ABSTRACT _Fusobacterium nucleatum_ is an oral anaerobe recently found to be prevalent in human colorectal cancer (CRC) where it is associated with poor treatment outcome. In mice,

hematogenous _F. nucleatum_ can colonize CRC tissue using its lectin Fap2, which attaches to tumor-displayed Gal-GalNAc. Here, we show that Gal-GalNAc levels increase as human breast cancer

progresses, and that occurrence of _F. nucleatum_ gDNA in breast cancer samples correlates with high Gal-GalNAc levels. We demonstrate Fap2-dependent binding of the bacterium to breast

cancer samples, which is inhibited by GalNAc. Intravascularly inoculated Fap2-expressing _F. nucleatum_ ATCC 23726 specifically colonize mice mammary tumors, whereas Fap2-deficient bacteria

INTRACELLULAR BACTERIA IN CANCER—PROSPECTS AND DEBATES Article Open access 09 October 2023 THE ADHESIN RADD ENHANCES _FUSOBACTERIUM NUCLEATUM_ TUMOUR COLONIZATION AND COLORECTAL

CARCINOGENESIS Article 21 August 2024 KILLING TUMOR-ASSOCIATED BACTERIA WITH A LIPOSOMAL ANTIBIOTIC GENERATES NEOANTIGENS THAT INDUCE ANTI-TUMOR IMMUNE RESPONSES Article 25 September 2023

INTRODUCTION _Fusobacterium nucleatum_ is a common oral non-spore forming gram-negative anaerobe that has long been known to be associated with the development of periodontal disease. More

recently, however, genomic studies provided evidence that _F. nucleatum_ may also be prevalent in colorectal carcinoma1,2. In support of this proposed link with cancer, subsequent studies

demonstrated that _F. nucleatum_ promotes tumorigenesis3,4, affects infiltration of tumor-infiltrating lymphocytes5,6,7, inhibits killing of cancer cells by Natural Killer (NK) cells and

tumor-infiltrating T cells8, and induces resistance to chemotherapy in colon cancer9,10. Moreover, treatment of mice bearing a colon cancer xenograft with antibiotics to eliminate _F.

nucleatum_ concomitantly reduced cancer cell proliferation and overall tumor growth11. Together, these CRC-related fusobacterial effects provide a rational for the observation that high

numbers of _F. nucleatum_ in CRC tissue is inversely correlated with overall survival12 (also reviewed in ref. 13). In addition to CRC, a high burden of _F. nucleatum_ is also associated

with poor prognosis in esophageal cancer14,15. CRC-associated fusobacteria originate from the oral microbial community, and we have previously hypothesized that they reach the colon via the

hematogenous rather than the gastrointestinal route16. In this model, _F. nucleatum_ will enter the bloodstream during transient bacteremia, which is frequent in periodontal disease, and

specifically dock to CRC tissue through its surface-exposed lectin Fap2. Fap2 recognizes Gal-GalNAc, which is a sugar that is abundantly displayed by CRC cells16. We hypothesized that, if

oral _F. nucleatum_ can translocate to colon tumors hematogenously, this bacterium may also reach Gal-GalNAc-displaying tumors in other organs via this route. To address this, we recently

screened different cancer tissue samples for abundant Gal-GalNAc. This screen identified breast cancer as a strong candidate, which was in agreement with previous studies that had detected

Gal-GalNAc in breast cancer17,18,19,20 or observed high accumulation of DNA from the genera _Fusobacterium_ in the microbiome of malignant but not benign breast tissue samples21. Breast

cancer is the most commonly diagnosed cancer and the leading cause of cancer mortality in women22. Therefore, it is important to understand if _F. nucleatum_ has the same profound impact on

the progression and outcome of breast cancer as it has in CRC. Our present study provides several lines of molecular and experimental evidence for this to be the case. We demonstrate that

Gal-GalNAc levels increase along the progression of human breast cancer and that _F. nucleatum_ DNA is overabundant in human breast cancer samples, particularly in those with high Gal-GalNAc

signals. Using two different murine orthotropic models, we show that _F. nucleatum_ colonizes mammary tumors via abundant Gal-GalNAc on tumor cells, with the same Fap2 lectin whereby

recognizes CRC cells. Furthermore, we find that the colonization of mammary tumors by _F. nucleatum_ accelerates breast cancer progression and metastatic development, most likely through

suppression of T cell accumulation in the tumor microenvironment. Importantly, breast tumor exacerbation by _F. nucleatum_ in mice can be counteracted by antibiotic treatment with

metronidazole. RESULTS GAL-GALNAC IS OVERDISPLAYED IN BREAST CANCER We have shown previously that CRC-specific attachment and colonization by _F. nucleatum_ is facilitated by high Gal-GalNAc

levels on CRC cells, and that the Fap2-mediated recognition of this sugar is inhibited with soluble GalNAc or removal of the cell-exposed sugar with O-glycanase16. In addition,

Fap2-deficient _F. nucleatum_ showed impaired attachment to CRC samples, and much reduced CRC colonization in a mouse model16. Since Gal-GalNAc (also known as the Thomsen–Friedenreich

antigen) was known to be displayed in breast cancer17,18,19,20, we hypothesized that _F. nucleatum_ would target breast cancer tissue in a similar manner. To this end, we stained tissue

microarrays containing human breast cancer and matched adjacent non-tumor tissue samples using fluorescein isothiocyanate (FITC)-labeled peanut agglutinin (PNA), a Gal-GalNAc specific

lectin. In 25 of 30 tested paired samples, Gal-GalNAc levels in the tumor samples were higher than those in the matched normal tissue (Fig. 1a). In addition, breast cancer samples showed

significantly higher (_p_ = 0.0003) Gal-GalNAc levels than the matched adjacent non-tumor tissue (Fig. 1a). We then compared Gal-GalNAc levels in samples from normal human breast tissue,

breast tumor tissue including benign tumors, and from different stages along the progression of the disease. Gal-GalNAc signals were lowest in normal [median florescence intensity (MFI) 7.12

 × 105] and hyperplasia tissues (MFI 7.55 × 105). Atypical hyperplasia (MFI 1.73 × 107) and malignant (MFI 3.50 × 106) tissue displayed 24- and 4.7-fold, respectively, higher levels of

Gal-GalNAc than did normal tissue (Fig. 1b). Note that the high median of Gal-GalNAc levels in atypical hyperplasia might be also due to the low number of available samples. Interestingly,

benign tumors (MFI 1.89 × 104) showed significantly lower Gal-GalNAc levels than did the different malignant tumors (all with an MFI higher than 1.68 × 105), and overall did not

significantly differ from normal tissue (MFI 4.7 × 104) (Fig. 1c). Comparing by subgroups of breast cancer, mucinous adenocarcinoma displayed at least two orders of magnitude higher amounts

of Gal-GalNAc over the other five cancer subgroups tested (Fig. 1c). Of the latter, mucinous breast cancer showed the highest Gal-GalNAc levels in the mucin layers, whereas in ductal breast

cancer high Gal-GalNAc levels were observed in necrotic regions, ducts, and in the ductal lumen (Fig. 1d–g). _F. NUCLEATUM_ GDNA IS OVERABUNDANT IN BREAST CANCER Next, we tested whether _F.

nucleatum_ occurred in breast cancer, and if so, whether its presence correlates with high Gal-GalNAc levels. For this purpose, we searched for _F. nucleatum_-specific sequences in 50 deep

sequencing libraries of PCR-amplified bacterial 16S rDNA from different breast cancer samples. Since breast tumors have low bacterial biomass, we included as negative controls for possible

signals from contaminating DNA, 50 empty DNA extractions (controls) and 50 no-template PCR controls (NTCs). DNA from 21 CRC samples was included as positive controls. We detected _F.

nucleatum_ in 30% and 57% of the analyzed breast tumors and CRC samples, respectively (Fig. 2a, b). Importantly, _F. nucleatum_ was predominantly found in those breast cancer samples that

displayed high Gal-GalNAc levels (Fig. 2c). These results not only confirm the previously observed prevalence of the _Fusobacterium_ genera in the human breast cancer microbiome21, they also

indicate a significant role of Fap2-Gal-GalNAc interactions in fusobacterial colonization of breast cancer tissue. FAP2 BINDS THROUGH GAL-GALNAC DISPLAYED ON BREAST CANCER In our previous

work establishing the Fap2 outer membrane protein as a major bacterial Gal-GalNAc lectin, we generated two Fap2-deficient mutants, K50 and D22, of _F. nucleatum_ ATCC 2372616,23. Here, we

used these strains to assay whether _F. nucleatum_ through Fap2 specifically attaches also to breast cancer cells. Using microarrays containing samples of malignant and non-cancer (normal

and benign) human breast tissue, we found that the Fap2-proficient wild-type strain (WT) ATCC 23726 exhibited significantly higher attachment to malignant breast cancer sections (median

21.96 Fn/mm2, Fig. 3a) than to normal (median 7.26 Fn/mm2) or benign breast tumor (median 8.59 Fn/mm2) sections. By contrast, the Fap2-deficient K50 mutant showed significantly impaired

attachment to malignant breast cancer tissue (median 12.03 Fn/mm2), compared with the wild-type parental strain (median 21.96 Fn/mm2), while its attachment to normal breast sections (median

6.20 Fn/mm2) or benign tissue (median 5.41 Fn/mm2) was similar to WT (Fig. 3a). Of note, while the Fap2-deficient K50 mutant still attached better to breast cancer tissue, as compared with

normal or benign sections, this tumor-specific attachment was not statistically significant. We speculate that other _F. nucleatum_ tumor-binding factors such as FadA3, or the

CEACAM1-binding CbpF outer-surface protein24 may enable _F. nucleatum_ to recognize breast cancer cells even in the absence of Fap2. Next, we used flow cytometry to compare attachment of WT

versus Fap2-deficient _F. nucleatum_ to several breast cancer cell lines, i.e., murine AT3 and 4T1, and human MCF-7. Mouse melanoma cell line, B16, served as negative control. Based on PNA

binding, all three tested breast cancer cell lines displayed higher Gal-GalNAc levels than the B16 melanoma cell line (Fig. 3b, Supplementary Fig. 4). Similarly, _F. nucleatum_ was found to

attach only to the breast cancer cell lines. Supporting the predicted Fap2-mediated recognition of Gal-GalNAc, wild-type _F. nucleatum_ ATCC 23726 showed significantly higher attachment than

the K50 and D22 mutants (Fig. 3b). Addition of soluble GalNAc suppressed attachment, most likely by saturation of the Fap2 lectin (Fig. 3b). Interestingly, both wild-type and Fap2-deficient

bacteria invariably attached better to AT3 cells than to MCF-7 or 4T1 cells, suggesting that the AT3 cell line possesses additional (surface) factors for fusobacterial attachment. In

conclusion, the above results strongly argue _F. nucleatum_ primarily uses its Fap2 lectin to home to breast cancer tissue with high levels of Gal-GalNAc. _F. NUCLEATUM_ PREFERENTIALLY

COLONIZES BREAST CANCER Transient bacteremia is common in periodontal disease13,25,26, and provides an opportunity for oral fusobacteria to enter the circulatory system not only during

dental treatment but also during daily routine. Once in the bloodstream, previously oral _F. nucleatum_ might translocate to breast cancers hematogenously. To test this, we simulated

transient bacteremia by intravascular injection of _F. nucleatum_ in the 4T1 orthotropic BALB/c mouse model of mammary cancer. In this model, 4T1 cells are transplanted into the mammary fat

pad of female BALB/c mice where they are highly tumorigenic and invasive. As illustrated in Fig. 4a, we transplanted 1 × 106 4T1 cells into the mammary fat pad, and when tumors reached a

size of 500 mm3, mice were inoculated once with 5 × 107 _F. nucleatum_ ATCC 23726 by tail vein injection. Analysis of tissue 24 h post inoculation revealed that, consistent with our human

breast cancer samples (see above), Gal-GalNAc levels (measured using FITC-labeled PNA) in the mouse cancer sections were higher (6.8-fold when comparing the medians) than in sections of

matching normal tissues (Fig. 4b, right panel). We observed an at least two orders of magnitude higher median abundance of the bacteria, using _F. nucleatum_ gDNA as a proxy, in the tumor

sections than in the matching normal tissue (Fig. 4c). By contrast, we failed to detect bacteria in the mammary of control tumor-free mice (No tumor, Fig. 4c) treated the same way,

indicating that blood-borne _F. nucleatum_ require the presence of a tumor to localize to breast tissue. FAP2 MEDIATES BREAST CANCER COLONIZATION BY _F. NUCLEATUM_ To validate the observed

mammary cancer colonization by _F. nucleatum_ in a different animal model and evaluate the role of Fap2 in the process, we used the AT3 orthotropic mammary cancer model in C57BL/6 mice. Mice

with tumors were generated as described above for the 4T1 model, yet this time injected with either wild-type _F. nucleatum_ ATCC 23726, Fap2-deficient _F. nucleatum_ K50, or the

Gram-negative, anaerobic periodontal bacterium _Porphyromonas gingivalis_ (ATCC 33277) as a control. Note that the latter species has been reported to be overabundant in oral squamous cell

carcinoma27,28,29,30. Tumors and unaffected tissue from an adjacent mammary were analyzed 24 h post inoculation (Fig. 4d). As in the BALB/c model above, _F. nucleatum_ ATCC 23726 was

enriched in the tumor compared with adjacent normal tissue, as determined by both, colony counting and qPCR (Fig. 4e, left and right panels respectively). Fap2 deficiency (mutant K50)

resulted in an at least a three orders of magnitude drop in mammary tumor colonization, as compared with WT bacteria (Fig. 4e). Interestingly, these results closely match those obtained with

a CRC mouse model16. They imply that while neoplastic tissue certainly plays a critical role in enriching _F. nucleatum_ in tumors, breast cancer-specific enrichment by _F. nucleatum_ is

crucially driven by Fap2. Also in agreement with CRC colonization by _F. nucleatum_20, breast tumors are not colonized by just any oral anaerobic bacterium associated with periodontitis.

This is to say that in mice inoculated with _P. gingivalis_, bacterial numbers in tumors were below the detection limit of culturing (∼2 CFU/g tissue) or qPCR (Fig. 4e). Thus, _F. nucleatum_

likely possess distinctive features for tumor colonization, such as Fap2 (the focus of this work), FadA3 or CbpF24,31. Finally, in order to directly visualize fusobacterial breast cancer

colonization, we tracked FITC-labeled _F. nucleatum_ ATCC 23726 in animals by multiphoton microscopy. To this end, we inoculated mammary tumor-bearing C57BL/6 (AT3 orthotropic mammary cancer

model, see above) with 5 × 107 FITC-labeled bacteria or injected PBS (the vehicle used to inject the bacteria) as control. Twenty-four hours post inoculation, we detected signals of

fusobacteria in all tumor samples, whereas there was no signal in any of the adjacent normal mammary samples, nor in samples harvested from the sham-inoculated mice (Fig. 4f). ANTIBIOTICS

INHIBIT _F. NUCLEATUM_-INDUCED TUMOR EXACERBATION Given that colonization by _F. nucleatum_ accelerates the formation of colonic tumor3,4,11, we used the AT3 orthotropic mammary cancer model

to test whether it affects tumor progression in breast cancer as well, following the scheme in Fig. 5a. When tumors reached 500 mm3 in size (day 1), mice were randomly divided into five

treatment (intravascular injection or inoculation) groups: PBS vehicle (V), wild-type _F. nucleatum_ ATCC 23726 (726) or a Fap2 mutant (K50), _F. nucleatum_ ATCC 23726 and additional

treatment with metronidazole (726+MTZ.), or metronidazole alone (MTZ.). Starting with metronidazole (MTZ.) or PBS (V), bacteria were inoculated 30 min later. Treatment with metronidazole or

PBS vehicle was repeated after 8 h, twice a day (morning and evening) in the following 2 days (days 2–3), and daily for 4 more days. On day 8, mice were sacrificed, and tumor and lungs were

harvested to weigh breast cancer tissue and count lung metastases, respectively. As shown in Fig. 5b, mice inoculated with _F. nucleatum_ ATCC 23726, but not with Fap2-deficient K50

bacteria, displayed significantly larger tumors than those in the control group. Metronidazole treatment alone did not affect tumor size (Fig. 5b), but did prevent tumor enlargement in mice

inoculated with fusobacteria (Fig. 5b, c). The median number of lung metastases in mice inoculated with _F. nucleatum_ ATCC 23726 was at least two orders of magnitude higher than in the

sham-inoculated mice (Fig. 5e). The _F. nucleatum_-driven increase in metastases numbers and size was also readily visible in mice bearing GFP-expressing AT3 tumors (Fig. 5f). By contrast,

Fap2-deficient bacteria (K50), while promoting some increase in the number of metastases, did not cause a statistical significant higher burden of metastases. As with tumor size,

metronidazole treatment prevented the pro-metastasis effect of _F. nucleatum_ (Fig. 5e). _F. NUCLEATUM_ MODULATES IMMUNITY AGAINST MAMMARY TUMORS We considered several different mechanisms

whereby _F. nucleatum_ induces an increase in breast tumor size: (i) enhancing proliferation of the cancer cells; (ii) inhibiting apoptosis of the cancer cells; or (iii) manipulating

anti-tumor immunity. Immunohistochemistry staining for the Ki67 proliferation marker or the apoptosis marker CC3 cleaved caspase 3 revealed no differences between _F. nucleatum_-infected and

sham-infected tumors (Supplementary Fig. 1). Similarly, we found no indication for increased proliferation or reduced apoptosis in an RNA-seq based comparison of AT3 cells incubated for 24 

h with or without _F. nucleatum_ ATCC 23726 (Supplementary Fig. 3, Supplementary Data 1 and 2). These results suggested that breast tumor acceleration by _F. nucleatum_ in the AT3 mouse

model might be immune-mediated. We previously discovered that _F. nucleatum_ can inhibit anti-tumor immunity by activating two human (though not the mouse homologs of) immune-suppression

checkpoint receptors, TIGIT and CEACAM18,31. In addition, tissue load of _F. nucleatum_ was previously found to be inversely correlated with density of tumor-infiltrating lymphocytes in

colorectal carcinoma tumors5,6,7. Therefore, to determine whether immune cell accumulation might be affected by _F. nucleatum_, we used flow cytometry to compare levels of NK cells, CD4+ T

cells and CD8+ T cells between AT3 mammary tumors from _F. nucleatum_-infected and uninfected mice. This analysis showed that inoculation by _F. nucleatum_ resulted in fewer CD4+ and CD8+ T

cells (Fig. 6a, Supplementary Fig. 5a–c). To evaluate the involvement of T, B, and NK cells in fusobacterial acceleration of the progression of the AT3 tumor, we repeated our _F. nucleatum_

inoculation scheme in SCID-beige mice, which lack T, B, and NK cells. Unlike in C57BL/6 mice, AT3 tumors grown in uninfected SCID-beige mice were not smaller than those in the infected mice

(Fig. 6b). We interpret this finding to suggest that in the immune-competent C57BL/6 mice, growth of AT3 breast tumor is restricted by T, B, or NK cells, and that _F. nucleatum_ directly or

indirectly dampens this immune-mediated tumor control. Searching the above-mentioned RNA-seq data for additional potential mechanisms whereby the presence of _F. nucleatum_ might accelerate

development of breast tumors, we noticed an overexpression of matrix metalloproteinase 9 in the AT3 cells incubated with _F. nucleatum_ (Supplementary Fig. 3). Proteases of the matrix

metalloproteinase (MMPs) family play vital roles in many biological processes that involve matrix remodeling. In particular, MMP-9 activity has been related to cancer pathology including

invasion, angiogenesis, and metastasis32. While many MMPs expressed in breast cancer are produced by stromal cells, MMP-9 is produced mainly by the tumor cells themselves32. Echoing this

general patterns, a gel zymography assay with gelatin as substrate showed unaltered secretion of MMP-2 by cell lines AT3 (murine mammary tumor) or MDA-MB-231 (human breast cancer) incubated

with _F. nucleatum_ ATCC 23726 or with _F. nucleatum_ ATCC 25586; by contrast, the secretion of MMP-9 was significantly and invariably increased when both cell lines where incubated with

each of the two _F. nucleatum_ strains (Fig. 6c). Therefore, in addition to immune modulation as the putative major mechanism of _F. nucleatum_ in the AT3 mouse model in C57BL/6 mice,

induction of MMP might be another mechanism whereby _F. nucleatum_ accelerates breast tumor progression. DISCUSSION The involvement of viruses in tumor development has been investigated for

over a century, whereas the interest in potentially similar roles of bacteria in cancer biology has been rather recent, with the primary focus being the carcinogenic role of _Helicobacter

pylori_ in gastric cancer33,34. Periodontitis is a common bacterial-induced inflammatory disease. Existing data provide support for positive association between periodontitis and risk of

cancer35,36, particularly oral, lung, and pancreatic cancers37. A recent meta-analysis also predicted that periodontal disease increased the risk of breast cancer by 1.22-fold38. Of the many

different bacteria presently linked to cancer development, _F. nucleatum_ has so far been associated only with colorectal and esophageal cancers. Being an oral microbe, it was plausible

that _F. nucleatum_ reaches CRC tissue by descending through the digestive tract4. However, our recent finding that oral _F. nucleatum_ might probably colonize CRC via the hematogenous

route, and its selectivity for tumors that display Gal-GalNAc16,20, suggested that additional tumors might be colonized by this bacterium. As to a specific candidate cancer, we have

demonstrated here that Gal-GalNAc levels increase as breast cancer progresses (Fig. 1a, b), supporting earlier reports of Gal-GalNAc detection in breast cancer samples17,18,19,20. Breast

cancer represents a sequence of events, starting with non-neoplastic epithelium and subsequent progress through the stages of hyperplasia, atypical hyperplasia, carcinoma in situ and

invasive adenocarcinoma. It has been hypothesized that the conversion between benign hyperplasia and carcinoma in situ (the stage preceding invasive carcinoma) is made at the transition from

hyperplasia to atypical ductal hyperplasia39. Interestingly, we observed the sharpest rise in Gal-GalNAc levels in this very transition phase (Fig. 1b). In addition, Gal-GalNAc levels in

breast cancer were 4.7-fold higher than in normal tissue (Fig. 1b), but only 1.4-fold higher than in matched normal adjacent tissue (Fig. 1a). These results suggest that the proximal tumor

also affects the adjacent non-tumor tissue, causing aberrant Gal-GalNAc levels in the latter. These results are in line with previous ones demonstrating that normal tumor adjacent tissue

represents an intermediate state between healthy and tumor40. The sum of our in vitro and in vivo results strongly support a model whereby _F. nucleatum_ colonizes not only colorectal

cancer16, but also breast cancer through recognition of Gal-GalNAc by Fap2. They also imply, that as in CRC3,4,16, colonization of breast cancer by _F. nucleatum_ is secondary to tumor

initiation: in the absence of tumors, no _F. nucleatum_ bacteria were detectable in the mouse mammary glands. Colonization likely occurs as blood supply is attracted to the tumor, enabling

sufficient bacterial trafficking from the mouth, and after tumor Gal-GalNAc levels raised. Importantly and in agreement with its role in CRC3,4, colonization by _F. nucleatum_ accelerated

the progression of mammary tumor, yet strictly in a Fap2-dependent manner (Fig. 5b, c). That is, tumors were not enlarged in mice inoculated with Fap2-deficient bacteria. Since _F.

nucleatum_ only activates human TIGIT and CEACAM1 and not their murine homologs8,31, tumor acceleration in the mouse models used here cannot involve impaired T cell and NK cell

killing-activity via the activation of these important checkpoints. Nonetheless, our observation of reduced CD4+ and CD8+ T cell levels in _F._ _nucleatum_-infected tumors (Fig. 6a), and the

lack of acceleration of AT3 tumors by _F. nucleatum_ in SCID-beige mice (Fig. 6b), both indicate a role of immunity in fusobacterial tumor acceleration in the AT3 mouse model. Thus, it

appears that in the immune-competent C57BL/6 mice, growth of AT3 breast tumor is normally restricted by NK, B, or T cells, but in the presence of _F. nucleatum_ T cells become reduced,

resulting in tumor growth acceleration. The reduction of immune cells might involve apoptosis. Apoptosis was shown to be induced by _F. nucleatum_ in lymphocytes, through its lectin Fap241.

Importantly, we expect the pro-tumorigenic effect of _F. nucleatum_ to be much stronger in humans because the activity of the NK and few T cells present in the tumor will be further weakened

by inhibitory interactions of Fap2 with TIGIT and of CbpF with CEACAM1. There are several important similarities of our results with previous observations with CRC. First, lower numbers of

T cells have also been reported in CRC samples containing _F. nucleatum_5,6,7. Second, metronidazole treatment to target _F. nucleatum_ reduces tumor growth not only in CRC11, but also

prevents fusobacteria-associated acceleration of breast cancer (Fig. 5b, c). Since the metronidazole treatment had no effect on tumor development in the absence of _F. nucleatum_, it must be

the tissue sterilization by the antibiotic that nullifies the fusobacterial-mediated tumor acceleration (Fig. 5b). Metastasis causes as much as 90% of cancer-associated mortality in

general, and is also the main cause of death by breast cancer. Whereas patients with localized breast cancer have a 5-year survival rate of 98%, this drops to 26% in patients with metastatic

breast cancer42. In our mouse model, we observed not only an enlargement of primary AT3 mammary tumors by _F. nucleatum_, but also a significant increase in the number of lung metastases

(Fig. 5e, f). The fusobacterial-driven increase in metastasis was clearly dependent on Fap2, i.e., it was not statistically significant with the Fap2-deficient K50 mutant (Fig. 5e). Further

work is needed to delineate the pro-metastasis mechanisms of _F. nucleatum_, for example, whether the increase in metastasis load is due to _F. nucleatum_ increasing the size of the primary

tumors. However, if we normalize metastases number by tumor weight, we see a higher metastasis burden in the lungs of mice with _F. nucleatum_ than in those without bacterial inoculation

(Supplementary Fig. 2). In other words, there are more lung metastases in the presence of _F. nucleatum_, even if the primary tumors are of the same size as in non-inoculated mice. In

addition, 8 days post _F. nucleatum_ infection may be too short to detect new metastasis with H&E staining used here. We therefore conclude for now that it was the _F. nucleatum_-driven

growth acceleration of pre-infection metastases that led us to observe more metastases at the end of the experiment. This hypothesis is also supported by the visualization of lungs

metastases in mice implanted with GFP-expressing AT3 cells, where the metastases in mice infected with _F. nucleatum_ are clearly larger than those in the sham-infected mice (Fig. 5f).

Finally, our current study supports the previous important observation of overabundant DNA of the genus _Fusobacterium_ in the human breast cancer microbiome21. Moreover, we show here that

_F. nucleatum_ DNA signals in human breast cancer correlate with the extent of Gal-GalNAc displayed by the tumors (Fig. 2c). Together, these results provide strong evidence for a model

whereby _F. nucleatum_ generally reaches tumors via the hematogenous route and specifically attaches to them via a bacterial lectin-host sugar (Fap2-Gal-GalNAc) interaction. This new

knowledge, combined with our demonstration that metronidazole treatment counteracted acceleration of development and metastatic progression in mammary tumors (Fig. 5), suggests that

targeting _F. nucleatum_ might benefit treatment not only of CRC11 but also of breast cancer. METHODS HUMAN SAMPLE ACQUISITION AND DNA EXTRACTION Tumor samples were collected and analyzed

according to IRB-approved protocols by the Sheba Medical Center and the Maccabi Health Care Services. Fifty formalin-fixed paraffin-embedded (FFPE) samples from breast tumors and 21 fresh

frozen colon tumor samples were collected after informed consent was obtained. DNA was extracted with the UltraClean Tissue & Cells DNA Isolation kit (MoBio) according to the

manufacturer’s instructions with a 10 min bead beating step (Vortex adapter –Qiagen) before proteinase K digestion. Extraction of DNA from FFPE samples was done as described above with some

adaptations. Briefly: samples were deparaffinized prior to digestion with proteinase K. Lysates were bead beated with 0.1 mm zirconia/silica beads (Biospec) for 10 min. Fifty empty tubes

were added to the DNA extraction and were subjected to 16S sequencing together with sample DNA in order to detect and monitor contaminating bacterial DNA. 16S SEQUENCING AND DATA ANALYSIS

16S-rDNA amplification and sequencing were performed using a novel method that allows the processing of DNA from FFPE samples and increases the resolution of the assay because it amplifies

five regions on the 16S rRNA gene. The method is described in detail in ref. 43. Fifty no-template controls (NTC) were added to the PCR amplification and further processed into libraries to

differentiate (together with the extraction controls) contaminating bacterial DNA from true signal in the samples. Five regions of the 16S rRNA gene were amplified using 100 ng DNA as an

input and a set of 10 multiplexed primers (Supplementary Table 1). Sequencing libraries were loaded together with 20% of PhiX on an Illumina Hi-seq or Mi-seq system. Reads were demultiplexed

per sample, filtered and aligned to each of the five amplified regions based on the primers’ sequences. The SMURF package was applied to combine read counts from the five regions into a

coherent profiling results solving a maximum likelihood problem44. The Greengenes database (May 2013 version) was used as a reference. BACTERIAL STRAINS AND GROWTH CONDITIONS _F. nucleatum_

strains ATCC 25586, ATCC 23726, the isogenic Fap2-inactivated mutants K50, D22 and _P. gingivalis_ ATCC 33277 were grown in Wilkins Chalgren broth (Oxoid, UK) or on Columbia agar plates

(Oxoid, UK) supplemented with 5% defibrinated sheep blood (Novamed, Israel). The bacteria were grown in an anaerobic chamber (Bactron I–II Shellab, USA) in an atmosphere of 90% N2, 5% CO2,

and 5% H2 at 37 °C. Thiamphenicol (2.5 µg/ml) was added for K50 and D22. TISSUE MICROARRAY ANALYSIS Breast cancer tissue array BR1006, array BR1003a, and HBre-Duc060CS-01 (US Biomax) were

used in these studies. Details about the cases for each core on the array are available on the US Biomax Web site (https://www.biomax.us/). FLUORESCENCE MICROSCOPY AND SECTION PREPARATION

TMA slides were stained with H&E or processed for immunofluorescence microscopy. TMA slides went through deparaffinization as follows: xylene twice 5 min, xylene 1 min, xylene/ethanol

(50%/50%) 1 min, ethanol twice 10 min, 90% ethanol 2 min, 80% ethanol 2 min, 70% ethanol 2 min and then washed in DDW. Slides went under heat-induced epitope retrieval using sodium citrate

10 mM pH 6.0 in a microwave for 20 min, then 40 min in room temperature and washed 5 min in PBS. For PNA (Sigma-Aldrich), anti-CK-7 (Abcam) and anti-von willebrand factor (Dako) binding,

sections were blocked with PBS supplemented with 10% BSA, 10% FBS, and 5% Triton for 2 h at room temperature followed by incubation with FITC-labeled PNA (50 µg/ml in PBS), anti-CK-7

(1:200), or anti-von willebrand factor (1:500) overnight at 4 °C. The slides were then washed three times with PBS for 15 min and for anti-CK-7 and anti-von willebrand factor a second

antibody Cy5 and Cy3 was added, respectively (Jackson, 1:100), for 2 h, washed three times with PBS for 15 min and then incubated with Hoechst 33258 diluted 1:5000 for 15 min at room

temperature. Fluorescence intensity FITC-labeled PNA was evaluated using the ImagePro Analyzer 7.0 software (Cybernetics, USA). For _F. nucleatum_ binding, bacteria were labeled with Cy5 or

Cy3 (PA25001 Life Sciences GE) solution diluted 0.1 mg/ml in PBS. Sections were blocked with TBS (0.05 M Tris-HCl [pH 7.8], 0.1 M NaCl) supplemented with 20% BSA, 20% FBS, and 5% Triton for

6 h at room temperature, followed by incubation with the labeled bacteria (3 × 107 bacteria/ml blocking solution) for 6 h at room temperature and then overnight at 4 °C. The slides were then

washed once with PBS + TWEEN 0.5% followed by two washes with PBS for 15 min each, and then incubated with Hoechst 33258 diluted 1:5000 for 15 min at room temperature. IMMUNOHISTOCHEMISTRY

Two slides 10-µm thick and 10 µm apart were prepared from the center of each formalin-fixed paraffin-embedded tumor. Slides were stained with Hematoxylin and one for ki67 (ɑ

ki67—ThermoScientific (SP6) RM—9106-SO, diluted x 200) and the other for CC3 (ɑ CC3—Cell Signaling—9661S, diluted x 300). 10 randomly areas from each slide were captured in ×20 lens. Binary

area (µm2) of ten areas were averaged for both DAB (+) and Hematoxylin (+). Each symbol represents the ratio between the average of DAB (+) to Hematoxylin (+) in the tumor. CELL LINES AND

TISSUE CULTURE The human breast cancer cell line MCF-7 and MDA-MB-231, mouse BALB/c breast cancer model cell line 4T1, mouse C57BL/6 breast cancer model cell line AT3, and mouse C57BL/6

melanoma model cell line B16, were cultured with DMEM with 10% fetal bovine serum, 1% L-Glutamine, penicillin–streptomycin and amino acid (Biological Industries). FLOW CYTOMETRY _F.

nucleatum_ strains ATCC 23726 and the isogenic Fap2-inactivated mutants K50, D22 (109 CFU/ml) were labeled with fluorescein isothiocyanate (FITC, 0.1 mg/ml in PBS; Sigma-Aldrich) for 30 min

at room temperature and washed three times in PBS. FITC-labeled bacteria were used at a multiplicity of infection of 3. Bacteria were incubated with cells in 96-well plates, for 30 min at

room temperature and washed twice prior to flow cytometry (LSRFortessa Analyzer, BD, USA). Analysis was performed using FlowJo 10.0.8 software (Tree Star, Ashland, OR, USA). FITC-labeled PNA

lectin (Sigma-Aldrich) was incubated at a final concentration of 140 nM per 2.5 × 105 cells per well. For competition experiments, bacteria were incubated with GalNAc (concentration range:

0, 25, 100, and 400 mM) for 30 min prior to incubation with cells. ANIMAL MODELS All mouse experiments were carried out under protocol MD-17-15239-5 approved by the Hebrew University of

Jerusalem Ethics Committee and signed by the chairman Prof. Sara Eyal. Six- to seven-week-old female Balb/C, C57BL/c, and SCID-beige mice were purchased from Envigo (Israel). Mice were kept

at a relative humidity 40–70%, 20–24 °C, and in 12 h dark/light cycles (07:00–19:00 light). IN VIVO BREAST CANCER COLONIZATION Female BALB/c mice were orthotopically (mammary fat pad)

injected with 1 × 106 4T1 tumor cells. When tumor reached 500 mm3 size, mice were injected intravenously with 5 × 107 _F. nucleatum_ ATCC 23726. Female C57BL/c mice were orthotopically

(mammary fat pad) injected with 1 × 106 AT3 tumor cells. At tumor size of 500 mm3, mice were randomly divided to three groups and injected intravenously with 5 × 107 _F. nucleatum_ ATCC

23726, _F. nucleatum_ MUT K50 and _P. gingivalis_ ATCC 33277. After 24 h, breast tumor and normal tissue from an adjacent mammary were harvested from each mice and homogenized under sterile

conditions. QUANTIFICATION OF BACTERIA USING PLATING AND QPCR Tumor and non-tumor adjacent tissue samples were homogenized in 500 µl of PBS for 45 s at 4.5 m/s using a FastPrep (MP

Biomedicals, USA) and plated on Columbia agar plates supplemented with 0.15% (final concentration) crystal violet and 5% (final concentration) defibrinated sheep blood45. Colonies were

enumerated after 7 days of incubation under anaerobic conditions. DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Germany) according to manufacturer’s instructions. A

custom TaqMan primer/probe set was used to amplify _F. nucleatum_ and _P. gingivalis_ DNA. The cycle threshold (Ct) values were normalized to the amount of murine gDNA in each reaction by

using a primer/probe set for the reference gene (Gapdh). Each reaction contained 1 ng of DNA and was assayed in triplicate in 20 µL reactions containing 2× qPCRBIO Lo-ROX Probe Mix as

appropriate for individual qPCR machines. Reaction conditions were as follows: 2 min at 50 °C, 10 min at 95 °C, and 40 cycles of 15 s at 95 °C, and 1 min at 60 °C16. The sequences of all

primers can be found in Supplementary Table 1. MULTIPHOTON MICROSCOPY OF FITC-LABELED _F. NUCLEATUM_ AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old female

C57BL/6 mice. At tumor size of 500 mm3, mice were randomly divided to two groups; one group injected intravenous with 5 × 107 FITC-labeled _F. nucleatum_ ATCC 23726 (_n_ = 5) and the second

control group, with PBS vehicle (_n_ = 3). After 24 h, breast tumor tissue was harvested from each mice. A slice from the fresh tissue was cut and placed on a slide with a drop of water and

covered with a cover slip. Image sections were taken using a Nikon Multiphoton A1MP set to 740 nm wavelength using ×25 objectives. EFFECT OF _F. NUCLEATUM_ ON THE PROGRESSION OF MAMMARY

TUMOR AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old female C57BL/6 mice. When tumor reached 500 mm3 size (day 1), mice were randomly divided into five IV

injected treatment groups: PBS (V), 5 × 107 _F. nucleatum_ ATCC 23726 and treated with metronidazole (726+MTZ.), metronidazole only (MTZ.), 5 × 107 _F. nucleatum_ ATCC 23726 treated with PBS

vehicle (726), or with 5 × 107 Fap2-deficient _F. nucleatum_ K50 treated with PBS (K50). For metronidazole or vehicle treatment, mice were injected with metronidazole or PBS 30 min before

IV inoculation with _F. nucleatum_. Treatment with metronidazole or vehicle was repeated after 8 h, twice a day (morning and evening) in the following 2 days (days 2–3), and daily for 4 more

days. On day 8 mice were sacrificed, and tumors and lungs were harvested. Breast cancer tissue was weighed and metastases were counted by histological examination (experimental scheme can

be seen Fig. 4a). When the GFP-expressing AT3 breast cancer cell line was implanted in C57BL/6 mice, relatively few mice developed cancer presumably due to anti-GFP immunity. In mice in

which breast tumor was developed, metastases were visualized using Nikon SMZ25 fluorescent binocular in an ×0.5 objectives. CHARACTERIZATION OF AT3 TUMOR IMMUNE INFILTRATE Lymphocytes were

purified from homogenized primary tumors by centrifugation on Lymphoprep (STEMCELL Technologies, Cat number 07851). Lymphocytes were characterized using anti-mouse CD3-allophyco-cyanin clone

145-2C11 (Cat. number 100312, BioLegend) for T cells together with either anti-mouse CD4 PE clone GK1.5 (Cat. number 100408, BioLegend) or anti-mouse CD8 PE clone 53-6.7 (Cat. number

100708, BioLegend). For the characterization of NK cells we used anti-mouse NKp46 (CD335, NCR1) APC clone 29A1.4 (Cat. number 137608, BioLegend). RNA-SEQ Infection of AT3 cells was carried

out as describe before46 with the following modifications. Two days prior infection 2 × 105 AT3 cells were seeded in six-well plates and 2 ml complete DMEM. The plates were transferred to an

incubator with O2-control set to 1% O2 and the media exchanged after 24 h with complete DMEM without antibiotics. A day before infection, _F. nucleatum_ ATCC 23726 was inoculated in

Columbia broth under anaerobic conditions. On the next day, the culture was diluted 1:50 in fresh Columbia broth and grown to an OD600 of 0.2. Bacterial cells were harvested (5 min at 4400 ×

 _g_ in room temperature) resuspended in fresh, reduced complete DMEM (lacking antibiotics) and added to the AT3 cells at a multiplicity of infection (m.o.i.) of 10. The plates were

centrifuged for 5 min at 250 × _g_ at room temperature and placed back into the O2-controlled incubator. Twenty-four hours after infection, the cells were washed once with PBS before

extracting total RNA using the mirVana kit (Ambion) following the manufacturer’s instructions. The infection was performed in three biological replicates. cDNA libraries for Illumina

sequencing were generated by the Core Unit SysMed (University Würzburg). Five hundred nanograms of total RNA were used for the library generation using oligo-dT capture beads for poly-A-mRNA

enrichment via the TruSeq Stranded mRNA Library Preparation Kit (Illumina) according to manufacturer’s instructions. The success of the library preparation was controlled by electrophoresis

on Agilent High Sensitivity Bioanalyzer microfluidic chips prior to sequencing of pooled libraries (including 1% PhiX control library). For sequencing the single-end mode on the NextSeq 500

platform (Illumina) with the High-Output Kit v2.5 (75 cycles) was used. The generated RNA-seq data was deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series

accession number GSE144143. FASTQ files were generated and quality filtered using the local run manager software from Illumina (v2.2.0) and FastQC (v.0.11.7;

https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Mapping was performed using the READemption47 tool (v0.4.3) using the _Mus muculus_ reference sequence provided by ENSEMBL

(GRCm38.p6). After mapping, strand-specific gene-wise quantification was performed using READemption based upon the annotation provided by ENSEMBL (GRCm38.p6). Only uniquely mapped reads

were used as input for differential expression analysis via the edgeR package (v3.24.3)48,49 using an upper-quartile normalization and a prior count of 1. Only gene showing at least 10

uniquely mapped reads were considered for the analysis and all genes with an adjusted _p-_value of 0.05 and passing the log2 fold change threshold (−2 ≤ log2FC ≥ 2) were considered

significant differentially expressed. In order to assess enrichment of genes involved in different processes a gene set enrichment analysis (GSEA50,51 v4.0.3) was performed using the curated

Molecular Signatures Database (MSigDB 7.0) for GO terms (biological processes) available via GSEA. The log2 fold changes reported by the edgeR analysis were used as input for the GSEA which

was run in ranked list mode (statistic setting: classic) and limited to gene sets with 15–500 genes. An overview of all GO terms reported with an FDR-corrected _p_-value ≤0.05 are reported

in Supplementary Data 1 and the top 15 based upon the normalized enrichment score are shown in Supplementary Fig. 3. MMPS DETECTION USING GEL ZYMOGRAPY AT3 and MDA-MB-231 cells were cultured

on 6-well plate at 37 °C in DMEM medium supplemented with 10% serum and 1% L-glutamine for 24 h to reach subconfluent density under microaerobic conditions using the Oxoid CampyGen

atmosphere generating system. _F. nucleatum_ strains ATCC 25586 and ATCC 23726 were brought to OD600 = 1 and added to the cell cultures at cell to bacterial ratio of 1:100. The cultures were

then grown for 24 h in the absence of serum. Growth media were collected and conditioned by passing through 0.22 µm filter to remove bacteria and cancer cells, and concentrated 10X using 50

 kDa membrane filters (Amicon Ultra 50k). For zymogram analysis52, 20 µg of conditioned media were dissolved in sample buffer (192 mM Tris-HCl [pH 6.8], 30% glycerol, 9% SDS) without

β-mercaptoethanol and without boiling, and subjected to SDS-PAGE using 7.5% gels containing 1 mg/ml Gelatin from porcine skin (Sigma-Aldrich, Germany). Following electrophoresis, the gels

were washed twice for 30 min at room temperature with washing buffer (50 mM Tris-HCl [pH 8.0], 5 mM CaCl2, 1 µM ZnCl2, 0.016% NaN3), containing 2.5% Triton X-100, then incubated for 10 min

in incubation buffer (washing buffer containing 1% Triton X-100) and incubated overnight at 37 °C with fresh incubation buffer. Proteolytic activity was visualized as a clear band against a

blue background after staining with Coomassie brilliant blue R-250. STATISTICAL ANALYSIS The various groups were compared against each other in pairs: the exact Wilcoxon test was used for

pairs that were matched, the exact Mann–Whitney test for independent samples (Nonparametric Tests, IBM SPSS Statistics for Windows, Version 25, 2017, Armonk, NY: IBM Corp.). When the same

hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding _p_-values were combined by Fisher’s method53. Wherever the pairs were not

independent, the multiple comparisons were adjusted for using the Holm modification54 of the Bonferroni correction. In all presented boxplots, whiskers represent extrema, box bounds

represent upper and lower quartiles, and center-line represents the median value. REPORTING SUMMARY Further information on research design is available in the Nature Research Reporting

Summary linked to this article. DATA AVAILABILITY The raw 16S sequencing data have been deposited in the NCBI BioProject database- accession # PRJNA624822. The RNA-seq data that support the

findings of this study has been deposited in GEO with the accession code GSE144143. The source data underlying Figs. 1a–c; 2a–c; 3a, b; 4b, c, e; 5b, e; 6a–c; Supplementary Figs. 1 and 2 are

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procedure. _Scand. J. Stat._ 6, 65–70 (1979). MathSciNet  MATH  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Professor Norman Grover for his valuable assistance in

statistics, Dr. Zakhariya Manevitch and Dr. Yael Feinstein-Rotkopf for their valuable help in microcopy, Ms. Noam Koren and Dr. Oded Heyman for their useful discussion. This work was

supported by the Israel Cancer Research Fund Project grant, the Israel Science Foundation Moked grant and the Israel Ministry of Science and Technology Personalized Medicine grant. We thank

the Vogel Stiftung Dr. Eckernkamp for supporting F.P. with a Dr. Eckernkamp Fellowship, and the Core Unit SysMed and IZKF (project Z-6), University of Würzburg, for RNA-seq data generation.

AUTHOR INFORMATION Author notes * These authors contributed equally: Lishay Parhi, Tamar Alon-Maimon. AUTHORS AND AFFILIATIONS * The Institute of Dental Sciences, The Hebrew

University-Hadassah School of Dental Medicine, Jerusalem, Israel Lishay Parhi, Tamar Alon-Maimon, Asaf Sol, Amjad Shhadeh, Jawad Abed, Naseem Maalouf & Gilad Bachrach * Department of

Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel Deborah Nejman & Ravid Straussman * Department of Developmental Biology and Cancer Research, Institute for Medical

Research Israel Canada (IMRIC), Hebrew University-Hadassah Medical School, Jerusalem, Israel Tanya Fainsod-Levi, Olga Yajuk & Zvi Granot * Department of Immunology and Cancer Research,

Institute for Medical Research Israel Canada (IMRIC), Hebrew University-Hadassah Medical School, Jerusalem, Israel Batya Isaacson & Ofer Mandelboim * Department of General and

Oncological Surgery-Surgery C, The Chaim Sheba Medical Center, Tel Hashomer, Ramat Gan, Israel Aviram Nissan * The Pathology Institute, Maccabi Healthcare Services, Rehovot, Israel Judith

Sandbank & Einav Yehuda-Shnaidman * Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Center for Infection Research (HZI), Würzburg, Germany Falk Ponath & Jörg

Vogel * Institute for Molecular Infection Biology, Medical Faculty, University of Würzburg, Würzburg, Germany Jörg Vogel Authors * Lishay Parhi View author publications You can also search

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CONTRIBUTIONS G.B, L.P., T.A.M., and Z.G. conceived and designed the study. L.P. and T.A.M. performed cell culture experiments, microscopy work, and animal experiments. A.S. performed data

analysis. R.S. conceived and designed the collection of clinical samples by A.N., J.S., and E.Y.S., and D.N. supervised the processing of samples and performed the 16S-rDNA amplification and

analysis. A.S. performed gel zonography. J.A. and N.M. handled bacterial cultures and performed part of the fluorescence microscopy. T.F.L. and O.Y. assisted in the animal experiments and

performed mouse histology and immunohistochemistry. J.V. designed RNA-seq experiments and analysis and supervised F.P. who performed them. O.M. contributed to the study design and analysis

and supervised B.I. who profiled tumor associated immune cells. G.B. wrote the paper together with L.P., T.A.M., and J.V. CORRESPONDING AUTHOR Correspondence to Gilad Bachrach. ETHICS

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Alon-Maimon, T., Sol, A. _et al._ Breast cancer colonization by _Fusobacterium nucleatum_ accelerates tumor growth and metastatic progression. _Nat Commun_ 11, 3259 (2020).

https://doi.org/10.1038/s41467-020-16967-2 Download citation * Received: 05 May 2019 * Accepted: 02 June 2020 * Published: 26 June 2020 * DOI: https://doi.org/10.1038/s41467-020-16967-2

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