We thank Dr. Andrew Olson (Stanford Neuroscience Microscopy Service) for discussions and equipment use. We thank Dr. Juergen K. Willmann for his mentorship. This study was supported by NIH R21EB022214 grant (KEW), NIH R25CA118681 training grant (KEW), and NIH K99EB023279 (KEW). The Stanford Neuroscience Microscopy Service was supported by NIH NS069375.

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
<ol>
	<li>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 spontaneous...

<ol>
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D., Winter, G., Chiswell, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+antibodies:+filamentous+phage+displaying+antibody+variable+domains.">Phage antibodies: filamentous phage displaying antibody variable domains.</a> Nature. 348 (6301), 552-554 (1990).</li><li>Ferlay, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+incidence+and+mortality+worldwide:+sources,+methods+and+major+patterns+in+GLOBOCAN+2012.">Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012.</a> International Journal of Cancer. 136 (5), E359-E386 (2015).</li><li>Reichert, J. M., Valge-Archer, V. 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G., Suria, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodistribution+Mechanisms+of+Therapeutic+Monoclonal+Antibodies+in+Health+and+Disease.">Biodistribution Mechanisms of Therapeutic Monoclonal Antibodies in Health and Disease.</a> The AAPS Journal. 12 (1), 33-43 (2009).</li><li>Duraiyan, J., Govindarajan, R., Kaliyappan, K., Palanisamy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+immunohistochemistry.">Applications of immunohistochemistry.</a> Journal of Pharmacy &amp; Bioallied Sciences. 4 (Suppl 2), S307-S309 (2012).</li><li>Gambhir, S. 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The authors have nothing to disclose.

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...

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 K&#246;hler (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 therapeutics4,5.
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 system12,13. 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 models8,15. 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&#160;fluorescence and photoacoustic molecular imaging is explored18. This application&#39;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.

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			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Humanized anti-netrin-1 antibody&#160;</td>
			<td style="width:196px;">Netris Pharma</td>
			<td style="width:166px;"></td>
			<td>contact@netrispharma.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Rabbit anti-Mouse CD276 (B7-H3)</td>
			<td style="width:196px;">Abcam</td>
			<td style="width:166px;">ab134161</td>
			<td>EPNCIR122 Clone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Rat anti-Mouse CD31</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:166px;">550274</td>
			<td>MEC 13.3 Clone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Reagents</td>
			<td style="width:196px;"></td>
			<td style="width:166px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:166px;">A2153-50G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Clear Nail Polish</td>
			<td style="width:196px;"></td>
			<td style="width:166px;"></td>
			<td>Any local drug store</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Indocyanine Green - NHS</td>
			<td style="width:196px;">Intrace Medical</td>
			<td style="width:166px;">ICG-NHS ester</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Mounting Medium</td>
			<td style="width:196px;">ThermoFisher Scientific</td>
			<td style="width:166px;">TA-006-FM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Normal Goat Serum</td>
			<td style="width:196px;">Fisher Scientific</td>
			<td style="width:166px;">ICN19135680</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Paraformaldehyde (PFA)</td>
			<td style="width:196px;">Fisher Scientific</td>
			<td style="width:166px;">AAJ19943K2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Sterile Phosphate Buffered Saline (PBS)</td>
			<td style="width:196px;">ThermoFisher Scientific</td>
			<td style="width:166px;">14190250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Triton-X 100</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:166px;">T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Supplies</td>
			<td style="width:196px;"></td>
			<td style="width:166px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Adhesion Glass Slides</td>
			<td style="width:196px;">VWR</td>
			<td style="width:166px;">48311-703</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Desalting Columns</td>
			<td style="width:196px;">Fisher Scientific</td>
			<td style="width:166px;">45-000-148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Glass Cover Slips</td>
			<td style="width:196px;">Fisher Scientific</td>
			<td style="width:166px;">12-544G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Hydrophobic Barrier Pen</td>
			<td style="width:196px;">Ted Pella</td>
			<td style="width:166px;">22311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Microcentrifuge Tubes</td>
			<td style="width:196px;">Fisher Scientific</td>
			<td style="width:166px;">05-402-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Slide Staining Tray</td>
			<td style="width:196px;">VWR</td>
			<td style="width:166px;">87000-136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Software</td>
			<td style="width:196px;"></td>
			<td style="width:166px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">FIJI</td>
			<td style="width:196px;">LOCI, UW-Madison.</td>
			<td style="width:166px;">Version 4.0</td>
			<td>https://fiji.sc/</td>
		</tr>
	</tbody>
</table>

in vivo immunofluorescence localization for assessment of therapeutic and diagnostic antibody biodistribution in cancer research

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&#160;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.

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 ...

Watch this Scientific Journal Video about In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research at JoVE.com

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

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.

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

This protocol for in vivo immunofluorescence localization, or IVIL, assesses the in vivo biodistribution of diagnostic and therapeutic antibodies or antibody-based agents for cancer research, detection, and treatment. In contrast to conventional immunofluorescent staining where cellular expression of antigens is revealed ex vivo, the IVIL method elucidates how antibodies and antibody-based agents distribute in the living animal. This video will aid understanding of the overall workflow of the IVIL method.It shows important details for successful reproduction of the protocol for other applications and antibodies. Observe mice from the desired cancer model for the appropriate tumor growth via palpation or caliper measurement before proceeding. Purify rabbit anti-mouse B7-H3 and rabbit IgG isotype control antibodies on a desalting column to remove preservatives and storage buffers following the manufacturer instructions.Aliquot dosages of 33 micrograms of each antibody conjugate in individual microcentrifuge tubes. Following anesthetization as described in the text protocol, 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 seconds. Avoid heating the entire animal. Using a 27-gauge tail vein catheter, insert the butterfly needle into one of the two lateral tail veins.Visualize blood backflow into the catheter to ensure that the needle is properly placed so that the solutions will fully enter the animal versus being captured in the tail. Use a piece of surgical tape to carefully fix the tail with the inserted needle to the stage. Flush the catheter with 25 microliters of sterile phosphate-buffered saline.Then, inject the antibody solution into the catheter using insulin syringes. Flush the catheter once again with 25 microliters of sterile PBS. Remove the needle from the tail, and apply pressure to stop any bleeding.Perform humane euthanasia of the mouse as described in the text protocol, and lay the mouse in the supine position. Use surgical scissors and forceps to excise tumor tissues. Use forceps to grasp only the outer layer of skin between the set of mammary glands closest to the tail, and make a small incision with a pair of surgical scissors.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. Make a vertical incision up the abdomen, continuing to separate the skin from the inner membrane. Between the third and fourth mammary glands, make a horizontal cut across the abdomen to allow retraction of the skin and visualization of the mammary glands.Grasping each tumor or normal gland with forceps, carefully trim away the attached skin using surgical scissors. Place excised tissues into tissue disposable base molds that are prelabeled and filled with optimal cutting temperature embedding medium. Quickly freeze the molds by placement on dry ice.In order to study off-target delivery, excise other tissues or organs of interest. Using a cryostat, section frozen tissue blocks at 10-micron thickness, and place adjacent sections onto prelabeled adhesion glass slides. Rinse frozen tissue slides with room temperature PBS for five minutes to remove the embedding medium.Demarcate tissue sections with a hydrophobic barrier pen to reduce the volume of solutions needed during staining. Fix the tissue sections with 4%paraformaldehyde solution for five minutes. After rinsing the slides in PBS for five minutes, permeabilize tissue sections with 0.5%Triton X-100 in PBS for 15 minutes.Rinse the slides again in PBS for five minutes. Block the tissues with PBS containing 3%weight per volume bovine serum albumin and 5%volume per volume goat serum for one hour at room temperature. After rinsing the slides in PBS for five minutes, incubate the sections with record-keeping primary antibodies such as a common nuclear, vascular, or cytoplasmic marker.Here, rat anti-mouse CD31 is used at a one-to-100 dilution in the blocking solution. The slides with primary antibody are left overnight at four degrees Celsius, protected from dehydration on a slide tray. Following incubation, rinse the slides in PBS for five minutes three times.Change the PBS each time. Incubate the slides with secondary antibodies to label the primary antibodies. For this application, visualize anti-B7-H3 antibody using Alexa Fluor 546 conjugated goat anti-rabbit antibody, and visualize CD31 with Alexa Fluor 488 goat anti-rat secondary antibody in blocking solution.Protect the slides from light and dehydration on a slide tray for one hour at room temperature. After rinsing the slides in PBS as before, apply one drop of the mounting medium into the center of the tissue slice. Carefully place a coverslip, avoiding entrapment of air bubbles.Seal the edges of the coverslip with clear nail polish, and allow to dry. Proceed with confocal microscopy imaging and quantitative image analysis as described in the text protocol. Representative confocal micrographs show the comparison of specific B7-H3 antibody-ICG conjugate and nonspecific isotype control antibody-ICG conjugate localization in murine mammary glands containing normal or carcinoma tissues.Normal murine mammary glands from an animal intravenously injected with Iso-ICG or B7-H3-ICG and counterstained with CD31 show no staining of the antibody conjugates. Normal tissues were shown to have no expression of B7-H3 by standard ex vivo immunofluorescence staining. In invasive mammary tumors, B7-H3-ICG strongly binds to the vasculature, the first point of contact in vivo, and then is able to extravasate from the vasculature to heterogeneously stain the tumor epithelium.Iso-ICG shows nonspecific accumulation within the tumor tissues. Standard ex vivo immunofluorescent staining shows uniform expression of the B7-H3 marker on epithelial and endothelial cells. Representative confocal micrographs show the in vivo immunofluorescence localization method to detect netrin-1 expression or isotype control expression in murine carcinoma and normal mammary glands.In vivo immunofluorescence localization confirms epithelial signal for netrin-1 in MMTV-PyMT tumors but not in normal mammary glands. Furthermore, there is a strong netrin-1 signal on endothelial cells in breast tumors, as indicated by the colocalized yellow signal. The signal is significantly weaker in normal mammary glands.The IVIL method will need to be optimized in terms of antibody dosage, tissue collection timing, and secondary antibody dilution for successful implementation. IVIL allows targeting of conformational epitopes that cannot be detected using standard immunofluorescence staining, which requires tissue processing. Also, healthy organs of tumor-bearing mice can be collected to assess off-target delivery of therapeutic antibodies.With increased use of antibody-based therapeutics and contrast agents in cancer research and management, the IVIL method is highly applicable to pharmaceutical research and development. Researchers in cancer therapies, molecular imaging, inflammation, and other areas may find that the IVIL method provides useful information.

in-vivo-immunofluorescence-localization-for-assessment-therapeutic

Research

JoVE Journal

Cancer Research

8.7K 조회수.  INSERM U1052-CNRS UMR5286, Centre Léon Bérard. 생체 내 면역형광국화 또는 IVIL을 위한 이 프로토콜은 암 연구, 검출 및 치료를 위한 진단 및 치료 항체 또는 항체 기반 제제의 생체 내 생체 분포를 평가한다. 항원의 세포 발현이 밝혀진 종래의 면역형성 염색과는 대조적으로, IVIL 방법은 살아있는 동물에서 항체 및 항체 기반 제제가 어떻게 분배되는지 해명한다. 이 비디오는 IVIL 메서드의 전반적인 워크플로우를 이해하는 ?...