Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

Summary

This protocol was developed to quantitatively identify tumor microenvironment components in glioblastoma patient resections using chromogenic immunohistochemistry and ImageJ.

Abstract

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within patient samples of glioblastoma, the deadliest and most invasive brain cancer. Not only are quantitative methods beneficial for accurately describing diseased tissues, they can also potentially contribute to more accurate prognosis, diagnosis, and the development of tissue-engineered systems and replacements. In glioblastoma, glial cells, such as microglia and astrocytes, have been independently correlated with poor prognosis based on pathologist grading. However, the state of these cells and other glial cell components has not been well-described quantitatively. This can be difficult due to the large processes that mark these glial cells. Furthermore, most histological analyses focus on the overall tissue sample or only within the bulk of the tumor, as opposed to delineating quantifications based on regions within the highly heterogeneous tissue. Here, we describe a method for identifying and quantitatively analyzing the populations of glial cells within the tumor bulk and adjacent regions of tumor resections from glioblastoma patients. We used chromogenic immunohistochemistry to identify the glial cell populations in patient tumor resections and ImageJ to analyze percent coverage of staining for each glial population. With these techniques we are able to better describe the glial cells throughout regions of the glioma tumor microenvironment.

Introduction

Glioblastoma (GBM), the most common and malignant brain cancer, is characterized by highly diffuse invasion from the primary tumor bulk into the surrounding healthy brain parenchyma^1,2. This diffuse invasion makes the tumor particularly difficult to resect fully, and the invading cancer cells that remain post-therapy is the most common reason for inevitable recurrence^2,3,4. Previously, we found inhibiting the diffuse glioma cell invasion to be therapeutically beneficial⁵, however little is known about the complex mechanisms contributing to GBM invasion. The tumor microenvironment, or tissue surrounding the cancer, has been implicated in the progression of tumors in multiple cancers^6,7. The glioblastoma tumor microenvironment, in particular, is relatively under-characterized and is uniquely complex, composing of multiple glial cells, such as astrocytes, microglia, and oligodendrocytes, as well as extracellular matrix, soluble factors, and biophysical factors. Experimentally, astrocytes and microglia have been shown to increase glioma progression and invasion^8,9,10, but the composition of all glial cells in the native human brain microenvironment is unknown.

We previously showed microenvironmental components can predict patient survival by quantitatively analyzing cellular components of the glioblastoma microenvironment and incorporating our analyses into a proportional hazards model¹¹. Here, we describe the quantitative analysis method for identifying the populations of glial cells within the tumor bulk and adjacent regions of tumor resections from glioblastoma patients. We used chromogenic immunohistochemistry to identify the glial cell populations and ImageJ to analyze percent coverage of staining for each glial population. Assessing percent coverage creates a simple measurement for determining the morphological differences of cells, particularly those affected by interactions with cancer cells. Previous studies for quantifying histopathological staining use standard staining such as hematoxylin and eosin¹² or Masson's trichrome¹³, which do not take advantage of the specificity of antibody-based immunohistochemistry staining. Our method was developed to directly quantify the glial populations within glioblastoma patient tumor resections, which we aim to use to elucidate the complex glioblastoma microenvironment.

Protocol

This protocol identifies cellular components in formalin-fixed paraffin embedded (FFPE) samples, as is typical for banked clinical patient samples. Paraffin embedding allows for the best maintenance of cellular and tissue morphology as well as has better longevity of sections. The samples used for this analysis were accessed through the University of Virginia Biorepository and Tissue Research Facility. Patient samples were selected by a neuropathologist based on a definitive diagnosis of glioblastoma (astrocytoma, WHO grade IV) who had completed tumor resections at the University of Virginia between 2010 and 2013, and were de-identified prior to this analysis¹¹.

1. FFPE Sample Deparaffinization and Rehydration

NOTE: This portion of the protocol is specific to FFPE samples. While paraffin-embedded samples can be more useful for this analysis because of the preservation of cellular and tissue morphology, this analysis can also be done with frozen sections. If using frozen sections, this portion can be omitted and proceed directly to chromogenic immunohistochemistry.

1. Perform the following washes for 5 min each: Xylene, Xylene, 100% Ethanol, 100% Ethanol, 95% Ethanol, 95% Ethanol, 70% Ethanol, Deionized water, Deionized water

2. Antigen Retrieval

NOTE: This portion of the protocol is necessary to break methylene bridges formed during formalin fixation of FFPE samples and expose antigen sites for antibodies to bind.

1. Dilute Tris-based high pH antigen unmasking solution at manufacturer recommendation in distilled water.
2. Perform heat-mediated antigen retrieval using a microwave. Other forms of heat-mediated retrieval (such as pressure cooker, vegetable steamer, or boiling water) would also suffice.
   1. Add the diluted unmasking solution into a non-sealed microwaveable vessel. Place slides into vessel. Place slides into microwave.
   2. Boil for 20 min at high power. Monitor liquid levels for evaporation, and replenish with distilled water as necessary.
3. Allow samples to cool in solution for 1 h at room temperature.

3. Chromogenic Immunohistochemistry

1. Outline tissue sample with a hydrophobic pen to minimize volume of reagents necessary to cover sample.
   NOTE: Be sure to keep tissue samples hydrated and do not let them dry out as this will affect staining efficacy.
2. Pipet enough permeabilization solution (Tris-buffered saline (TBS) + 0.01% Triton-X) to cover tissue sample (typically about 100 - 200 µL).
3. Remove and discard solution and repeat step 3.2.
4. Incubate samples at room temperature with blocking solution (2.5% horse serum + permeabilization solution).
5. Incubate samples overnight in 4 ºC with primary antibodies diluted in blocking solution.
   NOTE: All primary antibodies used here are diluted at 1:200, but optimal dilutions can be determined using serial dilutions starting with manufacturer recommendation. Detailed information on antibodies used in this protocol was previously published¹¹.
6. Detect primary antibodies using a horseradish peroxidase polymer reagent corresponding with the primary antibody host animal, following manufacturer protocol.
7. Pipet enough permeabilization solution to cover tissue sample and incubate for 5 min.
8. Remove and discard solution and repeat Step 3.7.
9. Incubate slides in 0.3% H₂O₂ in 1x TBS for 15 min.
10. Develop samples with a peroxidase diaminobenzidine (DAB) substrate for 2 - 10 min until desired stain intensity is achieved.
11. Counterstain samples to identify cell nuclei, such as with hematoxylin, following manufacturer protocol.
12. Dehydrate samples with 100% ethanol and xylene.
13. Mount samples permanently with mounting media.

4. Regions of Interest Identification

1. Image slides under brightfield microscopy capable of high resolution images.
   NOTE: Use imaging cellular components at a minimum of 20x resolution.
2. Move camera to specific regions of interest throughout tissue samples.
3. Save images as TIFF files for quantification.

5. Image Analysis

1. Open images in ImageJ for quantification of percent coverage.
2. Use the Threshold Colour plugin to remove purple color from hematoxylin stained nuclei.
3. Convert image to 8-bit.
4. Add threshold without dark background.
5. Process image using one of the 17 pre-loaded ImageJ threshold filters (i.e. MaxEntropy) so only DAB stained portions are included in the threshold.
   NOTE: Select the optimal pre-loaded threshold filter that minimizes inclusion of background staining. This can depend on the quality of staining and specificity of antibody. Use the same threshold for all technical replicates within each patient sample.
6. Apply threshold.
7. Measure percent area of thresholded image.
8. Average percent area coverage for multiple regions within each sample.

Results

For this analysis, two regions of interests within our tumor resections - the primary tumor bulk and the adjacent regions, primarily composed of healthy tissue with diffuse invading cancer cells (Figure 1A, 1B) - were identified by collaborating neuropathologists on hematoxylin and eosin stained patient samples. Within each patient sample, positive staining for astrocytes (Figure 1C), microglia (

Discussion

Our method proposed here is a quantitative approach to analyzing histological samples stained using traditional chromogenic immunohistochemistry. Current methodology for this type of analysis includes similar staining protocols followed by grading by independent pathologists. This method has been reliable, yet for a number of applications, a more precise understanding of the cellular make-up is required, such as better understanding of the heterogeneity associated with tumors and accurate recapitulation of tumors for in ...

Materials

Name	Company	Catalog Number	Comments
Xylene	Fisher Chemical	X3P
Ethanol
High pH antigen unmasking solution	Vector Labs	H-3301
TBS
Triton-X	Amresco	9002-93-1
Horse serum
Anti-ALDH1L1	abcam	ab56777
Anti-Iba1	abcam	ab5076
Anti-Oligodendrocyte Specific Protein1	abcam	ab53041
ImmPRESS anti-goat	Vector Labs	MP-7405
ImmPRESS Universal (anti-mouse/rabbit)	Vector Labs	MP-7500
Hydrogen peroxide	Sigma Aldrich	216763
ImmPACT DAB substrate	Vector Labs	SK-4105
Hematoxylin counterstain	ThermoScientific	72404
Histochoice Mounting Media	Amresco	H157-475
Aperio Scanscope	Leica Biosystems
Image Scanscope	Leica Biosystems
Super HT PAP Pen	Research Products International	195506