1. The Pancreatic Cancer Microbiome Promotes Oncogenesis by Induction of Innate and Adaptive Immune Suppression
Cancer discovery
Gut Microbiota Promotes Tumor Growth in Mice by Modulating Immune Response
Gastroenterology
오늘 목요 세미나의 주제는 Gut microbiota 와 Pancreatic cancer 입니다.
Cancer discovery
Gastroenterology
Anti-Programmed Death-1 Antibody (αPD-1) 
R3 강 승경 / Pf. 류 지 곤

2. INTRODUCTION
Pancreatic ductal adenocarcinoma (PDA) : 3rd most lethal cancer in USA, 85% of pancreatic malignancies
Peripancreatic inflammation
paramount for induction of oncogenesis
Innate / adaptive immune cell subsets : cooperate to promote tumorigenesis

3. INTRODUCTION Pattern Recognition Receptors (PRR)
Transmit inflammation, tumorigenesis↑
Deficient in select PRR signaling
Toll-like receptor 4 (TLR4), TLR7, TLR9, Mincle
Slower progression of PDA
Protumorigenic effects of PRR ligation in PDA
Bacterial dysbiosis influences PDA progression.

4. INTRODUCTION Microbiome
Microbial dysbiosis, Disrupted epithelial barrier function
Translocation of bacteria
Neoplastic transformation
Microbiome
Contributor to oncogenesis in intestinal tract malignancies
laryngeal, esophageal, gastric, colorectal, primary liver cancer
Direct contact, recipient of portal venous drainage(liver cancer)
Remote from the gastrointestinal lumen or its drainage.
Etiologic relationship?
intrapancreatic microbiota immune-suppressive inflammation in PDA

6. METHOD Cell lines Flow Cytometry 16S Ribosomal RNA Gene Sequencing
KPC cells
spontaneous pancreatic tumors originating in KrasG12D/þ; Trp53R172H/þ; Pdx-1cre mice
Braf-Pten melanoma cells
topical tamoxifen induced tumors arising in Tyr-CreER; BrafV600E/þ; Ptenfl/fl mice
MC38 colon cancer cells
Flow Cytometry
16S Ribosomal RNA Gene Sequencing
Statistical Analyses
Unpaired Student t test with Welch’s correction
Mann-Whitney test, and 1-way analysis of variance with Tukey’s multiple comparison test for comparisons

7. Establish cancer models
By subcutaneous injection of KPC pancreatic cancer cells or melanoma cells
By splenic injection of KPC cells, B16-F10 melanoma cells, or MC38 colon cancer cells to induce liver metastases.
Objective
Evaluate the Impact of gut microbiome depletion on tumor growth

8. RESULT Gut microbiome depletion
Subcutaneous tumor burden ↓↓ in pancreatic cancer and melanoma models
Liver metastases burden ↓↓ in pancreatic cancer, colon cancer, and melanoma models
그림1.

9. RESULT
그림1.

10. RESULT
그림1. D, E/ Supplementary Figure 1.

11. RESULT
Abolished tumor-suppressing effect of gut microbiome depletion in Rag1 knockout mice
Active participation of adaptive immunity, not an off-target cytotoxic action
Figure 2A/ Supplementary Figure 2.

12. RESULT Impact of gut microbiome depletion on T cells in TME
naïve helper T cells (Th0)  mature into Th1, Th2, regulatory T cell, Th17 lineage
Th1 - cytokine (IFN-ɣ): anti-tumorigenic role
Th2/ Treg - cytokines IL4, IL5, IL10 : pro-tumorigenic role
High Th1/Th2 ratio in TME : survival ↑ in pancreatic cancer
Th17 cells
Pro-tumorigenic in pancreatic cancer, melanoma, colorectal cancer.
IL17a
Linked to the gut microbiome and plays a key role in defending against fungal and bacterial pathogens.

13. RESULT
Th1
Tc1
(Figure 2)

14. RESULT
(Figure 2)

15. RESULT-subcutaneous KPC mic
Ablation of 16S ribosomal DNA
Decrease in relative abundance of the 2 major phyla
- Bacteroidetes , Firmicutes
Supplementary Figure 3.

16. Supplementary Figure 3.

17. DISCUSSION
Mechanism of interaction with microbiome and immune system : Unclear , some inferences
Gut microbes interact with immune system via PRR in pancreatic and other cancers
Rag1 knockout / IL17a-neutralizing antibody not cytotoxic effect of antibiotics in anti-tumor phenomenon
Depleting the gut microbiome  ↑ infiltration of pancreatic tumors with effector T cells.
Future studies
gut microbial modulation strategy
 potentiate the efficacy of checkpoint inhibitors or cytotoxic drugs

18. Cancer discovery April, 2018
1Department of Basic Science and Craniofacial Biology, New York University College of Dentistry, New York, New York.
2S. Arthur Localio Laboratory, Department of Surgery, New York University School of Medicine, New York, New York.
3National Gnotobiotic Rodent Research Center, University of North Carolina, Chapel Hill, North Carolina.
4Department of Epidemiology and Health Promotion, NYU College of Dentistry, New York, New York.
5Department of Biology, Brooklyn College and the Graduate Center (CUNY), Brooklyn, New York, New York.
6Department of Medicine, New York University School of Medicine, New York, New York.
7Department of Medicine, Microbiology, and Immunology, University of North Carolina, Chapel Hill, North Carolina.
8Department of Cell Biology, New York University School of Medicine, New York, New York.

19. METHOD Animals and in vivo models
KC mice: develop spontaneous pancreatic neoplasia by targeted expression of mutant Kras in the pancreas
KPC mice: express mutant intrapancreatic KrasG12D/þ; Trp53R172H/þ; Pdx-1cre mice
Antibiotic treatment, fecal, and bacterial transfer experiments
Ablation of the gut microbiome
6-week-old WT or KC mice , antibiotic cocktail by oral gavage daily for 5d. Controls-PBS.
* Broad-spectrum antibiotics cocktail
: vancomycin, neomycin, metronidazole, ampicillin and amphotericin B
PBS: Phophate-buffered saline

20. METHOD Murine cellular isolation, Flow cytometry, In vitro experiments
Histology, Immunohistochemistry, RNA analysis
Quantitative PCR
FISH
Statistical considerations for tumor size and immunologic analyses
measurements of tumor size : Student t test

21. RESULT CFSE-labeled E. faecalis immune fluorescence microscopy
Mice
CFSE-labeled E. faecalis
Human
그림 1.
immune fluorescence microscopy
16S rRNA FISH

22. RESULT
(Supplementary Fig. S2A)

23. (Supplementary Fig. S2C). (Supplementary Fig. S2E).

24. RESULT
16S rRNA gene sequencing PDA tumors, 12 pt.

25. RESULT
qPCR
Increased bacterial abundance in PDA compared with normal pancreas in mice and humans
Repopulation in a antibiotic-treated WT mice
gut microbiome from KPC mice; higher capacity for translocation to the pancreas
그림 1.

26. slowly progressive KC model
H&E stain
trichrome-stain
slowly progressive KC model
Germ Free KC mice
Delayed acinar effacement
Reduced pancreatic dysplasia
Diminished intratumoral fibrosis
Lower pancreatic weights
그림 1.
50%

27. RESULT repopulated Bacterial ablation KPC repopulation 그림2. KC mice
Protective against disease progression
KPC repopulation
Progression of tumor
KC mice
GF KC(6wks)
(8wks)
그림2.
repopulated

28. T-cell ↓
FISH
RESULT
그림2.

29. M1-associated chemokine
RESULT
(Supplementary Fig. S6C-E)
M1-associated chemokine

30. MØ
M2-like TAM
Splenic MØ, cell-free extract from KC mice
그림2.

31. RESULT Treg Intratumoral CD8:CD4 T-cell ratio ↑
(Supplementary Fig. S6F)
(Supplementary Fig. S6G,H,I,J)
Treg

32. RESULT
PDA microbiome T-cell suppression
그림3. MØ

33. RESULT Ova pulsed MØ from PDA host  capacity of T-cell activation ↓↓
TAMs from PDA tumor in Abx ablated mice capacity of T-cell activation ↑↑
그림3.

34. RESULT
Macrophage neutralization  intratumoral T-cell activation↓ associated with microbial ablation in PDA
50%
그림3.
Synergy
Enhanced adaptive immunity in microbial ablation
T cells from orthotopic KPC tumors- transferred to cohorts of mice challenged with subcutaneous KPC tumor.
T cells derived from antibiotic-treated mice reduced tumor burden by ∼50%

35. RESULT Antibiotic with αPD-1 CD4+ , CD8+ T-cell ↑
T cells  CXCR3 ↑ and LFA1↑
(Supplementary Fig. S7B-E)

36. RESULT
PDA microbiome  differential TLR activation immune suppression
Antibiotic-ablated hosts : markedly lower expression of PRRs and associated signaling molecules
Control : x fold
그림4.

37. RESULT MØ MØ
그림4.
Innate & Adative immune suppression

38. RESULT Inhibition of TLR signaling blocking TRAF6
PDA-promoting effects↓
Inhibition of TLR signaling blocking Myd88
CD4+ T-cell activation ↑
그림4.
PDA microbiome programs TAMs via TLR signaling
* TRAF6, Myd88: signal transducer in TLR signaling pathways

39. DISCUSSION
Distinct stagespecific gut , pancreatic microbiome  intratumoral immune suppression  disease progression
Species or signatures that promotes disease pathogenesis : B. pseudolongum
abundant in gut and tumor, accelerated oncogenesis via TLR
Targeting the microbiome
Protected against PDA
Enhanced antitumor immunity
Susceptibility ↑ to immunotherapy
Elements of the microbiome : useful in early diagnosis and risk stratification.
Microbial-targeted therapies(oral antibiotics, probiotics)
reduce risk in preinvasive disease
adjuvant to standard therapies / synergy with checkpoint directed immunotherapy in invasive disease.

40. CONCLUSION Bi-aspect of antibiotics Protumorigenic?
Change of diversity
Additional research
FMT of favorable gut microbiome
Antibiotics : dose, duration of use
Synergistic effect in Cytotoxic chemotherapy?

41. Thank You