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 ...

Research

JoVE Journal

Cancer Research

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

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 ...

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

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 ...

Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced ...

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 ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

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 ...

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

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 ...

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 ...

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

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment (Video) | JoVE 

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

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

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 ...