Experimental design.

A - Overview of the experimental design and procedures. B - Using technical replicates at the cell suspension level to account for sampling bias in the clonotype frequencies.

Repertoire overlap, clonality and isotype composition.

A - repertoire overlap between pairs of tissues, by F2 metric, repertoires split by immunoglobulin isotype; B - network representation of Ig repertoires from PBMC, LN, and tumor of mp3 patient (melanoma); individual clonotypes of the same origin (PBMC, tumors, or LN) are shown as bubbles connected with the edges to one anchor node. Clonotypes shared between tissues were connected with two or three edges to the corresponding anchor nodes and were located between them. The size of the bubbles represents the relative frequency of clonotypes within a sample, and the color represents the isotype. The relative distance between anchor nodes corresponded to the similarity of repertoires (the number of shared clonotypes). C, D - Clonality of Ig repertoires in PBMC, LN, and tumors of N cancer patients. This reflects the presence of clonal expansion. Calculated as in 58: [1-normalized Shannon-Wiener index]; C–total IGrepertoire; D–IgM repertoire; E–LN/tumor overlap for IgA and IgG repertoires; F– PBMC/tumor overlap for IgA and IgG repertoires; G, H–isotype fraction correlation between PBMC and tumor repertoires (G), or between LN and tum repertoires (H).

CDR3 amino acid properties

A - Mean amino CDR3 length of top 100 most frequent clonotypes from tumor, lymph node and PBMC tissues irrespective of isotype CDR3 are on average significantly higher in tumor than PBMC for total repertoire (p<0.01, two-sided t-test, Bonferroni correction); B - Comparison of mean amino acid CDR3 length of 100 most frequent clonotypes for colon, lung and melanoma cancer samples from, tumor. CDR3s of tumor-infiltrating clonotypes were shorter for colorectal cancer patients compared to melanoma in IgA repertoires (p=0.02); C - Comparison of amino acid properties in the central region of CDR-H3, for total repertoire (C-left) or IGHG repertoire (C-right), all cancers (significantly increased - red, significantly decreased - blue) two-sided t-test, Bonferroni-Holm correction; D - Comparison of amino acid properties in the central region of CDR-H3, for IGHM repertoire, lung cancer (significantly increased - red, significantly decreased - blue); (p<0.01, two-sided t-test, Bonferroni-Holm correction)

Immunoglobulin hypermutation analysis across tissues and isotypes

A - Schematic representation of analysis and triangle plot visualization of clonal group distribution between tissues; B, C, D - clonal group distribution between tissues for colorectal (B), lung (C), and melanoma (D); stars represent non-weighted by size mean center of triangle coordinates. Chi-2 test for goodness of fit was used to test whether each tissue equally contributed to clonal group formation.

LN-to-LN difference of BCR repertoires in colorectal cancer.

A - repertoire overlap between tumor and three separate lymph nodes from the draining lymph node pool; Mann-Whitney test, Bonferroni correction. B: Network representation of Ig repertoires from tumor and three separate lymph nodes, with circles representing individual CDR3 sequences, size of the circles corresponding to the clone frequency, and color corresponding to the isotype; C - triangle plot visualization of clonal group distribution between three different lymph nodes, with size corresponding to the number of individual CDR3 sequences (clonotypes) within a given clonal group, and color corresponding to the percentage of tumor-derived clonotypes–within the clonal group; D–example of a clonal lineage consisting of CDR3 sequences derived from tumor (blue) and all three LNs (shades of red) from patient ccp2, shapes representing isotypes and size representing frequency of a given sequence in a given sample.

Intra-LN and intratumoral heterogeneity

A - repertoire overlap between tumor and lymph node fragments for colorectal cancer patient ccp6 and lung cancer patient lcp3; Mann-Whitney test, Bonferroni corr. B - triangle plot visualisation of clonal group distribution between lymph node fragments, with size corresponding to the number of individual CDR3 sequences (clonotypes) within a given clonal group, and color corresponding to the percentage of tumor-derived clonotypes within the clonal group; C - network representation of Ig repertoires from tumor fragments, with circles representing individual CDR3 sequences, size of the circles corresponding to the clone frequency, and color corresponding to the isotype, edges connect clonotypes to their fragment of origin; D - triangle plot visualization of clonal group distribution between tumor fragments, with size corresponding to the number of individual CDR3 sequences (clonotypes) within a given clonal group, and color representing dominant clonotype; Stars represent non-weighted by size mean center of triangle coordinates. Chi-2 test for goodness of fit is used to test if each tumor segment equally contributes to clonal groups formation.

Expanded clonotypes and hypermutation analysis.

A, B - Visualization of expanded clonotypes on frequency correlation plots for pairs of tumor fragments of melanoma tumor from patient mp3; C - Cytoscape network visualization of top 300 most frequent individual Ig CDR-H3 clonotypes, colored by isotype, with size of circles proportional to the frequency of a given clonotype; D - example of a clonal lineage containing expanded CDR-H3 sequence; G - examples of the biggest clonal lineages, none of which contain expanded CDR-H3 sequences; F - average number of mutations in expanded and non-expanded clonotypes; Mann-Whitney test.