| Objective:Early diagnosis of colorectal cancer is an effective strategy to reduce mortality.In recent years,the incidence of colorectal cancer in China has increased annually,with both the incidence and mortality rates ranking second among all malignant tumors.Moreover,the age of onset is gradually becoming younger,making it a heavy burden for cancer prevention and control in China.The compliance of the Chinese population with colonoscopy and fecal screening is low,and obviously,the compliance with blood sampling methods is higher than that of fecal sampling.Therefore,it is of great significance to find blood-based diagnostic markers for primary and secondary prevention of colorectal cancer.Existing colorectal cancer liquid biopsy markers,such as cell-free tumor DNA methylation,gene mutations,etc.,have defects such as high detection costs,insensitivity to early tumors,and complex detection techniques.Proteins are the molecules most directly related to diseases and can be detected based on conventional ELISA technology,making them a good source of blood markers.Tumor proteins include two major categories:proteins that respond to tumors and proteins produced by tumor cells.This study aims to explore the role of the two important tumor-related protein markers,namely tumor-specific autoantibodies and oncofetal chondroitin sulfate proteoglycans,in colorectal cancer liquid biopsy,and to evaluate the potential of their combined use as markers for colorectal cancer screening and early diagnosis.Methods:（1）Through HuprotTM high-throughput human whole protein chip analysis,we identified the tissue-infiltrating lymphocytes and blood-derived autoantibody spectrum of colorectal cancer,characterizing the differences in IgG and IgM autoantibody repertoire from blood and tissue sources,and screening for colorectal cancer-specific autoantibody combinations.Through in vitro recombinant expression,we purified the candidate autoantibody-reactive tumor-associated antigens,constructed an ELISA detection system,evaluated the detection efficacy of specific tumor autoantibodies for colorectal cancer in a case-control cohort of colorectal cancer,and assessed the relationship between tumor autoantibody levels and clinical indicators.（2）Based on the binding specificity of Plasmodium falciparum protein VAR2CSA with oncofetal chondroitin sulfate glycosaminoglycans,the level of oncofetal chondroitin sulfate and their modified proteoglycans CD44,CSPG4,and SDC1 in colorectal cancer cells was evaluated through immunofluorescence.An ELISA detection system based on recombinant VAR2CSA protein was constructed to detect the sensitivity and specificity of its detection for intracellular and plasma proteoglycans in the colorectal cancer cell line SW480.The levels of cell free oncofetal sulfated modified CD44,CSPG4,and SDC1 were detected in a case-control cohort of colorectal cancer to evaluate their potential for detecting colorectal cancer.（3）The diagnostic capabilities of tumor autoantibodies and oncofetal sulfated glycosaminoglycan proteins for colorectal cancer are validated in a new case-control cohort,and the ability of combined markers in the diagnosis of colorectal cancer was analyzed.Results:（1）IgM was the predominant of tumor autoantibody in tissue and blood,while tumor-specific autoantibodies in the peripheral blood of colorectal cancer patients were mainly of the IgG subtype;A high-throughput human whole protein chip had screened 19 candidate tumor-associated antigens reactive IgG antibodies as colorectal cancer-specific autoantibodies;In vitro expression technology had successfully purified the RPRD1A autoantigen,and constructed an ELISA detection system with recombinant RPRD1A as the capture molecule and HRP-modified IgG antibody as the detection molecule;In the retrospective case-control cohort,the level of IgG autoantibodies against RPRD1A antigen in the peripheral blood of colorectal cancer patients was significantly higher than that of healthy controls（with a p value less than 0.0001）,and the level of tumor autoantibodies was negatively correlated with pathological staging,which could be used as a tumor marker for assisting in the diagnosis of colorectal cancer,with an area under the curve of 0.7（95%CI=0.61-0.78）,at a cut-off value of 0.764,the sensitivity was 87%,the specificity was47.1%,and it had a certain potential for early diagnosis.（2）Based on recombinant VAR2CSA,oncofetal chondroitin sulfate-modified proteoglycans,including CD44,CSPG4,and SDC1,were found to be highly expressed in colorectal cancer.The detection of free tumor-specific sulfated glycosaminoglycans based on recombinant VAR2CSA protein exhibited good sensitivity and specificity.The levels of cell free CD44,CSPG4,and SDC1 proteoglycans in colorectal cancer patients were significantly higher than those in normal individuals with p value<0.0001,P=0.0015,P=0.0111,respectively.Oncofetal chondroitin sulfate modified CD44,CSPG4,and SDC1 can be used as colorectal cancer biomerkers,with respective areas under the curve AUC:0.84（95%CI:0.769-0.906）;0.69（95%CI:0.584-0.802）;0.77（95%CI:0.676-0.873）.Notably,the detection efficiency was highest for CD44 proteoglycan.The combined analysis of the three markers yields an AUC of 0.86,95%CI=0.781-0.931,without significantly improving the overall performance.（3）In a new case-control study cohort,the AUC of tumor-specific autoantibody anti-RPRD1A for colorectal cancer detection is 0.628（95%CI=0.504-0.751）,with a sensitivity of 80%and specificity of 44.7%.The AUC of oncofetal sulfated glycosaminoglycan-modified CD44 protein for colorectal cancer detection is 0.767（95%CI=0.664-0.870）,with a sensitivity of 88%and specificity of 57.3%.Combining the tumor-specific autoantibody anti-RPRD1A and CD44 proteoglycan significantly enhanced the diagnostic specificity of colorectal cancer,and significantly improved the efficiency of diagnosing colorectal cancer.Conclusion:This study systematically characterized the differences in autoantibody spectra derived from colorectal cancer tissues and blood,and found that the autoantibodies derived from tumor tissues in the peripheral blood of colorectal cancer patients are mainly IgG.We screened and identified a colorectal cancer-specific autoantibody group composed of 19 autoantibodies and established a method for efficient detection of tumor autoantibody anti-RPRD1A.Based on the oncofetal chondroitin sulfate proteoglycan detection method established in the laboratory,this study verified that colorectal cancer cells highly express oncofetal chondroitin sulfate proteoglycans CD44,CSPG4,SDC1,and clarified the detection sensitivity and specificity of the detection system.In the case-control cohort of colorectal cancer,we confirmed that anti-RPRD1A tumor autoantibodies and oncofetal chondroitin sulfate-modified CD44,CSPG4,SDC1 have detection efficacy for colorectal cancer.In a new validation cohort,it was found that the combined use of anti-RPRD1A and oncofetal chondroitin sulfate-modified CD44 can serve as tumor markers for the auxiliary diagnosis of colorectal cancer.The combined analysis of the two types of targets can significantly improve the diagnostic specificity for colorectal cancer and enhance the overall diagnostic performance,providing a new direction for the screening and diagnosis of colorectal cancer. |
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