Clinical characteristics of RBCs associates with the progression of HCC tumorigenesis
To explore the relationship between the tumorigenesis of HCC and RBCs in patients, we collected the clinicopathologic characteristics of LC (N=17), HCC (N=30) and healthy controls (HC, N=25) (Table S1). The tested ALT, AST and AFP level in peripheral blood are extremely high in HCC comparing to LC and HC, indicating the functional damages in liver with the onset of HCC. The AFP level in HCC fluctuates extremely largely (Fig. 1A), and over 50% of HCC patients are included in the normal range, which could result in the missed detection of HCC cases. The AFP level in LC is similar to HC, indicating that AFP could not be used as a biomarker for early diagnosis of HCC. The level of hemoglobin (Fig. 1B) and RBCs (Fig. 1C) in peripheral blood are significantly lower in LC than HC, suggesting that RBCs start to be influenced with the occurance of LC. Interestingly, this disturbance is gradually recovering in established HCC, approaching to HC, indicating the association of molecular characteristics in RBCs with the process of HCC tumorgenesis. Tumor-educated blood platelets were previously characterized to distinguish six types of cancers from healthy controls using mRNA profiling besides HCC . We first observed that platelets are also associated with HCC tumorogenesis, since the number of platelets in peripherial blood changes with the similar trend as RBCs (Fig.1D). Leukocyte changes slightly during the tumorogenesis of HCC. Taken together, our results demonstrate that the molecular characteristics of RBCs during tumorigenesis could reflect the progression of HCC and provide a novel strategy for early diagnosis.
LC is a crucial stage developing into HCC
RBCs could clinically indicate the progression of HCC, however, the molecular characteristics in RBCs through the whole process of HCC tumorigenesisis has been largely unknown. Here we utilized DIA mass spectrometry to comprehensively analyze the protein profiles in RBCs from the cohort including HCC (N=30), LC patients (N=17) and HC (N=25) (Table 1). 659 proteins were identified for characterization (Table S2), of which most proteins are present in vesicles and cytosol of RBCs (Fig. 2A), and fulfill a varity of cellular responses and metabolism related functions (Fig 2. B). The hierarchical clustering analysis showed that protein expression profiles of RBCs in LC and HCC patients are quite different from HC, and LC is closer to HCC (Fig. 2C), which was also revealed by PCA analysis (Fig. 2D). In terms of proteome in RBCs, we demonstrated that the pathology of LC is a developmental stage towards HCC, and LC is a crucial stage developing into HCC. The dynamic changes of proteins in LC could facilitate the early diagnosis of HCC.
Erythroid-specific proteins could indicate the pathological process of HCC
The clinical characteristics of RBCs and hemoglobin in peripheral blood from the cohort flunctuates through the tumoregeneis of HCC. We next wonder the relationship between the expression profiles of erythroid-specific proteins and the progression of HCC, which might suggest the biomarkers for early diagnosis of HCC. Hemoglobin is the most abundant protein in RBCs, combines and transports oxygen to organs in the body through its tetrameric structure. Two alpha chains together with two gamma chains constitute fetal hemoglobin (HbF: a2γ2) which is normally replaced by adult hemoglobin (HbA: a2β2) at birth. We observed that the components of hemoglobin, including HBA1, HBE1, HBG2 an HBB, showed differentiated expression during the tumorigenesis of HCC (Fig 3. A-D). Specifically, HBA1 expression increases significantly at the stage of LC, while HBB expression decreases at the same stage. HBG2, that is predominant fetal globin at birth, remains similar expression level as HC, but increases the expression at HCC. As an embryonic globin, HBE1 expression increases at both the stage of LC and HCC.
PRM is a targeted proteomics technology based on high-resolution, high-precision mass spectrometry, which can selectively detect target proteins and target peptides to achieve absolute quantification of the target protein/peptide. In this study, we verified the expression of differentially expressed globins in another cohort of LC, HCC and healthy controls with RPM technology (Table S3-4), and observed that the expression pattern of HBA1 and HBE1 is similar with the expression pattern in proteomic data (Fig. E, F).
Consistently, we observed decreased expression of GYPA, a specific marker of mature erythrocytes, in RBCs of HCC (Fig. 3G), and the nucleated erythroid cells were observed in the peripheral blood of HCC patients (Fig. 3H). These results indicate that the immature erythroid cells could exist in the peripheral blood of HCC. To test this hypothesis, we counted the erythroid cells respectively expressing HbF and HbA in peripheral blood from another cohort of HCC, LC and HC by flow cytometry analysis. Interestingly, we observed that the number of erythroid cells expressing HbF is significantly higher in LC patients than HC (Fig 3. I), indicating that the production of erythroid cells is initially affected at the stage of LC, and the immature erythroid cells could be released into peripheral blood at this stage. Consistently, we observed the lowest number of erythroid cells expressing HbA consisting of two alpha chains together with two beta chains at the stage of LC comparing with HC and HCC (Fig. 3J), even though no statistically significant difference was observed among them. Together, our results demonstrated that the expression changes of erythroid-specific proteins in the process of HCC tumorigenesis could indicate the pathological process. The increase of HbF and the decrease of HbA in LC could be used for early diagnosis for HCC.
Impairments in RBCs on the onset of liver cirrohosis
We next explored the disturbance to RBCs with the occurance of LC, which might be a sign of the development into HCC. A total of 157 DEPs (79 up-regulated, 78 down-regulated) were identified in RBCs with the onset of LC compared with HC (Table S5, Fig4. A, B). The altered proteins are much more than the process from LC to HCC (57 DEPs), indicating that the occurance of LC leads to more drastic changes in RBCs durng the tumorigenesis of HCC.
Autophagy is a major player in LC and considered as an anti-fibrosis pathway, because it provides survival signals for hepatocytes and acts as the gate keeper of HCC . In this study, we also observed the significantly disturbed autophagy pathway with the onset of LC in RBCs. The impairment of m-TOR and tight junction pathway are firstly characterized in LC (Fig4. C).
Interestingly, we observed two proteins, SMIM1and ANXA7, that were associated with the cellular characteristics red blood cells [20-22] and the tumorigenesis of hematocellular carcinoma and functions of erythroid cells, respectively [23-26]. Since these two proteins are significantly changed proteins in RBCs on the occurance of LC (Fig. 4A-B), we speculate that they could be potential biomarkers for early diagnosis of HCC. These two proteins were further verified with clinical samples by RPM technology in this study. In summary, these identified disturbed proteins or pathways in RBCs with the occurance of LC could facilitate the early diagnosis of HCC.
RBCs molecular characteristics changes from LC to HCC during tumorigenesis
Liver cirrhosis is a developmental stage that develops into HCC, during which the the maturation of RBCs in peripheral blood are influenced. We next explored what happened to RBCs during LC-HCC transition by analyzing the differentially expressed proeins (DEPs) between LC and HCC patients. We identified 57 DEPs (26 up-regulated, 31 down-regulated) in HCC compared to LC （Table S6, Fig. 5 A）, and these disturbed DEGs were also enriched in autophagy pathway (Fig. 5B) that is necessary for the suppression of spontaneous tumorigenesis through a cell-intrinsic mechanism, and the impairment of autophagy initiates spontaneous liver tumorigenesis in aged mice [27, 28]. This finding suggested the disruption of the gate keeper of HCC during this process. The estrogen pathway (Fig. 5B), another HCC associated pathway, was also disturbed during this process . However, the disturbed oxytocin and GnRH pathways were first identified during this progress.
During this transition, we observed SMIM1 is down-regulated with the onset of LC and gradually increases towards normal level at HCC (Fig. C), and ANXA7 gradually increases during the whole process of HCC tumorigenesis (Fig. 5D). PRM assay confirmed the dynamic changes of SMIM1 and ANXA7 in RBCs during the whole process of HCC tumorigenesis with clinical samples (Fig. 4E, F; Table S3-4). SMIM1 dramatically decreases in LC and gradually increases in HCC, while ANXA7 continuously increases from HC to HCC, both of which could indicate the tumorigenesis of HCC. The dramatic decrease of SMIM1 expression in RBCs from LC patietns could act as early diagnosis biomarker for HCC. Moreover, we selected SMIM1 to be tested with HCC tissue. The current result showed that it is highly expressed in HCC tissue but not in precancerous lesions (Fig.5G), suggesting it is probably associated with the production of erythroid cells or progression of HCC. The underlying mechanism needs to be further explored in the future.
A few pathways involving erythropoiesis are affected between HCC and HC
By comparing with HC, we next wonder the alterations in RBCs that could be caused by HCC tumorigenesis. The DEGs between HC and HCC can clearly distinguish these the two stages (Fig. 6A). The disturbance of oxygen transport, folate metabolic pathway, HIF-1 pathway and glycolysis pathway in RBCs of HCC patients that are closely related to erythroid differentiation [30-33], suggesting erythropoiesis is abnormal in established HCC (Fig. 6B, Table S7). Interestingly, the disturbed mTOR pathway, that is first identified in LC in this study, was also identified in established HCC (Fig. 6B). A few cancer-related pathways are also altered in HCC, including autophagy, cell death, protein degradation, proteolysis, response to tumor necrosis, and a variety of metabolic pathways that are enriched by the down-regulated proteins (Fig. 6C), demonstrating that cancer-associated dysfunctions can also be revealed in RBCs in established HCC.