Name: expanding the comprehension of the tumor microenvironment using mass spectrometry imaging of formalin-fixed and paraffin-embedded tissue samples
Uploaded: 2022-06-29T13:00:00
Description: Watch this Scientific Journal Video about Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples at JoVE.com

The authors acknowledge Don Norwood from Editing Services, Research Medical Library at MD Anderson for editing this article and the Multiplex Immunofluorescence and Image Analysis Laboratory at the Department of Translational Molecular Pathology at MD Anderson. This publication resulted in part from research facilitated by the scientific and financial support for the Cancer Immune Monitoring and Analysis Centers-Cancer Immunologic Data Commons Network (CIMAC-CIDC) provided through the National Cancer Institute (NCI) Cooperative Agreement (U24CA224285) to The University of Texas MD Anderson Cancer Center Cancer Immune Monitoring and Analysis Center (CIMAC).

Tissue samples were obtained for research purposes in accordance with the Institutional Review Board of The University of Texas MD Anderson Cancer Center, and samples were further de-identified.
1. Antibody selection
<ol>
	<li>Define the research questions to choose the best available antibody clones. Use the Human Protein Atlas, published research, and antibody manufacturer websites as research resources. 
	NOTE: The selection of adequate human tissue controls and the ...

<ol>
	<li>Laplane, L., Duluc, D., Bikfalvi, A., Larmonier, N., Pradeu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+the+tumour+microenvironment.">Beyond the tumour microenvironment.</a> International Journal of Cancer. 145 (10), 2611-2618 (2019).</li><li>Galli, 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=Relevance+of+immune+cell+and+tumor+microenvironment+imaging+in+the+new+era+of+immunotherapy.">Relevance of immune cell and tumor microenvironment imaging in the new era of immunotherapy.</a> Journal of Experimental &amp; Clinical Cancer Research. 39 (1), 89 (2020).</li><li>Liu, C. C., Steen, C. B., Newman, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+approaches+for+characterizing+the+tumor+immune+microenvironment.">Computational approaches for characterizing the tumor immune microenvironment.</a> Immunology. 158 (2), 70-84 (2019).</li><li>Eisenstein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+sorting:+Divide+and+conquer.">Cell sorting: Divide and conquer.</a> Nature. 441 (7097), 1179-1185 (2006).</li><li>Scognamiglio, G., 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=Multiplex+immunohistochemistry+assay+to+evaluate+the+melanoma+tumor+microenvironment.">Multiplex immunohistochemistry assay to evaluate the melanoma tumor microenvironment.</a> Methods in Enzymology. 635, 21-31 (2020).</li><li>Guo, N., 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=A+34-marker+panel+for+imaging+mass+cytometric+analysis+of+human+snap-frozen+tissue.">A 34-marker panel for imaging mass cytometric analysis of human snap-frozen tissue.</a> Frontiers in Immunology. 11, 1466 (2020).</li><li>Fu, 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=Spatial+architecture+of+the+immune+microenvironment+orchestrates+tumor+immunity+and+therapeutic+response.">Spatial architecture of the immune microenvironment orchestrates tumor immunity and therapeutic response.</a> Journal of Hematology &amp; Oncology. 14 (1), 98 (2021).</li><li>Rost, 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=Multiplexed+ion+beam+imaging+analysis+for+quantitation+of+protein+expresssion+in+cancer+tissue+sections.">Multiplexed ion beam imaging analysis for quantitation of protein expresssion in cancer tissue sections.</a> Laboratory Investigation. 97 (8), 992-1003 (2017).</li><li>Keren, 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=A+structured+tumor-immune+microenvironment+in+triple+negative+breast+cancer+revealed+by+multiplexed+ion+beam+imaging.">A structured tumor-immune microenvironment in triple negative breast cancer revealed by multiplexed ion beam imaging.</a> Cell. 174 (6), 1373-1387 (2018).</li><li>Mathieson, W., Thomas, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+formalin-fixed,+paraffin-embedded+biospecimens+must+be+used+in+genomic+medicine:+An+evidence-based+review+and+conclusion.">Why formalin-fixed, paraffin-embedded biospecimens must be used in genomic medicine: An evidence-based review and conclusion.</a> The Journal of Histochemistry and Cytochemistry. 68 (8), 543-552 (2020).</li><li>Baghban, R., 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=Tumor+microenvironment+complexity+and+therapeutic+implications+at+a+glance.">Tumor microenvironment complexity and therapeutic implications at a glance.</a> Cell Communication and Signaling. 18 (1), 59 (2020).</li><li>Cai, S., Allam, M., Coskun, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+spatial+bioimaging+for+combination+therapy+design.">Multiplex spatial bioimaging for combination therapy design.</a> Trends in Cancer. 6 (10), 813-818 (2020).</li><li>Allam, M., Cai, S., Coskun, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+bioimaging+of+single-cell+spatial+profiles+for+precision+cancer+diagnostics+and+therapeutics.">Multiplex bioimaging of single-cell spatial profiles for precision cancer diagnostics and therapeutics.</a> NPJ Precision Oncology. 4, 11 (2020).</li><li>Rad, H. 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=The+Pandora's+box+of+novel+technologies+that+may+revolutionize+lung+cancer.">The Pandora's box of novel technologies that may revolutionize lung cancer.</a> Lung Cancer. 159, 34-41 (2021).</li><li>Tan, W. C. 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=Overview+of+multiplex+immunohistochemistry/immunofluorescence+techniques+in+the+era+of+cancer+immunotherapy.">Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy.</a> Cancer Commununications. 40 (4), 135-153 (2020).</li><li>Ptacek, 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=Multiplexed+ion+beam+imaging+(MIBI)+for+characterization+of+the+tumor+microenvironment+across+tumor+types.">Multiplexed ion beam imaging (MIBI) for characterization of the tumor microenvironment across tumor types.</a> Laboratory Investigation. 100 (8), 1111-1123 (2020).</li></ol>

The comprehensive elucidation of the complex, intricate interactions among the multiple components of a TME remains a pivotal objective of cancer research. Manufacturers have introduced numerous multiplexed assays as part of this effort, especially over the past 5 years. Multiplexed spatial analysis is a versatile and powerful tool that facilitates the simultaneous categorization of several targets while preserving structural morphology in tumor samples. Spatial analysis techniques can be performed using fluorescence ima...

The importance of the numerous cell types surrounding clonal tumor populations is a crucial element in the categorization of carcinogenesis. Interest in elucidating this tumor microenvironment (TME) composition and interactions has risen continuously following the establishment of immune-based therapy as part of the cancer treatment arsenal. Therefore, treatment strategies have shifted from a tumor-centric approach to a TME-centric one1.
Efforts to elucidate the roles of immune cells in tumor surveillance and cancer development have increased strikingly in recent years2,3. In medical research, a plethora of methods, including cytometry-based methods and singleplex and multiplex imaging technologies, arose as part of this attempt to decipher the unique interactions of multiple elements of TMEs.
Pioneering methods such as flow cytometry (invented in the 1960s), fluorescence-activated cell sorting, and mass cytometry are focused mainly on identifying and quantifying TME components4. Even though cytometry-based quantitative techniques allow for immune landscape phenotyping, determining the cellular spatial distribution is impossible. Conversely, methods such as standard singleplex immunohistochemistry preserve the tissue architecture and enable researchers to analyze cellular distribution, though a reduced number of targets in a single tissue section is a limitation of these methods5,6. Over the past several years, multiplexed imaging technologies for single-cell resolution such as multiplex immunofluorescence, barcoding fluorescence imaging, and imaging mass spectrometry have emerged as comprehensive strategies for acquiring information on simultaneous marker staining using the same tissue section7.
Here we present a technology that couples metal tagged antibodies and secondary ion mass spectrometry and enables single-cell resolution quantification, marker co-expression (phenotyping), and spatial analysis using formalin-fixed, paraffin-embedded (FFPE), and fresh frozen (FF) tissue samples8,9. FFPE samples are the most widely used materials for tissue archiving samples and represent a more readily available resource for multiplexed imaging technologies than fresh frozen samples10. Additionally, this technology offers the possibility of reacquiring images months after. Herein, we discuss our staining and image processing protocols using FFPE tissue samples.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100% Reagent Alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>R8382</td>
			<td></td>
		</tr>
		<tr>
			<td>95% Reagent Alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>R3404</td>
			<td></td>
		</tr>
		<tr>
			<td>80% Reagent Alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>R3279</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Reagent Alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>R315</td>
			<td></td>
		</tr>
		<tr>
			<td>20X TBS-T</td>
			<td>Ionpath</td>
			<td>567005</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Low-Barium PBS pH 7.4</td>
			<td>Ionpath</td>
			<td>567004</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Tris pH 8.5&#160;</td>
			<td>Ionpath</td>
			<td>567003</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#176;C Refrigerator</td>
			<td>ThermoScientific</td>
			<td>REVCO</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerosol Barrier Pipette Tips P10</td>
			<td>Olympus</td>
			<td>24-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerosol Barrier Pipette Tips P20</td>
			<td>Olympus</td>
			<td>24-404</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerosol Barrier Pipette Tips P200</td>
			<td>Olympus</td>
			<td>24-412</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerosol Barrier Pipette Tips P1000</td>
			<td>Olympus</td>
			<td>24-430</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugal Filter Ultrafree-MC</td>
			<td>Fisher Scientific</td>
			<td>UFC30VV00</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized H2O</td>
			<td>Ionpath</td>
			<td>567002</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma-Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyDip Slide Staining Jar, Green</td>
			<td>Electron Microscopy Sciences</td>
			<td>71385-G</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyDip Slide Staining Jar, Yellow</td>
			<td>Electron Microscopy Sciences</td>
			<td>71385-Y</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyDip Slide Staining Kit (Jar+Rack), White</td>
			<td>Electron Microscopy Sciences</td>
			<td>71388-01</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyDip Stainless Steel Holder</td>
			<td>Electron Microscopy Sciences</td>
			<td>71388-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde 70% EM Grade</td>
			<td>Electron Microscopy Sciences</td>
			<td>16360</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Induced Epitope Retrieval (HIER) buffer: 10X Tris with EDTA, pH 9</td>
			<td>Dako</td>
			<td>S2367</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat resistant slide chamber</td>
			<td>Electron Microscopy Sciences</td>
			<td>62705-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic barrier pen</td>
			<td>Fisher</td>
			<td>50-550-221</td>
			<td></td>
		</tr>
		<tr>
			<td>MIBI/O software</td>
			<td>Ionpath</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>MIBIcontrol software</td>
			<td>Ionpath</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>MIBIslide</td>
			<td>Ionpath</td>
			<td>567001</td>
			<td></td>
		</tr>
		<tr>
			<td>MIBIscope</td>
			<td>Ionpath</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5415D</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica</td>
			<td>RM2135</td>
			<td></td>
		</tr>
		<tr>
			<td>Moisture Chamber (Humid Chamber)</td>
			<td>Simport</td>
			<td>M922-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS) Tablets</td>
			<td>Fisher Scientific</td>
			<td>BP2944100</td>
			<td></td>
		</tr>
		<tr>
			<td>PT Module</td>
			<td>Thermo Scientific</td>
			<td>A80400012</td>
			<td></td>
		</tr>
		<tr>
			<td>Rapid-Flow Sterile Disposable Filter Units</td>
			<td>Fisher Scientific</td>
			<td>097403A</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>BioRocker</td>
			<td>S2025</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin column (Ultrafree-MC Spin Filter, 0.5mL 0.1&#956;m )</td>
			<td>MillQ</td>
			<td>UFC30VV00</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide oven</td>
			<td>Fisher Scientific</td>
			<td>6901</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaccum Cabinet Desiccator</td>
			<td>VWR</td>
			<td>30621-076</td>
			<td></td>
		</tr>
		<tr>
			<td>Task-whipe</td>
			<td>Kimberly Clark</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sigma-Aldrich</td>
			<td>534056-4L</td>
			<td></td>
		</tr>
	</tbody>
</table>

expanding the comprehension of the tumor microenvironment using mass spectrometry imaging of formalin-fixed and paraffin-embedded tissue samples

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the tumor immune landscape. Although standard immunohistochemistry is suitable for studying tissue architecture, it is limited to the analysis of a small number of markers. Conversely, techniques such as flow cytometry can evaluate multiple markers simultaneously, although information about tissue morphology is lost. In recent years, multiplexed strategies that integrate phenotypic and spatial analysis have emerged as comprehensive approaches to the characterization of the tumor immune landscape. Herein, we discuss an innovative technology combining metal-labeled antibodies and secondary ion mass spectrometry focusing on the technical steps in assay development and optimization, tissue preparation, and image acquisition and processing. Before staining, a metal-labeled antibody panel must be developed and optimized. This hi-plex image system supports up to 40 metal-tagged antibodies in a single tissue section. Of note, the risk of signal interference increases with the number of markers included in the panel. After panel design, particular attention should be given to the metal isotope assignment to the antibody to minimize this interference. Preliminary panel testing is performed using a small subset of antibodies and subsequent testing of the entire panel in control tissues. Formalin-fixed, paraffin-embedded tissue sections are obtained and mounted on gold-coated slides and further stained. The staining takes 2 days and closely resembles standard immunohistochemical staining. Once samples are stained, they are placed in the image acquisition instrument. Fields of view are selected, and images are acquired, uploaded, and stored. The final stage is image preparation for the filtering and removal of interference using the system&#39;s image processing software. A disadvantage of this platform is the lack of analytical software. However, the images generated are supported by different computational pathology software.

Tonsil and lung adenocarcinoma TMA tissue sections (5 mm thick) were obtained and placed on the middle of gold-coated slides following the specifications regarding tissue size and secure margins of the slides. Free glass margins of 5 mm and 10 mm between the edge of the tissue and the lateral and inferior borders of the glass slides, respectively, are necessary for optimal staining. The tissue sections were baked overnight in an oven prior to staining to assure proper adherence of the section to the slide. The antibody p...

Watch this Scientific Journal Video about Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples at JoVE.com

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the ...

These protocols enable single cell resolution quantification, marker co-expression and a special analysis using a formalin-fixed and paraffin-embedded and fresh frozen tissue samples to elucidate the roles of immune cells in cancer. This technique is a powerful tool that facilitate the simultaneous characterization of a 40 marker on a single tissue section, widely preserve the structure of the tissue in the samples. The critical step is antibody optimization and validation.Failure to complete the step, can result in low quality images that may compromise a retaining high quality reproducible data and results. Begin by spinning down all the antibody containing tubes at 10, 000 x G for five minutes, add individual antibodies to the blocking buffer and mix well. Do not disturb the bottom of the antibody tube when pipetting it.Filter the antibody panel by pre-wetting a 0.1 micrometer centrifugal filter device with 100 microliters of blocking buffer. Spin the filter at 10, 000 x G for two minutes and remove the blocking buffer flow-through with a pipette. Then transfer the antibody panel into a spin column and spin the filter at 10, 000 x G for two minutes.Discard the spin column and use the flow-through as the filtered antibody panel. For the antibody staining, remove the blocking buffer from the slides by tipping each slide on its side and gently tapping the edges against a task wiper. Place the slides in a moisture chamber and add 100 to 200 microliters of antibody master mix to the tissue.Add positive and negative control slides to the staining batch and incubate the slides at 4 degrees Celsius overnight. To set up the instrument, login to the control software, and click on Wake Up, if the instrument is in sleep mode. Click on Exchange Sample to load a slide and then click on Continue to open the door.Put the slide in the loading slot with a sample facing up and the label on the right, click on Continue to close the door. Select the project and then select the slide name for the loaded sample. Visualize the panoramic image on the slide optical image pane for the field of view or FOV selection, click on the Mode menu and select Secondary Electron Detector, then click on the Imaging Mode menu and pick QC 300 micrometer.Click on Jog Stage to control the FOV location and then click on the arrows to navigate and select the position of the FOV. Click add FOV, set 400 micrometer by 400 micrometer as the field size and select Fine as the Imaging Mode and 1 millisecond of Dwell Time, click on Confirm. After creating an FOV list, proceed to the image focus and adjust the Stigmation by moving the beam to a tissue region that will not be imaged.Adjust the parameters until a clear central image is obtained, click on Start Run. The acquired images will be automatically uploaded and stored in the web-based image management application. The matrix shown here describes the percent crosstalk derived from the probe purity and oxides.Here blue boxes indicate greater than or equal to 0.5%crosstalk and clear boxes indicate less than 0.5%crosstalk. For the Mass tags, probes that contributed at least 0.5%crosstalk into no channels, one or two channels, or more than two channels are shown here. Mass channels receiving at least 0.5%crosstalk from no probes, one or two probes, or more than two probes are also shown.The representative staining images in different tissues like lung adenocarcinoma, non-neoplastic kidney and tonsil are depicted here. An image of a tonsil tissue section stained using this technique and visualized using a third party digital image analysis software program before filtering and correction is presented here. The image of the same section under the same conditions after filtering and correction steps is shown here.This method generate images that can be uploaded to a third party digital image analysis software, allowing comprehensive analysis such as tissue compartmentalization, cell segmentation, marker co-expression, as well as neighbor analysis and distant analysis. Highly multiplex imaging methods are available through research. By constructing different panels, researchers can investigate cancer, normal, paraneoplastic, inflammatory and infectious samples.

expanding-comprehension-tumor-microenvironment-using-mass

Research

JoVE Journal

Cancer Research

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and eosin (H&amp;E) staining but can be visualized through elastic staining. One of the important benefits of elastic staining is that it makes it easy to determine whether tumor cells have invaded beyond the elastic lamina. This helps in examining the extent of peritoneal surface invasion, thereby distinguishing the pT3 and pT4 stages in gastrointestinal cancer. In this study, we present a protocol to identify elastic fibers in the formalin-fixed, paraffin-embedded pT3N0M0 gastric cancer tissue sections. We prepare 5-&#181;m paraffin sections fixed on slides, and then all the slides are deparaffinated and rehydrated during the staining procedure. Subsequently, all the sections are oxidized by potassium permanganate, bleached by oxalic acid, stained by elastic staining, and counterstained by Van Gieson's. Finally, we examine the effect of staining; elastic lamina is stained blue-black and collagen fibers are stained red, while the cancer cells are stained in varying shades of yellow. We also describe in detail the methods used for determining the positional relationship between cancer cells and elastic lamina. This method is simple, low-cost, and widely applicable in the identification of peritoneal surface invasion in gastrointestinal cancer because of its outstanding selectivity for elastic fibers and authentic results.

Here, we present a protocol of elastic staining to identify elastic fibers in pT3N0M0 gastric cancer tissues on formalin-fixed, paraffin-embedded sections. Subsequently, we describe a method to determine whether tumor cells invade beyond the elastic lamina.

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and ...

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength and flexibility and allows it to fulfill its functions. Bone is continuously exposed to a variety of stimuli, which in pathological conditions can deregulate bone remodeling. To study bone biology and diseases and evaluate potential therapeutic agents, it has been necessary to develop in vitro and in vivo models.
This manuscript describes the dissection process and culture conditions of calvarias isolated from neonatal mice to study bone formation and the bone tumor microenvironment. In contrast to in vitro and in vivo models, this ex vivo model allows preservation of the three-dimensional environment of the tissue as well as the cellular diversity of the bone while culturing under defined conditions to simulate the desired microenvironment. Therefore, it is possible to investigate bone remodeling and its mechanisms, as well as the interactions with other cell types, such as the interactions between cancer cells and bone.
The assays reported here use calvarias from 5-7 day old BALB/C mice. The hemi-calvarias obtained are cultured in the presence of insulin, breast cancer cells (MDA-MB-231), or conditioned medium from breast cancer cell cultures. After analysis, it was established that insulin induced new bone formation, while cancer cells and their conditioned medium induced bone resorption. The calvarial model has been successfully used in basic and applied research to study bone development and cancer-induced bone diseases. Overall, it is an excellent option for an easy, informative, and low-cost assay.

The ex vivo culture of bone explants can be a valuable tool for the study of bone physiology and the potential evaluation of drugs in bone remodeling and bone diseases. The presented protocol describes the preparation and culture of calvarias isolated from newborn mice skulls, as well as its applications.

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength ...

Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample types have been analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to visualize the distribution of proteins, lipids, and metabolites across the biological sample of interest. We have developed a novel sample preparation method that combines the strengths of the three previous applications to address an underexplored approach for identifying chemical communication in cancer, by seeding mammalian cell cultures into agarose in coculture with healthy tissues followed by desiccation of the sample. Mammalian tissue and cells are cocultured in close proximity allowing chemical communication via diffusion between the tissue and cells. At specific time points, the agarose-based sample is dried in the same manner as microbial colonies prepared for IMS analysis. Our method was developed to model the communication between high grade serous ovarian cancer derived from the fallopian tube as it interacts with the ovary during metastasis. Optimization of the sample preparation resulted in the identification of norepinephrine as a key chemical component in the ovarian microenvironment. This newly developed method can be applied to other biological systems that require an understanding of chemical communication between adjacent cells or tissues.

A novel method of sample preparation was developed to accommodate cell and tissue coculture to detect small molecule exchange using imaging mass spectrometry.

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample ...

Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and Fixed Tissue Analysis

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary sites. Recently, we established that cancer cell dissemination in primary breast cancer and at metastatic sites in the lung occurs only at doorways called Tumor MicroEnvironment of Metastasis (TMEM). TMEM doorway number is prognostic for distant recurrence of metastatic disease in breast cancer patients. TMEM doorways are composed of a cancer cell which over-expresses the actin regulatory protein Mena in direct contact with a perivascular, proangiogenic macrophage which expresses high levels of TIE2 and VEGF, where both of these cells are tightly bound to a blood vessel endothelial cell. Cancer cells can intravasate through TMEM doorways due to transient vascular permeability orchestrated by the joint activity of the TMEM-associated macrophage and the TMEM-associated Mena-expressing cancer cell. In this manuscript, we describe two methods for assessment of TMEM-mediated transient vascular permeability: intravital imaging and fixed tissue immunofluorescence. Although both methods have their advantages and disadvantages, combining the two may provide the most complete analyses of TMEM-mediated vascular permeability as well as microenvironmental prerequisites for TMEM function. Since the metastatic process in breast cancer, and possibly other types of cancer, involves cancer cell dissemination via TMEM doorways, it is essential to employ well established methods for the analysis of the TMEM doorway activity. The two methods described here provide a comprehensive approach to the analysis of TMEM doorway activity, either in na&#239;ve or pharmacologically treated animals, which is of paramount importance for pre-clinical trials of agents that prevent cancer cell dissemination via TMEM.

We describe two methods for assessing transient vascular permeability associated with tumor microenvironment of metastasis (TMEM) doorway function and cancer cell intravasation using intravenous injection of high-molecular weight (155 kDa) dextran in mice. The methods include intravital imaging in live animals and fixed tissue analysis using immunofluorescence.

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

Industrialized, Artificial Intelligence-guided Laser Microdissection for Microscaled Proteomic Analysis of the Tumor Microenvironment

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell populations. To characterize proteome-level variation and tumor heterogeneity at scale, high-throughput methods are needed to selectively isolate discrete cellular populations in solid tumor malignancies. This protocol describes a high-throughput workflow, enabled by artificial intelligence (AI), that segments images of hematoxylin and eosin (H&#38;E)-stained, thin tissue sections into pathology-confirmed regions of interest for selective harvest of histology-resolved cell populations using laser microdissection (LMD). This strategy includes a novel algorithm enabling the transfer of regions denoting cell populations of interest, annotated using digital image software, directly to laser microscopes, thus enabling more facile collections. Successful implementation of this workflow was performed, demonstrating the utility of this harmonized method to selectively harvest tumor cell populations from the TME for quantitative, multiplexed proteomic analysis by high-resolution mass spectrometry. This strategy fully integrates with routine histopathology review, leveraging digital image analysis to support enrichment of cellular populations of interest and is fully generalizable, enabling harmonized harvests of cell populations from the TME for multiomic analyses.

This protocol describes a high-throughput workflow for artificial intelligence-driven segmentation of pathology-confirmed regions of interest from stained, thin tissue section images for enrichment of histology-resolved cell populations using laser microdissection. This strategy includes a novel algorithm enabling the transfer of demarcations denoting cell populations of interest directly to laser microscopes.

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell ...

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 ...