Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination

Summary

Near-infrared photoimmunotherapy (NIR-PIT) is an emerging cancer therapeutic strategy that utilizes an antibody-photoabsorber (IR700Dye) conjugate and NIR light to destroy cancer cells. Here, we present a method to evaluate the antitumor effect of NIR-PIT in a mouse model of pleural disseminated lung cancer and malignant pleural mesothelioma using bioluminescence imaging.

Abstract

The efficacy of photoimmunotherapy can be evaluated more accurately with an orthotopic mouse model than with a subcutaneous one. A pleural dissemination model can be used for the evaluation of treatment methods for intrathoracic diseases such as lung cancer or malignant pleural mesothelioma.

Near-infrared photoimmunotherapy (NIR-PIT) is a recently developed cancer treatment strategy that combines the specificity of tumor-targeting antibodies with toxicity caused by a photoabsorber (IR700Dye) after exposure to NIR light. The efficacy of NIR-PIT has been reported using various antibodies; however, only a few reports have shown the therapeutic effect of this strategy in an orthotopic model. In the present study, we demonstrate an example of efficacy evaluation of the pleural disseminated lung cancer model, which was treated using NIR-PIT.

Introduction

Cancer remains one of the leading causes of mortality despite decades of research. One reason is that radiation therapy and chemotherapy are highly invasive techniques, which may limit their therapeutic benefits. Cellular- or molecular-targeted therapies, which are less invasive techniques, are receiving increased attention. Photoimmunotherapy is a treatment method that synergistically enhances the therapeutic effect by combining immunotherapy and phototherapy. Immunotherapy enhances tumor immunity by increasing the immunogenicity of the tumor microenvironment and reducing immunoregulatory suppression, resulting in the destruction of tumors in the body. Phototherapy d....

Protocol

All in vivo experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animal resources of Nagoya University Animal Care and Use Committee (approval #2017-29438, #2018-30096, #2019-31234, #2020-20104). Six-week-old homozygote athymic nude mice were purchased and maintained at the Animal Center of Nagoya University. When performing the procedure in mice, they were anesthetized with isoflurane (introduction: 4-5%, maintenance 2-3%); the paw was pressed with tweezers to confirm the depth of anesthesia.

1. Conjugation of IR700 with mAb

1. Incubate mAb (1 mg, 6.8 n....

Results

Anti-podoplanin antibody NZ-1 was conjugated with IR700 to generate NZ-1-IR700. We confirmed the binding of NZ-1 and IR700 on an SDS-PAGE (Figure 8). Luciferase-expressing H2373 (H2373-luc) was prepared by transfecting malignant mesothelioma cells (H2373) with a luciferase gene¹⁰.

We anesthetized 8-12-week-old female homozygote athymic nude mice and injected 1 × 10⁵ H2373-luc cells into the thoracic cavity. The day of injection o.......

Discussion

In this study, we demonstrated a method for measuring the therapeutic effect of NIR-PIT on the pleural dissemination model of MPM. Highly selective cell killing was performed with NIR-PIT; thus, the normal tissue was hardly damaged^23,24,25. With this type of selective cell killing, NIR-PIT was demonstrated to be safe in disseminated models^9,26<.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25w/v% Trypsin-1mmol/l EDTA 4Na Solution with Phenol Red	Wako	209-016941	for cell culture
1mL syringe	TERUMO	SS-01T	for mice experiment
30G needle	Nipro	1907613	for mice experiment
BALB/cSlc-nu/nu	Japan SLC
Collidal Blue Staining Kit	Invitrogen	LC6025	use for gel protein staining
Coomassie (bradford) Plus protein assay	Thermo Fisher Scientific Inc (Waltham, MA, USA)	PI-23200	for measuring the APC concentration
Dimethyl sulfoxide (DMSO)	Wako	043-07216	use for conjugation of IR700
D-Luciferin (potassium salt)	Cayman Chemical	14681	for bioluminescence imaging and DLIT
GraphPad Prism7	GraphPad software		for statistical analysis
Image Studio	Li-Cor Biosciences		for analyzing 700 nm fluorescent image
IRDye 700DX Ester Infrared Dye	LI-COR Bioscience (Lincoln, NE, USA)	929-70011
isoflurane	Wako	095-06573	for mice anesthesia
IVIS Spectrum CT	PerkinElmer		for capturing bioluminescent image and DLIT
Living Image	PerkinElmer		for analyzing bioluminescent image and DLIT
Na2HPO4	SIGMA-ALDRICH (St. Louis, MO, USA)	S9763	use for conjugation of IR700
NIR Laser	Changchun New Industries Optoelectronics Technology	MRL-III-690R	for NIR irradiation
Novex WedgeWell 4 to 20%, Tris-Glycine, 1.0 mm, Mini Protein Gel, 12 well	Invitrogen	XP04202BOX	use for SDS-PAGE
NuPAGE LDS Sample Buffer (x4)	Invitrogen	NP0007	use for SDS-PAGE
Optical power meter	Thorlabs (Newton, NJ, USA)	PM100	for measuring the output of the NIR laser
PBS(-)	Wako	166-23555
Pearl Trilogy imaging system	Li-Cor Biosciences		for capturing 700 nm fluorecent image
Penicilin-Streptomycin Solution (x100)	Wako	168-23191	for cell culture
Puromycin Dihydrochloride	ThermoFisher	A1113803	for luciferase transfection
RediFect Red-Fluc-Puromycin Lentiviral Prticles	PerkinElmer	CLS960002	for luciferase transfection
RPMI-1640 with L-glutamine and Phenol Red	Wako	189-02025	for cell culture
Sephadex G25 column (PD-10)	GE Healthcare (Piscataway, NJ, USA)	17-0851-01	use for conjugation of IR700
UV-1900i	Shimadzu		for measuring the APC concentration