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
- Incubate mAb (1 mg, 6.8 nmol) with IR700 NHS ester (66.8 mg, 34.2 nmol, 5 mmol/L in DMSO) in 0.1 mol/L Na2HPO4 (pH 8.6) at 15-25 °C for 1 h.
- Purify the mixture using a column (e.g., Sephadex). Prepare and wash the column with PBS. Then, apply the mixture onto the column and collect the drop, which contains the purified IR700-conjugated antibody. This IR700-conjugated antibody is referred to as the antibody photosensitizer conjugate (APC).
- Measure the protein and IR700 concentration in the APC.
- Prepare calibration curves for protein and IR700 using a spectrophotometer.
- Mix standard concentrations of albumin with a protein assay kit following the kit protocol (see Table of Materials, Coomassie Brilliant Blue (CBB) protein staining). Measure the absorbance of albumin at 595 nm wavelength, and plot the calibration curve (a linear approximation formula) for the protein using the following equation: y = ax + b (x: concentration, y: absorbance).
- Obtain calibration curves for IR700 with absorption at 690 nm using the same procedure. The standard concentration of IR700 is recommended at 0.1-5 µM (0.1954-9.77 µg/mL).
- Measure the protein concentration and IR700 concentration in the APC using a calibration curve [x = (y-b)/a (x: concentration, y: absorbance)].
- Determine the number of IR700 dyes bound per mAb with the results of the molar concentration.
NOTE: It is important to determine the optimal conjugation number of IR700 molecules per mAb molecule. Generally, approximately three IR700 molecules bound on a single mAb molecule would be effective both in vitro and in vivo. Many IR700 bound per antibody (e.g., six) makes it easier to be trapped in the liver during in vivo experiments. The ratio of antibody bound to IR700 was in the range of 1:2-1:4. The proportion of IR700 was reduced, if necessary.
- Perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a confirmation for the formation of an APC. Image the gel at 700 nm using a fluorescent imager, and stain the protein in the gel using a protein staining kit following the kit protocol (see Table of Materials, CBB protein staining).
2. Generation of a pleural dissemination model
- Prepare luciferase-expressing target cells and suspend 1.0 × 106 target cells in 100 µL of phosphate-buffered saline (PBS)
NOTE: Intrathoracic cancer cells such as lung cancer and MPM are suitable as target cells. Luciferase-expressing cells were prepared via luciferase gene transfection, and high expression of luciferase was confirmed after > 10 cell passages. Cells were cultured in a medium supplemented with 10% fetal bovine serum and penicillin (100 IU/mL) and streptomycin (100 mg/mL). The number of cells was adjusted according to the tumor growth rate and the time course of treatment (1.0. × 105-6.0 × 106 cells/body weight).
- Prepare 8-12-week-old female homozygote athymic nude mice, with a preferable body weight of 19-21 g.
- Anesthetize mice during the procedure with isoflurane (introduction: 4-5%, maintenance 2-3%); press the tail with tweezers to confirm that there is no reaction.
- Make a stopper with polystyrene foam and attach the stopper to the 30 G needle so that the tip remains at 5 mm to prevent lung injury. Bend the needle tip by pressing it against a hard object to avoid pneumothorax (Figure 1).
CAUTION: Be careful not to pierce oneself. Use forceps to bend the needle. Do not hold the stopper when attaching it to the needle. It is safer to stick the stopper before filling the cells into the syringe.
- Fill a syringe (1 mL) with target cells, and attach a 30G needle with a stopper.
- Pierce a needle into the chest of the mouse through the intercostal space. Owing to the resistance while hitting against the ribs at that time, the needle tip moved up and down. After passing through the intercostal space, press the syringe against the mouse and inject 100 µL of target cells (Figure 2).
NOTE: The mouse breaths deeply when the needle properly enters the chest cavity. With the bending of the needle tip, pneumothorax and inappropriate injection of cells into the lung could be avoided.
- Roll the mouse 2-3 times to spread the cells throughout the thoracic cavity.
- Return the mouse to the cage. After the procedure, the mouse will wake up from anesthesia and behave normally.
3. Measurement of bioluminescence
NOTE: The software used for data acquisition is listed in the Table of Materials.
- To confirm the generation of the pleural dissemination model, evaluate the bioluminescence images every day after injecting the cells into the thoracic cavity.
- Anesthetize mice (step 2.3) and inject intraperitoneally with D-luciferin (15 mg/mL, 200 µL).
- Ten minutes after the injection, set the mouse in the bioluminescence imaging (BLI) measuring equipment. For image acquisition, open the Acquisition Control Panel of the software. Select Luminescent, Photograph, and Overlay (Figure 3).
- Set exposure time as Auto. Set Binning as small.
- Set f/stop as 1 for luminescent and 8 for photograph; f/stop controls the amount of light received by the charged-coupled device detector.
- Set the Field of View as C.
- Once the mouse sample is ready for imaging, click Acquire for imaging acquisition. Mice with sufficient luciferase activity were selected for further studies.
NOTE: A suitable pleural dissemination model shows strong luminescence on the diffused site in the chest when viewed from the ventral side. If the BLI images are not diffused in the thorax, and only at the injection site, the tumor may be transplanted subcutaneously.
- After displaying the image, set the display format to Radiance. Open the Tool Palette panel (Figure 4A).
- Select ROI Tools. We recommend using the Circle to range the bioluminescent area on images.
- Click Measure ROIs to measure the surface bioluminescent intensity (Figure 4B).
- Use Configure Measurement on the left corner of the ROI measurement panel to select the values/information needed. Export this data table as a .csv file (Figure 4C).
- Use the values of Total Flux (p/s) as the bioluminescent intensity quantification in the .csv file.
4. Diffuse luminescence imaging tomography (DLIT)
NOTE: The software used for data acquisition is listed in the Table of Materials.
- Turn on the X-ray Armed button.
- Anesthetize the mice (step 2.3) and then inject D-luciferin (15 mg/mL, 200 µL) intraperitoneally into the mice. To shoot DLIT continuously from 3.2 to 3.7, skip this step.
- Ten minutes after injection, set the mouse in the BLI equipment.
- Open the Acquisition Control Panel of the software. Select Luminescent, Photograph, CT, Standard-One Mouse, and Overlay. Other settings were the same as in 3.4-3.6 (Figure 5A).
- Select the Imaging Wizard on the Acquisition Control Panel.
- Select Bioluminescence and then DLIT (Figure 5B).
- Select Firefly as the wavelength to measure (Figure 5C).
- 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 (Figure 5D).
- Push the X-rays will be produced when energized. Acquire.
- Open the CT sequential image data.
- Open Surface Topography on the Tool Palette. Select Show (Figure 6A).
- Adjust the threshold as the purple display shows only the body surface (Figure 6B). Then, select the Subject Nude Mouse and click the Generate Surface. Make sure that the outline of the mouse is accurately drawn (Figure 6C).
- Open the Tool Palette, DLIT 3D Reconstruction Properties tab. Select Tissue Properties as Mouse Tissue and Source Spectrum as Firefly (Figure 6D). Next, open the Analyze tab and confirm the data for each selected wavelength data. Finally, click the Reconstruct button (Figure 6E).
- Confirm the presence of BLI in the chest cavity in the configured DLIT image.
5. NIR-PIT for in vivo pleural dissemination model
- Measure the light dose of 690 nm wavelength (NIR) laser with a power meter, and adjust the output to 100 mW/cm2.
NOTE: The laser light is coherent with a precise coil size; thus, the light energy hardly changes regardless of the distance within 50 cm. If there are many adverse events, such as burns, reduce the output within the range of 40 mW/cm2.
- Intravenously inject APC (100 µg) via the tail vein 24 h before NIR irradiation.
NOTE: Adjust the volume of APC to 50-200 µL for injection.
- Anesthetize the mice (step 2.3), and lay them on their back. To avoid NIR irradiation to the non-target site, shield other sites with aluminum foil (Figure 7A). Irradiate with NIR light with a laser of 100 J/cm2; if the tumor is disseminated back to the belly, the NIR-light irradiation dose could be divided in multiple directions (Figure 7B).
NOTE: Adjust the dose at 30-150 J/cm2 depending on the in vitro results and adverse events such as burns.
- When the NIR irradiation is complete and the mouse is awake, return it to the cage.
- Observe the BLI and measure the ROI over time (every day) (See 3.2-3.12).
- For ex vivo imaging, euthanize mice with carbon dioxide 24 h after the APC injection, immediately before the NIR irradiation.
- To observe the inside of the chest of the mouse, remove the thorax and cut the ribs and sternum. Capture the fluorescence image (700 nm) alongside the control without APC administration. Then, apply D-luciferin (150 µg/mL) over the exposed thorax, and the BLI was taken (refer 3.2-3.7).
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 declare that they have no competing financial interests.
|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|
|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|
|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|
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