Re-evaluation of the relationship between PrPc expression and patient prognosis in primary esophageal squamous cell carcinoma and primary hepatocellular carcinoma
PrPc is a GPI-anchored glycoprotein encoded by PRNP and exists in multiple cleavage forms – . PrPc is believed to be associated with neurodevelopment and can lead to bovine spongiform encephalopathy (BSE) if misfolded – . PrPc has also been found to be highly expressed in various cancers and linked to cancer development, including gastric cancer – , breast cancer , , pancreatic cancer – , colorectal cancer , , lung cancer , glioma . However, there have been conflicting reports regarding the relationship between PrPc expression levels in gastric cancer tissues and patient prognosis. Zhou et al. used the anti-PrPc antibody 3F4 to study a microarray of gastric cancer tissues from 238 patients and found that high PrPc expression was associated with poorer prognosis compared to low PrPc expression . In contrast to Zhou et al., Tang et al. found that negative PrPc expression, as detected by the anti-PrPc antibody 8H4, was associated with poor overall survival . 3F4 and 8H4 are two of the most commonly used anti-PrPc antibodies in related studies. Tang et al. suggested that differences between the 3F4 and 8H4 antibodies might account for the conflicting results. Our team previously reported that anti-PrPc antibodies inhibited tumor initiation and liver metastasis of PrPc-positive human colorectal cancer (CRC) cells in mouse model experiments . In a recent study, we cloned a new mouse anti-PrPc antibody (Clone 6) that can inhibit the migration of colorectal cancer stem cells (CCSC) derived from colorectal cancer patients, as well as the growth of CCSCs in nude mice. We further affinity-matured Clone 6, resulting in a new clone named 4AA . The affinity of 4AA for full-length PrPc is 100 times higher than that of Clone 6, with a dissociation constant 2.03 × 10 −10 M. 4AA-m (a variant of 4AA with its human constant region replaced by a mouse constant region) is highly specific in both Western blot analysis and immunohistochemistry, and is far more sensitive than the commonly used anti-PrPc antibodies 8H4 and 3F4 . Additionally, the recognition epitope of the 4AA-m antibody differs from that of 8H4 and 3F4, potentially enabling it to bind a different cleaved form of PrPc , . Therefore, re-evaluating reported cancer prognoses using the 4AA-m antibody may provide additional insights. In this study, we used 4AA-m-stained tissue microarrays of esophageal squamous cell carcinoma (ESCC) and hepatocellular carcinoma (HCC) to analyze the relationship between PrPc expression and prognosis. Currently, relatively few studies have examined the correlation between PrPc and these two cancers. Two separate studies on these two cancers have been published , . Our findings on ESCC are opposite to those in the article authored by Zhao et al. , but are consistent with those in the article authored by Kim et al. . We have provided a detailed discussion of the conflicting results in the immunohistochemistry of PrPc, particularly the potential contribution of various anti-PrPc antibodies to these discrepancies.   The expression of PrPc in ESCC cell lines and patient tissues First, we labeled four esophageal carcinoma cell lines with 4AA-m for flow cytometry. We found that the three cell lines—KYSE-150, KYSE-450, and KYSE-510—expressed PrPc, while no PrPc expression was detected in TE-1 cell line (Fig. A). We also examined PrPc expression in tissues from patients with ESCC. Positive PrPc expression was observed in basal cells of para-carcinoma tissue, consistent with previous findings . PrPc was expressed at variable levels across ESCC patient tissues (Fig. B). Thus, only some ESCC cell lines and patient cancer samples express PrPc. PrPc expression is negatively correlated with lymph node metastasis in ESCC patients and ESCC patients with high PrPc expression have a better prognosis To investigate the correlation between PrPc expression and cancer progression, we conducted immunohistochemistry on tissue microarrays containing samples from 179 primary ESCC patients. The Tumor-DAB-H-score, which semi-quantitatively represents PrPc expression levels in cancer tissues (refer to Materials and methods for their calculation), was used to classify patients into two groups: high and low PrPc expression. This score was further analyzed to assess correlations between PrPc expression and various clinical and pathological outcomes. Using chi-square analysis, we found no significant association between PrPc expression and patient gender, age, histologic grade, tumor size, morphological type, T classification, or M classification. However, PrPc expression was significantly correlated with lymph node ratio (LNR) ( P < 0.001) and log odds of positive lymph nodes (LODDS) ( P < 0.001), two parameters related to lymph node metastasis that are important prognostic indicators for ESCC – (Table ). Further, by rank analysis, the patients’ PrPc expression is negatively correlated with both LNR (Rank correlation coefficient = − 0.166, P = 0.032) (Fig. A) and LODDS (Rank correlation coefficient = − 0.234, P = 0.002) (Fig. B). The results of these two analyses support that PrPc expression at high levels in ESCC predicts good prognosis. We then used Kaplan-Meier analysis to evaluate the impact of PrPc expression on overall survival (OS). Patients with high PrPc expression had significantly better OS compared to those with low expression. The median OS was 30 months for the high PrPc group and 18 months for the low PrPc group, with 3-year survival rates of 44.4% and 28.8%, respectively (log-rank test: P = 0.044) (Fig. C). Univariate Cox analysis revealed that PrPc expression, along with gender, tumor size, and tumor stage, was associated with OS (Table ). Multivariate Cox regression analysis confirmed that high PrPc expression was an independent prognostic factor for OS (HR = 0.545, 95% CI 0.316–0.941, P = 0.029) (Table ). Additionally, the Tumor-DAB-H-score, reflecting semi-quantitative PrPc expression, was also identified as an independent prognostic factor for OS (HR = 0.995, 95% CI 0.991–1.000, P = 0.035), with high PrPc expression indicating better prognosis (Table ). In summary, these analyses indicate that in primary ESCC, high PrPc expression is correlated with favorable prognosis. The expression of PrPc in liver cancer cell lines Similarly, we assessed PrPc expression in liver cancer cell lines using the 4AA-m antibody by flow cytometry (Fig. A). PrPc was highly expressed in RBE, HCCLM3, and SMMC7721 cells and positively expressed at lower levels in HepG2, BEL7407, and HepB3 cells. No PrPc expression was detected in PLC/PRF/5, HuH-7, and SK-HEP-1 cell lines. Overall, PrPc is expressed in the majority of liver cancer cell lines tested. Positive PrPc expression is an independent prognostic factor for poor recurrence-free survival in hepatocellular carcinoma Immunohistochemistry using the 4AA-m antibody was performed on tissue microarrays from 92 patients with primary HCC. Among these, 13 patients showed positive PrPc expression in their tumor tissues, while all para-carcinoma hepatocytes were negatively stained. Typical examples are shown in Fig. B. In all para-carcinoma specimens, hepatic stellate cells showed high PrPc expression, consistent with previous reports that activated hepatic stellate cells express PrPc , . Patients were classified into PrPc-positive and PrPc-negative groups for further analysis. Chi-square analysis indicated that PrPc expression was not associated with factors such as gender, age, grade of differentiation, cirrhotic nodule number, liver cirrhosis, clinical stage, tumor size, vascular invasion, tumor capsule, hepatitis B and C markers, total bilirubin (TB), alanine transaminase (ALT), albumin (ALB), alpha-fetoprotein (AFP), or gamma-glutamyltransferase (GGT) (see Supplementary Table 1). Using Kaplan–Meier survival curve and log-rank test, patients with positive PrPc expression had a shorter relapse-free survival (RFS) than the group without PrPc expression (log-Rank test: P = 0.03) (Fig. A). The median RFS was 48 months for the PrPc-negative group and 19 months for the PrPc-positive group, with 3-year RFS rates of 52.6% and 38.5%, respectively. Univariate Cox analysis (Supplementary Table 2) and multivariate Cox analysis (Table ) further confirmed that positive PrPc expression was an independent prognostic factor for RFS (HR = 2.646, 95% CI 1.271–5.509, P = 0.009). However, PrPc expression did not show statistical significance for OS (log-Rank test: P = 0.564) (Fig. B).   First, we labeled four esophageal carcinoma cell lines with 4AA-m for flow cytometry. We found that the three cell lines—KYSE-150, KYSE-450, and KYSE-510—expressed PrPc, while no PrPc expression was detected in TE-1 cell line (Fig. A). We also examined PrPc expression in tissues from patients with ESCC. Positive PrPc expression was observed in basal cells of para-carcinoma tissue, consistent with previous findings . PrPc was expressed at variable levels across ESCC patient tissues (Fig. B). Thus, only some ESCC cell lines and patient cancer samples express PrPc.   To investigate the correlation between PrPc expression and cancer progression, we conducted immunohistochemistry on tissue microarrays containing samples from 179 primary ESCC patients. The Tumor-DAB-H-score, which semi-quantitatively represents PrPc expression levels in cancer tissues (refer to Materials and methods for their calculation), was used to classify patients into two groups: high and low PrPc expression. This score was further analyzed to assess correlations between PrPc expression and various clinical and pathological outcomes. Using chi-square analysis, we found no significant association between PrPc expression and patient gender, age, histologic grade, tumor size, morphological type, T classification, or M classification. However, PrPc expression was significantly correlated with lymph node ratio (LNR) ( P < 0.001) and log odds of positive lymph nodes (LODDS) ( P < 0.001), two parameters related to lymph node metastasis that are important prognostic indicators for ESCC – (Table ). Further, by rank analysis, the patients’ PrPc expression is negatively correlated with both LNR (Rank correlation coefficient = − 0.166, P = 0.032) (Fig. A) and LODDS (Rank correlation coefficient = − 0.234, P = 0.002) (Fig. B). The results of these two analyses support that PrPc expression at high levels in ESCC predicts good prognosis. We then used Kaplan-Meier analysis to evaluate the impact of PrPc expression on overall survival (OS). Patients with high PrPc expression had significantly better OS compared to those with low expression. The median OS was 30 months for the high PrPc group and 18 months for the low PrPc group, with 3-year survival rates of 44.4% and 28.8%, respectively (log-rank test: P = 0.044) (Fig. C). Univariate Cox analysis revealed that PrPc expression, along with gender, tumor size, and tumor stage, was associated with OS (Table ). Multivariate Cox regression analysis confirmed that high PrPc expression was an independent prognostic factor for OS (HR = 0.545, 95% CI 0.316–0.941, P = 0.029) (Table ). Additionally, the Tumor-DAB-H-score, reflecting semi-quantitative PrPc expression, was also identified as an independent prognostic factor for OS (HR = 0.995, 95% CI 0.991–1.000, P = 0.035), with high PrPc expression indicating better prognosis (Table ). In summary, these analyses indicate that in primary ESCC, high PrPc expression is correlated with favorable prognosis.   Similarly, we assessed PrPc expression in liver cancer cell lines using the 4AA-m antibody by flow cytometry (Fig. A). PrPc was highly expressed in RBE, HCCLM3, and SMMC7721 cells and positively expressed at lower levels in HepG2, BEL7407, and HepB3 cells. No PrPc expression was detected in PLC/PRF/5, HuH-7, and SK-HEP-1 cell lines. Overall, PrPc is expressed in the majority of liver cancer cell lines tested.   Immunohistochemistry using the 4AA-m antibody was performed on tissue microarrays from 92 patients with primary HCC. Among these, 13 patients showed positive PrPc expression in their tumor tissues, while all para-carcinoma hepatocytes were negatively stained. Typical examples are shown in Fig. B. In all para-carcinoma specimens, hepatic stellate cells showed high PrPc expression, consistent with previous reports that activated hepatic stellate cells express PrPc , . Patients were classified into PrPc-positive and PrPc-negative groups for further analysis. Chi-square analysis indicated that PrPc expression was not associated with factors such as gender, age, grade of differentiation, cirrhotic nodule number, liver cirrhosis, clinical stage, tumor size, vascular invasion, tumor capsule, hepatitis B and C markers, total bilirubin (TB), alanine transaminase (ALT), albumin (ALB), alpha-fetoprotein (AFP), or gamma-glutamyltransferase (GGT) (see Supplementary Table 1). Using Kaplan–Meier survival curve and log-rank test, patients with positive PrPc expression had a shorter relapse-free survival (RFS) than the group without PrPc expression (log-Rank test: P = 0.03) (Fig. A). The median RFS was 48 months for the PrPc-negative group and 19 months for the PrPc-positive group, with 3-year RFS rates of 52.6% and 38.5%, respectively. Univariate Cox analysis (Supplementary Table 2) and multivariate Cox analysis (Table ) further confirmed that positive PrPc expression was an independent prognostic factor for RFS (HR = 2.646, 95% CI 1.271–5.509, P = 0.009). However, PrPc expression did not show statistical significance for OS (log-Rank test: P = 0.564) (Fig. B).   We recently generated a highly specific and sensitive anti-PrPc antibody 4AA-m . In this study 4AA-m was used to carry out immunohistochemistry on ESCC and HCC tissue chips and compare the correlations between the PrPc expression level and prognosis of the cancer patients. We found that PrPc expression in ESCC patients was positively correlated with prognosis, a finding that contrasted with the results of Zhao et al. . In HCC, however, PrPc expression was negatively correlated with prognosis, consistent with the findings of Pan et al. . Several factors may explain the divergent results between our study and Zhao et al.’s in the context of ESCC. First, Zhao et al. used a rabbit polyclonal antibody generated against a peptide from residues G70-A120 of human PrPc (DF7034), with limited available data on its specificity and sensitivity for immunohistochemistry. In contrast, our antibody, 4AA-m, is a monoclonal human-mouse chimeric antibody that targets residues 127–161 of PrPc, with a K D value of 2.03 × 10 −10 M . This antibody has demonstrated superior specificity and sensitivity in immunohistochemistry compared to commonly used monoclonal antibodies 3F4 and 8H4, particularly in testing on P6C (a human colorectal cancer cell line) and its PrPc-knockout variant, PRNP -deleted P6C, used as a negative control . Second, Zhao et al.’s immunofluorescence data showed no significant PrPc expression in the basal cells of para-cancerous tissues , while our results indicated strong PrPc expression in these cells, in agreement with Pammer et al. . Third, Zhao et al. suggested that PrPc is involved in iron metabolism and ferroptosis, impacting patient prognosis. In our study, we found that PrPc expression was negatively correlated with lymph node metastasis, a key prognostic factor, and specifically with LNR and LODDS – , suggesting that high PrPc expression may support a more favorable prognosis in ESCC. Conflicting data also exist regarding PrPc expression in gastric cancer prognosis , . Studies using the commercial monoclonal antibodies 3F4 and 8H4 have reported opposite findings: with 8H4, negative PrPc expression was linked to poor survival , while with 3F4, positive expression was associated with poor survival . In our previous experiments, 4AA-m proved to be significantly more sensitive than 3F4 and 8H4, recognizing PrPc across different molecular weights in a Western blot of overexpressed 293T cells . For immunohistochemistry on human cerebellum tissue, 4AA-m was 29 and 166 times more sensitive than 3F4 and 8H4, respectively . PrPc has multiple cleavage sites . The alpha site is a major cleavage site (at amino acid site 110/111 or 111/112) , and the binding regions of 3F4 and 8H4 are located on amino acid residues 109–112 and 145–180 of human PrPc, respectively. If PrPc underwent cleavage, DF7034 and 3F4 antibodies could not recognize PrPc on the membrane, while 4AA-m and 8H4 still could (Fig. ). The N1 fragment from cleavage is associated with LLPS and important for physiological functions . However our data show a complicated situation that 8H4 has better binding specificity in immunohistochemistry on P6C cells as well as human cerebellum while 3F4 binds PrPc better than 8H4 in Western blot on PrPc-overexpressed 293T cells . Obviously, the locations of the epitopes of these antibodies could be one factor that contributes to the conflicting results. The other features of the antibodies could also play roles. As for the role of PrPc in the prediction of prognosis of patients with HCC, the data presented in this study were largely consistent with the conclusions by Kim et al. who used 8H4 as anti-PrPc antibody and Wang et al. who analysed through TCGA database . In our study, PrPc expression detected by 4AA-m was found to be an independent prognostic factor for RFS in HCC (HR = 2.646, 95% IC: 1.271–5.509). While there was a trend toward poorer overall survival in PrPc-positive HCC patients, it did not reach statistical significance (Fig. B). Notably, the PrPc-positive rate in our study was only 14%, much lower than that reported by Kim et al. , which may account for the lack of statistical significance. This low positive rate was consistent with Zheng et al.’s observation that PrP mRNA expression in HCC was lowest among various tumor tissues . Currently, ESCC and HCC are two cancers in which the prognostic role of PrPc expression remains contentious. It is not unusual for the same protein to play different roles in different cancer types , , as seen with Notch1. In hepatocellular carcinoma, NOTCH1 is a proto-oncogene linked to poor prognosis , , whereas in some studies of esophageal cancer, reduced Notch1 expression has been associated with increased lymph node metastasis – , which has similarities with the manifestation of PrPc. In pancreatic ductal carcinoma, Notch1 and PrPc can form a complex that enhances tumor cell viability and invasiveness . Given the complex role of PrPc across cancer types, further exploration of the interaction between PrPc and Notch1 in cancer progression would be valuable. Discrepancies in the literature could arise from several factors, including differences in antibody choice, cancer type, tissue source, and immunohistochemistry methods. Various post-translational modifications and cleavage patterns of PrPc may differ across cancer tissues, and these factors can influence antibody binding. The four antibodies discussed target different regions of PrPc (DF7034: 70–120; 3F4: 109–112; 4AA-m: 127–161; 8H4: 145–180), and both cleavage location and post-translational modifications within these regions may affect immunohistochemical outcomes. To address these inconsistencies, fixing one condition (such as the same cell lines or tissue sample) and testing the others (such as two different antibodies) are necessary. An ideal approach would involve cell lines with identical genetic backgrounds except for PrPc expression levels. Flow cytometry, Western blotting, and immunohistochemistry could then be used to compare antibody performance. Additionally, generating organoids from these cell lines may offer more nuanced insights, as they more closely mimic tumor behavior in vivo and could be valuable for immunohistochemical comparison of antibody performance.   Patients and tissue microarrays The cancer tissue microarrays were purchased from Shanghai OUTDO Biotechnology Co LTD. Tissue microarray blocks were cut into 4 μm thick slices. The primary hepatocellular carcinoma tissue microarray item number is HLivH180Su17 (Ethics number: SHYJS-CP-1710004). The patients were treated with surgical resection from June 2007 to November 2008, and were followed up until July 2012.The esophageal cancer tissue microarrays are HEsoS180Su06 and HEsoS180Su11 (Ethics number: SHYJS-CP-1507001 and SHYJS-CP-2004003). The patients in HEsoS180Su06 were treated with surgical resection from January 2011 to April 2013, and followed up until June 2015. The patients in HEsoS180Su11 were treated with surgical resection from 2006.7 to 2008.9 and followed up until July 2015. Some patients were uncertain whether they were primary squamous esophageal carcinoma, so they were excluded, and 179 cases of primary squamous esophageal carcinoma were finally analyzed. TNM staging of all patients was performed according to the 7th edition of the AJCC Cancer Staging Manual. This study gained approval from the Ethics Committee of Shanghai Outdo Biotech Company (Ethics number: SHYJS-CP-1710004, SHYJS-CP-1507001 and SHYJS-CP-2004003) and was performed in accordance with the relevant guidelines. Informed consent was obtained from all participants. Cell culture and flow cytometry KYSE-150 (RRID: CVCL_1348) cells and KYSE-510 (RRID: CVCL_1354) cells were donated by Runsheng Chen’s research group (Institute of Biophysics, Chinese Academy of Sciences). KYSE-450 (RRID: CVCL_1353) cells, TE-1 (RRID: CVCL_1759) cells, SMMC7721 (RRID: CVCL_0534) cells, RBE (RRID: CVCL_4896) cells, HuH-7 (RRID: CVCL_0336) cells, HCCLM3 (RRID: CVCL_6832) cells, BEL-7404 (RRID: CVCL_6568) cells, SK-HEP-1 (RRID: CVCL_0525) cells were donated by Xiyun Yan’s research group (Institute of Biophysics, Chinese Academy of Sciences). HepG2 (RRID: CVCL_0027) cells, PLC/PRF/5 (RRID: CVCL_0485) cells and Hep3B (RRID: CVCL_0326) cells were donated by Pengyuan Yang’s research group (Institute of Biophysics, Chinese Academy of Sciences). KYSE-150, KYSE-450, KYSE-510, TE-1, SMMC7721, RBE, BEL-7404, SK-HEP-1 cell lines were cultured in RPMI (HyClone, item no. SH30809.01) with 10% FBS (HyClone). HepG2, HuH-7, Hep3B cell lines were cultured in DMEM (HyClone, item no. SH30243.01) with 10% FBS. PLC/PRF/5 cell line were cultured in DMEM with 20% FBS. HCCLM3 cell line are cultured in MEM (Thermo, item no. C11095500BT) with 10% FBS. In the cells mentioned above, no positive mycoplasma infection was found. Flow cytometry detection was performed using PE Mouse IgG2a, κ Isotype Ctrl (FC) (Biolegend, item no. 400213) and PE labeled 4AA-m. 4AA-m antibody was prepared by our laboratory. We used the activator SMCC (Pierce, catalog number 22360) to react with the amine of PE to generate a maleimide-labeled PE. The disulfide bond of 4AA-m was reduced using DTT. PE is covalently coupled to the antibody through its maleimide group, which reacts with the free thiol groups in the antibody. 500,000 cells were resuspended in 100 µL of the system and 0.05 µg of antibody was added to the system and incubated on ice for 30 min. All flow cytometry was performed on FACS AriaIII (BD). Immunohistochemical staining Immunohistochemistry was performed using a fully automated immunohistochemistry with in situ hybridization stainer Bond RX. 4AA-m antibody working concentration was 0.6 µg/mL and incubated for 1 h at room temperature. The chip arrays were scanned by the pathology section scanner CS2. Data analysis was performed using the HALO ® high-throughput full-slice pathology image analysis platform developed and manufactured by Indica Labs, Inc. Tumor tissue PrPc staining intensity was analyzed using the Multiplex IHC v3.1 module and tumor-DAB-H-Score was calculated. Tumor-DAB-H-Score was used as a score for PrPc expression level in cancer tissues, and used for various analyses related to pathological and prognostical information . [12pt]{minimal} 				 				  				  				  				 				 				 				 				$$ {} & = ( {{}  1} ) \\ &  + ( {{}  2} ) \\ &  + ( {{}  3} ) \\ $$ Statistical analysis All statistical analyses were performed using SPSS 26.0 for Windows (SPSS, Chicago, IL). The correlation of PrPc expression with pathological factors was determined using chi-square test and rank correlation analysis. Correlation of PrPc expression and other pathological factors with patient survival was determined using log rank test and Cox regression analysis. Variables whose P value were under 0.2 were included in multivariate survival analysis using the Cox proportional hazards model. P < 0.05 was considered statistically significant and all P values were two-sided.   The cancer tissue microarrays were purchased from Shanghai OUTDO Biotechnology Co LTD. Tissue microarray blocks were cut into 4 μm thick slices. The primary hepatocellular carcinoma tissue microarray item number is HLivH180Su17 (Ethics number: SHYJS-CP-1710004). The patients were treated with surgical resection from June 2007 to November 2008, and were followed up until July 2012.The esophageal cancer tissue microarrays are HEsoS180Su06 and HEsoS180Su11 (Ethics number: SHYJS-CP-1507001 and SHYJS-CP-2004003). The patients in HEsoS180Su06 were treated with surgical resection from January 2011 to April 2013, and followed up until June 2015. The patients in HEsoS180Su11 were treated with surgical resection from 2006.7 to 2008.9 and followed up until July 2015. Some patients were uncertain whether they were primary squamous esophageal carcinoma, so they were excluded, and 179 cases of primary squamous esophageal carcinoma were finally analyzed. TNM staging of all patients was performed according to the 7th edition of the AJCC Cancer Staging Manual. This study gained approval from the Ethics Committee of Shanghai Outdo Biotech Company (Ethics number: SHYJS-CP-1710004, SHYJS-CP-1507001 and SHYJS-CP-2004003) and was performed in accordance with the relevant guidelines. Informed consent was obtained from all participants.   KYSE-150 (RRID: CVCL_1348) cells and KYSE-510 (RRID: CVCL_1354) cells were donated by Runsheng Chen’s research group (Institute of Biophysics, Chinese Academy of Sciences). KYSE-450 (RRID: CVCL_1353) cells, TE-1 (RRID: CVCL_1759) cells, SMMC7721 (RRID: CVCL_0534) cells, RBE (RRID: CVCL_4896) cells, HuH-7 (RRID: CVCL_0336) cells, HCCLM3 (RRID: CVCL_6832) cells, BEL-7404 (RRID: CVCL_6568) cells, SK-HEP-1 (RRID: CVCL_0525) cells were donated by Xiyun Yan’s research group (Institute of Biophysics, Chinese Academy of Sciences). HepG2 (RRID: CVCL_0027) cells, PLC/PRF/5 (RRID: CVCL_0485) cells and Hep3B (RRID: CVCL_0326) cells were donated by Pengyuan Yang’s research group (Institute of Biophysics, Chinese Academy of Sciences). KYSE-150, KYSE-450, KYSE-510, TE-1, SMMC7721, RBE, BEL-7404, SK-HEP-1 cell lines were cultured in RPMI (HyClone, item no. SH30809.01) with 10% FBS (HyClone). HepG2, HuH-7, Hep3B cell lines were cultured in DMEM (HyClone, item no. SH30243.01) with 10% FBS. PLC/PRF/5 cell line were cultured in DMEM with 20% FBS. HCCLM3 cell line are cultured in MEM (Thermo, item no. C11095500BT) with 10% FBS. In the cells mentioned above, no positive mycoplasma infection was found. Flow cytometry detection was performed using PE Mouse IgG2a, κ Isotype Ctrl (FC) (Biolegend, item no. 400213) and PE labeled 4AA-m. 4AA-m antibody was prepared by our laboratory. We used the activator SMCC (Pierce, catalog number 22360) to react with the amine of PE to generate a maleimide-labeled PE. The disulfide bond of 4AA-m was reduced using DTT. PE is covalently coupled to the antibody through its maleimide group, which reacts with the free thiol groups in the antibody. 500,000 cells were resuspended in 100 µL of the system and 0.05 µg of antibody was added to the system and incubated on ice for 30 min. All flow cytometry was performed on FACS AriaIII (BD).   Immunohistochemistry was performed using a fully automated immunohistochemistry with in situ hybridization stainer Bond RX. 4AA-m antibody working concentration was 0.6 µg/mL and incubated for 1 h at room temperature. The chip arrays were scanned by the pathology section scanner CS2. Data analysis was performed using the HALO ® high-throughput full-slice pathology image analysis platform developed and manufactured by Indica Labs, Inc. Tumor tissue PrPc staining intensity was analyzed using the Multiplex IHC v3.1 module and tumor-DAB-H-Score was calculated. Tumor-DAB-H-Score was used as a score for PrPc expression level in cancer tissues, and used for various analyses related to pathological and prognostical information . [12pt]{minimal} 				 				  				  				  				 				 				 				 				$$ {} & = ( {{}  1} ) \\ &  + ( {{}  2} ) \\ &  + ( {{}  3} ) \\ $$   All statistical analyses were performed using SPSS 26.0 for Windows (SPSS, Chicago, IL). The correlation of PrPc expression with pathological factors was determined using chi-square test and rank correlation analysis. Correlation of PrPc expression and other pathological factors with patient survival was determined using log rank test and Cox regression analysis. Variables whose P value were under 0.2 were included in multivariate survival analysis using the Cox proportional hazards model. P < 0.05 was considered statistically significant and all P values were two-sided.   Below is the link to the electronic supplementary material. Supplementary Material 1   