Article
Fusobacterium nucleatum Promotes
Chemoresistance to Colorectal Cancer byModulating AutophagyGraphical Abstract
Highlights
d Specific gut microbes track with post-chemotherapy
recurrence of colorectal cancer
d F. nucleatum orchestrates the Toll-like receptor, microRNAs,
and autophagy network to control cancer chemoresistance
d Measuring and targeting F. nucleatum may be useful for
patient prognosis and management
Yu et al., 2017, Cell 170, 548–563July 27, 2017 ª 2017 Elsevier Inc.http://dx.doi.org/10.1016/j.cell.2017.07.008
Authors
TaChung Yu, FangfangGuo, Yanan Yu, ...,
Jie Hong, Weiping Zou, Jing-Yuan Fang
[email protected] (Y.C.),[email protected] (H.C.),[email protected] (J.H.),[email protected] (W.Z.),[email protected] (J.-Y.F.)
In Brief
Reducing a specific gut microbe in
colorectal cancer patients may improve
their response to chemotherapy and
reduce cancer recurrence.
Article
Fusobacterium nucleatum Promotes Chemoresistanceto Colorectal Cancer by Modulating AutophagyTaChung Yu,1,3 Fangfang Guo,1,3 Yanan Yu,1 Tiantian Sun,1 Dan Ma,1 Jixuan Han,1 Yun Qian,1 Ilona Kryczek,2
Danfeng Sun,1,2 Nisha Nagarsheth,2 Yingxuan Chen,1,* Haoyan Chen,1,* Jie Hong,1,* Weiping Zou,2,4,*and Jing-Yuan Fang1,*1State Key Laboratory for Oncogenes and RelatedGenes, Key Laboratory of Gastroenterology andHepatology, Ministry of Health, Division ofGastroenterology and Hepatology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Cancer Institute,Shanghai
Institute of Digestive Disease, 145 Middle Shandong Road, Shanghai 200001, China2Department of Surgery, the University of Michigan Comprehensive Cancer Center, Graduate programs in Immunology and Cancer Biology,
University of Michigan School of Medicine, Ann Arbor, MI, USA, 481093These authors contributed equally4Lead Contact
*Correspondence: [email protected] (Y.C.), [email protected] (H.C.), [email protected] (J.H.),
[email protected] (W.Z.), [email protected] (J.-Y.F.)http://dx.doi.org/10.1016/j.cell.2017.07.008
SUMMARY
Gutmicrobiota are linked to chronic inflammation andcarcinogenesis. Chemotherapy failure is the majorcause of recurrence and poor prognosis in colorectalcancer patients. Here, we investigated the contribu-tion of gut microbiota to chemoresistance in patientswith colorectal cancer. We found that Fusobacterium(F.) nucleatum was abundant in colorectal cancertissues in patients with recurrence post chemo-therapy, and was associated with patient clinico-pathological characterisitcs. Furthermore, our bio-informatic and functional studies demonstrated thatF. nucleatum promoted colorectal cancer resistanceto chemotherapy. Mechanistically, F. nucleatum tar-geted TLR4 and MYD88 innate immune signalingand specific microRNAs to activate the autophagypathway and alter colorectal cancer chemothera-peutic response. Thus, F. nucleatum orchestrates amolecular network of the Toll-like receptor, micro-RNAs, and autophagy to clinically, biologically, andmechanistically control colorectal cancer chemore-sistance. Measuring and targeting F. nucleatum andits associated pathway will yield valuable insight intoclinical management and may ameliorate colorectalcancer patient outcomes.
INTRODUCTION
Colorectal cancer (CRC) is the third most common cancer and
the second leading cause of cancer-related death worldwide
(Cartwright, 2012; Siegel et al., 2013). In advancedCRCpatients,
the purpose of chemotherapy is to shrink tumor size, reduce
tumor growth, and inhibit tumor metastasis. In general, active
cytotoxic drugs, including 5-fluorouracil (5-FU) and capecita-
bine, inhibit the enzyme activity of thymidylate synthase during
548 Cell 170, 548–563, July 27, 2017 ª 2017 Elsevier Inc.
DNA replication (Walko and Lindley, 2005). Oxaliplatin, another
chemotherapy drug, inhibits tumor cell growth and causes cell
G2 phase arrest by covalently binding DNA and forming plat-
inum-DNA adducts (Kelland, 2007). The combination of these
chemotherapeutic agents is widely used in the treatment of
CRCs (Cartwright, 2012). The majority of patients with advanced
CRC are initially responsive to the combined chemotherapy.
However, the patients eventually experience tumor recurrence
due to drug resistance, and the 5 year survival rate is lower
than 10% in advanced CRC patients (Dahan et al., 2009). Unfor-
tunately, colon cancer patients are generally not responsive
to novel immune checkpoint therapy (Zou et al., 2016). Thus,
it is of paramount importance to elucidate the mechanism of
chemotherapy resistance in CRC patients.
Cancer chemoresistance results from a complex interplay be-
tween gene regulation and the environment. The microbiota is
linked to CRC initiation and progression via affecting intestinal
inflammation (Arthur et al., 2012; Garrett, 2015; Man et al.,
2015; Zitvogel et al., 2015) and tumor-related signaling pathways
(Schwabe and Jobin, 2013). Recent mouse studies have shown
that the gut microbiota may modulate local immune responses
and in turn affect chemotherapy (Iida et al., 2013; Viaud et al.,
2013) and immunotherapy (Sivan et al., 2015; Vetizou et al.,
2015). Human gutmicrobiota are linked to inflammatory cytokine
production (Schirmer et al., 2016). Two groups have recently
shown that the abundance of F. nucleatum is gradually increased
from normal tissues to adenoma tissues and to adenocarcinoma
tissues in colorectal carcinogenesis (Castellarin et al., 2012; Kos-
tic et al., 2012).Moreover, the amount of F. nucleatum in CRC tis-
sues is associated with shorter survival (Mima et al., 2016).
F. nucleatum adhesin FadA may bind to the E-cadherin protein
and promote colorectal carcinogenesis (Rubinstein et al.,
2013). In addition, F. nucleatum lectin Fap2 may recognize the
host Gal-GalNAc and help this bacterium localize abundantly in
colon cancer epithelial cells (Abed et al., 2016). However, the po-
tential effect of F. nucleatum on chemotherapy is not examined in
human literature. Here, we tested whether and how F. nucleatum
affected chemotherapy in CRC patients. We have found that
the amount of F. nucleatum is increased in CRC patients with
(legend on next page)
Cell 170, 548–563, July 27, 2017 549
recurrence post-chemotherapy, compared with those with non-
recurrence post-chemotherapy. We have demonstrated that
F. nucleatum plays a critical role in mediating CRC chemoresist-
ance in response to small drug chemotherapeutics via a selec-
tive target loss of miR-18a* and miR-4802, and activation of
the autophagy pathway.
RESULTS
F. nucleatum Is Associated with Colorectal CancerRecurrence and Patient OutcomeTo examine the potential relationship between the gut micro-
biota alteration and CRC recurrence, we re-analyzed our previ-
ous data (Yu et al., 2015) and compared the pyrosequenced
data by using a Roche 454 GS FLX in 16 CRC tissues from pa-
tients with recurrence and 15 CRC tissues without recurrence
(Cohort 1, Figure 1A, Table S1). We used the LEfSe algorithm
(Segata et al., 2011) to define the potential differential bacterium
patterns between recurrent and non-recurrent CRC patients
in Cohort 1. We found that Fusobacterium, Anaerosporobacter,
Parvimonas, Peptostreptococcus, and Prevotella were enriched
in recurrent CRC tissues as compared to non-recurrent CRC
tissues (Figure 1B). Anaerosporobacter is rarely associated
with human disease (Jeong et al., 2007). We further studied
Fusobacterium nucleatum, Prevotella intermedia, Parvimonas
micra, Peptostreptococcus anaerobius. Real-time PCR showed
that F. nucleatum was the most enriched bacterium among
the four bacteria in patients with recurrent CRC (Figure S1A),
compared with patients without recurrence. This suggests that
F. nucleatum may play a role in CRC recurrence. F. nucleatum
is the most dominant phylotype in CRC (Kostic et al., 2012).
We quantified the amount of F. nucleatum in 48 CRC tissues
from patients without recurrence (group 1) and 44 CRC tissues
from patients with recurrence (group 2) (Cohort 2, Table S2).
In agreement with our data in Cohort 1 and previous reports
(Castellarin et al., 2012; Kostic et al., 2012), the amount of
F. nucleatum in CRC tissues was higher in recurrent patients
than that in non-recurrent patients (Figure 1C). Furthermore,
there was an enrichment of F. nucleatum in CRC tissues
compared with adjacent normal tissues in both recurrence and
non-recurrence groups (Figure 1C). CRC recurrence is attributed
to chemoresistance. Thus, F. nucleatum correlates with CRC
Figure 1. F. nucleatum Is Associated with Cancer Recurrence and Pat
(A) A cladogram representation of data in CRC patients with recurrence (16) vers
recurrence (Red) and without recurrence (Blue). The brightness of each dot is pr
(B) Linear discriminant analysis (LDA) coupled with the effect sizemeasurements id
and non-recurrent (Blue) patients are indicated with negative (Red) or positive
are shown.
(C) Statistical analysis of the amount of F. nucleatum in Cohort 2, nonparametric
(D) Recurrence-Free Survival (RFS) was compared between patients with low an
(E) Receiver operating characteristic (ROC) analysis was conducted based on th
(F) Univariate analysis was performed in Cohort 2. The bars correspond to 95%
(G) Multivariate analysis was performed in Cohort 2. The bars correspond to 95%
(H) Statistical analysis was conducted based on the amount of F. nucleatum an
Cohort 2, Chi-square test.
(I) RFS was compared between patients with low and high abundance of F. nucl
F. nucleatum defined in Cohort 2, Log-rank test.
See also Figure S1.
550 Cell 170, 548–563, July 27, 2017
recurrence. The high amount of F. nucleatum may potentially
promote CRC chemoresistance.
We next evaluated the relationship between the amount of
F. nucleatum and different clinicopathological features in Cohort
2. The amount of F. nucleatumwas positively associated with the
American Joint Committee on Cancer (AJCC) stage and tumor
size (Figure S1B). A high amount of F. nucleatum was strongly
associated with shorter recurrence free survival (RFS) (Fig-
ure 1D). The five-year recurrence survival was substantially
shorter in the F. nucleatum-high group than the F. nucleatum-
low group. Receiver operating characteristic (ROC) curve anal-
ysis was conducted to predict the potential CRC recurrence
using either AJCC stage or the amount of F. nucleatum (Fig-
ure 1E, Table S3). We observed that the area under curve
(AUC) of F. nucleatum-based prediction was higher than that
of the AJCC-stage based model (0.776 versus 0.646, p =
0.039). Youden Index was used to determine the optimal cut-
off point and �10.3 [-deltaCT value] was selected based on
the abundance of F. nucleatum that provided the best balance
between the sensitivity and the specificity to predict CRC recur-
rence (Table S3). In addition, univariate (Figure 1F) and multivar-
iate (Figure 1G) regression analyses of Cohort 2 demonstrated
that the amount of F. nucleatum was an independent predictor
of CRC aggressiveness with significant hazard ratios for predict-
ing clinical outcome. Its predictive value was comparable to that
of the AJCC stage. Thus, the data in Cohort 2 not only confirm
our observation in Cohort 1 but also define the potential value
of the amount of F. nucleatum in predicting CRC recurrence.
To further validate if F. nucleatum had a similar prediction
value in cancer recurrence in a different and large patient popu-
lation, we analyzed an additional cohort with 173 patients
(Cohort 3, Table S4). The samples in Cohort 3 were classified
into high and low-risk subsets according to the cut-off value
(�10.3[-deltaCT value]) of F. nucleatum abundance derived
from Cohort 2 (Table S3). We found that the recurrence rate in
the high-risk group was significantly higher than the low-
risk group (73.4% versus 30.9%, p = 2.436e-8) (Figure 1H).
Again, the amount of F. nucleatum was higher in recurrent pa-
tients than non-recurrent patients in Cohort 3 (Figure S1C). We
confirmed that the high amount of F. nucleatum was associated
with shorter RFS (Figure 1I). Univariate (Figure S1D) andmultivar-
iate (Figure S1E) Cox regression analyses in Cohort 3 revealed
ient Outcome
us no recurrence (15) by 16S rDNA sequencing. Taxa enriched in patients with
oportional to its effect size.
entifies the significant abundance of data in A. Taxa enriched in recurrent (Red)
(Blue) LDA scores, respectively. Only taxa greater than LDA threshold of 3.5
Mann–Whitney test.
d high amount of F. nucleatum in Cohort 2, Log-rank test.
e amount of F. nucleatum and AJCC in colorectal cancer.
confidence intervals.
confidence intervals.
d recurrence rate in Cohort 3 by the cut off value of F. nucleatum defined in
eatum in 173 patients with colorectal cancer (Cohort 3) by the cut off value of
(legend on next page)
Cell 170, 548–563, July 27, 2017 551
that the amount of F. nucleatum was an independent predictor
of CRC aggressiveness. Our data indicate that F. nucleatum is
pathologically and clinically associated with cancer recurrence
and patient outcome.
F. nucleatum Promotes Cancer Autophagy ActivationWe hypothesized that F. nucleatum was biologically involved
in the development of colon cancer chemoresistance. To
test this hypothesis, we co-cultured colon cancer cells with
F. nucleatum, performed a RNA-seq analysis, and compared
the gene expression profiles between the colon cancer cells
co-cultured with or without F. nucleatum. Co-culture with
F. nucleatum downregulated 992 gene expressions and upregu-
lated 1,466 gene expressions in HT29 cells (adjusted p value <
0.05, raw data accessible via GEO: GSE90944) (Table S5). Single
sample gene set enrichment analysis (ssGSEA) revealed that the
gene sets including MizushimaI_Autophagosome_Formation,
KEGG_Lysosome, KEGG_regulation_of_Autophagy, and Hall-
mark_apoptosis were enriched in CRC cells co-cultured with
F. nucleatum (Figure 2A). Given the role of the autophagy
pathway in cellular survival (Song et al., 2009), our data suggest
that F. nucleatummay cause autophagy pathway activation and
potentially support cancer chemoresistance. In line with this,
western blot analysis showed that F. nucleatum increased the
expression of multiple autophagy signaling elements including
pAMPK, pULK1, ULK1, and ATG7 in HCT116 cells and HT29
cells. These cells exhibited low LC3 protein cleavage level at
the basal condition (Figures S2A and S2B). Real-time PCR
confirmed that F. nucleatum affected the ULK1 and ATG7
mRNA levels (Figures 2B and 2C). These data suggest that
F. nucleatum may drive autophagy activation in CRC cells.
To examine this possibility, we performed autophagy functional
assays in CRC cells co-cultured with F. nucleatum. Increased
LC3-II and decreased p62 expression was detected in the
F. nucleatum-co-cultured HCT116 cells (Figure 2D) and HT29
cells (Figure S2C) in a concentration-dependent manner (Fig-
ure S2D). These effects were not found in the CRC cells co-
cultured with P. intermedia, P.micra, P. anaerobius, Escherichia
coli, Enterococcus faecalis, or Bacteroides fragilis (Figure 2D,
Figures S2C, S2E and S2F). Furthermore, we treated HCT116
cells (Figure 2E) and HT29 cells (Figure S2G) with Chloroquine
(CQ), an autophagy lysosomal inhibitor. Addition of CQ blocked
the autophagic flux in the F. nucleatum-cultured cells (Figure 2E).
We established HCT116 cells and HT29 cells that stably ex-
pressed a tandem mRFP-EGFP-LC3 construct. We found
that F. nucleatum induced the autophagic flux in HCT116 cells
Figure 2. F. nucleatum Promotes Cancer Autophagy Activation
(A) ssGSEA analysis was conducted to show the relationship between the amou
(B, C) Real-Time PCR was performed in HCT116 cells (B) and HT29 (C) cells cult
(D) Western blot was performed on autophagy element expression in HCT116 ce
(E) Western blot was performed in HCT116 cells co-cultured with F. nucleatum in
(F and G) HCT116 cells (F) and HT29 cells (G) that stably expressed mRFP-EGFP
analysis is shown (2000 3 magnification). Bar scale, 5 mm.
(H) Autophagosomes were observed by transmission electron microscopy (175
F. nucleatum. Bar scale, 1 mm.
(I and J) Statistical analysis was performed to calculate the number of autophag
microscopy, nonparametric Mann–Whitney test.
See also Figure S2.
552 Cell 170, 548–563, July 27, 2017
(Figure 2F) and HT29 cells (Figure 2G). In addition, autophago-
somes were evaluated in HCT116 cells and HT29 cells co-
cultured with or without F. nucleatum (Figure 2H). Transmission
electron microscopy showed an increase in the formation of
autophagic vesicles in the F. nucleatum-co-cultured HCT116
cells (Figure 2I) and HT29 cells (Figure 2J). Collectively, our
data indicate that F. nucleatum activates the autophagy pathway
in CRC cells.
F. nucleatum Induces Cancer Chemoresistance via theAutophagy PathwayWe next hypothesized that F. nucleatum induced cancer
chemoresistance via the autophagy pathway. To initially deter-
mine whether F. nucleatum induces cancer chemoresistance,
different multiplicity of infection (MOI) of F. nucleatum was used
in the co-culture with CRC cells. We observed that F. nucleatum
(MOI = 100) had no effect on HCT116 cell and HT29 cell pro-
liferation (Figures S3A and S3B). We examined the potential role
of this dose of F. nucleatum in CRC chemoresistance. As ex-
pected, Oxaliplatin (Figure 3A) and 5-FU (Figure 3B) induced
HCT116 cell apoptosis. Co-culture with F. nucleatum, but not
with P. intermedia, P. micra, P. anaerobius, and medium control
(Figures S3C–S3F), reduced HCT116 cell apoptosis induced by
these chemotherapeutic agents. Moreover, F. nucleatum had no
protective effect on HCT116 cells and HT29 cells treated with
Doxorubicin (Figures S3G and S3H). The data indicate that
F. nucleatum inuduces CRC resistance to Oxaliplatin and 5-FU.
SW480 cells are relatively sensitive to Oxaliplatin treat-
ment (Moutinho et al., 2014). To quantitatively evaluate the po-
tential effect of F. nucleatum on Oxaliplatin-induced SW480
cell chemoresistance, we generated the Oxaliplatin-resistant
SW480 cells from the parential SW480 cells by continuous
exposure to gradually increased concentrations of Oxaliplatin.
We compared Oxaliplatin-induced cell apoptosis among the
parental SW480 cells, the Oxaliplatin-resistant SW480 cells,
and the F. nucleatum-co-cultured parental SW480 cells in
the presence of different concentrations of Oxaliplatin (Fig-
ures S3I and S3J). We found that 109.9 mM Oxaliplatin resulted
in 50%, 32.9%, and 35.5% cell apoptosis in the parental
SW480 cells, the Oxaliplatin-resistant SW480 cells, and the
F. nucleatum-co-cultured parental SW480 cells, respectively
(Figure S3I). Given that the Oxaliplatin-resistant SW480 cells
and the F.nucleatum-cultured parental SW480 cells were
similarly resistant to Oxaliplatin treatment, the data suggest
that F. nucleatum efficiently enables chemoresistance to the
parental SW480 cells. In further support of this possibility, the
nt of F. nucleatum and autophagy-related pathways in CRC tissues.
ured with F. nucleatum, nonparametric Mann–Whitney test.
lls co-cultured with F. nucleatum, E. coli, E. faecalis or B. fragilis.
the presence of CQ.
-LC3 fusion protein were co-cultured with F. nucleatum. Confocal microscopic
00 3 magnification) in HCT116 cells (left) and HT29 cells (right) cultured with
osomes in HCT116 cells (I) and HT29 cells (J) shown by transmission electron
Figure 3. F. nucleatum Induces Chemoresistance in Colorectal Cancer Cells via Activation of the Autophagy Pathway
(A–D) Apoptosis was detected by flow cytometry in HCT116 cells (A, B) and HT29 cells (C, D). The cells were co-culturedwith F. nucleatum or treatedwith CQ, and
different concentrations of Oxaliplatin (A and C) and 5-FU (B, D). nonparametric Mann–Whitney test.
(E and F) Cleaved caspases and p-H2AX expression were detected by western blot in HCT116 cells (E) and HT29 cells (F). The cells were co-cultured with
F. nucleatum or treated with CQ, and different concentrations of Oxaliplatin and 5-FU.
(G and H) Apoptosis was detected by flow cytometry in HCT116 cells (G) and HT29 cells (H). The cells were transfected with ULK1 and ATG7 siRNAs, and
subsequently co-cultured with F. nucleatum and different concentrations of Oxaliplatin, nonparametric Mann–Whitney test.
See also Figure S3.
Cell 170, 548–563, July 27, 2017 553
Figure 4. F. nucleatum Activates Cancer Autophagy via Downregulation of miR-18a* and miR-4802
(A) The predicted binding sequences for miR-18a* (left) and miR-4802 (right) within the human ULK1 and ATG7 30UTR, respectively. Seed sequences are
highlighted.
(legend continued on next page)
554 Cell 170, 548–563, July 27, 2017
EC50ofOxaliplatinwas109.9mM,190.9mM,and177.2mMfor the
parental SW480 cells, the Oxaliplatin-resistant SW480 cells, and
the F.nucleatum-cultured parental SW480 cells, respectively
(Figure S3J). In addition, we inoculated the parental SW480 cells
into nude mice and treated the mice with different doses of Ox-
aliplatin with or without F. nucleatum. F. nucleatum alone had
no effect on tumor growth (Figures S3K and S3L). As expected,
tumor growth was significantly inhibited by both low and high
doses ofOxaliplatin, and these effectswere efficiently aborgated
by F. nucleatum (Figures S3M and S3N). The data suggest that
F. nucleatum endows potent chemoresistance to several
CRC cells.
To address whether F. nucleatum modulates CRC chemore-
sistance via the autophagy pathway, we co-cultured HCT116
cells and HT29 cells with F. nucleatum and treated these cells
with Oxaliplatin and 5-FU in the presence of CQ. We found
that the F. nucleatum-induced chemoresistant effect was abol-
ished by CQ treatment in HCT116 cells (Figures 3A and 3B)
and HT29 cells (Figures 3C and 3D). Moreover, western blotting
showed that Oxaliplatin and 5-FU induced the cleavage of cas-
pase 9, caspase 3, caspase 6, caspase 7, PARP, and p-H2AX,
and these effects were blocked by F. nucleatum co-culture
in HCT116 cells (Figure 3E) and HT29 cells (Figure 3F). Thus,
F. nucleatum may prevent CRC cells from chemotherapy-
induced apoptosis via the autophagy pathway.
To examine whether the autophagy elements such as ULK1
andATG7may participate in F. nucleatum-induced chemoresist-
ance in CRC cells, we analyzed LC3 cleavage status in ULK1 and
ATG7 siRNA-transfected CRC cells cultured with F. nucleatum.
As expected, ULK1 and ATG7 siRNAs decreased the two gene
expression in CRC cells (Figures S3O and S3P). F. nucleatum
co-culture increased the cleavage of LC3, ULK1, and ATG7
expression in wild-type ULK1 and ATG7 expressing CRC
cells. ULK1 and ATG7 siRNAs blocked F. nucleatum-induced
LC3 cleavage (Figures S3Q and S3R). The data indicate that
F. nucleatum may induce autophagy activation via increasing
ULK1 and ATG7 expression. Next, we treated CRC cells with
chemotherapy agents. We found that F. nucleatum decreased
CRC cell apoptosis in response to Oxaliplatin (Figures 3G and
3H) and 5-FU treatment (Figures S3S and S3T). This effect was
abolished in ULK1 or ATG7-siRNA-transfected cells (Figures
3G and 3H; Figures S3S and S3T). Thus, the data strongly sug-
gest that F. nucleatum may promote CRC chemoresistance by
(B) Luciferase activity wasmeasured in HCT116 cells transfected withmiR-18a*m
human ULK1 30UTRs were used. The luciferase activity was normalized based o
(C) Luciferase activity was measured in HCT116 cells transfected with miR-480
mutant human ATG7 30UTRs were used.
(D) Real-time PCR was performed in HCT116 cells to detect the expression of U
Mann–Whitney test.
(E) Real-time PCR was performed in HCT116 cells to detect the expression of A
Mann–Whitney test.
(F and G) HCT116 cells were transfected with mimics or inhibitor of miR-18a* (F) an
target proteins were detected by western blot in HCT116 cells.
(H) HCT116 cells that stably expressedmRFP-EGFP-LC3 fusion protein were tran
autophagosomes were observed under confocal microscope (2000 3 magnifica
(I) Autophagosomes were observed by transmission electron microscopy (17500
(right) mimics, and then co-cultured with F. nucleatum. Bar scale, 1 mm.
See also Figure S4.
activating the autophagy pathway, and the autophagy elements
ULK1 and ATG7 participate in the F. nucleatum-mediated che-
moresistance in CRC cells.
F. nucleatum Activates Cancer Autophagy via aSelective Loss of miR-18a* and miR-4802To explore the mechanism by which F. nucleatum induced
upregulation of pULK1, ULK1, and ATG7 at both the mRNA
and protein levels, we constructed the recombination luciferase
reporter plasmids, pGL3-ULK1p and pGL3-ATG7p, containing
the promoter region of ULK1 or ATG7. Luciferase assay showed
that F. nucleatum co-culture had no effect on transcriptional
activity of pGL3-ULK1p and pGL3-ATG7p in CRC cells (Figures
S4A and S4B). This suggests that F. nucleatum-increased ULK1
orATG7mRNA is not dependent on the transcriptional activation
of ULK1 or ATG7 promoter.
MiRNAs often regulate gene expression by binding to the
RISC complex and directing sequence-specific cleavage of
target mRNA or repressing the target mRNA translation (Bartel,
2009; Krek et al., 2005). We hypothesized that dysregulated
miRNAs may contribute to F. nucleatum-increased ULK1 and
ATG7 expression. To test this hypothesis, we performed a global
miRNA expression profiling of CRC tissues with a high amount
of F. nucleatum from six recurrent patients, and of CRC tissues
with a low amount of F. nucleatum from six non-recurrent
patients (Cohort 1, Figure S4C, left). Sixty-eight miRNAs were
significantly downregulated in the CRC tissues with a high
amount of F. nucleatum as compared to that with a low amount
of F. nucleatum (Figure S4C, right; Table S6). Next, we used the
FindTar3 (http://bio.sz.tsinghua.edu.cn/) and miRDB databases
(http://mirdb.org/miRDB/) to identify potential miRNAs, which
may regulate ULK1 and ATG7. After overlapping these potential
ULK1- and ATG7-regulatory miRNAs with the identified 68
downregulatedmiRNAs, we found four and threemiRNAs, which
may regulate ULK1 and ATG7, respectively (Figure S4C, right).
We validated these seven miRNAs with real-time PCR.We found
that miR-4802 and miR-18a* were the most significantly down-
regulated miRNAs in response to F. nucleatum intervention in
HCT116 cells (Figure S4D) and HT29 cells (Figure S4E). Target
prediction programs and sorting algorithm suggested potential
specific targets for miR-18a* and miR-4802 in the seed regions
within the 30UTR regions of ULK1 and ATG7 genes, respectively
(Figure 4A). Luciferase reporter assays demonstrated that
imics or control miRNA. The luciferase reporters expressingwild-type ormutant
n the control miRNA transfection. n.s., not significant.
2 mimics or control miRNA. The luciferase reporters expressing wild-type or
LK1 gene after transfected with miR-18a* mimics or inhibitor, nonparametric
TG7 gene after transfected with miR-4802 mimics or inhibitor, nonparametric
dmiR-4802 (G), respectively. After culturing with F. nucleatum, autophagy and
sfected with miR-18a* andmiR-4802mimics. After culturing with F. nucleatum,
tion) in HCT116 cells. Bar scale, 5 mm.
3magnification) in HCT116 cells transfected with miR-18a* (left) and miR-4802
Cell 170, 548–563, July 27, 2017 555
Figure 5. miR-18a* and miR-4802 Regulate F. nucleatum-Mediated Chemoresistance
(A–D) Apoptosis was detected by flow cytometry in HCT116 cells. HCT116 cells were transfectedwith mimics (A, B) or inhibitors (C, D) of miR-18a* andmiR-4802,
co-cultured with F. nucleatum, and treated with different concentrations of Oxaliplatin (A, C) and 5-FU (B, D). nonparametric Mann–Whitney test.
(legend continued on next page)
556 Cell 170, 548–563, July 27, 2017
miR-18a* and miR-4802 suppressed the luciferase activity in
HCT116 cells (Figures 4B–4C) and HT29 cells (Figures S4F and
S4G) transfected with wild-type ULK1 and ATG7 reporter plas-
mids, but not with the mutant reporter ones. Real-time PCR
showed that miR-18a* and miR-4802 decreased ULK1 and
ATG7 mRNA levels, and the mRNA expression levels of ULK1
and ATG7 were rescued by miR-18a* and miR-4802 inhibitors
in HCT116 cells (Figures 4D and 4E) and HT29 cells (Figures
S4H and S4I). In addition, western blotting revealed that overex-
pression of miR-18a* or miR-4802 suppressed F. nucleatum-
induced conversion of LC3-I to LC3-II and simultaneously
increased p62 protein expression in HCT116 cells (Figures 4F
and 4G) and HT29 cells (Figures S4J and S4K). Accumulation
of autophagosomes was reduced in miR-18a* and miR-4802
overexpressing HCT116 cells (Figures 4H and 4I) and HT29 cells
(Figures S4L and S4M) co-cultured with F. nucleatum, compared
to controls. Thus, the data indicate that F. nucleatum acti-
vates cancer autophagy via a selective loss of miR-18a* and
miR-4802.
MiR-18a* and miR-4802 Regulate F. nucleatum-Mediated ChemoresistanceThe selective loss of miR-18a* and miR-4802 expression in
F. nucleatum-cultured cell lines led us to hypothesize that miR-
18a* and miR-4802 may regulate F. nucleatum-mediated che-
moresistance. To test this hypothesis, miR-18a* and miR-4802
mimics or inhibitors were transfected in CRC cells cultured
with F. nucleatum. These microRNA mimics and inhibitors had
no effect on CRC proliferation (Figure S5A). However, miR-
18a* and miR-4802 mimics increased Oxaliplatin- and 5-FU-
induced apoptosis in HCT116 cells (Figures 5A and 5B) and
HT29 cells (Figures S5B and S5C) cultured with F. nucleatum,
compared with controls. Consistent with these data, a loss-of-
function study revealed that anti-miR-18a* and anti-miR-4802
increased chemoresistance in HCT116 cells (Figures 5C and
5D) and HT29 cells (Figures S5D and S5E) cultured with
F. nucleatum. Western blotting showed that the inhibitory effects
of F. nucleatum on the chemotherapy-induced caspase and
PARP cleavage and p-H2AX were abolished by miR-18a*
and miR-4802 mimics transfection in HCT116 cells (Figure 5E)
and HT29 cells (Figure S5F).
In the CRC xenograft mouse models, HCT116 cells and HT29
cells were inoculated into nude mice, followed by treatment
with miRNAs, chemotherapeutic agents, F. nucleatum-co-cul-
ture, and other manipulations. There was no difference in tumor
weight (Figures S5G and S5H) and tumor growth (Figure S5I)
among different control groups. Interestingly, tumor growth
was significantly decreased by Oxaliplatin (Figures S5J–S5L)
(E) Western blot was performed in HCT116 cells. HCT116 cells were transfec
F. nucleatum, and treated with different concentrations of Oxaliplatin (left) and 5
(F) Representative data of tumors in mice under different conditions. Figure 5 (F)
(G and H) Statistical analysis of tumor weights (G) and volumes (H) in different gr
(I) TUNEL assays were performed to detect tumor cell apoptosis in xenograft tum
(J) Transmission electron microscopy was performed to show the autophagosom
magnification). Bar scale, 1 mm.
(K) Statistical analysis of autophagosomes. Autophagosomes were detected by
Mann–Whitney test.
See also Figure S5-6.
and 5-FU treatment (Figures S5M–S5O), and these decreases
were blocked by F. nucleatum treatment in vivo. Thus,
F. nucleatum participates in CRC chemoresistance in response
to Oxaliplatin and 5-FU therapy. Furthermore, F. nucleatum-
induced CRC chemoresistance was rescued by CQ, an
autophagy pathway inhibitor (Figures S5J–S5O). In addition,
F. nucleatum-induced CRC chemoresistance was reversed by
miR-18* and miR-4802 adenovirus transduction in tumor
bearing mouse models treated with Oxaliplatin (Figures 5F–
5H) or 5-FU (Figures S6A–S6C). Overexpression of ULK1 and
ATG7 blocked miR-18a* and miR-4802-mediated reversion of
F. nucleatum-stimulated chemoresistance in tumor tissues
(Figures 5F–5H, Figures S6A–S6C). Furthermore, western blot-
ting confirmed that miR-18a* and miR-4802 rescued the inhib-
itory effects of F. nucleatum on the apoptotic gene expression
(Figure S6D), tumor apoptosis (Figure 5I, Figure S6E), and auto-
phagosome formation (Figures 5J and 5K, Figures S6F and
S6G) in response to chemotherapy. These data support that
miR-18a* and miR-4802 regulate F. nucleatum-mediated che-
moresistance by blocking F. nucleatum-induced autophagy
activation.
TLR4 and MYD88 Pathway Is Involved in F. nucleatum-Mediated ChemoresistanceThe TLR4 and MYD88 innate immune signaling pathway
is activated in response to F. nucleatum intervention (Abreu
and Peek, 2014; Liu et al., 2007). In line with this, we found
that the levels of TLR4 and MYD88 transcripts (Figures S7A
and S7B) and proteins (Figures S7C and S7D) were enhanced
in response to F. nucleatum treatment in HCT116 cells and
HT29 CRC cells. To examine whether the TLR4 and MYD88
pathway participated in F. nucleatum-induced autophagy acti-
vation, we transfected CRC cells with TLR4 and MYD88 siRNA
and co-cultured the cells with F. nucleatum. Western blotting
showed that F. nucleatum-mediated autophagy activation
was reduced in TLR4 or MYD88 siRNA-transfected CRC cells
(Figures S7C and S7D). Knockdown of TLR4 or MYD88
expression blocked F. nucleatum-induced ULK1 and ATG7
upregulation, F. nucleatum-induced miRNA-18a* and miRNA-
4802 loss, and F. nucleatum-induced chemoresistance in
HCT116 cells (Figures 6A–6F) and HT29 cells (Figures 6G–
6L). Furthermore, F. nucleatum-induced CRC chemoresistance
was rescued by knocking down TLR4 or MYD88 in the CRC
xenograft mouse model, as shown by reduced tumor weight
(Figures 6M and 6N, Figures S7E and S7F) and tumor volume
(Figure 6O, Figure S7G). The data indicate that F. nucleatum-
induced loss of miRNA-18a* and miRNA-4802, F. nucleatum-
activated autophagy pathway, and F. nucleatum–mediated
ted with mimics or inhibitors of miR-18a* and miR-4802, co-cultured with
-FU (right).
and Figure S5G shared experimental controls.
oups, n = 8/group nonparametric Mann–Whitney test.
or tissues. The mice received different treatments.
es in xenograft tumor tissues. The mice received different treatments (175003
transmission electron microscopy in xenograft tumor tissues, nonparametric
Cell 170, 548–563, July 27, 2017 557
(legend on next page)
558 Cell 170, 548–563, July 27, 2017
chemoreistance are dependent on the TLR4 and MYD88
signaling pathway.
The Levels of F. nucleatum, MicroRNAs, and AutophagyComponents Correlate and Are Clinically Relevant inCRC PatientsTo investigate clinical significance of F. nucleatum, microRNAs
(miR-18a* and miR-4802), and autophagy components (ULK1
and ATG7) in CRC patients, we studied CRC tissues and normal
colorectal tissues adjacent to cancer lesions in Cohort 2. We
quantified F. nucleatum and the expression of miR-18a* and
miR-4802 with real-time PCR and detected the protein expres-
sion of pULK1, ULK1, and ATG7 by immunohistochemistry in
CRC tissues and normal tissues. We found that the high amount
of F. nucleatum, the high expression of p-ULK1, ULK1, and
ATG7, and the low levels of miR18a* and miR-4802 expression
were more likely detected in patients with recurrence, compared
with patients without recurrence (Figures 7A–7C). The amount of
F. nucleatum in CRC tissues negatively correlated with the levels
of miR-18a* and miR-4802 and positively correlated with ULK1
and ATG7 expression (Figure 7D). The data demonstrate that
the amount of F. nucleatum and the expression levels of miR-
18a* and miR-4802 positively and negatively correlate with
autophagy pathway activation status, respectively. Altogether,
we reason that F. nucleatum may act on CRC via TLR4 and
MYD88, cause a selective loss of miR-18a* and miR-4802
expression, subsequently result in autophagy activation, and
consequently promote chemoresistance in patients with colo-
rectal cancer (Figure 7E).
DISCUSSION
Capecitabine (and 5-FU) in combination with platinum-based
chemotherapy has been widely used to treat different types of
cancer including CRC (Kelland, 2007). Although CRC patients’
initial responses to surgical debulking and chemotherapy is often
effective, relapse with drug-resistant cancer usually occurs and
patients succumb to disease (Bertotti and Sassi, 2015; Jemal
et al., 2009). Unfortunately, CRC patients are generally not
responsive to novel immune checkpoint therapy (Zou et al.,
2016). Conventional chemotherapy remains the first line therapy
for patients with CRC. Thus, understanding the mechanisms of
chemoresistance in CRC is essential to optimizing current ther-
apeutic strategies.
Cancer genetic and epigenetic alterations in CRC chemother-
apeutic response have been extensively reported (Bardelli and
Figure 6. TLR4 and MYD88 Pathway Is Involved in F. nucleatum-Media
(A–D) Real-time PCR was performed to detect ATG7 (A), ULK1 (B), miR-18a* (C),
with F. nucleatum and transfected with TLR4 and MYD88 siRNAs, respectively.
(E and F) Apoptosis was detected by flow cytometry in HCT116 cells. The cells w
and subsequently treated with different concentrations of Oxaliplatin (E) or 5-FU
(G–J) Real-time PCR was performed to detect expression of ATG7 (G), ULK1 (
co-cultured with F. nucleatum and transfected with TLR4 and MYD88 siRNAs, re
(K and L) Apoptosis was detected by flow cytometry in HT29 cells. The cells were
subsequently treated with different concentrations of Oxaliplatin (K) or 5-FU (L),
(M) Representative data of tumors in nude mice bearing HCT116 cells in differen
(N and O) Statistical analysis of mouse tumor weights (N) and volumes (O) in diff
See also Figure S7.
Siena, 2010; Dallas et al., 2009; Esteller, 2008; Linardou et al.,
2008; Van Geelen et al., 2004; Weichert et al., 2008). Recent
mouse studies have shown that the gut microbiota may modu-
late local immune responses and in turn affect chemotherapy
(Iida et al., 2013; Viaud et al., 2013) and immunotherapy (Sivan
et al., 2015; Vetizou et al., 2015). Human studies demon-
strate that the adaptive immune system can also regulate che-
mosensitivity of human ovarian cancer (Wang et al., 2016).
However, the potential role of the gut microbiota in CRC
chemoresistance is poorly understood. Through a combination
of genomic, bioinformatic, biological, in vivo models and clinical
studies, we have demonstrated that autophagy-related path-
ways are enriched and activated in CRC patients with a high
amount of F. nucleatum, and that F. nucleatum promotes CRC
chemoresistance.
Metagenomic and transcriptomic analyses have revealed
that intestinal microbes, especially F. nucleatum, are involved
in CRC development (Castellarin et al., 2012; Kostic et al.,
2012). F. nucleatum attaches to the host epithelial E-cadherin
and promotes colorectal carcinogenesis via the fusobacterial
adhesin FadA(Rubinstein et al., 2013). The interaction between
a host polysaccharide, Gal-GalNAc with fusobacterial lectin
(Fap2) facilitates F. nucleatum enrichment in CRC tissues
(Abed et al., 2016). However, it is unknown whether and
how F. nucleatum may mediate chemoresistance in CRC. We
report that F. nucleatum induces LC3-II expression, autophagic
flux, and autophagosome synthesis in CRC cells. Accordingly,
F. nucleatum stimulates expression of the autophagy-related
proteins, pULK1, ULK1, and ATG7 in CRC, and biochemical
and genetic autophagy inhibition enhances the sensitivity
of F. nucleatum-treated CRC cells to 5-FU and Oxaliplatin.
Thus, we conclude that autophagy contributes to F. nucleatum-
mediated CRC resistance to Oxaliplatin and 5-FU regimens.
Our data may explain why CRC patients with a high amount of
F. nucleatum experience poor outcomes (Mima et al., 2016).
We have dissected the mechanisms by which F. nucleatum
mediates the ULK1 and ATG7 pathway activation. F. nucleatum
does not affect the transcription of ULK1 and ATG7. It has been
reported that adherent-invasive E. coli (AIEC) may regulate
human intestinal epithelial cell autophagy via modulating the
expression of miR-30C and miR-130A (Nguyen et al., 2014).
Our bioinformatic and functional studies have elucidated that
miR-18a* and miR-4802 target ULK1 and ATG7, respectively,
and are selectively lost due to F. nucleatum co-culture, and can
biologically modulate CRC chemoresistance in vitro and in vivo.
Thus, F. nucleatum medicates chemoresistance via selectively
ted Chemoresistance
and miR-4802 (D) expression in HCT116 cells. HCT116 cells were co-cultured
ere co-cultured with F. nucleatum after TLR4 and MYD88 siRNAs transfection,
(F), nonparametric Mann–Whitney test.
H), miR-18a* (I), and miR-4802 (J) expression in HT29 cells. HT29 cells were
spectively.
co-cultured with F. nucleatum after TLR4 andMYD88 siRNAs transfection, and
nonparametric Mann–Whitney test.
t groups. Figure 6M and Figure S6A shared experimental controls.
erent groups, n = 8/group, nonparametric Mann–Whitney test.
Cell 170, 548–563, July 27, 2017 559
Figure 7. The Levels of F.nucleatum, miR-18a*, miR-4802, and Autophagy Components Correlate and Are Relevant in CRC Patients
(A) Representative immunohistochemistry of p-ULK1 (upper), ULK1 (middle), and ATG7 (lower) proteins in CRC tissues from patients without recurrence and with
recurrence (Cohort 2). NR, non-recurrence; R, recurrence.
(legend continued on next page)
560 Cell 170, 548–563, July 27, 2017
targeting specific miRNAs and autophagy elements. Given that
the TLR4 and MYD88 innate immune signaling pathway is
essential for F. nucleatum infection (Abreu and Peek, 2014; Liu
et al., 2007), we have demonstrated that F.nucleatum-induced
genomic loss of miR-18a* and miR-4802 depends on the TLR4
and MYD88 signaling pathway. Therefore, F. nucleatum orches-
trates the TLR4-MYD88,miR18a* andmiR4802, andULK1/ATG7
autophagy network to biologically control CRC chemoresistance
(Figure 7E).
In addition to its biological importance, our work may be
relevant in clinical management of CRC patients. As the
amount of F. nucleatum is associated with the risk of CRC
recurrence, the measurement of F. nucleatum post-surgery
may be an effective approach to predict patient outcome.
Furthermore, our data raise an important clinical question:
are conventional chemotherapeutic regimens including Cape-
citabine plus Oxaliplatin suitable for CRC patients with a
high amount of F. nucleatum? Alternatively, we suggest that
CRC patients with a high amount of F. nucleatum may be
treated with conventional chemotherapy in combination with
anti-F. nucleatum treatment and/or an autophagy inhibitor.
Thus, it is important to detect F. nucleatum and its associated
pathway and differentially manage patients with different levels
of F. nucleatum.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d CONTACT FOR REAGENT AND RESOURCE SHARING
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
(B)
pro
(C)
(D)
(E)
B Mice
B Bacterial Strains and Growth Conditions
B Cell Lines
B Patients and Clinical Specimens
d METHOD DETAILS
B High-Throughput Sequencing
B RNA Extraction and Real-Time PCR
B Detection of F. nucleatum
B Western Blot and Chemical Reagents
B Cell Proliferation Assay, Apoptosis Detection, and
TUNEL Assay
B Oligonucleotide Transfection
B Luciferase Assay
B Electron Microscopy
B Confocal Microscope
B Adenovirus and Plasmids Construction
d QUANTIFICATION AND STATISTICAL ANALYSES
B Statistical Analysis
B Data and Software Availability
Statistical analysis of immunohistochemical immunoreactive score of Remme
teins in Cohort 2. NR, non-recurrence; R, recurrence.
Statistical analysis of miR-18a* (left) and miR-4802 (right) expression by real-
Correlations among F. nucleatum, miR-18a*, miR-4802, ULK1, and ATG7 lev
Schematic diagram of the relationship among F. nucleatum, autophagy and c
SUPPLEMENTAL INFORMATION
Supplemental Information includes seven figures, seven tables and can be
found with this article online at http://dx.doi.org/10.1016/j.cell.2017.07.008.
AUTHOR CONTRIBUTIONS
Conceptualization, W.Z., J.H., J.-Y.F.; Methodology, I.K., N.N., J.H., H.C.;In-
vestigation, T.Y., F.G., Y.Y., T.S., D.M., J.H., Y.Q., D.S.; Writing-Original Draft,
J.H., H.C., F.G.; Statistical analyses, H.C., F.G.; Writing-Reviewing & Editing,
I.K., N.N., Y.C., W.Z., J.-Y.F.; Funding Acquisition, J.-Y.F., J.H., H.C.,Y.C.,
W.Z.; Supervision, J.-Y.F., W.Z., J.H., and H.C.
ACKNOWLEDGMENTS
This project was supported in part by grants from the National Natural Science
Foundation of China (81421001, 81320108024, 81530072, 81522008
31371273, 31371420, 81572303, and 81001070), the National Key Technology
R&D Program (2014BAI09B05), the Program for Professor of Special Appoint-
ment (Eastern Scholar No. 201268 and 2015 Youth Eastern Scholar
NO.QD2015003) at Shanghai Institutions of Higher Learning, the Shanghai
Municipal Education Commission—Gaofeng Clinical Medicine Grant (no.
20152512, 20161309), the Chenxing Project of Shanghai Jiao-Tong University
(H. Chen and J. Hong), and the National Cancer Institute (CA211016, W.Z). We
thank Dr. Ming Zhong for collecting colocretal cancer tissues and patient
information for this work. We thank Dr.Tingting Yan for graphic abstract
conception.
Received: January 22, 2017
Revised: May 11, 2017
Accepted: July 10, 2017
Published: July 27, 2017
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Cell 170, 548–563, July 27, 2017 563
STAR+METHODS
KEY RESOURCES TABLE
REAGENTS or RESOURCES SOURCE IDENTIFIER
Antibodies
Anti-ACTB-HRP Sigma-Aldrich Cat#A1978
Anti-LC3B Sigma-Aldrich Cat#L7543
Anti-P62 Cell Signaling Technology Cat#8025
Anti-Cleaved Caspase9 Cell Signaling Technology Cat#9505
Anti-Cleaved Caspase3 Cell Signaling Technology Cat#9664
Anti-Cleaved Caspase6 Cell Signaling Technology Cat#9761
Anti-Cleaved Caspase7 Cell Signaling Technology Cat#8438
Anti-Cleaved PARP Cell Signaling Technology Cat#5625
Anti-p-H2AX(Ser139) Cell Signaling Technology Cat#9718
Anti-p-AMPK(a)(Thr172) Cell Signaling Technology Cat#2535
Anti-Beclin1 Cell Signaling Technology Cat#3495
Anti-ATG5 Cell Signaling Technology Cat#12994
Anti-ATG12 Cell Signaling Technology Cat#4180
Anti-ATG16L1 Cell Signaling Technology Cat#8089
Anti-ATG3 Cell Signaling Technology Cat#3415
Anti-MYD88 Cell Signaling Technology Cat#3699
Anti-ULK1 Abcam Cat#ab65056
Anti-p-ULK1(Ser556) Abcam Cat#ab203207
Anti-ATG7 Abcam Cat#ab52472
Anti-TLR4 Abcam Cat#ab13556
Biological Samples
Fresh colorectal cancer tissues Renji Hospital affiliated with Shanghai
Jiaotong University School of Medicine
N/A
Formalin-fixed paraffin-embedded colorectal
cancer tissues
Renji Hospital affiliated with Shanghai
Jiaotong University School of Medicine
N/A
Chemicals, Peptides, and Recombinant Proteins
Oxaliplatin Selleck Chemicals Cat#S1224
5-FU Selleck Chemicals Cat#S1209
Doxorubicin Selleck Chemicals Cat#S1208
Chloroquine Sigma-Aldrich Cat#C6628
Critical Commercial Assays
FITC Annexin V Apoptosis Detection Kit I BD Biosciences Cat#556547
Fixable Viability Stain 510 BD Biosciences Cat#564406
QIAamp DNA FFPE Tissue Kit QIAGEN Cat#56404
miRNease FFPE Kit QIAGEN Cat#217504
In Situ Cell Death Detection Kit, POD Roche Ca#11684817910
Deposited Data
Raw and analyzed data This paper GSE90944
Experimental Models: Cell Lines
FHC ATCC CRL-1831
HCT116 ATCC CCL-247
HT29 ATCC HTB-38
SW480 ATCC CCL-228
(Continued on next page)
e1 Cell 170, 548–563.e1–e5, July 27, 2017
Continued
REAGENTS or RESOURCES SOURCE IDENTIFIER
SW1116 ATCC CCL-233
Caco2 ATCC HTB-37
LoVo ATCC CCL-229
RKO ATCC CRL-2577
DLD1 ATCC CCL-221
Experimental Models: Organisms/Strains
Fusobacterium Nucleatum strain 25586 ATCC Cat#59899827
Bacteroides fragilis strain 43860 ATCC Cat#5038385
Enterococcus faecalis strain 47077 ATCC Cat#5091158
Peptostreptococcus anaerobius strain 27337 ATCC Cat#63229810
Parvimonas micra strain 33270 ATCC Cat#62282361
Prevotella intermedia strain 49046 ATCC Cat#63032740
Escherichia coli strain DH5a TIANGEN Cat#CB101
BALB/c nude mice Experimental Animal Centre of SIBS N/A
Sequences of miRNA mimics, miRNA inhibitors,
and mRNA siRNAs, see Table S7.
Genepharma N/A
DNA primer sequences, See Table S7 Sangon Biotech N/A
miRNA and U6 Primers, See Table S7 GeneCopoeia Included in Table S7
Software and Algorithms
ImageJ National Institutes of Health https://imagej.nih.gov/ij/
FindTar3 School of Life Science, Tsinghua University http://bio.sz.tsinghua.edu.cn/
miRDB Department of Radiation Oncology,
Washington University School of Medicine
http://mirdb.org/miRDB/
FlowJo FlowJo LLC https://www.flowjo.com/
ZEN 2011 Light Edition ZEISS https://www.zeiss.com/microscopy/
R R Development Core Team https://www.r-project.org/
TopHat2 Kim et al., 2013 http://tophat.cbcb.umd.edu
DeSeq2 Love et al., 2014 https://www.bioconductor.org/
HTSeq Anders et al., 2015 http://www-huber.embl.de/HTSeq/
GSVA Hanzelmann et al., 2013 https://www.bioconductor.org/
CONTACT FOR REAGENT AND RESOURCE SHARING
As Lead Contact, Weiping Zou is responsible for all reagent and resource requests. Please contact Weiping Zou at at wzou@med.
umich.edu with requests and inquiries.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
MiceFor the xenograft experiments, four-week-old male BALB/c nude mice were housed in laminar flow cabinets under specific path-
ogen-free conditions with food and water provided ad libitum. Colorectal cancer cells (1 3 107 SW480-male cells or 5 3 106
HCT116-male cells) were injected subcutaneously into the right axilla of each mouse to establish the CRC xenograft model. Six
days after subcutaneous inoculation, mice were randomly divided into different groups for different sets of experiments. The relevant
viral vectors and F. nucleatum were given by multipoint intratumoral injection, twice per week for three weeks. Chemotherapeutic
agents and CQ were administered by intraperitoneal injection, twice per week for three weeks.
Toexplore the roleofF.nucleatum in chemoresistance inOxaliplatin-sensitive tumor invivo,weusedSW480-malecells (13107) in the
xenograft experiments. There were six groups: i) Saline (Control); ii) F. nucleatum bacteria solution; iii) low-dose Oxaliplatin (5 mg/kg);
iv) low-dose Oxaliplatin (5 mg/kg) and F. nucleatum; v) high-dose Oxaliplatin (10 mg/kg); vi) high-dose Oxaliplatin(10 mg/kg) and
F. nucleatum.Mice received intratumoral injectionwithF. nucleatum, and intraperitoneal injectionwithOxaliplatin twice aweek for three
weeks.
Cell 170, 548–563.e1–e5, July 27, 2017 e2
In the following experiments, we used HCT116-male cells (5 3 106) to establish the xenograft models, and CQ (30 mg/kg),
Oxaliplatin (7.5 mg/kg), and 5-FU (50 mg/kg) to treat the mice.
To explore the role of F. nucleatum, miRNAs, and autophagy in CRC chemoresistance in vivo, we usedHCT116-male cells (53 106)
in the xenograft experiments. There were eight groups: i) saline (Control); ii) F. nucleatum bacteria solution; iii) miRNA vector virus; iv)
miR-18a* virus; v) miR-4802 virus; vi) control virus; vii) ATG7 overexpression virus; and viii) ULK1 overexpression virus.
To explore the role of autophagy in F. nucleatum-mediated chemoresistance in vivo, we designed seven groups: i) Control group;
ii) F. nucleatum group; iii) CQ group; iv) Oxaliplatin (or 5-FU) group; v) Oxaliplatin (or 5-FU) and F. nucleatum group; vi) Oxaliplatin (or
5-FU) and CQ group; vii) Oxaliplatin (or 5-FU), F. nucleatum and CQ group.
To explore the role of specific miRNAs and autophagy elements in F. nucleatum-mediated chemoresistance in vivo, we designed
seven groups: i) Control group; ii) Oxaliplatin (or 5-FU) group; iii) Oxaliplatin (or 5-FU) and F. nucleatum group; iv) Oxaliplatin (or 5-FU),
F. nucleatum and miR-18a* virus group; v) Oxaliplatin (or 5-FU), F. nucleatum and miR-4802 virus group; vi) Oxaliplatin (or 5-FU),
F. nucleatum, miR-18a* virus and ULK1 overexpression virus group; vii) Oxaliplatin (or 5-FU), F. nucleatum, miR-4802 virus and
ATG7 overexpression virus group.
To explore the role of the TLR4 signaling pathway in F. nucleatum-mediated chemoresistance in vivo, we designed eight groups:
i) Control group; ii) control shRNA group; iii) TLR4 shRNA virus group; iv) MYD88 shRNA virus group; v) Oxaliplatin (or 5-FU) group;
vi) Oxaliplatin (or 5-FU) and F. nucleatum group; vii) Oxaliplatin (or 5-FU), F. nucleatum, and TLR4 shRNA virus group; viii) Oxaliplatin
(or 5-FU), F. nucleatum, and MYD88 shRNA virus group.
The length and width of the tumors (in millimeters) were measured every three days with calipers. Tumor volume was calculated
using the formula (A3 B2) /2, where A and B are the long and short dimensions, respectively. After three weeks, all mice were sacri-
ficed and subcutaneous tumors were collected and weighed. The tumor volume and weight are presented as means ± SEM (n = 8).
Mouse experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Labo-
ratory Animals. The study procedures were approved by the Institutional Animal Care and UseCommittee of Renji Hospital, School of
Medicine, Shanghai Jiaotong University.
Bacterial Strains and Growth ConditionsFusobacterium nucleatum strain ATCC 25586 (Castellarin et al., 2012), Bacteroides fragilis strain 43860, Enterococcus faecalis strain
47077,Peptostreptococcus anaerobius strain 27337,Parvimonasmicra strain 33270, andPrevotella intermedia strain 49046were pur-
chased fromAmericanTypeCultureCollection (ATCC,Manassas,VA).F.nucleatumandP.micrawereculturedovernightat 37�Cunder
anaerobic conditions (DG250, DonWhitley Scientific,West Yorkshire, UK) in brain heart infusion (BHI) broth supplementedwith hemin,
K2HPO4, Vitamin K1, and L-Cysteine(Rhee et al., 2009). P. anaerobius and P. intermedia were cultured in Wilkins-Chalgren anaerobe
broth (CM0643, Thermo Fisher Scientific, West Palm Beach, FL) at 37�C under anaerobic conditions. B.fragiliswas cultured overnight
at 37�C under anaerobic conditions in ATCC 1490 Modified chopped meat medium. E. faecalis and the commensal Escherichia coli
strain DH5a (Tiangen, China) were cultured in Luria-Bertani (LB) medium overnight at 37�C in shake cultivation at 220 rpm/min.
Cell LinesHuman colorectal cancer cell lines RKO-NA, SW1116-male, DLD1-male, SW480-male, Caco2-male, LOVO-male, HT29-female, and
HCT116-male (ATCC) were cultured in RPMI-1640medium (GIBCO, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS)
at 37�C in a humidified 5%CO2 atmosphere. Human colonic epithelial cell FHC (from 13weeks gestation) was cultured in DMEM/F12
medium (GIBCO, Carlsbad, CA) supplemented with 25mM HEPES, 10 ng/ml cholera toxin, 0.005mg/ml insulin, 0.005 mg/ml trans-
ferrin, 100 ng/ml hydrocortisone and 10% FBS at 37�C in a humidified 5% CO2 atmosphere. To establish the Oxaliplatin-resistant
SW480 cells, we cultured SW480 cells with gradually increased Oxaliplatin for two months. The EC50 of Oxaliplatin was 190.9 mM
and 109.9 mM for the established Oxaliplatin-resistant SW480 cells and the parental SW480 cells, respectively.
Patients and Clinical SpecimensWe studied 3 cohorts of patients with colorectal cancer from Renji Hospital affiliated to Shanghai Jiaotong University School of Med-
icine between 2012 and 2015. Cohort 1 and cohort 2 were from theWestern Campus of Renji Hospital. Cohort 3 was from the Eastern
Campus of Renji Hospital. There were 31 fresh tissues in Cohort 1 and 92 and 173 formalin-fixed paraffin-embedded tissues (FFPE) in
Cohorts 2 and 3, respectively. We performed 16 s RNA sequencing studies in Cohort 1 to define which bacterium is predominant
(and/or different) in the recurrent colorectal cancer tissues compared to the non-recurrent colorectal cancer tissues. We used Cohort
2 as a discovery clinical sample set to determine which levels of F. nucleatum are linked to chemoresistance-associated cancer
recurrence. We used Cohort 3 as a validation dataset to evaluate whether the cut-off value generated fromCohort 2 could be applied
and validated in an independent cohort with known clinical information such as recurrent or no-recurrent cancer. Thus, Cohorts 1, 2,
and 3 serve as the models for our research exploration, discovery, and validation, respectively. All the patients information could be
found in Tables S1-S2 and S4.
Patients were pathologically and clinically diagnosed with colorectal cancer. After surgical debulking, patients had undergone
XELOX regimen therapy (Oxaliplatin 130 mg/m2 IV over two hours on the first day; Capecitabine 850�1000 mg/m2, twice
daily p.o. for 14 days; repeated every three weeks). Informed consent was obtained from the patients before sample collection
in accordance with institutional guidelines. Recurrence was monitored by imaging examination systems (Chest X-ray and CT),
e3 Cell 170, 548–563.e1–e5, July 27, 2017
gastrointestinal endoscopy with biopsy, and telephone follow-up. The Ethics Committees in the Renji Hospital approved the study
protocols. Written informed consents were obtained from all participants in this study. All the research was carried out in accordance
with the provisions of the Helsinki Declaration of 1975.
METHOD DETAILS
High-Throughput SequencingFor RNA sequencing, each sample was cleaned up on an RNeasyMini Column (QIAGEN, Hilden, Germany), treated with DNase, and
analyzed for quality on an Agilent 2100 Bioanalyzer. Samples were run on an Illumina HiSeq 3000 for 2 3 150-bp paired-end
sequencing. The RNA-seq data analysis was performed according to the TopHat- HTSeq-DeSeq2 frame (Anders et al., 2013). Briefly,
reads were mapped to the human genome (hg19) using TopHat v2.0.11(Kim et al., 2013) (http://tophat.cbcb.umd.edu) with the
default options with a TopHat transcript index built from Ensembl_GRCh37. Count files of the aligned sequencing reads were gener-
ated by the htseq-count script from the Python package HTSeq with union mode, using the GTF annotation file(Anders et al., 2015).
The read counts from each sequenced sample were combined into a count file, which was subsequently used for the differential
expression analysis. Differential analyses were performed to the count files using DESeq2 packages, following standard normaliza-
tion procedures(Love et al., 2014). Genes with less than 5 total counts in both conditions were removed from further analysis. The
RNA sequence data have been deposited in NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are
accessible through GEO Series accession number GSE90944.
RNA Extraction and Real-Time PCRTotal RNA was extracted from the CRC lines using Trizol reagent (Invitrogen, Carlsbad, CA), and 1 mg of total RNA was reverse tran-
scribed using the PrimeScript RT Reagent Kit (Perfect Real Time; Takara, Japan) to detect relative mRNAs. For the miRNAs, 1 mg of
total RNAwas reverse transcribed using All-in-OneMiRNAQ-PCRDetection Kit (GeneCopoeia, Rockville, MD) according tomanufac-
turer’s instructions in a total reaction volume of 25 ml. Quantitative real-time PCRwas performed in triplicates on an Applied Biosystem
7900 quantitative PCR system (Applied Biosystems, Foster City, CA) as previously described (Sun et al., 2015). The Ct values obtained
from different samples were compared using the 2-DDCt method. b-actin and U6 served as internal reference genes, respectively.
Detection of F. nucleatumThe primer sequences of the reference gene, prostaglandin transporter (PGT) and the method for F. nucleatum detection were
described previously (Castellarin et al., 2012; Chen et al., 2013). gDNA was extracted from fresh colorectal cancer tissue with the
QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) and from FFPE with QIAamp DNA FFPE Tissue Kit (QIAGEN, Hilden, Germany).
gDNA from each specimen was subjected to qPCR to determine the amounts of F. nucleatum by detecting the 16S genes. Each re-
action contained 40 ng of gDNA and was assayed in triplicate in 10 mL reactions containing 13 Power SYBR Green PCRMaster Mix
(Thermo Fisher Scientific, West PalmBeach, FL),0.4 mMeach primer andwas placed in a 96-well optical PCR plate. Amplification and
detection of DNA was performed with the ABI StepOne Plus Real-Time PCR System (Applied Biosystems, Foster City, CA) under the
following reaction conditions: 10 min at 95�C, followed by 40 cycles of denaturation at 95�C for 15 s and at 60�C for 1 min. The cycle
threshold (Ct) values for F. nucleatum were normalized to the amount of human biopsy gDNA in each reaction by using PGT as a
reference gene (Castellarin et al., 2012).
Western Blot and Chemical ReagentsWestern blot was performed using standard techniques as described previously (Xiong et al., 2012). Cell extracts were collected and
quantified using BCA Protein Assay Kit (Thermo Fisher Scientific, West Palm Beach, FL). 40 mg of protein was electrophoresed
through 12% SDS polyacrylamide gels and were then transferred to PVDF membranes (Bio-Rad, Hercules, CA). The membranes
were blocked in 5% fat-free milk for one hour and then incubated with primary antibodies at 4�C overnight. Secondary antibodies
were labeled with HRP (KangChen, China) and the signals were detected using ECL Kit (Pierce Biotech, Rockford, IL). Subsequently,
the images were analyzed by ImageJ 1.43 software. A b-actin antibody was used as a control for whole-cell lysates. The information
on all antibodies is listed in RESOURCES TABLE.
Cell Proliferation Assay, Apoptosis Detection, and TUNEL AssayCell proliferation was assessed byCell Counting Kit-8 (Dojindo, Japan) assay. Cells were seeded at 2000 cells/well into 96-well plates
with 100 mL culture medium. The 10 mL of CCK-8 solution was added to the cells at specific time points and cells were incubated for
2 hr at 37�C. The reaction product was quantified according to the manufacturer’s instructions.
Apoptosis was examined by flow cytometric analysis. An Annexin V FITC/PI double stain assay (BD Biosciences, San Jose, CA)
was performed following the manufacturer’s protocol. When the cells were treated with Doxorubicin, we performed FVS510 and
Annexin V-FITC double staining to avoid the fluorescent signal of the drug. Tumor cell apoptosis in the xenograft tumor tissues
was detected by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) technology using the in Situ
Cell Death Detection Kit (Roche Molecular Biochemicals, Mannheim, Germany). The negative control was incubated with label
solution (without terminal transferase) instead of the TUNEL reaction mixture.
Cell 170, 548–563.e1–e5, July 27, 2017 e4
Oligonucleotide TransfectionsiRNAs, miRNAmimics, and inhibitors were purchased fromGenepharma (Shanghai, China) (Table S7). Oligonucleotide transfection
was performed using the DharmaFECT 1 siRNA transfection reagent (Invitrogen, Carlsbad, CA), while nonspecific siRNA or miRNA
was used as negative controls.
Luciferase AssayHCT116 cells or HT29 cells were co-transfected with 80 ng of the luciferase reporter plasmid, 10 ng of the pRL-TK-Renilla-luciferase
plasmid (Promega, Madison, WI), and the indicated RNAs (final concentration of 20 nmol/L). Twenty-four hours after transfection,
firefly and Renilla luciferase activities were quantified using the Dual-Luciferase Assay Kit (Promega, Madison, WI). Each transfection
was performed in triplicates and repeated twice.
Electron MicroscopyCells were treated as indicated and fixed with 2.5% glutaraldehyde containing 0.1 mol/L sodium cacodylate. Samples were fixed
using 1%osmium tetroxide, followed by dehydration with an increasing concentration gradient of ethanol and propylene oxide. Sam-
ples were then embedded, cut into 50-nm sections, and stained with 3%uranyl acetate and lead citrate. Images were acquired using
a JEM-1200 electron microscope (JEOL, Tokyo, Japan).
Confocal MicroscopeHCT116 cells and HT29 cells were plated in 6-well plates and allowed to reach 50%–70% confluence at the time of transfection.
MRFP-GFP-LC3 adenoviral vectors were purchased from HanBio Technology (Shanghai, China). The principle of the assay is based
on different pH stability of green and red fluorescent proteins. The fluorescent signal of EGFP could be quenched under the acidic
condition (pH below 5) inside the lysosome, and the mRFP fluorescent signal has no significant change under the acidic condition. In
green and red-merged images, autophagosomes are shown as yellow puncta (i.e., RFP+GFP+), while autolysosomes are shown as
red puncta (i.e., RFP+GFP�). Autophagic flux is increased when both yellow and red puncta are increased in cells, while autophagic
flux is blocked when only yellow puncta are increased without red puncta alteration, or when both yellow and red puncta are
decreased in cells (Zhou et al., 2012).
Adenoviral infection was performed according to the manufacturer’s instructions. CRC cells were incubated in growth medium
with the adenoviruses for 2 hr at 37�C, and were grown in medium containing F. nucleatum and miRNAs at the indicated concentra-
tions for 0-24 h at 37�C. LC3 puncta were examined with Zeiss LSM710 confocal microscope (Carl Zeiss) fitted with a 63 3 oil
immersion objective.
Adenovirus and Plasmids ConstructionThe control, control shRNA, control miRNA, miR-18a*, miR-4802, TLR4 shRNA, MYD88 shRNA adenovirus, as well as ULK1 and
ATG7 overexpression adenovirus, and all plasmids were constructed by Shanghai Obio Techonology Company, Shanghai, China.
QUANTIFICATION AND STATISTICAL ANALYSES
Statistical AnalysisStatistical analyses were carried out using the programR (www.r-project.org). Data from at least three independent experiments per-
formed in triplicates are presented as themeans±SE. Error bars in the scatterplots and the bar graphs represent SE.Datawere exam-
ined to determine whether they were normally distributed with the One-Sample Kolmogorov-Smirnov test. If the data were normally
distributed, comparisons of measurement data between two groups were performed using independent sample t test and the com-
parisons among three ormore groupswere first performed by one-way ANOVA test. If the results showed significant difference, when
thedatawere skeweddistribution, comparisonswereperformedbynonparametric test.Measurement data between twogroupswere
performed using nonparametric Mann-Whitney test. To generate the ROC curves, patients were classified as recurrence time either
longer or shorter than themedian recurrence free survival, excluding patients whowere alive for durations less than themedian recur-
rence free survival at last follow-up. Single-sample gene set enrichment analysis (ssGSEA) was used to assess gene set activation
scores in gene expression profiling data. ssGSEA calculates a sample level gene set score by comparing the distribution of gene
expression ranks insideandoutside thegene set. The ssGSEAscorewascalculated byGeneSet VariationAnalysis (GSVA)Rpackage
(Hanzelmann et al., 2013). Statistical tests were two-tailed and a p value of less than 0.05 was considered statistically significant.
Data and Software AvailabilityData from this study have been deposited in the Gene Expression Omnibus (GEO) databases under the following accession:
GSE90944.
e5 Cell 170, 548–563.e1–e5, July 27, 2017
Supplemental Figures
A
C
B
Cohort 3
F. n
ucle
atum
abu
ndan
ce(-
Ct)△
P<0.01
E
D
F. nucleatum
yes no
AJCC stage(III) P=0.022Tumor size(>15cm ) 3
P=0.018Penetration (Serosa)P=0.335
High F. nucleatum Low F. nucleatum
0 20 40 60 80
NR R(n=87)(n=86)
-20
-15
-10
-5
0
0.1 1 10
PHR (95% CI)
3.667(2.336-5.756) <0.001
1.699(1.108-2.604) 0.015
1.253(0.787-1.994) 0.342
1.19(0.766-1.846) 0.439
1.909(0.831-4.388) 0.128
0.917(0.585-1.807) 0.708
1.305(0.838-2.033) 0.238
Univariable risk factor
Cohort 3
Cohort 3
Multivariable risk factor
0.1 1 10
HR (95% CI) P
0.015
<0.001
1.697(1.106-2.604)
3.662(2.333-5.764)
Cohort 2Cohort 1
P. intermediaF. nucleatumP. micraP. anaerobius
-30
-20
-10
0NR R NR R NR R NR R
Rel
ativ
e ab
unda
nce(
- C
t)△
P<0.01 P<0.05 P<0.05n.s.
AJCC stage (III vs II)
(high vs low)
Penetration(Serosa vs Muscular)
F. nucleatum abundance
AJCC stage (III vs II)
(high vs low)F. nucleatum abundance
Gender(Male vs Famale)
Pathological differentiation(Poor vs Well & Moderate)
Age(≥60 vs <60)
Tumor size (>15cm³ vs ≤ 15cm³)
Figure S1. F. nucleatum Is Associated with Cancer Recurrence and Patient Outcome, Related to Figure 1
(A) Statistical analysis of P. intermedia, F. nucleatum, P. micra, P. anaerobius amounts in CRC tissues from patients without recurrence (n = 15) and with
recurrence (n = 16), nonparametric Mann–Whitney test. The bars represent SE.
(B) Comparing tumor size, positive or negative serosal invasion, and AJCC stage between F. nucleatum high-expression and low-expression tumors of Cohort 2.
The heatmap illustrates the association of different clinical characters with F. nucleatum high- and low-expression tumors. Statistical significance was performed
by Chi-square test.
(C) Statistical analysis of F. nucleatum amount in CRC tissues from patients without recurrence (n = 86) and with recurrence (n = 87) (Cohort 3), nonparametric
Mann–Whitney test. The bars represent SE.
(D) Univariate analysis was performed in the Cohort 3. The bars correspond to 95% confidence intervals.
(E) Multivariate analysis was performed in the Cohort 3. The bars correspond to 95% confidence intervals. NR, non-recurrence; R, recurrence.
P<0.05
P<0.05
P<0.05
P<0.05
P<0.05
P<0.05
P<0.05
P<0.05
F. nucleatumControl
F. nucleatumControl
ULK1
ATG12
ATG5
ATG16L1
ATG7
ATG3
Beclin1
ACTB
Control F. nucleatum Control F. nucleatum
p-ULK1
p-AMPK
ULK1
ATG12
ATG5
ATG16L1
ATG7
ATG3
Beclin1
ACTB
p-ULK1
p-AMPK
0 1 2 3 4 5Relative intensity
0 1 2 3 4Relative intensity
C
E
D
A
B
LC3 ILC3 II
LC3 ILC3 II
LC3 ILC3 II
LC3 ILC3 II
LC3 ILC3 II
P62
ACTB
P62
ACTB
2h 4h 2h 4h 2h 4hTime
E. coli
E. faec
alis
Contro
l
F. nuc
leatum
ACTB
LC3 I
LC3 II
HCT116
FHCRKO
SW11
16
HT29SW
480
DLD1
Caco2
LoVo
B. frag
ilis
2h 4h
Contro
l
F. nuc
leatum
P. ana
erobiu
s
P. micr
a
P. inter
media
HCT116
HCT116
Contro
l
F. nuc
leatum
P. ana
erobiu
s
P. micr
a
P. inter
media
HT29
HT29
HT29
F G
- - + +- + - +
HT29
CQF. nucleatum
P62
ACTB
P62
ACTB
+F. nucleatum (MOI)
0 100 500 1000
HT29
P62
ACTB
+F. nucleatum (MOI)
0 100 500 1000HCT116
Figure S2. F. nucleatum Promotes Cancer Autophagy Activation, Related to Figure 2
(A) The basic levels of LC3 protein in nine colorectal epithelial cell lines were detected by western blot assays.
(B) Western blot was performed to detect autophagy-related protein expression in HCT116 cells (left) and HT29 cells (right) co-cultured with F. nucleatum,
nonparametric Mann–Whitney test. The relative density of each band was analyzed by ImageJ software.
(legend continued on next page)
(C) Western blot was used to detect LC-3I, LC-3II, and p62 expression in HT29 cells co-cultured with F. nucleatum, or E. coli, or E. faecalis, or B. fragilis,
respectively.
(D) Western blot was used to detect LC-3I, LC-3II, and p62 expression in HCT116 cells (left) and HT29 (right) cells co-cultured with F. nucleatum at different
multiplicity of infection (MOI).
(E and F) Western blot was used to detect LC-3I and LC-3II expression in HCT116 cells (E) and HT29 cells (F) co-cultured with F. nucleatum, P. anaerobius,
P. micra or P. intermedia, respectively.
(G) Western blot was performed to detect LC-3I, LC-3II, and p62 expression in HT29 cells co-cultured with F. nucleatum and CQ.
A BControlF. nucleatum
(MOI 100)F. nucleatum (MOI 500)F. nucleatum (MOI 1000)
010203040506070
Rel
ativ
e ce
ll vi
abili
ty
0 1 2 3 4 5Days
Rel
ativ
e ce
ll vi
abili
ty
0 1 2 3 4 5Days
0
2
4
6
8
10ControlF. nucleatum (MOI 100)F. nucleatum
(MOI 500)F. nucleatum (MOI 1000)
G H
HCT116 HT29
Q
R
O P1.5
0.5
0.0
1.0
Contro
lRel
ativ
e U
LK1
mR
NA
expr
essi
on
HCT116
0.5
0.0
1.0
1.5
siATG7-1
siATG7-2
siATG7-1
siATG7-2
Rel
ativ
e A
TG7
mR
NA
expr
essi
on
0.5
0.0
1.0
1.5
Contro
lCon
trol
siULK
1-1
Rel
ativ
e U
LK1
mR
NA
expr
essi
on HT29
0.5
0.0
1.0
1.5
Contro
lRel
ativ
e A
TG7
mR
NA
expr
essi
on HT29
P<0
.05
P<0
.05
HCT116
P<0
.05
P<0
.05
P<0
.05
P<0
.05
P<0
.05
P<0
.05
siULK
1-2
siULK
1-1siU
LK1-2
Contro
l
L-Oxa
liplat
in
L-Oxa
liplat
in
+F. n
uclea
tum
Contro
l
H-Oxa
liplat
in
H-Oxa
liplat
in
+F. n
uclea
tum0
1
2
3Tu
mor
wei
ght(g
)
0
1
2
3
Tum
or w
eigh
t(g)
P<0
.05
P<0
.05
K L M
Control
F. nucleatum
Control F. nucleatum0.00.51.01.52.02.5
Tum
or w
eigh
t(g) n.s.
N
S T
5-Fu (μM) 5-Fu (μM)
ControlF. nucleatumF. nucleatum+siULK1-1F. nucleatum+siULK1-2F. nucleatum+siATG7-1F. nucleatum+siATG7-2
ControlF. nucleatumF. nucleatum+siULK1-1F. nucleatum+siULK1-2F. nucleatum+siATG7-1F. nucleatum+siATG7-2
P<0
.05
0 400 8000
20
40
60
Apop
tosi
s(%
)
P<0
.05
0 400 8000
20
40
60
Apop
tosi
s(%
)
P<0
.05 P
<0.0
5
+F. nucleatum
+F. nucleatum
ULK1
siULK1-1
0 4h 0 4h
HCT116Control
ACTB
P62
P62
siATG7-1
0 4h 0 4h
ATG7
ACTB
Control
4h 0 4h
HT29
HT29
0
siATG7-1
0 4h 0 4h
Control
LC3 ILC3 II
LC3 ILC3 II
HCT116
siULK1-1Control
HCT116 HT29
Control
L-Oxaliplatin
H-Oxaliplatin
L-Oxaliplatin+F. nucleatum
H-Oxaliplatin+F. nucleatum
C
0 64 1280
20
40
60
Apop
tosis
(%)
Oxaliplatin (μM)
Control
P. intermediaP. micraP. anaerobius
HCT116
5-Fu (μM)Oxaliplatin (μM)0 64 128
01020304050
Apop
tosis
(%)
0 400 8000
10
20
30
40
Apop
tosis
(%)Control
P. intermediaP. micraP. anaerobius
Control
P. intermediaP. micraP. anaerobius
FEHT29 HT29
D
0 400 8000
1020304050
Apop
tosis
(%)
Apop
tosis
(%)
Apop
tosis
(%)
5-Fu (μM)
Control
P. intermediaP. micraP. anaerobius
ControlF.nucleatum
ControlF.nucleatum
HCT116
HCT116
0
20
40
60 HT29
0 02 400 02 40 0
20
40
60
Doxorubicin(μM) Doxorubicin(μM)
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
I J
Oxaliplatin concentration(μM)
Apo
ptos
is(%
)
0 1000
1020304050
50 150 200
Parental SW480 cells
SW480 resistant cellsParental SW480 cells + F. nucleatum 0
50
100
150
200
250
109.9
177.2 190.9
ParentalSW480 cells
SW480ParentalSW480 cells resistant
cells+F. nucleatum
EC50 value of different SW480 cells
Oxa
lipla
tin c
once
ntra
tion(μM
)
(legend on next page)
Figure S3. F. nucleatum Induces Chemoresistance in Colorectal Cancer Cells via Activation of the Autophagy Pathway, Related to Figure 3
(A and B) Cell proliferation was detected in HCT116 cells (A) and HT29 cells (B) co-cultured with F. nucleatum at different MOIs, non-parametric Mann-Whit-
ney test.
(C–F) Apoptosis was detected by flow cytometry in HCT116 cells (C, D) and HT29 cells (E, F). The cells were independently co-cultured with F. nucleatum,
P. anaerobius, P. micra, or P. intermedia and treated with different concentrations of Oxaliplatin (C, E) and 5-Fu (D, F), nonparametric Mann–Whitney test.
(G and H) Apoptosis was detected by flow cytometry in HCT116 cells (G) and HT29 cells (H). The cells were co-cultured with F. nucleatum and treated with
different concentrations of Doxorubicin, nonparametric Mann–Whitney test.
(I) Apoptosis was detected by flow cytometry in parental SW480 cells, Oxaliplatin-resistant SW480 cells, and parental SW480 cells co-cultured with F. nucleatum
in the presence of different concentrations of Oxaliplatin. Each point is the mean of 3 replicates. All analyses were performed using R software using the Analysis
of Dose-Response Curves (DRC) package.
(J) The EC50 value in different SW480 cells treated with Oxaliplatin.
(K–N) Representative data of tumors in nude mice bearing SW480 cells in different experimental conditions (K, M). Statistical analysis of mouse tumor weights in
different groups (L, N), n = 8/group, nonparametric Mann–Whitney test. L-Oxaliplatin, low concentration of Oxaliplatin; H-Oxaliplatin, high concentration of
Oxaliplatin.
(O) Real-time PCR was performed to detect ULK1 mRNA level in HCT116 cells (left) and HT29 cells (right) transfected with ULK1 siRNAs. n = 3, non-parametric
Mann-Whitney test.
(P) Real-time PCR was performed to detect ATG7 mRNA level in HCT116 cells (left) and HT29 cells (right) cells transfected with ATG7 siRNAs. n = 3, non-
parametric Mann-Whitney test.
(Q)Western blot was performed to detect ULK1, LC-3I, LC-3II, and p62 expression in HCT116 cells (left) and HT29 cells (right) transfected with ULK1 siRNA-1 and
co-cultured with F. nucleatum.
(R)Western blot was performed to detect ATG7, LC-3I, LC-3II, and p62 expression in HCT116 cells (left) and HT29 cells (right) transfected with ATG7 siRNA-1 and
co-cultured with F. nucleatum.
(S and T) Apoptosis was detected by flow cytometry in HCT116 cells (S) and HT29 cells (T). The cells were co-cultured with F. nucleatum after ULK1 and ATG7
siRNAs transfection and subsequently treated with different concentrations of 5-Fu, nonparametric Mann–Whitney test.
A
HCT116 HT290.0
0.4
0.8
1.2
F. nucleatumF. nucleatum
ATG7
pro m
o ter
luci f
eras
eac
tivi ty
Bn.s. n.s.
HCT116 HT290.0
0.4
0.8
1.2Control Control
ULK1
pro m
o ter
luci fe
rase
activ
i ty
n.s. n.s.
C
(
D
ULK1
ULK1
ATG7
ATG7
E
Downregulated miRNAs(n=68) in high F. nulceatum group
Potential target miRNAs of ULK1 (n=4) and ATG7 (n=3)
qPCR confirmation
hsa−miR−3907hsa−miR−3614−5phsa−miR−4763−3phsa−miR−485−5phsa−miR−4540hsa−miR−3197hsa−miR−3150b−3phsa−miR−4794hsa−miR−4747−5phsa−miR−4464hsa−miR−4657hsa−miR−6069hsa−miR−423−5phsa−miR−4716−3phsa−miR−4507hsa−miR−762hsa−miR−1233−1−5phsa−miR−3198hsa−miR−4768−3phsa−miR−3676−3phsa−miR−3130−3phsa−miR−4448hsa−miR−4632−5phsa−miR−3622b−5phsa−miR−340−3phsa−miR−6723−5phsa−miR−3676−5phsa−miR−5100hsa−miR−4778−5phsa−miR−21−5phsa−miR−29b−3phsa−miR−6075hsa−miR−5195−5phsa−miR−3185hsa−miR−421hsa−miR−1287hsa−miR−1208hsa−miR−583hsa−miR−5003−3phsa−miR−223−3phsa−miR−605hsa−miR−4713−3phsa−miR−658hsa−miR−4648hsa−miR−103bhsa−miR−4494hsa−miR−509−3−5phsa−miR−4425hsa−miR−514b−5phsa−miR−542−5phsa−miR−198hsa−miR−509−5phsa−miR−183−3phsa−miR−4539hsa−miR−4538hsa−miR−1247−3phsa−miR−564hsa−miR−4711−5phsa−miR−4422hsa−miR−4773hsa−miR−4684−3phsa−miR−513c−5phsa−miR−513a−5phsa−miR−4802−5phsa−miR−4635hsa−miR−4481hsa−miR−4790−3phsa−miR−18a−3phsa−miR−4647hsa−miR−610hsa−miR−5696hsa−miR−3131hsa−miR−4514hsa−miR−4788hsa−miR−4436ahsa−miR−616−3phsa−miR−602hsa−miR−6086hsa−miR−149−3phsa−miR−193b−5phsa−miR−188−3phsa−miR−548az−5phsa−miR−3939hsa−miR−4497hsa−miR−4270hsa−miR−125b−2−3phsa−miR−1471hsa−miR−4665−5phsa−miR−939−5phsa−miR−4321hsa−miR−325hsa−miR−3928hsa−miR−6501−3phsa−miR−187−5phsa−miR−1273chsa−miR−4496hsa−miR−4664−3phsa−miR−4662bhsa−miR−4694−3phsa−miR−3616−3phsa−miR−23b−5phsa−miR−3161hsa−miR−3934−5phsa−miR−4722−5phsa−miR−3714hsa−miR−3944−5phsa−miR−513bhsa−miR−3122hsa−miR−4530
group
Group
High F. nucleatumLow F. nucleatum
−2
−1
0
1
2
HCT116
HT29
0.0
0.5
1.0
1.5
2.0
0.0
0.5
1.0
1.5
2.0
Rel
ativ
e m
icro
RN
A ex
pres
sion
Rel
ativ
e m
icro
RN
A ex
pres
sion
Rel
ativ
e m
icro
RN
A ex
pres
sion
Rel
ativ
e m
icro
RN
A ex
pres
sion
0.0
0.5
1.0
1.5
2.0
0.0
0.5
1.0
1.5
miR-45
15
miR-14
9*
miR-10
3b
miR-12
5b-2*
miR-18
a*
miR-42
70
miR-48
02
miR-45
15
miR-14
9*
miR-10
3b
miR-12
5b-2*
miR-18
a*
miR-42
70
miR-48
02
P<0
.05
P<0
.05
P<0
.05
P<0
.05
ControlF. nucleatum
ControlF. nucleatum
ControlF. nucleatum
ControlF. nucleatum
G
0.00.20.40.60.81.01.2
Rel
ativ
e lu
cife
rase
act
ivity n.s.
0.00.20.40.60.81.01.2
Rel
ativ
e lu
cife
rase
act
ivity n.s.
F
3
0
1
2
Rela
tive
ULK
1 m
RNA
expr
essi
on
H
3
0
1
2
Rela
tive
ATG
7 m
RNA
expr
essi
on
HT29 HT29 HT29 HT29I
P<0.05P<0.05
P<0
.05
P<0
.05
P<0
.05
P<0
.05
Contro
l inhib
itor
miR-18
a* in
hibito
r
Contro
l mim
ics
miR-18
a* m
imics
Contro
l inhib
itor
miR-48
02 in
hibito
r
Contro
l mim
ics
miR-48
02 m
imics
Contro
l miR
NA+ULK
1 3' U
TR-Wt
miR-18
a*+U
LK1 3
' UTR-W
t
Contro
l miR
NA+ULK
1 3' U
TR-Mut
miR-18
a*+U
LK1 3
' UTR-M
ut
Contro
l miR
NA+ATG7 3
' UTR-W
t
miR-48
02+A
TG7 3' U
TR-Wt
miR-48
02+A
TG7 3' U
TR-Mut
Contro
l miR
NA+ATG7 3
' UTR-M
ut
+F. nucleatum 0 4h 0 4h 0 4h 0 4h
HT29
ULK1
ACTB
P62
P62
J
ATG7
ACTB
+F. nucleatum 0 4h 0 4h 0 4h 0 4h
KL M
LC3 ILC3 II
LC3 ILC3 II
Controlinhibitor
miR-18a*inhibitor
Controlmimics
miR-18a*mimics
miR-4802mimics
Controlmimics
Controlinhibitor
miR-4802inhibitor
HT29 HT29Control F. nucleatumControl F. nucleatum
F. nucleatum+miR-18a*
F. nucleatum+miR-4802F. nucleatum
+miR-18a*F. nucleatum+miR-4802
(legend on next page)
Figure S4. F. nucleatum Activates Cancer Autophagy via Downregulation of miR-18a* and miR-4802, Related to Figure 4
(A and B) Luciferase assays were performed in HCT116 and HT29 cells. The cells were co-cultured with F. nucleatum after transfection of ULK1 (A) and ATG7 (B)
promoter plasmids. n.s., not significant.
(C) Six pairs of CRC tissueswere chosen formiRNAChip array (left). The abundance of F. nucleatumwas used as the basis for grouping, n = 6. Schematic of target
miRNA candidate screening process is shown (right).
(D and E) Expression of candidate miRNAs was quantified by real-time PCR in HCT116 cells (D) and HT29 cells (E). n = 3.
(F and G) Luciferase activity was measured in HT29 cells. The luciferase reporters expressing wild-type or mutant human ULK1 30UTRs (F) and ATG7 30UTRs (G)
were used. The cells were co-transfected with miR-18a* mimics, miR-4802 mimics, or control miRNA.
(H and I) Real-time PCR was performed in HT29 cells to detect the expression of ULK1 (H) and ATG7 (I) genes. The cells were transfected with miR-18a* mimics,
miR-4802 mimics, or inhibitor, nonparametric Mann–Whitney test.
(J and K) Autophagy-related proteins were detected by western blot in HT29 cells. The cells were transfected with mimics or inhibitor of miR-18a* (J) and miR-
4802 (K), then co-cultured with F. nucleatum.
(L) Autophagosomes were detected with confocal microscope (2000 3 magnification) in HT29 cells. The cells expressing mRFP-GFP-LC3 fusion protein were
transfected with miR-18a* (left) and miR-4802(right) mimics, then cultured with F. nucleatum, Bar scale, 5 mm.
(M) Autophagosomes were observed by transmission electron microscopy (175003magnification) in HT29 cells. The cells were transfected with miR-18a* (left)
and miR-4802 (right) mimics, then co-cultured with F. nucleatum. Bar scale, 1 mm.
Oxaliplatin (μM)
HT29
A
HT29
5-Fu (μM)
ControlF. nucleatumF. nucleatum+miR-18a* mimicsF. nucleatum+miR-4802 mimics
B
C
Oxaliplatin (μM)
HT29
5-Fu (μM)
HT29ControlF. nucleatum
D
F
E
0 1 2 3 4 5Days Days
0 1 2 3 4 50
5
10
15
20 Negative control miR-18a* mimicsmiR-4802 mimics
Rel
ativ
e ce
ll vi
abili
ty
miR-18a* inhibitormiR-4802 inhibitor
0
1
2
3
4
5
Rel
ativ
e ce
ll vi
abili
ty
Negative control miR-18a* mimicsmiR-4802 mimicsmiR-18a* inhibitormiR-4802 inhibitor
ControlF. nucleatumF. nucleatum+miR-18a* mimicsF. nucleatum+miR-4802 mimics
miR-18a* inhibitormiR-4802 inhibitor
ControlF. nucleatummiR-18a* inhibitormiR-4802 inhibitor
HT29HCT116
P<0
.05
P<0
.05
0 64 1280
20
40
60
Apop
tosi
s(%
)
P<0
.05
P<0
.05
0 400 8000
20
40
60
Apop
tosi
s(%
)
P<0
.05
P<0
.05
0 64 1280
20
40
60
Apop
tosi
s(%
)
P<0
.05
P<0
.05
0 400 8000
20
40
60
Apop
tosi
s(%
)
OxaliplatinHT29 5-Fu
miR-18a*miR-4802
Cleaved Caspase-9
CleavedCaspase-3
Cleaved Caspase-6
Cleaved Caspase-7
CleavedPARP
ACTB
F. nucleatum
p-H2AX
F. nucleatum
Control
Control miRNAadenovirus
miR-18a*adenovirus
miR-4802 adenovirus
Control adenovious
ULK1 adenovious
ATG7 adenovious
Contro
l
F. nuc
leatum
Contro
l miR
NA
aden
oviou
s
miR-18
a* ad
enov
irus
miR-48
02 ad
enov
irus
Contro
l ade
novir
us
ULK1 a
deno
virus
ATG7 ade
novir
us0.0
0.5
1.0
1.5
2.0
2.5
Tum
or w
eigh
t(g)
G H
I
Control miRNAadenoviousmiR-18a* adenovirusmiR-4802 adenovirusControl adenovirusULK1 adenovirusATG7 adenovirus
ControlF. nucleatum
Days6 9 12 15 18 21
500
1000
1500
2000
2500
Tum
or v
olum
e(m
m3 )
J K
L
M N
O
Control
F. nucleatum
CQ
Oxaliplatin
F. nucleatum+
Oxaliplatin
Oxaliplatin
F. nucleatum+
Oxaliplatin
CQ+
0
1
2
3
Tum
or w
eigh
t(g)
P<0.05
P<0.05P<0.05
n.s.n.s.
P<0.05n.s.
Contro
l
F. nuc
leatum CQ
Oxalip
latin
F. nuc
leatum
+Oxa
liplat
in
Oxalip
latin
F. nuc
leatum
+Oxa
liplat
in
CQ+
6 9 12 15 18 21 24
500
1000
1500
2000
2500
Days
Tum
or v
olum
e(m
m3 )
Control F. nucleatum CQ F. nu mutaelc
Control QCF. nucleatum+CQ
Oxaliplatin
CQ+
Control
5-Fu
F. nucleatum+
5-Fu
5-Fu
F. nucleatum+
5-Fu
F. nucleatum
CQ
0
1
2
3
Tum
or w
eigh
t(g)
Contro
l
F. nuc
leatum CQ
5-Fu
F. nuc
leatum
+5-F
u5-F
u
F. nuc
leatum
+5-F
uCQ+
P<0.05
P<0.05P<0.05 P<0.05
n.s.n.s.
n.s.
6 9 12 15 18 21 24
500
1000
1500
2000
2500
Days
Tum
or v
olum
e(m
m3 )
lortnoCF. nu mutaelcCQ F. nu mutaelc
lortnoCCQ F. nucleatum+CQ
5-Fu
(legend on next page)
Figure S5. MiR-18a* and miR-4802 Regulate F. nucleatum-Mediated Chemoresistance, Related to Figure 5
(A) Cell proliferationwas detected in HCT116 cells (left) and HT29 (right) cells after transfectionwithmimics or inhibitor ofmiR-18a* andmiR-4802, non-parametric
Mann-Whitney test.
(B–E) Apoptosis was detected by flow cytometry in HT29 cells. The cells were independently transfected with themimics (B, C) or inhibitors (D, E) of miR-18a* and
miR-4802, then co-cultured with F. nucleatum, and treated with different concentrations of Oxaliplatin (B, D) and 5-Fu (C, E). Nonparametric Mann–Whitney test.
(F) Western blot was performed to detect apoptosis-related protein levels in HT29 cells with different treatments. Oxaliplatin (left) or 5-Fu (right).
(G) Representative data of tumors in nude mice bearing colorectal cancer cells in eight control groups.
(H and I) Statistical analysis of mouse tumor weights (H) and volumes (I) in control groups, n = 8/group, nonparametric Mann–Whitney test.
(J) Representative data of tumors in nude mice bearing colorectal cancer cells in different groups.
(K–L) Statistical analysis of mouse tumor weights (K) and volumes (L) in different groups, n = 8/group, nonparametric Mann–Whitney test.
(M) Representative data of tumors in nude mice bearing colorectal cancer cells in different groups. (J) and (M) shared the experimental controls.
(N and O) Statistical analysis of mouse tumor weights (N) and volumes (O) in different groups, n = 8/group, nonparametric Mann–Whitney test.
A B C
F. nucleatum+
5 -Fu
Control
5-Fu
F. nucleatum+
5 -Fu
miR-18a* adenovirus
miR-4802 adenovirus
miR-18a*+ULK1adenovirus
miR-4802 +ATG7 adenovirus
0.0
0.5
1.0
1.5
2.0
2.5
Contro
l5-F
u
F. nuc
leatum
+5-F
u
F. nucleatum +5-Fu
miR-18
a*
aden
oviru
s
miR-48
02
aden
oviru
s
miR-18
a*+U
LK1
aden
oviru
s
miR-48
02+A
TG7
aden
oviru
s
E
F
DOxaliplatin
- - + + + + + +
- - - - + + - -
- - - - - - + +
miR-18a*miR-4802
Cleaved Caspase-9
Cleaved Caspase-3
Cleaved Caspase-6
Cleaved Caspase-7
Cleaved PARP
ACTB
ULK1
ATG7
F. nucleatum +++ + ++--
+ +- - - - + + - -- - - - - -
5-Fu
5-Fu+F. nucleatum5-Fu
5-Fu+F. nucleatum+miR-18a* adenovirus
5-Fu+F. nucleatum+miR-4802 adenovirus
P<0.05
P<0.05 P<0.05
P<0.05
P<0.05
P<0.05
LC3 ILC3 II
5-Fu
0
2
4
6
Num
ber o
f aut
opha
goso
mes
/ ce
ll
P<0
.05
P<0
.05
Control
+miR-18a* adenovirus+miR-4802 adenovirus
F. nucleatumF. nucleatumF. nucleatum
P<0
.05
G
Tum
or w
eigh
t(g)
5-Fu
5-Fu+F. nucleatum
5-Fu+F. nucleatum
miR-18a*+ULK1 adenovirus
miR-4802+ATG7
Control
Control
miR-18a* adenovirusmiR-4802 adenovirus
adenovirus
6 9 12 15 18 21 24
500
1000
1500
2000
2500
Days
Tum
or v
olum
e(m
m3 )
5-Fu+F. nucleatum5-Fu
5-Fu+F. nucleatum+miR-18a* adenovirus
5-Fu+F. nucleatum+miR-4802 adenovirus
Figure S6. MiR-18a* and miR-4802 Regulate F. nucleatum-Mediated Chemoresistance In Vivo, Related to Figure 5
(A) Representative data of tumors in nude mice bearing colorectal cancer cells in different groups.
(B and C) Statistical analysis of mouse tumor weights (Q) and volumes (R) in different groups, n = 8/group, nonparametric Mann–Whitney test.
(D) Western blot was performed to detect autophagy and apoptosis-related proteins in xenograft tumors after different treatments.
(E) TUNEL assays were performed to detect the apoptosis in the xenograft tissues after 5-Fu and F. nucleatum treatment with or without miR-18a* and miR-4802
overexpression.
(F) Transmission electron microscopy was performed to detect autophagosomes in xenograft tissues after 5-Fu and F. nucleatum treatment with or without miR-
18a* and miR-4802 overexpression (17500 3 magnification). Bar scale, 1 mm.
(G) Statistical analysis of autophagosomes in the xenograft tissues detected by transmission electron microscopy, nonparametric Mann–Whitney test.
Control
5-Fu
F. nucleatum
F. nucleatum+
F. nucleatum+
A B
C D
0
1
2
3
5-Fu TLR4 shRNA
MYD88 shRNA
P<0.05
P<0.05 P<0.05
P<0.05
5-Fu
5-Fu+F. nucleatum
F. nucleatumTLR4 shRNAMYD88 shRNA
Control
5-Fu+
Tum
or w
eigh
t(g)
5-Fu+F. nucleatum
5-Fu5-F
u+
TLR4 s
hRNA
MYD88 sh
RNA
Contro
l
F. nuc
leatum
6 9 12 15 18 21 24
500
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Days
Tum
or v
olum
e(m
m3 )
F GE
TLR4 MYD880
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2
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4R
elat
ive
mR
NA
expr
essi
onHCT116
ControlF. nucleatum
TLR4 MYD880
1
2
3HT29
Rel
ativ
e m
RN
A ex
pres
sion
ControlF. nucleatum
P<0
.05
P<0
.05
P<0
.05
P<0
.05
P62
TLR4
Control siTLR4
0 4h 0 4h
Control siTLR4
0 4h 0 4h+F. nucleatum
P62
ACTB ACTB
MYD88
Control
0 4h 0 4h
Control siMYD88
0 4h 0 4h+F. nucleatum
siMYD88
LC3 ILC3 II
LC3 ILC3 II
HCT116 HCT116HT29 HT29
Figure S7. TLR4 and MYD88 Pathway Is Involved in F. nucleatum-Mediated Chemoresistance, Related to Figure 6
(A andB) Real-time PCRwas performed in HCT116 cells (A) and HT29 cells (B) to detect the expression of TLR4 andMYD88 genes, the cells were co-culturedwith
F. nucleatum, nonparametric Mann–Whitney test.
(C) Western blot was performed in HCT116 cells (left) and HT29 cells (right). The cells were co-cultured with F. nucleatum and transfected with TLR4 siRNAs.
(D) Western blot was performed in HCT116 (left) and HT29 (right) cells. HT29 cells were co-cultured with F. nucleatum and transfected with MYD88 siRNAs.
(E) Representative data of tumors in nude mice bearing colorectal cancer cells in different groups.
(F and G) Statistical analysis of mice tumor weights (F) and volumes (G) in different groups, n = 8/group, nonparametric Mann–Whitney test.