Spatial interpretation | comprehensive analysis of spatial structure of primary liver cancer

Spatial transcriptome in 20 By the nature methods Rated as technology of the year , It may become the next wave of craze after single cell analysis , The first spatial transcriptome article in the field of liver cancer has finally been published in the near future , stay 2021 year 12 month 17 Published on SCIENCE ADVANCES, titled ：Comprehensive analysis of spatial architecture in primary liver cancer, Let's study together today .

Heterogeneity is a major challenge in cancer prevention and treatment . The study begins with Constructed primary liver cancer (PLC) High resolution spatial transcriptome map spectrum , Include from 7 Famous patients 21 In an organization 84,823 individual spot. Space tumor microenvironment (TME) Features from non tumor areas 、 Progressive comparison from marginal area to tumor area , It is found that the tumor capsule may affect the continuity of spatial clusters in the tumor 、 Transcriptome diversity and immune cell infiltration . In part , The study found that in 100mm Wide cluster - Bidirectional ligands on the cluster boundary - Receptor interactions help maintain the structure within the tumor , and PROM1+ and CD47+ Tumor stem cell niche and TME Remodeling is related to tumor metastasis . Last , The study proposes a kind of TLS-50 Mark , It is used to accurately locate the tertiary lymphoid structure in space (TLS), And reveals TLS Their unique composition is determined by their distance from tumor cells . This study provides previously unknown insights into the multiple tumor ecosystems of primary liver cancer , It also provides potential guidance for cancer intervention .

The results ：

Using spatial transcriptome to explore the structure of primary liver cancer

For a comprehensive analysis PLC Spatial heterogeneity of , The study collected 7 Of a patient 21 Example organization , Include 5 Cases of hepatocellular carcinoma (HCC-1 HCC-5), 1 Cases complicated with hepatocellular and cholangiocarcinoma (cHC-1), And a ICC (ICC-1) In patients with , utilize 10X Genomics Space technology sequenced the space transcriptome . The whole exome sequencing was also done for all large pieces of tissue in all sections (WES), use PBMC As a contrast .

In this study, a single spot（ contain 8 To 20 About cells ） The diameter is 55um, In the capture area , Every slice （6.5mm x 6.5mm） contain 5000 individual spot. For average sequencing depth , Single spot It contains about 30000UMI and 3000 A gene , And tumor tissue area UMI The number is higher than that of normal tissue .

Different phenotypes of spatial heterogeneity of primary liver cancer

To characterize the spatial diversity of primary liver cancer , The study will come from different parts of each patient spot Cluster analysis is carried out , And use UMAP Exhibition .

Pictured 2A Shown , The study found that HCC-1T、HCC-3T and HCC-4T The clustering in has the characteristics of regional distribution , and HCC-2T and cHC – 1T Clustering in is intertwined .HCC-3L Medium Cluster 5 Is a unique Cluster, stay HCC-3N and HCC-3T None of them appear .

Next , The study also used a gene based strategy to compare the scores of different cell types in different subgroups . It is worth noting that , Fibroblasts and endothelial cells in the interstitial region scored significantly higher , Immune cell scores in different tumor clusters showed a high degree of diversity .

By fitting the change curve of cell type score （ Include T cells 、B cells 、 Natural killer cell (NK)、 Myeloid cells 、 Endothelial cells and fibroblasts ）, This study found that clusters of cells associated with matrix regions formed fibroblasts 、 Variant phenotypes of endothelial cells and even immune cells .

Microenvironment characteristics of tumor marginal areas

Pictured 2A Shown , The spatial distribution of tumor clusters presents two different patterns . One is block , The boundary between clusters is clear ( Such as HCC-1T、HCC-3T), The other is discontinuous 、 mixed type ( Such as HCC-2T). In order to quantitatively evaluate this feature , The study introduced a new concept called “ Spatial continuity ” The measurement , It is calculated by comparing the clustering consistency between each point and its adjacent points . Combine another measure “ Transcriptome diversity ” Together , This study quantitatively found transcriptome heterogeneity in tumor margins and tumor regions , also HCC-1、HCC-3 and HCC-4 The slices of patients have high spatial continuity and low transcriptome diversity .

Internal heterogeneity of primary liver cancer

In order to explore the heterogeneity of tumor area on the edge and tumor section , The study examined each of the tumor areas spot To calculate the Hallmark Channel performance , And finally determined two modules . modular 1 It shows the high activity of cell cycle and metabolic related pathways ( for example ,MYC Target point v1, G2-Mcheckpoint, E2F Target point , Cholesterol homeostasis , Bile acid and fatty acid metabolism ), And modules 2 In inflammation , Angiogenesis and epithelium - There is higher activity in interstitial transformation (EMT) access .

Heterogeneous tumor stem cells in primary liver cancer （CSC） Functional analysis of

CSCs It is considered to be an important factor in the heterogeneity of liver cancer . Different cell subpopulations defined by different tumor stem cell markers may contain different carcinogenic drivers . ad locum , This study focused on five commonly used liver cancer stem cell related markers (CD47、EPCAM、KRT19、PROM1 and SOX9). For each marker （marker）, Put each patient's tumor spot The former with the highest expression in 5% Of spot Defined as marker positive CSC Niche .

By comparison L/T/P part CSC Niche proportion , The study found that in HCC-2 in ,CD47-、SOX9- and PROM1 The proportion of positive gradually increases , and EPCAM or KRT19 Genes do not show such a phenomenon .

A complete HCC The overall heterogeneity within the nodule

In order to better study the heterogeneity within tumors on a whole scale , The study collected 1 The diameter is about 1cm Of HCC, use 4 Three sections covering its central plane were analyzed for spatial transcriptome . The study will be divided into four parts spot Integration , By unsupervised clustering 6 Sub groups . These six subgroups have different scores on each section , according to H&E Staining and different gene expression patterns , It can be marked as normal area (cluster 4)、 Stromal cell area (cluster 3/6) And tumor area (cluster 1/2/5).

The third grade lymphatic structure （TLS） Spatial distribution and clinical characteristics of

TLS It is an ectopic lymphatic structure , by DC Maturity 、 Antigen presentation and T and B The activation and differentiation of cells provide a region , This is related to anti-tumor immune response and potential immunotherapeutic response . ad locum , The study notes that HCC-5 Of cluster 6 High expression CXCL13、CCL19、CCL21、LTF、LTB、CD79A/B And so on , These are all TLS Necessary molecules for formation . Through and with H&E Staining combined with analysis ,cluster6 Identified as TLS Area . The study chose cluster6 The former with medium to high specific expression 50 A gene （TLS-50） As a characteristic gene , And tested its recognition TLS The ability of . Pathologists confirmed , And other signals ( Including breast cancer 、12- Chemokines 、T Auxiliary 1 and B cells 、T Follicular helper cells 、CXCL13 And plasma cell signals ) comparison ,TLS-50 It can identify those with higher signal-to-noise ratio TLS Area . By using TLS-50 Locate different slices TLS spot , The study found that they mainly exist in L Slice instead of T and N section , Especially in L The non tumor area of the slice .

summary

This study adopts 10X Space transcriptome technology in 55mm Comprehensive analysis under the resolution 7 Genome wide transcriptome heterogeneity in patients with primary liver cancer . Through the normal 、 Make a gradual comparison between the edge and the tumor area , This study found that the tumor capsule may affect the continuity of spatial clusters in the tumor 、 Transcriptome diversity 、 Immune cell infiltration and tumor marker pathway activity . meanwhile , This study comprehensively analyzed several local spatial characteristics of liver cancer , Include 100mm Wide boundaries between tumor subpopulations 、 Cancer stem cell niche and tertiary lymphocyte composition .