Name: multiplexed immunofluorescence analysis and quantification of intratumoral pd-1+ tim-3+ cd8+ t cells
Uploaded: 2018-02-08T12:30:00
Description: Watch this Scientific Journal Video about Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells at JoVE.com

This work was supported by grants from Institut National du Cancer (INCA) (ET), Ligue contre le Cancer (ET), Universit&#233; Sorbonne Paris Cit&#233; (ET), ANR (Selectimmunco) (ET), Labex Immuno-Oncology (ET), SIRIC CARPEM (CG, ET). EdG was funded by a fellowship of Fondation ARC. EV and CD were funded by a fellowship of APHP (bourse ann&#233;e recherche). ChB is funded by a fellowship of Universit&#233; Sorbonne Paris Cit&#233; (contrat doctoral). The authors thank Bristol Myers Squibb for their funding in this project. The authors thank the department of Pathology of Hopital Europ&#233;en Georges Pompidou and Necker (Laurianne Chambolle, Elodie Michel and Gis&#232;le Legall). The authors thank the Histology platform of PARCC, Hopital Europ&#233;en Georges Pompidou (Corinne Lesaffre).

This study was conducted in accordance with the Declaration of Helsinki and was approved by the local ethics committee (CPP Ile de France nr. 2012-05-04). Informed consent was obtained from the participant included in the cohorts.
1. Tissue Material
<ol>
	<li>Collect RCC tissue samples on the day of surgery. Handle the surgical specimen at room temperature (pathology department).</li>
	<li>Collect a tumor sample of approximately 0.5 cm x 0.5 cm x 0.5 cm size in a dry tube. Snap freeze in liq...

<ol>
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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=Nivolumab+for+Metastatic+Renal+Cell+Carcinoma:+Results+of+a+Randomized+Phase+II+Trial.">Nivolumab for Metastatic Renal Cell Carcinoma: Results of a Randomized Phase II Trial.</a> J Clin Oncol. 33 (13), 1430-1437 (2015).</li><li>Robert, C., 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=Nivolumab+in+previously+untreated+melanoma+without+BRAF+mutation.">Nivolumab in previously untreated melanoma without BRAF mutation.</a> N Engl J Med. 372 (4), 320-330 (2015).</li><li>Robert, C., 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=Ipilimumab+plus+dacarbazine+for+previously+untreated+metastatic+melanoma.">Ipilimumab plus dacarbazine for previously untreated metastatic melanoma.</a> N Engl J Med. 364 (26), 2517-2526 (2011).</li><li>Rosenberg, J. E., 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=Atezolizumab+in+patients+with+locally+advanced+and+metastatic+urothelial+carcinoma+who+have+progressed+following+treatment+with+platinum-based+chemotherapy:+a+single-arm,+multicentre,+phase+2+trial.">Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial.</a> Lancet. 387 (10031), 1909-1920 (2016).</li><li>Schadendorf, D., 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=Pooled+Analysis+of+Long-Term+Survival+Data+From+Phase+II+and+Phase+III+Trials+of+Ipilimumab+in+Unresectable+or+Metastatic+Melanoma.">Pooled Analysis of Long-Term Survival Data From Phase II and Phase III Trials of Ipilimumab in Unresectable or Metastatic Melanoma.</a> J Clin Oncol. 33 (17), 1889-1894 (2015).</li><li>Ribas, A., Hu-Lieskovan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+does+PD-L1+positive+or+negative+mean?.">What does PD-L1 positive or negative mean?.</a> J Exp Med. 213 (13), 2835-2840 (2016).</li><li>Rizvi, N. A., 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+immunology.+Mutational+landscape+determines+sensitivity+to+PD-1+blockade+in+non-small+cell+lung+cancer.">Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer.</a> Science. 348 (6230), 124-128 (2015).</li><li>Roussel, H., 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=Composite+biomarkers+defined+by+multiparametric+immunofluorescence+analysis+identify+ALK-positive+adenocarcinoma+as+a+potential+target+for+immunotherapy.">Composite biomarkers defined by multiparametric immunofluorescence analysis identify ALK-positive adenocarcinoma as a potential target for immunotherapy.</a> OncoImmunology. 6 (4), e1286437 (2017).</li><li>Pages, F., Granier, C., Kirilovsky, A., Elsissy, C., Tartour, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarqueurs+pr&#233;dictifs+de+r&#233;ponse+aux+traitements+bloquant+les+voies+de+costimulation+inhibitrices.">Biomarqueurs pr&#233;dictifs de r&#233;ponse aux traitements bloquant les voies de costimulation inhibitrices.</a> Bull Cancer. 103, S151-S159 (2016).</li><li>Tumeh, P. C., 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=PD-1+blockade+induces+responses+by+inhibiting+adaptive+immune+resistance.">PD-1 blockade induces responses by inhibiting adaptive immune resistance.</a> Nature. 515 (7528), 568-571 (2014).</li><li>Fourcade, 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=Upregulation+of+Tim-3+and+PD-1+expression+is+associated+with+tumor+antigen-specific+CD8++T+cell+dysfunction+in+melanoma+patients.">Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen-specific CD8+ T cell dysfunction in melanoma patients.</a> J Exp Med. 207 (10), 2175-2186 (2010).</li><li>Koyama, S., 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=Adaptive+resistance+to+therapeutic+PD-1+blockade+is+associated+with+upregulation+of+alternative+immune+checkpoints.">Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints.</a> Nat Commun. 7, 10501 (2016).</li><li>Wherry, E. J., Kurachi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+cellular+insights+into+T+cell+exhaustion.">Molecular and cellular insights into T cell exhaustion.</a> Nat Rev Immunol. 15 (8), 486-499 (2015).</li><li>Cai, C., 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=Tim-3+expression+represents+dysfunctional+tumor+infiltrating+T+cells+in+renal+cell+carcinoma.">Tim-3 expression represents dysfunctional tumor infiltrating T cells in renal cell carcinoma.</a> World J Urol. 34 (4), 561-567 (2016).</li><li>Sakuishi, K., 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=Targeting+Tim-3+and+PD-1+pathways+to+reverse+T+cell+exhaustion+and+restore+anti-tumor+immunity.">Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity.</a> J Exp Med. 207 (10), 2187-2194 (2010).</li><li>Stack, E. C., Wang, C., Roman, K. A., Hoyt, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+immunohistochemistry,+imaging,+and+quantitation:+a+review,+with+an+assessment+of+Tyramide+signal+amplification,+multispectral+imaging+and+multiplex+analysis.">Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis.</a> Methods. 70 (1), 46-58 (2014).</li><li>Bethmann, D., Feng, Z., Fox, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoprofiling+as+a+predictor+of+patient's+response+to+cancer+therapy-promises+and+challenges.">Immunoprofiling as a predictor of patient's response to cancer therapy-promises and challenges.</a> Curr Opin Immunol. 45, 60-72 (2017).</li><li>Badoual, C., 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=PD-1-expressing+tumor-infiltrating+T+cells+are+a+favorable+prognostic+biomarker+in+HPV-associated+head+and+neck+cancer.">PD-1-expressing tumor-infiltrating T cells are a favorable prognostic biomarker in HPV-associated head and neck cancer.</a> Cancer Res. 73 (1), 128-138 (2013).</li><li>Nizard, M., 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=Induction+of+resident+memory+T+cells+enhances+the+efficacy+of+cancer+vaccine.">Induction of resident memory T cells enhances the efficacy of cancer vaccine.</a> Nat Commun. 8, 15221 (2017).</li><li>Nghiem, P. T., 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=PD-1+Blockade+with+Pembrolizumab+in+Advanced+Merkel-Cell+Carcinoma.">PD-1 Blockade with Pembrolizumab in Advanced Merkel-Cell Carcinoma.</a> N Engl J Med. 374 (26), 2542-2552 (2016).</li><li>Roussel, H., 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=Composite+biomarkers+defined+by+multiparametric+immunofluorescence+analysis+identify+ALK-positive+adenocarcinoma+as+a+potential+target+for+immunotherapy.">Composite biomarkers defined by multiparametric immunofluorescence analysis identify ALK-positive adenocarcinoma as a potential target for immunotherapy.</a> Oncoimmunology. (4), (2017).</li><li>Woods, K., 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=Mismatch+in+epitope+specificities+between+IFNgamma+inflamed+and+uninflamed+conditions+leads+to+escape+from+T+lymphocyte+killing+in+melanoma.">Mismatch in epitope specificities between IFNgamma inflamed and uninflamed conditions leads to escape from T lymphocyte killing in melanoma.</a> J Immunother Cancer. 4, 10 (2016).</li><li>Granier, C., 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=Tim-3+Expression+on+Tumor-Infiltrating+PD-1+CD8++T+Cells+Correlates+with+Poor+Clinical+Outcome+in+Renal+Cell+Carcinoma.">Tim-3 Expression on Tumor-Infiltrating PD-1+CD8+ T Cells Correlates with Poor Clinical Outcome in Renal Cell Carcinoma.</a> Cancer Res. 77 (5), 1075-1082 (2017).</li><li>Feng, Z., 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=Multispectral+Imaging+of+T+and+B+Cells+in+Murine+Spleen+and+Tumor.">Multispectral Imaging of T and B Cells in Murine Spleen and Tumor.</a> J Immunol. 196 (9), 3943-3950 (2016).</li><li>Feng, Z., 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=Multispectral+imaging+of+formalin-fixed+tissue+predicts+ability+to+generate+tumor-infiltrating+lymphocytes+from+melanoma.">Multispectral imaging of formalin-fixed tissue predicts ability to generate tumor-infiltrating lymphocytes from melanoma.</a> J Immunother Cancer. 3, 47 (2015).</li><li>Fridman, W. H., Pages, F., Sautes-Fridman, C., Galon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+contexture+in+human+tumours:+impact+on+clinical+outcome.">The immune contexture in human tumours: impact on clinical outcome.</a> Nat Rev Cancer. 12 (4), 298-306 (2012).</li><li>Nizard, M., Roussel, H., Tartour, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+Memory+T+Cells+as+Surrogate+Markers+of+the+Efficacy+of+Cancer+Vaccines.">Resident Memory T Cells as Surrogate Markers of the Efficacy of Cancer Vaccines.</a> Clin Cancer Res. 22 (3), 530-532 (2016).</li><li>Sandoval, F., 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=Mucosal+imprinting+of+vaccine-induced+CD8(+)+T+cells+is+crucial+to+inhibit+the+growth+of+mucosal+tumors.">Mucosal imprinting of vaccine-induced CD8(+) T cells is crucial to inhibit the growth of mucosal tumors.</a> Sci Transl Med. 5 (172), 172ra120 (2013).</li><li>Carstens, J. 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Modifications and Troubleshooting:
The tissue quality is an important parameter; it can be easily checked by hematoxylin and eosin coloration.
One advantage of the technique is the possibility of multiplexed stainings, but to avoid fluorophore spillover, it recommended to choose emission wavelengths with delta of 10 nm minimum. Here, because we had 4 stainings including DAPI, we chose to spread out the wavelengths (DAPI: 460 nm, AF488: 519 nm, Cyani...

In the past few years, immunotherapy has emerged as a very promising treatment for many types of cancers, including RCC. Particularly, immunotherapy based on the inhibition of inhibitory checkpoints like PD-1 and CTLA-4 has been reported to be clinically effective1,2,3,4,5,6. Monoclonal antibodies against CTLA-4, PD-1, or Program Death Ligand 1 (PD-L1) are already approved in several cancers and lead to long lasting clinical responses in more than 20% of patients7. Nevertheless, not all patients are responders, the cost of the treatment is high, and these treatments are toxic, leading to potential serious autoimmune-like side effects. Therefore, the current challenge is to identify predictive markers to those new immunotherapies. The rate of mutations in the tumor, the expression of PD-L1, or the levels of intratumoral CD8+ T cell infiltration have been reported to correlate with clinical response. However, this association is still too weak to recommend the use of these clinical biomarkers in clinical practice except for the companion test for PD-L1 before the administration of Pembrolizumab in non-small cell lung cancer (NSCLC) patients8,9,1011,12. It has been demonstrated that the co-expression of many inhibitory receptors like PD-1, Tim-3, Lag-3, and CTLA-4, induces a cell exhaustion phenotype and resistance to therapy13,14,15. Since peripheral blood is not representative of the tumor microenvironment, it is of high interest to analyze the phenotypic features of the cells in situ. PD-1 and Tim-3 co-expressing T cells are known to be functionally impaired cells in several contexts13,16,17. In this study, the prognostic impact of the co-expression of the two inhibitory receptors PD-1 and Tim-3 on CD8+ T cells was assessed.
Up until now, studying the co-expression of multiple markers on tumor infiltrating lymphocytes (TILs) has mainly been performed by flow cytometry analysis, making it necessary to work on fresh tumors and therefore precluding retrospective analyses. With conventional in situ staining, only one staining at a time can be performed, and the characterization of the cell type that co-expresses the markers is not possible. For example, PD-L1 is expressed by many cell types of the tumoral microenvironment, making it difficult to define by conventional immunohistochemistry analysis which cells expressing PD-L1 are the more relevant for correlative studies. In this work, we developed an innovative in situ multiparametric immunofluorescence method with computer-counting to correlate the co-expression of PD-1 and Tim-3 by tumor infiltrating CD8+ T-cells with clinical outcomes in RCC. This technique has several advantages, including the possibility to analyze at a single cell level and multiple markers at the same time using a multispectral camera that can capture restricted intervals &#62;10 nm through liquid crystal filters18. Moreover, the procedure is automatic which enables an inter-operator reproducibility and a shortened analysis compared to manual techniques19. In the cancer field, several studies reported convincing multiple stainings of immune molecules like PD-1, PD-L1, and CD8 in Merkel-cell carcinoma, lung cancer, and head and neck cancer20,21,22,23. The automated cell count is possible with training by the user (phenotyping step). The fluorescence is measured in the different cell compartments (nuclei, cytoplasm, and membrane).
Here, different subsets of tumor-infiltrating CD8+ T cells expressing PD-1 and/or Tim-3 in a large cohort of RCC were counted and the results were correlated with clinical gravity scores and survival parameters. It was also possible to analyze membrane fluorescence intensity at the cellular resolution with Mean Fluorescence Intensity (MFI) data like in cytometry. As far as we know, this represents the first study reporting prognostic results using this multispectral imaging based count technique.

<table>
	<tbody>
		<tr>
			<td>Vectra 3 Automated Quantitative Pathology Imaging&#160;</td>
			<td>Perkin Elmer</td>
			<td>CLS142338</td>
			<td></td>
		</tr>
		<tr>
			<td>inForm cell analysis 2.1.</td>
			<td>Perkin Elmer</td>
			<td>CLS135781</td>
			<td></td>
		</tr>
		<tr>
			<td>R software</td>
			<td>https://www.r-project.org</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dakopen delimiting pen</td>
			<td>Dako</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Buffer Salin TBS Tablets</td>
			<td>Takara, Bio Inc.</td>
			<td>TAKT9141Z</td>
			<td>pH7.6 100 tablet</td>
		</tr>
		<tr>
			<td>Tris Buffer Salin Tween 20 TBS(+Tween20)</td>
			<td>Takara, Bio Inc.</td>
			<td>TAKT9142Z</td>
			<td>pH 7.6 100 tablets</td>
		</tr>
		<tr>
			<td>Biotin blocking system</td>
			<td>Dako</td>
			<td>X0590</td>
			<td>Avidin 0.1% and Biotin 0.01%</td>
		</tr>
		<tr>
			<td>normal donkey serum</td>
			<td>Jackson Immunoresearch</td>
			<td>017-000-001</td>
			<td>5% vol./vol. concentration</td>
		</tr>
		<tr>
			<td>Fluoroshield with DAPI</td>
			<td>Sigma-aldrich</td>
			<td>F6057</td>
			<td>1.5 &#181;g/mL concentration&#160;</td>
		</tr>
		<tr>
			<td>Knittel glass coverslip</td>
			<td>Knittel&#160;Gl&#228;ser,</td>
			<td>100039</td>
			<td>24x60 mm 100 cover slips</td>
		</tr>
		<tr>
			<td>Rabbit anti-CD8 Clone P17-V</td>
			<td>novus</td>
			<td>NBP1-79055</td>
			<td>use at 4&#181;g/mL</td>
		</tr>
		<tr>
			<td>Mouse anti-PD-1 Clone NAT</td>
			<td>abcam</td>
			<td>ab52587</td>
			<td>use at 2 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Goat anti-Tim-3</td>
			<td>R&#38;D</td>
			<td>AF2365</td>
			<td>use at 3 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Rabbit anti-PD-L1 Clone SP142</td>
			<td>Roche</td>
			<td>7309457001</td>
			<td>use at 1 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Mouse AF647 labeled pan- Keratin Clone C11&#160;</td>
			<td>Cell Signalling</td>
			<td>4528</td>
			<td>use at 0.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Goat anti-human gal9&#160;</td>
			<td>R&#38;D</td>
			<td>AF2045</td>
			<td>use at 0.3 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Cyan 5 conjugated donkey anti-rabbit&#160;</td>
			<td>Jackson Immunoresearch</td>
			<td>711-175-152</td>
			<td>use at 5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Biotinylated F(ab&#8217;2) donkey anti-mouse IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>715-066-150</td>
			<td>use at 3 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Alexa Fluor488 conjugated donkey anti-goat IgG</td>
			<td>abcam</td>
			<td>ab150133</td>
			<td>use at 5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Cy3 labeled streptavidin&#160;</td>
			<td>Amersham</td>
			<td>PA43001</td>
			<td>use at 3 &#181;g/mL</td>
		</tr>
		<tr>
			<td>negative control mouse IgG1</td>
			<td>Dako</td>
			<td>X0931</td>
			<td>use at 2 &#181;g/mL</td>
		</tr>
		<tr>
			<td>IgG from goat serum</td>
			<td>Sigma-aldrich</td>
			<td>I5256</td>
			<td>use at 3 &#181;g/mL</td>
		</tr>
		<tr>
			<td>IgG from rabbit serum</td>
			<td>Sigma-aldrich</td>
			<td>I5006</td>
			<td>use at 4&#181;g/mL</td>
		</tr>
	</tbody>
</table>

multiplexed immunofluorescence analysis and quantification of intratumoral pd-1+ tim-3+ cd8+ t cells

Immune cells are important components of the tumor microenvironment and influence tumor growth and evolution at all stages of carcinogenesis. Notably, it is now well established that the immune infiltrate in human tumors can correlate with prognosis and response to therapy. The analysis of the immune infiltrate in the tumor microenvironment has become a major challenge for the classification of patients and the response to treatment.
The co-expression of inhibitory receptors such as Program Cell Death Protein 1 (PD1; also known as CD279), Cytotoxic T Lymphocyte Associated Protein 4 (CTLA-4), T-Cell Immunoglobulin and Mucin Containing Protein-3 (Tim-3; also known as CD366), and Lymphocyte Activation Gene 3 (Lag-3; also known as CD223), is a hallmark of T cell exhaustion. We developed a multiparametric in situ immunofluorescence staining to identify and quantify at the cellular level the co-expression of these inhibitory receptors. On a retrospective series of frozen tissue of renal cell carcinomas (RCC), using a fluorescence multispectral imaging technology coupled with an image analysis software, it was found that co-expression of PD-1 and Tim-3 on tumor infiltrating CD8+ T cells is correlated with a poor prognosis in RCC. To our knowledge, this represents the first study demonstrating that this automated multiplex in situ technology may have some clinical relevance.

Using the general protocol described above, we aimed to quantify intratumoral CD8+ T cells co-expressing the inhibitory receptors PD-1 and Tim-3 in frozen tissues from patients with RCC, and to correlate the results with clinical outcomes25.
Optimization of the CD8/PD-1/Tim-3 Staining:
Different species of antibodies ...

Watch this Scientific Journal Video about Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells at JoVE.com

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells

Studying tumor microenvironment may identify prognostic or predictive biomarkers of clinical response to immunotherapy. Presented here, is an innovative method based on in situ fluorescence multispectral imaging to analyze and count automatically various subpopulations of CD8+ T cells. This reproducible and reliable technique is suitable for large cohort analyses.

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells

Immune cells are important components of the tumor microenvironment and influence tumor growth and evolution at all stages of carcinogenesis. Notably, it ...

The overall goal of this multiplexed immunofluorescence analysis is to automatically count cells based on their phenotype. This method allows multifluorescence and automatic cell counting to analyze the phenotype and function of tumor immune cells within a tumor microenvironment. This technology facilitated the generation of two types of composite prognosis for predicted by your markers derived directly from the tumor microenvironment.Generally, individuals new to this method may struggle because the pheno typing procedure is very long and the resulting raw data requires further processing. We first had the idea for this method when we attempting to characterize the phenotype and immunological interactions of annhodge miranty cells. After thawing, use a paper towel to carefully dry the slides around the tissue sections and then circle the tissues with a hydrophobic barrier pen.When the barrier has dried, fix the samples in 100%acetone for five minutes, followed by two minutes of air drying, then wash the slides in TBS for 10 minutes. Pretreat the slides with 3 drops of 0.1%Avidin for 10 minutes in the dark, then tap or flick the slides to cover the tissue with the Avidin solution. Add 3 drops of 01%biotin to each sample, then tap or flick the biotin across the samples followed by a TBS wash as just demonstrated.Then block any nonspecific binding with 100 microliters of 5%normal serum in TBS for 30 minutes at room temperature. At the end of the incubation, tap the slides to remove the excess serum and incubate the slides in 100 microliters of the primary antibody cocktail of interest for one hour in a humidified chamber. Wash the antibodies labeled slides in TBST for five minutes.After drying, label the cells with 100 microliters of the secondary antibody cocktail of interest for 30 minutes in the humidified chamber, followed by a 10 minute wash in TBS. Incubate the dried secondary antibody labeled slides with 100 microliters of the tertiary antibody cocktail of interest for 30 minutes followed by 10 minutes of a TBS wash. At the end of the wash, dry the slides and mount the tissues with a dapi supplemented mounting medium.Then clover each slide with a cover slip and let the mounting medium set for at least two hours. To scan the slides, open the microscope software and load the image scanning protocol. Load the first slide onto the microscope stage.Then in the control bar, click set exposure and adjust the exposure time for each filter until a sufficient but not saturating signal is obtained. Click start in the upper panel and open the LabID folder. Enter the ID of the first slide and click next.To acquire the overview of the slide under bright field light at a 4x magnification, click monochrome imaging and find specimen. To select the are of tissue to be scanned, press the control key and use the cursor to select or deselect the regions of interest to be scanned at the 4x magnification. To obtain a fluorescent red/blue/green image of each region, click low power imagine.For high power field selection, press the control key and select at least five regions of interest that correspond to the areas of the tissue that will be scanned at the 20x magnification. Then, to obtain a multi spectral image acquisition of each field, click high power imagine, and click data storage to store the images in the appropriate lab ID folder. To create a new project under the file menu, select new project, under the find feature menu, select cell segmentation, and under the pheno typing menu, select pheno typing, then click configure.In image format, select multi spectral. In sample format, choose fluorescence. To integrate the representative images into the project, under the file menu click open image and select 10-30 representative images of the whole series.To remove the auto florescence, open the unstained slide, and select the library source and the appropriate fluorophores. Click auto florescence and select the area of auto florescence on the blank slide. To generate a composite image, select prepare all to integrate the fluorescence library and the auto fluorescent spectra.Click the eye icon to open the view editor panel and deselect autoflourescence to trim the autoflourescence signal. Select an appropriate color for each marker. To segment the cells, in the compartment menu, select nuclei and membrane.Under the nuclei menu, set DAPI as the nuclear counter stain. Click segment cells and check the segmentation of the nuclei in the cell membranes. To phenotype the cells, under the phenotype menu, click add.To create the cell phenotype categories, select more than five representative for Fluorophor and Fluorophor combination, and click train classifier. The classifier is creating an algorithm that attributes a phenotype for each cell with a confidence interval. Zoom and check the phenotypes of the cells.Then click phenotype all to save the phenotype algorithm and the image project, under the file menu select save project. Then name the project and save it. To perform a batch analysis of the series, select bath analysis and select the saved project.Select a folder for storing the batched data. Add the images to be analyzed then click run. At the end of the analysis check the quality of the composite image and verify the pheno typing of all of the images within the series.Analysis of the tumor infiltrating CD8 positive T-cells, on a clear renal cell carcinoma specimen by fluorescence multi spectral imaging reveals approximately half of the CD8 positive T-cells to be single positive for PD-1 and about 1/3 to be double positive for PD-1 and Tim-3. After integrating these various cell signals on a tissue section, measurement of the PD-1 florescence intensity on PD-1 positive Tim-3 positive versus PD-1 positive, Tim-3 negative CD8 positive T-cells, reveals that the PD-1 MFI is higher when Tim-3 is co expressed reinforcing the functional relevance of Tim-3 expression. In this representative experiment, 66%of the renal tumor patients were also positive for PD-1 Ligand expression and 100%were positive for the Tim-3 ligand Galactin-9.Clinical scoring of the tumor tissues from renal cell carcinoma patients, also demonstrated the positive correlation between the number and percentage of tumor infiltrating CD-8 positive T-cells expressing PD-1 and Tim-3 and the prognostic scoring parameters but not PD-1 positive CD-8 not expressing Tim-3. Interestingly, renal cell carcinoma patients with CD-8 positive T-cells co expressing PD1 with and Tim 3 above the median percentage were more likely to relapse while the expression of PD-1 without Tim-3 co expression was found not to be correlated with patient relapse. While I attempted this procedure it's important to remember to review the phenotype of the cells with two separated investigators including a pathologist if possible.Following this procedure other matters like for cytometry can be performed to answer additional questions about the functional impact of self phenotype after x-vivo stimulation. After it's development, this technique paved the way for researchers in the field of cancer to explore immune responses in effected organs.

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Research

JoVE Journal

Cancer Research

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

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 ...

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded&#160;tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image&#160;analysis technique.

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...

Multiplex Immunofluorescence and Spatial Image Analysis of the Tumor Microenvironment

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending on its composition and the interactions between cancer cells and peri-tumoral cells, the TME may affect cancer progression. The characterization of tumors and their complex microenvironment could improve the understanding of cancer diseases and may help scientists and clinicians to discover new biomarkers.
We recently developed several multiplex immunofluorescence (mIF) panels based on tyramide signal amplification (TSA) for the characterization of the TME in colorectal cancer, head and neck squamous cell carcinoma, melanoma, and lung cancer. Once the staining and scanning of the corresponding panels are completed, the samples are analyzed on an image analysis software. The spatial position and the staining of each cell are then exported from this quantification software into R. We developed R scripts that allow us not only to analyze the density of each cell type in several tumor compartments (e.g. the center of the tumor, the margin of the tumor, and the stroma) but also to perform distance-based analyses between different cell types.
This particular workflow adds a spatial dimension to the classical density analysis already routinely performed for several markers. mIF analysis could allow scientists to have a better understanding of the complex interaction between cancer cells and the TME and to discover new predictive biomarkers of response to treatments, such as immune checkpoint inhibitors, and targeted therapies.

Author Spotlight: Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment  

In this article, a protocol for manual tyramide signal amplification (TSA) multiplex immunofluorescence (mIF) combined with image analysis and spatial analysis is described. This protocol can be used with formalin-fixed paraffin-embedded (FFPE) sections for the staining of two to six antigens per slide depending on the slide scanner available in the laboratory.

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending ...

Flow Analysis of PDL1-Vaxx Antibody Blockade Using Magnetic Fluorescent Beads

Magnetic Fluorescent Bead-Based Dual-Reporter Flow Analysis of PDL1-Vaxx Peptide Vaccine-Induced Antibody Blockade of the PD-1/PD-L1 Interaction

The inhibition of checkpoint receptors (PD-1, PD-L1, and CTLA-4) with monoclonal antibodies has shown great benefit in clinical trials for treating cancer patients and has become a mainstay approach in modern cancer immunotherapy. However, only a subset of patients respond to checkpoint monoclonal antibody immunotherapy. Therefore, it is urgent to develop new therapeutic strategies against cancer. A novel B-cell peptide epitope PDL1 (programmed death ligand 1) cancer vaccine has been developed, with amino acids 130-147 linked to the MVF peptide (&#34;promiscuous&#34; T-cell measles virus fusion protein) via a GPSL linker. Preclinical testing has indicated that this PDL1 vaccine (PDL1-Vaxx) effectively stimulates highly immunogenic antibodies in animals. Animals immunized with PDL1-Vaxx show reduced tumor burden and extended survival rates in various animal cancer models. The mechanisms of action indicate that vaccine-elicited antibodies inhibit tumor cell proliferation, induce apoptosis, and block the PD-1/PD-L1 interaction. This manuscript introduces a magnetic bead-based assay that uses a dual-reporter flow analysis system to evaluate the PD-1/PD-L1 interaction and its blockade by the anti-PDL1 antibodies raised against the PDL1-Vaxx.

Author Spotlight: Magnetic Fluorescent Bead-Based Dual-Reporter Flow Analysis of PDL1-Vaxx Peptide Vaccine-Induced Antibody Blockade of the PD-1/PD-L1 Interaction 

Checkpoint inhibitors are important targets in developing therapies for the battle against cancer. This report introduces a novel PDL1 peptide-based cancer vaccine, PDL1-Vaxx, which induces neutralizing polyclonal antibody production that blocks PD-1/PDL1 complex formation. This work also details the development and testing of a fluorescent bead-based assay for analyzing this activity.

The inhibition of checkpoint receptors (PD-1, PD-L1, and CTLA-4) with monoclonal antibodies has shown great benefit in clinical trials for treating cancer ...

Automated Kinetic Imaging for Drug Assessment in Patient-Derived Tumor Organoids

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

Author Spotlight: Developing Multiplexed Kinetic Assays for Organoid-Based Drug Response Analysis

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell ...

﻿PD-L1 Magnetic Bead-Based PD-1/PD-L1 Blocking Assay

PD-L1 Magnetic Bead-Based PD-1/PD-L1 Blocking Assay  

Start by preparing stock solutions of all antibodies at 2, 000 micrograms per milliliter. Then for each antibody, label the dilution tubes with their respective concentrations, including the name of the antibody. Also include a blank tube for each antibody as PBS-TBN vehicle only control.Next, add 75 microliters of PBS-TBN to all antibody dilution tubes labeled as 500 micrograms per milliliter and below, including the vehicle only control tubes. For each antibody, pipette 150 microliters of the stock solution into the tube labeled as 1, 000 micrograms per milliliter. To create a complete dilution series for each antibody, transfer 75 microliters from the 1, 000 micrograms per milliliter tube to the subsequent lower dilution in the series.Afterward, close the newly completed dilution tube and briefly vortex it. Then add 25 microliters of diluted antibodies into the designated wells of a 96-well microtiter plate, followed by adding 25 microliters of biotinylated recombinant human PDL1 to every reaction well. Cover the microtiter plate with a foil or plastic adhesive seal and incubate at room temperature for one hour with shaking on an orbital plate shaker at 600 RPM.Next, dilute the recombinant human PD1 coupled beads to a concentration of 50, 000 beads per milliliter with PBS-TBN. Remove the 96-well microtiter reaction plate from the shaker and peel the adhesive plate seal. Add 50 microliters of the recombinant human PD1 coupled bead mixture to each well.Seal the plate tightly and incubate for one hour in the dark at room temperature on an orbital shaker set at 600 RPM. After incubation, carefully remove the adhesive plate seal. Then place the plate on the magnetic plate separator for two minutes to immobilize the beads.Confirm that the magnet and microtiter plate are securely connected. Flip the plate over and discard the supernatant carefully. To wash the excess reaction reagents from the beads, remove the microtiter plate from the magnetic plate separator and add 150 microliters of PBS-TBN to each well.After removing the sealer and immobilizing the beads as demonstrated, confirm that the magnet and microtiter plate are secured and dumped the supernatant. Now add 100 microliters of SAPE working solution to each reaction well and resuspend the beads by pipetting. Seal the plate and incubate it for one hour at room temperature in the dark.Then carefully remove the adhesive plate sealer once the beads are immobilized as well as the magnet and plate are secured together. Gently dump the supernatant by inverting the plate. To wash the excess SAPE off the beads, remove the microtiter plate from the magnetic plate holder and add 150 microliters of PBS-TBN to each well to resuspend the beads.Then immobilize the beads by placing the microtiter plate on a magnetic plate separator for two minutes. After confirming that the magnet and microtiter plate is secured, invert the plate and remove the supernatant. Next, add 100 microliters of diluted Brilliant Violet 421 conjugated anti-human or anti-rabbit immunoglobulin G to their respective wells.Seal the plate and incubate it for one hour in the dark at room temperature. Then immobilize the beads and remove the adhesive plate sealer. Confirm that the magnet and microtiter plate are secured and dump the supernatant.Wash the excess secondary antibodies from the beads by adding 150 microliters of PBS-TBN to each well to resuspend the beads. Next, place the microtiter plate on the magnetic plate separator for two minutes to immobilize the beads. Confirm that the magnet and microtiter plate are securely together and then invert the plate and dump the supernatant.Once the fourth and final wash buffer is removed, take out the microtiter plate from the magnetic plate separator. Using a pipette, resuspend the beads in 100 microliters of PBS-TBN per well. Finally, to determine the median fluorescent intensity of each reaction, read the plate on the dual reporter flow analysis system with the aspiration volume set to 50 microliters, minimum bead count to 100 beads, the timeout setting to 40 seconds, gating range to 7, 000 to 17, 000, and operating mode to the dual reporter.All four polyclonal anti-PDL1 peptide antibodies blocked the recombinant human PDL1 interaction with PD1. The percentage of inhibition of the different anti-PDL1 peptide antibodies ranged from 48%to 74%at the maximum tested concentration of 1, 000 micrograms per milliliter. The positive control monoclonal antibody atezolizumab achieved 92%blockade of the PD1-PDL1 interaction at the maximum tested concentration of four micrograms per milliliter.Comparative binding analysis of PDL1 vax-induced candidate antibodies to recombinant human PDL1 indicated that all four polyclonal anti-PDL1 peptide antibodies bound to recombinant human PDL1 in a concentration-dependent manner. The anti-PDL1(130)antibody showed the highest RH PDL1 binding signal of the four PDL1 vax-induced antibody candidates.