Revised Preparation of a Mimetic Tissue Model for Quantitative Imaging Mass Spectrometry CURRENT STATUS: POSTED

A revised methodology is presented for the preparation of a mimetic tissue model for use in quantitative imaging mass spectrometry (IMS). Tissue homogenates spiked with known concentrations of a compound standard are serially added to a mold and are snap frozen. The resulting mimetic tissue plug is then cryosectioned and thaw-mounted on the same slide as the tissue(s) to be imaged and quantified. From here, the sample tissue(s) and mimetic tissue model undergo identical sample preparation conditions for IMS. Once IMS is performed, the known analyte concentrations can be correlated with the average intensity of each layer of the mimetic model to generate a calibration curve. This standard curve can then be used to estimate the quantity of analyte that is present in the tissue(s) of interest. The matrix-matched standards applied in this approach account for common IMS issues such as ion suppression and extraction efficiency.


Introduction
Imaging Mass Spectrometry (IMS) combines spatial information with the highly selective and sensitive nature of mass spectrometry detection to yield a powerful molecular histology technique. In brief, this is achieved by rastering a sample surface as a 2-dimensional pixel array where at each position a mass spectrum is generated. By relating the intensity of any ion in the mass spectra with its corresponding x-y coordinates, a false color map can be generated which represents that ion's distribution in the sample. Over the past decade, significant contributions have been made to develop quantitative IMS which is the topic of several reviews. [1][2][3][4] Quantitative IMS has demonstrated promise in drug discovery and development where it can be used to assess drug distribution in tissue which has implications towards drug safety and efficacy. 5,6 The presentation of a quantified tissue distribution has distinct benefits over the common approach of quantifying the tissue homogenate especially if tissue distribution is heterogeneous (localized). Two of the major challenges in IMS which can impact the accuracy of quantification are the suppression of ionization primarily caused by the endogenous biological matrix as well as the efficiency of analyte extraction from the tissue. There are several common approaches to building a calibration curve for quantitative IMS proposed in the literature which are outlined nicely by Porta. 7 The first involves 3 spotting the calibration standards directly onto the sample substrate (typically glass slide) adjacent to the tissue that is to be quantified. While this is a straightforward process, it fails to account for the suppression and extraction efficiency associated with the analyte in the sample tissue. A secondary approach is to spot the calibration standards on a control or surrogate tissue section to account for tissue suppression, but it is unclear how this approach relates to the analyte extraction from the tissue. A third method that was previously proposed by our group utilizes tissue homogenates spiked with known concentrations of analyte to yield matrix-matched standards. This method is designed to more closely replicate the suppression and extraction conditions experienced by the analyte in the sample tissue yielding a more accurate quantification. 8 One drawback with this approach had been the elaborate process involved to generate the mimetic tissue model.
After several iterations, a more efficient method has emerged to produce the mimetic tissue model as is outlined in this protocol and is summarized in the flow diagram below. Briefly, a range of known concentration standard solutions are spiked into a series of tissue homogenates. These spiked homogenates are then serially frozen into a mold, producing a plug of tissue with a stepped concentration gradient of the analyte of interest. This mimetic tissue model can then be cryosectioned and thaw-mounted onto the sample substrate (microscope slide) along with the tissue(s) to be imaged and quantified. Once IMS is performed, the average intensity of each layer of the mimetic tissue model can be correlated with the known spiked concentration to generate a calibration curve. This curve can then be used to quantify regions of interest (ROIs) on the target tissue. The following were utilized for imaging mass spectrometry

Procedure
In accordance with the USFDA industry guidance for bioanalytical method validation, 9 the ideal calibration curve should have at least seven points including a blank. The remaining six points should cover the intended concentration range and be evenly spaced along the x-axis (concentration) to avoid issues with leverage. The blank is included to more accurately assess the detection capability, limit of blank (LoB) and limit of detection (LoD), as this provides a means of determining background relating not only to electronic and chemical noise but also from potential isobaric species inherent to the tissue matrix. 10 Therefore, the mimetic tissue model will consist of six levels of spiked homogenate and one non-spiked homogenate for assessment of the detection capability.

Homogenization (10 minutes)
By first intent, the homogenate of the mimetic tissue model should be made from the same tissue type as that which is being quantified. This, however, is not always going to be possible given that certain tissue types do not homogenize well (i.e. skin). Furthermore, appropriate control tissue does not always exist (i.e. tumor or xenograft tissue), and it may not always be possible to obtain control tissue from the same animal species. In these situations, we have found that rat liver tissue can be used as an effective surrogate. While the use of surrogate tissue can mimic the ion suppression and extraction from the tissue being quantified, the potential for unforeseen isobaric species in either the model or the target sample tissue should be noted. If an isobar exists in the sample tissue and not in the model tissue, the limit of detection can be underestimated and vice versa for the inverse situation. When preparing the tissue homogenate for the mimetic model, the use of a bead homogenizer without the addition of solvent is critical to yield a more morphologically relevant homogenate. 8,11 1.
Determine the minimum amount of homogenate that will be needed. --1.1. The aim is to have about 300-400 mg of homogenate for each layer.

2.
Prepare the tissue for homogenization. --2.1. If performing a large-scale homogenization (i.e. whole rat liver) choose an appropriate sized tube/vial.
--2.2. Cut small enough portions of the tissue that will fit into your tube of choice.
----2.2.1. Portion enough tissue to at least satisfy the minimum amount required.
--2.3. Do not overfill the tube (only fill about ¾ to leave room for beads & homogenization).
--2.4. Add stainless steel homogenizing beads to the tube (MP Metal Bead Lysing Matrix).
--2.5. Wrap the top of the tube with parafilm to prevent loss of tissue.
--3.2. Let the homogenate settle or spin down at low speed (1000 x g for ~ 30 s) to collect as much as possible from the tube walls.
----3.2.1. The intention of spinning down is to collect the material off the tube walls but it may result in phase separation. If using the homogenate directly, gently stir to reincorporate the mixture.
--3.3. If the tissue was large enough to be spread over several tubes, it is suggested that these homogenates be combined to avoid potential bias in sampling from the tissue.
--3.4. Aliquot the homogenate into smaller single use containers (~2 g each) to avoid multiple freeze/thaw cycles.

Preparing the Mold (5 minutes)
The mold for the mimetic model is a modified 3 mL syringe (BD 3 mL Syringe REF 309657).
Regardless of the exact type or brand of syringe, there are a few important attributes to the syringe choice. Given that the volume of the syringe ultimately dictates the amount of homogenate that is 7 needed as well as the height of each layer of the model, the 3 mL syringe has a large enough inner diameter that can accommodate the tip for the positive displacement pipette and is small enough to limit the amount of homogenate required for each layer. Additionally, the plunger seal should be rubber-like material (avoid hard plastic) to limit the penetration of ethanol into the barrel during the freezing step. --4.2. Try to limit the amount of time between freezing sequential layers.

11
----4.2.1. Once the fresh homogenate layer is added, it should be frozen as quickly as possible to prevent the previous layer from melting and mixing with the new layer.

5.
Once the last layer has been added and frozen, remove the handle (allen key), wrap the mold in foil, and place in a -80°C freezer for at least 3 hours or overnight to ensure that it is thoroughly frozen.

Sectioning the Mimetic Tissue Model (10 minutes)
Once the mimetic model is constructed for a given standard, applying the quantification of that standard to any tissue merely requires collecting a section of the mimetic model onto the same slide with the tissue to be quantified.

1.
Equilibrate the model to the cryostat temperature as you would any other tissue.  This will allow for a better assessment of the average intensity as well as the standard deviation of the intensity at each level which is used for the weighted regression. An example is shown below of the ion image from the mimetic tissue model and a section of dosed tissue along with the associated calibration curve.

Concluding Remarks
The mimetic tissue model represents an approach to IMS quantification which accounts for the major IMS challenges of ion suppression and extraction efficiency through the use of matrix-matched standards. The tissue homogenate used for the mimetic model represents the average ion suppression that would be expected from the corresponding target tissue. The protocol presented here is an improved methodology for a more efficient preparation of the mimetic tissue model.
Because the model is placed on the same slide with the tissue(s) to be quantified, it undergoes the same sample preparation and acquisition conditions as the tissue(s) of interest. Due to the potential for variation in sample preparation, it is key that the mimetic tissue model be applied only within an acquisition.
14 While the mimetic tissue model is seemingly a more involved preparation compared to the spotting approaches, there are a number of applications where the mimetic tissue model is more efficient.
When replication is necessary or if the quantification is to be applied to multiple tissues, this merely requires a single preparation followed by the collection of another section of the tissue model rather than collecting a control tissue section and hand-spotting the calibration curve for each replicate. This is often the case in pharmaceutical analysis where tissues are available from multiple animals in different dosing groups or from various time points. Figure 3 Trimming the syringe barrel Figure 4 Trimming the plunger seal Homogenate spiking and mixing Figure 7 Preparing the Mimetic Tissue Model plug Figure 8 Removing the model from the mold 19 Figure 9 Mounting the Mimetic Tissue Model for cryosectioning Figure 10 Cryosectioning the Mimetic Tissue Model Mimetic_Tissue_Model_Calculations.xlsm