This work was supported by grants fromthe Belgian Fund for Scientific Research (FNRS), Les Amis de l&#8217;Institut Bordet, FNRS-Op&#233;ration T&#233;l&#233;vie, Plan Cancer of Belgium, Fonds Lambeau-Marteaux, Fonds J.C. Heuson and Fonds Barsy.

NOTE: All specimens were acquired using a protocol approved by the Medical Ethics Committee of the Institute Jules Bordet with written informed consent obtained from each patient.
1. Preparation of the Tissue Homogenate
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	<li>Dissect resected tissues (malignant and normal tissue resected from the operating room) are in the pathology lab by trained personnel for immediate pickup. Tumor, NANT (taken the furthest distance from the tumor as possible) and normal tissue fragments are routinely...

<ol>
	<li>Chen, D. S., Mellman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncology+meets+immunology:+the+cancer-immunity+cycle.">Oncology meets immunology: the cancer-immunity cycle.</a> Immunity. 39 (1), 1-10 (2013).</li><li>Boudreau, A., van't Veer, J. L., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+'elite+hacker':+breast+tumors+exploit+the+normal+microenvironment+program+to+instruct+their+progression+and+biological+diversity.">An 'elite hacker': breast tumors exploit the normal microenvironment program to instruct their progression and biological diversity.</a> Cell Adh Migr. 6 (3), 236-248 (2012).</li><li>Gajewski, T. F., Schreiber, H., Fu, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+and+adaptive+immune+cells+in+the+tumor+microenvironment.">Innate and adaptive immune cells in the tumor microenvironment.</a> Nat Immunol. 14 (10), 1014-1022 (2013).</li><li>Quezada, S. 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=Limited+tumor+infiltration+by+activated+T+effector+cells+restricts+the+therapeutic+activity+of+regulatory+T+cell+depletion+against+established+melanoma.">Limited tumor infiltration by activated T effector cells restricts the therapeutic activity of regulatory T cell depletion against established melanoma.</a> J Exp Med. 205 (9), 2125-2138 (2008).</li><li>Grange, 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=Phenotypic+characterization+and+functional+analysis+of+human+tumor+immune+infiltration+after+mechanical+and+enzymatic+disaggregation.">Phenotypic characterization and functional analysis of human tumor immune infiltration after mechanical and enzymatic disaggregation.</a> J Immunol Methods. 372 (1-2), 119-126 (2011).</li><li>McCauley, H. A., Guasch, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+orthotopic+transplantation+of+epithelial+tumors+in+single-cell+suspension.">Serial orthotopic transplantation of epithelial tumors in single-cell suspension.</a> Methods Mol Biol. 1035, 231-245 (2013).</li><li>Gros, 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=Myeloid+cells+obtained+from+the+blood+but+not+from+the+tumor+can+suppress+T-cell+proliferation+in+patients+with+melanoma.">Myeloid cells obtained from the blood but not from the tumor can suppress T-cell proliferation in patients with melanoma.</a> Clin Cancer Res. 18 (19), 5212-5223 (2012).</li><li>Zirakzadeh, A. A., Marits, P., Sherif, A., Winqvist, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+B+cell+characterization+in+blood,+lymph+nodes,+and+tumors+from+patients+with+malignancies.">Multiplex B cell characterization in blood, lymph nodes, and tumors from patients with malignancies.</a> J Immunol. 190 (11), 5847-5855 (2013).</li><li>Gu-Trantien, 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=CD4(+)+follicular+helper+T+cell+infiltration+predicts+breast+cancer+survival.">CD4(+) follicular helper T cell infiltration predicts breast cancer survival.</a> J Clin Invest. 123 (7), 2873-2892 (2013).</li><li>Buisseret, L., 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=Lymphocytes+Infiltrating+Breast+Cancer+:+Density,+Composition+And+Organization.">Lymphocytes Infiltrating Breast Cancer : Density, Composition And Organization.</a> Annals of Oncology. 25 (1), 17 (2014).</li><li>Garaud, 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=Characterization+of+B+Cells+Infiltrating+Human+Breast+Cancer.">Characterization of B Cells Infiltrating Human Breast Cancer.</a> Annals of Oncology. 25 (1), 18 (2014).</li><li>Gu-Trantien, 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=Cxcl13-Producing+Follicular+Helper+T+Cells+In+Human+Breast+Cancer.">Cxcl13-Producing Follicular Helper T Cells In Human Breast Cancer.</a> Annals of Oncology. 25 (1), 17 (2014).</li><li>Yee, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+endogenous+T+cells+for+adoptive+transfer.">The use of endogenous T cells for adoptive transfer.</a> Immunol Rev. 257 (1), 250-263 (2014).</li><li>Butler, M. O., 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=Ex+vivo+expansion+of+human+CD8++T+cells+using+autologous+CD4++T+cell+help.">Ex vivo expansion of human CD8+ T cells using autologous CD4+ T cell help.</a> PLoS One. 7 (1), 30229 (2012).</li><li>Ye, Q., 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=Engineered+artificial+antigen+presenting+cells+facilitate+direct+and+efficient+expansion+of+tumor+infiltrating+lymphocytes.">Engineered artificial antigen presenting cells facilitate direct and efficient expansion of tumor infiltrating lymphocytes.</a> J Transl Med. 9, 131 (2011).</li></ol>

This study describes an optimized method for the rapid preparation of normal and malignant breast tissue homogenates without enzymatic digestion for subsequent cell sorting, extraction, cryopreservation and/or phenotypic analysis of CD45+ subpopulations. The goal of this experimental approach is to produce images of the TIL that closely reflect their in vivo state and compare them to normal tissues with minimal manipulation of the tissues fresh from the operating room. To date, our laboratory has used...

The tumor microenvironment is composed of various cell types with numerous studies showing they each play distinct and important roles in tumorigenesis1,2. These include, but are not limited to, infiltrating immune cells, stromal cells, endothelial cells and tumor cells3. Ex vivo studies of tumor infiltrating lymphocytes (TIL; CD45+ cells or leukocytes, which are predominantly lymphocytes in breast tumors) from fresh human tissue samples is made difficult by their low frequency, the small sample sizes often available for research and the potential for loss of viability during extraction. Because immune cells infiltrating tumors are usually present as passengers rather than permanent residents in general they are easier to release from the tissue matrix.
Dissociating tumor tissue while maintaining cellular integrity is technically challenging and has traditionally been performed using a combination of mechanical and enzymatic steps to prepare single cell suspensions4-8. This approach involves lengthy incubation periods and is associated with a significant reduction in cell viability as well as the loss of cell surface receptors by enzymatic cleavage. High quality flow cytometric studies characterizing TIL in the tumor microenvironment as well as clean purifications of CD45+ subpopulations by flow cytometry or antibody-coated beads are more difficult to achieve from enzyme-digested tumor tissue. In addition, the supernatant (SN) from the resulting tumor homogenate is not amenable to further analysis including quantification of secreted proteins (cytokines, chemokines, immunoglobulins or tumor antigens) or experimental treatment of normal cells, because of the potential for protein degradation in the enzymatic digests.
In our search for a method to prepare single cell homogenates from breast tissues [including tumor, non-adjacent non-tumor (NANT) and normal (from mammary reductions) breast tissues] without enzymatic digestion, we tested a variety of mechanical homogenization techniques. Homogenates prepared using a mechanical dissociator had increased cell viability (2-fold) and total cell recovery (2-fold) while preserving surface receptor expression. Enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells suggesting they were all released in the initial homogenate. Thus, this rapid and simple approach allows both qualitative and quantitative assessment of the CD45+ subpopulations present in various normal and malignant human tissues. An added advantage of this approach is that the SN from the initial homogenate (primary tissue SN) can be collected and stored for further analysis or experimentation.

<table border="0" cellpadding="0" cellspacing="0" width="980">
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;">Equipment</td>
			<td style="width:163px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:409px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">GentleMacs Dissociator&#160;</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-093-235</td>
			<td>BD Medimachine is somewhat equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Centrifuge 5810 R</td>
			<td style="width:163px;">Eppendorf</td>
			<td style="width:137px;">N/A</td>
			<td>or other standard table top centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Centrifuge 5417 R</td>
			<td style="width:163px;">Eppendorf</td>
			<td style="width:137px;">N/A</td>
			<td>or other standard microcentrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Esco Class II A2 Biosafety Cabinet</td>
			<td>ESCO global</td>
			<td style="width:137px;">N/A</td>
			<td>or other standard BSL2 hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Inverted Microscope</td>
			<td style="width:163px;">Nikon eclipse TS100</td>
			<td style="width:137px;">N/A</td>
			<td>or other microscope compatible for a hemacytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">B&#252;rker Chamber</td>
			<td style="width:163px;">Marienfield&#160;</td>
			<td style="width:137px;">640210</td>
			<td>or other standard hemacytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Navios Flow Cytometer</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:137px;">N/A</td>
			<td>or other flow cytometer (8-10 color recommended)</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;"></td>
			<td style="width:163px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;">Materials</td>
			<td style="width:163px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">GentleMacs C-Tube</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-096-344</td>
			<td>BD Medimachine uses Filcon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Cell Culture Dish</td>
			<td style="width:163px;">Sarstedt</td>
			<td style="width:137px;">72,710</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Disposable Scalpel</td>
			<td style="width:163px;">Swann-Morton</td>
			<td style="width:137px;">0510</td>
			<td>or standard single use sterile scalpel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD Cell Strainer 40&#181;m</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">734-0002</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD Falcon Tube 50mL</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">352070</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD Falcon Tube 15mL</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">352097</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD FACS Tube 5mL</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">352008</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Sterile Pasteur Pipette 5 mL&#160;</td>
			<td style="width:163px;">VWR</td>
			<td style="width:137px;">612-1685</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Microfuge Tube 1.5 mL</td>
			<td style="width:163px;">Eppendorf</td>
			<td style="width:137px;">7805-00</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;"></td>
			<td style="width:163px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;">Reagents</td>
			<td style="width:163px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">X-Vivo 20</td>
			<td style="width:163px;">Lonza</td>
			<td style="width:137px;">BE04-448Q</td>
			<td>serum-free medium recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Phosphate buffered saline</td>
			<td style="width:163px;">Lonza</td>
			<td style="width:137px;">BE17-516F</td>
			<td>standard physiological PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Trypan blue&#160;</td>
			<td style="width:163px;">VWR</td>
			<td style="width:137px;">17942E</td>
			<td>or other vital stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">VersaLyse</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:137px;">A09777</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Fixable viability Dye eFluor 780&#160;</td>
			<td style="width:163px;">eBioscience</td>
			<td style="width:137px;">65-0865-14</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD3 FITC</td>
			<td style="width:163px;">BD Biosciences</td>
			<td style="width:137px;">345763</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD3 Vio Blue</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-094-363</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD4 PE</td>
			<td style="width:163px;">BD Biosciences</td>
			<td style="width:137px;">345769</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD4 APC</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-091-232</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD8 ECD</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:137px;">737659</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD8 PerCP</td>
			<td style="width:163px;">BD Biosciences</td>
			<td style="width:137px;">345774</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD19 APC-Vio770</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-096-643</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD45 VioGreen</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-096-906</td>
			<td>for flow cytometry experiments</td>
		</tr>
	</tbody>
</table>

a simple and rapid protocol to non-enzymatically dissociate fresh human tissues for the analysis of infiltrating lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

Enzymatic digestion of tissue fragments with either commercially available tissue dissociation solutions or various laboratory mixtures of collagenase, DNase and/or hyaluronidase inhibitors, cleave a wide variety of receptors on the surface of cells. Our studies, initially focused on CD4+ T cells infiltrating breast tumors, were quickly presented with a major technical problem due to cleavage of surface CD4 receptors using standard enzymatic digestion protocols4-8. We tested a variety of collagenase...

Watch this Scientific Journal Video about A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes at JoVE.com

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

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Immunology and Infection

19.9K vistas.  Université Libre de Bruxelles. This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.