Human colorectal cancer cells frequently express IgG and display unique Ig repertoire

Figure 1 Proportions of Ig classes in colon cancer cells and normal epithelial cells. A: The process of immunoglobulin heavy chain (IgH) gene rearrangement and the structure of rearranged IgH; B: Design of the primers for the arm-polymerase chain reaction (PCR) technology used to amplify the immune repertoire. During the first round of PCR, multiple forward primers Fo (forward-out) and Fi (forward-in) were used to target V genes. The reverse primers Ro (reverse-out) and Ri (reverse-in) were targeted to the 5 classes of IgH. The Fi and Ri primers included sequencing adaptors. The second round PCR was carried out with communal primers B and A. The barcodes were in between primer A and the C gene specific primers; C: Proportions of the five IgH classes in cancer and normal cells; D: The proportion of IgG in the 5 patients. Small horizontal lines indicate the mean ± SD. Statistical significance was determined by a two-tailed unpaired Student’s t-test. ^bP < 0.01.

Figure 2 The restricted VDJ patterns and distribution of immunoglobulin heavy chain in cancer and normal cells. A: V-J-CDR3 map of immunoglobulin heavy chain (IgH) expressed in normal B cells[26], colon cancer cells and normal epithelial cells. Each rectangle represents a unique V-J-CDR3 nucleotide sequence and the size denotes its relative frequency. Colors for each rectangle are chosen randomly and, thus, do not match between plots; B: The distribution of V_Hs expressed in cancer and normal epithelial cells; C: The utilizations of V_Hs in cancer and normal epithelial cells. The order of V_Hs on the X-axis corresponds to its position on a chromosome; D: Utilizations of 7 V_H and 6 J_H families in cancer and normal cells from patients with colon cancer (red and blue columns), and B cells from peripheral blood of a healthy donor[28] (orange columns). Small horizontal lines indicate the mean ± SEM. All data comparing cancer with normal cells were determined by the two-tailed unpaired Student’s t-test and none of the differences were significant (P > 0.05). The percentage of V_H and J_H families in B cells (D) was derived from the data from other sources[28]; thus, statistical analysis was not performed.

Figure 3 Different V_H3-23/D/J_H usages in cancer and normal cells. The top three V_H3-23/D/J_H and their percentage to total V_H3-23 rearrangements in cancer cells and normal epithelial cells of the first 3 patients. The same color represents the same V, D or J segment.

Figure 4 Somatic hypermutation in colon cancer and normal epithelial cells. A: Mutation rates of V_H3-23 in cancer and normal cells. Dotted lines represent the cut-off value 2%; B: Mutant positions and corresponding frequencies of V_H3-23 in different patients. P1: Patient-1. P2: Patient-2. The X-axis represents the 1^st to 354^th nucleotides using IMGT-numbering; C: Representative mutant positions of V_H3-74/D6-19/J_H4 in cancer and normal cells of patient-3 compared to the germline sequence of V_H3-74. Small horizontal lines indicate the mean ± SEM. All data comparing cancer with normal cells were determined by the two-tailed unpaired Student’s t-test. ^cP < 0.001. ^dP < 0.0001.