In Vivo Imaging Techniques

Our lab leverages a number of newly developed in vivo imaging techniques to gain unique insight into the growth and spatial-temporal migration behavior of transplanted stem cells, effector cell populations, and tumors in living mice.

These novel imaging approaches to our GVHD, GVT, and immune reconstitution studies offer unique means to better understand the physiological processes involved for many of our studies.

In collaboration with a number of research groups here at Memorial Sloan-Kettering Cancer Center (e.g., Drs. Gelovani and Larson), we have developed techniques to employ bioluminescence imaging (BLI) and positron emission tomography (PET) to monitor hematopoietic progenitor cells and alloreactive T cells.

These techniques allow us to perform highly specific and sensitive tempo-spatial bioluminescence and PET imaging (alone or in combination with other imaging modalities such as CT) of genetically labeled bone marrow progenitor cells and other cell types of interest.

For example, by genetically labeling bone marrow cells with an SFG vector, containing a fusion reporter gene coding for HSV1-TK, enhanced green fluorescent protein (eGFP), and luciferase (Luc) under the control of a viral long terminal repeat (LTR) that constitutively drives the expression of the reporter gene, we are able to monitor the growth and migration of these cells in vivo.

[Enlarge Image] Figure 1 Spatial-temporal In vivo Imaging by BLI of Transplanted HPCs in a Lethally Irradiated Recipient.

Donor-derived whole bone marrow cells were retrovirally transduced with an efficiency of 30 percent. Transduced cells express constitutively the HSV1 TK-EGFP-Luc fusion protein. Following transduction, 5 x 10⁵ of the partially transduced whole bone marrow cells were injected into a lethally irradiated recipient mouse via the penile vein. The control animal received 1 x 10⁶ untransduced whole bone marrow cells. Over 4 weeks post transplantation, the whole body distribution of transduced cells was monitored using bioluminescence imaging.

At the indicated time points, mice received intravenous injections of 150 mg/kg D-luciferin. Subsequently, mice were imaged in supine followed by prone position for 5 min each. Color bar settings (Photons/sec/cm²/steradian) for each position are given first, followed by the images. Italics indicate a change in the color bar settings for a specific time point. In this study, a progressive reconstitution with labeled cells of especially the femurs, skull, and ribs and, to a lesser extent, spine can be seen.

[Enlarge Image] Figure 2 In vivo Analyses of Alloreactive T cells during GVHD.

Whole Ly5.1 congenic BM cells (1X10⁶ cells) were injected (0.250ml/mouse) into a lethally irradiated (1100 cGy split) recipient where the BM had been transduced with a SFG vector, containing a fusion reporter gene coding for HSV1-TK, enhanced green florescent protein (EGFP), and luciferase. These mice were tracked for in vivo luminescence, and subsequently were harvested 81 days later, and 10X10⁶ whole BM were injected intravenously (0.250 ml/mouse) into lethally irradiated C57BL/6 recipients.

Mice were imaged weekly, and at 8 weeks, spleens were harvested and 4X10⁶ donor derived CD4⁺ and CD8⁺ T cells were given in conjunction with 5 x 10⁶ TCD C57BL/6 BM into a lethally irradiated (1300 cGy split) CBA/J 10-week-old recipient.

Prior to imaging, mice were injected intravenously with 0.200 ml firefly luciferin at 150 mg/kg and anesthetized for approximately 2 minutes via isofluorane, prior to being imaged in the supine position. Mouse was imaged on days 0, 1, 2, 5, 6, 7, 8, 9, and 12. On day 13, the mouse died of acute GVHD.

Recently, our lab has transduced a series of tumor cell lines, including B16 melanoma, P815 and A20, with a transgene containing luciferase (Luc) under the control of the viral long terminal repeat (LTR). The viral LTR promotor drives the expression of the reporter gene.

Figure 3 below represents a series of images obtained from a recipient of genetically labeled P815 cells after tail vein injection. Bioluminescence images of the same animal were taken over a period of weeks after injection. Analysis of the total photon count measured on a given day allows a quantitative analysis of tumor growth.

[Enlarge Image] Figure 3 Spatial-temporal In vivo Imaging by BLI of P815 mastocytoma Cells.

B6D2F1 mice were lethally irradiated and subsequently received 5x10⁶ TCD B6 BM cells alone or TCD B6 BM and 1x10³ P815 TGL mastocytoma cells and 3x10⁶ B6 splenic CD8⁺ T cells. Whole body bioluminescent signal intensity was determined twice weekly and pseudocolor images overlayed on conventional photographs are shown.