with AML cells and the stroma, migrating with high average velocities (~10 µm/min) and slowing down to ~3 µm/min in late-stage disease.
 After 2 days of BL-8040 treatment, disease symptom scores improved from 3 to 1 while the untreated controls progressed from 3 to 4 (range 0-6). TPM revealed a 4-fold reduction of AML cellularity in BM. Cellular velocities of both AML and T cells were unchanged by BL-8040 treatment. After acute drug administration, a fraction of stromal AML cells begun entering capillary vessel lumens by amoeboid movement. The intravasated AML cells adhered to vessel wall for 1-2 minutes before rapid detachment. Some cells remained tethered while already loose in the blood stream.
 CONCLUSIONS: A novel, brightly cyan-fluorescent syngeneic AML1-mTurq2 AML model is advantageous for 6-color intravital microscopy of cell trafficking and immune surveillance in optimal compatibility with green, yellow and red reporters of cell lineages and tissue architecture. Using this model, we show that CXCR4 inhibitor BL-8040 decreases AML BM cellularity by increasing the frequency of intravasation without increasing AML migratory velocity.
 
 
 Zal: Daiichi-Sankyo: Research Funding; NIH-CTEP: Research Funding; BioLineRx: Research Funding; VueBio.com: Equity Ownership; NIH/NCI: Research Funding; CPRIT: Research Funding; Moleculin Biotech, Inc.: Research Funding. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; Aptose: Equity Ownership; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy.
