Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 immunotherapy^1,2. 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 old^4,5. 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 photosensitizers^6,7. 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 reported^{9,10,11,12,13,14,15,16,17}. However, only a few reports have described the efficacy of NIR-PIT in pleural disseminated MPM or lung cancer models^9,10,11,12. 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 phenotype^19,20,21,22. 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.

Protokół

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 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.
2. 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).
3. Measure the protein and IR700 concentration in the APC.
   1. Prepare calibration curves for protein and IR700 using a spectrophotometer.
   2. 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).
   3. 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).
   4. Measure the protein concentration and IR700 concentration in the APC using a calibration curve [x = (y-b)/a (x: concentration, y: absorbance)].
   5. 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.
4. 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

1. 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).
2. Prepare 8-12-week-old female homozygote athymic nude mice, with a preferable body weight of 19-21 g.
3. 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.
4. 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 with clean forceps or by pressing it against a hard object cleaned with 70% EtOH 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.
5. Fill a syringe (1 mL) with target cells, and attach a 30G needle with a stopper.
6. Disinfect the chest of the animal with 70% EtOH before the procedure.
7. 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.
   NOTE: Right chest wall puncture is recommended to avoid the risk of cardiac wall puncture.
8. Roll the mouse 2-3 times to spread the cells throughout the thoracic cavity.
9. Return the mouse to a clean and warm cage and monitor until ambulatory. 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.

1. To confirm the generation of the pleural dissemination model, evaluate the bioluminescence images every day after injecting the cells into the thoracic cavity.
2. Anesthetize mice (step 2.3) and inject intraperitoneally with D-luciferin (15 mg/mL, 200 µL).
3. Ensure that the mouse is breathing normally before and after imaging. Use a heater if necessary to prevent hypothermia during anesthesia. 
4. 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).
5. Set exposure time as Auto. Set Binning as small.
6. 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.
7. Set the Field of View as C.
8. 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.
9. After displaying the image, set the display format to Radiance. Open the Tool Palette panel (Figure 4A).
10. Select ROI Tools. We recommend using the Circle to range the bioluminescent area on images.
11. Click Measure ROIs to measure the surface bioluminescent intensity (Figure 4B).
12. 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).
13. 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.

1. Turn on the X-ray Armed button.
2. 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.
3. Ten minutes after injection, set the mouse in the BLI equipment.
4. 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).
5. Select the Imaging Wizard on the Acquisition Control Panel.
6. Select Bioluminescence and then DLIT (Figure 5B).
7. Select Firefly as the wavelength to measure (Figure 5C).
8. 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).
9. Push the X-rays will be produced when energized. Acquire.
10. Open the CT sequential image data.
11. Open Surface Topography on the Tool Palette. Select Show (Figure 6A).
12. 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).
13. 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).
14. Confirm the presence of BLI in the chest cavity in the configured DLIT image.

5. NIR-PIT for in vivo pleural dissemination model

1. 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.
2. Clean the tail with 70% EtOH and intravenously inject APC (100 µg) via the tail vein 24 h before NIR irradiation. Apply pressure to the injection site to control bleeding.
   NOTE: Adjust the volume of APC to 50-200 µL for injection.
3. 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.
4. When the NIR irradiation is complete and the mouse is awake, return it to the cage.
5. Observe the BLI and measure the ROI over time (every day) (See 3.2-3.12).
6. For ex vivo imaging, euthanize mice with carbon dioxide 24 h after the APC injection, immediately before the NIR irradiation.
   1. 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).

Wyniki

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 i...

Dyskusje

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<...

Materiały

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