Lymphocyte Function-Associated Antigen-1 (LFA-1; CD18/CD11a) is one of the main adhesion molecules used by immune cells to infiltrate the liver under inflammatory conditions. Recently, the expression of this integrin has also been reported on several solid tumors, including colorectal cancer. However, its functional role in the metastatic progression to the liver remains unknown. Using in vitro assays and an experimental orthotopic in vivo model of liver metastasis, we aimed to elucidate the role of tumor LFA-1 in the metastatic progression by means of the partial depletion of the β2 subunit of LFA-1, required for integrin activation, firm adhesion and signaling. To do so, we evaluated the effects of β2 reduction on the murine colon carcinoma C26 cell line on their pro-metastatic features in vitro and their metastatic potential in vivo in a mouse model of colon carcinoma metastasis to the liver. The reduction in β2 integrin expression correlated with a slower proliferation, and a reduced adhesion and migration of C26 cells in an in vitro setting. Additionally, tumor cells with a reduced in β2 integrin expression were unable to activate the liver sinusoidal endothelial cells (LSECs). This resulted in a recovery of the cytotoxic potential of liver lymphocytes which is compromised by LSECs activated by C26 cells. This was related to the abrogation of RNA expression of inflammatory and angiogenic cytokines by C26 cells after their activation with sICAM-1, the main ligand of β2αL. Furthermore, in vivo tumor cell retention and metastasis were profoundly reduced, along with a decrease in the recruitment and infiltration of myeloid derived suppressor cells (MDSCs) and lymphocytes to the liver. Taken together, our findings uncovered the modulatory role for the tumor β2 subunit of the LFA-1 integrin in the metastatic progression of colorectal cancer to the liver by impairing activation of liver endothelium and thus, the local immune response in the liver. Besides, this integrin also showed to be critical in vivo for tumor cell retention, cytokine release, leukocyte recruitment and metastasis development. These data support a therapeutical potential of the integrin LFA-1 as a target for the treatment of colorectal liver metastasis.

Background: Activated HSCs play a major role in tissue repair, extracellular matrix regulation, immune response, and inflammation. However, their contributions to the hepatic tumor microenvironment are underexplored and need to be clarified. Methods: In vitro, we analyzed the responses of freshly isolated LSECs and HSCs to tumor cell supernatants and secretome-driven activation of both primary cell types. For in vivo HSC depletion, transgenic mice expressing the herpes simplex virus-thymidine kinase (HSV-Tk) gene driven by the mouse glial fibrillary acidic protein promoter were used. MC38 colon carcinoma or B16 melanoma was intrasplenically injected to generate liver metastasis to further analyze metastatic growth, collagen accumulation, angiogenesis, and immunosuppression. Results: Metastatic tumor cells arrest and adhere in the liver 48 hours after intrasplenic injection. The 65% of arrested tumor cells were surrounded by α-smooth muscle actin–expressing cells. In vitro, tumor-activated LSEC-derived secretomes stimulated α-smooth muscle actin expression, migration, VEGF, and LSEC promigratory factor release by HSCs. Tumor cell secretomes stimulated HSC proliferation and the secretion of proangiogenic and protumoral mediators. HSC depletion reduced the foci number and metastatic area in colorectal cancer and melanoma models. Moreover, livers from transgenic mice showed reduced key tumor microenvironment parameters, including intratumoral collagen accumulation, neoangiogenesis, and recruitment of myeloid-derived suppressor cells. Conclusions: Depletion of tumor-reactive proliferating HSCs implicates these cells as the required spark for the initiation and progression of liver metastasis, making them a good candidate for new therapies targeting the tumor microenvironment to treat liver metastasis of different primary origins. Export

Primary and secondary liver cancer are the third cause of death in the world, and as the incidence is increasing, liver cancer represents a global health burden. Current treatment strategies are insufficient to permanently cure patients from this devastating disease, and therefore other approaches are under investigation. The importance of cancer-associated fibroblasts (CAFs) in the tumour microenvironment is evident, and many pre-clinical studies have shown increased tumour aggressiveness in the presence of CAFs. However, it remains unclear how hepatic stellate cells are triggered by the tumour to become CAFs and how the recently described CAF subtypes originate and orchestrate pro-tumoural effects. Specialized in vitro systems will be needed to address these questions. In this review, we present the currently used in vitro models to study CAFs in primary and secondary liver cancer and highlight the trend from using oversimplified 2D culture systems to more complex 3D models. Relatively few studies report on the impact of cancer (sub)types on CAFs and the tumour microenvironment, and most studies investigated the impact of secreted factors due to the nature of the models.