The efforts on the researches about postoperative adjuvant therapy of stage Ⅱ CRC have been decades. However, the guideline remains unclear, the controversies remain exist. “Who”, “how”, and “duration time” are always the interest topics, and Intergroup 0089,21 GENCORE tria,22, 23 QUASAR,24 IDEA Collaboration,25 and the researches from Chau et al,26 Kumar et al,27 Tsai et al28 are the representative of large samples. Undoubtedly, “who” is the foundation of “how” and “duration time”, so we have been committed to research at the source.
It is important to note that this study does not contradict - but rather extends our previous study. The 5-year survival rate for stage II CRC is about 80%, and the improvement in survival following adjuvant chemotherapy is less than 5%.29 Facing such a relatively narrow window of benefit for adjuvant therapy and combined its well-recognized heterogeneous disease. The more accurate of the model for the prediction of the recurrence is, the better guide the clinical practice is. In this study, macrophage infiltration related-GRM (AUC = 0.906) provide a better performance for the prediction of the recurrence than previous GRM (AUC = 0.882). Which put the goal of further improving the diagnostic efficacy of the GRM of stage II CRC into practice. In addition, high risk patients with recurrence should receive targeted surveillance and more active adjuvant therapy.
As we all known, iron is necessary to maintain cell growth and proliferation. The faster the cells grow and proliferate, such as tumor cells, the greater the demand for iron. transferrin is an important chelator with a primary function of iron transportation. TF, transferrin receptor (TFR), and Fe3 + form the TF-TRF-Fe3 + complex on the cell surface, then Fe3 + is transported into the cell by endocytosis, subsequently, Fe3 + is reduced to Fe2 + to perform biological functions. TF-TRF complex return to the cell surface through exocytosis. Continue to form the TF-TRF-Fe3 + complex. The cycle then repeats itself. Higher transferrin saturation was associated with increased cancer risk (HR 1.68 (95% CI: 1.18, 2.38; P < 0.01)) and increased risk of cancer death (HR: 0.65; 95% CI: 0.42, 0.99; P < 0.05).30 Based on the biological properties of intracellular transportation and tumor selectivity, TF, TFR andTF-TRF-Fe3 + complex has been widely developed as active-target drug delivery platforms for cancer therapy.31, 32 Simultaneous assay of feces hemoglobin (Hb) and TF showed high accuracy and effectiveness for colorectal cancer screening, which with a sensitivity and specificity of 68.2% and 92.6%, respectively.33 Previously, we reported TF is a hub gene which play an important role in promoting the recurrence of stage II CRC. In this study, we further validated the macrophage infiltration level was significant negative correlated with the recurrence-related hub gene TF. Which indicated macrophage may defend against the recurrence of stage II CRC by targeting recurrence-related hub gene TF.
As macrophage is one of the most abundant cells in the TME.34 And the infiltrated macrophage was associated with poor clinical outcome in numerous solid tumors, including bladder,35 breast,36 renal,37 prostate,38 gastric,39 glioma, endometrial and cervical cancers.40 Hence, significant attention has been drawn towards development of cancer immunotherapies targeting macrophage. However, macrophage reported plays inconsistent even contradictory roles in CRC.41, 42 In this study, still focus on stage Ⅱ CRC, we found the macrophage infiltration level in the nonrecurrence group was significantly higher than that in the recurrence group, the macrophage infiltration level was significantly negative correlated with the recurrence-related hub gene TF and was significantly positive correlated with the improved DFS and OS. Which means macrophage infiltrated in the TME could defends against recurrence and then improves the prognosis of stage II CRC.
Macrophage is one of the earlier and more amount infiltrated immune cells in the TME of CRC,11 and exhibit different phenotypes and functions in response to various microenvironmental signals which generated from tumor and stromal cells.43 According to their activation, macrophages are divided into three subtypes (M0, M1 and M2). M0 is the unactivated subtype and without inflammatory or tumor-related function. Depending on the different activation pathways, M0 can differentiate into two activated subtypes, M1 and M2, which exhibit pro-tumor or anti-tumor roles respectively. As the evidence indicated that the infiltrated macrophage could recruited by tumor cell through several different mechanisms.44, 45 During inflammation, cancer and other pathological processes, macrophage is among the first responders, recognizing pathogen-associated patterns (PAMPs) like LPS or IFN- γ.46 LPS engages the Toll-like receptor 4 (TLR-4) on the surface of macrophage to activate transcription factors e.g. interferon regulatory factors (IRFs) and nuclear factor kappaB (NF-κB), then activates the inflammatory response and also participates in the host innate immunity.45, 46, 47 Releasing a variety of cytokines, including interleukin (IL)-1β, IL-6, IL-12, IL-16, tumor necrosis factor-a (TNF-a),48 nitric oxide,49 interleukin-1 h,50, 51 and reactive oxygen intermediates,52, 53 facilitating the local recruitment of more macrophage and leukocyte infiltrate to fight against pathogenic insult.54 Finally, killing the tumor in the TME.55
According to the analysis results from GSEA, we speculate the macrophage defends against recurrence maybe a dynamic spatiotemporal process as Chen and Mellman56 proposed cancer-immunity cycle (CIC) [Figure 6]: 1. Tumor cells release (LPS (GSE14000, GSE14769) or IFN- γ (GSE1925)) antigens. 2. Macrophages (M1) receive the stimulus signals (LPS or IFN- γ) and activation. 3. Trafficking of macrophages (M1) to tumor. 4. Macrophages (M1) infiltrate into tumor. 5. Macrophages (M1) recognize tumor then secrete IL-6 (GSE411) to directly or indirectly act on TF or create anti-tumor TME to kill tumor cells. Finally, inhibit the recurrence of stage II CRC. In additional, according to the result of GSE411 (PMID: 12754506),57 suppressor of cytokine signaling (SOCS)protein is feedback inhibitor of the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) signaling pathway.58 When Socs3-deficient, IL-6 induces a wider transcriptional response in Socs3-deficient macrophage than in wild-type cell. Thus, SOCS3 functions to control the quality of the response to IL-6. In our study, the nonrecurrence group was significantly enriched in this up-regulated gene set, which means regulation of IL-6 during the suppression of the recurrence of the stage II CRC was of high quality.
However, in fact, TME is so complex which with multiple cells interacting and numerous mechanisms intersecting, that it is difficult to explain any biological process such as the recurrence of stage II CRC from a single perspective. On the one hand, the macrophages(M1) shapes the anti-tumor microenvironment by providing immunosuppressive effect through a myriad of mechanisms, However, on the other hand, to gain fitness advantages, cancer cells can actively engage in constructing ecological niches by escaping the killing of macrophages (M1) through circumvent these inhibitory signals. These macrophage (M1)-tumor-microenvironment interactions can have significant implications for the recurrence of stage II CRC. Unfortunately, there is no significant immunologic signatures gene set enriched in the recurrence group, to some extent, its limited our comprehensive exploration of the possible mechanisms for the recurrence of stage II CRC.
However, our current study has the following limitations: 1. Our study is the retrospective analysis from TCGA database, therefore, the evidence level is imperfect. 2. Our study was based on mRNA evaluations from the TCGA database, therefore, it is not as persuasive as the level of protein expression. Due to the restrictions on the type of data run by TIMER, this immune risk score model could not be verified in GEO database. And we have not use clinical samples to further verify its authenticity. 3. As the clinical information of TCGA database was partial absence. It was not possible to implement a comparison of predictive effectiveness between our macrophage infiltration related-GRM and high-risk factors defined by the NCCN guidelines. While intriguing and hypothesis generating, our study requires further validation with a prospective, multicenter, randomized controlled clinical trial. As not only the quantity, but also the high ratio of macrophages to tumor cells and the position especially the tumor front positively influences the prognosis in CRC.59 These are all what we need to pay attention to in order to ensure the accuracy of the research in the future. In addition, at present, immunotherapy is mostly used in patients with advanced CRC. However, the clinical value of immune cell infiltration in the recurrence and prognosis of early stage CRC has been increasingly discovered.9, 17 Which providing a novel immunological perspective to improve clinical therapy performance and broadening the treatment thoughts of the early stage CRC, as well as assist clinicians in selecting targets for immunotherapies and individualize treatment strategies for these early patients. We wish this study will provide a novel immunological perspective to explore the mechanism of recurrence and improve clinical therapy practice in stage II CRC.