Ligand-dependent Wnt signaling promotes gastric cancer metastasis through hyaluronan expression in microenvironment

Domain: neuroscience
Published: 2026-02-14
Editorial outcome: major_revision

Abstract
The majority of gastric cancer cells proliferate in a Wnt ligand-dependent manner. To investigate this, we generated mice harboring Kras, Tgfbr2, and Trp53 (KTP) as well as with the same mutations plus Wnt1 expression (WKTP) in gastric mucosa. While KTP mice develop gastric metaplasia, WKTP mice develop dysplastic tumors, highlighting the role of ligand-dependent Wnt signaling in primary tumorigenesis. Organoids derived from WKTP mice form liver metastases following splenic transplantation, whereas KTP organoids do not. Notably, Apc disruption fails to induce metastasis of KTP cells, suggesting that stromal Wnt signaling promotes metastasis. Mechanistically, tumor-derived Wnt ligands cooperate with TGFβ signaling to induce Has2 expression in cancer-associated fibroblasts (CAFs), leading to hyaluronan accumulation in the metastatic microenvironment. Strikingly, hyaluronidase expression in WKTP cells significantly suppresses liver metastasis. Here we show the critical role of ligand-dependent Wnt signaling and Has2-mediated hyaluronan deposition in metastasis, offering potential therapeutic strategy against gastric cancer metastasis.

Introduction
Gastric cancer is the fifth most common cancer worldwide and ranks third in cancer-related mortality1,2. The survival rate for patients with metastatic gastric cancer remains dismal, with a median survival of approximately three months, highlighting the urgent need for the development of preventive and therapeutic strategies against gastric cancer metastasis3,4. However, the mechanisms underlying gastric cancer metastasis are not fully understood.

Comprehensive genomic analyses have identified gene mutations involved in gastric cancer development, leading to its classification into four subtypes: Epstein-Barr virus-positive, microsatellite instability, genomically stable, and chromosomal instability (CIN) types5. Unlike colorectal cancer, where multi-step tumorigenesis is well-characterized, gastric cancer exhibits significant genetic heterogeneity6,7. While mutations in APC or CTNNB1, which activate canonical Wnt signaling, are present in 86% of non-hypermutated colorectal cancers, these mutations are less common in gastric cancer5,7. Nevertheless, Wnt signaling plays a critical role in maintaining normal tissue stem cells and is thought to be important for the development of various types of cancers, including gastric cancer8,9.

Recent analyses using gastric cancer-derived organoids have shown that Wnt signaling is activated in approximately 60% of gastric cancers through an exogenous ligand-dependent mechanism, despite the absence of APC or CTNNB1 mutation10. Elevated expression of Wnt ligand family genes, such as Wnt1, Wnt3a, and Wnt5a, as well as aberrant expression of Wnt receptors and co-receptors, has been identified in gastric cancer. These contribute to constitutive Wnt signaling activation9,11. The role of ligand-dependent Wnt signaling in gastric tumorigenesis has been previously reported. For instance, KLF3 is upregulated in gastric cancer, leading to increased Wnt1 expression, which promotes proliferation, migration, and metastasis of gastric cancer cells12. Gastric cancer cells disseminated in the peritoneal cavity express Wnt3a and Wnt5a in ascitic fluid, enhancing the colony-forming ability of dormant cancer cells13. Moreover, Wnt1 transgenic mice have been shown to develop glandular-type gastric tumors when inflammatory responses are present14,15. Despite these findings, the detailed role of ligand-dependent Wnt signaling in gastric cancer, particularly in metastasis, remains unclear.

Recently, a genetic mouse model for CIN-type gastric cancer was developed by introducing mutations in Kras, Tgfbr2, and Trp53 simultaneously in gastric epithelial cells using Anxa10 gene promoters16. This model develops invasive gastric adenocarcinomas with histological features similar to human CIN-type gastric cancers. However, the role of Wnt signaling was not examined in this model.

In this work, we analyze the role of ligand-dependent Wnt signaling in gastric cancer development and metastasis by generating novel mouse and organoid models. Using this system, we demonstrate that Wnt ligand expression in tumor cells is crucial for liver metastasis development. Tumor cell-secreting Wnt ligands activate stromal cells to express Has2, leading to the production and deposition of hyaluronic acid (hyaluronan) in the tumor microenvironment. This hyaluronan deposition is critical for the survival and colonization of disseminated cancer cells in the liver. These results suggest that targeting the ligand-dependent Wnt signaling or HAS2 expression in the stromal cells could serve as a potential preventive or therapeutic strategy against gastric cancer metastases.

Results
Ligand-dependent Wnt signaling promotes gastric tumor formation
Kras activation, TGFβ suppression, and p53 mutation are major driver pathways in CIN-type or intestinal-type gastric cancer5,7. We generated compound mouse models, in which Kras+/G12D, Tgfbr2-/-, or Trp53+/R270H mutations were introduced in the gastric epithelial cells using Claudin18-CreERT2 mice17. In this study, we designated these model mice as K (Kras+/G12D), KT (Kras+/G12D, Tgfbr2-/-), KP (Kras+/G12D, Trp53+/R270H), and KTP (Kras+/G12D, Tgfbr2-/-, Trp53+/R270H) (Fig. 1a). It has been shown that Wnt signaling is activated in a ligand-dependent manner in more than half of gastric cancers9,10. We found upregulation of at least one of the Wnt ligand family member genes except Wnt8A in more than 80% of gastric cancers (Supplementary Fig. 1). In this study, we selected Wnt1 based on previous reports demonstrating a strong correlation between WNT1 expression and advanced grade of gastric cancer, and showing that WNT1-mediated signaling promotes the gastric tumor growth and metastasis12,18. We thus crossed the KT, KP, and KTP mice with K19-Wnt1 transgenic mice, which express Wnt1 in gastric epithelia, to generate WKT, WKP, and WKTP mice, respectively (Fig. 1a). K19-Wnt1 mice exhibit only limited dysplastic lesions in the gastric mucosa14,15.

All mutant mice except the simple Kras mutant group showed significantly increased gastric mucosal thickness compared with wild-type mice (Fig. 1b, c). In wild-type mice, Ki67-positive proliferating cells were confined to the gland neck, while in KT, KP, and KTP mice, they were significantly increased at the gland bottom of the glandular stomach (Fig. 1d). In WKT, WKP, and WKTP mice, Ki67-positive cells spanned entire glands, with a significantly increased Ki67 labeling index (Fig. 1d, e). These results indicate that the KrasG12D with Tgfbr2-/- or Trp53R270H mutations induce mucosal hyperplasia, with concurrent Wnt signaling activation further enhancing proliferation.

The KrasG12D mutation also impacted cell differentiation: H+/K+ ATPase-expressing parietal cells were lost in all mice carrying KrasG12D mutation, while Muc5AC-expressing mucous cells increased in the K, KT, KP, and KTP mouse glandular stomachs (Fig. 1f). Furthermore, dysplastic epithelial cells proliferated predominantly in the gastric mucosa of WKT, WKP, and WKTP mice, which was associated with decreased Muc5AC-expressing cells (Fig. 1b). Collectively, these results indicate that KrasG12D mutation induces hyperplasia and mucous cell metaplasia, whereas the combination of KrasG12D mutation and Wnt signaling activation leads to suppression of epithelial differentiation and the formation of dysplastic tumors (Supplementary Table 1).

KT, KTP, WKT, and WKTP mice that had KT mutations in their genotypes developed invasive tumors in the submucosa of the stomach, while such invasive tumors were not found in K, KP, WKP mice (Fig. 1b, g). Aggressive submucosal tumors with a collagen-rich fibrotic microenvironment formed in both KTP and WKTP mice (Fig. 1h). These results suggest that a combination of KrasG12D and Tgfbr2-/- (KT) mutations is sufficient for submucosal invasion, independent of Wnt ligand expression (Supplementary Table 1).

KTP mutations are sufficient for invasive tumor formation
We next established organoids from gastric epithelial cells of tamoxifen-untreated KTP and WKTP mice, and Kras, Tgfbr2, and Trp53 mutations were introduced in the organoids via tamoxifen treatment (Fig. 2a). As expected, KTP organoids could not survive or proliferate when Wnt ligands were removed from the culture medium, while WKTP organoids continued to proliferate, even without Wnt ligands in the medium (Fig. 2b, c). Notably, inhibition of Wnt ligand secretion using the PORCN inhibitor, C59, significantly suppressed proliferation of WKTP organoid cells. These results indicate that both KTP and WKTP cells require exogenous Wnt ligand stimulation for survival and proliferation, but WKTP cells utilize self-secreted Wnt ligands via autocrine or paracrine mechanisms (Fig. 2d).

We then injected KTP and WKTP organoid cells orthotopically into the submucosal area of the immunodeficient NSG mouse stomach (Fig. 2e). In vivo imaging analysis indicated that both KTP and WKTP cells developed tumors of similar sizes in the stomach (Fig. 2f, g). Histologically, dysplastic KTP and WKTP tumor cells proliferated in the submucosa of the stomach, and a fibrotic microenvironment was generated with αSMA-expressing fibroblasts (Fig. 2h). When KTP and WKTP organoid cells were transplanted subcutaneously, both organoids formed similar dysplastic tumors associated with a fibrotic microenvironment like orthotopic tumors (Supplementary Fig. 2). These results suggest that the combination of KTP driver mutations is sufficient to induce dysplastic tumor formation in both the submucosal and subcutaneous environments.

Wnt ligand expression is required for liver metastasis
We next evaluated the role of Wnt ligands in liver metastasis. In the orthotopic transplantation model of WKTP organoids, three of five mice exhibited signs of peritoneal dissemination by day 28, accompanied by clinical deterioration, which limited the observation window for assessing liver metastasis (Supplementary Fig. 3). We therefore performed spleen transplantation of KTP and WKTP organoids in NSG mice to examine the liver metastatic tumors (Fig. 3a).

Importantly, WKTP organoid cells formed large metastatic tumors in the liver, whereas KTP organoid cells did not develop detectable tumors in the liver (Fig. 3b, c). Histologically, metastatic tumors of the WKTP cells consisted of dysplastic and proliferating tumor cells, and a fibrotic microenvironment with αSMA-expressing cells was generated in the stroma (Fig. 3d, right). In contrast, KTP cells formed only small, scattered lesions, lacking a fibrotic microenvironment (Fig. 3d, left). No metastatic lesions were detected outside the liver in mice transplanted with either WKTP or KTP organoids.

Furthermore, treatment of mice with PORCN inhibitor ETC-159 significantly suppressed liver metastasis formation by WKTP cells (Fig. 3e, f). Another inhibitor, GNF-6231, also reduced tumor area, although the difference was not statistically significant. Histological analysis revealed that the remaining WKTP cells in the ETC-159-treated mouse livers were differentiated, forming enlarged cystic structures with decreased Ki67 labeling (Fig. 3g). These results, taken together, indicate that Wnt ligand expression is critical for the development of liver metastases in gastric cancer.

We next performed histological analysis to examine the early stages of liver metastasis development using GFP-labeled KTP and WKTP organoids. On day 3 after organoid transplantation to the spleen, both KTP and WKTP cells were detected in the liver sinusoids with similar efficiency (Fig. 3h). On day 7 after transplantation, numerous small metastatic lesions of WKTP cells were detected in the liver, accompanied by activated fibroblasts and infiltrating CD206+ M2-like macrophages (Fig. 3h, i, and Supplementary Fig. 4). In contrast, such metastatic lesions were rarely detected in livers transplanted with KTP cells. Therefore, it is possible that ligand-dependent Wnt signaling is critical for the colonization of the disseminated tumor cells during the early stage of liver metastasis.

Cell-intrinsic Wnt signaling is insufficient for metastasis
The Wnt ligands secreted by WKTP cells may activate Wnt signaling not only in tumor cells but also in stromal cells. To examine whether Wnt signaling activation in tumor cells alone is sufficient for metastasis, we disrupted the Apc gene in KTP cells using CRISPR/Cas9 to generate Apc-KTP cells (Fig. 4a, Supplementary Fig. 5). In Apc-KTP cells, Wnt signaling is expected to be constitutively activated in a ligand-independent manner due to homozygous Apc gene disruption, while Wnt signaling in surrounding stromal cells remains unaffected (Fig. 4b). As a control, we constructed Wnt1-expressing KTP cells, Wnt-KTP, by transfecting a Wnt1 expression vector into KTP cells.

Loss of APC suppresses phosphorylation-dependent β-catenin degradation, thereby increasing the unphosphorylated (active) form of β-catenin. The stabilized β-catenin translocates to the nucleus and functions with TCF/LEF to drive Wnt-target gene transcription. Consistent with this rationale, immunoblotting showed a significant increase in unphosphorylated β-catenin in Apc-KTP cells compared with KTP and WKTP cells (Fig. 4c), and the TOPFlash reporter assay demonstrated significantly higher β-catenin/TCF transcriptional activity in Apc-KTP cells, which is comparable to the Apc-disrupted intestinal tumor-derived organoids, AKTP19 (Fig. 4d). The Wnt activation level in Wnt-KTP cells was also increased, comparable to that in WKTP cells. However, proliferation rates and invasive capacities of Apc-KTP and Wnt-KTP cells were not changed from those of parental KTP cells (Supplementary Fig. 6a, b).

Subsequently, the Apc-KTP and Wnt-KTP cells were transplanted into the mouse spleen (Fig. 4e). We confirmed multiple metastatic tumors of WKTP cells in the livers, while no tumors were found in the KTP cell-transplanted mice (Fig. 4f, g). Among the three independently established Apc-KTP organoid lines, only line #2 developed limited, small macroscopic liver metastasis (Fig. 4f, arrowheads, Fig. 4g), whereas other lines showed no grossly detectable liver metastasis. Moreover, histological analysis revealed rare, small clusters of tumor cells in #2 and #8 Apc-KTP-transfected livers (Supplementary Fig. 6c), which were below the threshold of gross detection. In contrast, Wnt-KTP organoid cells formed visible liver tumors. These results indicate that cell-intrinsic Wnt signaling activation in tumor cells is insufficient for metastasis, suggesting that Wnt signaling activation in stromal cells is essential for liver metastasis.

Wnt signaling activation in CAFs of liver metastatic tumors
To investigate Wnt signaling activation in the tumor stroma, we performed spatial transcriptomic analysis (Visium) on histological sections of WKTP cell liver metastasis (Fig. 5a). RNA sequence data at 3,634 spots identified seven clusters, with cluster 7 excluded as it corresponded to necrotic areas (Fig. 5b). We next examined the expression of cell-type-specific marker genes. The hepatocyte marker Alb was significantly enriched in cluster 3 and 6, whereas Prom1 and Muc1, which are highly expressed in WKTP cells, were predominantly expressed in cluster 2 (Fig. 5c). On the other hand, myofibroblast markers, Acta2 and Tagln, were increased in cluster 4 and 5, and fibroblast markers, Pdgfrb and Col6a2 were increased in Cluster 5. H&E staining indicates that the major cell type in the cluster 5 area is fibroblastic cells of tumor stroma (Fig. 5d, enclosed with a white line in A), while both fibroblasts and tumor epithelial cells are mixed in the cluster 4 area (Fig. 5d, enclosed with a yellow line in B), indicating that CAFs are predominantly enriched in cluster 5. We confirmed αSMA expression in fibroblastic cells of cluster 5 area by immunohistochemistry (Supplementary Fig. 7a). Notably, cluster 5 was enriched with extracellular matrix (ECM) components, Eln, Dpt, Fbn1, Fbn2, Col1a2, Col3a1, Col6a2, Col8a1, and Col15a1, and ECM remodeling enzymes, Has2, Mmp2, Mmp23, and Adam19 (Supplementary Data 1). These results suggest dynamic remodeling of the fibrotic microenvironment in the liver metastatic tumors.

Ingenuity pathway analysis (IPA) using the cluster 5 differentially expressed genes (DEGs) revealed significant activation of Wnt signaling pathways including CTNNB1 and WNT3A, and TGFβ pathways, such as TGFB1, SMAD3, and SMAD4 (Fig. 5e). Consistently, we found nuclear accumulation of phosphorylated Smad2 in stromal cells of liver metastatic tumors (Supplementary Fig. 7b). We also confirmed both WKTP tumor cells and CAFs express TGFβ1 (Supplementary Fig. 7c). These findings suggest that WKTP cells serve as the primary source of TGFβ ligands, which in turn activate stromal cells to express TGFβ themselves, resulting in sustained activation of the TGFβ signaling pathway. Since TGFβ is crucial for generating a fibrotic microenvironment in the liver19,20, these findings suggest that tumor cell-secreting Wnt ligands activate Wnt signaling in CAFs, cooperating with TGFβ to promote metastatic microenvironment formation.

Wnt ligands activate stromal fibroblasts
It has been reported that CAFs in gastric cancer tissues can be classified into myofibroblastic CAFs (myCAFs) and inflammatory CAFs (iCAFs) based on distinct gene expression profiles21,22. Moreover, recent findings indicate that hepatic stellate cells (HSCs) have the potential to differentiate into both myCAFs and iCAFs23. However, it is also established that CAFs exhibit considerable heterogeneity in their cellular origins24. Therefore, to assess whether Wnt ligands activate stromal fibroblasts in the metastatic niche, we conducted experiments using both HSCs and CAFs, treating them with afamin-bound Wnt3a, which maintains stable canonical Wnt ligand activity25. (Fig. 6a). Afamin-Wnt3a treatment significantly increased Wnt signaling activation in both the HSCs and CAFs (Fig. 6b), and it also upregulated Wnt-target genes such as Axin2, Lef1, and Cd44 in HSCs (Fig. 6c). Additionally, expression of Acta2 encoding αSMA was also upregulated in HSCs by Afamin-Wnt3a, indicating that Wnt ligand-signaling activates stromal fibroblasts (Fig. 6d, e). Furthermore, combined treatment with afamin-Wnt3a and TGFβ synergistically enhanced HSC contraction in ECM gels (Fig. 6f), suggesting that Wnt and TGFβ signaling cooperatively activate HSCs, leading to activation of CAFs in the microenvironment.

Hyaluronan promotes metastatic tumor development
Among the genes upregulated in cluster 5, Has2, which encodes hyaluronan synthase, was identified (Supplementary Data 1). We confirmed that Has2 expression is specifically increased in cluster 5 of liver metastatic tumors of WKTP cells (Fig. 7a). A recent study indicates that hyaluronan plays an important role in the malignant progression of colon cancer26. Furthermore, Has2 expression in primary HSCs, imHSCs, and CAFs was significantly increased upon treatment with either afamin-Wnt3a or TGFβ. Notably, Has2 expression levels were dramatically elevated when HSCs and CAFs were simultaneously stimulated with both afamin-Wnt3a and TGFβ, indicating a synergistic effect of Wnt and TGFβ signaling in Has2 induction in stromal cells (Fig. 7b). We further examined HAS2 expression in human gastric cancer using the cBioPortal database (https://www.cbioportal.org/). As expected, there was a positive correlation between HAS2 and ECM component genes, including collagens (Supplementary Fig. 8a). Moreover, we observed a modest but positive correlation between HAS2 and canonical Wnt ligands such as WNT2, WNT2B, and WNT6 (the latter has been reported to act predominantly through the canonical Wnt signaling), suggesting a potential role for canonical Wnt signaling in the regulation of HAS2 expression in human gastric cancer (Supplementary Fig. 8b). Interestingly, there is also a positive correlation the non-canonical Wnt ligand WNT5A, suggesting a possible involvement of non-canonical Wnt signaling in HAS2 induction. We also detected significant hyaluronan deposition in the stroma of liver metastatic tumors of mouse and human gastric cancer (Fig. 7c, Supplementary Fig. 8c). It is of note that hyaluronan deposition was found from the early stage (day 7) of metastasis development.

We then examined the role of hyaluronan in liver metastasis development. To suppress hyaluronan deposition in the tumor stroma, we transfected expression vectors of hyaluronidases, Hyal1 and Hyal2, into WKTP cells to generate Hyal-WKTP cells (Fig. 7d). We anticipated that Hyal-WKTP cells, which co-express Hyal1 and Hyal2, would degrade both high molecular weight (HMW)- and low molecular weight (LMW)-hyaluronan within the tumor microenvironment27. Immunoblotting confirmed the expression of both Hyal1 and Hyal2 in Hyal-WKTP cells, and their expression did not affect the proliferation rate of WKTP cells (Supplementary Fig. 9a). Subsequently, Hyal-WKTP cells were transplanted into mouse spleens, and liver tissues were examined (Fig. 7e). Notably, metastatic tumor development was markedly suppressed in mice transplanted with Hyal-WKTP cells compared to those transplanted with parental WKTP cells (Fig. 7f, g). Interestingly, Hyal-WKTP cells formed tumors of a similar size at the spleen transplantation site as parental WKTP cells (Supplementary Fig. 9b). These results collectively indicate that hyaluronan deposition in the tumor stroma is crucial for the development of liver metastatic tumors in gastric cancer.

Methods
Ethics statement
All experiments were conducted in compliance with all relevant ethical regulations. All animal experiments were performed with the protocol approved by the Committee on Animal Experimentation of Kanazawa University (approval no. AP-214223). The approved humane endpoints were based on clinical signs of deterioration or severe distress. Because tumor burden could not be measured in the orthotopic/splenic transplantation model, animals were euthanized if they exhibited ≥35% loss of body weight from baseline or inability to remain upright. For animals exhibiting 25–35% body weight loss, health checks were performed at least twice daily, and water-soaked food was provided on the cage floor to support hydration and nutrition. Animals meeting these criteria were euthanized immediately. All experiments using human samples were approved by the Human Genome/Gene Analysis Research Ethics Committee of Kanazawa University, and written informed consent was obtained from all patients.

Mouse strains
Tgfbr2flox/flox, Trp53+/LSL-R270H, and Kras+/LSL-G12D mice were obtained from the Mouse Repository (NCI-Frederick, Frederick, MD)42–44. K19-Wnt1 mice were previously constructed14,15. For induction of driver mutations in gastric epithelial cells, Claudin18-CreERT2 mice were used17. The genetic background of all mutant strains used in this study is C57BL/6. Mice were administered tamoxifen intraperitoneally (i.p.) at 4 mg/mouse at 6 weeks of age, and tumor phenotypes in the glandular stomach were evaluated 28 days after administration (n=3 mice for each genotype). Both male and female mice were used for examination. No significant sex-related differences were observed in tumor phenotypes.

Human samples
Three liver metastasis samples of gastric cancer were obtained from three male patients aged 45–78 years. No participant compensation was provided. These tissues were fixed in 10% buffered formalin, embedded in paraffin, and 4-μm sections were used for histological assessment and hyaluronan expression analysis. Human samples were limited to three male patients; therefore, sex-based analyses were not performed.

Histology and Immunohistochemistry
Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 4-µm thickness. These sections were stained with H&E or processed for immunohistochemistry. Leica DM5000B (Leica) and BZ-9000 Biorevo (Keyence) were used to examine histology, immunohistochemistry (IHC), and fluorescence IHC.

The height of the gastric mucosa was measured on the H&E sections, and the relative mucosal thickness to the wild type was calculated. Tumor areas on the digital images of H&E sections were colored in yellow using an All-in-One microscope and BZ-II Analyzer (Keyence, Kanagawa, Japan), and the tumor areas were measured per field.

Primary antibodies used were anti-Ki67 (#ab16667, Abcam, Cambridge, UK; 1:1,000), anti-H+/K+-ATPase β subunit (#D032-3, MBL, Tokyo, Japan; 1:1000), anti-Muc5AC (#MS-145, Thermo Fisher Scientific, Grand Island, NY; 1:200), anti-αSMA (#A2547, SIGMA, St. Louis, MO; 1:800), anti-E-cadherin (#AF748, R&D Systems, Minneapolis, MN; 1:100), anti-GFP (#2956, Cell Signaling, Danvers, MA; 1:500), anti-CD206 (#24595, Cell signaling, Danvers, MA; 1:200), anti-F4/80 (#MCA497, Bio-Rad, Hercules, CA; 1:500), and anti-phospho-Smad2 (#44-244G, Thermo Fisher Scientific; 1:400). The staining signals were visualized using an ImmPACT DAB Peroxidase Substrate Kit (#SK-4105, Vector Laboratories, Burlingame, CA).

The number of Ki67 staining positive cells and total epithelial cells was counted in 3~5 independent microscopic fields per mouse (n=3 mice), and the Ki67 labeling index was calculated. Submucosal invasion efficiency was assessed on the E-cadherin immunostaining sections. Masson trichrome staining was performed using Trichrome Stain (Masson) Kit (#HT15, SIGMA, St. Louis, MO). Detection of hyaluronan was performed as previously described45. Briefly, paraffin sections were incubated with Avidin/Biotin Blocking kit (#SP-2001, Vector Laboratories, Burlingame, CA), followed by 5μg/mL of biotinylated hyaluronan binding protein (#BC41, Hokudo CO., LTD, Sapporo, Japan) for 1 h. Staining signals were visualized using an ImmPACT DAB Peroxidase Substrate Kit (#SK-4105, Vector Laboratories) or Alexa Fluor 594-conjugated Streptavidin (#016-580-084, Jackson ImmunoResearch, West Grove, PA).

Immunocytochemistry
Cultured cells were fixed with 4% paraformaldehyde, and incubated with antibody for αSMA (#A2547, SIGMA, St. Louis, MO; 1:800), followed by Alexa Fluor 488-conjugated antibody (#A-21206, Thermo Fisher Scientific, Grand Island, NY; 1:200). DyLight 594-Phalloidin (#12877, Cell Signaling, Danvers, MA; 1:20) was used to detect F-actin.

Organoid culture
Organoids were established from mouse gastric tumors and cultured according to the previously described methods46. For each genotype, organoids were derived from both male and female mice; however, sex was not considered as a biological variable in this study because no sex-dependent differences were observed in the primary tumor phenotypes. In brief, dissected glandular stomach tissues were soaked in 5 mM EDTA at 4°C for 2 h, and the isolated glands were embedded in growth-factor-reduced (GFR) Matrigel (#356231, Corning, Corning, NY), and cultured in advanced DMEM/F-12 medium with 10 mM HEPES, 2 mM Glutamax, 1×N2, 1×B27 (Invitrogen, Carlsbad, CA), 1 mM N-acetyl-cysteine (Sigma-Aldrich, St. Louis, MO), supplemented with 50 mg/ml EGF (Invitrogen), 100 ng/ml FGF10 (Peprotech, Rocky Hill, NJ), 10 nM gastrin (Sigma-Aldrich), 5 μM GSK3 inhibitor (CHIR-99021; Tocris Bioscience, Bristol, UK), 10 μM ROCK inhibitor (Y-27632; Sigma-Aldrich), and 50% of L-WRN-conditioned medium47. L-WRN cells express Wnt3a, R-spondin, and Noggin in the medium. In the absence of WRN-CM (WRN0%), 100 ng/ml Noggin (Peprotech, Cranbury, NJ) was added to the medium. Established organoid cells were treated with 1 μM of 4-hydroxy (4-OH) tamoxifen (Sigma-Aldrich) for 14 days to induce KTP mutations. The cell proliferation rate was examined using a CellTiter-Glo 3D Cell Viability Assay (#G9682; Promega, Madison, WI).

To inhibit Wnt ligand signaling, organoids were treated with 100 nM PORCN inhibitor, Wnt-C59 (C59) (S7037; Selleckchem, Houston, TX).

To label organoids, GFP cDNA was subcloned into the pPB-CAG-IP PiggyBac transposon expression vector (a gift from Dr. Hitoshi Niwa) and co-transfected with a transposase expression vector into organoid cells using Lipofectamine (Thermo Fisher Scientific). For the invasion assay, cells were seeded in Matrigel-coated Fluoroblok culture inserts with 8 μm pores (Corning). The invaded cell areas were quantified using an All-in-One microscope and BZ-II Analyzer (Keyence, Kanagawa, Japan).

Transfection and gene disruptions
To establish luciferase-expressing KTP and WKTP cells, a luciferase expression vector, pPB-CAG-Luc-IB piggyBac48, was co-transfected with a transposase expression vector (a gift from Dr. Hitoshi Niwa) using Lipofectamine LTX (#15338100, Thermo Scientific, Waltham, MA). The luciferase-expressing cells were selected by culture in 1 μg/mL blasticidin for 7 days.

To establish Apc-KTP cells, the pSpCas9(BB)-2A-Puro (PX459) V2.0 plasmid (#62988, Addgene) was used to package guide oligonucleotides targeting Apc. Cells were transfected with the constructed plasmid, and selected by 1 μg/mL puromycin culture for 2 days. The single-guide RNA (sgRNA) sequence is as follows: ACGGAAGTCGGGAAGGATCT.

To establish Wnt-KTP cells, mouse Wnt1 cDNA was excised from pUSEamp-Wnt1 (Upstate, Charlottesville, VA) and subcloned into pPB-CAG-IP piggyBac (a gift from Dr. Hitoshi Niwa) to construct the pPB-CAG-Wnt1-IP vector. To establish Hyal1/2-KTP cells, Hyal1 and Hyal2 cDNAs were amplified by RT-PCR. Primer sequences for RT-PCR are as follows: mHyal1-F: GCC’AAG’ACA’TGC’TTG’GGC’TTA, mHyal1-R: GTT’GGG’TGC’AGC’TAA’TCA’TCA, mHyal2-F: GAG’CTG’CTA’CCA’GGC’AGG’TA, mHyal2-R: TCC’AGG’GGG’AGA’GAT’CCC’TCA. After sequence confirmation, the Hyal1 and Hyal2 cDNA fragments were subcloned into pPB-CAG-IP piggyBac and pPB-CAG-IB piggyBac, respectively. These piggyBac vectors were co-transfected with transposase expression vector using Lipofectamine LTX (#15338100, Thermo Scientific, Waltham, MA), and Hyal1/Hyal2-expressing cells were selected by 1 μg/mL puromycin and 1 μg/mL blasticidin cultures for 7 days.

Organoid transplantation in mouse experiments
For orthotopic transplantation, 5×105 each of KTP or WKTP cells were transplanted into submucosa of the glandular stomach of female 6 weeks of age NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (NSG mice; The Jackson Laboratory, Japan) (n=3 for each genotype), and tumor phenotypes were histologically examined at 21 days after transplantation.

For liver metastasis analysis, 5×105 each of KTP, WKTP, Apc-KTP, Wnt-KTP, or Hyal-WKTP cells were transplanted into the spleens of female 6 weeks of age NSG mice, and liver tissues were examined at 3 and 7 days (for early stage analysis), 14 days (for PORCN inhibitor treatment experiments), and 21 days after transplantation. For subcutaneous (s.c.) transplantation, 5×105 each of KTP or WKTP cells were transplanted s.c. to female 6 weeks of age Crlj:SHO-Prkdcscid Hrhr mice (SHO mice, The Jackson Laboratory, Japan), and tumor phenotypes were histologically examined at 28 days after transplantation. We used female mice for these transplantation experiments.

For inhibitor treatment experiments, WKTP cells were transplanted into the spleen, and mice were treated with PORCN inhibitors, ETC-159 (Selleckchem, Houston, TX) (30 mg/kg/day for 14 days, oral administration, p.o., n=5) or GNF-6231 (Selleckchem, Houston, TX) (3 mg/kg/day for 14 days, p.o., n=5) according to a previously described method49,50. Liver tissues were examined at 14 days after splenic transplantation with no-drug control mice (n=8).

In vivo luciferase imaging
In vivo imaging analyses were performed at 1, 2, and 3 weeks after transplantation of luciferase-expressing KTP and WKTP cells. Mice were intraperitoneally injected with 150 mg/kg D-luciferin (Fujifilm, Osaka, Japan), and liver bioluminescence was measured using IVIS Lumina LT and Living Image Software (PerkinElmer, Waltham, MA).

Wnt signaling reporter assay
For measurement of Wnt signaling activity, the cells were transfected with Super 8×TOPFlash or Super 8×FOPFlash (Addgene, Cambridge, MA) using Lipofectamine LTX (Thermo Fisher). After 24 h of transfection, the luciferase activity was measured using a Dual-Luciferase Reporter assay system (Promega, Madison, WI).

Immunoblotting
Cultured cells were lysed in TNE buffer with cOmplete Mini Protease Inhibitor Cocktail (#11836170001, Roche, Basel, Switzerland), and 10 μg of the protein samples were separated using 10% SDS-polyacrylamide gel. Primary antibodies used were anti-β-catenin (#C2206, Sigma-Aldrich; 1:5000), anti-active β-catenin (#05-665, Sigma-Aldrich; 1:500), anti-TGFβ1 (#ab179695, Abcam; 1:1000), anti-Hyal1 (#25179-1-AP, Proteintech, Rosemont, IL; 1:1000), and anti-Hyal2 (#15115-1-AP, Proteintech, Rosemont, IL; 1:1000). Antibody against GAPDH (#016-25523, FUJIFILM Wako Pure Chemicals, Japan; 1:2000) was used for internal control. The ECL detection system (#RPN2235, GE Healthcare, Buckinghamshire, UK) was used to detect immunoblotting signals. Band intensities of active β-catenin were measured using ImageJ software (v1.52, NIH, Bethesda, MD) and normalized with those of GAPDH. Uncropped and unprocessed scans of blots are provided as a Source Data file.

HSC and CAF culture experiments
Hepatic stellate cells (HSCs) were isolated from C57BL/6 or Trp53 LSL-R270H mouse livers according to the protocol51. Trp53 LSL-R270H-derived HSCs were established as immortalized HSCs (imHSCs). Cancer-associated fibroblasts (CAFs) were isolated from liver metastasized tumors in Trp53 LSL-R270H mice transplanted with intestinal tumor-derived AKTP cells19. The primary HSCs, imHSCs, and CAFs were stimulated with 10% Afamin-Wnt3a (#J2-001, MBL, Tokyo, Japan) and/or 20 ng/mL recombinant mouse TGFβ1 (#7666-MB, R&D, Minneapolis, MN) for 48 h.

For PCR Array analysis, the cDNA was synthesized using RT2 First Strand Kit (#330401, Qiagen GmbH, Hilden, Germany), and expression of Wnt-target genes was examined using the RT2 Profiler PCR Array Mouse WNT Signaling Targets (#PAMM-243ZD, Qiagen GmbH, Hilden, Germany). The statistical significance of the expression data was analyzed using the GeneGlobe Data Analysis Center (https://geneglobe.qiagen.com/us/analyze/).

For contraction assay, 1×106 HSCs were suspended in 1 mL ECM gel [Cell matrix Type I-A (Nitta gelatin, Japan): Matrigel (Corning) = 3:1], transferred to a 24-well plate, and allowed to solidify at 37°C for 30 min. Then, 10% Afamin-Wnt3a and/or TGFβ1 were added to the culture medium, and the size of the ECM disks was measured at 48 h.

RT-PCR analysis
Total RNA was extracted from the primary HSCs, imHSCs, and CAFs using Isogen (Nippon Gene, Toyama, Japan), reverse-transcribed using a PrimeScript RT reagent Kit (＃RR081, Takara Bio, Otsu, Japan), and amplified using TB Green Premix Ex Taq II (＃RR820, Takara Bio, Otsu, Japan) in an AriaMx real-time thermocycler (Agilent Technologies). Relative mRNA levels of Has2 and Acta2 to those of Gapdh were calculated. The primer sequences are indicated as follows: Alb-F: TTGCAAGGCTGCTGACAAGG, Alb-R: TTTAGGCTAAGGCGTCTTTGCATC, Prom1-F: TGTTAATGGCATTCTGTGTGGCTA, Prom1-R: CCTCTGAATCCATCCTGCGATA, Muc1-F: AAGAGCTATGGGCAGCTGGAC, Muc1-R: TCACCACAGCTGGGTTGGTATAA, Acta2-F: GACAATGGCTCTGGGCTCTGTA, Acta2-R: TTTGGCCCATTCCAACCATTA, Has2-F: TGTGAGAGGTTTCTATGTGTCCT, Has2-R: ACCGTACAGTCCAAATGAGAAGT, Gapdh-F: GGCACAGTCAAGGCTGAGAATG, Gapdh-R: ATGGTGGTGAAGACGCCAGTA

Spatial transcriptomic analysis
Formalin-fixed, paraffin-embedded (FFPE) samples passing the RNA quality control (DV200>50%) were used for spatial transcriptomic construction and sequencing. The tissue was prepared according to the Visium CytAssist Special Gene Expression for FFPE-Tissue Preparation Guide (10X Genomics, Pleasanton, CA). The sequencing was performed by the Research Institute for Microbial Diseases at Osaka University. Libraries were sequenced on an MGI DNBSEQ-G400RS (MGI Tech Co, Shenzhen, China). Raw spatial transcriptomics data were processed using the Space Ranger pipeline (v2.0.1, 10x Genomics, Pleasanton, CA), aligned to the mm10-2020-A mouse reference genome, and analyzed from the resulting FASTQ files. For downstream analysis, including UMAP visualization, violin plots, and gene expression analysis, we used Loupe Browser v6.0 (10x Genomics). Principal component analysis (PCA) was performed using 100 dimensions.

The cell-type-specific marker genes were extracted using the PanglaoDB database (https://panglaodb.se/)52. Upstream regulators were examined using the Ingenuity Pathway Analysis (IPA) (Ingenuity Systems, QIAGEN Digital Insights). A Z-score of ≥2 and a p-value of <0.05 were considered to indicate activation with statistical significance.

Database analysis
The genetic alterations, including mutations, structural variants, copy number variations, and mRNA overexpression in WNTs and HAS2, were analyzed in 440 stomach adenocarcinoma (STAD) samples from the TCGA PanCancer Atlas using the cBioPortal database (https://www.cbioportal.org/). Samples with an mRNA expression Z-score of 1 or higher across all STAD samples in the genomic profile were considered to exhibit overexpression. Expression levels in these samples were confirmed to be more than twice as high as those in the other samples. The distribution of genetic alterations in WNTs and HAS2 was analyzed using OncoPrint in cBioPortal. Additionally, co-expression analysis between HAS2 and ECM components, as well as between HAS2 and WNTs, was performed using cBioPortal, with Spearman’s correlation applied. Genes related to extracellular matrix (ECM) components and the collagen family were extracted using QuickGO (https://www.ebi.ac.uk/QuickGO/).

Statistical analyses
The data are presented as the mean ± standard deviation (s.d.). The statistical analysis was performed using two-sided unpaired t-tests or a one-way ANOVA, followed by Tukey’s post-hoc test. Excel (version 16.94, Microsoft) and GraphPad Prism 9 were used for these analyses. A one-sided Fisher’s exact test was used to calculate P-values of overlap in Ingenuity Pathway Analysis. P-values of <0.05 were considered statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors identified a new role for WNT ligands in promoting liver metastasis of gastric cancer, utilizing genetically engineered organoids, transplantation models, and spatial transcriptomics (Visium). It was proposed that canonical Wnt ligands (specifically Wnt1) secreted by tumor cells upregulate hyaluronan synthase, Has2, in cancer-associated fibroblasts (CAFs) derived from hepatic stellate cells, thereby promoting liver metastasis. This is an interesting study that unveils an unexpected role of canonical WNT ligands in establishing a tumor-favoring metastatic niche. However, several points warrant further attention through new experimental results, discussion, and tone adjustments.\n\nMajor comments\n1. Orthotopic vs. intrasplenic transplantation. With the exception of gastric tumor analysis (Figure 2), the authors performed intrasplenic injection of organoids instead of orthotopic transplantation. Consequently, this study does not provide direct evidence on whether orthotopic injection of KTP or WKTP organoids into the stomach would result in metastasis to other organs. It is necessary to assess whether orthotopically injected organoids/cells give rise to distant metastases, particularly to the liver, followed by repeating key experiments shown in Figures 3-7 using orthotopic models.\n2. Immunocompetent vs. immunocompromised mice: The authors utilized NSG and SHO mice for transplantations. Given that KTP and WKTP organoids were derived from C57BL/6 mice, syngeneic transplantation in C57BL/6 mice could offer a more pathophysiologically relevant immune environment. Could the authors clarify their rationale for choosing an immunodeficient background instead? Considering the aforementioned point (#1), performing orthotopic transplantation of organoids (or organoid-derived cells) in C57BL/6 mice is necessary. Alternatively, investigating liver metastasis in the genetically engineered mouse models (GEMMs) (Figure 1) could also be considered, as the authors described in the Introduction (lines 92-96; notably, this GEMM was not used to study metastasis in this manuscript).\n3. Mechanisms: How does WNT/β-catenin signaling convert HSCs into CAFs and upregulate Has1/2? More discussion is needed.\n4. HAS2 blockade in tumor cells (performed in this manuscript) vs. CAFs (not presented): Instead of Hyal expression in WKTP, HAS2 blockade in CAFs would be ideal. Thus, tone adjustment or further discussion is recommended.\n5. Pathological relevance: The study focuses on liver metastasis, but GC is known to metastasize to various organs, including the peritoneum, lymph nodes, and lungs. The authors must clarify whether liver metastasis is the most clinically relevant metastatic site for GCs expressing canonical Wnt ligands.\n\nMinor comments\n1. The Rationale for selecting Wnt1 (over other ligands, including Wnt3a and Wnt5a). This study mentioned the role of ligand-dependent Wnt signaling in GC, and Wnt1 was specifically chosen for transgenic expression in the mouse model. However, it remains unclear why Wnt1 was selected over Wnt3a or Wnt5a. The authors need to provide literature, dataset, or experimental results-based justification for prioritizing Wnt1. Additionally, is there any transcriptomic or patient-derived data supporting WNT1 as the dominant Wnt ligand in liver-metastatic gastric cancer or a CIN-subtype? It should be noted that Wnt5a is a non-canonical WNT ligand. Similarly, in line 278, it is necessary to separate canonical vs. non-canonical WNT ligands since this study suggests the crucial role of canonical WNT ligands in liver metastasis.\n2. Figures 6 and 7: What is the primary source of TGF-beta to activate CAFs and upregulate HAS in conjunction with canonical WNT ligands?", "reviewer_2": "The manuscript demonstrates that Wnt ligand-dependent signaling promotes metastasis of chromosomal instability (CIN) subtype gastric cancer to liver through increasing hyaluronan expression in the metastatic tumor microenvironment. This study used multiple mouse models with mutations driven by Claudin18-IRES-CreERT2, inducing Tgfbr2flox/flox, Trp53LSL·R270H, KrasLSL·G12D, and K19-Wnt1. The organoids derived from these mouse models highlighted that Wnt1 signaling activation is required for efficient liver metastasis in stromal cells.\n\nThe current manuscript could be the first study detailing the potential mechanism of how stromal Wnt1 promotes CIN gastric cancer metastasis via Has2. The experiments are very nicely illustrated, and the data are almost all of high quality. The paper is also clearly written. However, there are several concerns that should be addressed:\n1. The study used a tamoxifen (Tam) concentration of 4mg/mouse to induce CreERT2 at 6 weeks of age. Tam at this concentration can cause parietal cell loss and induce chief cells to go through spasmolytic polypeptide-expressing metaplasia (SPEM). Authors should discuss the possible stomach epithelial injury resulting from use of Tam at this concentration and its potential impacts on their gastric cancer genetic engineered mouse model and cite the relevant papers: PMID: 22001866, 27246044, 31233898. It is possible the metaplasia caused by Tam helps give a headstart to the lesions/tumors.\n2. How did the authors activate CreERT2 in vitro? Tamoxifen is usually not miscible in tissue culture media, so most people use 4-hydroxy tamoxifen instead of tamoxifen.\n3. In Figure 1A, authors should clarify the time needed for Tam to induce KTP mutations in organoids. Also, “KP” is an error, as it is the same as “KT”.\n4. For the invasive lesions in Fig. 1, how confident are the authors that the glandular forms found, especially solitary ones are actually invasive? Because when there is marked hyperplasia of mouse stomach, the tissue can be distorted such that glands can twist around and be cut in cross-section, appearing pseudo-invasive. Although the image is small, it does look as though the single gland invasion in 1G is invasive, because there is distortion of surrounding stromal cells, and there seems to be an actual tumor (mass) formed, but this call can be tricky in many cases.\n5. There seems to be a loss of Muc5AC-positivity in the pit zone of Wnt-expressing tumor mice. What makes up the pit in these mice? Is there a reason why overexpression of Wnt would block foveolar cell differentiation?\n6. The mouse genetics are a bit unclear. Is the Trp53R270H allele kept as a heterozygote with the other, endogenous Trp53 allele wild-type? Presumably, the Kras gain of function is an LSL?\n7. Figure 2C is missing the statistical analysis.\n8. Figure 3 and Figure 4: for the KTP/WKTP/Apc-KTP/Wnt-KTP spleen injection experiment, besides in the liver, is there any other organ metastasis? Especially in the WKTP group, the liver metastasis phenotype is very strong as shown in Figure 4F.\nFigure 3A: typo, “speen” should be “spleen”.\n9. Figure 3F: needs to show the representative H&E images of tumor area/field. Moreover, how are we to interpret reproducibility/statistics here? One control mouse is lower than all 3 in GNF. Are the datapoints means from many tumor areas/fields in a single mouse? There is no stat analysis here either.\n10. Figure 3H: authors used GFP to trace tumor cells. But where is the GFP coming from? Authors did not mention the use of GFP in the mouse strains. Authors should clarify all the detailed genotypes of the mouse strains used in current manuscript (W, K, KT, KP, KTGP, WKT, WKP, WKTP).\n11. Figure 4C needs to label the protein size (kDa). Why not western blot for APC and Wnt1?\n12. Figure 4F/Supplementary Figure 5B experiment need the β-catenin IHC staining results. Are there more nuclear β-catenin in stromal cells in the Wnt-KTP group compared to KTP or Apc-KTP groups?\n13. The labels among the groups in Figure 4F and Figure 4G are confusing. There is one group of KTP mice in Figure 4F, but with two columns for KTP mice (#1,2) shown in Figure 4G; and one group of WKTP mice in Figure 4F but with three columns shown in Figure 4G (#1,2,3). In Figure 4F, the KTP and WKTP image section is missing a scale bar.\n14. The authors should perform a transwell assay to test the cell invasion ability among KTP, WKTP, Apc-KTP, and Wnt-KTP groups.\n15. Supplementary Figure 5A is missing Wnt-KTP cell data. How did authors reach the conclusion that “the proliferation rates of Apc-KTP and Wnt-KTP cells were unchanged compared to parental KTP cells”?\n16. Figure 5B, 5D and Supplementary Figure 6 are missing a scale bar.\n17. “Hepatocyte marker Alb was significantly increased in cluster 3 and 6, while epithelial markers, Ceacam1 and Muc1, were increased…” Technically, liver is epithelium also, so this should be clarified, because it is not clear which epithelial markers were used or why the ones shown are the representative epithelial cell markers.\n18. The authors should show representative IHC image results (translational level) to validate the spatial transcriptomic data (transcriptional level), for CTNNB1, WNT3A, TGFB1, SAMD3, and SMAD4.\n19. In Figure 7A, Has2 is not limited to cluster 5, which raises the question of whether the authors have performed the filtering before analyzing the spatial transcriptomic data by using Loupe Browser? The authors did not mention how they processed the spatial transcriptomic data. This should be detailed.\n20. The authors mention HA interacting with CD44 on stem cells and hypothesize the same sort of interaction in gastric cancer, but there is a paper that does show that metaplasia in stomach expands CD44+ progenitor cells that as HA expands, so this paper should be cited and potentially discussed: PMID:23589310. There are other papers detailing CD44 role in metaplasia and stemness in stomach (eg, PMID: 31071489), so the CD44/xCT/HA pathway may be important in both metaplasia and metastasis.\n21. A visualization of cell-cell communication, using differentially expressed genes (DEGs), would greatly improve analysis, as one would expect important cell-cell communication between cluster 5 and the rest of the clusters. For example, as the authors state, CD44 in tumor cells could be communicating with HA in stroma.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "Furutani et al engineered a mouse model of gastric cancer expressing mutated Kras, Tgfbr2, and Trp53 (KTP mice) specifically in the gastric mucosa. These mice displayed gastric metaplasia with parietal cell loss. In a second GEMM the same driver mutations were expressed together with Wnt1 (WKTP mice) and these mice developed dysplastic tumors. To investigate the role of ligand-dependent Wnt signaling in gastric cancer, they established organoids from the gastric epithelia of these mice, transplanted them into the spleens of NSG mice and observed that while both KTP and WKTP organoids formed local splenic tumors, only WKTP organoids could form liver metastases. Using a combination of approaches, including spatial transcriptomic, they found that in mice implanted with WKPT organoids, liver metastases were associated with a significant stromal response and identified hyaluronan synthase (Has2) as a potential mediator of this response. Furthermore, they found a significant hyaluronan deposition in liver metastases of both mouse and human gastric cancers. The authors concluded that paracrine Wnt signaling activation in hepatic stellate cells in conjunction with TGFβ results in increased Has2 expression and this promotes metastatic tumor development through hyaluronan deposition and increased ECM production. This study follows an earlier one by the same group where the role of these driver mutations in formation of intestinal tumors was analyzed (Kok et al, 2021).\n\nA similar approach to generating gastric cancer GEMMs was published by Seidlitz T. et al in 2019 so the strategy used is not entirely novel. However, the present study adds novelty, by identifying a major role for Wnt ligands in shaping the liver microenvironment and thereby, enabling gastric cancer liver metastases.\n\nTechnically, the experiments were well executed, and the manuscript is (with a few exceptions) well written. However, there are several issues that need to be addressed\n\nGeneral comment:\nIn Seidlitz et al, a similarly constructed GEMM based on driver mutations KrasG12D/+;Tp53R172H/+;Smad4fl/f in the gastric epithelium resulted in gastric tumors that were metastatic to liver and lung even in the (apparent) absence of Wnt production. These findings are at variance with the results obtained in the present study with the KTP mice and the authors should discuss potential differences that could explain the different phenotypes.\n\nSpecific points\nFigure 2 (E-H). This model of orthotopic implantation best mimics the clinical pathology. Did the authors analyze progression to metastatic disease in these mice? Do WKTP tumors spontaneously metastasize to the liver in this setting? If not, a discussion is warranted.\nFigure 3F - Were the differences between the different group statistically significant? If not, this should be stated. There appears to be a high variability in the data obtained for the control group and analysis of additional mice may be necessary to support the conclusion that these inhibitors had an effect.\nFigure 3 (H&I) – Among the earliest events in liver metastases formation is the recruitment of innate immune cells, among them neutrophils and monocytes which are still functional in NSG mice. Wnt1 is known to affect immune cell function including macrophage polarization (e.g. Staal et al, 2008, Haseeb M et al 2109). Given the results shown this figure, the authors should analyze or at least comment on the potential role of myeloid-derived cells in promoting establishment of metastases of WKPT cells.\nFig 4C- The ratio of activated:total beta-catenin appears to be higher in KTP than in WKTP cells. This is surprising. Is there no autocrine effect of Wnt1 in the WKTP cells? Does Wnt1 only act in a paracrine fashion and specifically only in the liver? Did the authors quantify Wnt1 production levels in the three models (KPT, WKPT and Wnt-KTP)? These data are important for interpretation of the results.\nFigure 4F- How do the authors explain the striking difference in the extent of liver metastatic disease in WKPT and Wnt-KTP implanted mice? Were Wnt1 production levels in these cells compared?\nFigure 5- To determine whether the extensive stromal response observed in these livers is a cause or consequence of metastatic expansion, livers should also be analyzed at earlier time points. It is also of interest to know whether the spatial transcriptomic analysis identified any myeloid/innate immune cell clusters and whether liver sections of KTP -implanted mice were also analyzed for comparison.\n\nDiscussion and data interpretation\nThe authors propose that the ability of KTP and WKPT organoid to form tumors and grow equally well in the orthotopic (and subcutaneous) site is due to local Wnt1 production by stromal cells. This is speculative and should be experimentally tested, for example by using the PORCN Wnt1 inhibitor C59. This also raises the question why the authors switched from an orthotopic to an intra-splenic model to evaluate liver metastasis. If WKPT cells do not metastasize following intra-mucosal implantation (or metastasis is a very late event) this should be stated and discussed.\nThe functional significance of hyaluronan in metastatic expansion in the liver in this model has not been defined. Is it utilized by WKPT cells to attach via CD44?\nWhether the fibrotic response is a cause or consequence of tumor expansion in the liver should be resolved experimentally by analyzing the response on day 7 not at late stages of metastasis as demonstrated in Fig 5.\nLanguage editing is required. For example see line 329.", "reviewer_5": "Furutani and colleagues investigated the role of ligand-dependent Wnt signaling in gastric cancer development and metastasis by creating genetically modified mice (KTP and WKTP) with or without Wnt1 expression. They found that WKTP mice developed dysplastic tumors and liver metastases, while KTP mice only showed gastric metaplasia, highlighting Wnt signaling's role in both tumor initiation and metastasis. Using organoid transplantation and spatial transcriptomics, they showed that stromal Wnt signaling, together with TGFβ, activates hepatic stellate cells into cancer-associated fibroblasts that express Has2, promoting hyaluronan accumulation and metastasis. Importantly, inhibiting Has2 or Wnt signaling reduced liver metastases, suggesting a potential therapeutic approach for gastric cancer.\n\nGeneral Remark: While the study is interesting, there is very little evidence that the findings could be translated to human (very little has been done to validate the findings in human). While the study uses an experimentally sophisticated system (many transgenic animals) there is an important number of issues that needs to be carefully addressed. When talking about Wnt-signaling and paracrine communication between cancer cells and CAF it is important to note that specific Wnt ligands will be secreted by specific cells while specific receptors will be expressed on certain cells. This is where the study in reviewers opinion is limited and to certain extent far from realistic situation encountered in human.\n\nMajor Remark 1: In human gastric cancer HAS2 is not expressed on stellate cell derived CAF. Namely, there are generally 2 types of CAF (in the liver) depending on their origin, stellate cell derived (RGS5+) or resident fibroblast derived (PDGFRA+). Hence the model that is used in vitro (employing immortalised HSC) is unfortunately irrelevant. This goes further, HYAL2 is not expressed by cancer cells, but by endothelial cells while Wnt5A is expressed by cancer cells.\nI am attaching in support some meta-analysis that I have conducted based on previously published data (DOI:10.1002/ctm2.730)\nIn conclusion, the authors need to be very careful about which ligand is expressed on which cell (lay out their model based on human data, either single cell if clear enough or IHC). Then according to this design their experimental strategy. Concretely, which cells express WNT1 in human gastric cancer and what is the target receptor(s), where are these receptors situated in humans?\n\nMajor Remark 2: The part of the data that is related to CAF needs thorough revision. The authors state \"It has been shown that CAFs in the liver metastasis are originated from activated hepatic stellate cells (HSCs)\", which is simply wrong in many aspects (the cited literatures is not relevant, please see doi: 10.1038/s41586-019-1631-3.). Again, CAFs in the liver metastasis are derived from at least 3 different precursor cells - and in contrast to what the authors are claiming, those CAF that reorganise the ECM are not stellate cell derived. I further refer to the attached meta-analysis where on primary gastric cancer samples from human we can clearly see at least 2 groups of CAF (stellate cells do exist also in stomach and many other organs next to liver) to begin with.\n\nMajor Remark 3: Provided that the authors sort out the cellular identities according to the remarks made above, what is the link between Wnt-signalling and HAS2 expression? The authors switch without any explanation from Wnt1 (which is their initial model) to Wnt3. What is the reason for this? What is the downstream signalling and how Wnt1 (and not 3) could cause the HAS2 over-expression?\n\nMajor Remark 4: Hyaluronidase inhibitors have been shown to inhibit cancer metastasis (https://www.nature.com/articles/s41598-024-64924-6). The authors here show the contrary. It seems that high molecular weight hyaluronic acid (HMW-HA) is tumor protective, while low molecular weight hyaluronic acid (LMW HA) is tumor promotive. Authors need to clarify better which type of HA they are dealing with and relate this to what is observed in human samples."}, "author_response": "Response to the reviewer comments\nOur point-by-point responses to the comments made by the Reviewers are included\nbelow. The comments are quoted “verbatim”, followed by our responses.\nReviewer #1 (Remarks to the Author)\nThe authors identified a new role for WNT ligands in promoting liver metastasis of\ngastric cancer, utilizing genetically engineered organoids, transplantation models, and\nspatial transcriptomics (Visium). It was proposed that canonical Wnt ligands\n(specifically Wnt1) secreted by tumor cells upregulate hyaluronan synthase, Has2, in\ncancer-associated fibroblasts (CAFs) derived from hepatic stellate cells, thereby\npromoting liver metastasis. This is an interesting study that unveils an unexpected role\nof canonical WNT ligands in establishing a tumor-favoring metastatic niche. However,\nseveral points warrant further attention through new experimental results, discussion,\nand tone adjustments.\nResponseWe have responded to all comments by Reviewer #1 as follows.\n1. Orthotopic vs. intrasplenic transplantation. With the exception of gastric tumor\nanalysis (Figure 2), the authors performed intrasplenic injection of organoids instead of\northotopic transplantation. Consequently, this study does not provide direct evidence\non whether orthotopic injection of KTP or WKTP organoids into the stomach would\nresult in metastasis to other organs. It is necessary to assess whether orthotopically\ninjected organoids/cells give rise to distant metastases, particularly to the liver, followed\nby repeating key experiments shown in Figures 3-7 using orthotopic models.\nResponseWe thank the reviewer for this important comment. In our orthotopic\ntransplantation experiments, we monitored gastric tumor development using in vivo\nimaging up to day 21, as shown in Figure 2g. At this time point, no clear evidence of\nmetastasis was observed. However, by day 28, some mice began to exhibit signs of\nperitoneal dissemination, as demonstrated in the imaging data provided below.\nAlthough histological analysis at this stage did not reveal liver metastases, the mice\nshowed signs of clinical deterioration, limiting our ability to extend observation further.\nTo establish a more quantitative and\nreproducible model for assessing liver\nmetastasis, we therefore employed the\nintrasplenic injection approach. As the\nreviewer rightly noted, this model\nbypasses early steps of the metastatic\ncascade, particularly invasion and\n1\nintravasation from the primary site. Nonetheless, it enables robust evaluation of the role\nof Wnt ligands in supporting metastatic outgrowth in the liver microenvironment.\nTo clarify this point in the manuscript, we have revised the sentence in the Results\nsection (page 7) as follows: “To investigate the role of ligand-dependent Wnt signaling\nin liver metastasis by disseminated tumor cells, we transplanted KTP and WKTP\norganoid cells into the spleen of mice and analyzed liver tissues.” We hope this\nexplanation and the complementary use of both models address the reviewer’s\nconcerns and support the rationale behind our experimental design.\n2. Immunocompetent vs. immunocompromised mice: The authors utilized NSG and\nSHO mice for transplantations. Given that KTP and WKTP organoids were derived\nfrom C57BL/6 mice, syngeneic transplantation in C57BL/6 mice could offer a more\npathophysiologically relevant immune environment. Could the authors clarify their\nrationale for choosing an immunodeficient background instead? Considering the\naforementioned point (#1), performing orthotopic transplantation of organoids (or\norganoid-derived cells) in C57BL/6 mice is necessary. Alternatively, investigating liver\nmetastasis in the genetically engineered mouse models (GEMMs) (Figure 1) could also\nbe considered, as the authors described in the Introduction (lines 92-96; notably, this\nGEMM was not used to study metastasis in this manuscript).\nResponseIn our previous study (Sakai et al., Cancer Res, 2018), we established\nAKTP organoids derived from intestinal tumors in C57BL/6 (B6) mice, harboring\nmutations in Apc, Kras, Tgfbr2, and Trp53. In that study, we found that intrasplenic\ninjection of AKTP cells into syngeneic B6 mice resulted in liver metastases in\napproximately 40% of the animals, whereas injection into immunodeficient NSG mice\nled to liver metastasis in nearly 100% of recipients. This discrepancy is likely due to the\nNK cell-mediated surveillance in B6 mice, although it remains to be elucidated. Based\non this finding, we selected NSG mice for the current metastasis experiments to ensure\nhigh reproducibility and efficiency in detecting metastatic phenotypes, particularly in\ncomparing the metastatic potential of KTP and WKTP organoids.\nIn response to the reviewer’s suggestion, we performed intrasplenic\ntransplantation of WKTP organoid cells into immunocompetent B6 mice. As shown in\n2\nthe representative photograph below, multiple liver metastasis developed in 2 out of 5\nmice, and low-multiplicity liver tumors were observed in additional 2 mice. In contrast,\nconsistent with our previous observation, WKTP cells give rise to multiple liver\nmetastatic in 100% of NSG (Fig. 3f, 4f, 4g). These results support the enhanced\nsensitivity of the NSG mice for studying Wnt ligand-associated metastasis mechanism.\nRegarding the potential use of GEMMs to study spontaneous metastasis, we\nconducted long-term observation (up to 90 days) of both KTP and WKTP mice. While\nboth genotypes eventually developed lung tumors and became moribund likely due to\nCreER expression in the lung, no spontaneous liver metastases were observed during\nthis period. Histological images of lung tumors and corresponding survival curves are\nprovided below. Thus, to directly assess the metastatic potential of KTP and WKTP\ncells, transplantation-based models were necessary. We hope this explanation clarifies\nthe rationale behind our use of NSG mice for the analysis and highlights the technical\nchallenges associated with spontaneous metastasis in GEMMs.\n3. Mechanisms: How does WNT/β-catenin signaling convert HSCs into CAFs and\nupregulate Has1/2? More discussion is needed.\nResponseIt has been reported that TGFβ signaling activates fibroblasts and induces\nECM components in part by downregulating the Wnt antagonist DKK1, thereby\nenhancing Wnt/β-catenin signaling and contributing to TGFβ-induced CAF activation\n(Akhmetshina et al., Nat Commun, 2012, Ref. 33). Moreover, Wnt/β-catenin signaling\nhas been shown to directly regulate Has2 expression through TCF7L2 binding to the\nHas2 gene promoter (Sun et al., Biomolecules, 2022, Ref. 34). These findings support\na model in which Wnt and TGFβ signaling synergistically promote the conversion of\nHSCs into CAFs and induction of Has2 expression. We have added these mechanistic\ninsights into the Discussion section, along with newly added references (page 15).\n4. HAS2 blockade in tumor cells (performed in this manuscript) vs. CAFs (not\npresented): Instead of Hyal expression in WKTP, HAS2 blockade in CAFs would be\n3\nideal. Thus, tone adjustment or further discussion is recommended.\nResponseAs the reviewer pointed out, assessing the effect of Has2 inhibition\nspecifically in CAFs would indeed provide more direct evidence of its role in the tumor\nmicroenvironment. However, generating a conditional Has2 knockout model in stromal\nfibroblasts, such as HSCs, would require a substantial amount of time. In line with the\nreviewer’s recommendation, we have accordingly moderated the tone of our\nconclusions (page 15) and added a discussion in the revised manuscript, highlighting\nthe need for future studies to investigate the functional relevance of Has2 in CAFs\nusing more targeted approaches (page 16).\n5. Pathological relevance: The study focuses on liver metastasis, but GC is known to\nmetastasize to various organs, including the peritoneum, lymph nodes, and lungs. The\nauthors must clarify whether liver metastasis is the most clinically relevant metastatic\nsite for GCs expressing canonical Wnt ligands.\nResponseWe thank the reviewer for pointing out the organotropism of gastric cancer\n(GC) metastasis. It is well established that GC metastasizes to various organs,\nincluding the liver, peritoneum, lymph nodes, and lungs. Among these, liver and\nperitoneal metastases are the most frequent.\nTo address the reviewer’s comment, we analyzed publicly available single-cell\nRNA-seq datasets of GC (GSE163558, GSE239676, and GSE246662) and examined\nthe expression of Wnt ligand family genes in primary tumor cells (PT), metastatic tumor\ncells from liver (Li), lymph nodes (LN), ovary (Ov), and peritoneum (PC), and cancer-\nassociated fibroblasts (CAFs). As shown in the figure below, WNT2–a canonical Wnt\nligand–is highly expressed in the liver\nmetastasis, while WNT2B expression is\nincreased in LN, Ov, and PC metastasis.\nThus, no clear correlation can be\nestablished between canonical Wnt\nligand expression and liver metastasis by\nthis analysis.\nAccording to TCGA classifications, GC exhibits significant genomic heterogeneity\nand is categorized into four molecular subtypes. Our current mouse and organoid\nmodels are based on the CIN subtype, which is more frequently associated with liver\nmetastasis compared to the diffuse-type, which often harbors CDH1 mutations and\nprone to peritoneal dissemination (Lee JE, et al., Br J Cancer, 129, 672-682, 2023).\nFurthermore, a previous study using GC-derived organoids (Nanki et al, Cell,\n4\n2018, Ref.10) did not identify a significant association between Wnt ligand expression\nand GC histological subtype. Thus, whether Wnt ligand-expressing GC cells\npreferentially metastasize to the liver remains unsolved and is likely influenced by\nspecific driver gene alterations.\nTo further investigate this, we are currently generating Wnt1-expressing, CDH1-\ndeficient GC organoids to assess the role of Wnt ligand signaling in peritoneal\ndissemination. Given these considerations, we believe it would be inappropriate to\ngeneralize that GC cells expressing Wnt ligands preferentially metastasize to the liver\nin the present study. We hope the reviewer understands our rationale.\nMinor comments\n1. The Rationale for selecting Wnt1 (over other ligands, including Wnt3a and Wnt5a).\nThis study mentioned the role of ligand-dependent Wnt signaling in GC, and Wnt1 was\nspecifically chosen for transgenic expression in the mouse model. However, it remains\nunclear why Wnt1 was selected over Wnt3a or Wnt5a. The authors need to provide\nliterature, dataset, or experimental results-based justification for prioritizing Wnt1.\nAdditionally, is there any transcriptomic or patient-derived data supporting WNT1 as\nthe dominant Wnt ligand in liver-metastatic gastric cancer or a CIN-subtype? It should\nbe noted that Wnt5a is a non-canonical WNT ligand. Similarly, in line 278, it is\nnecessary to separate canonical vs. non-canonical WNT ligands since this study\nsuggests the crucial role of canonical WNT ligands in liver metastasis.\nResponseAs indicated in Supplementary Fig.1, all Wnt ligand family genes-except\nWnt8A-are upregulated in more than 10% of gastric cancers. The upregulation\nfrequencies are comparable among several canonical Wnt ligands, including Wnt1,\nWnt2, Wnt3, and Wnt3a. Among these, we selected Wnt1 for this study based on\nseveral supporting findings. Previous studies have reported a strong correlation\nbetween Wnt1 expression and advanced tumor grade in gastric cancer, and Wnt1-\nmediated signaling has been shown to promote gastric tumor growth and metastasis\n(Li et al, Lab Invest, 2023, Ref. 12; Mao et al, Cell Death and Disease, 2014, Ref. 18).\nIn response to the reviewer’s comment, we have included this rationale in the Results\nsection, along with newly added reference (page 5).\nRegarding the distinction between canonical and non-canonical Wnt ligands as\nraised by the reviewer, we have revised the explanation for Supplementary Fig. 8b to\nclearly distinguish the canonical from non-canonical Wnt pathway, highlighting the\nspecific focus of this study on canonical Wnt signaling in the context of liver metastasis\nof gastric cancer (page 12).\n5\n2. Figures 6 and 7: What is the primary source of TGF-beta to activate CAFs and\nupregulate HAS in conjunction with canonical WNT ligands?\nResponse We confirmed the expression of TGF-β1 in both WKTP tumor cells and\nCAFs by Western blotting. Moreover, analysis of the VISIUM spatial transcriptomic\ndata revealed that TGF-β1 expression is highly enriched in clusters 1 and 2 (tumor\ncells), as well as clusters 4 and 5 (stromal fibroblasts). Therefore, we consider WKTP\ntumor cells to be a primary source of TGF-β ligands in the metastatic lesions. In\naddition, activated stromal cells subsequently express TGF-β, contributing to sustained\nactivation of TGFβ signaling pathway. These findings have been included\nSupplementary Figure 7c and described in the Results section (page 11).\nReviewer #2 (Remarks to the Author)\nThe manuscript demonstrates that Wnt ligand-dependent signaling promotes\nmetastasis of chromosomal instability (CIN) subtype gastric cancer to liver through\nincreasing hyaluronan expression in the metastatic tumor microenvironment. This study\nused multiple mouse models with mutations driven by Claudin18-IRES-CreERT2,\ninducing Tgfbr2flox/flox, Trp53LSL·R270H, KrasLSL·G12D, and K19-Wnt1. The organoids derived from these mouse models highlighted that Wnt1 signaling activation\nis required for efficient liver metastasis in stromal cells.\nThe current manuscript could be the first study detailing the potential mechanism of\nhow stromal Wnt1 promotes CIN gastric cancer metastasis via Has2. The experiments\nare very nicely illustrated, and the data are almost all of high quality. The paper is also\nclearly written. However, there are several concerns that should be addressed:\nResponseWe would like to thank the Reviewer #2 for constructive comments on our\nmanuscript. We have responded to all comments as follows.\n1. The study used a tamoxifen (Tam) concentration of 4mg/mouse to induce CreERT2\nat 6 weeks of age. Tam at this concentration can cause parietal cell loss and induce\nchief cells to go through spasmolytic polypeptide-expressing metaplasia (SPEM).\nAuthors should discuss the possible stomach epithelial injury resulting from use of Tam\nat this concentration and its potential impacts on their gastric cancer genetic\nengineered mouse model and cite the relevant papers: PMID: 22001866, 27246044,\n31233898. It is possible the metaplasia caused by Tam helps give a headstart to the\nlesions/tumors.\nand regenerative colonic mucosa of DSS-treated mice promotes the development of\ninvasive tumors (Oshima et al, Cancer Res, 2015, Ref. 30). As Reviewer #2 pointed\nout, it is therefore possible that tamoxifen-induced epithelial injury in the gastric\nmucosa and subsequent regenerative responses contributed to the acquisition of\ninvasive potential in gastric tumor cells harboring Tgfbr2 mutations. In response, we\nhave added this discussion in the revised manuscript (page 14), including citation of\none of the suggested references (Huh et al, Gastroenterology, 2012, Ref. 31).\n2. How did the authors activate CreERT2 in vitro? Tamoxifen is usually not miscible in\ntissue culture media, so most people use 4-hydroxy tamoxifen instead of tamoxifen.\nResponse We used 4-hydroxy tamoxifen to induce KTP mutations in the established\ngastric organoid cell lines. We apologize for lack of this information in the original\nMethod section. The relevant details have now added the relevant details to the revised\nmanuscript. (page 19).\n3. In Figure 1A, authors should clarify the time needed for Tam to induce KTP\nmutations in organoids. Also, “KP” is an error, as it is the same as “KT”.\nResponse Mice were administered tamoxifen intraperitoneally (i.p.) at 4 mg/mouse\nat 6 weeks of age, and tumor phenotypes in the glandular stomach were evaluated 28\ndays after administration. Organoids were treated with 1 μΜ of 4-ΟΗ tamoxifen for 14\ndays to induce KTP mutations. These tamoxifen treatment schedules for both mice and\norganoids have been added to in the Method section (page 17, 19). In addition, the\nlabeling error in the Figure 1a has been corrected as pointed out by the Reviewer #2.\n4. For the invasive lesions in Fig. 1, how confident are the authors that the glandular\nforms found, especially solitary ones are actually invasive? Because when there is\nmarked hyperplasia of mouse stomach, the tissue can be distorted such that glands\ncan twist around and be cut in cross-section, appearing pseudo-invasive. Although the\nimage is small, it does look as though the single gland invasion in 1G is invasive,\nbecause there is distortion of surrounding stromal cells, and there seems to be an\nactual tumor (mass) formed, but this call can be tricky in many cases.\nResponseTo distinguish pseudo-invasion, such as glandular herniation, from true\ntumor invasion, we followed the criteria described for mouse intestinal tumors (Boivin et\nal, Gastroenterology, 124: 762-777, 2003). Specifically, in the submucosa of mice\ncarrying KT mutations (i.e., KT, KTP, WKT, and WKTP), we observed invasive tumors\n7\ncharacterized by irregular or angulated glands with dysplastic features, accompanied\nby desmoplastic stromal reactions (Fig. 1g top and Fig. 1h). However, as Reviewer #2\npointed out, the single round-shaped gland (Fig. 1g top left in the original manuscript)\nmight represent herniation of a hyperplastic gland rather than true tumor invasion.\nAccordingly, we have removed the data corresponding to this “single gland invasion”\nfrom the original Fig. 1g. This modification does not affect our conclusion that KT\nmutations promote the invasive phenotype. To more clearly illustrate the dysplastic\nfeatures of the invasive tumors, we have added higher-magnification images of E-\ncadherin IHC in the insets in Figure 1g. Figure legend has been updated accordingly.\n5. There seems to be a loss of Muc5AC-positivity in the pit zone of Wnt-expressing\ntumor mice. What makes up the pit in these mice? Is there a reason why\noverexpression of Wnt would block foveolar cell differentiation?\nResponseAs suggested by Reviewer#2, differentiation of gastric epithelial cells\nappears to be suppressed in Wnt1-expressing compound mutant mice (WKT, WKP,\nand WKTP), as most epithelial cells are proliferative, and Muc5AC-positive foveolar\ncells as well as H+/K+ ATPase-positive parietal cells are absent. However, such\nchanges were not observed in the stomachs of simple Wnt1 transgenic mice.\nTherefore, it is possible that the combination of Wnt activation and Kras mutation leads\nto suppression of gastric epithelial differentiation and promotes the formation of\ndysplastic tumors. We have added this comment to the Results section (page 6).\n6. The mouse genetics are a bit unclear. Is the Trp53R270H allele kept as a\nheterozygote with the other, endogenous Trp53 allele wild-type? Presumably, the\nKras gain of function is an LSL?\nResponseWe apologize for not providing a detailed information regarding the mouse\ngenotypes. We have now included the precise description of mouse genotypes in the\nMethod section as Tgfbr2flox/flox, Trp53+/LSL-R270H and Kras+/LSL-G12D mice (page 17). In\naddition, we have provided the corresponding genotypes in the Results section as\nfollows: K (Kras+/G12D), KT (Kras+/G12D, Tgfbr2-/-), KP (Kras+/G12D, Trp53+/R270H), and KTP\n(Kras+/G12D, Tgfbr2-/-, Trp53+/R270H) (page 5).\n7. Figure 2C is missing the statistical analysis.\nResponseWe have added the results of statistical analyses in Figure 2c and\nconfirmed a significant decrease in proliferation of WRN-0% KTP cells compared to\nWRN-50% KTP cells. A significant reduction was also confirmed in WRN-0% + C59\n8\n(PORCN inhibitor) WKTP cells compared to WRN-0% WKTP cells. Figure legend has\nbeen updated accordingly.\n8. Figure 3 and Figure 4: for the KTP/WKTP/Apc-KTP/Wnt-KTP spleen injection\nexperiment, besides in the liver, is there any other organ metastasis? Especially in the\nWKTP group, the liver metastasis phenotype is very strong as shown in Figure 4F.\nA: typo, “speen” should be “spleen”.\nResponseBased on routine pathological examination, no metastatic lesions were\ndetected outside the liver in any of the spleen transplantation models, including those\nwith WKTP cells. We have added this clarification to the Results section (page 8).\nWe apologize for a typographical error in Figure 3a and have corrected “speen” to\n“spleen”.\n9. Figure 3F: needs to show the representative H&E images of tumor area/field.\nMoreover, how are we to interpret reproducibility/statistics here? One control mouse is\nlower than all 3 in GNF. Are the datapoints means from many tumor areas/fields in a\nsingle mouse? There is no stat analysis here either.\nResponseTo show representative H&E images of tumor area/fields, we have added\nlow-magnification H&E images of liver tissues from both control and ETC-159-treated\nmice in Figure 3g (left). These images illustrate that the tumor area/field was markedly\nreduced following ETC-159 treatment.\nAs the reviewer pointed out, each datapoint in the graph represents the mean\ntumor area/field per mouse. In response to this comment, we increased the number of\nmice in each experimental group to n=8 for control, n=5 for ETC-159 treatment, and\nn=5 for GNF-6231 treatment, and performed statistical analysis. As shown in the\nrevised Figure 3f (right), ETC-159 treatment significantly suppressed liver metastasis\nof WKTP organoid cells compared to control. GNF-6231 treatment also resulted in a\nreduction in tumor aria/field, although the difference was not statistically significant.\nGiven the variability in tumor area/field observed among control mice, we concluded\nthat Wnt ligand expression is critical for the development of liver metastasis in this\nmodel of gastric cancer. These new data have been included in Figure 3f (right) and\ndescribed in the Results section (page 8). Figure legend has been updated\naccordingly.\n10. Figure 3H: authors used GFP to trace tumor cells. But where is the GFP coming\nfrom? Authors did not mention the use of GFP in the mouse strains. Authors should\nclarify all the detailed genotypes of the mouse strains used in current manuscript (W, K,\nKT, KP, KTGP, WKT, WKP, WKTP).\nResponseWe apologize for not providing information regarding the GFP-expressing\ntumor organoids. GFP-expressing KTP and WKTP organoids were generated by\ntransfecting a GFP expression vector. We have now added a description of the GFP-\nlabeling procedure in the Method section (page 19) and clarified that GFP-labeled\norganoids were used in the relevant experiments in the Results section (page 8).\nIn accordance with the reviewer’s suggestion, we have also provided detailed\ngenotypes of all mouse strains used in this study, including W, K, KT, KP, KTP, WKT,\nWKP, WKTP, in Supplementary Fig. 2.\n11. Figure 4C needs to label the protein size (kDa). Why not western blot for APC and\nWnt1?\nResponseWe have added molecular weight markers (kDa) to the immunoblotting\ndata in Figure 4c. Detection of APC protein by Western blotting is technically\nchallenging due to its high molecular weight and poor transfer efficiency to the\nmembrane. Additionally, most Wnt ligands are difficult to detect by Western blotting\ndue to the lack of reliable antibodies. Therefore, we assessed Wnt pathway activity by\ndetecting the active form of β-catenin via immunoblotting and by performing the\nTOPFLASH reporter assay. These approaches are widely used and considered reliable\nfor evaluating Wnt signaling activity.\n12. Figure 4F/Supplementary Figure 5B experiment need the β-catenin IHC staining\nresults. Are there more nuclear β-catenin in stromal cells in the Wnt-KTP group\ncompared to KTP or Apc-KTP groups?\nResponseIn response to the reviewer’s suggestion, we performed\nimmunohistochemistry (IHC) for β-catenin in liver metastatic lesions derived from\nWKTP, Apc-KTP, and Wnt-KTP cells. Accumulation of β-catenin was observed in\ntumor epithelial cells across all groups; however, we did not detect nuclear β-catenin\naccumulation in the stromal compartment (See figure below).\nWnt3-stimulated hepatic stellate cells (imHSCs). Although increased TOPFLASH\nactivity and induction of Wnt-target genes were confirmed in Wnt3-stimulated imHSCs\n(Fig. 6b, c), nuclear β-catenin accumulation was not observed in these cells (See figure\nbelow).\nThese findings suggest that Wnt-activated stromal fibroblasts may not\naccumulate nuclear β-catenin to a level detectable by conventional IHC or ICC, unlike\ntumor epithelial cells. Nonetheless, considering potential technical limitations in\ndetecting nuclear β-catenin in fibroblasts, we have chosen not to further discuss this\npoint in the manuscript. We hope that the reviewer understand our decision.\n13. The labels among the groups in Figure 4F and Figure 4G are confusing. There is\none group of KTP mice in Figure 4F, but with two columns for KTP mice (#1,2) shown\nin Figure 4G; and one group of WKTP mice in Figure 4F but with three columns shown\nin Figure 4G (#1,2,3). In Figure 4F, the KTP and WKTP image section is missing a\nscale bar.\nResponseWe apologize for confusing presentation in the original Figure 4f and 4g.\nWe examined two KTP organoid lines (#1 and #2) and three WKTP organoid lines (#1,\n#2, and #3) in spleen transplantation experiments, as shown in Figure 4g and\nSupplementary Figure 6c. In the original Figure 4f, we showed representative liver\ntissue photographs of one KTP line (KTP#1) and one WKTP line (WKTP#2) without\nindicating the organoid line numbers, which may have caused confusion. To clarify this,\nwe have now added the organoid line numbers for both KTP and WKTP samples in\nFigure 4f. Additionally, to illustrate the heterogeneity in tumor phenotypes among the\nWKTP lines, we have added liver photographs from WKTP#1 in Figure 4f. Figure\nlegend has been updated accordingly.\nWe also apologize for missing a scale bar in the Figure 4f image; this has now\nbeen corrected.\n11\n14. The authors should perform a transwell assay to test the cell invasion ability among\nKTP, WKTP, Apc-KTP, and Wnt-KTP groups.\nResponseAs suggested by the reviewer, we have performed transwell assay using\nKTP, WKTP, Apc-KTP, and Wnt-KTP organoid cells to assess their invasive potential.\nWe found no significant differences in invasiveness among these four organoid lines.\nThese data have been added to Supplementary Fig. 6a and 6b, and described in the\nResults section (page 9).\n15. Supplementary Figure 5A is missing Wnt-KTP cell data. How did authors reach the\nconclusion that “the proliferation rates of Apc-KTP and Wnt-KTP cells were unchanged\ncompared to parental KTP cells”?\nResponseWe have added representative culture images and CTG assay results for\nWnt-KTP cells in Supplementary Fig. 6a and 6b (original Supplementary Fig. 5a).\nBased on these new data, we concluded that the proliferation rate of Apc-KTP and\nWnt-KTP cells are comparable to those of parental KTP cells. This conclusion has\nbeen indicated in the Results section (page 9).\n16. Figure 5B, 5D and Supplementary Figure 6 are missing a scale bar.\nResponseWe apologize for the absence of scale bars in Figure 5b, 5d, and\nSupplementary Figure 7 (original Supplementary Fig. 6). We have now added scale\nbars in these images.\n17. “Hepatocyte marker Alb was significantly increased in cluster 3 and 6, while\nepithelial markers, Ceacam1 and Muc1, were increased…” Technically, liver is\nepithelium also, so this should be clarified, because it is not clear which epithelial\nmarkers were used or why the ones shown are the representative epithelial cell\nmarkers.\nResponseAs Reviewer#2 correctly pointed out, hepatocytes are epithelial cells;\ntherefore, it was inappropriate to use the term “epithelial markers” to distinguish gastric\ntumor cells (WKTP cells) from hepatocytes. Initially, we referred to the PanglaoDB\npublic database and selected Ceacam1 and Muc1 from the list of “epithelial markers”.\nFor the revision, we reanalyzed the mRNA expression levels of Ceacam1, Muc1,\nand Alb in WKTP cells and normal liver tissue by RT-PCR. Among them, Muc1 showed\nsignificantly higher expression in WKTP cells, while Ceacam1 did not. In contrast, Alb\nexpression was significantly elevated in liver tissues. To further refine the distinction,\nwe searched for additional WKTP-specific marker genes using database analysis and\n12\nconfirmed by RT-PCR that Prom1 expression is markedly increased in WKTP cells\ncompared to hepatocytes. Accordingly, in the revised manuscript, we replaced the term\n“epithelial markers” in the original Figure5c, and now present violin plots of Muc1 and\nProm1 as “WKTP cell markers” in the updated Figure 5c. These findings are described\nin Results section (page 10). RT-PCR data for Muc1, Prom1, and Alb have also been\nadded to Figure 5c. Figure legend has been updated accordingly.\n18. The authors should show representative IHC image results (translational level) to\nvalidate the spatial transcriptomic data (transcriptional level), for CTNNB1, WNT3A,\nTGFB1, SAMD3, and SMAD4.\nResponseThe results shown in Figure 5e indicate activation of Wnt signaling and\nTGFβ pathways based on IPA analysis using differentially expressed genes in cluster\n5. However, this does not necessarily imply that individual pathway components—such\nas Ctnnb1, Wnt3A, Tgfb1, Smad3, and Smad4—are transcriptionally upregulated.\nAs Reviewer #2 correctly pointed out, validation of the spatial transcriptomic data\nat the protein level is important. However, as noted in our response to comment #12,\ndetecting nuclear β-catenin in fibroblasts by IHC is technically challenging. Instead,\nWnt signaling activation in HSCs and CAFs was confirmed through in vitro experiments\n(Figure 6).\nIn contrast, we were able to detect phosphorylated Smad2 (P-Smad2) by IHC in\nfibroblastic cells within the metastatic tumor stroma, indicating activation of TGFβ\npathway. These IHC results for P-Smad2 have been added to Supplementary Figure\n7b and described in the Results section (page 11).\n19. In Figure 7A, Has2 is not limited to cluster 5, which raises the question of whether\nthe authors have performed the filtering before analyzing the spatial transcriptomic data\nby using Loupe Browser? The authors did not mention how they processed the spatial\ntranscriptomic data. This should be detailed.\nResponseHas2 expression is significantly enriched in cluster 5, as shown in the\nviolin plot in Figure 7a and in Supplementary Table1. However, as the reviewer\npointed out, its expression is also detected at lower levels in other clusters. In the\nVISIUM platform, each spot with 55 μm in diameter contains more than 10 cells, which may\naccount for the presence of lower Has2 expression in other clusters that include\nstromal cells.\nWe apologize for not providing sufficient methodological details regarding spatial\ntranscriptomic data processing in the original manuscript. Raw spatial transcriptomics\n13\ndata were processed using the Space Ranger pipeline (v2.0.1, 10x Genomics,\nPleasanton, CA), aligned to the mm10-2020-A mouse reference genome, and analyzed\nfrom the resulting FASTQ files. For downstream analysis, including UMAP\nvisualization, violin plots, and gene expression analysis, we used Loupe Browser v6.0\n(10x Genomics). Principal component analysis (PCA) was performed using 100\ndimensions. These methodological details have now been included in the Methods\nsection (page 24-25).\n20. The authors mention HA interacting with CD44 on stem cells and hypothesize the\nsame sort of interaction in gastric cancer, but there is a paper that does show that\nmetaplasia in stomach expands CD44+ progenitor cells that as HA expands, so this\npaper should be cited and potentially discussed: PMID:23589310. There are other\npapers detailing CD44 role in metaplasia and stemness in stomach (eg, PMID:\n31071489), so the CD44/xCT/HA pathway may be important in both metaplasia and\nmetastasis.\nResponseAs pointed out by Reviewer #2, the hyaluronan receptor CD44 has been\nshown to play a critical role in the proliferation of metaplastic gastric epithelial\nprogenitor cells (Khurana et al, JBC, 2013, Ref. 37). In addition, CD44 and xCT are\nrequired for the reprogramming of gastric epithelial cells following mucosal injury\n(Meyer et al, Cell Mol Gastroenterol Hepatol, 2019, Ref. 38). Accordingly, we have\ncited these studies and discussed the possibility that hyaluronan may enhance the\nstem-like properties of gastric cancer cells through CD44-mediated signaling, thereby\ncontributing to metastatic tumor formation. This discussion has been added in the\nDiscussion section (page 16).\n21. A visualization of cell-cell communication, using differentially expressed genes\n(DEGs), would greatly improve analysis, as one would expect important cell-cell\ncommunication between cluster 5 and the rest of the clusters. For example, as the\nauthors state, CD44 in tumor cells could be communicating with HA in stroma.\nResponseIn this study, we identified that tumor cell-derived Wnt ligands activate\nWnt signaling in surrounding stromal cells and induce Has2 expression in cooperation\nwith TGFβ signaling. Furthermore, we demonstrated that hyaluronan (HA) deposition in\nthe tumor stroma is critical for the development of liver metastatic tumors.\nAs suggested by Reviewer #2, cell-cell communication analysis using spatial\ntranscriptomic data could provide important mechanistic insights into HA-associated\nmetastasis development. While we agree that this approach is highly valuable, we\n14\nbelieve that such analyses are beyond the scope of the present study. Nonetheless, we\ngreatly appreciate the reviewer’s constructive suggestion and intend to pursue this line\nof investigation in future study.\nReviewer #3 (Remarks to the Author)\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nReviewer #4 (Remarks to the Author)\n........The authors concluded that paracrine Wnt signaling activation in hepatic stellate\ncells in conjunction with TGFβ results in increased Has2 expression and this promotes\nmetastatic tumor development through hyaluronan deposition and increased ECM\nproduction. This study follows an earlier one by the same group where the role of these\ndriver mutations in formation of intestinal tumors was analyzed (Kok et al, 2021).\nA similar approach to generating gastric cancer GEMMs was published by\nSeidlitz T. et al in 2019 so the strategy used is not entirely novel. However, the present\nstudy adds novelty, by identifying a major role for Wnt ligands in shaping the liver\nmicroenvironment and thereby, enabling gastric cancer liver metastases.\nTechnically, the experiments were well executed, and the manuscript is (with a\nfew exceptions) well written. However, there are several issues that need to be\naddressed\nResponseWe sincerely thank Reviewer #4 for the constructive comments on our\nmanuscript. We have carefully addressed all the comments as detailed below.\nGeneral comment:\nIn Seidlitz et al, a similarly constructed GEMM based on driver mutations\nKrasG12D/+;Tp53R172H/+;Smad4fl/f in the gastric epithelium resulted in gastric\ntumors that were metastatic to liver and lung even in the (apparent) absence of Wnt\nproduction. These findings are at variance with the results obtained in the present\nstudy with the KTP mice and the authors should discuss potential differences that could\nexplain the different phenotypes.\nResponseAs the reviewer correctly pointed out, the GEMM model used by Seidlitz\n15\net al, (Ref. 16), which harbors KrasG12D, Smad4-/-, Trp53R172H mutations, developed\ngastric cancer with liver metastasis despite in the apparent absence of transgenic Wnt\nligand expression. Several key differences between their model and ours (KTP mice)\nmay account for the distinct metastatic phenotypes.\nFirst, the CreER drivers used to induce recombination may target different\nepithelial cell populations: Anxa10-CreER in the Seidlitz model versus Claudin18-\nCreER in our model. These possible target cell differences could influence the\ntumorigenicity and metastatic potential of the resulting tumors.\nSecond, the specific mutations in the TGFβ pathway and p53 differ: Seidlitz et al.\nused Smad4 deletion and Trp53R172H, while our model includes Tgfbr2 deletion and\nTrp53R270H. These molecular differences may affect the regulation of Wnt signaling in\ntumor cells. We have briefly discussed these points in the Discussion section (page\n15).\nSpecific points\n1. Figure 2 (E-H). This model of orthotopic implantation best mimics the clinical\npathology. Did the authors analyze progression to metastatic disease in these mice?\nDo WKTP tumors spontaneously metastasize to the liver in this setting? If not, a\ndiscussion is warranted.\nResponse We thank the reviewer for this important comment. In our orthotopic\ntransplantation experiments, we monitored gastric tumor development using in vivo\nimaging up to day 21, as shown in Figure 2g. At this time point, no clear evidence of\nmetastasis was observed. However, by day 28, some mice began to exhibit signs of\nperitoneal dissemination, as demonstrated in the imaging data provided below.\nAlthough histological analysis at this stage did not reveal liver metastases, the mice\nshowed signs of clinical deterioration, limiting our ability to extend observation further.\nThus, we could not examine whether WKTP\ntumors spontaneously metastasize by long\nterm analysis.\nTo establish a more quantitative and\nreproducible model for assessing liver\nmetastasis, we therefore employed the\nintrasplenic injection approach in this study. Although this model bypasses early steps\nof the metastatic cascade, it enables robust evaluation of the role of Wnt ligands in\nsupporting metastatic outgrowth in the liver. To clarify this point in the manuscript, we\nhave revised the sentence in the Results section (page 7) as follows: “To investigate\n16\nthe role of ligand-dependent Wnt signaling in liver metastasis by disseminated tumor\ncells, we transplanted KTP and WKTP organoid cells into the spleen of mice and\nanalyzed liver tissues.”\n2. Figure 3F - Were the differences between the different group statistically significant?\nIf not, this should be stated. There appears to be a high variability in the data obtained\nfor the control group and analysis of additional mice may be necessary to support the\nconclusion that these inhibitors had an effect.\nResponseIn response to this comment, we increased the number of mice for each\nexperiment group to n=8 for control, n=5 for ETC-159 treatment, and n=5 for GNF-\n6231 treatment, and performed statistical analysis. As shown in the revised Figure 3f,\nETC-159 treatment significantly suppressed liver metastasis of WKTP organoid cells\ncompared to the control. GNF-6231 treatment also resulted in a reduction in tumor\naria/field, although the difference was not statistically significant. Given the variability in\ntumor area/filed observed among control mice, we concluded that Wnt ligand\nexpression is critical for the development of liver metastasis in this model of gastric\ncancer. These new data have been included in Figure 3f, and described in the Results\nsection (page 8).\n3. Figure 3 (H&I) – Among the earliest events in liver metastases formation is the\nrecruitment of innate immune cells, among them neutrophils and monocytes which are\nstill functional in NSG mice. Wnt1 is known to affect immune cell function including\nmacrophage polarization (e.g. Staal et al, 2008, Haseeb M et al 2109). Given the\nresults shown this figure, the authors should analyze or at least comment on the\npotential role of myeloid-derived cells in promoting establishment of metastases of\nWKPT cells.\nResponseAs the reviewer suggested, we performed immunohistochemistry for\nαSMA and F4/80 to detect activated fibroblasts and macrophages, respectively, using\nliver sections at day 3 and 7 after spleen transplantation of KTP or WKTP cells.\nNotably, we observed fibroblast activation and macrophages infiltration in the stroma of\nWKTP micro-metastatic tumors at day 7 WKTP. These IHC results have been added to\nSupplementary Figure 4 and described in the Results section (page 8). As the\nreviewer suggested, it has been reported that Wnt/β-catenin signaling promotes M2\npolarization of tumor-associated macrophages (TAMs) (Tigue et al, Cancer Res, 2023,\nRef. 32). We have now discussed the potential role of Wnt-induced M2 TAMs in\nfacilitating metastasis development in the Discussion section (page 14, 15).\n17\n4. Fig 4C- The ratio of activated:total beta-catenin appears to be higher in KTP than in\nWKTP cells. This is surprising. Is there no autocrine effect of Wnt1 in the WKTP cells?\nDoes Wnt1 only act in a paracrine fashion and specifically only in the liver? Did the\nauthors quantify Wnt1 production levels in the three models (KPT, WKPT and Wnt-\nKPT)? These data are important for interpretation of the results.\nResponseWe quantified the band intensity of active β-catenin in our immunoblotting\ndata using ImageJ and normalized the values to GAPDH levels. While active β-catenin\nlevels in KTP cells were not higher than those in WKTP cells, they were rather\ncomparable. These results are presented as bar graph in Figure 4c. In contrast,\nTOPFLASH reporter assay (Figure 4d) demonstrated that Wnt signaling activity is\nsignificantly higher in WKTP cells compared to KTP cells. These data suggest that\nWnt1 ligands may activate Wnt signaling in WKTP cells via an autocrine mechanism,\nwithout leading to a detectable increase in active β-catenin levels by immunoblotting.\nWe previously observed similar results in RNF43-mutant (Wnt ligand-dependent)\ncolon cancer cells, where exogenous Wnt ligands activated Wnt signaling without\nsignificantly increasing active β-catenin levels. Therefore, in response to the\nReviewer’s question, we believe that WKTP cells utilize self-secreted Wnt1 ligands to\nsupport their survival and proliferation, as supported by the data shown in Figure 2b\nand 2C.\nRegarding Wnt1 expression, Wnt ligands undergo palmitoleoylation by PORCN in\nthe endoplasmic reticulum, which is essential for their secretion. Therefore, to\naccurately compare the levels of biologically active Wnt1, it is necessary to measure\nsecreted Wnt1. However, due to the lack of reliable antibodies suitable for\nquantification by ELISA or Western blotting, this is currently not feasible. Importantly,\nour TOPFLASH reporter assay (Figure 4d) showed that Wnt signaling activities in\nWKTP and Wnt-KTP cells are significantly higher than that in KTP cells.\n5. Figure 4F- How do the authors explain the striking difference in the extent of liver\nmetastatic disease in WKPT and Wnt-KTP implanted mice? Were Wnt1 production\nlevels in these cells compared?\nResponseAs shown in the bar graph of Figure 4g, there is variation in tumor area\nper field among the three independently established WKTP organoid lines, and even\namong replicates of the same line. In the original Figure 4f, we presented liver\nmetastasis images from the WKTP#2 line, which exhibited a markedly aggressive\nphenotype compared to Wnt-KTP, as the Reviewer pointed out. However, the levels of\n18\ntumor area per field for the WKTP#1 line was comparable to that of Wnt-KTP organoids\n(Figure 4g). Therefore, the striking difference in liver metastasis observed between\nWKTP#2 and Wnt-KTP may reflect biological variability among organoid lines with the\nsame genotype. To address this, we have added representative images of liver tumors\nderived from WKTP#1 in addition to WKTP#2 in the revised Figure 4f.\nRegarding the detection of Wnt1 expression, we have responded to the\ncomment#4. TOPFLASH reporter assay (Figure 4d) showed no significant difference in\nWnt signaling activity between WKTP and Wnt-KTP cells.\n6. Figure 5- To determine whether the extensive stromal response observed in these\nlivers is a cause or consequence of metastatic expansion, livers should also be\nanalyzed at earlier time points. It is also of interest to know whether the spatial\ntranscriptomic analysis identified any myeloid/innate immune cell clusters and whether\nliver sections of KPT -implanted mice were also analyzed for comparison.\nResponseDetermining whether the formation of a fibrotic microenvironment in the\nliver is a cause or consequence of metastatic colonization is one of the major goals of\nour research. In our previous study (Kok et al., Nat Commun, 2021, Ref. 19), we\ndemonstrated that metastasis was not developed when host TGFβ signaling was\ngenetically disrupted, as the stromal response was not induced even after\ndissemination of metastatic tumor cells. In the present study, we also showed that\nhyaluronan production in the stroma is essential for metastatic tumor formation. Taken\ntogether, these findings suggest that the stromal activation in the liver is a prerequisite\nfor the development of metastatic lesions. In the conclusion of the current manuscript,\nwe have highlighted the therapeutic potential of targeting CAF-mediated Wnt signaling\nand Has2 expression in the tumor stroma (page 16).\nIn line with Reviewer Comment #3, we performed immunohistochemical analyses\nof liver tissues at days 3 and 7 after splenic transplantation of organoids. These\nanalyses revealed that prominent infiltration of F4/80-positive macrophages in the\nstroma of WKTP cell-derived tumors, whereas such infiltration was less evident in KTP-\nderived lesions. Given that Wnt/β-catenin signaling has been reported to promote M2\npolarization of TAMs, it is plausible that Wnt-induced M2 TAMs may contribute to\nmetastatic progression. This possibility is now added in the Discussion section (page\n14). We plan to perform spatial transcriptomic analyses as part of our future\ninvestigations. We hope the reviewer understands our rationale.\n19\n7. Discussion and data interpretation\nThe authors propose that the ability of KPT and WKPT organoid to form tumors and\ngrow equally well in the orthotopic (and subcutaneous) site is due to local Wnt1\nproduction by stromal cells. This is speculative and should be experimentally tested,\nfor example by using the PORCN Wnt1 inhibitor C59. This also raises the question why\nthe authors switched from an orthotopic to an intra-splenic model to evaluate liver\nmetastasis. If WKPT cells do not metastasize following intra-mucosal implantation (or\nmetastasis is a very late event) this should be stated and discussed.\nResponseTo address this comment, we performed subcutaneous transplantation of\nKTP organoids and treated the recipient mice with the PORCN inhibitor ETC-159.\nWhile ETC-159 treatment significantly suppressed liver metastasis formation of WKTP\ncells (Figure 3f), it did not inhibit subcutaneous tumor development. Although it is\npossible that KTP cells do not require exogenous Wnt ligands for proliferation in the\nsubmucosal and subcutaneous environments, we currently lack direct mechanistic data\nto support this hypothesis. Therefore, we have softened the wording in the Results\nsection (page 7) to: “These results suggest that the combination of KTP driver\nmutations is sufficient to induce dysplastic tumor formation in both the submucosal and\nsubcutaneous environments.”\nAlthough our current experiments do not directly test stromal Wnt ligand\ndependency in the subcutaneous setting, this does not impact the main conclusions of\nour study.\nRegarding the metastasis analysis in orthotopic models, we have described\nabout limitation of orthotopic models for long term analysis in our response to the\nComment#1.\n8. The functional significance of hyaluronan in metastatic expansion in the liver in this\nmodel has not been defined. Is it utilized by WKPT cells to attach via CD44?\nWhether the fibrotic response is a cause or consequence of tumor expansion in the liver\nshould be resolved experimentally by analyzing the response on day 7 not at late stages\nof metastasis as demonstrated in Fig 5. Language editing is required. For example see\nline 329.\nResponseAs the reviewer pointed out, hyaluronan receptor CD44 plays a key role in\nthe proliferation of gastric progenitor cells (Khurana et al, JBC, 2013, Ref. 37).\nFurthermore, CD44 and xCT have been shown to promote the reprogramming of\ngastric epithelial cells (Meyer et al, Cell Mol Gastroenterol Hepatol, 2019, Re. 38).\nThese findings suggest that hyaluronan may enhance the stem-like properties of\n20\ngastric cancer cells via CD44-mediated signaling, thereby contributing to metastatic\ntumor formation. We have included this point in the Discussion section (page 16).\nAdditionally, it has been reported that low molecular weight (LMW)-hyaluronan\npromotes tumor progression, whereas high molecular weight (HMW)-hyaluronan exerts\ntumor-suppressive effects (Liu et al, Front Immunol, 2019, Ref. 35). Moreover, a recent\nstudy showed that reducing LMW-hyaluronan levels with hyaluronidase inhibitor\nsuppresses cancer cell metastasis (McGuire et al, Sci Rep, 2024, Ref. 36). These\nresults, together with our findings, support the notion that LMW-hyaluronan plays a\ncritical role in promoting gastric cancer metastasis. We have discussed this aspect in\nthe Discussion section (page 15-16).\nRegarding whether the fibrotic niche is a cause or consequence of tumor\nexpansion in the liver, our data shown in the Supplementary Figure 4 demonstrate that\na fibrotic microenvironment marked by αSMA+ fibroblasts is already established at day\n7 in WKTP cell micrometastasis, while such a fibrotic reaction is absent in KTP cell\nclusters. These results suggest that fibrotic niche formation is a consequence of WKTP\ncell colonization. On the other hand, degradation of stroma cell-derived hyaluronan\nsignificantly suppressed development of metastasis (Figure 7f, g), indicating that\nstromal remodeling is a driving factor in tumor progression. Thus, stromal activation\nmay act both as a cause and consequence of metastatic growth.\nWe acknowledge that the interaction between disseminated tumor cells and host\nstromal responses is complex. Therefore, we plan to conduct more detailed\nmechanistic studies in future projects to further elucidate this relationship.\nWe have carefully revised the manuscript to improve English usage throughout,\nincluding the point noted by the reviewer (line 329 in original manuscript).\nReviewer #5 (Remarks to the Author)\nGeneral Remark: While the study is interesting, there is very little evidence that the\nfindings could be translated to human (very little has been done to validate the findings\nin human). While the study uses an experimentally sophisticated system (many\ntransgenic animals) there is an important number of issues that needs to be carefully\naddressed. When talking about Wnt-signaling and paracrine communication between\ncancer cells and CAF it is important to note that specific Wnt ligands will be secreted by\nspecific cells while specific receptors will be expressed on certain cells. This is where\nthe study in reviewers opinion is limited and to certain extent far from realistic situation\nencountered in human.\nimportant to clarify which specific Wnt ligands and Frizzled receptors mediate\nmetastatic progression in human gastric cancer. However, the current understanding of\nthe pairing between the 19 Wnt ligands and 10 Frizzled receptors remains incomplete.\nNotably, Nanki et al. (Cell, 2018, Ref. 10) reported substantial heterogeneity among\ngastric cancer organoids derived from 36 patients, particularly regarding their\ndependency on exogenous Wnt ligands or R-spondin, as well as endogenous Wnt\nligand expression patterns by the cancer cells themselves. Furthermore, TCGA data\nshows that 18 Wnt ligands are each upregulated in more than 10% of gastric cancer\nsamples, and that such Wnt ligand upregulation is found in approximately 85% of\ncases (Supplementary Fig. 1), suggesting that multiple Wnt ligands may be functionally\nrelevant. Thus, whether specific ligand–receptor pairs are dominant, or if redundant\nsignaling mechanisms exist in gastric cancer, remains an open question likely requiring\nlarge-scale single-cell transcriptomic analysis.\nOur current study demonstrates that Wnt ligand-mediated signaling promotes the\ndevelopment of metastatic lesions in gastric cancer. While we acknowledge that further\ninvestigation is necessary to define the precise ligand–receptor combinations involved\nin human metastasis, we believe that our findings provide important insights and a\nstrong foundation for such future research. We hope the reviewer understands the\nscope and significance of our present work in this context.\nMajor Remark 1: In human gastric cancer HAS2 is not expressed on stellate cell\nderived CAF. Namely, there are generally 2 types of CAF (in the liver) depending on\ntheir origin, stellate cell derived (RGS5+) or resident fibroblast derived (PDGFRA+).\nHence the model that is used in vitro (employing immortalised HSC) is unfortunately\nirrelevant. This goes further, HYAL2 is not expressed by cancer cells, but by\nendothelial cells while Wnt5A is expressed by cancer cells.\nI am attaching in support some meta-analysis that I have conducted based on\npreviously published data (DOI:10.1002/ctm2.730)\nIn conclusion, the authors need to be very careful about which ligand is\nexpressed on which cell (lay out their model based on human data, either single cell if\nclear enough or IHC). Then according to this design their experimental strategy.\nConcretely, which cells express WNT1 in human gastric cancer and what is the target\nreceptor(s), where are these receptors situated in humans?\nResponseThank you for your important comment regarding the origin of CAFs. As\ncited by the reviewer, CAFs in gastric cancer can be categorized into two major\n22\npopulations: iCAFs (expressing PDGFRA and CXCL12) and mCAFs (myCAFs)\n(expressing RGS5 and ACTA2). GSEA results suggest that iCAFs primarily promote\nstromal remodeling and cancer cell proliferation and invasion. We fully understand the\nreviewer’s concern that using HSCs for in vitro experiments may not completely reflect\nthe functional CAF populations found in human gastric cancer.\nHowever, the origin and classification of CAFs within the tumor stroma remain\ncontroversial. For example, a recent review by Geng and Schwabe (Hepatology, 2025,\nRef. 23) indicates that HSCs can differentiate into both iCAFs and myCAFs. Moreover,\nmyCAFs expressing HAS2 have been reported to promote tumor progression through\nhyaluronan-mediated signaling (Cogliati et al., Nat Rev Gastroenterol Hepatol, 2023).\nThese findings support the relevance of using HSCs for in vitro studies, especially in\nthe context of HAS2-driven metastatic promotion by myCAFs.\nAs the reviewer rightly pointed out, multiple cellular origins of CAFs likely exist\nbeyond HSCs. To address this, we conducted additional experiments using a CAF line\nisolated from liver metastatic tumors. These data demonstrate that Wnt ligands activate\ncanonical Wnt signaling also in CAFs, and that Wnt ligands synergize with TGF-β to\ninduce Has2 expression in CAFs. These new findings have been included in the\nrevised Figures 6b and 7b. Figure legends have been updated accordingly.\nRegarding HYAL1 and HYAL2, we transfected expression vectors for these\ngenes into WKTP cells specifically to degrade stromal hyaluronan in order to evaluate\nits functional role in metastasis. We did not assume that HYAL1 or HYAL2 are\nendogenously expressed by cancer cells in vivo.\nConcerning the reviewer’s question on Wnt ligand and Frizzled receptor\nexpression, we have addressed this point in our reply to the General Remark from the\nsame reviewer. Briefly, our study focuses on how exogenous Wnt ligands, expressed\nby tumor cells, influence the surrounding tumor microenvironment during metastasis. In\nour model, Wnt ligands are expressed by cancer cells, and the corresponding Frizzled\nreceptors for Has2 induction are thought to express on CAFs. We selected WNT1\nbased on previous reports demonstrating its association with gastric cancer\naggressiveness and metastasis (Mao et al., Cell Death Dis, 2014, Ref. 18; Li et al., Lab\nInvest, 2023, Ref. 12). We have included this rationale for use of Wnt1 in the Results\nsection, along with newly added reference (page 5).\nMajor Remark 2: The part of the data that is related to CAF needs thorough revision.\nThe authors state \"It has been shown that CAFs in the liver metastasis are originated\nfrom activated hepatic stellate cells (HSCs)\", which is simply wrong in many aspects\n23\n(the cited literatures is not relevant, please see doi: 10.1038/s41586-019-1631-3.).\nAgain, CAFs in the liver metastasis are derived from at least 3 different precursor cells\n- and in contrast to what the authors are claiming, those CAF that reorganise the ECM\nare not stellate cell derived. I further refer to the attached meta-analysis where on\nprimary gastric cancer samples from human we can clearly see at least 2 groups of\nCAF (stellate cells do exist also in stomach and many other organs next to liver) to\nbegin with.\nResponseWe appreciate the Reviewer’s important comment regarding the origin of\nCAFs in liver metastases. In the original text, we stated that “CAFs in the liver\nmetastasis are originated from activated hepatic stellate cells (HSCs).” However, as the\nReviewer correctly pointed out, CAFs can arise from multiple precursor cell types, and\nHSCs represent one of several possible sources. We also realized that the cited\nliteratures were not appropriate, and have therefore deleted both the statement and\nassociated references from the manuscript.\nIn the revised Results section (page 11), we now state that CAFs in gastric\ncancer tissues can be classified into myCAF and iCAF subtypes based on distinct gene\nexpression profiles, as described by Jiang et al. (Clin Transl Med, 2022, Ref. 21), as\nreferenced in the Reviewer’s comment #1. We also mention that HSCs have the\npotential to differentiate into both myCAFs and iCAFs. However, consistent with current\nunderstanding, we emphasize that CAFs exhibit substantial heterogeneity in their\ncellular origins, citing relevant literatures including Yamamoto et al. (Cancer Sci., 2022,\nRef. 24).\nIn response to the Reviewer’s suggestion that the use of only HSCs is insufficient\nto assess CAF function, we performed additional in vitro experiments using a CAF line\nisolated from liver metastatic tumors. These new results have been incorporated into\nFigures 6b and 7b in the revised manuscript.\nMajor Remark 3: Provided that the authors sort out the cellular identities according to\nthe remarks made above, what is the link between Wnt-signalling and HAS2\nexpression? The authors switch without any explanation from Wnt1 (which is their initial\nmodel) to Wnt3. What is the reason for this? What is the downstream signalling and\nhow Wnt1 (and not 3) could cause the HAS2 over-expression?\nResponseWe appreciate the Reviewer’s insightful comment regarding the\nmechanistic link between Wnt signaling and HAS2 expression, as well as the rationale\nfor using Wnt3 in our in vitro experiments.\ngastric cancer cells induce HAS2 expression in CAFs in a manner synergistic with\nTGF-β signaling. This conclusion is supported by spatial transcriptomic analysis, in\nvitro experiments using HSCs and CAFs, histological analysis of human and mouse\ngastric cancer liver metastases, and analysis of public databases showing a positive\ncorrelation between Wnt ligand and HAS2 expression (Supplementary Fig. 8).\nRegarding the use of Wnt3 instead of Wnt1 in our in vitro experiments, we\nemployed afamin-bound Wnt3a because it has been reported to be water-soluble and\nto provide stable and potent canonical Wnt signaling activity (Mihara et al., eLife, 2016,\nRef. 25). Since both Wnt1 and Wnt3a activate the canonical Wnt/β-catenin signaling\npathway, afamin-Wnt3a serves as a functional surrogate for Wnt1 in our experimental\nsystem. To clarify this point, we added the following sentence to the Results section\n(page 11): “we conducted experiments using both HSCs and CAFs, treating them with\nafamin-bound Wnt3a, which preserves stable canonical Wnt ligand activity (25).”\nWith respect to downstream signaling, both Wnt1 or Wnt3a bind to Frizzled\nreceptors, leading to inhibition of GSK3-mediated phosphorylation of β-catenin. This\nresults in β-catenin stabilization and nuclear translocation, where it interacts with TCF\ntranscription factors to induce expression of canonical Wnt target genes. Notably,\nTCF7L2, a key Wnt pathway transcription factor, has been reported to directly bind the\nHas2 promoter and induces its expression (Sun et al., Biomolecules, 2022, Ref. 34).\nThis provides a possible mechanism by which both Wnt1 and Wnt3a may drive Has2\nexpression via canonical Wnt signaling. We have included this mechanistic insight and\nsupporting reference in the revised Discussion section (page 15).\nMajor Remark 4: Hyaluronidase inhibitors have been shown to inhibit cancer\nmetastasis (https://www.nature.com/articles/s41598-024-64924-6). The authors here\nshow the contrary. It seems that high molecular weight hyaluronic acid (HMW-HA) is\ntumor protective, while low molecular weight hyaluronic acid (LMW HA) is tumor\npromotive. Authors need to clarify better which type of HA they are dealing with and\nrelate this to what is observed in human samples.\nResponseWe appreciate the Reviewer’s insightful comment. As the Reviewer noted,\nlow molecular weight (LMW) hyaluronan has been reported to promote tumor\nprogression, whereas high molecular weight (HMW) hyaluronan exhibits tumor-\nsuppressive effects (Liu et al., Front Immunol, 2019, Ref. 35). In the study cited by the\nReviewer (McGuire et al., Sci Rep, 2024, Ref. 36), treatment with delphinidin, a\n25\npotential hyaluronidase inhibitor, led to the accumulation of HMW-hyaluronan and was\nassociated with suppression of cancer invasion and metastasis.\nIn our study, we simultaneously overexpressed two hyaluronidases, Hyal1 and\nHyal2, in WKTP tumor cells. Hyal2 initiates to breakdown of HMW-hyaluronan into\nLMW-fragments, while Hyal1 further degrades LMW-hyaluronan into smaller\noligosaccharides, promoting its clearance (Bourguignon and Flamion, FASEB J, 2016,\nRef. 27). Thus, in both McGuire et al. and our studies, the level of tumor-promoting\nLMW-hyaluronan is reduced, albeit through different mechanisms-one by inhibiting its\ngeneration, the other by enhancing its degradation and clearance. Therefore, we\nbelieve that our findings are not contradictory to those reported in the cited literature.\nTo clarify this point, we have added the following statement to the Results section\n(page 13): \" We anticipated that Hyal-WKTP cells, which co-express Hyal1 and Hyal2,\nwould degrade both high molecular weight (HMW)- and low molecular weight (LMW)-\nhyaluronan within the tumor microenvironment (27).\"\nAdditionally, we have discussed the possibility that LMW-hyaluronan may play an\nimportant role in promoting gastric cancer metastasis in the Discussion section, along\nwith newly added references (page 15-16).\n26"}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have done a very scholarly job of addressing reviewer concerns. Here are some minor comments:\n1. It would be better to mention in the manuscript why authors chose to assess Wnt pathway activity by detecting the active form of β-catenin via immunoblotting and by performing the TOPFLASH reporter assay.\n2. In manuscript lines 213–214, the authors stated: “Importantly, all three independently established Apc-KTP organoid cell lines did not develop liver metastasis, and only rare small clusters of tumor cells were detected.” However, in the current Figure 4f (Apc-KTP#2), liver metastases are visible at multiple locations: in the first liver (5 o’clock and 11 o’clock), the second liver (1 o’clock), and the third liver (3 o’clock and 11 o’clock), with tumors present at the liver edges. In addition, Supplementary Figure 6C clearly shows liver metastases in the Apc-KTP group. Therefore, the statement in lines 213–214 is inconsistent with the presented data and should be revised.\n3. The reason for using a splenic transplant (vs true orthotopic) model as well as not using C57/Bl6 mice as hosts might be mentioned in the final text and not just in the response to reviewers, as some readers may also be curious.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The authors satisfactorily addressed most of my concerns. A better characterization of the polarization state of recruited macrophages in their GEMMs could further improve the understanding of the role of immune cells in liver metastasis in these models.", "reviewer_5": "Furutani et al have provided the revised manuscript. Overall reviewers comments regarding the consideration of relevant literature, CAF-related language have been corrected. However, the key problems have not been experimentally addressed (except the new CAF in vitro experiments in the Fig. 6B and 7B). Unfortunately, two key points remain unanswered and that is:\n1) relevance to human: only very limited data, with reduced statistical significance, have been provided from human samples to indicate the some of the observations might hold true in human samples.\n2) take more care about the cell of origin. It is not OK to use cancer cells to express stromal genes even if these are supposed to be secreted and exert their activity outside the cell. One does not know if these proteins may have functions in the cytoplasm. Hence, if HYAL1 is expressed in endothelial cells, then a model must account for that and expressing HYAL1 in cancer cells will not be considered as relevant.\nNext to this, there are several points that need to be addressed to clarify if the data from the manuscript could be reproduced in the future. In particular the authors should:\n1) make sure that reviewers now and other scientist later can access the OMICS data. The link that has been provided does not work.\n2) the WKTP gastric cancer model, as well as all other models that are not publicly available yet used in the present manuscript, need to be outlined in the \"Material Availability\" section, where the conditions and accessibility for sharing these models should be clearly stated.", "reviewer_6": "Furutani and colleagues present a compelling study identifying a novel role for Wnt1-dependent signaling in conjunction with hyaluronan-expressing cancer-associated fibroblasts as a key mediator of gastric cancer liver metastasis. Using diverse methodologies, they demonstrate that cell-intrinsic Wnt signaling alone is insufficient for metastasis induction; instead, synergistic Wnt and TGFβ signaling induces hyaluronan in stromal cells. The study is strengthened by robust genetic control populations and inhibition models. The authors adequately addressed previous reviewer concerns, with manuscript revisions enhancing the overall quality, and I support its publication in Nature Communications.\nMinor concerns are outlined below.\n1) Lines 113–114 claim all mutant mice showed significantly increased thickness, but the statistical comparison of WT vs. K is missing in Figure 1c.\n2) Supplementary Figure 8b shows a correlation between HAS2 and WNT6, but WNT6 is not mentioned in the text.\n3) Figures 6 and 7 label “Afamin-Wnt3,” while the text uses “Afamin-Wnt3a.” Relabel figures as “Afamin-Wnt3a” for consistency.\n4) The response to Reviewer #1’s concern about using immunodeficient vs. immunocompetent mice is well-articulated. The referenced study (Sakai et al., Cancer Res, 2018) should be cited in the paper, and including parts of this explanation in the discussion would strengthen the experimental design and conclusions."}, "author_response": "Response to the reviewer comments\nOur point-by-point responses to the comments made by the Reviewers are included below.\nThe comments are quoted “verbatim”, followed by our responses.\nReviewer #2 (Remarks to the Author):\nThe authors have done a very scholarly job of addressing reviewer concerns. Here are some\nminor comments:\n1. It would be better to mention in the manuscript why authors chose to assess Wnt pathway\nactivity by detecting the active form of β-catenin via immunoblotting and by performing the\nTOPFLASH reporter assay.\nWe thank the reviewer for this helpful suggestion. In the revised manuscript, we now clarify\nour rationale for using two complementary assays to assess Wnt pathway activity in the text\nas follows. “Loss of APC suppresses phosphorylation-dependent β-catenin degradation,\nthereby increasing the unphosphorylated (active) form of β-catenin. The stabilized β-catenin\ntranslocates to the nucleus and functions with TCF/LEF to drive Wnt target gene\ntranscription. Consistent with this rationale, immunoblotting showed a significant increase in\nunphosphorylated β-catenin in Apc-KTP cells compared with KTP and WKTP cells (Fig. 4c),\nand the TOPFlash reporter assay demonstrated significantly higher β-catenin/TCF\ntranscriptional activity in Apc-KTP cells, which is comparable to the Apc-disrupted intestinal\ntumor-derived organoids, AKTP (19) (Fig. 4d)” (page 9).\n2. In manuscript lines 213–214, the authors stated: “Importantly, all three independently\nestablished Apc-KTP organoid cell lines did not develop liver metastasis, and only rare small\nclusters of tumor cells were detected.” However, in the current Figure 4f (Apc-KTP#2), liver\nmetastases are visible at multiple locations: in the first liver (5 o’clock and 11 o’clock), the\nsecond liver (1 o’clock), and the third liver (3 o’clock and 11 o’clock), with tumors present at\nthe liver edges. In addition, Supplementary Figure 6C clearly shows liver metastases in the\nApc-KTP group. Therefore, the statement in lines 213–214 is inconsistent with the presented\ndata and should be revised.\nWe thank the reviewer for pointing out this inconsistency. We have revised the Results to\naccurately reflect the data shown in Fig. 4f and Supplementary Fig. 7c (former\nSupplementary Fig. 6c) as follows. “Among the three independently established Apc-KTP\norganoid lines, only line #2 developed limited, small macroscopic liver metastasis (Fig. 4f,\narrowheads, Fig. 4g), whereas other lines did not show grossly detectable liver metastasis.\nMoreover, histological analysis revealed rare, small clusters of tumor cells in #2 and #8 Apc-\ndetection” (page 10).\n3. The reason for using a splenic transplant (vs true orthotopic) model as well as not using\nC57/Bl6 mice as hosts might be mentioned in the final text and not just in the response to\nreviewers, as some readers may also be curious.\nWe thank the Reviewer #2 for this helpful suggestion. We have added an explanation in\nthe Results describing our use of a splenic transplantation model with showing in vivo\nimaging data (Supplementary Fig. 4), and in the Discussion, our rationale for using NSG\nrather than C57BL/6 mice as hosts.\nResults:\n“We next evaluated the role of Wnt ligands in liver metastasis. In the orthotopic\ntransplantation model of WKTP organoids, three of five mice exhibited signs of peritoneal\ndissemination by day 28, accompanied by clinical deterioration, which limited the observation\nwindow for assessing liver metastasis (Supplementary Fig. 4). We therefore performed\nspleen transplantation of KTP and WKTP organoids in NSG mice to examine the liver\nmetastatic tumors. (Fig. 3a)” (page 7).\nDiscussion:\n“In our previous study, spleen transplantation of intestinal tumor-derived AKTP organoids,\nharboring mutations in Apc, Kras, Tgfbr2, and Trp53 into syngeneic C57BL/6 (B6) mice\nyielded liver metastasis in ~40% of recipients, whereas injection into immunodeficient NSG\nmice resulted in nearly 100% penetrance (37). This discrepancy is likely due to the\nNK cell-mediated surveillance in B6 mice, although it remains to be elucidated. Based on these data,\nwe used NSG mice for the current study to maximize reproducibility and detection sensitivity\nwhen comparing the metastatic potential of KTP and WKTP organoids” (page 16).\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #4 (Remarks to the Author):\nThe authors satisfactorily addressed most of my concerns. A better characterization of the\n2\npolarization state of recruited macrophages in their GEMMs could further improve the\nunderstanding of the role of immune cells in liver metastasis in these models.\nWe appreciate Reviewer #4’s suggestion to further characterize macrophage polarization.\nAccordingly, we performed immunohistochemistry for CD206, an M2 macrophage marker, on\nserial sections alongside F4/80, and found CD206+ M2-like macrophages in the early stage\nof WKTP cell liver metastatic tumors. We have added these results in the Supplementary\nFig. 5, and revised the Results section as “On day 7 after transplantation, numerous small\nmetastatic lesions of WKTP cells were detected in the liver, accompanied by activated\nfibroblasts and infiltrating CD206+ M2-like macrophages (Fig. 3h, i, and Supplementary Fig.\n5)” (page 8). Moreover, we have briefly discussed the role of Wnt signaling in M2 polarization\nin Discussion as “Consistent with prior reports that Wnt/β-catenin signaling promotes M2\npolarization of tumor-associated macrophages (TAMs) (32), we observed CD206+ M2-like\nmacrophages in the liver micrometastatic lesions. These results suggest that Wnt-induced\nM2 TAMs may facilitate metastatic progression” (page 15).\nReviewer #5 (Remarks to the Author):\nFurutani et al have provided the revised manuscript. Overall reviewers comments regarding\nthe consideration of relevant literature, CAF-related language have been corrected.\nHowever, the key problems have not been experimentally addressed (except the new CAF in\nvitro experiments in the Fig. 6B and 7B). Unfortunately, two key points remain unanswered\nand that is:\n1) relevance to human: only very limited data, with reduced statistical significance, have\nbeen provided from human samples to indicate the some of the observations might hold true\nin human samples.\nWe appreciate the Reviewer’s emphasis on human relevance. Our study was designed to\nmechanistically interrogate ligand-dependent Wnt signaling in gastric cancer liver metastasis\nusing genetically controlled mouse models and organoid transplantation systems.\nWe agree that definitive validation in human tissues is essential. While our current\ndataset includes only limited human evidence, the convergence of multiple independent,\ngenetically defined models provides robust proof-of-concept for a previously\nunderappreciated mechanism of metastasis.\nIn recognition of this limitation, we revised the Discussion to state: “Our present study\nsupports a model in which ligand-dependent Wnt signaling and CAF-derived Has2 promote\nhyaluronan deposition in the tumor stroma to facilitate gastric cancer metastasis. While these\nresults were obtained in genetically controlled mouse and organoid models, prospective\n3\nvalidation in human gastric cancer liver metastasis is warranted, and will be an important\nfocus of our future work” (page 17).\n2) take more care about the cell of origin. It is not OK to use cancer cells to express stromal\ngenes even if these are supposed to be secreted and exert their activity outside the cell. One\ndoes not know if these proteins may have functions in the cytoplasm. Hence, if HYAL1 is\nexpressed in endothelial cells, then a model must account for that and expressing HYAL1\nin cancer cells will not be considered as relevant.\n We appreciate the reviewer’s insightful comment regarding the importance of considering\nthe cell of origin when assessing the function of stromal genes.\nIn our study, we ectopically expressed hyaluronidases (Hyal1 and Hyal2) in WKTP\ncancer cells to experimentally degrade hyaluronan in the tumor microenvironment of liver\nmetastases. This strategy was adopted to functionally assess the role of extracellular\nhyaluronan in metastatic niche formation. Our data showed that expression of these\nenzymes significantly suppressed liver metastasis, suggesting a critical role for hyaluronan\ndeposition in the metastatic microenvironment.\nWe acknowledge the reviewer’s concern that expressing stromal genes in cancer cells\nmay potentially introduce intracellular effects unrelated to their physiological function. To\naddress this, we have confirmed that expression of Hyal1/Hyal2 in WKTP cells did not affect\ntheir in vitro proliferation as well as in vivo tumorigenic potential, indicating that their primary\neffect is likely exerted extracellularly (see Supplementary Fig. 10) (page 13).\nThat said, we fully agree that evaluating the role of stroma-derived hyaluronan more\nphysiologically—such as by targeting Has2 in cancer-associated fibroblasts (CAFs)—will\nprovide more definitive insights. Accordingly, we have clarified this point in the revised\nmanuscript as follows: \"This hypothesis warrants further investigation, including studies\nspecifically targeting Has2 in stromal fibroblasts to delineate its functional role in the\nmetastatic process\" (page 16).\nNext to this, there are several points that need to be addressed to clarify if the data from the\nmanuscript could be reproduced in the future. In particular the authors should:\n1) make sure that reviewers now and other scientist later can access the OMICS data.\nThe link that has been provided does not work.\nAt the time we submitted the revision, the DDBJ records were still under hold, which\ncaused the temporary dead link. We have since released the records to public access, and\nwe have verified that all datasets cited in the Data Availability section are now accessible via\ntheir accession numbers.\n4\n2) the WKTP gastric cancer model, as well as all other models that are not publicly available\nyet used in the present manuscript, need to be outlined in the \"Material Availability\" section,\nwhere the conditions and accessibility for sharing these models should be clearly stated.\nAccording to the Reviewer #5’s suggestion, we have added the information of availability\nof mouse and organoid lines used in this study in Materials Availability section as “WKTP and\nKTP mouse models, and WKTP, KTP, Apc-KTP, and Hyal-KTP organoids are available from\nthe corresponding author upon reasonable request and completion of a Material Transfer\nAgreement” (page 27).\nReviewer #6 (Replacing Reviewer #1, Remarks to the Author):\nFurutani and colleagues present a compelling study identifying a novel role for Wnt1-\ndependent signaling in conjunction with hyaluronan-expressing cancer-associated fibroblasts\nas a key mediator of gastric cancer liver metastasis. Using diverse methodologies, they\ndemonstrate that cell-intrinsic Wnt signaling alone is insufficient for metastasis induction;\ninstead, synergistic Wnt and TGFβ signaling induces hyaluronan in stromal cells. The study\nis strengthened by robust genetic control populations and inhibition models. The authors\nadequately addressed previous reviewer concerns, with manuscript revisions enhancing the\noverall quality, and I support its publication in Nature Communications.\nWe thank the Reviewer #6 for the positive assessment of our manuscript and support for\npublication.\nMinor concerns are outlined below.\n1) Lines 113–114 claim all mutant mice showed significantly increased thickness, but the\nstatistical comparison of WT vs. K is missing in Figure 1c.\nThank you for pointing this out. We re-checked the statistics using a one-way ANOVA\nfollowed by Tukey’s post-hoc test across all genotypes. The comparison between wild-type\n(WT) and the simple Kras mutant (K) was not significant; all other mutant groups showed\nsignificantly increased gastric mucosal thickness vs WT. We have updated Fig. 1c to label\nthe WT vs K comparison as n.s., and we revised the text as “All mutant mice except the\nsimple Kras mutant group showed significantly increased gastric mucosal thickness\ncompared with wild-type mice (Fig. 1b, c)” (page 5)\n2) Supplementary Figure 8b shows a correlation between HAS2 and WNT6, but WNT6 is not\nmentioned in the text.\nWNT6 has been reported to act predominantly through the canonical β-catenin pathway.\nThus, we have revised the Results as “Moreover, we observed a modest but positive\ncorrelation between HAS2 and canonical Wnt ligands such as WNT2, WNT2B, and WNT6\n(the latter has been reported to act predominantly through the canonical Wnt signaling),\nsuggesting a potential role for canonical Wnt signaling in the regulation of HAS2 expression\nin human gastric cancer (Supplementary Fig. 8b)” (page 12).\n3) Figures 6 and 7 label “Afamin-Wnt3,” while the text uses “Afamin-Wnt3a.” Relabel figures\nas “Afamin-Wnt3a” for consistency.\nAs the Reviewer #6 suggested, we have relabeled “Afamin-Wnt3” as “Afamin-Wnt3a” in\nFigures 6 and 7.\n4) The response to Reviewer #1’s concern about using immunodeficient vs.\nimmunocompetent mice is well-articulated. The referenced study (Sakai et al., Cancer Res,\n2018) should be cited in the paper, and including parts of this explanation in the discussion\nwould strengthen the experimental design and conclusions.\nWe thank the Reviewer #6 for this helpful suggestion. We have added an explanation in of\nour rationale for using NSG mice rather than syngeneic C57BL/6 mice with citation of our\nprevious study (Sakai et al, Cancer Res, 2018) in Discussion as “In our previous study,\nspleen transplantation of intestinal tumor-derived AKTP organoids, harboring mutations in\nApc, Kras, Tgfbr2, and Trp53 into syngeneic C57BL/6 (B6) mice yielded liver metastasis in\n~40% of recipients, whereas injection into immunodeficient NSG mice resulted in nearly\n100% penetrance (37). This discrepancy is likely due to the NK cell-mediated surveillance in\nB6 mice, although it remains to be elucidated. Based on these results, we used NSG mice\nfor the current study to maximize reproducibility and detection sensitivity when comparing the\nmetastatic potential of KTP and WKTP organoids” (page 16).\n6"}, {"round": 3, "reviewer_comments": {"reviewer_5": "I note the text modifications the authors have made. The reviewer believes that experiments would have been more appropriate. The reviewer has no further comments."}, "author_response": ""}]

Decision letter