Mapping the spreading routes of lymphatic metastases in human colorectal cancer

Abstract

Lymphatic metastases are closely associated with tumor relapse and reduced survival in colorectal cancer (CRC). How tumor cells disseminate within the lymphatic network remains largely unknown. Here, we analyze the subclonal structure of 94 tumor samples, covering the primary tumors, lymph node metastases (LNMs), and liver metastases from 10 CRC patients. We portray a high-resolution lymphatic metastatic map for CRC by dividing LNMs into paracolic, intermediate, and central subgroups. Among the 61 metastatic routes identified, 38 (62.3%) are initiated from the primary tumors, 22 (36.1%) from LNMs, and 1 from liver metastasis (1.6%). In 5 patients, we find 6 LNMs that reseed 2 or more LNMs. We summarize 3 diverse modes of metastasis in CRC and show that skip spreading of tumor cells within the lymphatic network is common. Our study sheds light on the complicated metastatic pattern in CRC and has great clinical implications.

Fig. 1. Research strategy.

a Case screening pipeline. Ten treatment-naive CRC patients with multiple LNMs were selected for this study. b Schematic showing CRC patients with LNMs and LMs. Spatial distribution and classification of paracolic (purple), intermediate (green), and central (brown) LNMs are shown on the left. Lymph nodes without tumor cell are colored gray. Multiple tumor regions (2–5 regions according to tumor size) were isolated from the primary tumor and liver metastasis. c Two representative whole-slide LNM sections stained with hematoxylin and eosin showing the tumor regions isolated by laser capture microdissection. Tumor regions (marked by red dashed lines) were marked by pathologists. Scale bar = 2.5 mm.

Fig. 2. Genomic landscape of 10 CRC patients.

a The top panel shows the number of mutations identified in each tumor sample. The spatial location of each tumor sample is denoted below. The second panel details seven recurrent driver genes (mutated in ≥2 patients) with the mutation type indicated. The third panel details recurrent genes associated with putative neoantigens. The bottom panel shows recurrent CNAs: dark red for amplifications (CN ≥ 4), light red for gains (2 < CN < 4), dark blue for deletions (CN = 0), and light blue for losses (0 < CN < 2). b Percentage of mutations shared by all the tumor samples, two or more tumor samples, and private to only one tumor sample in each patient, respectively. c Somatic mutations and d Neoantigens shared between the primary tumors and metastases.

Fig. 3. Clonal evolutionary history and parsimonious metastatic map of Patient_8.

a Oval plots showing the subclonal structure of tumor samples from Patient_8. Each row represents a sample. Ovals in the same color represent the same mutation clusters and are denoted by numbers. The area of each oval is proportional to its CCF value. Subclones are shown with solid borders. Subclonal structures are illustrated by the nested ovals to the left. The mutation cluster clonal in all lesions (cluster 1) is the trunk cluster, representing the most recent ancestor clone (MRCA). Other mutation clusters (clusters 2–7) shared by two or more lesions are defined as branch clusters, representing branch subclones. White asterisks denote monoclonal metastases. b Clonal evolutionary tree inferred from the subclonal structure. Lengths of lines are proportional to the number of substitutions in each cluster. Selected aberrations are labeled accordingly. LOH, loss of heterozygosity. c Clonal evolutionary history. The dashed box shows the predicted historical states of the primary tumor. Horizontal arrows denote acquisition of new subclones and tumor progression. Vertical fishbone-like arrows denote metastases, which are labeled with involved clone/subclones. Each colored line corresponds to a clone/subclone. d Parsimonious metastatic map based on the clonal evolutionary history. Each metastasis is colored by the smallest involved subclone.

Fig. 4. Clonal structures of 9 CRC cases.

a Oval plots for 9 CRC cases. Clones and subclones are denoted as in Fig. 3. b Phylogenetic trees were constructed from the subclonal structure of each patient. The trees were rescaled to fit the plot and are denoted as in Fig. 3b. Detailed genomic events of each patient are shown in Supplementary Figs. 3–11.

Fig. 5. Parsimonious metastatic maps and three modes of metastasis in CRC.

a, b Parsimonious metastatic routes for nine patients. Metastatic routes between lesions are denoted by arrows and colored according to the smallest involved subclone (see Fig. 4). For instance, in Patient_2, P2 is seeded by the red subclone in the primary tumor, thus this route is labeled in red. P3 was then seeded by the red and green subclones from P2, so this route is labeled in green, since the green subclone was smaller. Three metastatic modes were extracted from 61 metastatic events: c inter-layer sequential spread, d inter-layer skip spread, and e intra-layer spread. The metastatic network is dissected into five layers, spanning from T to P, I, C, and LM. The arrows indicate metastatic routes.