“THE EFFECT OF GROWTH DIFFERENTIATION FACTOR 11 (GDF11) ON THE RESPONSE OF TUMOR-ASSOCIATED MACROPHAGES IN HEPATOCELLULAR CARCINOMA DERIVED CELLS”

Abstract

Hepatocellular carcinoma (HCC) is the most common and aggressive form of liver cancer. Until 2020, worldwide, it has been ranked 5 th and 3 rd in terms of incidence and mortality, respectively. HCC has various etiologies, but tumors formed by high cholesterol intake induce an aggressive phenotype and high tumor-associated macrophages (TAM) infiltration. TAM acquires an alternative polarity (M2) with immunosuppressive and pro-tumoral activities. HCC patients with high TAM recruitment have a poor prognosis with a low free-survival rate. The design of therapies represents a challenge. The growth differentiation factor 11 (GDF11) has been proposed as a promising molecule for treating HCC, favors a reduction in proliferation, invasion, and lipid metabolism, and therefore reduces the aggressive phenotype. In our research group, we have found that GDF11 also has various effects on cells belonging to the tumor microenvironment, particularly in TAM. To evaluate TAM polarity and their intercommunication with HCC-derived cells during treatment with GDF11. THP-1-macrophages cell line was used; lipopolysaccharides were used for the M1 polarity acquisition and IL-4/13 or conditioned medium (CM) derived from Huh-7 cells for M2 polarity acquisition, flow cytometry (FCM) was used. GDF11 (50 ng/mL) was applied every 24 h up to 3 days. We used anti-Smad2 and 3 antibodies in Western blot (WB). Cell viability and proliferation studies were performed using crystal violet. The quantification of cholesterol was performed using o-Phthaldialdehyde and free radicals’ detection was performed using dihydroethidium. Data were presented as mean ± standard error of the mean (SEM). The analysis of variance (ANOVA) test was used to compare the mean values ​​between groups. Each result has at least 3 independent experiments. Statistical significance was indicated with an asterisk (*P<0.05). GDF11 treatments induced Smad proteins activation by specific phosphorylation, indicating that THP-1 macrophages respond to treatment in times from 5 to 60 min, but maintaining their response up to 72 h. This could suggest that the difference in the subsequent response may be involved in the binding or activation of additional adapter proteins of this signaling pathway. Furthermore, it was observed that GDF11 does not affect the cell viability, proliferation, and morphology studied in their different polarities. Macrophage re-education or re-polarization assays were carried out by adding GDF11 for an additional 72h. The data obtained show that TAM populations lost CD206 marker, going from 90% to 40% of the population, suggesting a loss of this pro-tumor polarity and a re-education towards anti-tumor macrophages responses. However, experiments show a dual role of GDF11 in macrophages due to the increase of specific markers in non-activated cells, indicating that it has a different effect depending on the activation state. Since GDF11 compromises cholesterol metabolism, which is observed in a decrease in total cholesterol levels in non-activated macrophages, which is observed in other studies using HCC cell lines. Also, it was possible to corroborate that GDF11 is behaving like a statin used as a positive control in macrophages and Huh-7 cells. Finally, GDF11 increases ROS levels, specifically superoxide anion, characteristic of phagocytic M1 macrophages. To evaluate response on HCC cells, wound-healing assays indicated that macrophage secretion is a key factor for cell displacement rather than viability and proliferation. In the present study, we found that GDF11 has immunomodulatory effects in TAM, decreasing pro-tumoral markers and lipid content that could be important to design HCC therapies. The authors declare that there is no conflict of interest.