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. 2008 Dec 2;105(48):18936-41.
doi: 10.1073/pnas.0803943105. Epub 2008 Nov 18.

Evidence for a Human-Specific Mechanism for Diet and Antibody-Mediated Inflammation in Carcinoma Progression

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Free PMC article

Evidence for a Human-Specific Mechanism for Diet and Antibody-Mediated Inflammation in Carcinoma Progression

Maria Hedlund et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Patients with cancer have circulating heterophile antibodies that agglutinate animal red cells via recognition of the mammalian cell surface sialic acid N-glycolylneuraminic acid (Neu5Gc), which was long considered an oncofetal antigen in humans. However, humans are genetically deficient in Neu5Gc production and instead metabolically accumulate Neu5Gc from dietary sources, particularly red meats and milk products. Moreover, mice with a human-like defect showed no alternate pathway for Neu5Gc synthesis and even normal humans express anti-Neu5Gc antibodies. We show here that human tumors accumulate Neu5Gc that is covalently attached to multiple classes of glycans. The paradox of human tumor Neu5Gc accumulation in the face of circulating anti-Neu5Gc antibodies was hypothesized to be due to facilitation of tumor progression by the resulting low-grade chronic inflammation. Indeed, murine tumors expressing human-like levels of Neu5Gc show accelerated growth in syngeneic mice with a human-like Neu5Gc deficiency, coincident with the induction of anti-Neu5Gc antibodies and increased infiltration of inflammatory cells. Transfer of polyclonal monospecific syngeneic mouse anti-Neu5Gc serum also enhanced growth of transplanted syngeneic tumors bearing human-like levels of Neu5Gc, with tumors showing evidence for antibody deposition, enhanced angiogenesis and chronic inflammation. These effects were suppressed by a cyclooxygenase-2 inhibitor, a drug type known to reduce human carcinoma risk. Finally, affinity-purified human anti-Neu5Gc antibodies also accelerate growth of Neu5Gc-containing tumors in Neu5Gc-deficient mice. Taken together, the data suggest that the human propensity to develop diet-related carcinomas is contributed to by local chronic inflammation, resulting from interaction of metabolically-accumulated dietary Neu5Gc with circulating anti-Neu5Gc antibodies.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Enhanced growth of Neu5Gc-expressing tumors in Cmah −/− mice with a human-like deficiency in Neu5Gc production. (A) Neu5Gc expressing B16 melanoma cells were injected s.c. into the flank of C57Bl6 wild-type or Cmah−/− mice. Tumors were removed from the flank, weighed, measured, and processed for immunostaining as described under Methods. Pooled data from four experiments, where each dot represents a single animal (P = 0.06). (B) Neu5Gc-expressing tumors growing in Cmah−/− mice show evidence of increased leukocyte infiltration by immunofluorescence staining using anti-Mac-1 antibody (see Inset for an example). There was increased Mac-1 staining of tumors grown in Cmah−/− mice compared to wild-type mice (P = 0.08). (C) Tumor-bearing mice show variable anti-Neu5Gc antibody response in serum collected at the time of tumor harvest. The binding specificity was examined using ELISA with human versus chimpanzee serum (Neu5Gc negative and positive respectively), as described in the Methods. Both anti-Neu5Gc IgG and IgM were significantly elevated in Cmah−/− mice (P < 0.05 and P < 0.005 respectively).
Fig. 2.
Fig. 2.
Generation and characterization of a polyclonal mouse anti-Neu5Gc serum for passive transfer studies. Anti-Neu5Gc antibodies were generated by immunization of Cmah−/− mice using cell membrane preparations from chimpanzee RBC or human RBC (control) in Freund's complete adjuvant. The resulting sera were then adsorbed against human RBCs. (A) Adsorbed mouse anti-Neu5Gc antisera show a polyclonal response against two different Neu5Gc-containing epitopes, as shown here by ELISA using microtiter plates coated with Neu5Gcα-polyacrylamide (Neu5GcαPAA) or Neu5Gcα2–6GalNAc-PAA, (see Methods for further details). (B) Adsorbed anti-Neu5Gc antibodies selectively react with MC-38 tumor cells, but there was no binding of adsorbed serum control to these cells. (C) Passively transferred anti-Neu5Gc antibodies stimulate the growth of transplanted Neu5Gc-positive syngeneic tumors. Cmah−/− mice were injected s.c. with MC-38 adenocarcinoma cells and on day 5 injected I.P. with syngeneic anti-Neu5Gc containing mouse serum. Data were pooled from two experiments, and each dot represents a single animal (P < 0.05).
Fig. 3.
Fig. 3.
Passive transfer of anti-Neu5Gc antibodies induces COX-2 dependent tumor growth, inflammation and increased vascular density. (A) Inhibition of antibody effects on tumor growth by the COX-2 inhibitor NS-398. Tumor-bearing Cmah−/− mice were injected i.p. with 10 mg NS-398 per kg of body weight every 3 days after injection of tumor cells. On day 14–18 tumors were harvested, weighed, and processed for immunohistochemistry, as described under Methods. Data from one representative experiment is shown, with each dot representing a single animal. There was a significant difference between control and anti-Neu5Gc serum treated mice (P < 0.001) and between NS-398-treated or untreated mice (both receiving anti-Neu5Gc serum), (P < 0.005). (B) Evidence of antibody deposition, increased vascular density, and leukocyte infiltration after passive transfer of adsorbed anti-Neu5Gc containing serum. Harvested tumors were analyzed by immunofluorescence using anti-mouse IgG (detecting Ig deposition), anti-CD31 (detecting blood vessels), or anti-Gr-1 (detecting leukocyte infiltration). (C) Positive staining of antibody deposition, angiogenesis, and leukocyte infiltration was quantified using Adobe Photoshop, analyzing three random high power fields of each tumor. Tumors from mice receiving anti-Neu5Gc antibodies displayed significantly increased IgG deposition (P < 0.05), Gr-1 staining (P < 0.01), and increased CD31 staining (P = 0.063). COX-2 inhibition using NS-398 reduced the vascular density (P = 0.058) and leukocyte infiltration (P = 0.052), but left IgG antibody deposition unaltered.
Fig. 4.
Fig. 4.
Human anti-Neu5Gc antibodies enhance tumor growth. (A) Affinity purified human anti-Neu5Gc IgG antibodies were biotinylated and binding to MC-38 cells (intrinsically expressing Neu5Gc) was detected with Cy5-streptavidin by flow cytometry. We used Cy5-streptavidin (without anti-Neu5Gc antibodies) as the control for binding. Data shown is representative of two independent experiments. (B) MC38 cells were injected s.c. into Cmah−/− mice. Five days later, affinity purified human anti-Neu5Gc antibodies in PBS were injected i.p. at 1 μg/g or 0.5 μg/g mouse body weight (n = 8 per group), control mice were injected with PBS (n = 7). On day 18, tumors were harvested and weighed, showing that mice receiving the higher antibody dose had larger tumors relative to the lower dose or control. (*, P = 0.06, **, P = 0.3 in an unpaired t test). (C) Tumor volumes were calculated from daily tumor measurements. The higher dose of antibodies promoted the growth of tumors (two-way-ANOVA; P < 0.01 on day 18) at a higher rate then the lower dose (P > 0.05 on day 18), compared to the control.

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