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.
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.
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 destroys primary tumors with a combination of photosensitizers and light rays, and tumor-specific antigens released from the tumor cells enhance tumor immunity. Tumors can be selectively treated using photosensitizers as they are specific and selective for the target cells. The modality of phototherapy includes photodynamic therapy (PDT), photothermal therapy (PTT), and photochemistry-based therapies1.
Near-infrared photoimmunotherapy (NIR-PIT) is a recently developed method of antitumor phototherapy that combines photochemical-based therapy and immunotherapy1,2. NIR-PIT is a molecularly targeted therapy that targets specific cell surface molecules through the conjugation of a near-infrared silicon phthalocyanine dye, IRdye 700DX (IR700), to a monoclonal antibody (mAb). The cell membrane of the target cell is destroyed upon irradiation with NIR light (690 nm)3.
The concept of using targeted light therapy by combining conventional photosensitizers and antibodies or targeted PDT is over three decades old4,5. Previous studies have attempted to target conventional PDT agents by conjugating them to antibodies. However, there was limited success because these conjugates were trapped in the liver, owing to the hydrophobicity of the photosensitizers6,7. Moreover, the mechanism of NIR-PIT is completely different from that of conventional PDT. Conventional photosensitizers generate oxidative stress that results from an energy conversion that absorbs light energy, dislocates to an excited state, transitions to the ground state, and causes apoptosis. However, NIR-PIT causes rapid necrosis by directly destroying the cell membrane by aggregating photosensitizers on the membrane through a photochemical reaction8. NIR-PIT is superior to conventional targeted PDT in many ways. Conventional photosensitizers have low extinction coefficients, requiring the attachment of large numbers of photosensitizers to a single antibody molecule, potentially reducing binding affinity. Most conventional photosensitizers are hydrophobic, making it difficult to bind the photosensitizers to antibodies without compromising their immunoreactivity or in vivo target accumulation. Conventional photosensitizers typically absorb light in the visible range, reducing tissue penetration.
Several studies on NIR-PIT targeting intrathoracic tumors such as lung cancer and malignant pleural mesothelioma (MPM) cells have been reported9,10,11,12,13,14,15,16,17. However, only a few reports have described the efficacy of NIR-PIT in pleural disseminated MPM or lung cancer models9,10,11,12. Subcutaneous tumor xenograft models are thought to be standard tumor models and are currently widely used to evaluate the antitumor effects of new therapies18. However, the subcutaneous tumor microenvironment is not permissive for the development of an appropriate tissue structure or a condition that properly recapitulates a true malignant phenotype19,20,21,22. Ideally, orthotopic disease models should be established for a more precise evaluation of the antitumor effects.
Here, we demonstrate a method of efficacy evaluation in a mouse model of pleural disseminated lung cancer, which was treated using NIR-PIT. A pleural dissemination mouse model is generated by injecting tumor cells into the thoracic cavity and confirmed using luciferase luminescence. The mouse was treated with an intravenous injection of mAb conjugated with IR700 and NIR irradiation to the chest. The therapeutic effect was evaluated using luciferase luminescence.
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
2. Generation of a pleural dissemination model
3. Measurement of bioluminescence
NOTE: The software used for data acquisition is listed in the Table of Materials.
4. Diffuse luminescence imaging tomography (DLIT)
NOTE: The software used for data acquisition is listed in the Table of Materials.
5. NIR-PIT for in vivo pleural dissemination model
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 of tumor cells into the mice was indicated as day 1.
At day 4, BLI and DLIT were performed after D-luciferin (15 mg/mL, 200 µL) was injected intraperitoneally, and mice with sufficient luciferase activity in the chest cavity were selected for further studies (Figure 9). Hundred micrograms of NZ-1-IR700 (100 µL) was intravenously injected via the tail vein. The control group was injected with PBS (100 µL).
At day 5, two mice were sacrificed using carbon dioxide asphyxiation for ex vivo. The NZ-1-IR700 injected mouse showed both high IR700 fluorescence and luciferase activities in thoracic tumors, indicating that intravenously injected NZ-1-IR700 reached the disseminated pleural tumor sites (Figure 10).
For the evaluation of the effect of NIR-PIT in the pleural disseminated mouse model, the NIR light was applied at 15 J/cm2 from two directions (total of 30 J/cm2) at 40 mW/cm2 transcutaneously on day 5 (the NIR light was irradiated externally), followed by serial BLI. The control group was not irradiated with NIR light.
After treatment of mice with NIR-PIT, the treated group showed decreased luciferase activity. However, the relative light unit in the control group showed a gradual increase (*p < 0.05 versus control, t-test) (Figure 11).
Figure 1: Easy hand-made device for cell transplantation. Attach the stopper made with polystyrene foam to the 30G needle so that the tip remains at 5 mm. The tip of the needle should be bent to avoid pneumothorax. Please click here to view a larger version of this figure.
Figure 2: Injection of target cells into the thoracic cavity. Turn the mouse sideways and pierce the needle into the mouse toward the lung. Since the stopper and needle tip are bent, the needle enters the thoracic cavity without sticking to the lungs. Inject target cells while pressing the needle against the mouse. Please click here to view a larger version of this figure.
Figure 3: Acquisition Control Panel. Select Luminescent, Photograph, and Overlay. Set Exposure Time as Auto, Binning as Small, f/stop as 1 for luminescent and 8 for photograph, and Field of View as C. Once the mouse sample is ready for imaging, click Acquire for imaging acquisition. Please click here to view a larger version of this figure.
Figure 4: Measurement (BLI). (A) Tool Palette panel. Select ROI Tools. We recommend the Circle to range the bioluminescent area on images. (B) BLI quantification. After selecting the ROI in each image, click Measure ROIs to analyze. (C) Quantification information. Use Configure Measurement on the left corner of the ROI measurements panel to select the values/information needed. Export this data table as .csv file. Please click here to view a larger version of this figure.
Figure 5: Acquisition of DLIT. (A) Acquisition Control Panel for DLIT. Select Luminescent, Photograph, CT, Standard-One Mouse, and Overlay. Other settings are the same as 3.4-3.6 (Figure 3). (B) Imaging Wizard panel. Select Bioluminescence, and DLIT. (C) Select measurement wavelength. Select the wavelength as firefly. (D) Set the Imaging Subject as Mouse, Exposure Parameters as Auto Settings, Field of View as C-13.4 cm, and Subject Height as 1.5 cm. Then Click the X-rays will be produced when energized. Acquire. Please click here to view a larger version of this figure.
Figure 6: Reconstruction of DLIT. (A) Tool Palette panel. Open Surface Topography on the Tool Palette. Select Show. (B) Adjusting mouse surface recognition. Adjust the Threshold as the purple display shows only the body surface. Select the subject Nude Mouse, then click the Generate Surface. Make sure that the outline of the mouse is accurately drawn. (C) Tool Palette. Open the DLIT 3D reconstruction Properties tab, select Tissue Properties as Mouse Tissue and Source Spectrum as Firefly. (D) Open the Analyze tab and select the data for each wavelength data. (E) Click the Reconstruct button. Please click here to view a larger version of this figure.
Figure 7: NIR irradiation. (A) Shield its belly with aluminum foil to prevent NIR irradiation to belly. (B) Irradiate NIR light using laser where BLI is strong; in some cases, NIR laser is divided in multiple directions. Please click here to view a larger version of this figure.
Figure 8: SDS-PAGE. Successful confirmation of conjugated NZ-1-IR700 on an SDS-PAGE gel (left, colloidal blue staining; right, fluorescence at 700 nm channel). Diluted NZ-1 served as the control. Please click here to view a larger version of this figure.
Figure 9: DLIT. Confirmation of the luciferase-expressing tumor cells in the thoracic cavity. Please click here to view a larger version of this figure.
Figure 10: Ex vivo imaging. Characterization of the pleural disseminated MPM model 24 h after NZ-1-IR700 injection with BLI. Please click here to view a larger version of this figure.
Figure 11: Antitumor effect of NIR-PIT on pleural disseminated model. (A) The podoplanin-targeted NIR-PIT regimen is shown in a line. Podoplanin-targeted NIR-PIT with NZ-1-IR700 on pleural disseminated model with H2373-luc tumors. BLI of the pleural disseminated model is shown. (B) While luciferase activities measured with BLI did not increase in the NIR-PIT group, the control group showed a gradual increase along with tumor growth. (n ≥ 3 in both groups, t-test). Please click here to view a larger version of this figure.
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 damaged23,24,25. With this type of selective cell killing, NIR-PIT was demonstrated to be safe in disseminated models9,26. However, alternative methods are possible in some steps. Various methods have been reported for the pleural dissemination model27,28,29,30. We selected the injection model because it is a simple procedure that is least burdensome to mice. We used BLI to measure the therapeutic effect of NIR-PIT because we can evaluate quantitatively with live mice. For example, positron emission tomography/computed tomography (PET/CT)27 could be an alternative way to evaluate the tumor volume of the pleural dissemination model. NIR-PIT with BLI in other orthotopic models has been reported; NIR-PIT can be used for the abdominal dissemination model, lung multiple metastatic tumor model, and brain tumor12,26,31,32,33,34,35,36. Even small metastatic tumor foci can be observed with BLI, and NIR-PIT can be performed11,12.
We described some parts of the protocol based on preliminary experiments. First, the time from APC administration to NIR irradiation. The pharmacokinetics were evaluated in advance using a subcutaneous tumor model. APC peaks in the tumor 24 h after intervention via the tail vein using mAb; NIR irradiation can be performed 24 h after the APC administration9,10,11,12. Second, the NIR light dose required for killing tumor cells in NIR-PIT differs depending on the antibody and target cell lines, which is predicted using the in vitro results.
This study has a few limitations. First, tumors were widely distributed in the thoracic cavity, and NIR-irradiated energy could not be measured precisely. The wavelength of the NIR excitation light (peak at 690 nm) allows penetration of at least 2-3 inches into the tissue37. Therefore, in the case of mice, NIR light reaches the thoracic cavity even externally. Currently, NIR laser devices are used for the mouse pleural dissemination model9,10,11,12. However, in actual clinical use, we intend to use precise fiber optics to irradiate the entire intrathoracic cavity via the thoracic drainage tube34. Second, the NIR-irradiation dose is limited depending on the specificity of the mAb to antigens expressed on tumor cells. Non-specific binding of APC could cause unexpected organ damage.
In conclusion, we presented a method to evaluate the therapeutic effect of NIR-PIT with BLI in the pleural dissemination model of MPM.
The authors have nothing to disclose.
None
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 |