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Revealing lymphoma growth and the efficacy of immune cell therapies using in vivo bioluminescence imaging

Matthias Edinger, Yu-An Cao, Michael R. Verneris, Michael H. Bachmann, Christopher H. Contag and Robert S. Negrin

Article Figures & Data

Figures

  • Fig. 1.

    In vivo imaging of BCL1 lymphoma.

    (A) BCL1 lymphoma cells were transduced with the pGC-gfp/luc fusion construct, sorted for GFP, and injected intravenously into syngeneic BALB/c mice. GFP+cells were passaged 4 times through BALB/c animals and finally through SCID mice to avoid lymphocyte contamination at time of cryoconservation. The final BCL1-gfp/luccells are CD19+ and show a homogeneous GFP expression. (B) 7 × 103 BCL1-gfp/luccells were injected intravenously into syngeneic BALB/c mice. Three hours after injection (day 0) a bioluminescent signal (ventral and lateral views) was detectable from the lungs (first 2 images), and after 4 to 5 days tumor engraftment in liver and spleen could be visualized (second 2 images). Tumor growth leading to massive hepato-splenomegaly could be followed over the next 5 weeks (third set of images), and an infiltration of lungs and lymph nodes was observed in final disease stages (5 weeks; final image). The scale to the left of the images describes the color map for the photon count. (C) Quantification of tumor growth by measuring light emission from BALB/c mice after intravenous injection of 2 × 103BCL1-gfp/luc cells (●, n = 5) compared with a disease-free control animal (■).

  • Fig. 2.

    Visualization of leukemic bone marrow infiltration by BLI.

    (A) Leukemic infiltration of the BM of sublethally irradiated BALB/c mice as revealed by whole body BLI one week after intravenous injection of 2 × 104 A20-luc/yfp cells into sublethally irradiated BALB/c mice (4 Gy). The scale to the left of the images describes the color map for the photon count. (B) Tumor expansion as quantified by measuring the photon counts of individual animals (n = 4) emitted at the time points indicated. (C) FACS analysis of BM cells from the femur 21 days after tumor inoculation revealed that 61% of the BM cells were CD19+, YFP+ tumor cells.

  • Fig. 3.

    Correlation of bioluminescent signal intensity with tumor burden.

    (A) Linear correlation (r = 0.989) between bioluminescent signal intensity determined in vivo and tumor burden as determined by FACS analysis of isolated cells from the liver and spleen of tumor-bearing animals (n = 15) 1 to 4 weeks after intravenous injection of 2 × 103 BCL1-gfp/luccells. (B) FACS plots represent analyses of the hematopoietic cell fraction (CD45+) of liver and spleen from an animal with high (▪ in panel A, top row) and one with low tumor load (▴ in panel A, lower row). Numbers represent the percentage of cells within quadrants.

  • Fig. 4.

    Tumor regression in response to chemotherapy and irradiation therapy.

    BALB/c mice were engrafted with 2 × 103BCL1-gfp/luc lymphoma cells, and tumor growth was quantified by BLI 14 days after tumor inoculation. Groups of animals received either (A) no further treatment (n = 3), (B) 2 × 40 mg/kg cyclophosphamide intraperitoneally within 24 hours (n = 5), or (C) total body irradiation with 8 Gy followed 24 hours later by a syngeneic bone marrow transplantation (n = 4). Tumor growth, regression, and relapse were determined by BLI 2 days and 4 days after treatment and in weekly intervals thereafter.

  • Fig. 5.

    Inhibition of tumor relapse by CIK cells after allogeneic bone marrow transplantation.

    BALB/c mice (H-2d) received 2 × 103BCL1-gfp/luc lymphoma cells intravenously, and tumor engraftment was monitored and quantified by BLI on day 7 as described in “Materials and methods.” Following BLI, animals were lethally irradiated (8 Gy) and underwent transplantation within 24 hours with 5 × 106whole BM cells from C57BL/6 animals (H-2b). Treatment groups received either (A) BM alone (n = 11), (B) BM + 2.5 × 106 splenocytes (n = 9), or (C) BM + 2.5 × 106 CIK cells (n = 13). Tumor regression following irradiation was determined 2 or 3 days after irradiation, and animals were monitored by BLI for tumor relapse in weekly intervals thereafter (A-C). On the x-axes are days after tumor cell injection. The y-axes are relative light emission from individual animals over a 5-minute integration time. (D) Survival of animals from treatment groups A (■), B (▵), and C (○); on the x-axis are days after tumor cell injection. On the y-axis is the proportion of recipients surviving. (P < .0001 between all treatment groups.) (E) Tumor distribution pattern of one representative animal prior to irradiation (day 7), 2 days after irradiation (day 9), and 9 days after irradiation (day 16).

  • Fig. 6.

    Trafficking of CIK cells.

    BALB/c splenocytes were retrovirally transduced with the pGC-gfp/luc vector, FACS sorted, and expanded under CIK culture conditions as described in “Material and methods.” Transduced CIK cells were injected intravenously into syngeneic animals bearing a macroscopically visible A20-lymphoma subcutaneously (n = 6 in 2 separate experiments). Shown in the figure is the repetitive imaging of one representative animal. Day 0: early localization of CIK cells to the lungs; day 1: distribution to other sites, including liver and spleen; day 3: preferential infiltration of subcutaneous tumor site (shaved area, lower right quadrant, and shaved control area in the middle of the back of the animal); day 12: regression of tumor and minimal signal from remaining CIK cells. For days 3 and 12, tangential photographs (grayscale) are shown for a better tumor localization and the bioluminescent overlay (bioluminescence) for CIK cell localization.