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Abstract
Background Near-infrared photoimmunotherapy (NIR-PIT) is a novel cancer therapy based on a monoclonal antibody conjugated with a photosensitizer (IR700Dye) that is activated by near-infrared light. When exposed to light irradiation, IR700Dye undergoes photochemical ligand reactions, releasing hydrophilic side chains. This chemical transformation damages the cell membrane of targeted cancer cells, leading to necrotic cell death. Furthermore, NIR-PIT rapidly induces immunogenic cell death, thereby triggering the activation of the adaptive immune response through dead cell-associated antigens.
In this study, we examined the efficacy of NIR-PIT utilizing a small protein mimetic (Affibody, 6–7 kDa) in lieu of a monoclonal antibody for addressing brain metastasis of HER2-positive breast cancer. Due to its compact size, the Affibody demonstrates efficient penetration through the blood-brain barrier (BBB) when the BBB is partially disrupted. Our innovative NIR-PIT system has the potential to target brain metastasis and eliciting an immune response within the brain.
Methods In the in vitro study, we employed the HER2-positive breast cancer cell line (MDA-MB361), which is derived from brain metastases, alongside the HER2-negative cell line (MDA-MB231). Cell viability was assessed to gauge the impact of NIR-PIT. For the in vivo investigation, MDA-MB361 cells were injected into the brain of mice (BALB/cSlc-nu/nu) to establish a brain metastasis model. Two weeks after cell injection, the HER2 Affibody-IR700Dye conjugate was injected into the tail vein. Subsequently, the brain was monitored fluorescence intensity, and the brain tumors were irradiated with near-infrared light. The brain tissues were observed using fluorescence microscopy, hematoxylin and eosin (HE) staining, and immunohistochemical (IHC) analysis of microglial cells and HER2 protein.
Results MDA-MB361 cells exhibited stronger fluorescent signals of IR700Dye (figure 1). Upon measuring cell viability, only MDA-MB361 cells exposed to NIR-PIT with the HER2 Affibody-IR700Dye conjugate maintained low viability (figure 2).
The fluorescence intensity in the head experienced a significant surge immediately following the administration of the HER2 Affibody-IR700Dye conjugate. Within an hour after the injection, the HER2 Affibody-IR700Dye conjugate delineated the tumor mass. This observation aligned with HE staining of consecutive sections and IHC of HER2 protein (figure 3). Following NIR-PIT, HE staining revealed necrotic cell death within the brain tumor, with microglial cells localized inside the tumor mass (figure 4).
Conclusions The proposed approach holds the potential for facilitating effective NIR-PIT for brain metastases in breast cancer. Subsequently, we plan to utilize humanized mice that were established human immune system being to investigate immune responses within the brain following NIR-PIT.
Fluorescence images of the cells. Fluorescence image of the cells bound the HER2 Affibody-IR700Dye conjugate. MDA-MB361 cells exhibited stronger fluorescent signals of IR700Dye than MDA-MB231 cells. Scale bar: 50 µm
Cell viability (alamarBlue Assay). When cell viability was measured over an extended period of time of 5 days, only the HER2-overexpressing cells (MDA-MB361) exposed to NIR irradiation and HER2 Affibody–IR700Dye conjugate were were maintained statically low
Fluorescence images of the brain. An intense fluorescence signal was emitted from the site of MDA-MB361 tumor on the sliced brain images. The HER2 Affibody-IR700Dye conjugate clearly delineated tumor mass from the surrounding normal tissues which correlated well with H&E staining
IHC before and after NIR-PIT using the HER2 Affibody IR700Dye conjugate. Immunostaining of microglia revealed that without NIR-PIT treatment, microglia were concentrated around the tumor area, whereas 24 h after NIR-PIT treatment, microglia were observed mostly inside the tumor
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