Bioluminescent Imaging Tracks Responses

ORLANDO—Bioluminescent imaging may provide a noninvasive method to monitor the effect of new biologic and immunomodulatory treatments for cancer, Matthias G. Edinger, MD, said at the 43rd Annual Meeting of the American Society of Hematology (abstract 1817). Dr. Edinger is in the Division of Bone Marrow Transplantation at Stanford University School of Medicine.

In this study, bioluminescent imaging of tumor responses and effector cells showed that adoptive transfer of expanded CD8+ NKT cells can improve long-term survival and reduce graft-vs-host disease (GVHD) in an animal model of lymphoma treated with allogeneic bone marrow transplantation (BMT). The new method also showed that cytotoxic cells trafficked to the tumor site and remained there until the tumor was eradicated.

Michael A. Caligiuri, MD, who introduced the presentation, said that new approaches to cancer therapy are taking advantage of T-cell characteristics, specifically "the receptors they use to see what is going on in the body." Dr. Caligiuri is director for clinical research at Ohio State University’s Comprehensive Cancer Center, Columbus.

In allogeneic BMT, the donor T cell "sees" different major histocompatibility complex (MHC) antigens on the tumor cell surface and launches a graft-vs-leukemia effect that eliminates some tumors left behind even by high-dose chemotherapy. The downside is that this also increases the risk of GVHD.

Natural killer (NK) cells, unlike T cells, "see" the absence of MHC class I molecules. NKT cells are midway between the two. Dr. Caligiuri said that they detect MHC components, as T cells do, but also can detect the absence of MHC, as NK cells do.

The Animal Models

"Since light is transmitted through mammalian tissues at low levels, cells expressing the bioluminescent reporter gene luciferase (Luc) can be detected within living mice using low light imaging CCD [charged couple device] cameras," Dr. Edinger said. Luc uses the substrate luceriferin, a small, water-soluble molecule that rapidly penetrates cells and tissues after IV or IM injection. "Using this technique," he said, "we established animal models of leukemia and lymphoma that allow us to localize and quantify tumor cells noninvasively in vivo."

The animal models were made by retrovirally transducing tumor cell lines with a dual optical reporter gene containing Luc together with green or yellow fluorescent protein as a fusion construct. The bioluminescent Luc allowed the detection and quantification of the cells in vivo, and the fluorescent reporter genes were used to re-isolate tumor cells from the organs using flow cytometry.

"You can follow tumor growth within an individual animal," he said. "Only living cells contribute to the signal, so it is possible to follow both response to therapy and the development of relapse."

Luc-expressing cells could be detected in internal organs such as spleen, liver, lung, and lymph nodes, and even within the bone marrow of living mice. Signal intensity correlated with tumor load. The high sensitivity of the method allowed evaluation of tumor cell trafficking and expansion even in minimal residual disease stages.

The investigators used the new approach to assess the therapeutic application of a cytotoxic cell population known as cytokine-induced killer (CIK) cells. These cells were generated from splenocytes by stimulation with interferon-gamma, followed by anti-CD3-antibody and IL-2 stimulation and expansion. About half of the resulting cells are "CD8+ NKT" cells that are CD3+, CD8+, and co-express the NK marker NK 1.1. The antitumor activity of these CD8+ NKT cells was tested in syngeneic tumor models as well as after allogeneic BMT.

Dr. Edinger said that all animals treated only with allogeneic BMT died of relapsed BCL-1 lymphoma, as shown by large increases in bioluminescent signal intensity. Mice treated only with allogeneic splenocytes died even sooner.

Mice treated with CIK cells after allogeneic BMT showed inhibition of lymphoma regrowth and a reduced incidence of GVHD, compared with those receiving allogeneic splenocytes.

CIK cells expressing Luc were also injected into syngeneic animals bearing subcutaneous lymphomas. Within 3 days, Dr. Edinger found that the CIK cells had "profoundly infiltrated the subcutaneous tumor mass." They remained detectable for 9 days, during which time the tumor disappeared. Five of the six animals treated remain tumor free.

"We conclude that this imaging technology allows for noninvasive examination of complex biological processes in real time with high sensitivity," Dr. Edinger said.