Introduction

Gastric Cancer (GC) is one of the most common carcinomas world-wide (1) characterised by a peculiar contribution of the tumour microenvironment to the aggressive biology of this malignancy (2). GC progression is characterised by striking changes in lymphatic vessels (LVs) and lymphatic metastasis is the most common feature of GC spread, representing the most significant prognostic factor determining clinical outcomes (3). Recent publications have demonstrated a functional link between the Extra-Cellular Matrix protein EMILIN-1, tumor growth, and lymphangiogenesis. EMILIN-1 is a key structural and functional element in the maintenance of a competent lymphatic vasculature and, as demonstrated in skin and colitis-associated cancer mouse models, exhibits anti-proliferative and oncosuppressive properties through the interaction with α4β1 integrin (4,5,6).

Aims & Methods

We have observed that EMILIN1 is present at lower levels in human gastric malignant and pre-neoplastic samples and this correlates with the presence of abnormal LVs (unpublished preliminary data). The aim of this study is to investigate the role of EMILIN-1 in gastric tumor onset and progression using mouse models of GC.
We used the syngeneic YTN16 gastric cancer cell line (7) in genetically modified EMILIN-1 mice (KO, knock out mice; E933A KI, E933A-mutated Emilin1 Knock-In animals expressing an EMILIN-1 mutant that is unable to interact with α4/α9β1 integrins). KI animals were included to specifically define the effects related to integrin engagement. Cells were injected intraperitoneally (i.p.), sub-cutaneously (s.c.) or into the footpad of 8-week-old C57BL/6 mice; tumors were analyzed after 3 weeks. A protocol using N-methyl-N-nitrosourea (MNU) was also performed on the same animal models to better investigate the role of EMILIN-1 when a chemically-induced gastric carcinogenesis approach was used. Mice were given drinking water ad libitum containing 240 ppm MNU on alternate weeks for a total exposure of 5 weeks and sacrificed after 30 weeks.

Results

To assess tumor growth, mice were injected with the syngeneic GC cell line YTN16 subcutaneously into both flanks. KO and E933A KI mice developed larger and earlier tumors; tumor samples were stained with Lyve-1 and CD31 to identify lymphatic and blood vessels, respectively. While no differences were observed for CD31-positive structures, a statistically significant increase in intratumoral lymphangiogenesis was observed in KO and E933A KI animals. Lymphatic spread was analyzed in detail by means of intra-footpad injection; enhanced lymph node (LN) metastasis of YTN16 transplanted tumors was observed in KO and E933A KI mice. In a model of peritoneal carcinomatosis and dissemination (by intra-peritoneal injection of YTN16), EMILIN-1 mutant animals also had higher histopathologic scores for invasion;
The use of the MNU protocol, that represents an interesting approach to investigate how deficiency of the ECM molecule EMILIN-1 may affect the incidence and frequency of tumour development, further showed that KO mice had higher tumour incidence, bigger adenomas and lower survival after MNU treatment. Similar results were obtained with the E933A KI model.

Conclusion

These results clearly demonstrate that EMILIN-1, exerts a pivotal role in GC onset, progression and dissemination through the involvement of α4/α9β1 integrin. Loss of EMILIN-1 may lead to alterations in the microenvironment able to promote tumorigenesis in the stomach. Our study also provides a novel animal model that, for the first time, targets a component of the gastric tumor microenvironment.

References

(1) Smyth EC, Nilsson M, Grabsch HI, van Grieken NC, Lordick F. Gastric cancer. Lancet Lond Engl. 2020;396:635–48.
(2) Rojas A, Araya P, Gonzalez I, Morales E. Gastric Tumor Microenvironment.Adv Exp Med Biol. 2020;1226:23–35.8
(3) Deng J-Y, Liang H. Clinical significance of lymph node metastasis in gastric cancer. World J Gastroenterol. 2014;20:3967– 75.
(4) Capuano A, Pivetta E, Sartori G, Bosisio G, Favero A, Cover E, et al.Abrogation of EMILIN1-β1 integrin interaction promotes experimental colitis and colon carcinogenesis.Matrix Biol J Int Soc Matrix Biol. 2019;83:97–115.
(5) Capuano A, Pivetta E, Baldissera F, Bosisio G, Wassermann B, Bucciotti F, et al.Integrin binding site within the gC1q domain orchestrates EMILIN-1-induced lymphangiogenesis. Matrix Biol J Int Soc Matrix Biol. 2019;81:34–49.
(6) Danussi C, Petrucco A, Wassermann B, Pivetta E, Modica TM, Del Bel Belluz L, et al.EMILIN1-α4/α9 integrin interaction inhibits dermal fibroblast and keratinocyte proliferation. JCell Biol. 2011;195:131–45.
(7) Yamamoto M, Nomura S, Hosoi A, Nagaoka K, Iino T, Yasuda T, Saito T, Matsushita H, Uchida E, et al. Established gastric cancer cell lines transplantable into C57BL/6 mice show fibroblast growth factor receptor 4 promotion of tumor growth. Cancer Sci. 2018 May;109(5):1480-1492

Introduction

Pre-neoplastic lesions such as spasmolytic polypeptide-expressing metaplasia (SPEM) are early hallmarks of gastric carcinogenesis, arising in response to epithelial injury, inflammation, and extracellular matrix (ECM) remodeling¹. The ECM glycoprotein EMILIN-1 exerts a protective function in the gastric environment through its gC1q domain, which modulates epithelial–stromal signaling and immune homeostasis, by binding to α4β1/α9β1 integrins^2–4. This domain is specifically targeted and cleaved by neutrophil elastase (NE), resulting in functional inactivation of EMILIN-1⁵.

Aims & Methods

The aim of this study is to investigate the role of EMILIN-1 in preventing gastric pre-neoplastic lesions and to elucidate the biological consequences of its genetic or enzymatic inactivation.
We employed in vivo mouse models including wild-type (WT) mice expressing normal levels of EMILIN-1, EMILIN-1 knock out (KO) mice and transgenic mice carrying a point mutation (E955A) that renders the gC1q domain non-functional. Mice were treated either with Tamoxifen, which induces parietal cell loss, or with DMP-777, a selective NE inhibitor that induces gastric pre-neoplastic lesions and serves as a chemical model to prevent EMILIN-1 inactivation. Histological analyses were conducted to assess the severity and incidence of gastric lesions.

Results

Tamoxifen treatment induced SPEM in all genotypes. Although not statistically significant, lesion severity was greater in KO and E955A mice compared to WT mice, suggesting increased vulnerability in the absence of functional EMILIN-1. Similarly, DMP-777 treatment led to more pronounced pre-neoplastic alterations in EMILIN-1-deficient and mutant mice, whereas WT mice displayed milder changes, confirming the protective function of intact EMILIN-1.

Conclusion

Collectively, these findings demonstrate that EMILIN-1 actively suppresses gastric pre-neoplastic progression and that NE-mediated inactivation of EMILIN-1 is a critical pathogenic event. Pharmacological targeting of this axis with selective inhibitors may represent a novel preventive strategy in early gastric carcinogenesis.

References

1. Capuano A, Vescovo M, Canesi S, Pivetta E, Doliana R, Nadin MG, et al. The extracellular matrix protein EMILIN-1 impacts on the microenvironment by hampering gastric cancer development and progression. Gastric Cancer. 2024;27(5):1016–30.
2. Danussi C, Petrucco A, Wassermann B, Pivetta E, Modica TM, Del Bel Belluz L, et al. EMILIN1-α4/α9 integrin interaction inhibits dermal fibroblast and keratinocyte proliferation. JCell Biol. 3 ottobre 2011;195(1540-8140 (Electronic)):131–45.
3. Capuano A, Pivetta E, Sartori G, Bosisio G, Favero A, Cover E, et al. Abrogation of EMILIN1-β1 integrin interaction promotes experimental colitis and colon carcinogenesis. Matrix Biol. ottobre 2019;83:97–115.
4. Pivetta E, Capuano A, Vescovo M, Scanziani E, Cappelleri A, Rampioni Vinciguerra GL, et al. EMILIN-1 deficiency promotes chronic inflammatory disease through TGFβ signaling alteration and impairment of the gC1q/α4β1 integrin interaction. Matrix Biol. agosto 2022;111:133–52.
5. Maiorani O, Pivetta E, Capuano A, Modica TM, Wassermann B, Bucciotti F, et al. Neutrophil elastase cleavage of the gC1q domain impairs the EMILIN1-alpha4beta1 integrin interaction, cell adhesion and anti-proliferative activity. SciRep. 11 gennaio 2017;7(2045-2322 (Electronic)):39974.

Introduction

Gastric cancer remains a prevalent malignancy, with molecular drivers critical for its progression and therapeutic targeting. Recently, some malignancies have been linked to the lncRNA ZFAS1, but its mechanism of action and the reasons for its dysregulation in gastric cancer are unknown.

Aims & Methods

We evaluated ZFAS1 expression in gastric cancer cells (SGC7901) by RT-qPCR and compared it with GES-1 cells. The LnCAR database provided insight into ZFAS1 levels in STAD compared to normal tissue. The effect of ZFAS1 knockdown on cell invasion, migration and proliferation was evaluated using transwell invasion, wound healing assays, CCK8 and flow cytometry. In addition, ZFAS1 promoter regions were examined using the JASPAR database and subsequent ChIP assays to understand SP1 transcription factor binding. The effect of ZFAS1 on the AKT/mTOR pathway was clarified using Western blotting.

Results

SGC7901 cells were shown to have increased ZFAS1 expression, which was linked to a poor prognosis for gastric cancer. Knockdown of ZFAS1 in SGC7901 cells inhibited cell invasion, migration and proliferation and induced apoptosis. In addition, SP1 was found to upregulate ZFAS1 transcription by binding to its promoter region. ZFAS1 knockdown resulted in a significant reduction of key proteins (p-AKT, AKT, p-mTOR, and mTOR) of the AKT/mTOR pathway. When the AKT activator SC79 was introduced, the repressive effects of ZFAS1 knockdown on cell invasion, migration, proliferation, and AKT/mTOR signaling were partially reversed.

Conclusion

Our results highlight the pivotal role of ZFAS1 in gastric cancer cell malignancy, which is modulated by the AKT/mTOR pathway. The regulatory involvement of SP1 in ZFAS1 transcription provides a novel understanding of the molecular mechanisms driving cancer progression and offers potential therapeutic avenues.

Introduction

Early gastric cancer (EGC) represents a critical stage in preventing and controlling the progression from gastritis to advanced gastric cancer (AGC). Therefore, identifying the single-cell characteristics of EGC, particularly the cellular composition of the tumor microenvironment (TME), as well as identifying potential predictive markers and therapeutic targets, could significantly enhance the monitoring of gastric cancer and improve clinical cure rates.

Aims & Methods

We constructed a comprehensive single-cell RNA sequencing atlas for 184,426 high-quality gastric cancer cells from various stages, utilizing clinical biopsies and surgical samples. The cell composition, differential gene expression, lineage differentiation, intercellular communication, immune infiltration, and clinical correlations between tumor and microenvironment cells were analyzed. Furthermore, through the integration of clinical sample detection, ex vitro and in vivo cell function tests, organoid and endothelial cell co-culture, mouse tumorigenic models, ultrasonic vascular detection, and transcriptome sequencing, key target cell subsets and their molecular roles in EGC were identified.

Results

Our single-cell atlas highlights the cellular and molecular characteristics of EGC. Eight distinct cell lineage states were identified, and it was observed that the number of epithelial cell meta-clusters gradually decreased. In contrast, the number of T&NK, B, plasma, fibroblast, myeloid, and endothelial cells increased with disease progression. Certain epithelial subclusters ((metaplastic stem-like cells (MSCs), metaplastic stem-like cells (PMC-like), proliferating cells), T-cell subclusters (T_reg, CCR7⁺ naive, CH25H⁺ CD4⁺, TEM CD8⁺, and GFPT2⁺ CD8⁺ T cells), and endothelial subclusters (IL-33⁺ Venous-1 and AMAMTSL2⁺ Artery-2) were found to be increased in EGC. The Venous-1 subcluster was found to express high levels of IL-33. Mechanistically, it was revealed that IL-33 enhances the survival and angiogenesis of endothelial cells by upregulating the expression of adhesion proteins CD34 and PECAM1. Patient-derived EGC and AGC organoids were subsequently generated, and it was demonstrated that endothelial-derived IL-33 promoted the growth of both EGC and AGC organoids ex vitro and in vivo. Furthermore, IL-33 was found to increase the expression of KRT17 in EGC organoids. Notably, we also found that high expression of IL-33 was positively correlated with the depth of invasion and malignancy of EGC.

Conclusion

This study provides novel insights into the single-cell components involved in EGC and reveals the role of the IL-33⁺ endothelial subcluster in EGC progression.

Introduction

CKLF-like MARVEL transmembrane domain containing 4 (CMTM4) plays an important role in regulation of the immune function and is involved in tumor formation and progression, but its role in gastric mucosa-related diseases remains unclear.

Aims & Methods

The aim of this study is to explore the role and mechanism of CMTM4 in gastric cancer cells.
Immunohistochemistry (IHC) was used to analyze the expression of CMTM4 in gastric biopsy. The CCK8 cell proliferation assay and plate clone formation were applied to analyze the proliferation of gastric cells. The cell scratch and transwell assay was used to analyze the migration and invasion of gastric cells, respectively. TMT proteome analysis was used to explore the possible mechanism of CMTM4 in gastric cancer cells, and verified by western blot.

Results

CMTM4 expression levels in gastric mucosal biopsy specimens from patients with gastritis and gastric cancer were analyzed by immunohistochemistry, and the results showed that CMTM4 expression was lower in gastric cancer than that in gastritis tissues (P<0.05). To investigate the function of CMTM4 in gastric cancer cell lines, we constructed CMTM4 knockdown and overexpression cells from AGS cell line and CMTM4 overexpression cells from SGC7901 cell line. The CCK8 cell proliferation assay and plate clone formation assay showed that CMTM4 overexpression significantly inhibited the proliferation of gastric cancer cells while knockdown CMTM4 promoted the proliferation of gastric cancer cells. The results of cell scratch assay showed that overexpression of CMTM4 inhibited the migration of gastric cancer cells, and knockout CMTM4 promoted the migration of gastric cancer cells. Transwell assay showed that overexpression of CMTM4 inhibited migration of gastric cancer cells and knockdown of CMTM4 promoted migration of gastric cancer cells, while knockdown of CMTM4 inhibited invasion of gastric cancer cells. In addition, overexpression of CMTM4 increased the apoptosis of gastric cancer cells. The results of TMT proteome showed that STAT1 was significantly up-regulated in gastric cancer cells overexpressing CMTM4, which was also verified by western blot.

Conclusion

CMTM4 has an oncogenic effect in gastric cancer cells. CMTM4 may affect the growth, migration and invasion of gastric cancer cells mainly through STAT1 signaling pathways.

Introduction

Gastric cancer (GC) remains one of the most lethal malignancies worldwide, with limited treatment success largely due to immune evasion and tumor heterogeneity ¹. The advent of immune checkpoint inhibitors has revolutionized the treatment of solid tumors ²; however, only a subset of GC patients benefits from PD-1/PD-L1 blockade, suggesting the presence of intrinsic mechanisms regulating immune escape ³. Leukemia Inhibitory Factor (LIF), a multifunctional interleukin (IL)-6 cytokine family, plays an essential role in epithelial repair, embryonic stem cell pluripotency, and immune regulation ⁴. In several type of tumors, LIF is emerging as a key mediator of tumor progression and immune modulation. LIF signals through a heterodimeric receptor complex composed of LIF-receptor (LIFR) and GP130 and promotes JAK/STAT3 pathway activation ^5,6. Notably, oncogenic STAT3 signaling has been shown to drive PD-L1 expression and promote immune suppression across multiple tumor types, including breast cancer ⁷ and NK/T-cell lymphoma, supporting the broader relevance of this axis in tumor immune escape ⁸. Although LIF is known to activate JAK/STAT3 signaling ⁴, its role in regulating PD-L1 expression and establishing an immunosuppressive microenvironment in GC is not fully defined.

Aims & Methods

We aimed to dissect the role of LIF in promoting PD-L1 expression and immune escape in GC and to explore the therapeutic potential of LIF inhibition. Neoplastic and non-neoplastic gastric mucosa from 61 GC patients were analyzed by transcriptomic profiling, immunofluorescence (IF), and tSNE analysis. Expression levels of LIF, PD-L1, and key signaling molecules were quantified. In vitro mechanistic studies, employed human gastric cancer cells (MKN45). MKN45 were exposed to recombinant LIF, with or without LRI-305, a novel LIF receptor antagonist. Western blotting, RT-qPCR, IF, and chromatin immunoprecipitation (ChIP) assays were used to evaluate downstream pathway activation and transcriptional regulation of PD-L1.

Results

Transcriptome analysis revealed a significant upregulation of LIF and PD-L1 in tumor samples, which was confirmed at the protein level by Immunofluorescence analysis. Elevated LIF expression correlated with reduced overall survival and an immunosuppressive tumor microenvironment characterized by increased infiltration of PD1⁺ T cells, Tregs, and LIF⁺ TAM. Functional assays demonstrated that LIF stimulation activated the JAK1/STAT3 pathway, leading to enhanced PD-L1 transcription. This was supported by increased phosphorylation of JAK1 and STAT3 and ChIP data confirming STAT3 recruitment to the PD-L1 promoter. LIF also promoted cell proliferation and epithelial-mesenchymal transition (EMT), indicated by upregulation of SNAIL1. Importantly, LRI-305 treatment abolished these effects, downregulating PD-L1 expression, reducing EMT markers, and restoring pro-apoptotic responses. These findings confirmed that LIF sustains tumor growth and play a pivotal role in immune resistance through STAT3-mediated PD-L1 upregulation.

Conclusion

Our data identify the LIF/STAT3/PD-L1 axis as a crucial pathway driving immune evasion in GC. LIF promotes a tolerogenic tumor microenvironment, impairs anti-tumor immunity and correlates with aggressive tumor behavior and poor prognosis. Pharmacological blockade of LIF signaling using LRI-305 effectively reverses these effects, offering a novel combinatorial strategy to enhance the efficacy of immune checkpoint inhibitors in GC patients.

References

1. Smyth, E. C. et al. Gastric cancer. Lancet 396, 635–648 (2020).
2. Ribas, A. & Wolchok, J. D. Cancer immunotherapy using checkpoint blockade. Science 359, 1350–1355 (2018).
3. Fuchs, C. S. et al. Safety and Efficacy of Pembrolizumab Monotherapy in Patients With Previously Treated Advanced Gastric and Gastroesophageal Junction Cancer: Phase 2 Clinical KEYNOTE-059 Trial. JAMA Oncol. 4, e180013 (2018).
4. Di Giorgio, C. et al. Next-Generation Sequencing Analysis of Gastric Cancer Identifies the Leukemia Inhibitory Factor Receptor as a Driving Factor in Gastric Cancer Progression and as a Predictor of Poor Prognosis. Front. Oncol. 12, 939969 (2022).
5. Pascual-García, M. et al. LIF regulates CXCL9 in tumor-associated macrophages and prevents CD8(+) T cell tumor-infiltration impairing anti-PD1 therapy. Nat. Commun. 10, 2416 (2019).
6. Yu, S. et al. Tumor-derived LIF promotes chemoresistance via activating tumor-associated macrophages in gastric cancers. Exp. Cell Res. 406, 112734 (2021).
7. Zerdes, I. et al. STAT3 Activity Promotes Programmed-Death Ligand 1 Expression and Suppresses Immune Responses in Breast Cancer. Cancers (Basel). 11, (2019).
8. Song, T. L. et al. Oncogenic activation of the STAT3 pathway drives PD-L1 expression in natural killer/T-cell lymphoma. Blood 132, 1146–1158 (2018).