Structural expression of bovine milk and meat factors in tissues of colorectal, lung and pancreatic cancer patients

Chronic inflammation, linked to the presence of bovine milk and meat factors (BMMFs) and specific subsets of macrophages, results in oxygen radical synthesis and induction of mutations in DNA of actively replicating cells and replicating single stranded DNA. Cancers arising from this process have been characterized as indirect carcinogenesis by infectious agents (without persistence of genes of the agent in premalignant or cancers cells). Here, we investigate structural properties of pleomorphic vesicles, regularly identified by staining peritumor tissues of colorectal, lung and pancreatic cancer for expression of BMMF Rep. The latter represents a subgroup of BMMF1 proteins involved in replication of small single‐stranded circular plasmids of BMMF, but most likely also contributing to pleomorphic vesicular structures found in the periphery of colorectal, lung and pancreatic cancers. Structurally dense regions are demonstrated in preselected areas of colorectal cancer, after staining with monoclonal antibodies against BMMF1 Rep. Similar structures were observed in human embryonic cells (HEK293TT) overexpressing Rep. These data suggest that Rep or Rep isoforms contribute to the structural formation of vesicles.


| INTRODUCTION
Similar global incidence data of several human cancers including colorectal cancer (CRC) and breast cancer linked the consumption of bovine meat and dairy products to the etiology. [1][2][3] Additional studies linked lung and pancreatic cancer to milk consumption. 4,5 A search for infectious agents which could be involved, was conducted by fractionation of density gradient centrifugation of bovine serum and analyzing single fractions for structural particles by electron microscopy, as well as DNA extraction and amplification. Bovine meat and milk factors (BMMFs) were initially isolated from these fractions as plasmid-like, episomal DNA molecules. The main open reading frame of all isolates was identified in silico as a putative replication gene (Rep). These isolates were grouped as BMMF1 to BMMF4 based on DNA sequence homology. [6][7][8][9] More than 130 BMMF DNA genomes were subsequently isolated from dairy products. Detailed analyses of their genomes revealed characteristics of both viruses and bacterial plasmids, with genes needed for replication, transcription and translation, frequently all present in a single genome, thereby constituting a novel class of pathogens. 10 The potential role of BMMF as causative agent in CRC was indicated by positive staining of lamina propria in peritumor tissue with antibodies generated against the Rep protein of BMMF1. 4 The mechanistic role for BMMF as infectious agent in indirect carcinogenesis was further elucidated by demonstrating the presence of the Rep protein in CD68 + macrophages present in chronic inflammation, leading to oxygen radical synthesis and induction of mutations in actively replicating crypt cells. 4,10 Isolation of modified BMMF1 H1MSB.1 genomes directly from Rep-positive-stained tissue sections strengthened our data. These modifications led to putative genes involved in deamination and other mutagenic events. 10 Analyses to identify a possible indirect carcinogenic mechanism in the etiology of additional human cancers were conducted, mainly concentrating on tumors with decreased incidence rates during prolonged immune suppression. These included pancreas, lung, breast and prostate cancers which have all been associated with preceding chronic inflammation. 5 BMMF1 Rep monoclonal antibodies were generated and used to analyze several types of cancer tissues by immunohistochemistry (IHC) for the presence of BMMF infections. 11 Table S1). The HEK293TT cell line has been authenticated using short tandem repeat profiling within the last 3 years. All experiments were performed with mycoplasma-free cells.

| Western blot analysis
Western blotting was performed as described before, 12

| Pre-embedding IEM
Serial FFPE sections from human biopsies were used for IHC. Standard procedures for immunolabeling of FFPE material followed by resin-embedding for subsequent ultrathin sectioning were as follows:
Analysis of WGS data was performed using the D-ViSioN (Detection of Integration of Virus by Singletons) workflow. 18 In a first alignment, the Burrows-Wheeler Aligner BWA-MEM (bwa/0.7.15) aligns the WGS reads against a library of BMMF sequences. 10 In a second step, identified reads were aligned to the BMMF library again using Nucleotide BLAST (blastn, ncbi-blast/2.7.1). In a second analysis pipeline, reads that did not align to the human genome GRCh38.p14 (Bowtie2 v.2.3.5.1 19 ) were extracted and assembled de novo by MEGAHIT v. 1    In addition, smaller molecular weight bands, which had been described in a previous study, 12 Figure S4A) and in association with membranes ( Figure S4B). An isotype AB did not react with the target-structure. Transfection with a pcDNA3.1 (À) vector (no Rep insert) did not result in segregation complexes.
Conventional resin TEM, without immunolabeling of BMMFtransfected HEK293TT cells, allows for more effective investigation of transfection-induced segregation patterns ( Figure S4C). Apart from the focal concentration of material within the cytoplasm of cells, association along cell membranes is a regular appearance.

| Rep expression in macrophages and pleomorphic bodies in lung cancer
We performed IHC staining with AB3 to analyze any BMMF expression in LUAD tissue ( Figure 3A). Strong Rep expression was observed in almost the entire cytoplasm of cells in tumor adjacent tissue. These cells were subsequently identified by co-immunofluorescence staining as CD68 + macrophages ( Figure 3B).
In this analysis based on one patient, the majority of CD68 + cells exhibited a congruent CD68 + and Rep expression. We further subjected a consecutive section of the sample to pre-embedding IEM and were able to identify a high number of pleomorphic structures with the same dimensions as those identified in colorectal mucosa ( Figures 1B and 3A, on the right). Application of isotype AB in a consecutive tissue section did not result in comparable staining of cells in IHC ( Figure S5A) and did not stain any target structures in pre-embedding IEM ( Figure S6A).

| Rep expression and pleomorphic bodies are observed in endocrine and exocrine pancreatic tissue
Immunohistological staining with AB3 identified Rep expression in both endocrine and exocrine cells in pancreatic tissues ( Figure 3C).
The extent of the signal was more pronounced in the endocrine islets of Langerhans, which is also reflected by co-immunofluorescence staining for insulin ( Figure 3D

| DISCUSSION
BMMFs pose a novel class of infectious agents based on their similarities to both known viruses, as well as a cryptic bacterial plasmid. 10 Presently, more than 200 isolates have been described based on their DNA genome composition. 6,7,9,10,[21][22][23] BMMF infections have been described as responsible factors in indirect carcinogenesis in CRC and other cancers based on the induction of chronic inflammation. The latter is visualized by increased numbers of CD68 + macrophages. 4,11,12 Expression of BMMF1-encoded Rep was identified by immunohistochemistry in CD68 + macrophages in the peritumor mucosa of CRC patients tested. 12  Cryo-IEM as well as conventional resin TEM, which both allow for more delicate structural preservation compared to pre-embedding IEM of FFPE samples, show BMMF dependent segregation bodies in the cytoplasm of cells with potential to associate with membranes. In all three modes of sample preparation, the detailed view into focal accumulations reveal ultrafine structures like voids and filaments indicative for higher order organization. BMMFs were originally isolated by extracting DNA from density gradient fractions. Nucleic acid was amplified by rolling circle amplification followed by restriction digest. Resulting fragments were cloned and sequenced. 6,13 The failure to identify any structural components in these fractions by conventional electron microscopic examination, led to the speculation of whether these could be composed of a plasmid attached to protein molecules. The isolation and in silico characterization of a large number of other members of the BMMF group, provided additional information which indicated a possible similarity to RNA viruses as mentioned previously. 10

CONFLICT OF INTEREST
The authors declare no potential conflict of interests.

DATA AVAILABILITY STATEMENT
The WGS data generated in our study are available in EGA under accession number EGAS00001006744. Other data that support the findings of our study are available from the corresponding author upon request.

ETHICS STATEMENT
The CRC cohort obtained from the University Hospital Mannheim