IL‐6/STAT3 pathway intermediates M1/M2 macrophage polarization during the development of hepatocellular carcinoma
Authors: Zi Yin
Ye Lin
Xin Lu
Zhixiang Jian
Sheng Chen
Tingting Ma
Chuanzhao Zhang
Journal: Journal of Cellular Biochemistry (2018)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: chemotherapy resistance; hepatocellular carcinoma (HCC); interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signaling pathway; macrophage; metastasis.
Affiliations: General Surgery Department Guangdong General Hospital, Guangdong Academy of Medical Sciences Guangzhou China, Gynaecology and Obstetrics Department Sun Yat‐sen Memorial Hospital, Sun Yat‐sen University Guangzhou China

Abstract:  Human cancers, including hepatocellular carcinoma (HCC), are characterized by a high degree of drug resistance in chemotherapy. However, the underlying molecular mechanism remains unknown. To the role of interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in the regulation of macrophage polarization, M1-type and M2-type macrophages were separately induced using lipopolysaccharide and interleukin-4 (IL-4), and we found that the IL-6/STAT3 signaling pathway was inhibited in M1-type macrophages but activated in M2-type macrophages. After anti-IL-6-treated macrophages were separately induced by lipopolysaccharide and IL-4, we found that the inhibition of IL-6/STAT3 signaling pathway turned macrophages into M1-type. Co-culture with M1-type macrophages reduced HCC cell viability, proliferation, invasion, migration, drug resistance, but increased apoptosis. Co-culture with M2-type macrophages yielded reciprocal results. The inhibition of IL-6/STAT3 signaling pathway mediated by anti-IL6 was shown to significantly enhance the effects of M1-type macrophages on HCC cells and rescue HCC cells from co-culture with M2-type macrophages. Tumor xenografts of co-cultured HCC cells were established in nude mice and the results showed that the inhibition of IL-6/STAT3 signaling pathway mediated by anti-IL6 was found to reduce tumor formation of HCC cells co-cultured with M1- or M2-type macrophages and lung metastases. The current study reveals a novel mechanism of IL-6/STAT3 signaling pathway in the regulation of macrophage polarization, thus contributing to HCC metastasis and drug resistance in chemotherapy. 

Retracted: Yes

PubPeer comments:
 Fig 6.  The inhibition of ILu20106 STAT3 pathway mediated by antiu2010ILu20106 induces cell apoptosis in HCC cells cou2010cultured with LPSu2010treated macrophages or ILu20104u2010treated macrophages, demonstrated by flow cytometry detection . Areas are duplicated among panels, after a horizontal stretch in the case of the 2nd and 3rd panels. ![file] , 
 Fig 11A. Overlaps marked. ![file] , 
 Thank you very much for your attention and help. After receiving your question, our research group quickly re-investigated all the original data and pictures. All the original data are true and reliable. In the original publication, the resolution and quality are low for the FACS plots and HE staining, which caused some confusing to the readers. After discussion, our research group decided to correct the pictures in the Journal. Image correction is currently in progress. We would like to replace them with high quality images those are the other results in three times repeated experiments. We apologize for this error and we declare that this correction does not affect the description, interpretation, or the original conclusions of the manuscript. , 
 #3 The authors state that readers may be confused, but may they not also be potentially misled by a figure which on further inspection shows multiple overlaps among panels that are described as showing different experimental conditions? ![file] The legend for this figure states that it shows:  The inhibition of ILu20106 STAT3 signaling pathway mediated by antiu2010IL6 was found to reduces lung metastases in nude mice receiving subcutaneous injections of HCC cells cou2010cultured with M1u2010 or M2u2010type macrophages. Can the authors clarify whether this is not the main point of their paper, and hence key to their conclusions? , 
 #3 If the impression of recycled but modified flow-cytometry scatterplots is an illusion, caused by the low resolution of the images, isn t it better to dispel that impression by replacing the images with higher-resolution versions? , 
 Thank you very much for your attention and help. We have also found this problem of mistaken in the correction process. The experimental process of this part is to extract lung tissue for hematoxylin eosin staining, and identify tumor metastasis under the microscope. The number and size of lung metastases in nude mice were calculated. As far as the stained pictures are concerned, we found that due to the reasons of the experimenters, there are some errors caused by the upload process of pictures when taking the various fields of view of the stained pictures. We have sent the original and correct pictures to the magazine for correction. Of course, the correct pictures are the observation under different experimental conditions and drug effects, and are different stained slides. In the verification of molecular mechanism, we have clearly confirmed that after antiu2010ILu20106u2010treated macrophages were separately induced by lipopolysaccharide and ILu20104, we found that the inhibition of ILu20106 STAT3 signaling pathway turned macrophages into M1u2010type. Cou2010culture with M1u2010type macrophages reduced HCC cell viability, proliferation, invasion, migration, drug resistance, but increased apoptosis. Cou2010culture with M2u2010type macrophages yielded reciprocal results. The inhibition of ILu20106 STAT3 signaling pathway mediated by antiu2010IL6 was shown to significantly enhance the effects of M1u2010type macrophages on HCC cells and rescue HCC cells from cou2010culture with M2u2010type macrophages. In vivo animal experiments, tumor xenografts of cou2010cultured HCC cells were established in nude mice and the results showed that the inhibition of ILu20106 STAT3 signaling pathway mediated by antiu2010IL6 was found to reduce tumor formation of HCC cells cou2010cultured with M1u2010 or M2u2010type macrophages and lung metastases. This part of the experiment proved that the role and influence of IL-6 were explained in depth from the animal in vivo experiment. We have clearly confirmed the influence of IL-6 on the size, weight and growth of primary liver cancer in the original paper. Here the staining is to observe the influence on metastasis, which is an additional observation and verification. The reliability of the conclusion is not affected. , 
 As for the problem of low resolution of FACS in the original paper, considering the low resolution of the pictures in the original paper, the details of the pictures are easy to cause confusion and misreading of readers, high resolution pictures from the original three times experiment have been used to correct it. At present, it has been approved by the official submission system according to the requirements of the Journal. Thank you. , 
 We regret to point out there was an error in Fig. 11A, however, it s not that the first, third and fifth pictures you mentioned are repetitive. As shown in the original figure below. After our investigation and verification, only the first and third pictures are partially overlapped, in which the images of HE staining were inadvertently duplicated by using the same slide with different observation fields by mistake when processing and up-loading the pictures. A corrected version of different staining slides from different conditions have been used in progress. Erratum will be completed, thank you. , 
 ![file] The original HE staining section of the first picture (showing the surrounding tissue structure outside the paper picture) ![file] The fifth picture of the original HE staining complete section (showing the surrounding tissue structure outside the paper picture) , 
 #8 I respectfully beg to disagree with the author s explanation. The images speak for themselves, and indeed further inspection revealed other overlaps among panels in this figure (see below). It should be noted that the panels have been re-sized relative to one another in the final figure, which would appear to mitigate against a simple mistake in image assembly. ![file] , 
 #10 Thank you very much for pointing out the potential errors in our pictures. As mentioned before, we found the picture upload errors in the process of reviewing the original data and pictures. In the original experiment, after the staining under different conditions, we carried out a lot of shooting work of different vision of glass slide, which inevitably leads to overlapping of vision shooting of the same glass slide. But in the paper data statistics, that is, the statistical results of metastases are reliable. As you pointed out, we were careless in uploading and placing the pictures. There are some errors caused by the upload process of pictures when taking the various fields of view of the stained pictures. We have sent the original and correct pictures to the magazine for correction. Of course, the correct pictures are the observation under different experimental conditions and drug effects, and are different stained slides. In the final correction, we carefully checked each slide and shooting field of vision, and sent another batch of pictures of the three repeated experiments to the Journal for review and correction. Thank you. , 
 #11 With all due respect, I do not see how  uploading errors can account for a single image giving rise to 2 or more panels at different scales purporting to show different experimental conditions. Your proposed figure changes represent a major change to the presented data, and coupled with the flow cytometry diagram issues, it seems to me that it would be more appropriate for this study to be re-reviewed rather than mega-corrected. , 
 #10 The overlap between panels 1 and 3 (Hepal-6 and M2+Hepal-6) is large and to my mind, irrefutable. Marked here with blue rectangles. Yet the overlap does not extend to the rest of the M2+Hepal-6 panel, and the extended background of Hepal-6 provided in comment #9 - marked with orange boxes. ![file] , 
 I agree with Hoya s comments about unexpected overlaps in Figures 6 and 11. These have nothing to do with low resolution images, since I can clearly see the overlaps in the images. In particular, the similarities in Figure 6 s flow cytometry panels are not caused by a simple whole-panel duplication. Rather, it is a partial duplication in which certain areas appear to be exact copies, while other areas in the same panel are different. Such duplications-with-alterations are very unlikely to have occurred by honest errors. In my professional opinion, the trust in the remaining data in this paper has been lost, not in the least to the shifting explanations of the authors. Happy to discuss my opinions with the editors. , 
 Retracted [15 June 2022] .  The authors asked for a retraction and stated that unintentional errors occurred during the research process. Mistakes were found during figure compilation and duplications for this manuscript and so the conclusions of this manuscript are considered insufficiently supported. The authors apologize for these errors. ,