Recruitment Of Tumor Associated Macrophages (TAMs) By Sorafenib Contributes To Proliferative And Prometastatic Effects In Hepatocellular Carcinoma

Objectives Although Sorafenib was the first molecularly targeted agent approved for treating advanced HCC, it does not show the same effect in all HCC patients, and the response rate in HCC was modest. Antiangiogenic therapy with effects in malignant progression of tumors has been investigated in previous studies, in which increasing inflammation response may involve based on increasing evidence that increased systemic inflammation correlates with poorer cancer-specific survival. In recent years,molecular targeted therapies have demonstrated a survival benefit. In HCC, our recent studies showed that Sorafenib induced a significant increase in peripheral recruitment and intratumoral infiltration of F4/80- and CD11b-positive cells, which was accompanied with elevation of related chemokine in tumor and peripheral blood,promoting tumor progression, tumor angiogenesis, and lung metastasis compared with mice treated with Sorafenib alone, suggesting the role of macrophages in tumor progression under Sorafenib treatment. Furthermore, Sorafenib promotes invasiveness and the metastatic potential of orthotopic tumors from HCC cells by down-regulating expression of HTATIP2 via JAK-STAT3 signaling in mice. As tumor metastasis can be affected by tumor microenvironment, the present study was undertaken to determine whether the growth and metastasis of HCC were influenced receiving Sorafenib prior to implantation with tumors and demonstrated that the off-target effects of Sorafenib on host immunity. Moreover, we investigated the underlying mechanism and explored clinical available approach to improve the effect of Sorafenib in HCC.Methods1. The concentration of Sorafenib used in in vitro studies is 1–10 μmol/L. The effects of different concentrations of Sorafenib on the RAW264.7 viability and proliferation in experiment using MTT.2. RAW264.7 was cultured into two distinct states of polarized macrophage activation: the classically activated(M1) and the alternatively activated(M2) macrophage phenotypes. The polarized status of macrophage were determined by detecting expression of relevant cytokines in RT-PCR.3. RAW264.7 cultures will be treated with low dose Sorafenib(0-5umol/L). The cytokine levels secreted from Sorafenib treated macrophage were detected by ELISA and their m RNA levels will be measured by real time RT-PCR.4. The proliferation, migration and invasion ability of HCC cells(MHCC-97L)were explored by co-cultured with Sorafenib treated macrophages and related culture supernatant in experiments Wound Scratch Assay using and Transwell Assay.5. In cases of effects of Sorafenib on TAMs, the intracellular signalling pathways were detected by high-throughput genetic sequencing and further determined in experiments using real-time RT-PCR, Western blotting, and pharmacological blockers.Results1. The low dose of Sorafenib had little effects on viability and proliferation in experiment using MTT.2. LPS treated polarized macrophage activation-the classically activated(M1)macrophage and IL-4 treated polarized macrophage activation-the alternatively activated(M2) macrophage phenotypes was been confirmed by detecting expression of relevant cytokines in RT-PCR.3. Sorafenib promote macrophage polarize into M2 subtype by etecting expression of relevant cytokines in ELISA and their m RNA levels will be measured by real time RT-PCR.4. Sorafenib treated macrophages enhanced the proliferation, migration and invasion ability of HCC cells(MHCC-97L) in experiments Wound Scratch Assay using and Transwell Assay.5. Sorafenib regular the polarization of macrophage by activating the NF-KB signaling pathway by confirmation of western blot thechnology.6. The inhibitor of NF-k B signaling could reverse the suppression of M2macropahge and enhancement of M1 macrophage.Conclusion1. The low dose of Sorafenib had little effects on viability and proliferation in experiment using MTT.2. The expression of inflammation relevant cytokines of M1 macrophage and M2 macrophage phenotypes was totally different.3. Sorafenib promote macrophage polarize into M2 subtype.4. Sorafenib treated macrophages enhanced the proliferation, migration and invasion ability of HCC cells.5. Sorafenib regular the polarization of macrophage by activating the NF-KB signaling pathway, the inhibitor of which could reverse the suppression of M2 macropahge and enhancement of M1 macrophage.