Kras activation in endometrial organoids drives cellular transformation and epithelial-mesenchymal transition

KRAS, an oncogene, is frequently activated by mutations in many cancers. Kras-driven adenocarcinoma development in the lung, pancreas, and biliary tract has been extensively studied using gene targeting in mice. By taking the organoid- and allograft-based genetic approach to these organs, essentially the same results as in vivo models were obtained in terms of tumor development. To verify the applicability of this approach to other organs, we investigated whether the combination of Kras activation and Pten inactivation, which gives rise to endometrial tumors in mice, could transform murine endometrial organoids in the subcutis of immunodeficient mice. We found that in KrasG12D-expressing endometrial organoids, Pten knockdown did not confer tumorigenicity, but Cdkn2a knockdown or Trp53 deletion led to the development of carcinosarcoma (CS), a rare, aggressive tumor comprising both carcinoma and sarcoma. Although they originated from epithelial cells, some CS cells expressed both epithelial and mesenchymal markers. Upon inoculation in immunodeficient mice, tumor-derived round organoids developed carcinoma or CS, whereas spindle-shaped organoids formed monophasic sarcoma only, suggesting an irreversible epithelial-mesenchymal transition during the transformation of endometrial cells and progression. As commonly observed in mutant Kras-driven tumors, the deletion of the wild-type Kras allele was identified in most induced tumors, whereas some epithelial cells in CS-derived organoids were unexpectedly negative for KrasG12D. Collectively, we showed that the oncogenic potential of KrasG12D and the histological features of derived tumors are context-dependent and varies according to the organ type and experimental settings. Our findings provide novel insights into the mechanisms underlying tissue-specific Kras-driven tumorigenesis.


INTRODUCTION
Ras is a small guanosine-5'-triphosphate (GTP)-binding protein that transmits external stimuli to downstream signaling pathways, such as Raf, PI3K, and Ral-GEF. These effectors orchestrate various vital cellular processes, including gene transcription, cell proliferation, acquisition of cell motility, and inhibition of cell death [1]. Although Ras activation is normally regulated through GTPbinding and degradation in a transient and reversible manner, its constitutive activation is often observed in various types of cancer, mostly by missense mutations in the GTP-binding sites at codons 12, 13, and 61 or by gene amplification [2]. Epithelial malignancies have preferences for KRAS mutations among the three RAS genes, namely HRAS, NRAS, and KRAS; although KRAS mutation rate varies among tissues, it can be as high as~90% in pancreatic cancer; 20-50% in carcinomas of the colon, lung, and hepatobiliary tract; and much less frequent in other carcinomas [3]. Using genetically engineered mice (GEM) models, many studies have demonstrated the causal role of the Kras G12D mutation in tumor development, through cooperation with other common genetic aberrations in each cancer type [4]. Thus, Kras G12D has been established as a bona fide oncogene.
Because PTEN has the highest mutation rate across EC, the effect of its inactivation has been extensively investigated using GEM. Although Pten −/− mice died during embryogenesis [9], all Pten +/− mice developed precancerous lesions, 25% of which eventually developed EC within a year [10]. PR-Cre; Pten flox/flox mice, in which Pten was deleted by the Cre-recombinase induced in a uterine-specific manner, frequently developed invasive EC by 3 months of age [11]. After the local injection of adenovirus Cre into the uterus of Pten flox/flox mice, EC development was observed, albeit with partial penetrance [12,13,]. Concurrent Kras G12D accelerated Pten-dependent EC development [14,15,], suggesting that Kras G12D plays an oncogenic role in the endometrium. Alternatively, GEM with genitourinary tract-selective Trp53 deletion developed type 2 EC in 84% of mice at 58-68 weeks of age [16]. These findings suggest that the tumorigenic potential of endometrial cells and the histological features of EC may be genetically determined to a considerable extent, although more studies are required to fully elucidate its pathogenesis.
Organoid culture is an emerging technology that enables the long-term propagation of normal epithelial cells in a physiological μm. E Western blot analysis of transduced organoids. Both Pten knockdown and Akt phosphorylation were achieved by shPten. The induction of p16 Ink4a is prominent following lentiviral transduction with shLuc, but not with shPten. F Macroscopic findings of an isolated nodule. Pten knockdown alone did not result in the tumorigenic potential of endometrial organoids. Scale bar, 10 mm. G Histological findings of nodules derived from organoids with Pten knockdown. A few glands with mild atypia are observed after H&E staining. Scale bar, 50 μm.
setting [17,18,]. It is a three-dimensional culture method using the Matrigel, extracellular matrix that mimics the basement membrane, and serum-free medium supplemented with stem cell niche factors for each tissue. Owing to the high utility, its application has been expanding to many research fields, including infectious diseases [19], developmental biology [20], and tissue regeneration [21]. We reconstituted a combination of common genetic aberrations in organoids with lentiviral vectors encoding short hairpin RNA (shRNA) and Cre, which were inoculated into nude mice. Even without a tissue-specific microenvironment, certain combinations of genetic aberrations that are particularly common in certain cancers robustly give rise to subcutaneous tumors. For example, Apc inactivation and Kras activation in the intestinal organoids markedly accelerated tumorigenesis caused by Apc knockdown alone [22]. In organoids from the lung [23], hepatobiliary tract [24,25,], and pancreas [26], the development of full-blown tumors was achieved not by Kras activation alone, but by the concurrent inactivation of the p53 or Rb pathway. Since these outcomes are consistent with the results of earlier GEM studies [22,23,25,26,], we hypothesized that such organoid and allograft-based approach can establish an alternative carcinogenesis model in any organ.
To verify this notion, we investigated whether organoids from the murine endometrium could be transformed by reconstitution of genetic aberrations that developed type 1 EC in GEM. Through examination of transduced organoids, subcutaneous tumors, and derived organoids, we gained novel insights into multi-layered interactions that are involved in tumor development.

RESULTS
Pten knockdown allowed endometrial organoids to propagate after lentiviral infection To conduct an organoid-based tumorigenicity assay, we isolated and propagated primary epithelial cells from the murine uterine horn (Fig. 1A). Endometrial organoids that robustly propagated for at least a few months had rounded cystic structures, whereas stromal cells spontaneously disappeared within a few passages (Fig. 1B). Organoids consisted of columnar epithelial cells lined in a monolayer as verified by the cytokeratin + /vimentinstaining pattern (Fig. 1C). Efficient gene transduction was verified using a lentivirus vector encoding GFP (Fig. 1D).
Considering that most previous GEM models for EC were generated by the ablation of Pten [9][10][11][12][13], we first evaluated the tumorigenic potential of the organoids with Pten knockdown. By introducing shRNAs against Pten (hereafter, shPten) into the endometrial organoids, Pten knockdown and the subsequent Akt activation were verified compared to the shRNA against luciferase (shLuc) (Fig. 1E). Although organoids without infection or with shPten continued to proliferate, those with shLuc failed to propagate after the first passage and had a considerable elevation of the cell cycle inhibitor p16 Ink4a , suggesting the deleterious effects of lentiviral infection. Upon the inoculation of organoids with shPten into nude mice, only tiny nodules (Fig. 1F) containing a few glands with mild atypia of ductal or cystic shape developed (Fig. 1G). These observations suggest that despite the advantages of in vitro proliferation and unlike earlier in vivo studies, Pten knockdown alone is insufficient to induce tumorigenesis in endometrial organoids.
Endometrial organoids with Kras G12D and Pten knockdown did not develop tumors The activation of the Ras pathway has been implicated in human EC [7]. As Kras G12D accelerated the Pten-dependent EC development in mice [14,15,], we investigated whether this combination of genetic alterations could drive tumor development in endometrial organoids. We applied a two-step gene transduction protocol, namely, introduction of Cre and shRNA transduction ( Fig.  2A). Organoids from Kras LSL-G12D/+ mice were subjected to lentiviral infection, in which Kras G12D is transcribed upon the Cremediated excision of the STOP codon flanked by the two LoxP sequences (LSL) [28]. Subsequently, we detected a newly emerged Kras G12D amplicon slightly longer than the Kras WT amplicon by a single LoxP (Fig. 2B) and observed the induction of the Kras G12D protein (Fig. 2C), establishing successful recombination.
Although organoids with Cre steadily proliferated, those with the backbone vector pLKO.1 gradually stopped propagating over passages, similar to the organoids after transduction with shLuc. Because p16 Ink4a was induced after lentiviral infection and more prominently after Kras G12D induction, two potent shRNA clones against Cdkn2a encoding p16 Ink4a and p19 Arf were added to the second infection, along with shPten and shLuc, to avoid cell cycle arrest (Fig. 2D). No morphological changes were observed (Fig. 2E). Most Kras G12D organoids with shLuc did not develop tumors in the nine cases tested (Fig. 2F), with the exception of one case of CS, which was defined by the presence of both carcinoma and sarcoma components in the tumor (Fig. S1, Table S1). Kras G12D organoids with shPten clone #1 developed cysts in two out of three cases (Fig. 2F, G), in which the lumen was covered with a monolayer of columnar cells with mild atypia (Fig. 3A, B). Another shPten clone #2 did not induce tumors in two experiments, even after introduction into the Kras G12D organoids that gave rise to sarcoma upon the introduction of shLuc (Table S1, Fig. S1). These observations suggested the marginal tumorigenic potential of Kras G12D , which was not promoted by Pten knockdown.
Kras G12D organoids with Cdkn2a knockdown or Trp53 deletion developed CS In contrast, Kras G12D organoids with shCdkn2a developed solid tumors ( Fig. 2F) within eight weeks in all five cases (Table S1). The tumors partly contained cystic lesions or necrosis (Figs. 2G, 3A) and were predominantly occupied by spindle-like cells and partly by atypical glandular cells. Tiny nodules originating from the Kras G12D organoids with shLuc only contained a few glands with mild atypia (Fig. 3B). Among the five solid tumors, two were diagnosed with CS, two with monophasic sarcoma, and one with a combination of sarcoma and cyst (Table S1). In the CS cases, most carcinoma and sarcoma cells separately resided as cytokeratin + / vimentinand cytokeratin -/vimentin + populations, respectively (Fig. 3C). However, a subset of sarcoma cells showed cytokeratin + / vimentin + expression, indicating an intermediate state between typical carcinomas and sarcomas (Fig. 3C). This finding suggested an epithelial origin of the sarcoma cells. In the two experiments with another potent shCdkn2a clone, cysts or sarcomas were induced in Kras G12D organoids (Fig. 3D), thus eliminating the possibility of off-target effects by shRNA. These results suggest that Kras G12D expression and Cdkn2a knockdown cooperate for tumor development, preferentially toward the induction of sarcomatous differentiation. Kras G12D organoids with Cdkn2a knockdown developed CS. A Two-step protocol for organoid generation from Kras LSL-G12D/+ mouse. B Genomic PCR analysis of transduced organoids. The upper band depicted as Kras G12D indicates the successful recombination of the Kras locus by Cre-recombinase, whereas the lower band depicts an amplicon for the WT allele of Kras. C Western blotting analysis of endometrial organoids. Stepwise inductions of p16 Ink4a by infection itself and Cre transduction were observed. D Western blot analysis of Kras-activated endometrial organoids. The knockdown of the targeted gene products was achieved by the introduction shCdkn2a and shPten clones. shCdkn2a suppressed its two products p16 Ink4a and p19 Arf . Non-specific bands (*) and specific bands (arrowhead) are shown for the p19 Arf panel. E Transduced organoids visualized under phase-contrast microscope. Scale bar, 200 μm. F Tumor development in nude mice. Upper panel, subcutaneous tumors (arrowheads). Scale bar, 10 mm. Middle and lower panels, resected tumors. Representative results from two independent experiments (Exs. 1 and 2) are shown. Matrigel plugs (single asterisk) and cystic lesions (double asterisks) are classified as nontumorous lesions. Scale bar, 10 mm. G Macroscopic view of sliced tumors after formalin fixation. Scale bar, 10 mm. Cre + shCdkn2a #1: a solid tumor with cystic formation (arrowhead) in Ex. 1   In human uterine CS, TP53 is the most frequently mutated gene [29,30,]. Consistent with this finding, GEM with uterine-specific Trp53 deletion developed CS after a long latency and only in~10% of the tumors [16]. This suggested that other genetic alterations were required to accelerate CS development; therefore, we investigated whether Kras G12D and p53 loss could synergize tumorigenesis. After Cre transduction of endometrial organoids from Kras LSL-G12D/+ ; Trp53 flox/flox mice (Fig. 4A), the simultaneous   induction of Kras G12D expression and Trp53 deletion (Figs. 4B, 4C) was verified. We observed the steady proliferation of organoids (Fig. 4D), but not in organoids with pLKO.1 as predicted. Upon the inoculation into nude mice, solid tumors developed within eight weeks in all four cases (Fig. 4E). Notably, the tumors were invariably diagnosed as CS (Table S1, Fig. 4F). Thus, Trp53 deletion also cooperated with Kras G12D expression for CS development in endometrial organoids.
Tumor-derived organoids retained epithelial-mesenchymal transition (EMT) state Regardless of the presence of genetic alterations, endometrial organoids stereotypically maintained their round cystic shape. In contrast, those recovered from subcutaneous CS or sarcoma displayed diverse morphological features ranging from cystic, spindle-like, and combined types (Fig. 5A). Previously, we revealed that nude mice-derived stromal cells could not survive the current serum-free culture conditions over several passages [25,26,]. The presence of spindle-like cells in six out of eleven tumor-derived organoids (TDOs) strongly suggests that their transformed nature was stably retained during recovery from subcutaneous tumors. In addition, one combined type and two spindle-like type TDOs developed CS and monophasic sarcoma, respectively, whereas two cystic-type TDOs developed monophasic carcinoma or CS (Fig. 5A, Table S2). These observations indicated that spindle-like cells might have originated from epithelial cells and that carcinoma cells could have undergone EMT to sarcoma in an irreversible manner.
Tumor-derived organoids displayed heterogeneous statuses in the Kras loci For pancreatic and biliary tract organoids, TDOs generated from organoid-based Kras-driven carcinogenesis models were completely negative for the LSL cassette in the conditional allele [25,26,]. Considering that a fraction of cells retained the LSL at the time of inoculation, this finding suggested that Kras activation was a definite requirement for adenocarcinoma development in these organoids. Similarly, we examined the Kras locus in nine endometrial TDOs using genomic PCR analysis (Fig. 5B, Table S2). The emergence of the Kras G12D amplicon was detected in all TDOs as predicted, whereas the residual LSL cassette was unexpectedly detected in some cases (Fig. 5B, Table S2), indicating the survival of untransformed cells without Kras G12D within the CS. In addition, the intensity of Kras WT amplicon was clearly fainter than that of Kras G12D in four cases and was undetectable in five cases (Fig. 5B, Table S2), suggesting the spontaneous deletion of the Kras WT allele, as occasionally observed in the organoid-based pancreatic cancer model [26].
To evaluate the genetic instability comprehensively during CS development, we performed array CGH analysis on three pairs of transduced organoids and their corresponding TDOs. For Kras G12D organoids with both Cdkn2a knockdown (#4) and Trp53 deletion (#5, #6), the pre-inoculated organoids and TDOs exhibited genome stability, although no recurrent amplifications or deletions were detected (Fig. 5C). Thus, the Kras loci may have undergone unique alterations during the development of CS and sarcoma.

Significant transcriptomal changes in organoids underlay tumorigenesis
To better characterize the pathogenesis of CS from organoids, we conducted a transcriptome analysis. Based on hierarchical cluster analysis, organoids before inoculation and TDOs (#5 and #6) were relatively similar, although a spindle-type TDO (#4) was observed as an outlier (Fig. 6A). The Kyoto Encyclopedia of Genes and Genomes database was used to identify signaling pathways that were significantly altered during tumorigenesis. Among the upregulated gene functions, three out of the top ten were shared by all TDOs, including ECM-receptor interaction, the PI3K-AKT signaling pathway, and cytokine-cytokine receptor interaction (Fig. 6B). Considering that CS and sarcoma likely originated from epithelial organoids, we further examined the expression of EMT marker genes in TDOs. Spindle-type organoids (#4) showed high expression levels of several known mesenchymal markers, such as Vimentin, Cdh2, Zeb1, and Snai1, and low expression levels of epithelial markers, such as Krt7, Cdh1, Cldn7, and Ersp1 [31] (Fig.  6C). In contrast, the transcriptome profile of cystic-type organoids (#5 and #6) was similar to that of pre-inoculated organoids, except for a moderate increase in Vimentin (Fig. 6C). These results indicate that gene expression profiles reflect the morphology of TDOs.

DISCUSSION
We previously showed high concordance of tumorigenicity caused by certain combinations of genetic aberrations between GEM and organoid-based carcinogenesis models [22,25,26,]. Regarding Pten-driven tumorigenicity in the uterus, tumor development was not demonstrated in this study, even in the presence of Kras G12D . One possible explanation for the observed discrepancy is that the uterine-specific gene targeting in mice by PR-Cre mice [11] or the local injection of adeno-Cre [12,13,] may in fact have resulted in gene recombination in both epithelial and stromal cells of the uterus. In line with this notion, mice with uterine epithelialspecific ablation of Pten did not develop EC [32]. Considering that the resultant Pten knockdown in the stroma is not common in sporadic cases of human EC, the overestimated tumorigenic potential could be attributed to the interplay between the epithelia and the stroma of the uterus. Similar observations were previously documented in intestinal tumorigenesis, wherein Pten deletion in both the stroma and epithelia in mice led to tumor development, which was not observed when deletion was specific to the epithelium [33,34,] Another possibility is that even normal uterine stroma could play a pro-tumorigenic role. In a cell-based study, the introduction of myr-Akt or shPten into primary murine endometrial cells, followed by co-transplantation with the uterine stroma from neonatal WT mice, resulted in the development of adenocarcinoma in the kidney capsule [35], suggesting the importance of the microenvironment of the uterine stroma in EC development. Lastly, given that the MSI subtype of human EC preferentially harbors mutations in KRAS and PTEN [7], it is tempting to speculate that additional inactivation of mismatch repair genes in endometrial organoids could achieve type I EC development, which is worthy of further investigations.
Another unique property of endometrial organoids is the propagation arrest associated with lentiviral infection, which has never been observed in any gastroenterological organoids under almost the same culture conditions [22][23][24][25][26]. Considering the infinite propagation of uninfected organoids and p16 Ink4a induction following lentiviral infection, endometrial organoids may be particularly sensitive to DNA damage caused by viral genomic integration. Although the underlying mechanisms remain elusive, this finding prompted us to knockdown Cdkn2a in endometrial organoids, which unexpectedly led to the Kras-dependent development of CS or sarcoma. In human EC cases, KRAS mutations are rarely found in serous carcinoma (3%) but are more prevalent in endometrioid carcinoma (24%) and CS (12%); TP53 mutations are significantly more common in serous carcinoma (88%) and CS (91%) than in endometrioid carcinoma (21%) [36]. These findings are consistent with CS development in endometrial organoids upon Kras activation and p53 loss in this study. In GEM with identical genetic alterations, CS development was also observed in the ovary [37], while adenocarcinoma was always induced in the pancreatobiliary system for both in vivo and ex vivo [25,26,38,39,]. These findings suggest that gynecological organs may be predisposed to CS due to some inherent epigenetic regulation.
We further demonstrated the development of CS from epithelial cells in two ways: from transduced cystic organoids and from cystic-type TDOs originating from organoid-derived CS. In addition, we identified a fraction of CS cells that expressed both epithelial and mesenchymal markers. These findings are consistent with the notion that uterine CS is predominantly a monoclonal neoplasm of epithelial origin, rather than generated by the collision of two distinct cell populations [40,41,]. This indicates irreversible EMT during transformation and progression from carcinoma to sarcoma. Single-cell analysis at each step of this model may help clarify the molecular events involved in CS development. Further investigation on whether our results can be extrapolated to in vivo GEM is warranted. Also, our study might provide a unique resource for CS research, in which models are still few.
CS induced in this study was "homologous", which by definition demonstrates the presence of spindle-like sarcoma cells resembling inherent uterine stroma, such as fibroblasts and smooth muscle cells. In contrast, the uterine epithelial-specific ablation of Fbxw7 and Pten in mice resulted in the development of "heterologous" CS, having a sarcoma component of differentiation toward bone and cartilage in half of the cohorts in three months [42]. Interestingly, mutations in Trp53 and Kras were found in four and one cases, respectively, among 76 late-stage heterologous CS cases, suggesting the relevance of the deregulation in these two genes in the common pathway to the development of uterine CS. In line with this notion, tumors with concurrent mutation in KRAS and TP53 distributed across all four subtypes of uterine CS, which respectively correspond to the same four categories in EC [43]. We speculate that certain genetic alterations may determine the differentiation of cell lineages and molecular subtypes of CS, although further investigation is required.
The characterization of the Kras locus in organoids and TDOs revealed several aspects of CS pathogenesis. First, the copy number of the Kras WT allele frequently decreased in TDOs. We have previously documented the complete loss of the Kras WT allele, albeit at a low frequency, in Kras G12D -expressing TDOs originating from pancreatic organoids [26]. The frequent loss of KRAS WT has also been reported in tumors with mutant KRAS in humans [44]. Collectively, these findings are consistent with the notion that Kras WT acts as a relative tumor-suppressor gene in the presence of oncogenic Kras and that its deletion leads to the hyperactivation of the Kras pathway [45]. Second, the complete loss of Kras WT was observed in the TDOs. Because this is unlikely to occur spontaneously in normal cells, this observation negates the possibility that host-derived stromal cells can transform into CS cells. Last, the presence of residual cells without recombination in the Kras locus was revealed in TDOs. This finding is contrary to previous observations that TDOs originating from adenocarcinoma comprises only cells expressing Kras G12D , but not those that retain Kras WT , thus highlighting the critical roles of Kras activation in the development of adenocarcinoma [25,26,]. Consequently, based on this unexpected finding, we speculated that uterine CS cells might support the survival of normal epithelial cells to retain Kras WT , which might subsequently promote the proliferation of CS cells.
In conclusion, we established uterine CS using an organoid-based approach. Although patient-derived organoids (PDOs) for normal and cancer tissues in gynecologic organs have been recently established [27,[46][47][48][49], to the best of our knowledge, there are almost no reports on PDOs from CS cases. Therefore, our generated tumor organoids will be useful in preclinical studies of CS. In addition, future comparisons among organoids derived from ex vivo carcinogenesis models, GEM, and PDOs can provide further insights into the mechanisms underlying endometrial tumorigenesis.

MATERIALS AND METHODS Mice studies
Female mice of the C57BL/6 J strain and Balb/cA nu/nu (nude mice) at 5 weeks of age were purchased from CLEA Japan Inc. (Tokyo, Japan). Conditional knock-in mice heterozygous for the Lox-STOP-Lox-KrasG12D allele (hereafter referred to as Kras LSL-G12D/+ mice) and Trp53 flox/flox mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA) and maintained in a C57BL/6 J background. These mice were intercrossed to generate Kras LSL-G12D/+ ; Trp53 flox/flox mice. Genotyping was performed after weaning as previously described [28,50,]. Animal studies were carried out with the approval of the Chiba Cancer Center for Ethics in Animal Experimentation.

Tumorigenicity assays in nude mice
Transduced endometrial cells were propagated as organoids in Matrigel. Organoids corresponding to 5 × 10 5 cells were resuspended in 200 μL of medium mixed with Matrigel at a 1:1 ratio and inoculated into the dorsal skin of nude mice. After 8-9 weeks, palpable tumors or residual Matrigel plugs from the injected sites were isolated for histological examination or cell culture. The tumors were processed essentially in the same way as cell preparation for the primary organoid culture. As serum-free culture media do not support the survival of tumor-derived stromal cells, a pure population of epithelial cells is normally obtained within a few passages. In some cases, tumor-derived organoids were re-implanted into the dorsal skin.

Array-based comparative genomic hybridization analysis
To extract genomic DNA from organoids, a NucleoSpin Tissue Kit (Takara, Shiga, Japan) was used. DNA quantity and quality was assessed using a NanoDrop spectrophotometer and agarose gel electrophoresis (Thermo Fisher Scientific), respectively. A SureTaq DNA Labeling Kit (5190-03399, Agilent, Santa Clara, CA) was used to chemically label 500 ng of genomic DNA with either ULS-Cy5 or ULS-Cy3 dye. Hybridization with labeled DNA was performed using a SurePrint G3 mouse CGH microarray 4 × 180 K (G4826A, Agilent). Scanning and image analysis were performed using Agilent Feature Extraction ver.11.0 (Agilent) and a SureScan Microarray Scanner (G4900DA, Agilent). Agilent Genomic Workbench ver.7.0.4.0 software was used to visualize, detect, and analyze chromosomal patterns in the microarray profiles. Organoids without lentiviral infection were analyzed as reference for estimating copy number variations.

Transcriptome analysis
Total RNA was extracted using an RNeasy Mini Kit (Qiagen). The RNA quality and quantity of each sample were checked and measured, respectively, using an Agilent 2100 Bioanalyzer (Agilent). All samples had an RNA integrity number greater than 7.8. The cRNA was prepared from 200 ng of total RNA using a Low Input Quick Amp Labeling Kit, one-color (5190-2305, Agilent), and labeled with cyanin3. Hybridization was performed using the SurePrint G3 Mouse Gene Exp v2 Array Kit 8 × 60 K (G4852B, Agilent). Scanning and image analysis were performed on a SureScan Microarray Scanner (G4900DA, Agilent). Microarray data were analyzed using GeneSpring GX ver.13.1 software (Agilent). Organoids without lentiviral infection served as reference. Data are available at the GEO database (GSE175512).