Methods to Enable Spatial Transcriptomics of Bone Tissues

Summary

Here, we describe a method that allows for the decalcification of freshly obtained bone tissues and the preservation of high-quality RNA. A method is also illustrated for sectioning Formalin Fixed Paraffin Embedded (FFPE) samples of non-demineralized bones to obtain good quality results if fresh tissues are not available or cannot be collected.

Abstract

Understanding the relationship between the cells and their location within each tissue is critical to uncover the biological processes associated with normal development and disease pathology. Spatial transcriptomics is a powerful method that enables the analysis of the whole transcriptome within tissue samples, thus providing information about the cellular gene expression and the histological context in which the cells reside. While this method has been extensively utilized for many soft tissues, its application for the analyses of hard tissues such as bone has been challenging. The major challenge resides in the inability to preserve good quality RNA and tissue morphology while processing the hard tissue samples for sectioning. Therefore, a method is described here to process freshly obtained bone tissue samples to effectively generate spatial transcriptomics data. The method allows for the decalcification of the samples, granting successful tissue sections with preserved morphological details while avoiding RNA degradation. In addition, detailed guidelines are provided for samples that were previously paraffin-embedded, without demineralization, such as samples collected from tissue banks. Using these guidelines, high-quality spatial transcriptomics data generated from tissue bank samples of primary tumor and lung metastasis of bone osteosarcoma are shown.

Introduction

Bone is a specialized connective tissue comprised mainly of fibers of collagen type 1 and inorganic salts¹. As a result, bone is incredibly strong and stiff while being, at the same time, light and trauma-resistant. The great strength of bone derives from its mineral content. In fact, for any given increase in the percentage of mineral content, stiffness increases by five-fold². Consequently, investigators face significant problems when they analyze, by means of histological sectioning, the biology of a bone specimen.

Undecalcified bone histology is feasible and sometimes required, depending on the type of investigation (e.g., to study the micro-architecture of bone); it is, however, very challenging, especially if the specimens are large. In these cases, tissue processing for histological purposes requires several modifications of the standard protocols and techniques³. In general, to perform common histological evaluations, bone tissues are decalcified right after fixation, a process that may require a few days to several weeks, depending on the size of the tissue and the decalcifying agent utilized⁴. Decalcified sections are often used for the examination of bone marrow, the diagnosis of tumors, etc. There are three main types of decalcifying agents: strong acids (e.g., nitric acid, hydrochloric acid), weak acids (e.g., formic acid), and chelating agents (e.g., ethylenediaminetetracetic acid or EDTA)⁵. Strong acids can decalcify bone very rapidly, but they can damage the tissues; weak acids are very common and suitable for diagnostic procedures; chelating agents are by far the most used and appropriate for research application since, in this case, the demineralization process is slow and gentle, allowing for retention of high-quality morphology and preservation of gene and protein information, as required by many procedures (e.g., in situ hybridization, immunostaining). However, when the whole transcriptome needs to be preserved, such as for gene expression analyses, even a slow and gentle demineralization may be detrimental. Therefore, better approaches and methods are needed when the morphological analysis of the tissues needs to be paired with gene expression analyses of the cells.

Thanks to recent improvements in single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics, it is now possible to study the gene expression of a tissue specimen even when formalin fixation paraffin embedding (FFPE) was used to store the tissue samples^6,7,8. This opportunity has unlocked access to a larger number of samples, such as those stored in tissue banks worldwide. If scRNA-seq is to be employed, RNA integrity is the most important requirement; however, in the case of spatial transcriptomics of FFPE samples, both high-quality tissue sections and high-quality RNA are necessary to visualize the gene expression within the histological context of each tissue section. While this has been easily achieved with soft tissues, the same cannot be said for hard tissues like bone. In fact, to the best of our knowledge, no study using spatial transcriptomics has ever been performed on FFPE bone samples. This is because of the lack of protocols that can effectively process FFPE bone tissues while preserving their RNA content. Here, a method to process and decalcify freshly obtained bone tissue samples while avoiding RNA degradation is provided first. Then, recognizing the need for transcriptomics analysis of the FFPE samples collected in tissue banks worldwide, developed guidelines to properly handle FFPE samples of non-demineralized bones are also presented.

Protocol

All animal procedures described below were approved in compliance with the Guide for the Care and Use of Laboratory Animals at the University of Pittsburgh School of Dental Medicine.

1. Method to prepare FFPE blocks of bone tissue samples that require demineralization

1. Preparation of reagents and materials
   1. Prepare EDTA 20% pH 8.0. For 1 L, dissolve 200 g of EDTA in 800 mL of ultrapure water, adjust pH to 8.0 with sodium hydroxide 10 N and finally bring the volume to 1L with ultrapure water. Store at room temperature.
      NOTE: Always use freshly prepared EDTA 20% pH 8.0. Sodium hydroxide (NaOH) 10 N is prepared by dissolving 400 g of NaOH in 1 L of ultrapure water.
   2. Prepare paraformaldehyde (PFA) 4% in a fume hood utilizing 1x PBS without calcium and magnesium.
      NOTE: Designate an area in a fume hood for working with concentrated paraformaldehyde solutions, or paraformaldehyde solids, and label it as such. To avoid exposure, keep containers closed as much as possible. Use in the smallest practical quantities needed for the experiment being performed. If weighing paraformaldehyde powder and the weighing scale cannot be used in a fume hood, first take a container, then add PFA powder in the hood and cover before returning to the scale to weigh the powder. Always use freshly prepared 4% PFA.
   3. Use a suitable container for each specimen.
      NOTE: For instance, for 1 mg of bone tissue, use a container that allows for at least 1 mL of any solution to be in contact with the bone.
   4. Sterilize all the surgical equipment needed for dissection.
   5. Clean all the areas with a decontaminating solution to remove RNases.
   6. Collect the remaining experimental materials, including ice containers, 70% ethanol spray, and have access to an orbital shaker.
2. Isolation of bone tissue samples from mice
   1. Euthanize the mouse by CO₂ exposure, then perform cervical dislocation and lay it on its back. Spray the mouse skin with 70% ethanol.
   2. Using sterile dissecting scissors, open the skin and expose the leg muscle. Cut the muscle alongside the leg to obtain a clear view of the position of the bone, and gently disarticulate the bone to remove it without damage.
      NOTE: It is not necessary to remove entirely all the muscle attached to the bone. Try to be as fast as possible to limit RNA degradation.
   3. Immediately place the bone sample in a tube containing 4% PFA and place the tube on ice.
   4. Repeat the procedure to collect other bone samples as desired.
   5. Put samples with 4% PFA in agitation on an orbital shaker at 140 rpm at 4 °C for at least 24 h to allow proper fixation.
3. Decalcification of bone tissue samples
   1. Retrieve the bone tissue from the orbital shaker after fixation.
   2. Wash the bone tissue 2 times with 1x PBS for 3 min on an orbital shaker at 140 rpm to remove any PFA left.
   3. Using sterile dissecting scissors, remove any remaining muscles still attached to the bone.
   4. Wash the bone tissue one more time with 1x PBS for 3 min.
   5. Place the bone tissue in a new container with 20% EDTA pH 8.0. Then, place the container on an orbital shaker at 140 rpm for 30 min at room temperature (RT).
   6. Discard the solution, add fresh 20% EDTA pH 8.0 in the container, and place it on an orbital shaker at 140 rpm for 30 min at RT.
   7. Repeat step 1.3.6 10 times.
   8. Discard the solution, add fresh 20% EDTA pH 8.0 in the container, and place it at 4 °C in a cold room on an orbital shaker at 140 rpm overnight.
4. Dehydration of bone tissue samples
   1. Retrieve the bone tissue sample from the container.
   2. Inspect the tissue and verify the efficiency of decalcification by gently turning and twisting the bone. Alternatively, perform an X-ray of the bone using an X-ray machine.
      NOTE: If the specimen flexes easily, a good grade of decalcification has been achieved. If not, decalcification parameters (time, speed of agitation, volume of 20% EDTA pH 8.0. used, etc.) should be increased.
   3. Wash the bone tissue 2 times with 1x PBS for 3 min to remove residues of EDTA.
   4. Place the bone tissue in a new container and initiate dehydration with 70% ethanol. Incubate for 15 min at 140 rpm.
   5. Discard the 70% ethanol solution, put the sample in 90% ethanol solution, and incubate for 15 min at 140 rpm.
   6. Discard the 90% ethanol solution, put the sample in 100% ethanol, and incubate for 15 min at 140 rpm.
   7. Discard the 100% ethanol, put the sample in new 100% ethanol, and incubate for 15 min at 140 rpm.
   8. Discard the 100% ethanol, put the sample in new 100% ethanol, and incubate for 15 min at 140 rpm.
   9. Discard the 100% ethanol, put the sample in new 100% ethanol, and incubate for 30 min at 140 rpm.
   10. Discard the 100% ethanol, put the sample in new 100% ethanol, and incubate for 45 min at 140 rpm.
       ​NOTE: Dehydration can also be performed using an automatic processor. In this case, set up the program with the same conditions reported for the manual dehydration. The reported conditions are for specimens no thicker than 4 mm (i.e., bone tissue samples obtained from mice). For specimens thicker than 4 mm, dehydration timing must be determined experimentally.
5. Clearing of bone tissue samples
   1. Place the bone tissue sample in a new container and initiate the clearing process using xylene. Incubate for 20 min at 140 rpm.
   2. Discard the used xylene, add new xylene, and incubate for an additional 20 min at 140 rpm.
   3. Discard the used xylene, add new xylene, and incubate for 45 min at 140 rpm.
      ​NOTE: Clearing can also be performed using an automatic processor. In this case, set up the program with the same conditions reported for the manual clearing. The reported conditions are for specimens no thicker than 4 mm (i.e., bone tissue samples obtained from mice). For specimens thicker than 4 mm, the timing must be determined experimentally.
6. Infiltration and embedding of bone tissue samples
   1. Retrieve the bone tissue sample from the orbital shaker/automatic processor after clearing.
   2. Place the bone tissue sample in an embedding cassette and initiate infiltration with wax (see Table of Materials). Incubate for 30 min at 60 °C.
   3. Discard the used wax, add new wax, and incubate for 30 min at 60 °C.
   4. Discard the used wax, add new wax, and incubate for 45 min at 60 °C.
   5. Carefully place the bone tissue sample in a mold with the desired orientation.
   6. Allow the specimen block to solidify on a cold surface.
   7. Store the obtained FFPE at 4 °C until ready to start sectioning.
      NOTE: FFPE block can be stored for many years before sectioning is carried out.

2. Guidelines for sectioning un-demineralized FFPE samples

NOTE: The following guidelines provide valuable tips and instructions useful to greatly improve the quality of the tissue sections while avoiding RNA degradation especially in cases of small undecalcified bone specimens. These guidelines may also be applied to decalcified bone specimens when available. For larger bone tissue samples, demineralization should be performed to obtain better quality sections; in such cases, the method reported above should be followed, and parameters (timing, volumes of solutions, etc.) should be adjusted accordingly. The following guidelines are suitable either when FFPE bone tissues have already been processed (e.g., FFPE block collected from tissue banks or other labs) or when the collected sections will be utilized to perform any type of analysis that requires good quality RNA, high-quality tissue sections, or both.

1. Equipment preparation
   1. Spray all work surfaces and instruments with decontaminating solutions to remove RNases.
   2. Prepare a water bath with double distilled water and set the temperature to 42 °C.
   3. Take a microtome blade, spray it with 70% ethanol to remove oil residues, then spray it with decontaminating solutions to remove RNases.
   4. Secure the blade on the microtome. Ensure that the clearance angle is set to 10°.
      NOTE: Always use new blades for sectioning.
   5. Prepare two buckets of ice.
   6. Get tweezers, brushes, and probes, spray them with decontaminating solutions to remove RNases, and place them in one of the two ice buckets.
   7. Prepare an ice bath by adding double distilled water to the other ice bucket.
      NOTE: the FFPE block should not float in the added water; therefore, the amount of added water to the ice bucket must be just enough to allow the sample to be wet without floating.
2. Sectioning
   1. Retrieve the FFPE block from storage at 4 °C and place it on the microtome. Set the command to Trim or to 14 µm of scroll thickness, and start trimming the sample until the tissue is exposed.
      NOTE: If the FFPE block has already been trimmed and the tissue is already exposed, skip step 2.2.1. Verify that the block is without holes and cracks. If so, proceed directly to step 2.2.2. If not, cut a few sections to flatten the surface and avoid the holes and cracks, and then proceed to step 2.2.2.
   2. Place the FFPE block on the ice bath to hydrate the sample. The time of hydration must be determined experimentally.
      1. Start with 2 min and increase the time if hydration is not enough. Do not exceed 10 min. The hydration will expand the tissue and reduce the formation of "Venetian blinds" and lines. Longer hydration times could cause cracks in the paraffin block and, possibly, tissue detachment.
      2. If 10 min is insufficient to hydrate the sample, then perform hydration cycles no longer than 10 min and attempt sectioning again.
      3. If the surface of the block is not smooth and clean due to cracks or cuts, consider re-embedding the sample. RNA quality will not be affected by the re-embedding process.
   3. Put the sample in the specimen clamp of the microtome, align it with the blade, and start sectioning. Set the scroll thickness to 5 µm.
   4. Collect the section and place it in the water bath at 42 °C using cold tweezers and brushes.
      NOTE: Alternatively, if RNA isolation of tissue sections will be performed, collect the sections in a centrifuge tube and follow the manufacturing specifications for RNA preparation (see Table of Materials).
   5. Incubate the section in the water bath to stretch the tissue and eliminate any residual wrinkles and folds.
      NOTE: The time of floating in the water bath for each section must be determined experimentally. Consider leaving the bone sections an extra minute if tissue detachment issues occur. Do not leave the section floating too long since long floating times could damage the tissue.
   6. Place the section on a glass slide, then incubate it for 3 h at 42 °C.
   7. Place the glass slide in a desiccator or an oven overnight at RT for proper drying.
   8. Store the glass slide with the attached section at RT in a slide box.
      NOTE: Slides can be stored for many years at RT. If spatial transcriptomic will be performed, instead of a glass slide, place the section on the spatial gene expression slide provided by the manufacturer and follow the reported recommendations (see the Table of Materials).

Results

The method presented here describes how to process freshly isolated bones to obtain demineralized FFPE samples that can be easily sectioned with a microtome while preserving the RNA integrity (Figure 1). The method has been successfully employed on murine femurs but can be followed for other bone tissue samples of similar dimensions, or it can be adapted for larger bone specimens (e.g., human samples) by increasing all the parameters (timing, volumes of solutions, etc.).

Discussion

Here, a detailed method is provided to prepare FFPE blocks of decalcified bones and preserve RNA integrity for sequencing (i.e., next-generation sequencing (NGS)) or for other RNA-related techniques (i.e., in situ hybridization, quantitative reverse transcription polymerase chain reaction (qRT-PCR), etc.).

The method utilizes EDTA to decalcify bone tissue samples; the EDTA incubation allows for slow but fine demineralization of the samples, thus preserving the histological features of...

Materials

Name	Company	Catalog Number	Comments
Advanced orbital shaker	VWR	76683-470	Use to keep tissues under agitation during incubation as reported in the method instructions.
Camel Hair Brushes	Ted Pella	11859	Use to handle FFPE sections as reported in the guidelines.
Dual Index Kit TS Set A 96 rxns	10X Genomics	PN-1000251	Use to perform spatial transcriptomics.
Ethanol 200 Proof	Decon Labs Inc	2701	Use to perform tissue dehydration as reported in the method instructions.
Ethylenediaminetetraacetic Acid, Disodium Salt Dihydrate (EDTA)	Thermo Fisher Scientific	S312-500	Use to prepare EDTA 20% pH 8.0.
Fisherbrand Curved Medium Point General Purpose Forceps	Fisher Scientific	16-100-110	Use to handle FFPE sections as reported in the guidelines.
Fisherbrand Fine Precision Probe	Fisher Scientific	12-000-153	Use to handle FFPE sections as reported in the guidelines.
Fisherbrand Superfrost Plus Microscope Slides	Fisher Scientific	12-550-15	Use to attach sectioned scrolls as reported in the guidelines.
High profile diamond microtome blades	CL Sturkey	D554DD	Use to section FFPE blocks as reported in the guidelines.
Novaseq 150PE	Novogene	N/A	Sequencer.
Paraformaldehyde (PFA) 32% Aqueous Solution EM Grade	Electron Microscopy Sciences	15714-S	Dilute to final concentration of 4% with 1x PBS  to perform tissue fixation.
Phosphate buffered saline (PBS)	Thermo Fisher Scientific	10010-049	Ready to use. Use to dilute PFA and to perform washes as reported in the method instructions.
Premiere Tissue Floating Bath	Fisher Scientific	A84600061	Use to remove wrinkles from FFPE sections as reported in the guidelines.
RNase AWAY Surface Decontaminant	Thermo Fisher Scientific	7002	Use to clean all surfaces as reported in the method instructions.
RNeasy DSP FFPE Kit	Qiagen	73604	Use to isolate RNA from FFPE sections once they have been generated as reported in the guidelines.
Semi-Automated Rotary Microtome	Leica Biosystems	RM2245	Use to section FFPE blocks as reported in the guidelines.
Sodium hydroxide	Millipore Sigma	S8045-500	Prepare 10 N solution by slowly dissolving 400 g in 1 liter of Milli-Q water.
Space Ranger	10X Genomics	2.0.1	Use to process sequencing data output .
Surgipath Paraplast	Leica Biosystems	39601006	Use to perform tissue infliltration and embedding as reported in the method instructions.
Visium Accessory Kit	10X Genomics	PN-1000194	Use to perform spatial transcriptomic experiments.
Visium Human Transcriptome Probe Kit Small	10X Genomics	PN-1000363	Use to perform spatial transcriptomic experiments.
Visium Spatial Gene Expression Slide Kit 4 rxns	10X Genomics	PN-1000188	Use to place the sections if performing spatial transcriptomic experiments.
Xylene	Leica Biosystems	3803665	Use to perform tissue clearing as reported in the method instructions.