Introduction

Lymphoplasmacytic-rich and neutrophilic-rich tumour immune microenvironment (TIM) have been described in Epstein-Barr-virus-associated (EBV+) and microsatellite-unstable (MSI-high) gastric cancer (GC), respectively. Aim: to explore associations between EBV+/MSI-high status, TIM, peripheral blood cell ratios, tumour serum biomarkers and prognostic outcomes in GC.

Aims & Methods

Lymphoplasmacytic-rich and neutrophilic-rich tumour immune microenvironment (TIM) have been described in Epstein-Barr-virus-associated (EBV+) and microsatellite-unstable (MSI-high) gastric cancer (GC), respectively. Aim: to explore associations between EBV+/MSI-high status, TIM, peripheral blood cell ratios, tumour serum biomarkers and prognostic outcomes in GC.

Results

The series encompassed 7/140 EBV+ (5.0%) and 38/140 MSI-high (27.1%) GCs, characterised by lymphoplasmacytic-rich (EBV+:71.4% vs EBV-:5.3%, p<0.001) and neutrophilic-rich (MSI-high:28.9% vs MSS:2.0%, p<0.001) TMI, respectively. Prior to surgery, patients who underwent NAC harboured lower NLR compared to those submitted to surgery alone (3.05 vs 2.53, p=0.021). Pre-operative NLR and post-operative CRP levels were lower in neutrophilic-rich TMI GC (p=0.015 and p=0.006, respectively). Serum tumour biomarkers (CA125, CA19.9, CEA) were not related to morphological features or molecular subgroups. Overall, GC patients with lower CRP after surgery (<1.1mg/dl) showed better overall survival (p=0.032), even when stratified by type of therapy (NAC versus surgery alone), MSI status and presence of neutrophilic-rich TMI.

Conclusion

This study contributes to the growing body of evidence of blood-based biomarkers as predictive markers of GC prognosis. A reduction of systemic pro-inflammatory status after surgery (e.g. CRP<1.1md/dl) might contribute to better overall prognostic outcomes of GC patients. Moreover, we observed that GC patients with neutrophilic-rich TMI, a morphologic feature associated with MSI-high status, had lower levels of neutrophils in peripheral blood (low pre-operative NLR). Possible explanations include neutrophil sequestration within the tumour microenvironment and/or consumption by the tumour.

Introduction

Germline truncating variants in the CTNNA1 gene, encoding ⍺E-catenin, account for approximately 2% of hereditary diffuse gastric cancer (HDGC) cases. Signet-ring-cell (SRC) / poorly-cohesive (diffuse) gastric cancer (GC) with loss of ⍺E-catenin expression has been reported on a case-by-case basis in carriers of germline CTNNA1 variants. However, there is a knowledge gap regarding the detailed histopathological and immunohistochemical features of CTNNA1-associated GCs.

Aims & Methods

Eleven tumour samples, including six primary GCs and five (peritoneal and nodal) metastases, from nine CTNNA1 truncating carriers fulfilling the 2020 HDGC genetic testing criteria [doi:10.1016/S1470-2045(20)30219-9] were studied. Immunohistochemistry for ⍺E-catenin (clone EP1793Y) was performed on eight samples from seven patients.

Results

Median age at GC diagnosis was 48 years (range:37–75). One case was detected at early stage (pT1a) in a prophylactic total gastrectomy, while the remaining eight were diagnosed at advanced stage (≥pT2), including five with metastatic disease.
The pT1a carcinoma, adjacent to pagetoid spread of SRCs (pTis), showed classic SRCs. Advanced-stage GCs showed predominantly poorly-cohesive morphology with variable SRC component: <5% in five cases, 5–10% in two, and 10–50% in three. In two patients, the SRC proportion increased from 10% in primary tumours to 50% in corresponding metastases. All metastatic sites showed purely poorly-cohesive morphology with prominent desmoplastic stroma. In contrast, primary tumours displayed additional patterns, including nested, trabecular, rosetoid and true microglandular structures.
All tumours demonstrated aberrant ⍺E-catenin expression: complete loss in 63% (5/8) of samples analysed, and faint/incomplete membranous, paranuclear dotted, and/or cytoplasmic staining in the remaining 37% (3/8).

Conclusion

This is the largest cohort to date characterizing histopathological features and ⍺E-catenin expression in CTNNA1-associated GCs. The morphological diversity observed in primary tumours may represent a diagnostic pitfall in the recognition of HDGC cases. The possible utility of ⍺E-catenin expression as a marker for genetic triage in these cases warrants further investigation.

References

1. van der Sluis L, van Dieren JM, van der Post RS, Bisseling TM. Current advances and challenges in Managing Hereditary Diffuse Gastric Cancer (HDGC): a narrative review. Hered Cancer Clin Pract. 2024 Oct 8;22(1):21. doi: 10.1186/s13053-024-00293-5. PMID: 39379994
2. Lobo S, Benusiglio PR, Coulet F, Boussemart L, Golmard L, Spier I, Hüneburg R, Aretz S, Colas C, Oliveira C. Cancer predisposition and germline CTNNA1 variants. Eur J Med Genet. 2021 Oct;64(10):104316. doi: 10.1016/j.ejmg.2021.104316
3. Coudert M, Drouet Y, Delhomelle H, Svrcek M, Benusiglio PR, Coulet F, Clark DF, Katona BW, van Hest LP, van der Kolk LE, Cats A, van Dieren JM, Nehoray B, Slavin T, Spier I, Hüneburg R, Lobo S, Oliveira C, Boussemart L, Masson L, Chiesa J, Schwartz M, Buecher B, Golmard L, Bouvier AM, Bonadona V, Stoppa-Lyonnet D, Lasset C, Colas C. First estimates of diffuse gastric cancer risks for carriers of CTNNA1 germline pathogenic variants. J Med Genet. 2022 Dec;59(12):1189-1195. doi: 10.1136/jmg-2022-108740. Epub 2022 Aug 29. PMID: 36038258.
4. Gullo I, van der Post RS, Carneiro F. Recent advances in the pathology of heritable gastric cancer syndromes. Histopathology. 2021 Jan;78(1):125-147. doi: 10.1111/his.14228. PMID: 33382491.

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

Gastric cancer (GC) remains a challenging disease mainly due to its heterogeneity and complex tumour microenvironment[1]. GC has a 5-year survival rate of less than 40%, however, advanced GC has a much lower survival rate of around 5%. Standard treatment for resectable gastric cancer consists of surgical resection in combination with perioperative chemotherapy. Based on the FLOT4 trial, perioperative fluorouracil, leucovorin, oxaliplatin, and docetaxel (FLOT) chemotherapy improved overall survival in locally advanced, resectable esophagogastric adenocarcinoma compared to perioperative ECF/ECX[2]. Despite advances, around 50% of patients do not respond to any of these treatments, and the majority will have recurrence and treatment resistance. By suppressing the immune system and remodelling the extracellular matrix, cancer-associated fibroblasts (CAFs) have been found to promote drug resistance in several tumor types, including GC[3]. Given their high plasticity, the exact role of CAFs in tumor development and treatment resistance in GC remains unknown.

Aims & Methods

This project aims to establish mini-tumour (MT) models that accurately reflect the characteristics of gastric tumours, facilitating the evaluation of the role of stroma upon gastric cancer therapy. The study focuses on two key objectives: (1) evaluating whether CAF subsets in gastric MTs resemble those in parental tumour tissues, ensuring a reflective model for personalized treatment strategies, and (2) assess drug responses in MTs, identifying the most effective therapies, and evaluating CAF-tumor interactions in treatment resistance.
First, we isolate and culture patient-derived tumor organoids and CAFs from gastric cancer resected material, which forms our patient GC biobank. Then, we establish MTs consisting of co-cultured matched patient-derived organoids and CAFs. MT establishment includes mimicking parental tumor tissue tumour-stroma ratio, hotspot mutation data, and specific culture conditions. Once established, treatment assays are designed to study chemotherapy responses (single-drug and combination drugs), as well as CAF subset changes. Untreated and treated MTs are subsequently fixed and stained with cell proliferation, death, and tumor/stroma markers.

Results

Up to date, a total of n=3 MT models have been established, which recapitulate the primary tumour, based on mutation status (e.g. HER2, KRAS, CTNNB1, TP53) , tumor/stroma structures and CAF enrichment. Upon treatment, majority of chemotherapeutics used as single drug showed to be non-effective, and only a few combinations seemed to be clinically relevant (≤ 50% cell viability). Preliminary data shows that CAF sensitivity to chemotherapy differs between patients, particularly between treatment naïve and FLOT-treated, and seems to resemble patient responses.

Conclusion

Overall, MT models display patient-specific features, showcasing interpatient heterogeneity, and highlighting the need for tailored models to optimally evaluate therapy responses. Future experiments will evaluate intrinsic resistance mechanisms potentially associated with specific CAF subsets. To further look into these, spatial profiling of gastric MTs and primary tumours will be evaluated using a stromal 42-marker panel by Imaging Mass Cytometry. To evaluate the immune cell compartment in these MTs, incorporating cytomegalovirus (CMV)-stimulated T cells or patient-derived tumor infiltrating lymphocytes is planned.

References

1. Lei ZN, Teng QX, Tian Q, Chen W, Xie Y, Wu K, Zeng Q, Zeng L, Pan Y, Chen ZS, He Y. Signaling pathways and therapeutic interventions in gastric cancer. Signal Transduct Target Ther. 2022 Oct 8;7(1):358. doi: 10.1038/s41392-022-01190-w.
2. Möhring C, Mańczak A, Timotheou A, et al. Perioperative therapy with FLOT4 significantly increases survival in patients with gastroesophageal and gastric cancer in a large real-world cohort. Int J Cancer. 2023; 153(3): 609-622. doi:10.1002/ijc.34511
3. Ozmen, E., Demir, T. D., & Ozcan, G. (2024). Cancer-associated fibroblasts: protagonists of the tumor microenvironment in gastric cancer. Frontiers in Molecular Biosciences, 11. https://doi.org/10.3389/fmolb.2024.1340124

Disclosure

No conflict of interest.

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.