Protumor Steering of Cancer Inflammation by p50 NF-κB Enhances Colorectal Cancer Progression

p50 NF-κB promotes CAC by hampering M1/Th1 inflammation. To induce CAC, mice were treated with AOM and DSS. A, Top: Body weight monitored every 2 to 3 days during the experimental period (multi t test, starting from day 10; *, FDR < 0.05; N = 16). Bottom: Colon length measured at the time of mice sacrifice (day 100). Data shown are mean ± SEM of different mice from two independent experiments. ***, P < 0.001 by two-tailed Mann–Whitney test. Untreated WT: N = 6; untreated p50^–/–: N = 6; AOM/DSS-treated WT: N = 17; AOM/DSS-treated p50^–/–: N = 12. Center: Ulceration and degree of inflammation (colitis score) analyzed on colon swiss roll sections by H&E. Data shown are mean ± SEM of different mice from two independent experiments. *, P < 0.05 by two-tailed Mann–Whitney test. WT: N = 11; p50^–/–: N = 9. B, Colons were longitudinally opened and polyps were counted. Data shown are mean ± SEM of different mice from two independent experiments. ***, P < 0.001 by two-tailed Mann–Whitney test. WT: N = 11; p50^–/–: N = 9. Representative images are shown. The total number and the size of tumors were counted and tumor burden was calculated for each animal. Data shown are mean ± SEM of different mice or tumors from two independent experiments. *, P < 0.05; ***, P < 0.001 by two-tailed Mann–Whitney test. N = 11 different WT mice; N = 9 different p50^–/– mice; N = 52 WT tumors; N = 9 p50^–/– tumors). Representative images are shown. Scale bars: 1,000 μm. C, Top: Total RNA from tumors of AOM/DSS-treated WT and p50^–/– mice analyzed for the expression of the genes belonging to clusters 1 and 2; bottom: additional Th1/M1 genes. Results are expressed as fold induction over WT tumor expression. Data shown are mean ± SEM of different mice from two independent experiments. *, P < 0.05 by one-tailed Mann–Whitney test N = 13 WT mice; N = 15 p50^–/– mice. D, Top: FACS analysis of colorectal cancer lesions for selective M1 gene products in TAMs (CD11b⁺F4/80⁺ cells) and bottom: the frequency of IFNγ-expressing T cells. Intracellular expression of cytokines (IL12, TNFα, and IFNγ) was measured upon 3 hours of stimulation with 50 ng/mL PMA and 1 μg/mL ionomycin in presence of 5 μg/mL Brefeldin A. Data shown are mean ± SEM. *, P < 0.05; **, P < 0.01 by two-tailed t test. N = 6 WT mice; N = 5 p50^–/– mice. Center: Representative FACS histograms of M1 markers are shown.

Administration of M1 cytokines inhibits CAC development. A, Treatment regimen with IL12 and CXCL10 during CAC induction. B, Body weight loss during the treatment. *, FDR < 0.05 by multi t test of treated versus vehicle. N = 10 vehicle-treated mice; N = 7 IL12-treated mice; N = 8 CXCL10 treated mice. C, Colon length of mice treated with vehicle, IL12, or CXCL10, and D, analysis of tumor development (number of polyps) was performed at day 80 on longitudinally opened colons. Representative images are shown. Data shown are mean ± SEM. *, P < 0.05 by one-tailed Mann–Whitney test. N = 5 vehicle-treated mice; N = 6 IL12-treated mice; N = 6 CXCL10-treated mice.

Lack of p50 in Apc^Min mice inhibits spontaneous intestinal tumor development. A, Histological analysis of small gut and colons harvested from Apc^Min and Apc^Minp50^–/– mice at 12 or 18 weeks of age. Number and size of tumors was monitored, and tumor burden calculated for each mouse. Data shown are mean ± SEM of different mice or tumors. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-tailed Mann–Whitney test. N = 7 Apc^Min mice 12 weeks old; N = 8 Apc^Minp50^–/– mice 12 weeks old; N = 13 Apc^Min mice 18 weeks old; N = 15 Apc^Minp50^–/– mice 18 weeks old; N = 76 tumors from Apc^Min mice 12 weeks old; N = 39 tumors from Apc^Minp50^–/– mice 12 weeks old; N = 155 tumors from Apc^Min mice 18 weeks old; N = 133 tumors from Apc^Minp50^–/– mice 18 weeks old). B, Survival time of Apc^Min and Apc^Minp50^–/– mice. ***, P < 0.0001 by log-rank Mantel–Cox test. N = 54 Apc^Min mice; N = 26 Apc^Minp50^–/– mice. C and D, expression of gene clusters 1 and 2, and additional Th1/M1 genes in colorectal cancer lesions from Apc^Min mice versus C, the adjacent healthy colonic mucosa and D, colorectal cancer lesions from Apc^Minp50^–/– mice. Data shown are mean ± SEM. *, P < 0.05 by one-tailed, Mann–Whitney test. N = 6 Apc^Min mice; N = 6 Apc^Minp50^–/– mice.

Modulation of gut-associated leukocyte populations by p50 NF-κB. A, Formalin-fixed and paraffin-embedded colons from AOM/DSS-treated and untreated mice (WT and p50^–/–) were evaluated for the number of macrophages (F4/80⁺), monocytes (Ly6C⁺), neutrophils (Ly6G⁺), and T lymphocytes (CD3⁺) by immunohistochemistry. *, P < 0.05; **, P < 0.01; ***, P < 0.001 one-tailed Mann–Whitney test of WT (N = 5) versus p50^–/– (N = 4) mice. B, Immunofluorescence analysis of frozen colonic samples were performed to evaluate NK (CD3^–NKp46⁺) and NKT (CD3⁺NKp46⁺) cells. Data shown are mean ± SEM of different tumors (WT N = 15; p50^–/–N = 5) or fields (WT N = 23; p50^–/–N = 23) of different mice. *, P < 0.05; **, P < 0.01 one-tailed Mann–Whitney test of WT (N = 6) versus p50^–/– (N = 3) mice. C, FACS analysis of colorectal cancer lesions for the frequency of the depicted immune cell populations. Data shown are mean ± SEM of different mice. **, P < 0.01; ***, P < 0.001 two-tailed t test. N = 4 WT mice; N = 4 p50^–/– mice. D, Transcripts of genes encoding for markers of different leukocyte populations from healthy colons and tumors of untreated and AOM/DSS-treated mice. Results are shown as fold induction over healthy untreated WT mice. Data shown are mean ± SEM. *, P < 0.05 by one-tailed Mann–Whitney test. N = 4 untreated WT mice; N = 4 untreated p50^–/– mice; N = 5 AOM/DSS-treated WT mice; N = 6 AOM/DSS-treated p50^–/– mice). E, T cells were depleted from WT and p50^–/– mice during the entire experimental period by i.p. injections of anti-CD4 (α-CD4) and anti-CD8 (α-CD8). Control mice received vehicle (−) only. Tumor development was evaluated. Representative images of longitudinally opened colons at day 80 are shown. Data shown are mean ± SEM. *, P < 0.05 two-tailed Kruskal–Wallis test N = 11 vehicle-treated WT mice; N = 10 vehicle-treated p50^–/– mice; N = 10 anti-CD4/CD8–treated WT mice; N = 7 anti-CD4/CD8–treated p50^–/– mice).

Lack of p50 increases apoptosis of both colonic epithelial and tumor cells after AOM/DSS administration. A, Activated cleaved caspase-3 in colon sections from AOM/DSS-treated and untreated mice by immunohistochemistry and digital image analysis. Representative images are shown. Data shown are mean ± SEM of different fields. Scale bars, 20 μm. *, P < 0.05; **, P < 0.01 by one-tailed Mann–Whitney test. N = 4 fields from 1 untreated WT mice; N = 4 fields from 1 untreated p50^–/– mice; N = 16 total tumor fields and N = 14 total nontumor fields from 5 different AOM/DSS-treated WT mice; N = 8 total tumor fields and N = 14 total nontumor fields from 3 different AOM/DSS-treated p50^–/– mice. B, Transcripts of survival genes were evaluated in total RNA isolated from colon and tumor lesions of untreated and AOM/DSS-treated mice. Normalized qPCR results are shown as fold induction over healthy untreated WT mice. Data shown are mean ± SEM. *, P < 0.05 by one-tailed Mann–Whitney test. N = 2 untreated WT mice; N = 2 untreated p50^–/– mice; N = 5 AOM/DSS-treated WT mice; N = 6 AOM/DSS-treated p50^–/– mice.