In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Summary

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

Abstract

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

Introduction

Monoclonal antibodies (mAb) are large glycoproteins (approximately 150 kDa) of the immunoglobulin superfamily that are secreted by B cells and have a primary function in the immune system to identify and either inhibit the biological function of, or mark for destruction, bacterial or viral pathogens, and can recognize abnormal protein expression on cancer cells¹. Antibodies can have an extremely high affinity to their specific epitopes down to femtomolar concentrations making them highly promising tools in biomedicine². With the development of hybridoma technology by Milstein and Köhler (awarded the Nobel Prize in 1984), the production of mAbs became possible³. Later, human mAbs were generated using the phage display technology or transgenic mouse strains and revolutionized their use as novel research tools and therapeutics^4,5.

Cancer is a worldwide health issue and a major cause of death creating the need for novel approaches for prevention, detection, and therapy⁶. To date, mAbs have allowed extrication of the role of genes and their proteins in tumorigenesis and when directed against cancer biomarkers, can enable tumor detection and characterization for patient stratification. For cancer therapy, bispecific mAbs, antibody-drug conjugates, and smaller antibody fragments are being developed as therapeutics, and for the targeted drug delivery to enhance therapeutic efficacy⁷. Additionally, antibodies serve for the biomarker targeting of contrast agents for molecular imaging modalities such as fluorescence-guided surgery, photoacoustic (PA) imaging, ultrasound (US) molecular imaging, and clinically used positron emission tomography (PET) or single photon emission computed tomography (SPECT)⁸. Finally, antibodies can also be used as theranostic agents enabling stratification of patients and response monitoring for targeted therapies⁹. Therefore, novel mAbs are beginning to play a critical role in cancer detection, diagnosis, and treatment.

Despite critical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be rendered ineffective due to the complexity of the tumor environment. Antibody interactions are dependent on the type of epitope, i.e., whether it is linear or conformational¹⁰. In addition to the recognition of antigens, antibodies need to overcome natural barriers such as vessel walls, basal membranes, and the tumor stroma to reach target cells expressing the antigen. Antibodies interact with the tissue not only through the variable fragment antigen binding (Fab) domain but also through the constant crystalline fragment (Fc) which further leads to off-site interactions¹¹. Targeting is also complicated by the heterogeneous expression of tumor markers throughout the tumor bulk and heterogeneity in tumor vascularization and the lymphatics system^12,13. In addition, the tumor microenvironment is composed of cancer-associated fibroblasts which support tumor cells, tumor immune cells that suppress anti-tumor immune reactions, and the tumor endothelium which supports the transport of oxygen and nutrients, all of which interfere with the penetration, distribution, and availability of antibody-based therapeutics or diagnostics. Overall, these considerations can limit therapeutic or diagnostic efficacy, reduce treatment response, and may result in tumor resistance.

Therefore, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate within the tumor microenvironment. Currently, in preclinical studies, marker expression in tumor research models is analyzed ex vivo by immunofluorescence (IF) staining of tumor sections¹⁴. Standard IF staining is performed with primary marker-specific antibodies which are then highlighted by secondary fluorescently labeled antibodies on ex vivo tumor tissue slices that have been isolated from the animal. This technique highlights the static location of the marker at the time of tissue fixation and does not provide insight into how the antibody-based therapeutics or diagnostics might distribute or interact in physiological conditions. Molecular imaging by PET, SPECT, US, and PA can provide information about the antibody-conjugated contrast agent distribution in living preclinical models^8,15. As these imaging modalities are non-invasive, longitudinal studies can be performed and time-sensitive data can be collected with a minimal number of animals per group. However, these non-invasive molecular imaging approaches are not sensitive enough and do not have enough resolution for the localization of antibody distribution at the cellular level. Additionally, the physical and biological characteristics of the primary antibody may be drastically changed by the conjugation of a contrast agent¹⁶.

In order to take the in vivo physiological and pathological conditions into consideration of how antibody-based therapeutics and diagnostics interact within the tumor environment and to obtain high-resolution cellular and even sub-cellular distribution profiles of non-conjugated antibodies, we propose an IF approach, deemed In Vivo Immunofluorescence Localization (IVIL), in which the antigen-specific antibody is intravenously injected in vivo. The antibody-based therapeutic or diagnostic, acting as a primary antibody, circulates in functional blood vessels and binds to its target protein in the highly accurate, living tumor environment. After isolation of in vivo-labeled tumors with the primary antibody, a secondary antibody is used to localize accumulated and retained antibody conjugates. This approach is similar to a previously described IF histology approach injecting fluorescently labeled antibodies¹⁷. Though here, the use of non-conjugated antibodies avoids a potential change in biodistribution characteristics induced by antibody modification. Furthermore, ex vivo application of fluorescent secondary antibody avoids a possible loss of fluorescence signal during tissue collection and processing and provides amplification of fluorescence signal intensity. Our labeling approach reflects in vivo biodistribution of antibody-based drugs and targeted agents and can provide important insights for the development of novel diagnostic and therapeutic agents.

Here, we describe two applications of the IVIL method as applied in previous studies investigating the biodistribution and accessibility of antibody-based contrast agents for molecular imaging approaches for breast cancer detection. First, the biodistribution of an antibody-near infrared dye conjugate (anti-B7-H3 antibody bound to the near infrared fluorescence dye, indocyanine green, B7-H3-ICG) and the isotype control agent (Iso-ICG) for fluorescence and photoacoustic molecular imaging is explored¹⁸. This application's method is described in the protocol. Next, the biodistribution results of a conformationally sensitive antibody to netrin-1, typically not detectable with traditional IF imaging, used with ultrasound molecular imaging, is quantified and presented in the representative results¹⁹. At the conclusion of this protocol paper, readers should feel comfortable adopting the IVIL method for their own antibody-based research applications.

Protocol

All methods described here have been approved by the Institutional Administrative Panel on Laboratory Animal Care (APLAC) of Stanford University.

1. Transgenic mouse model of breast cancer development

1. Observe mice from the desired cancer model for the appropriate tumor growth via palpation or caliper measurement before proceeding.
   NOTE: Here, the transgenic murine model of breast cancer development (FVB/N-Tg(MMTV-PyMT)634Mul/J) (MMTV-PyMT) was used. These animals spontaneously develop invasive breast carcinomas between 6 and 12 weeks of age in each mammary gland²⁰. Normal mammary glands were used as controls from the transgene-negative, age-matched littermates.

2. Intravenous injection of specific and nonspecific antibody agents

1. Purify rabbit anti-mouse B7-H3 and rabbit IgG isotype control antibodies on a desalting column (e.g., PD-10) to remove preservatives and storage buffers following manufacturer instructions.
   NOTE: Some antibodies may need further purification with Protein-A agarose beads based protocol²¹.
2. Aliquot dosages of 33 µg of each antibody conjugate in individual microcentrifuge tubes.
   NOTE: Dosage of the administered agent may vary depending on the application and matches the dosages at which the antibody conjugate is routinely used. The concentration of the antibody solutions is required if the volume is more than 100 µL for animal safety.
3. At the desired time point before the tissue collection, here 96 h, anesthetize the tumor bearing animal with 2% isoflurane flowing in oxygen at 2 L/min, and place on a 37 °C heated stage. Do a toe pinch to make sure the proper level of anesthsia was reached prior to the procedure.
4. To prepare for the tail vein inoculation of the antibody solutions, disinfect the tail of the animal by wiping three times with an alcohol wipe. Dilate the tail veins by warming with a heat pad for approximately 30 s. Avoid heating the entire animal. Wipe the tail once more with an alcohol wipe after removing the heat pad.
5. Using a 27G tail vein catheter, insert the butterfly needle into one of the two lateral tail veins and carefully fix the tail with the inserted needle to the stage with a piece of surgical tape.
   NOTE: Visible blood backflow into the catheter indicates the proper location of the needle within the tail vein.
6. Flush the catheter with 25 µL of sterile phosphate buffered saline (PBS), then inject the antibody solution into the catheter using insulin syringes. Flush the catheter once again with 25 µL of sterile PBS.
7. Remove the needle from the tail and apply pressure to stop any bleeding.
8. Turn off anesthesia and observe the animal until fully awake for any signs of distress.

3. Collection and preparation of target tumor tissues

1. At the desired time point, humanely euthanize the animal according to the acceptable institutional procedure, here, by gradual inhalation of 100% CO₂ from a compressed gas tank with a chamber displacement flow rate of 10-30% volume/min.
   NOTE: Some applications of the IVIL technique require euthanasia by cardiac perfusion with PBS to remove freely circulating antibody^19,22.
2. After confirmation of humane euthanasia via cessation of respiratory and cardiac movement, lack of toe pinch response, and greying of mucous membranes, excise tumor tissues using surgical scissors and forceps as follows:
   1. Lay the mouse in the supine position and grasping only the outer layer of skin between the set of mammary glands closest to the tail (5^th) with forceps, make a small incision with a pair of surgical scissors.
   2. Introduce the closed scissors into the cut and slowly open the tip to carefully separate the skin from the underlying abdominal wall membrane keeping it intact.
   3. Make a vertical incision up the abdomen, continuing to separate the skin from the inner membrane. Between the 3^rd and 4^th mammary glands, make a horizontal cut across the abdomen to allow retraction of the skin and visualization of the mammary glands.
      NOTE: Mammary tumors and glands are located superficially under the skin.
   4. Grasping each tumor or normal gland with forceps, carefully trim away the attached skin using surgical scissors.
3. Place excised tissues into tissue disposable base molds, prelabeled and filled with optimal cutting temperature (OCT) embedding medium and freeze the molds quickly by placement on dry ice. In order to study off-target delivery, excise other tissues or organs of interest (e.g., the liver or lungs).
   NOTE: To pause the protocol at this point, store frozen tissue blocks at -80 °C until ready to proceed.
4. Using a cryostat, section frozen tissue blocks at 10 µm thickness and place adjacent sections onto prelabeled adhesion glass slides.
   NOTE: To pause the protocol at this point, store slides at -80 °C until ready to proceed.

4. Ex vivo staining protocol

NOTE: For the quantitative comparison between fluorescence microscopy images, all slides are stained at the same time with the same prepared solutions.

1. Rinse frozen tissue slides with room temperature PBS for 5 min to remove OCT.
2. Demarcate tissue sections with a hydrophobic barrier pen to reduce the volume of solutions needed during staining.
   NOTE: Be careful not to allow the pen to run over the tissue samples as it may either remove the tissue from the slide or prevent proper staining on the affected tissue portion. Do not allow the tissue sections to dehydrate at any point.
3. Fix the tissue sections with 4% paraformaldehyde solution for 5 min.
4. Rinse slides in PBS for 5 min.
5. Permeabilize tissue sections with 0.5% Triton-X 100 in PBS for 15 min.
6. Rinse slides in PBS for 5 min.
7. Block the tissues with 3% w/v bovine serum albumin (BSA) and 5% v/v goat serum, both in PBS (blocking solution) for 1 h at room temperature.
   NOTE: Match the blocking solution serum to the secondary antibody host animal.
8. Rinse slides in PBS for 5 min.
9. Incubate sections with record-keeping primary antibodies as desired, possibly a common nuclear (e.g., DAPI), vascular (e.g., CD31), or cytoplasmic marker (e.g., actin). Here, rat anti-mouse CD31 (vascular marker) at a 1:100 dilution was used according to manufacturer's instructions in the blocking solution overnight at 4 °C protected from dehydration on a slide tray.
   NOTE: Do not add additional primary antibody or antibody conjugate. The conjugate that was injected and allowed to accumulate in the tissues in vivo act as the primary antibody.
10. Rinse slides in PBS for 5 min three times, changing the PBS each time.
11. Incubate slides with secondary antibodies to label primary antibodies. For this application, visualize anti-B7-H3 antibody using AlexaFluor-546 conjugated goat anti-rabbit antibody (1:200 dilution, optimized according to manufacturer's instructions) and CD31 with AlexaFluor-488 goat anti-rat secondary antibody (1:200 dilution, optimized according to manufacturer's instructions) in blocking solution, protected from light and dehydration on a slide tray, for 1 h at room temperature.
    NOTE: Secondary antibodies are from the same host animal but match the affinity of the secondary antibodies to the host species of the respective primary antibody. Slides are protected from light from this point onwards.
12. Rinse slides in PBS for 5 min three times, changing the PBS each time.
13. Apply one drop of the mounting medium into the center of the tissue slice and carefully place a coverslip avoiding entrapment of air bubbles.
14. Seal the edges of the coverslip with clear nail polish and allow to dry.
    NOTE: To pause the protocol for up to a week at this point, store slides at -20 °C until ready to proceed.

5. Confocal microscopy imaging and quantitative image analysis

NOTE: Preparing the confocal microscope and imaging parameters will depend on the confocal system used. The microscope used here was purchased commercially (e.g., Zeiss LSM 510 Meta system) and the associated acquisition software was used (e.g., Zen 2009). However, many of these steps will apply to any confocal microscope and assume basic confocal microscopy knowledge.

1. After the system has been turned on and warmed up, select the desired objective; here a 20x (numerical aperture = 0.8) objective was used.
2. Load a positive control slide, coverslip down, to allow for setting the optimization for the brightest signal. Imaging in the red channel, focus the system on the sample in Live imaging mode.
3. For each laser channel used, optimize the laser intensity, master gain, and pinhole size as follows:
   1. Switch to Continuous mode for imaging.
   2. Optimize the Laser Intensity (which controls the laser power) and Gain (Master) (which controls the voltage for photomultiplier tube) slide bars while monitoring the Look Up Table (LUT) histogram. Adjust these two settings until the dynamic range of the histogram is filled without saturating pixels.
      NOTE: If the laser intensity is too high photobleaching will occur. If the Gain (Master) is too high, the image will become noisy. Ideally, the Gain (Master) will be in the middle of its range.
   3. Set the pinhole to 1 airy unit (AU), which gives the highest resolution and the thinnest z-slice.
   4. Slide the Digital Offset bar to minimize the noise floor on the LUT histogram for a true black background.
      NOTE: Once microscopy settings are optimized for each laser channel and objective, keep them constant throughout the imaging session and for imaging of all slides, to allow for the quantitative comparison between slides.
4. Under the Acquisition Mode tab, under Averaging, select the desired Number and Bit Depth. Click the Optimal button to set the optimal pixel size.
5. Use the Snap acquisition button to collect a high-quality image. Here, random fields of view were selected from within the tumor, but other areas of interest may apply for different applications (vessels, tumor margins, penetration depth, etc.)
6. Save image files in the format used by the confocal software (here, ".lsm") for offline processing and quantification.
   NOTE: If not all slides are imaged during the same session, save the settings and reload them on subsequent visits, though reimaging the same slide is not recommended due to photobleaching.
7. Perform the quantitative fluorescence intensity measurements. Open Fiji (Fiji Is Just ImageJ software)^19,23 and load an .lsm image file by dragging onto the status bar.
8. Split the color channel data. Go to Image > Stacks > Split Channels.
9. Preprocess the fluorescence images as needed, i.e., subtract the background signal (Process > Subtract Background) or reduce the noise via a filtering method.
10. Segment the color channel corresponding to the reference protein (vascular, nuclear, cellular stain) by setting a threshold on the signal intensity (Image > Adjust > Threshold).
    NOTE: Manual thresholding introduces subjectivity into the image analysis, therefore, using an automated thresholding algorithm or referencing image histograms makes image analysis results less biased.
11. Use this threshold to create a binary mask (Process > Binary > Convert to Mask).
12. Measure and label ROIs within the mask (Analyze > Analyze Particles > Check Add to Manager > OK).
13. Apply ROIs to the color channel corresponding to the antibody of interest (Click channel image, Analyze > Tools > ROI Manager > Measure). This will apply the mask ROIs to the antibody image and provide image measurements for label sections in the Results window. Save results window (File > Save).
14. Calculate the desired statistic of interest, such as the mean fluorescence intensity as described here²⁴.
15. Perform all processing steps identically to each image within a set of slides. Create a macro to automatically do this for large image batches²³.
16. For image display only after quantitative measurements, apply qualitative image adjustments to optimize visualization of biodistribution patterns by adjusting minimum, maximum, brightness, and contrast to the same levels on all slides (Image > Adjust > Brightness/Contrast).
17. Convert the image type (Image > Type > RGB Color) and save files in a lossless image type such as .tiff (File > Save As > Tiff…) for use in presentations and publications.

Results

The IVIL method was used here to examine the in vivo biodistribution and tissue interaction of B7-H3-ICG and Iso-ICG, by allowing the agents, after intravenous injection into a living animal, to interact with the target tissue for 96 h, and then once the tissues are harvested, to act as the primary antibodies during ex vivo immunostaining. The IVIL method was also compared to the standard ex vivo IF staining of the tissues for the B7-H3 marker. Normal murine mammary glands do not express ...

Discussion

This method has several critical steps and requires potential modifications to ensure successful implementation. First, the dosage and timing of the antibody/antibody conjugate intravenous injection must be tailored to the specific application. Generally, dosages should be used that are consistent with how the antibody conjugate will typically be used, i.e., matching dosages of the therapeutic antibody or antibody-based contrast agent. Also, the timing of the collection of target tissues should be carefully considered. A...

Materials

Name	Company	Catalog Number	Comments
Animal Model
FVB/N-Tg(MMTV-PyMT)634Mul/J	The Jackson Laboratory	002374	Females, 4-6 weeks of age
Animal Handling Supplies
27G Catheter	VisualSonics	Please call to order	Vevo MicroMarker Tail Vein Access Cannulation Kit
Alcohol Wipes	Fisher Scientific	22-246073
Gauze Sponges (4" x 4" 16 Ply)	Cardinal Health	2913
Heat Lamp	Morganville Scientific	HL0100
Isoflurane	Henry Schein Animal Health	29404
Ophthalmic Ointment	Fisher Scientific	NC0490117
Surgical Tape	3M	1530-1
Tissue Collection
Disposable Base Molds	Fisher Scientific	22-363-556
Optimal Cutting Temperature (OCT) Medium	Fisher Scientific	23-730-571
Surgical London Forceps	Fine Science Tools	11080-02
Surgical Scissors	Fine Science Tools	14084-08
Antibodies
AlexaFluor-488 goat anti-rat IgG	Life Technologies	A-11006
AlexaFluor-546 goat anti-rabbit IgG	Life Technologies	A-11010
AlexaFluor-594 goat anti-human IgG	Life Technologies	A11014
Human IgG Isotype Control	Novus Biologicals	NBP1-97043
Humanized anti-netrin-1 antibody	Netris Pharma		contact@netrispharma.com
Rabbit anti-Mouse CD276 (B7-H3)	Abcam	ab134161	EPNCIR122 Clone
Rat anti-Mouse CD31	BD Biosciences	550274	MEC 13.3 Clone
Reagents
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A2153-50G
Clear Nail Polish			Any local drug store
Indocyanine Green - NHS	Intrace Medical	ICG-NHS ester
Mounting Medium	ThermoFisher Scientific	TA-006-FM
Normal Goat Serum	Fisher Scientific	ICN19135680
Paraformaldehyde (PFA)	Fisher Scientific	AAJ19943K2
Sterile Phosphate Buffered Saline (PBS)	ThermoFisher Scientific	14190250
Triton-X 100	Sigma-Aldrich	T8787
Supplies
Adhesion Glass Slides	VWR	48311-703
Desalting Columns	Fisher Scientific	45-000-148
Glass Cover Slips	Fisher Scientific	12-544G
Hydrophobic Barrier Pen	Ted Pella	22311
Microcentrifuge Tubes	Fisher Scientific	05-402-25
Slide Staining Tray	VWR	87000-136
Software
FIJI	LOCI, UW-Madison.	Version 4.0	https://fiji.sc/