WMIC 2015: Exhibitor Poster Award 3

Selective cell elimination with near infrared photoimmunotherapy in 2D and 3D mixed cultures and in a mixed tumor model

Kazuhide Sato, National Cancer Institute

Innovation/ Impact/ Objectives: Cell cultures and tissues often contain cellular subpopulations that potentially interfere with or contaminate other cells of interest. However, it is difficult to eliminate unwanted cells without damaging the very cell population one is seeking to protect, especially in established tissue [1]. Near infrared photoimmunotherapy (NIR-PIT) is a new, highly selective cancer treatment that combines the specificity of intravenously injected antibodies that target tumors with the toxicity induced by photosensitizers activated by NIR-light [2]. Here, we present a method of eliminating specific subpopulations of cells from a mixed 2D or 3D cell culture or a tumor model with NIR-PIT. Using the optical reporters, RFP, GFP and luciferase, it could be demonstrated that the selected cell population could be eliminated by NIR-PIT without damaging adjacent non target cells.

Materials/ Methods: In vitro and in vivo experiments were conducted with an EGFR, luciferase and GFP expressing cell line (A431-luc-GFP) as a target, and EGFR negative, RFP expressing cell line (Balb/3T3-RFP) as background. An antibody-photosensitizer conjugate (APC) consisting of panitumumab and a phthalocyanine dye, IRDye-700DX, was synthesized (pan-IR700). NIR-PIT cytotoxicity for 2D and 3D A431-luc-GFP cell cultures was confirmed with dead staining, luciferase activity, and GFP fluorescence intensity. In a mouse model, the effect of NIR-PIT was confirmed with bioluminescence image (BLI), and fluorescence imaging (FLI). Specific cell elimination from almost confluent in vitro 2D mixed culture (A431-luc-GFP and 3T3-RFP) and in vitro mixed 3D (spheroids) were evaluated with luciferase activity and GFP/RFP fluorescence intensity. Specific cell elimination from in vivo mixed tumor was also assessed with BLI and FLI.

Result: In vitro NIR-PIT cytotoxicity for A431-luc-GFP cells followed a light-dose dependence. Repeated NIR-PIT eradicated whole cells within 3D spheroids and within in vivo tumors, which was confirmed with BLI and FLI. Elimination of A431-luc-GFP from an almost-confluent 2D mixed cell culture was demonstrated after NIR-PIT. Repeated NIR-PIT resulted in a decrease in size due to complete target cell elimination from the mixed 3D cell culture without damaging non-target cells. Repeated NIR-PIT also led to complete elimination of target-expressing cells from mixed tumors with minimal damage to non-target cells. All these data were demonstrated with BLI and FLI, and quantified by luciferase activity and fluorescence intensity. BLI and FLI of ex vivo tumor confirmed the results.

Conclusions: NIR-PIT is a practical method for eliminating a selective set of cells from cell culture or tissue without damaging the remaining cells. Local cell-specific elimination by NIR-PIT has the potential for applications in many fields, for instance, regenerative medicine, immunomodulation, and tumor immunity.

Ben-David, et al. Nat. Commun. 4, 1992 (2013).
Mitsunaga, M. et al. Nat. Med. 17, 1685–1691 (2011)

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