This study was supported by the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (no. 2022-RC350-04) and the CAMS Innovation Fund for Medical Sciences (nos. 2021-I2M-1-026, 2022-I2M-1-026-1, 02120101, 02130101, and 2022-I2M-2-002).

The animal experimental procedures were approved by the Animal Ethics Committee of Nanjing Medical University or the National Institutes of Quantum Science and Technology. Mice experiments were strictly performed in compliance with the institutional guidelines of the Committee for the Care and Use of Laboratory Animals.
1. Peptide synthesis
<ol>
	<li>Swell 100 mg of 4-methylbenzhydrylamine (MBHA) resin (loading capacity of 0.37 mmol/g) in 1 mL of&#160;N-methyl-2-pyrrolidone (NMP) for 30 min under mild N2 bubbling.</li>
	<li>Prepare a fresh stock buffer consisting of Fmoc-protected amino acids (5.0 equiv), HCTU (4.9 equiv), and DIPEA (10.0 equiv) in 1 mL of NMP, and add to the resin (100 mg). Allow the coupling reaction to proceed for 1.5-2 h.</li>
	<li>Prepare deprotection buffer consisting of 50% (vol/vol) morpholine in NMP. Wash the resin (100 mg) 2 x 30 min with 1 mL of the deprotection buffer in each wash to remove the Fmoc group on the amine group. Wash the resin with dichloromethane (DCM; 1 mL) for 1 min, remove the DCM, and rewash the resin with NMP (1 mL) for another 1 min. Next, remove the NMP and rewash the resin with DCM for 1 min. 
	NOTE: All the washing procedures are conducted under mild N2 bubbling. The above procedures are repeated until the last amino acid.</li>
	<li>For adding 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to the peptide, pre-activate the DOTA with N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC&#183;HCl) and N-hydroxysuccinimide (NHS). Incubate the stock buffer of DOTA/EDC&#183;HCl/NHS at a molar ratio of 1:1:1 in dimethyl sulfoxide (DMSO) for 3 h, with a final DOTA concentration of 1 M. Next, add the stock buffer (1 mL) to the resin (100 mg), and allow the reaction to proceed for 2 h with gentle N2 bubbling. 
	NOTE: 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) can also be utilized as a chelator for 68Ga complexation. The same procedures can be used for NOTA coupling.</li>
	<li>Rinse the resin thoroughly from the top using DCM and apply a vacuum to remove residual reaction solution simultaneously. Rinse the resin 3x using DCM (1.5 mL), and then rinse with MeOH (1.5 mL) to shrink the resin. Put the resin under nitrogen overnight, or under high vacuum for at least 4 h, until the resin becomes dry.</li>
	<li>Place the dried DPA peptide-containing resin in a 2 mL polypropylene container with a screw cap. Add the appropriate cleavage cocktail (95/2.5/2.5 TFA/TIS/H2O in volume; 1 mL/100 mg resin) and seal the container tightly using a screw cap. Gently agitate the reaction on an orbital shaker in a fume hood at 20-25 &#176;C for 2 h. 
	CAUTION: Prepare trifluoroacetic acid (TFA) in a fume hood wearing protective clothing, due to its highly corrosive nature.</li>
	<li>Remove most of the TFA by evaporation under nitrogen in the fume hood. Precipitate the peptides by adding diethyl ether (~1.5 mL/100 mg of resin).</li>
	<li>Vortex the mixture to triturate the peptides and centrifuge at room temperature (10,000 &#215; g, 5 min). Carefully pour the solvent out of the container. Repeat steps 1.7-1.8.</li>
	<li>Air-dry the residue in the open container for 10 min. Add 50% (vol/vol) aqueous acetonitrile, and vortex for 1-2 s to dissolve the products (1 mL/100 mg of resin).</li>
	<li>Remove the resin by filtering the mixture, and then wash the resin 2x using 0.2 mL of 50% (vol/vol) aqueous acetonitrile. Mix the filtrates for high-performance liquid chromatography (HPLC) purification. Use the following HPLC conditions. Column: YMC-Triat-C18 (4.6 mm internal diameter [i.d.], 150 mm, 5 mm); solvent gradient: solvent A-deionized water; solvent B-acetonitrile (0.1% TFA); flow time: 20 min, with acetonitrile from 10% to 90%; flow rate: 1 mL/min.</li>
	<li>Freeze the collected HPLC eluents overnight at -80 &#176;C and lyophilize (-50 &#176;C and &#60;1 pa). For radiolabeling, dissolve the solid peptide in sodium acetate buffer (100 mM, pH 5.0) at a final concentration of 1 mg/mL as a stock buffer.</li>
</ol>
2. 68Ga radiolabeling
NOTE 68Ga was generated in-house at Nanjing First Hospital (Nanjing, China) using a 68Ge/68Ga generator.
<ol>
	<li>Pipette 5 &#181;L of stock buffer into a 1.5 mL polypropylene container with a screw cap. Add 200 MBq [68Ga]GaCl3 (400 &#181;L) to the container.</li>
	<li>Vortex the mixture for 5 s. Measure the pH using pH test strips. Adjust the pH to 4-4.5 with NaOH (0.1 M). 
	NOTE: An appropriate pH is critical for complexation between 68Ga and DOTA.</li>
	<li>Incubate the solution at room temperature for 5-10 min. Subject the reaction mixture to radio-HPLC for radiolabeling yield analysis under the following conditions: column: YMC-Triat-C18 (4.6 mm i.d., 150 mm, 5 mm); solvent gradient: solvent A-deionized water; solvent B-acetonitrile (0.1% TFA); flow time: 20 min, with acetonitrile from 10% to 90%; flow rate: 1 mL/min. 
	&#8203;NOTE: Radio thin-layer chromatography (TLC) can be used as an alternative approach to examine radiolabeling yield. The suggested TLC elution buffer is 0.1 M Na3C6H5O7, pH 4.</li>
</ol>
3. Tracer stability test
<ol>
	<li>Tracer stability test in PBS
	<ol>
		<li>Add [68Ga]DPA (10 &#181;L, 3.7 MBq, in NaOAc) to the PBS (990 &#181;L). Incubate at 37 &#176;C for 1, 2, and 4 h with slight agitation.</li>
		<li>Collect 200 &#181;L of the solution at each time point. Inject it into the radio-HPLC for analysis.</li>
	</ol>
	</li>
	<li>Tracer stability in mouse serum
	<ol>
		<li>Add [68Ga]DPA (10 &#181;L, ~3.7 MBq, in NaOAc) to the mouse serum (90 &#181;L, freshly prepared). Incubate at 37 &#176;C for 1, 2, and 4 h with slight agitation.</li>
		<li>Collect 20 &#181;L of the solution at each time point. Add MeCN and water (100 &#181;L, 1:1, v/v).</li>
		<li>Centrifuge the mixture for 10 min (5,000 &#215; g, 25 &#176;C). Analyze the supernatant using radio-HPLC.</li>
	</ol>
	</li>
</ol>
4. Analysis of PD-L1 expression by flow cytometry
<ol>
	<li>Prepare culture medium by supplementing RPMI-1640 medium with 10% (vol/vol) fetal bovine serum and 1% penicillin-streptomycin (vol/vol). Resuspend the U87MG cells in culture medium, and seed in 12-well plates at a density of 105 cells/well. Place the cells in an incubator (5% CO2, 37 &#176;C), and culture for at least 24 h without disturbing.</li>
	<li>Wash the cells with 0.5 mL of PBS and add 250 &#956;L of trypsin-EDTA (0.25%). Place the cells back in the incubator (5% CO2, 37 &#176;C) for 2 min.</li>
	<li>Add 1 mL of culture medium to stop the cell dissociation. Add medium to the cells to detach them from the dishes by pipetting up and down and collect them in 1.5 mL tubes. Centrifuge the cells for 5 min (100 &#215; g).</li>
	<li>Remove the supernatant and resuspend the cells in 1 mL of PBS. Centrifuge the cells for 5 min (100 &#215; g). Repeat this step one more time.</li>
	<li>Dilute the fluorescein isothiocyanate (FITC)-conjugated PD-L1 antibody with 3% bovine serum albumin (BSA) to 20 nmol/L. Add to the cells and incubate at 4 &#176;C for 1 h.</li>
	<li>Centrifuge the cells for 5 min (100 &#215; g), followed by washing with cold PBS twice. Analyze the PD-L1-positive cells using a flow cytometer and analysis software.</li>
</ol>
5. Immunocytochemistry
<ol>
	<li>Seed the U87MG cells in glass bottom cell culture dishes (35 mm) at a density of 2.5 &#215; 105 cells per well. When 60% confluency is reached, aspirate the culture medium and add 1 mL of PBS. Gently shake several times and aspirate. Perform the washing step 3x.</li>
	<li>Add 500 &#181;L of 4% paraformaldehyde to the dishes. Place the cells at room temperature and fix for 30 min.</li>
	<li>Wash the cells 3x with PBS. Add 1 mL of 3% BSA (wt/vol, in PBS) and block the fixed cells for 2 h at room temperature.</li>
	<li>Remove the 3% BSA and directly incubate the cells with primary anti-PD-L1 antibody (mAb,1:100, diluted in 3% BSA) overnight at 4 &#176;C. 
	NOTE: After blocking the cells with BSA, do not wash with PBS.</li>
	<li>Wash the cells 3x with 3% BSA buffer. Incubate the cells with FITC-conjugated anti-human IgG Fc secondary antibody (1:500, diluted in PBS) for 1 h.</li>
	<li>Wash the cells 3x with PBS. Add 0.5 mL of DAPI (1 &#181;g/mL) to the cells and incubate at room temperature for 1 h. Wash the stained cells 3x with PBS, and observe using confocal fluorescence microscope.</li>
</ol>
6. Cellular uptake and inhibition experiment
<ol>
	<li>Cellular uptake experiment
	<ol>
		<li>Culture the U87MG cells in 12-well plates until 80% confluency is reached. Remove the medium and wash the cells with 0.5 mL of PBS.</li>
		<li>Dilute the [68Ga]DPA in fresh medium to a concentration of 74 KBq/mL. Add 0.5 mL of the diluted [68Ga]DPA buffer to each well.</li>
		<li>Incubate the cells with [68Ga]DPA at 37 &#176;C for different durations (10, 30, 40, and 120 min). Aspirate the medium using a pipette. Wash the cells with PBS (0.5 mL) three times.</li>
		<li>Add NaOH solution (0.5 M, 300 &#181;L per well) to lyse the cells. After 30 s, collect the viscous cell lysates in 1.5 mL tubes.</li>
		<li>Wash the plate with 0.4 mL of PBS twice. Collect the washing solution in the above 1.5 mL tube.</li>
		<li>Start the built-in computer of the automatic gamma counter; put the tubes into the built-in shelf. After loading all the samples onto the conveyor, press the START button. Results are calculated in the internal software. The read-out records the decay-correlated counts per minute (CPM) of each tube.</li>
	</ol>
	</li>
	<li>Competitive binding assay
	<ol>
		<li>Culture the U87MG cells in 12-well plates until 80% confluency is reached. Remove the medium and wash the cells with 0.5 mL of PBS.</li>
		<li>Dilute [68Ga]DPA in fresh medium to a concentration of 74 KBq/mL. Dissolve an appropriate amount of BMS202 compounds in 10% DMSO to yield a 10 mM concentration (400 &#181;L).</li>
		<li>Dilute 4 &#181;L of 10 mM BMS202 in 396 &#181;L of PBS to obtain a concentration of 100 &#181;M. Repeat this step to yield various concentrations of BMS202 (1 &#181;M, 10 nM, 100 pM, and 1 pM).</li>
		<li>Add 0.5 mL of the diluted [68Ga]DPA buffer to each well (0.37 MBq per well). Add 5 &#181;L of the BMS202 solutions to each well (three wells for each concentration). Incubate the cells in a cell incubator at 37 &#176;C for 120 min.</li>
		<li>Aspirate the medium using a pipette. Wash the cells with 3 x 0.5 mL of PBS.</li>
		<li>Add the NaOH solution (0.5 M, 300 &#181;L per well) to lyse the cells. After 30 s, collect the viscous cell lysates into 1.5 mL tubes. Wash the plate with 2 x 0.4 mL of PBS.</li>
		<li>Collect the washing solution in the above 1.5 mL tube. Put the tubes into the built-in shelf of the automatic gamma counter.</li>
		<li>Follow the same procedures as step 6.1.6.</li>
	</ol>
	</li>
</ol>
7. PET imaging
NOTE: Perform small animal PET imaging, using a micro PET scanner that provides 159 transverse axial sections spaced 0.796 mm apart (center-to-center), with a horizontal field of view of 10 cm and an axial field of view of 12.7 cm. All data collected in the list mode are organized into three-dimensional sinograms. The Fourier is then reassembled into two-dimensional sinograms (frame &#215; min: 4 &#215; 1, 8 &#215; 2, 8 &#215; 5).
<ol>
	<li>Use male, 5-8-week-old BALB/C nude mice in this study. Collect the U87MG cells following steps 4.1-4.4 and aspirate the cells into a 0.5 mL syringe. Subcutaneously inject the cells into the mice (1 &#215; 106 cells per tumor, two tumors per mouse). Monitor tumor growth after injection until the tumor volume is 100-300 mm3.</li>
	<li>Anesthetize the mice using 1%-2% (v/v) isoflurane (1 mL/min). Turn on the heating device and keep the animal bed of PET at 37 &#176;C.&#160;</li>
	<li>Put the anesthetized mice in the right position on the animal bed of the PET machine. Apply&#160;ophthalmic ointment to both&#160;eyes to prevent dryness. 
	CAUTION: Keep the mice in the prone position to avoid death during scanning. During the whole imaging process, administer isoflurane flow (1.0 mL/min) via the nose using a preinstalled tube.</li>
	<li>Adjust the animal bed position via the control panel. Inject the tracers (10-17 MBq/100-200 &#181;L) intravenously through a preinstalled tail vein catheter.</li>
	<li>Create a scanning workflow in a host computer using the referenced software (see Table of Materials) Create a study folder and set the acquisition protocol as per the manufacturer&#39;s protocol. Perform dynamic scans (60 min for each mouse) on all mice in 3D list mode.</li>
	<li>Define a histogram protocol and a reconstruction protocol following the manufacturer&#39; protocol. Use Hanning&#39;s filter with a Nyquist cutoff of 0.5 cycle/pixel to reconstruct PET dynamic images (25-30 min and 55-60 min) through filtered back-projection. Generate maximum intensity projection (MIP) images for all mice.</li>
	<li>Combine the protocols in a workflow and run the workflow. Analyze the resulting three-dimensional images using the software, following the manufacturer&#39;s protocol. 
	NOTE: Use the simulation software to select the volumes of interest. Radioactivity is decay-corrected to the injection time and presented as the percentage of the total injection dose/per gram tissue (% ID/g).</li>
</ol>
8. Ex vivo biodistribution
<ol>
	<li>Administer [68Ga]DPA (1.85 MBq/100 &#181;L) to the U87MG-bearing BALB/C nude mice through tail vein injection. Anesthetize the mice using 1%-2% (v/v) isoflurane (1 mL/min), then sacrifice&#160;three mice via cervical dislocation after injection for 5, 30, 60, and 120 min. 
	NOTE:&#160;The individuals demonstrating this procedure should be trained in the technical skills of cervical dislocation to ensure the loss of consciousness is rapidly induced. This will ensure that&#160;the euthanasia procedures in this experiment are all humanely performed.</li>
	<li>After death is confirmed, open the chest wall of the mice. Then, open the heart. Use a 1 mL syringe to draw blood; squeeze the blood from the syringe into a radioimmunoassay (RIA) tube (13 mm in diameter) for the gamma counter.</li>
	<li>Excise the major organs and tumors and place them in RIA tubes (13 mm in diameter) for the gamma counter. Major organs include the total blood, heart, thymus, liver, spleen, bone, stomach, kidneys, muscle, intestinal lymph node, small intestine, pancreas, testis, brain, and lungs. Weigh all the organs.</li>
	<li>Measure the radioactivity within the collected organs using an autogamma counter and decay-correct the values. Calculate the percentage of injected dose per gram of wet tissue (% ID/g).</li>
</ol>
9. Immunohistochemistry
<ol>
	<li>Collect the glioma tissues and wash them 3x with PBS. Put the fresh tissues in 4% paraformaldehyde and fix overnight at 4 &#176;C.</li>
	<li>Embed the fixed tissues in paraffin and section them to a thickness of 10 &#181;m. Place the sections in an incubator (60 &#176;C) and incubate for 2 h. Dewax and hydrate the sections by incubating for 10 min each in the following: xylene (twice), absolute alcohol, 95% alcohol, 90% alcohol, 80% alcohol, 75% alcohol.</li>
	<li>Put the sections in 0.01 M sodium citrate and heat them to 92-95 &#176;C. Maintain the temperature for 40 min to achieve antigen retrieval.</li>
	<li>Add 200 &#181;L of H2O2 solution (3%) to the sections and inactivate the endoperoxidases for 10 min at room temperature. Block the non-specific sites by treatment with 3% BSA for 2 h at room temperature.</li>
	<li>Incubate the sections in primary anti-PD-L1 antibody (mAb, 1:100, diluted in 3% BSA) overnight at 4 &#176;C. 
	NOTE: After blocking with BSA, do not wash with PBS.</li>
	<li>Wash the sections 3x with PBS. Incubate the sections with HRP-labeled goat anti-rabbit secondary antibody (1:500, diluted in PBS) at room temperature for 1 h.</li>
	<li>Wash the cells 3x with PBS. Add 200 &#181;L of 3,3-diaminobenzidine (DAB) working solution (solution A: solution B: solution C = 1:1:18) to the sections and incubate for 5 min in the dark.</li>
	<li>Wash the sections 3x with PBS. Add 500 &#181;L of hematoxylin solution (100%), and incubate for 5 min at room temperature. Wash the sections with water for 30 s. Put the sections into differentiation solution (75% alcohol:HCL = 99:1) for 30 s. Wash the sections using water for 1 min.</li>
	<li>Dehydrate the samples by sequentially incubating with 75% alcohol, 80% alcohol, 90% alcohol, 95% alcohol, absolute alcohol, and xylene (twice) (10 min each). Mount all the sections using neutral resin and observe them under an optical microscope.</li>
</ol>

No competing interests are declared.

The critical steps described in this method include the efficient labeling of 68Ga to DPA and choosing a suitable time window for PET imaging, which must perfectly match the pharmacodynamic pattern of DPA in the tumor.
In contrast to IHC, PET imaging enables real-time detection of whole-body PD-L1 expression in a noninvasive manner, allowing the visualization of each positive area in a heterogeneous tumor6,7. Peptides were c...

The discovery of immune checkpoint proteins was a breakthrough in tumor therapy, and has led to major advances in the development of immune checkpoint blockade therapy1. Programmed cell death-protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) are potential drug targets with several antibodies approved by the Food and Drug Administration (FDA). PD-1 is expressed by tumor-infiltrating immune cells, such as CD4+, CD8+ T cells, and regulatory T cells. PD-L1 is one of the PD-1 ligands, which is overexpressed in a variety of tumor cells2,3. The interaction between PD-1 and PD-L1 inactivates PD-1, thus suppressing the antitumor immune response4. These findings suggest that the inhibition of PD-L1 can improve the killing effect of immune cells and eliminate tumor cells5. Currently, chromogenic immunohistochemistry (IHC) is the most commonly used approach to identify patients who are most likely to respond to immune checkpoint therapy6,7. However, due to the heterogeneous expression of PD-L1 in tumor cells, IHC results from biopsies cannot provide accurate information about PD-L1 expression in patients8. Previous studies have reported that only 20%-40% of patients gain long-term benefits from immune checkpoint blockade therapy1,9,10. There is, therefore, an urgent need to develop a new method to circumvent the false-negative results caused by the heterogeneous expression of these immune checkpoint proteins.
Molecular imaging technology, such as positron emission tomography (PET), enables real-time visualization of the whole body in a noninvasive way, and thus can outperform the conventional IHC method11,12,13. Radiolabeled antibodies, peptides, and small molecules are promising tracers for monitoring PD-L1 expression in cancer patients14,15,16,17,18,19,20,21,22,23,24,25. The FDA has approved three PD-L1 therapeutic monoclonal antibodies: avelumab, atezolizumab, and durvalumab26. Immuno-PET tracers based on these antibodies have been well documented27,28,29,30,31,32. Early-phase clinical trials have revealed limited value for clinical application, because of the unfavorable pharmacokinetics30. Compared with antibodies, peptides exhibit faster blood and organ clearance from healthy organs, and can be easily chemically modified33. Multiple peptides with high affinities for PD-1/PD-L1 have been reported2; WL12 is a reported peptide that shows specific binding to PD-L134. Radiolabeled tracers, [64Cu]WL12, [68Ga]WL12, and [18F]FPyWL12, have been reported to show high in vivo specific tumor-targeting ability, which allows for the harvest of high-quality images of PD-L1 expression in tumors26,35,36,37. Moreover, the first in-human evaluation of radiolabeled WL12 has demonstrated that [68Ga]WL12 (chelated by NOTA) has a safe and efficient potential for clinical tumor imaging38. Due to its high hydrophobicity and high uptake in the healthy liver, WL12 has limited clinical use. Other radiolabeling peptides, such as TPP1 and SETSKSF, which specifically bind to PD-L1, have also showed potential stability and specificity to visualize whole-body PD-L1 expression39,40. However, unmodified peptides are easily degraded by proteases, and are rapidly metabolized by the kidney. Dextrorotary(D)-peptides have been widely used as effective mediators, due to the poor stability of left-handed (L)-peptides41,42,43. D-peptides are hyper-resistant to proteolytic degradation and have remarkably prolonged metabolic half-lives. Compared with their L-counterparts, D-peptides mostly show specific binding abilities44,45,46.
This study designed a new method to detect PD-L1 expression, based on PET imaging of a 68Ga-labeled PD-L1-targeted D-peptide, D-dodecapeptide antagonist (DPA), in a tumor-bearing mouse model47. The stability of [68Ga]DPA was first studied in phosphate-buffered saline (PBS) and mouse serum, after which the binding affinity of [68Ga]DPA in PD-L1-overexpressing tumors was tested. Thereafter, PET imaging was performed in glioblastoma xenograft models to confirm whether [68Ga]DPA was an ideal PET tracer to monitor PD-L1 expression in tumors. The combination of PET imaging and DPA not only provides a new approach to overcome challenges associated with the heterogeneous expression of PD-L1, but also lays the basis for the development of D-peptide-based radiotracers.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)</td>
			<td>Merck</td>
			<td>60239-18-1</td>
			<td>68Ga&#160; chelation</td>
		</tr>
		<tr>
			<td>3,3-diaminobenzidine (DAB) Kit</td>
			<td>Sigma-Aldrich</td>
			<td>D7304-1SET</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr>
			<td>anti-PD-L1 monoclonal antibody</td>
			<td>Wuhan Proteintech</td>
			<td>17952-1-ap</td>
			<td>Immunohistochemistry: primary antibody</td>
		</tr>
		<tr>
			<td>BMS202</td>
			<td>Selleck</td>
			<td>1675203-84-5</td>
			<td>Competitive binding assay: inhibitor</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Merck</td>
			<td>V900933</td>
			<td>Immunofluorescent : blocking&#160;</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Merck</td>
			<td>D9542</td>
			<td>Immunofluorescent: staining of nucleus</td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td>Merck</td>
			<td>34856</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>DIPEA</td>
			<td>Merck</td>
			<td>3439</td>
			<td>Peptide coupling</td>
		</tr>
		<tr>
			<td>EDC&#183;HCl</td>
			<td>Merck</td>
			<td>E6383</td>
			<td>Activation of DOTA</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10099</td>
			<td>Cell culture: supplement</td>
		</tr>
		<tr>
			<td>FITC-conjugated anti-human IgG Fc Antibody</td>
			<td>Biolegend</td>
			<td>409310</td>
			<td>Immunofluorescent: secondary antibody</td>
		</tr>
		<tr>
			<td>FITC-conjugated anti PD-L1 antibody</td>
			<td>Biolegend</td>
			<td>393606</td>
			<td>Flow cytometry: direct antibody</td>
		</tr>
		<tr>
			<td>HCTU</td>
			<td>Energy Chemical</td>
			<td>E070004-25g</td>
			<td>Peptide coupling</td>
		</tr>
		<tr>
			<td>HRP labeled goat anti-rabbit antibody</td>
			<td>Servicebio</td>
			<td>GB23303</td>
			<td>Immunohistochemistry: secondary antibody</td>
		</tr>
		<tr>
			<td>Hydroxysuccinimide (NHS)</td>
			<td>Merck</td>
			<td>130672</td>
			<td>Activation of DOTA</td>
		</tr>
		<tr>
			<td>MeCN</td>
			<td>Merck</td>
			<td>PHR1551</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Morpholine</td>
			<td>Merck</td>
			<td>8.06127</td>
			<td>Fmoc- deprotection</td>
		</tr>
		<tr>
			<td>NMP</td>
			<td>Merck</td>
			<td>8.06072</td>
			<td>Solevent</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Merck</td>
			<td>30525-89-4</td>
			<td>Fixation of tissues</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td>Cell culture: buffer</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin&#160;</td>
			<td>Gibco</td>
			<td>10378016</td>
			<td>Cell culture: supplement</td>
		</tr>
		<tr>
			<td>RIA tube</td>
			<td>PolyLab</td>
			<td>P10301A</td>
			<td>As tissue sample container</td>
		</tr>
		<tr>
			<td>RPMI-1640 medium</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td>Cell culture: basic medium</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Merck</td>
			<td>1.06264</td>
			<td>Salt for buffer</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td>Cell culture: dissociation agent</td>
		</tr>
		<tr>
			<td>U87MG cell line</td>
			<td>Procell Life Science &#38; Technology Co</td>
			<td>CL-0238</td>
			<td>Cell model</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>68Ge/68Ga generator</td>
			<td>Isotope Technologies Munich, ITM</td>
			<td>Not applicable</td>
			<td>Generation of [68Ga]</td>
		</tr>
		<tr>
			<td>Autogamma counter</td>
			<td>Perkin Elmer&#160;</td>
			<td>Wizard2</td>
			<td>Detection of radioactivity</td>
		</tr>
		<tr>
			<td>Confocal fluorescent microscopy</td>
			<td>Keyence</td>
			<td></td>
			<td>Observation of immunofluorescent results</td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>Becton Dickinson, BD</td>
			<td>LSRII</td>
			<td>Monitoring the PD-L1 positive cells</td>
		</tr>
		<tr>
			<td>High-performance liquid chromatography (HPLC)</td>
			<td>SHIMAZU</td>
			<td>LC-20AT&#160;</td>
			<td>Purification of DPA peptide</td>
		</tr>
		<tr>
			<td>PET scanner</td>
			<td>Siemens Medical Solutions</td>
			<td>Inveon MultiModality System</td>
			<td>PET imaging</td>
		</tr>
		<tr>
			<td>Optical microscopy</td>
			<td>Nikon&#160;</td>
			<td>Eclipse E100</td>
			<td>Observation of immunohistochemistry results</td>
		</tr>
		<tr>
			<td>Solid phase peptide synthesizer</td>
			<td>Promega Vac-Man Laboratory Vacuum Manifold</td>
			<td>LOT#11101</td>
			<td>Synthesis of DPA-DOTA peptide</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ASIPro</td>
			<td>Siemens Medical Solutions</td>
			<td>Not applicable</td>
			<td>Analysis of PET-CT results</td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>Becton Dickinson, BD</td>
			<td>FlowJo 7.6.1</td>
			<td>Analysis of the flow cytometer results</td>
		</tr>
		<tr>
			<td>Inveon Acquisition Workplace (IAW)</td>
			<td>Siemens Medical Solutions</td>
			<td>Not applicable</td>
			<td>Management of PET mechine</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>Graphpad</td>
			<td>Prism 8.0</td>
			<td>Analysis of the data&#160;</td>
		</tr>
	</tbody>
</table>

development of a 68gallium-labeled d-peptide pet tracer for imaging programmed death-ligand 1 expression

The development of immune checkpoint blockade therapy based on programmed cell death-protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) has revolutionized cancer therapies in recent years. However, only a fraction of patients responds to PD-1/PD-L1 inhibitors, owing to the heterogeneous expression of PD-L1 in tumor cells. This heterogeneity presents a challenge in the precise detection of tumor cells by the commonly used immunohistochemistry (IHC) approach. This situation calls for better methods to stratify patients who will benefit from immune checkpoint blockade therapy, to improve treatment efficacy. Positron emission tomography (PET) enables real-time visualization of the whole-body PD-L1 expression in a noninvasive way. Therefore, there is a need for the development of radiolabeled tracers to detect PD-L1 distribution in tumors through PET imaging.
Compared to their L-counterparts, dextrorotary (D)-peptides have properties such as proteolytic resistance and remarkably prolonged metabolic half-lives. This study designed a new method to detect PD-L1 expression based on PET imaging of 68Ga-labeled PD-L1-targeted D-peptide, a D-dodecapeptide antagonist (DPA), in tumor-bearing mice. The results showed that the [68Ga]DPA can specifically bind to PD-L1-overexpressing tumors in vivo, and showed favorable stability as well as excellent imaging ability, suggesting that [68Ga]DPA-PET is a promising approach for the assessment of PD-L1 status in tumors.

[68Ga]DPA radiolabeling and stability 
The model peptide, DPA, is an effective PD-L1 antagonist. DOTA-DPA was obtained with &#62;95% purity and a yield of 68%. The mass of DOTA-DPA is experimentally observed at 1,073.3 ([M+2H]2+). 68Gallium is considered a suitable radionuclide to label peptides for PET imaging, and therefore was chosen for this study. To radiolabel DPA with 68Ga (half-life: 68 min), DOTA-PEG3-DPA was synt...

Watch this Scientific Journal Video about Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression at JoVE.com

Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

This study developed a noninvasive and real-time method to evaluate the distribution of programmed death-ligand 1 in the whole body, based on positron emission tomographic imaging of [68Ga] D-dodecapeptide antagonist. This technique has advantages over conventional immunohistochemistry and improves the efficiency of identifying appropriate patients who will benefit from immune checkpoint blockade therapy.

Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

The development of immune checkpoint blockade therapy based on programmed cell death-protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) has revolutionized ...

development-68gallium-labeled-d-peptide-pet-tracer-for-imaging

Research

JoVE Journal

Cancer Research

1.4K Views.  Nanjing Medical University. This study developed a noninvasive and real-time method to evaluate the distribution of programmed death-ligand 1 in the whole body, based on positron emission tomographic imaging of [68Ga] D-dodecapeptide antagonist. This technique has advantages over conventional immunohistochemistry and improves the efficiency of identifying appropriate patients who will benefit from immune checkpoint blockade therapy.

Video: Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its excellent preservation of morphology. Personalized medicine increasingly provides individually adapted and targeted therapies for characterized individual diseases enabled by combined morphological and molecular analytical technologies and diagnostics. Performance of morphologic and molecular assays from the same FFPE specimen is challenging because of the negative impact of formalin due to chemical modification and cross-linking of nucleic acids and proteins. A non-cross-linking, formalin-free tissue fixative has been recently developed to fulfil both requirements, i.e., to preserve morphology like FFPE and biomolecules like cryo-preservation. Since FISH is often required in combination with histopathology and molecular diagnostics, we tested the applicability of FISH protocols on tissues treated with this new fixative. We found that formalin post-fixation of histological sections of non-cross-linking, formalin-free and paraffin-embedded (NCFPE) breast cancer tissue generated equivalent results to those with FFPE tissue in human epidermal growth factor receptor 2 (HER2) FISH analysis. This protocol describes how a FISH assay originally developed and validated for FFPE tissue can be used for NCFPE tissues by a simple post-fixation step of histological sections.

Fluorescence in-situ hybridization (FISH) is often required in combination with histopathology and molecular diagnostics for selection of therapy in personalized medicine. A novel non-cross-linking, formalin-free tissue fixative that allows high quality morphologic, molecular and FISH analyses from the same specimen by addition of a post-fixation step before FISH is presented.

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its ...

PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without the ability to target a specific or well-delineated tumor volume. The delivery of radiation was achieved using fixed radiation sources or linear accelerators producing megavoltage (MV) X-rays. These devices are unable to achieve sub-millimeter precision required for small animals. Furthermore, the high doses delivered to healthy surrounding tissue hamper response assessment. To increase the translation between small animal studies and humans, our goal was to mimic the treatment of human glioblastoma in a rat model. To enable a more accurate irradiation in a preclinical setting, recently, precision image-guided small animal radiation research platforms were developed. Similar to human planning systems, treatment planning on these micro-irradiators is based on computed tomography (CT). However, low soft-tissue contrast on CT makes it very challenging to localize targets in certain tissues, such as the brain. Therefore, incorporating magnetic resonance imaging (MRI), which has excellent soft-tissue contrast compared to CT, would enable a more precise delineation of the target for irradiation. In the last decade also biological imaging techniques, such as positron emission tomography (PET) gained interest for radiation therapy treatment guidance. PET enables the visualization of e.g., glucose consumption, amino-acid transport, or hypoxia, present in the tumor. Targeting those highly proliferative or radio-resistant parts of the tumor with a higher dose could give a survival benefit. This hypothesis led to the introduction of the biological tumor volume (BTV), besides the conventional gross target volume (GTV), clinical target volume (CTV), and planned target volume (PTV).
At the preclinical imaging lab of Ghent University, a micro-irradiator, a small animal PET, and a 7 T small animal MRI are available. The goal was to incorporate MRI-guided irradiation and PET-guided sub-volume boosting in a glioblastoma rat model.

In the past, small animal irradiation was usually performed without the ability to target a well-delineated tumor volume. The goal was to mimic the treatment of human glioblastoma in rats. Using a small animal irradiation platform, we performed MRI-guided 3D conformal irradiation with PET-based sub-volume boosting in a preclinical setting.

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without ...

Whole-body PET/MRI of Pediatric Patients: The Details That Matter

Integrated PET/MRI is a hybrid imaging technique enabling clinicians to acquire diagnostic images for tumor assessment and treatment monitoring with both high soft tissue contrast and added metabolic information. Integrated PET/MRI has shown to be valuable in the clinical setting and has many promising future applications. The protocol presented here will provide step-by-step instructions for the acquisition of whole-body 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) PET/MRI data in children with cancer. It also provides instructions on how to combine a whole-body staging scan with a local tumor scan for evaluation of the primary tumor. The focus of this protocol is to be both comprehensive and time-efficient, which are two ubiquitous needs for clinical applications. This protocol was originally developed for children above 6 years, or old enough to comply with breath-hold instructions, but can also be applied to patients under general anesthesia. Similarly, this protocol can be modified to fit institutional preferences in terms of choice of MRI pulse sequences for both the whole-body scan and local tumor assessment.

This article provides comprehensive step-by-step instructions for the acquisition of whole-body 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) PET/MRI scans for cancer staging of pediatric patients. The protocol was developed for children above 6 years, or old enough to comply with breath-hold instructions, but can be used for general anesthesia patients as well.

Integrated PET/MRI is a hybrid imaging technique enabling clinicians to acquire diagnostic images for tumor assessment and treatment monitoring with both ...

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology.

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography ...

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

Development and Angiographic Use of the Rabbit VX2 Model for Liver Cancer

The rabbit VX2 tumor is an animal model commonly utilized for translational research regarding hepatocellular carcinoma (HCC) in the field of Interventional Radiology. This model employs an anaplastic squamous cell carcinoma that is easily and reliably propagated in the skeletal muscle of donor rabbits for eventual harvest and allograft implantation into the liver of na&#239;ve recipients. This tumor graft rapidly grows within the liver of recipient rabbits into an angiographically identifiable tumor characterized by a necrotic core surrounded by a viable hypervascular capsule. The physical size of the rabbit anatomy is sufficient to facilitate vascular instrumentation allowing for the application and testing of various interventional techniques. Despite these benefits, there exists a paucity of technical resources to act as a concrete reference for researchers working with the model. Herein, we present a comprehensive visual outline for the technical aspects of development, growth, propagation, and angiographic utilization of the rabbit VX2 tumor model for use by novice and experienced researchers alike.

The goal of this article is to provide a primer for the development and use of the VX2 carcinoma rabbit model for liver cancer.

The rabbit VX2 tumor is an animal model commonly utilized for translational research regarding hepatocellular carcinoma (HCC) in the field of ...

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. Although heterogeneous in presentation, a common denominator among these tumors is the overexpression of somatostatin receptors. 68Ga-DOTATATE is a somatostatin analog labeled with the positron emitter gallium-68 (68Ga). For well-differentiated neuroendocrine tumors, 68Ga-DOTATATE positron emission tomography (PET)/computed tomography (CT) imaging is used for diagnosis, determination of disease burden, and therapy selection.
This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiolabeling of 68Ga-DOTATATE is performed with a fully automated labeling module coupled to a germanium-68 (68Ge)/68Ga generator. Quality control of the final product evaluates radiochemical purity with instant thin-layer chromatography and solid-phase chromatography, and pH prior to patient injection. Periodic quality control is performed to determine 68Ge breakthrough, sterility, and (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) content. Patient preparation includes patient instructions, a protocol for 68Ga-DOTATATE during treatment with somatostatin analogs, and intravenous administration of the radiopharmaceutical. For PET/CT imaging, the acquisition and reconstruction settings are described. For each step, radiation safety will be highlighted, as well as time constrictions due to the short half-life of 68Ga.
Fully automated in-house production and quality control of 68Ga-DOTATATE leads to very high success rates (95%) and produces two to four patient dosages per batch, depending on the yield of the generator. In conclusion, 68Ga-DOTATATE PET/CT imaging is a noninvasive and fast method of providing information on the tumor burden of neuroendocrine tumors (NETs) while also assisting in diagnosis and therapy selection.

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Well-differentiated neuroendocrine tumors overexpress somatostatin receptors which can be utilized for diagnostic imaging with the radiolabeled somatostatin analog 68Ga-DOTATATE. This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiation safety and time constrictions due to the short half-life of 68Ga are taken into account.

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. ...

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 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 ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.