Name: in vivo immunofluorescence localization for assessment of therapeutic and diagnostic antibody biodistribution in cancer research
Uploaded: 2019-09-16T12:45:00
Description: Watch this Scientific Journal Video about In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research at JoVE.com

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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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 height="21" style="height:21px;width:295px;">Animal Model</td>
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			<td height="21" style="height:22px;">FVB/N-Tg(MMTV-PyMT)634Mul/J</td>
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			<td>Vevo MicroMarker Tail Vein Access Cannulation Kit</td>
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			<td height="21" style="height:21px;width:295px;">Antibodies</td>
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			<td height="21" style="height:21px;">AlexaFluor-488 goat anti-rat IgG</td>
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			<td style="width:196px;">Life Technologies</td>
			<td style="width:166px;">A11014</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Human IgG Isotype Control</td>
			<td style="width:196px;">Novus Biologicals</td>
			<td style="width:166px;">NBP1-97043</td>
			<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

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates approaching 85% are common. Unfortunately, patients often become refractory to the treatment by altering their signal transduction pathways. An implementation of the expression profiling with microarrays can identify the overall mRNA-level changes, and proteomics can identify the overall changes in protein levels or can identify the proteins involved, but the activity of the signal transduction pathways can only be established by interrogating post-translational modifications of the proteins. As a result, the ability to identify whether a drug treatment is successful or whether resistance arose, or the ability to characterize any alterations in the signaling pathways, is an important clinical challenge. Here, we provide a detailed explanation of antibody arrays as a tool which can identify system-wide alterations in various post-translational modifications (e.g., phosphorylation). One of the advantages of using antibody arrays includes their accessibility (an array does not require either an expert in proteomics or costly equipment) and speed. The availability of arrays targeting a combination of post-translational modifications is the primary limitation. In addition, unbiased approaches (phosphoproteomics) may be more suitable for the novel discovery, whereas antibody arrays are ideal for the most widely characterized targets.

Here, we present a protocol for antibody arrays to identify alterations in signaling pathways in various cellular models. These changes, caused by drugs/hypoxia/ultra-violet light/radiation, or by overexpression/downregulation/knockouts, are important for various disease models and can indicate whether a therapy will be effective or can identify mechanisms of drugs resistance.

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates ...

Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer (NSCLC). Testing for PD-L1 expression with the PD-L1 immunohistochemistry (IHC) 22C3 companion diagnostic assay, which gives a tumor proportion score (TPS), has been validated on tumor tissue. We developed an optimized laboratory-developed test (LDT) that uses the 22C3 antibody (Ab) concentrate on a widely available IHC autostainer for biopsy and cytology specimens. The PD-L1 TPS was evaluated with 120 paired whole-tumor tissue sections and biopsy samples and with 70 paired biopsy and cytology samples (bronchial washes, n = 40; pleural effusions, n = 30). The 22C3 Ab concentrate-based LDT showed a high concordance rate between biopsy (~100%) and cytology (~95%) specimens when compared to PD-L1 IHC expression determined using the PD-L1 IHC 22C3 companion assay at both TPS cut points (&#8805;1%, &#8805;50%). The optimized LDT presented here, using the 22C3 Ab concentrate to determine the PD-L1 expression in both tumor tissue and in cytology specimens, will expand the ability of laboratories worldwide to assess the eligibility of patients with NSCLC for treatment with pembrolizumab monotherapy in a reliable and reproducible manner.

To expand the ability of laboratories worldwide to assess the eligibility of patients with lung cancer for treatment with pembrolizumab, in a reliable and reproducible manner, we developed an assay that uses the 22C3 antibody concentrate on a widely available immunohistochemical autostainer, for both biopsy and cytology specimens.

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few ...

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.