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levels in human gastric malignant and pre-neoplastic samples and this correlates with the presence of abnormal LVs (unpublished preliminary data).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
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(2) Rojas A, Araya P, Gonzalez I, Morales E. Gastric Tumor Microenvironment.Adv Exp Med Biol. 2020;1226:23–35.8
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(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
