Protocol for Imaging and Analysis of Mouse Tumor Models with CUBIC Tissue Clearing

Summary Tissue-clearing technologies have developed rapidly in the past decade, especially for use in neuroscience research. We previously reported that CUBIC, which is one tissue-clearing method, is useful for applications in cancer research. CUBIC cancer analysis can be used to detect cancer metastasis with single-cell resolution at whole mouse body/organ level. This approach can also analyze the tumor characteristics with high-quality 3D images. Here, we describe a detailed CUBIC cancer protocol from tissue clearing, capturing 3D images and post-immunohistochemistry. For complete details on the use and execution of this protocol, please refer to Kubota et al. (2017).


SUMMARY
Tissue-clearing technologies have developed rapidly in the past decade, especially for use in neuroscience research. We previously reported that CUBIC, which is one tissue-clearing method, is useful for applications in cancer research. CUBIC cancer analysis can be used to detect cancer metastasis with single-cell resolution at whole mouse body/organ level. This approach can also analyze the tumor characteristics with high-quality 3D images. Here, we describe a detailed CUBIC cancer protocol from tissue clearing, capturing 3D images and post-immunohistochemistry. For complete details on the use and execution of this protocol, please refer to Kubota et al. (2017).

BEFORE YOU BEGIN Establishment of Tumor-Bearing Mouse Models
Timing: 1 day to several months 1. Establishment of cancer metastasis mouse models using cancer cells stably expressing fluorescence proteins. a. Anesthetize mice with isoflurane if necessary. b. Inoculate cancer cells stably expressing fluorescence proteins (i.e., mCherry, tdTomato) or fluorescent dye (i.e., fluorescein isothiocyanate) into mice, intravenously, orthotopically, subcutaneously, or intracardially, dependent upon experiments. 2. Optional: When cancer cells are also expressing firefly luciferase gene, monitor tumor progression using in vivo bioluminescence imaging.

Representative inoculation mouse models
How to inject Metastatic organs (typically)
CRITICAL: For taking high-quality pictures, it is important that the refractive index of CU-BIC-R (N) and observation oil match.
Note: Pre-mixed and quality-controlled observation oil (Mounting solution for CUBIC-R+) is alternatively purchased.

Preparation of Reagents for Immunohistochemistry
Timing: 15 min 8. Endogenous peroxidase blocking solution a. Mix 270 mL methanol and 30 mL hydrogen peroxide. b. Store at 18 C-26 C in sealed container in the dark. 9. Antigen retrieval solution a. Dilute 30 mL Universal HIER antigen retrieval reagent (103) with 270 mL ultra-pure water. b. Store at 18 C-26 C in sealed container.
Note: Although it basically depends on the manufactural protocols, the antigen retrieval solution can be usually used for 4-5 times. Paraffin-embedded block manufacturing equipment n/a n/a Alternatives: Instead of Imaris software, images can be analyzed with ImageJ or FIJI, Big-Stitcher, NIS-Elements.

KEY RESOURCES TABLE
Alternatives: Rice steamer or automated device for antigen retrieval like LAB Vision PT Module (Thermo Scientific) can be also used instead of autoclave machine. (related to step 20) Note: Coagulation might be seen in the spleen after excision. The coagulated blood is very difficult to clear.

OPEN ACCESS
CRITICAL: From step 4 to step 12, samples should be protected from the light.
a. Exchange solution from PFA to PBS and shake gently for more than 2 h. b. Exchange PBS more than 2 times (total wash > 6 h). 6. Exchange solution from PBS to 50% CUBIC-L and shake gently at 37 C (50-100 rpm). 7. After over 6 h of incubation, exchange solution with 100% CUBIC-L, shake gently at 37 C for 1-7 days (until the exchanged CUBIC-L solution is not colored).
CRITICAL: In steps 6 and 7 (delipidation and decolorization process), the solution will be colored as shown in Figure 1A. Exchange CUBIC-L until the solution is no longer colored. Moreover, CUBIC-L might damage some kinds of tubes. Exchange tubes if it happens.
8. Wash samples with PBS to remove CUBIC-L. a. Exchange solution from CUBIC-L to PBS and shake gently for more than 2 h. b. Exchange PBS more than 2 times (total wash > 6 h). 9. Optional: In case of 3D staining (immunostaining including nuclear staining).
a. Staining with antibody.
(i) Immerse samples with antibody dilution in staining buffer (1:100-1:400 depending upon antibody). Shake gently at 22 C for 3 days. Protect from the light. b. Remove antibody solution and wash in PBS.
(i) Exchange solution from antibody solution to PBS and shake gently for more than 2 h. (ii) Exchange PBS more than 2-4 times (total wash > 6 h).
CRITICAL: Immerse entire organs in staining buffer. Use a container with a round bottom as shown in Figure 1B. We prefer to use fluorescent dye or fluorescence proteins conjugated primary antibodies for uniform deep staining.
(i) Exchange solution from antibody solution to PBS and shake gently for more than 2 h. (ii) Exchange PBS more than 2 times (total wash > 6 h). 11. Exchange solution to 50% CUBIC-R (N) and shake gently at 18 C-26 C for more than 6 h. Protect from the light. 12. Exchange solution to 100% CUBIC-R (N) and shake gently at 18 C-26 C for overnight (12-16 h).
Protect from the light.
Note: The liver might become white instead of clear in very rare cases. We are looking into the cause of this problem, but we have yet to solve it.
Optional: To avoid movements of organs while acquiring the images by LSFM, organs can be embedded in 2% gel before step 11.

Capture 3D Images
Timing: 1 day This step describes how to obtain images of cleared tissue samples with LSFM or confocal microscopy.
13. Take images with light sheet fluorescence microscopy or confocal microscopy. a. Wash samples in CUBIC-R (N) with observation oil. b. Put samples into a glass container fulfilled with observation oil. c. Collect images by light sheet fluorescent microscopy (LSFM) (z = 10 mm step). 14. Record images (TIFF format) and analyze data with Imaris software (see Quantification Analysis).

Timing: 2-4 days
This step describes how to prepare samples for immunohistochemistry.

Wash samples with PBS.
a. Put samples into PBS and shake gently for more than 2 h. b. Exchange PBS more than 2 times (total wash > 6 h). 16. Embed samples into paraffin using equipment (including dehydration and delipidation process with ethanol and xylene). 17. Make sections using standard histology microtome and mount on glass slide. Note: We showed avidin-biotin complex (ABC) method, but we can also apply other direct or indirect immunohistochemistry methods.
Alternatives: PBS can be used instead of TBS.
Optional: In case of H&E staining, after step 18, stain slides with hematoxylin and eosin.
Optional: When making frozen sections with samples after PBS wash (step 14), sucrose exchange process is needed.
a. Immerse samples in 5% sucrose in PB and shake gently for 30 min to 1 h. b. Exchange solution to 10% sucrose in PB and shake gently for 30 min to 1 h. c. Exchange solution to 15% sucrose in PB and shake gently for 30 min to 1 h. d. Exchange solution to 20% sucrose in PB and shake gently for 30 min to 1 h. e. Freeze samples with OCT compound.
Pause Point: After taking images (step 13), samples were preserved in CUBIC-R (N). However, leaving samples in CUBIC-R (N) might induce signal dropping. To preserve fluorescent signals, it is better to keep samples in PBS at 4 C after PBS wash to remove CUBIC-R (N).

EXPECTED OUTCOMES
Using this protocol, we can make various organs from mice transparent with high quality (Figures 1C  and 1D). With these transparent mouse organs, we can capture multiple 2D images without sectioning. Reconstitution of these 2D images makes us possible to get 3D images of the intact mouse organs. For example, A549 cells expressing mCherry pre-treated with TGF-b were inoculated intravenously and lungs were excised on day 10. After CUBIC procedures, cancer metastases were observed in deep tissue (Figure 2, Methods Video S1). OS-RC-2 cells expressing mCherry were inoculated intracardially and brains were excised on day 40. Using CUBIC procedures, cancer metastasis and a-SMA positive mature blood vessels in brain were observed (Figure 3, Methods Video S2). When CUBIC-cleared organs were washed with buffer, they can be processed for standard paraffin-embedded histological tissue processing and analysis. Post-H&E staining or post-immunohistochemistry is useful for further investigation (Figure 4).

QUANTIFICATION AND STATISTICAL ANALYSIS
1. Make tiff files to Imaris file by using Imaris File Converter. It is installed automatically when you install Imaris or Imaris Stitcher or you can download a free version.

OPEN ACCESS
2. Analyze Imaris file with Imaris software or analyze tiff files using Image J (Figures 2 and 3). 3. To evaluate the volume or the number of metastatic colonies with Imaris software, add new surface and decide threshold. The data is outputted as csv file.

LIMITATIONS
Autofluorescence sometimes becomes a problem, especially with green fluorescence proteins such as green fluorescent protein (GFP). When signals from cancer cells are low, autofluorescence can mask the positive signals. In our current protocol, delipidation process is essential and structure with lipid was damaged and disappeared. Various pigments such as heme can be removed with CU-BIC-L; however, some pigments such as melanin are not removed with CUBIC-L (Ueda et al., 2020). Moreover, CUBIC-R (N) reagent is not compatible with Alexa 488 and Dylight 488.
Perfusion with PBS and PFA process is needed to reduce autofluorescence signals; therefore, cancer cells in blood vessels are hardly detected in our current protocol.
This protocol is useful for detecting cancer cells expressing fluorescence proteins in mice, not optimized to 3D staining. However, 3D staining protocol is currently optimized and various antibodies can be used in CUBIC (Susaki et al., 2020). A549-mCherry-Luc2 cells were pre-treated with TGF-b1 for 2 days. These cells were injected intravenously (iv) into nude mice. Ten days after iv injection, mice were sacrificed and excised lungs were subjected to CUBIC procedures. The 3D images (A) and 2D images (B) were analyzed using Imaris software (A), (B) or ImageJ (B).

TROUBLESHOOTING Problem 1
Mouse organs do not become transparent.

Potential Solution
Increasing the temperature and the length of the tissue-clearing process, especially delipidation and decolorization process with CUBIC-L, improves the transparency of the samples, but might lead to a decrease in the signal.

Problem 2
Signals from fluorescence proteins cannot be detected.

Potential Solution
Check pH of CUBIC-L and CUBIC-R (N), because high or low pH can quench the signals from fluorescence proteins. To enhance the signals from fluorescence protein, immunostaining with antibody (i.e., anti-GFP antibody) should be done. It makes us possible to monitor the signals from quenched fluorescent signals.
Problem 3 3D staining does not work.

Potential Solution
Although depending on the antibodies, post-fixation of the stained organs after step 9 with PFA (4 C-22 C, 1-16 h, depending on samples) might improve the staining efficiency.

RESOURCE AVAILABILITY Lead Contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Kohei Miyazono (miyazono@m.u-tokyo.ac.jp) and Hiroki R. Ueda (uedah-tky@umin.ac.jp).

Materials Availability
This study did not generate new unique materials.

Data and Code Availability
The data of this study are available from the corresponding authors upon reasonable request.

ACKNOWLEDGMENTS
We thank Dr. Tainaka and Dr. Susaki for technical assistance and discussion. We also thank Mr. Morishita for his expert knowledge about immunohistochemistry. This work is supported by KAKENHI