Focus Biosciences II - HP-F13: Molecular and

Immunological Methods in Tumor Virology

Analysis of chronic inflammatory lesions of the colon

for BMMF Rep antigen expression and CD68
macrophage interactions
Timo Bund, Ekaterina Nikitina, … Ethel-Michele de Villiers
PNAS March 23, 2021

Supervisor:
Dr. Timo Bund

Student presenter:
Toros Taşgın

11/23/2021 1
Red Meat Consumption: Cancer Risk Factor

• Long time consumption of meat and dairy

products of bovine origin increases the risk of
colon cancer by approximately 20-30 %, and
linked to increased mortality rate.1

• Positively correlates with incidence of breast

cancer and Multiple Sclerosis (MS).2

• Carcinogenic aromatic carbohydrates and

nitrosamine derivatives (broiling or frying)
responsible for the increased cancer risk.3

Teresa Norat et al., (2007), doi: 10.1093/jnci/dji164

• What could be the leading factor(s) that explain
this strong association ?
Bovine Meat and Milk Factors (BMMF)
• Several studies have isolated numerous virus-like agents
from bovine serum, commercially available milk, human
blood and tissue samples from MS patients.4,5

• Grouped into four, according to their nucleotide similarity to

known DNA molecules.4

• BMMF1: Circular single-stranded episomal-DNA agents4

• All isolates contains:

- at least one ORF encoding Rep protein (putative replication

protein) which shows variable amino-acid similarity among
the isolates.
- AT-rich palindromic structure, and iteron-like repeat region
upstream of the Rep protein, which enable autonomous
replication.
Schematic view of the novel episomal circular DNAs isolated from milk, bovine serum
and tissue and blood samples from MS patients (adapted from zur Hausen, Bund and - Lack capsid protein
de Villiers 2017).
 Possible BMMF-pathogenesis
1. Bioactivity  replicative (persist in the cell) and
transcriptional activity in human cells.5
2. Host gene expression alteration (cell cycle
progression, and cell viability control) and
induction of local inflammation.5
3. DNA isolation from and detection of Rep protein in
colon cancer tissue samples.4
Persistent infections  induction of chronic inflammation
via promoting M2 macrophage differentiation (MDSCs) 
elevation of local ROS/RNS + other pro-tumorigenic factors
 increased DNA damage  increased carcinogenesis7,8,9
• BMMF were proposed to be indirect carcinogens by
inducing chronic inflammation, and subsequent DNA
damage in replicating cells, which provides room for
carcinogenic transformation.

4. Bacterial prion-like RepA protein  inducing

ROS/RNS dependent cellular stress, horizontally
transmissible.6
Estimated annual global cancer incidence due to infections, Haraldzur Hausen (2009),
https://doi.org/10.1016/j.virol.2009.06.001
Monoclonal Antibodies against Rep protein
Detection of BMMF Antigens in Colon Cancer Patient Tissue
Is there an association between BMMF infection and colon cancer?
Detection of BMMF Antigens in Colon Cancer Patient Tissue
Western Blot Analysis

Purified H1MSB.1
Rep protein

densitometrical
quantification
Detection of BMMF Antigens in Colon Cancer Patient Tissue
Western Blot Analysis

• Abs 2/3/5/10 revealed positive stained

bands, the rest is inconclusive.

• Stronger WB detection obtained in

peritumor tissues (majority) in comparison
to tumor samples.
• Experimental variation and insufficient
separation of both sample types unable
direct comparison.

• For 15 out of 16 CRC patients, and for all

adenoma polyp samples, Rep protein
was detected.
Detection of BMMF Antigens in Colon Cancer Patient Tissue
Is there an association between BMMF infection and colon cancer?
IHC
Detection of BMMF Antigens in Colon Cancer Patient Tissue
IHC
Detection of BMMF Antigens in Colon Cancer Patient Tissue

Immunohistochemistry (IHC) results

• Positive staining was observed in the

interstitial propria of all peritumor
samples
• No staining in epithelial cells at the
crypts of Lieberkühn.

• Both WB and IHC indicate the presence

of BMMF specifically in peritumor
tissue, and adenomatous polyps
(weaker staining).
Isolation of BMMF DNA from peritumor colon cancer tissue

Cloning

PCR
(with BMMF specific primers) Sequencing

Patients peritumor tumor

4798 ✅ -----
Rolling Circle Amplification 4799 ✅ ❌
(with random hexamers)
Laser Micro Dissection (LMD) 4808 ✅ ❌
4809 ✅ ❌
Association between BMMF and CRC - Summary
Association between BMMF and Local Infiltrating Immune Cells

Peritumor samples

Control Colon samples

Detection of BMMF Antigens in local Macrophages
CD68 1. Glycosylated transmembrane protein
2. Member of scavenger receptor family
3. Macrophage-specific inflammation marker
Detection of BMMF Antigens in local Lymphocytes

• No Rep+ B and T cell detected in

any of the peritumor samples

• Macrophage-driven inflammation
Detection of ROS/RNS dependent DNA damage

Downstream chronic
inflammation marker
Detection of ROS/RNS dependent DNA damage
Exposure of Stem and Progenitor cells to ROS/RNS
Summary

1. BMMF1 antigen (Rep) was detected in the lamina propria between the colon crypts in
peritumor colon cancer tissues via WB and IHC.

2. Presence of BMMF1 DNA was verified by LMD-PCR-sequencing in the same peritumor areas.

3. Colocalization of Rep protein with interstitial macrophages was shown and quantified via
fluorescence IHC. Elevated levels of Rep+/CD68+ inflammatory macrophages was detected
in peritumor samples in comparison to the cancer-free patients.

4. Association of Rep protein with chronic inflammation was further verified by the detection
of ROS/RNS dependent DNA damage marker in areas with increased concentration of Rep
protein.

5. Detection of Ki67+ crypt cells in the vicinity of Rep+ macrophages.

1. zur Hausen, H. (2012). Red meat consumption and cancer: Reasons to suspect involvement of bovine
infectious factors in colorectal cancer. International Journal Of Cancer 130, 2475-2483.
2. Pala., V. et al. Meat, eggs, dairy products, and risk of breast cancer in the european prospective investigation into cancer
and nutrition (EPIC) cohort. Am. J. Clin. Nutr. 90, 602–612 (2009).
3. Sugimura, T. et al., Mutagen carcinogens in food, with special reference to highly mutagenic pyrolytic products in broiled
foods. Cold Spring Harbor, 156177 (1977).
4. de Villiers, E., Gunst, K., Chakraborty, D., Ernst, C., Bund, T., and zur Hausen, H. (2019). A specific class of infectious agents
isolated from bovine serum and dairy products and peritumoral colon cancer tissue. Emerging Microbes & Infections 8,
1205-1218.
5. Eilebrecht, S., Hotz-Wagenblatt, A., Sarachaga, V., Burk, A., Falida, K., Chakraborty, D., Nikitina, E., Tessmer, C., Whitley, C.,
and Sauerland, C. et al. (2018). Expression and replication of virus-like circular DNA in human cells. Scientific Reports 8.
6. Revilla-García A, Fernández C, Moreno-del Álamo M, de los Ríos V, Vorberg IM, Giraldo R. 2020. Intercellular transmission
of a synthetic bacterial cytotoxic prion-like protein in mammalian cells. mBio 11:e02937-19.
https://doi.org/10.1128/mBio.02937-19.
7. Parisi, L., Gini, E., Baci, D., Tremolati, M., Fanuli, M., Bassani, B., Farronato, G., Bruno, A., and Mortara, L. (2018).
Macrophage Polarization in Chronic Inflammatory Diseases: Killers or Builders?. Journal Of Immunology Research 2018, 1-
25.
8. Veglia, F., Sanseviero, E. & Gabrilovich, D.I. Myeloid-derived suppressor cells in the era of increasing myeloid cell diversity.
Nat Rev Immunol (2021). https://doi.org/10.1038/s41577-020-00490-y
9. Benoit, M., Desnues, B., and Mege, J. (2008). Macrophage Polarization in Bacterial Infections. The Journal Of Immunology
181, 3733-3739.
Possible BMMF-pathogenesis
1. Recent studies demonstrated the replicative
(persist in the cell) and transcriptional activity of
BMMF1 isolates in human cells.5
2. BMMF (Rep) expression leads host gene expression
alterations (cell cycle progression, and cell viability
control), and induction of immune response.5
3. BMMF DNA isolation from and detection of Rep in
colon cancer tissue samples.4
4. A bacterial prion-like RepA protein was shown to
be capable of inducing ROS/RNS dependent cellular
stress, and being horizontally transmitted.6
• Possible mechanism7,8: promoting M2 macrophage
(MDSCs) differentiation ?  establishment of
chronic-inflammation + suppression of tumor
clearing lymphoid activity  elevated ROS/RNS
release
• BMMF were proposed to be indirect carcinogens by
inducing chronic inflammation, and subsequent
DNA damage in replicating cells, which provides
room for carcinogenic transformation.
Infection Agents: Cancer Risk Factor

• 20% of the global cancer burden can be linked to

infectious agents: viruses, bacteria, parasites.

• Schistosoma hematobium – a major cause of

bladder cancer in Egypt.

• What could be the link between bovine product

consumption and increased cancer incidence in
the context of infectious agents ?

Estimated annual global cancer incidence due to infections, Haraldzur Hausen

(2009), https://doi.org/10.1016/j.virol.2009.06.001
Possible BMMF-pathogenesis
1. Bioactivity  replicative (persist in the cell) and
transcriptional activity in human cells.5
2. Host gene expression alteration (cell cycle
progression, and cell viability control) and
induction of local inflammation.5
3. DNA isolation from and detection of Rep protein in
colon cancer tissue samples.4
Infections  induction M2 program9
• Link between BMMF and M2 macrophage differentiation7,8:
pro-tumorigenic TME  chronic inflammation lacking
lymphoid cell activity
• BMMF were proposed to be indirect carcinogens by
inducing chronic inflammation, and subsequent DNA
damage in replicating cells, which provides room for
carcinogenic transformation.

4. Bacterial prion-like RepA protein  inducing

ROS/RNS dependent cellular stress, horizontally
transmissible.6