Bovine Meat and Milk Factor (BMMF) Protein Is Expressed in Macrophages Spread Widely over the Mucosa of Colorectal Cancer Patients

Abstract

Red meat consumption is considered a risk factor for colorectal cancer (CRC) development and stimulated isolation of plasmid-like DNA molecules from bovine serum and milk, termed bovine meat and milk factors (BMMFs). BMMFs encode a conserved replication protein (Rep). Increased populations of Rep-expressing macrophages have been identified in the peritumor of CRC patients and pre-cancerous tissues when compared to the tissues of healthy individuals. This supports the concept that BMMFs increase cancer risk by indirect carcinogenesis, upon induction of chronic inflammation. However, the spread of Rep⁺ immune cells in tissues at greater distances from primary tumors has not yet been assessed. Here, we immunohistologically analyzed the presence of Rep⁺ immune cells in sets of tumor, peritumor and, additionally, distant tissues of CRC patients (n = 13). We identified consistently high numbers of BMMF-positive macrophages in mucosal tissues at distances of as much as 25 cm away from the primary tumors, at levels comparable to peritumors and associated with M2-like macrophage polarization. The broad distribution of BMMFs suggests that BMMF⁺ macrophages might already exist at stages of pre-cancerous dysplasia or before. Quantification of BMMF tissue expression during colonoscopy might help to preventively stratify individuals at risk of developing polyps/CRC and recommend them for enhanced surveillance and/or changes in dietary lifestyle.

Keywords: bovine meat and milk factors; chronic inflammation; colorectal cancer; diet; indirect carcinogenesis; macrophages; myeloid immune checkpoint.

Figure 1

Immunohistochemical detection of BMMF Rep protein expression in different tissue parts of CRC patients. Hematoxylin and eosin (H&E) and IHC DAB staining of FFPE tissue sections with anti-IgG1 (isotype control), anti-Rep (AB3) antibodies of tumor, peritumor and tumor-distant tissues (taken at 12, 17 or 25 cm distance from primary tumors) from 3 different representative CRC patients (representation of IgG isotype control staining: tumor distant tissue, scale bar: 100 μm, magnification of enlargements: 2×).

Figure 2

Immunohistochemical detection of BMMF Rep and CD68 in peritumor and tumor-distant tissues of CRC patients. IHC DAB staining with anti-Rep antibodies AB3 and AB10 or anti-CD68 antibodies applied on consecutive FFPE tissue sections of tumor-distant or peritumor tissue of 3 representative CRC patients (IgG Ct: isotype control, scale bar: 50 μm, magnification of enlargements: 2×).

Figure 3

Co-immunofluorescence (IF) staining of Rep⁺, CD68⁺ and CD163⁺ cells in tumor, peritumor and tumor-distant tissue regions of CRC patients. Representative images of IF staining for DAPI (blue), anti-Rep (AB3 and 10, red), anti-CD68 (green) and anti-CD163 (purple) of tumor-distant, peritumor, and tumor tissue from 2 representative CRC patients (scale bar: 50 μm, magnification of enlargements for dashed selection frames: 5×).

Figure 4

Quantification of Rep/CD68/CD163-positive cells by co-immunofluorescence staining in tumor-distant, peritumor and tumor tissues. Quantification of the number of Rep⁺ cells (% of all interstitial cells) (A), CD68⁺ macrophages (% of all interstitial cells) (B), Rep⁺CD68⁺ macrophages (MPs) (% of all interstitial cells) (C) and Rep⁺CD68⁺ MPs (% with respect to interstitial MPs) (D). (E) Quantification of Rep-positive M2-like (CD68⁺CD163⁺) or non-M2-like (CD68⁺CD163^-) macrophages (% with respect to all interstitial cells). (F) Log2 ratio of M2-like vs. non-M2-like macrophages (left) and Rep-positive M2-like vs. non-M2-like macrophages (right) (error bars indicating maximum/minimum). (G) Comparison of Rep⁺ cells, CD68⁺ MPs and Rep⁺CD68⁺ MPs (% with respect to all interstitial cells) in peritumor and tumor-distant tissues of 13 different CRC donors. Tumor-distant tissues were resected in regions at 12 cm (n = 4), 17 cm (n = 5) or 25 cm (n = 4) distance from the primary tumor. Correlation of Rep⁺ cells (H) and CD68⁺ macrophages (I) within pairwise comparisons of tumor, peritumor or tumor-distant tissues (Pearson’s correlation). Paired (two-sided) t-test used for statistical analysis of different tissue regions from individuals—tumor-distant (n = 13), peritumor (n = 12) and tumor (n = 10). ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 5

Summary of the fraction of BMMF Rep-positive macrophages among all macrophages identified in previous publications, labeled ‘PNAS’ [4], ‘MolOncol’ [12] and in the present study (‘Current’) (data presented as medians with interquartile range). Tissues were from healthy non-cancerous individuals (black, n = 8 and 10 donors, respectively), CRC tumors (red, n = 7, 26, and 10, respectively), CRC peritumors (blue, n = 7, 26, and 12, respectively), tissues distant to CRC primary tumors (green, n = 4, 5 and 4, respectively) or tissues adjacent to high- or low-grade dysplasia (grey, HGD, LGD, n = 14 and 9, respectively).