25.08 Depletion of the Gut Microbiome Decreases Pancreatic Cancer Metastases

Posted on January 31, 2019

S. Kurtom1,2, V. Sethi1, A. Ferrantella1, B. Giri1, H. K. Jacob1, S. Ramakrishnan1, A. Saluja1, V. Dudeja1  1University Of Miami,Surgery,Miami, FL, USA 2Virginia Commonwealth University,Surgery,Richmond, VA, USA

Introduction:

The gut is comprised of trillions of bacteria that play a crucial role in intestinal homeostasis. A growing body of evidence shows that the gut’s microbiome plays a role in modulating the immune system. Our study aimed to evaluate microbiome-driven immunological mechanisms, specifically toll-like receptor (TLR) activation, in metastatic murine pancreatic ductal adenocarcinoma (PDAC).  TLR2 and TLR4 are important mediators of the inflammatory response in cancer. We hypothesize that gut microbiome depletion decreases liver metastases, via abrogation of the TLR-induced inflammatory response.

Methods:
C57BL/6J mice received either oral saline or a gut sterilizing cocktail of poorly absorbable broad spectrum antibiotics (Vancomycin, Ampicillin, Amphotericin B, Metronidazole, and Neomycin). These mice then received intrasplenic injection of pancreatic cancer cells derived from KPC (Kras LSL.G12D/+; p53 R172H/+;Pdx::Cre) and PKT (Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox) mice. To evaluate the role of TLR activation, intrasplenic KPC injection was subsequently repeated in TLR2-/- or TLR4-/- mice. Mice were sacrificed and the tumors were immunophenotyped and immunostained for multiple antigens.

Results:
Gut microbiome depletion decreased hepatic metastases in KPC injected wild type and TLR4-/- mice and PKT injected WT mice. This tumor inhibitory effect of gut microbial depletion disappeared in the TLR2-/- KPC group. Antibiotic-treated mice had a decrease in intrametastatic IL-17+ CD4+ cells.

Conclusion:
Gut microbiota promote liver metastases in pancreatic cancer, potentially via modulating IL-17+ CD4 cells mediated through the TLR2 pathway. 
 

23.03 Gut Microbiota and Pancreatic Cancer Crosstalk via Immune System: Evidence of a Unique Symbiosis

Posted on January 31, 2019

V. Sethi1, S. Kurtom1, I. Fernandez2, J. Pignac-Kobinger2, A. Ferrantella1, C. Jacob1, P. Roy1, P. Sharma1, M. T. Abreu2, S. Roy1, S. Ramakrishnan1, A. Saluja1, V. Dudeja1  1University Of Miami,Surgical Oncology,Miami, FL, USA 2University Of Miami,Gastroenterology,Miami, FL, USA

Introduction:  Pancreatic ductal adenocarcinoma (PDAC) is characterized by a unique immunosuppressive milieu which makes it particularly resistant to modern checkpoint blocking drugs like anti-PD1 and anti-CTLA4.  Like many other cancers, PDAC is associated with a changed ‘dysbiotic’ gut microbiota. Whether this dysbiotic state promotes cancer or is a neutral-bystander is unclear. We aimed to study as well as investigate the immunotherapeutic implications of this relationship in mice. 

Methods: Wildtype mice-pups (guests) were co-housed with two different types of ‘host’ mice: cancer-bearing KPC (KrasG12D/+;Trp53R172H/+;Pdx-1-Cre) mice or with cancer-naïve mice. Due to coprophagy, the gut microbiota transferred horizontally from hosts to guests. After 2 months of co-caging, the guest mice were given subcutaneous KPC-PDAC and tumor progression was followed. In subsequent experiments, wildtype mice were depleted of their entire gut microbiome by oral antibiotics and growth of orthotopic, subcutaneous and metastatic PDAC was compared between them and controls. Experiments were repeated in various genetic backgrounds. Specific antibiotics were also combined with immune checkpoint-inhibitors in a therapeutic fashion. Pancreatic tumors and metastases were immunophenotyped and were probed for bacteria.

Results: Mice co-housed with KPC cagemates had more aggressive tumor-kinetics than mice co-caged with cancer-naïve mice. Eradicating the gut microbiota by oral antibiotics shrunk tumors in all models of PDAC. This effect disappeared in immunodeficient Rag1-/- mice but not in Tlr4-/- knockout mice. Antibiotics significantly enhanced the efficacy of anti-PD1 and anti-CTLA4 in decreasing tumor burden. Very interestingly, pancreatic tumors grew viable bacteria on culture medium and this was prevented by poorly-absorbable oral antibiotics. Similarly, 16S rRNA amplicon sequencing of PDAC metastases revealed  the presence of a metastatic microbiome similar to the gut, thereby suggesting a gut-to-tumor translocation of bacteria. Flowcytometry results indicated that gut bacteria induced a myeloid cell-mediated immunosupression inside the tumor and antibiotics induced a Th1/Tc1 cell mediated anti-tumor immunity. Neutralizing various nodes of the gut bacteria-induced immunosuppressive axis by monoclonal antibodies abrogated the tumor-suppressing effects of antibiotics thereby, revealing a possible mechanism. 

Conclusion: Our preclinical data presents evidence of the tumor-enhancing potential of gut dysbiosis in cancer-bearing hosts and highlights a potential role of oral antibiotics as novel immunotherapeutic agents against primary and metastatic pancreatic cancer.

21.02 Gut Microbiota and Pancreatic Cancer Crosstalk via Immune System: Evidence of a Unique Symbiosis

Posted on January 31, 2019

V. Sethi1, S. Kurtom1, I. Fernandez2, J. Pignac-Kobinger2, A. Ferrantella1, C. Jacob1, P. Roy1, P. Sharma1, M. T. Abreu2, S. Roy1, S. Ramakrishnan1, A. Saluja1, V. Dudeja1  1University Of Miami,Surgical Oncology,Miami, FL, USA 2University Of Miami,Gastroenterology,Miami, FL, USA

Introduction:  Pancreatic ductal adenocarcinoma (PDAC) is characterized by a unique immunosuppressive milieu which makes it particularly resistant to modern checkpoint blocking drugs like anti-PD1 and anti-CTLA4.  Like many other cancers, PDAC is associated with a changed ‘dysbiotic’ gut microbiota. Whether this dysbiotic state promotes cancer or is a neutral-bystander is unclear. We aimed to study as well as investigate the immunotherapeutic implications of this relationship in mice. 

Methods: Wildtype mice-pups (guests) were co-housed with two different types of ‘host’ mice: cancer-bearing KPC (KrasG12D/+;Trp53R172H/+;Pdx-1-Cre) mice or with cancer-naïve mice. Due to coprophagy, the gut microbiota transferred horizontally from hosts to guests. After 2 months of co-caging, the guest mice were given subcutaneous KPC-PDAC and tumor progression was followed. In subsequent experiments, wildtype mice were depleted of their entire gut microbiome by oral antibiotics and growth of orthotopic, subcutaneous and metastatic PDAC was compared between them and controls. Experiments were repeated in various genetic backgrounds. Specific antibiotics were also combined with immune checkpoint-inhibitors in a therapeutic fashion. Pancreatic tumors and metastases were immunophenotyped and were probed for bacteria.

Results: Mice co-housed with KPC cagemates had more aggressive tumor-kinetics than mice co-caged with cancer-naïve mice. Eradicating the gut microbiota by oral antibiotics shrunk tumors in all models of PDAC. This effect disappeared in immunodeficient Rag1-/- mice but not in Tlr4-/- knockout mice. Antibiotics significantly enhanced the efficacy of anti-PD1 and anti-CTLA4 in decreasing tumor burden. Very interestingly, pancreatic tumors grew viable bacteria on culture medium and this was prevented by poorly-absorbable oral antibiotics. Similarly, 16S rRNA amplicon sequencing of PDAC metastases revealed  the presence of a metastatic microbiome similar to the gut, thereby suggesting a gut-to-tumor translocation of bacteria. Flowcytometry results indicated that gut bacteria induced a myeloid cell-mediated immunosupression inside the tumor and antibiotics induced a Th1/Tc1 cell mediated anti-tumor immunity. Neutralizing various nodes of the gut bacteria-induced immunosuppressive axis by monoclonal antibodies abrogated the tumor-suppressing effects of antibiotics thereby, revealing a possible mechanism. 

Conclusion: Our preclinical data presents evidence of the tumor-enhancing potential of gut dysbiosis in cancer-bearing hosts and highlights a potential role of oral antibiotics as novel immunotherapeutic agents against primary and metastatic pancreatic cancer.

03.12 Bacterial Endotoxin Suppresses Recurrence and Metastasis in Pancreatic Cancer by Immune Activation

Posted on January 31, 2019

A. Ferrantella1, B. Giri1, S. Kurtom1, M. Tarique1, V. Sethi1, H. K. Jacob1, P. Sharma1, P. Roy1, S. Lavania1, A. Saluja1, V. Dudeja1  1University of Miami,Department Of Surgery,Miami, FL, USA

Introduction:  Preclinical studies have demonstrated that an immune response can be generated against pancreatic cancer, but current immunotherapeutic strategies have not been successful at changing the course of the disease. Thus, there is need to evaluate novel strategies to elicit an anti-tumor response. In the current study, we evaluate the bacterial outer membrane product lipopolysaccharide (LPS) as a therapeutic agent to provoke an immune response against pancreatic cancer.

Methods:  KPC pancreatic cancer cells (derived from the KrasG12D/+, Trp53R172H/+, Pdx-1-Cre model) were injected into the pancreata of C57BL/6 mice to induce tumors, and the orthotopic tumors were resected after 25 days.  Following resection, the mice were randomized to receive LPS (5mg/kg) or vehicle twice weekly by intraperitoneal injection, and the mice were followed for cancer recurrence.  In a separate experiment, KPC cancer cells were injected into the spleens of C57BL/6 and Rag1-knockout mice to induce liver metastases.  Following intra-splenic injection, the mice were randomized to receive low-dose LPS (1mg/kg), high-dose LPS (5mg/kg), or vehicle twice weekly.  Liver metastases were measured at the endpoint, and immunophenotyping was performed by flow cytometry.  Finally, subcutaneous tumors were induced in C57BL/6 mice using MC38 colon cancer and Braf-Pten melanoma cell lines.  After 15 days, the mice were randomized to receive either LPS or vehicle twice weekly, and the tumor volumes were serially measured.

Results: LPS treatment significantly reduced cancer recurrence following resection of pancreatic tumors, and the median survival for the LPS-treated mice was more than double that of the vehicle-treated mice. Both low-dose and high-dose LPS treatments drastically suppressed liver metastasis in C57BL/6 mice, but the effect of LPS was abrogated in the absence of an adaptive immune system in Rag1-knockout mice, which lack mature T and B cells. Intriguingly, we observed that, in addition to promoting the classically activated (M1) macrophage phenotype, there was a significant reduction in the pro-tumorigenic myeloid-derived suppressor cell (MDSC) populations, which are known to suppress T cell activity. LPS treatment decreased the growth of colon cancer and melanoma, suggesting that this strategy can be effective in other cancers as well.

Conclusion: Our findings demonstrate that LPS can stimulate the adaptive immune system to suppress the progression of pancreatic cancer. Elucidating the mechanism by which this anti-tumor response is triggered by LPS, and possibly even other pathogen-associated molecular patterns (PAMPs), could lead to identification of novel targets for activating the immune system against cancer, either alone or in combination with contemporary immunotherapeutic strategies.