Data on the evaluation of the relation between β-arrestin 2 and YAP phosphorylation in patient-derived colon cancer organoids

The data presented in this article is related to a rapid communication entitled “β-arrestin 2 suppresses the activation of YAP by promoting LATS kinase activity”. This article describes the correlation of β-arrestin 2 and YAP phosphorylation in patient-derived organoid models. Here, we analyzed 45 colon cancer organoids (CCOs) selected in the related research article to investigate the role of β-arrestin 2 in YAP phosphorylation. Hematoxylin and eosin (H&E) staining and immunohistochemistry data showed that the CCOs maintained tissue architecture and histological features of their original cancer tissues. Moreover, mutation data detected from RNA-seq (RNA-sequencing) analysis showed that these CCOs retained the genetic features of their original colon cancer tissues as well. We also confirmed at the protein level that organoids expressing β-arrestin 2 showed high expression of phosphorylated YAP. These organoid model studies strongly support the related research article that β-arrestin 2 suppresses the activation of YAP in colon cancer.

strongly support the related research article that β-arrestin 2

Value of the Data
• The data presented here show the suppressive role of β-arrestin 2 in YAP activation in colon cancers. This data provides accurate evidence obtained using patient-derived models to prove the molecular mechanism that β-arrestin 2 is associated with YAP phosphorylation in colon cancers. • The data contains results showing the similarity between organoid models and their original cancer tissues. Accordingly, this article may be useful for researchers searching for preclinical models that can faithfully recapitulate the characteristics of the original patient cancer tissues. • As YAP/TAZ is a key oncoprotein of the Hippo pathway in the nucleus of various tumor cells [3] , inhibition of nuclear YAP/TAZ is being highlighted as a potential therapeutic target [4] . The data may provide novel insights into targeting the Hippo pathway of colon cancers through β-arrestin 2 instead of YAP.

Data Description
The data presented in this article demonstrate that β-arrestin 2 promotes YAP phosphorylation in patient-derived colon cancer organoids (CCOs) to support the data in the associ-  ( Table 1 ). First, we verified the similarity between CCOs and their matched cancer tissues. Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) analyzes showed that our CCOs mimicked the tissue architecture of their original cancers and retained the expression of colorectal adenocarcinoma markers [5] cytokeratin 20 (CK20) and caudal-type homeobox 2 (CDX2) ( Fig. 1 A). Next, we compared the similarity of genomic features between the CCOs and their original cancer tissues using 43 CCOs that had matched tissues, as the original patient tissues of 2 CCOs did not have the quality for this analysis. We investigated somatic mutations in TP53, KRAS, APC, and FBXW7 genes, which are frequently found in colon cancers [6] , and most somatic mutations in tissues were detected in their matched CCOs, as the concordance of somatic mutations ranged from 72% to 90% for the 43 samples ( Fig. 1 B). In addition, we calculated the variant allele fraction (VAF) distribution to examine the correlation between tissues and CCOs on these 4 genes. As expected, the VAF values of these mutations in CCOs were correlated with those in tissues (  Table 1 Detailed information on the 45 patient-derived colon cancer organoids (CCOs) used in this article.
( continued on next page ) generally higher than those of original tissues, suggesting that CCOs were generated from pure cancer cells without non-tumor cells such as stromal and normal cells [7] . In conclusion, CCOs that we generated represent the cancer characteristics of their original tissues.
Based on the expression of β-arrestin 2  did not belong to any groups (non-group) ( Table 1 ). Next, to demonstrate the role of β-arrestin 2 in YAP activation, we investigated the correlation between β-arrestin 2 and phosphorylated YAP (pYAP) at the protein level; specifically, we focused on the phosphorylation at Ser 127 of YAP (pYAP S127), which is the form that blocks YAP transcriptional activity [8] . For Western Blotting analysis, we randomly selected 12 of 45 CCOs belonging to the AY1, AY2, or non-group ( Fig. 2 A). As a result, β-arrestin 2 and pYAP showed a positive correlation in CCOs ( Fig. 2 B; This result is consistent with our RNA expression data in the associated research article by Kim et al. that β-arrestin 2 shows a reverse correlation with YAP.
Collectively, our dataset shows that our CCOs maintained the nature of their original cancer tissues and that the CCOs were critically useful in investigating the role of β-arrestin 2 in YAP activation. Especially, CCOs with a strong expression of β-arrestin 2 were more likely to show the phosphorylation of YAP than other CCOs.

Experimental Design, Materials and Methods
Tissue preparation and culture of CCOs. The samples were surgically resected from colon cancer patients with patients' consent and directly transported to the laboratory on ice within To establish organoids, the resulting cell suspensions with 200 μL Matrigel (Corning, NY, USA) were allowed to solidify on 2 wells of pre-warmed 6-well culture plates (Corning) at 37 °C for 10 min. After gelation, 3 mL CCO MBM was added to the wells. The medium was changed every 3-4 days, and the organoids were passaged after 1-3 weeks. For passaging, solidified Matrigel drops containing the organoids were harvested using cold DPBS into a conical tube and centrifuged at 112 rcf for 3 min at 4 °C. The pellet was washed with cold DPBS and centrifuged at 250 rcf for 15 min at 4 °C. The pellet was divided into two layers, which were the Matrigel layer and the cell layer containing organoids. The Matrigel layer was removed from the pellet and organoids were resuspended in 2 mL TrypLE Express (Invitrogen) and incubated for 10 min at 37 °C for dissociation. After incubation, 10 mL of DMEM/F12 containing 10% FBS was added, and the samples were centrifuged at 112 rcf for 3 min. The pellet was washed with DPBS and centrifuged at 112 rcf for 3 min. The pellet was resuspended in CCO MBM + Matrigel (1:3) and reseeded at 1:3 to 1:4 ratios to allow the formation of new CCOs. Whole-transcriptome sequencing and data processing. To extract RNA from CCOs, we used CCOs ( > passage 3) cultured in 4-6 wells of 24-well culture plates (Corning). To obtain the CCOs, solidified Matrigel drops containing the CCOs were harvested in cold DPBS into a conical tube and centrifuged at 112 × g for 3 min at 4°C. The pellet was washed with cold DPBS and centrifuged at 250 × g for 15 min at 4 °C. Total RNA was extracted from the pellets using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. A cDNA library was constructed using the TruSeq RNA Access Library Prep Kit (Illumina, Inc., CA, USA) and 1 mg of total RNA. All cases passed the cDNA library quality assurance (minimum requirement: > 5 nM). Finally, 100-nt paired-end sequencing was performed using the HiSeq 2500 platform (Illumina, Inc.). Total RNA sequencing was performed using protocols described elsewhere [7] .
We analyzed the mutation profiles by using whole-transcriptome data obtained from 43 CCOs and matched tissue samples. To detect mutations from whole-transcriptome data, reads from RNA sequencing were mapped to the human reference genome (National Center for Biotechnology Information build 37) using STAR (2.7.3) [9] . After recalibration of the bases on the RNA-Seq reads, GATK HaplotypeCaller (3.8.0) and GATK Mutect2 (4.0.2) were run to search for mutations in the RNA BAM files. Mutations discovered using HaplotypeCaller were filtered based on the following criteria: FS > 30.0 and QD < 2.0. For variants detected by Mutect2, only the "PASS" from FilterMutectCalls was used [10] . RNA BAM depth at the mutated position was calculated using the "depth" option in samtools.
Hematoxylin and eosin (H&E) staining and immunohistochemistry. We performed H&E staining and immunohistochemistry using protocols described elsewhere [7] . Specifically, all samples containing tissues and matched organoids were prepared to paraffin block by fixing in 4% paraformaldehyde, dehydration, paraffin embedding, sectioning, and standard hematoxylin and eosin (H&E) staining. For immunohistochemical staining, samples were incubated with anticytokeratin 20 (CK20; K s 20. To investigate the similarity of somatic mutation analysis between CCOs and tissues, Spearman's correlation test was performed by using VAF values. To investigate the correlation between β-arrestin 2 and pYAP expression, a linear regression test was performed by using values of protein levels normalized to GAPDH. Statistical analysis was performed using R version 4.0.2 (R Foundation for Statistical Computing, Vienna, Austria) and SPSS Version 24. The graphs were generated using GraphPad Prism 5.0.1. P values < 0.05 were considered to indicate statistically significant differences.

Ethics Statement
Human specimens. This research has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). Small sections (approximately 1-4 cm 3 ) of colon cancer tissues were obtained from surgically resected colon cancer specimens at the Asan Bio-Resource Center (Seoul, Republic of Korea; Approval No. 2018-25(179)) with the patients' consent. The research protocol was approved by the Institutional Review Board of Asan Medical Center (Seoul, Republic of Korea; Approval No. 2018-0152). The entire experimental protocol was conducted in compliance with the institutional guidelines. Samples were determined as tumor or normal tissue on the basis of histopathological assessment. The diagnosis of each case was confirmed by the pathologists at Asan Medical Center.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that have or could be perceived to have influenced the work reported in this article.

Data Availability
Data on the evaluation of the relation between β-arrestin 2 and YAP phosphorylation in patient-derived colon cancer organoids (Original data) (DIB).