| BackgroundPrimary liver cancer(PLC)is a malignant tumor that endangers life and health of Chinese residents for a long time.More than 85%of primary liver cancer patients are hepatocellular carcinoma(HCC),ranking the fourth in morbidity and second in mortality in China.However,giant liver cancer is a special type of liver cancer with a tumor diameter of more than 10 cm.This type of liver cancer is characterized by low drug response rate,high postoperative recurrence rate and easy early progression due to its large diameter,tumor heterogeneity,immune escape of liver cancer cells and aggressive frontier area with "immune barrier" effect.Although surgical excision is still the preferred radical treatment at present,with the rapid development of tumor immunotherapy,traditional excision techniques can no longer fully meet patients’ expectations for prognosis.Therefore,it is particularly important for such patients to find appropriate downphase or neoadjuvant therapy on the basis of surgical resection,so as to improve the anti-tumor immune capacity of the body and form a new advantageous treatment mode while reducing the tumor load.Gelatin sponge microparticles transcatheter arterial chemoembolization(GSMs-TACE)is a technology that has been proved to be effective in controlling advanced liver cancer.Guided by the vascular digital subtraction platform,The tumor is killed directly by injecting chemotherapeut-laden particles directly into the artery supplying the tumor.We speculated that with the killing of the tumor,there would be subsequent release of tumor-specific antigens,and then chemotaxis and proliferation of immune cells in the tumor microenvironment,and finally completely change the process of tumor immune microenvironment.Although previous studies have proved that this treatment has the potential of immune enhancement,there have been no literature reports on whether the effect of microhepatic artery chemoembolization on immunity has anti-tumor specificity,and how endovascular local therapy represented by microhepatic artery embolization changes the tumor microenvironment and changes the proportion of immune cells in peripheral blood through what biological mechanism.This paper will be divided into two parts.The first part mainly discusses the reconstruction of tumor microenvironment by microparticle TACE from the perspective of cell composition,single-cell behavior and spatial transcriptome.The second part focuses on the changes of lymphocyte subsets related to systemic anti-tumor immunity and the clinical effects of enhancing the immune system to monitor tumors and prevent early recurrence.Method1.Designed the clinical study "A single-center,retrospective controlled study of GSMs-TACE combined surgical resection for massive liver cancer"and completed the procedures of ethical review,clinical study registration and patient enrollment.Full-layer tumor pathological specimens and blood samples at various time points of "direct resection of massive liver cancer" were obtained from this study as control group.Full-layer tumor pathological specimens and blood samples at various time points of "massive liver cancer resected one month after GSMs-TACE treatment" were obtained as control group and experimental group.2.immunohistochemistry of two groups was processed by Multiplex immunohistochemistry(mIHC).Image information was extracted from mIHC sections and single-cell location network was constructed.Through in-depth analysis of cell density and cell-cell interaction of each subgroup,the differences and similarities of cell distribution and cell behavior in the microenvironment between the two groups were expounded.3.Digital Spatial Profiler(DSP)was used to mark the special region of interest(ROI)of the two groups of pathological sections on the mIHC sections,and then full transcriptional amplification and sequencing with spatial information were carried out.To explore the transcriptome expression differences of tumor cells and infiltrating immune cells in the two groups of sections as a whole and in different types of areas such as "tumor center" and"tumor edge immune infiltrating zone",and then use biogenic analysis technology to analyze the differential genes of chemokine spectrum that affect the enrichment of immune cells,chemotaxis and behavior interpretation.The main pathway mechanism of GSMs-TACE microenvironment is described.4.Preoperative peripheral blood samples from 30 healthy volunteers and 60 patients receiving any antitumor therapy were collected for multistandard flow cytometry,and 24 detection items including various subsets of immune cells and immune checkpoint expression were analyzed for cluster dimensionality reduction,and peripheral blood "immune normalization model"was established.5.Based on the peripheral blood "immune normalization model",30 patients were evaluated to see whether they showed "immune normalization"after receiving GSMs-TACE.At the same time,progression-free survival time was used as the primary endpoint,and the "immune normalization model" was used as the grouping condition to evaluate the predictive value of this model for patients’ prognosis.Result1.A total of 10 samples of massive liver cancer after operation was performed on mIHC,including 5 patients in the experimental group and 5 patients in the control group.A month after the discovery of GSMs-TACE in large hepatoma,tumor antigen release and immune cell recruitment resulted in a new tumor microenvironment,which was different from that of the control group.2.After image information extraction and digital cell network establishment for a total of 100 ROIs of 5 cases of mIHC sections in the experimental group and 5 cases of control group,in-depth analysis of cell density and cell-cell interaction showed that,The density of immunoreactive T cells(Teff)in the new micro-environment after GSMs-TACE was higher than that in the control group.Although the comparison between the two groups showed no significant difference in the density of heterogeneous or silent immune cell subsets,such as regulatory T cells(Tregs),PD-1 high expression and TIGIT high expression Teff,in the microenvironment of the experimental group,each Treg was the center.The average number of effector T cells was increased in the 30μm radius area compared with the control group.With a single Treg as the starting point,the distance to the nearest CD8 group was close to the experimental group,indicating that the effect intensity of Treg competitive inhibition of normal Teff was reduced.3.DSP test was carried out on 100 ROI of mIHC sections of 5 patients in the experimental group and 5 patients in the control group respectively,and it was found that the tumor microenvironment of patients with massive liver cancer after GSMs-TACE intervention had a large number of gene differential mutations at the transcriptome level compared with the control group.Multiple mechanisms such as extracellular matrix,steroid metabolism pathway,interleukin-4 and interleukin-13 pathway are involved.The expression profiles of chemokines represented by CCL17 were significantly different from those of HCC samples without intervention,which provided a reasonable mechanism for the density and behavior changes of immunoinfiltrating cells.4.Flow cytometry was performed on blood samples of 30 patients with massive liver cancer treated with GSMs-TACE and 30 healthy volunteers before and after operation.The results showed that the proportion of Treg lymphocytes decreased and the proportion of helper T cells(TH)and natural killer cell(NK)lymphocytes increased in patients 1 month after GSMs-TACE treatment.There was no statistically significant change in the proportion of high expression cells to the total number of corresponding cells at most immune checkpoints.Clustering was performed after dimensionality reduction based on all the cell proportions above,and an "immune normalization model" was established.It was found that the proportion of peripheral blood immune cells in most patients was more similar to that of healthy volunteers one month after receiving GSMs-TACE treatment.After the first treatment,patients grouped according to the peripheral blood "immune normalization model" could predict the time of tumor progression to a certain extent.Conclusion1.GSMs-TACE therapy achieved the reconstruction of tumor immune microenvironment in patients with massive liver cancer through the intervention of signal channels such as CCL17.The density and cell behavior of some immune cell subsets were changed,and the "immune barrier" of the tumor margin region was broken,so that the direction of the microenvironment like more active anti-tumor immune effect was changed.2.GSMs-TACE restructures the tumor immune microenvironment and systematically improves the anti-tumor immune ability of patients’ peripheral blood.Finally,GSMs-TACE improves the long-term prognosis of patients by activating tumor immune monitoring in the multidisciplinary diagnosis and treatment model of liver cancer... |
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