Obesity enriches for tumor protective microbial metabolites and treatment refractory cells to confer therapy resistance in PDAC

Abstract

Obesity causes chronic inflammation and changes in gut microbiome. However, how this contributes to poor survival and therapy resistance in patients with pancreatic cancer remain undetermined. Our current study shows that high fat diet-fed obese pancreatic tumor bearing mice do not respond to standard of care therapy with gemcitabine and paclitaxel when compared to corresponding control diet-fed mice. C57BL6 mice were put on control and high fat diet for 1 month following with pancreatic tumors were implanted in both groups. Microbiome of lean (control) and obese (high fat diet fed) mice was analyzed. Fecal matter transplant from control mice to obese mice sensitized tumors to chemotherapy and demonstrated extensive cell death. Analysis of gut microbiome showed an enrichment of queuosine (Q) producing bacteria in obese mice and an enrichment of S-adenosyl methionine (SAM) producing bacteria in control diet-fed mice. Further, supplementation of obese animals with SAM sensitized pancreatic tumors to chemotherapy. Treatment of pancreatic cancer cells with Q increased PRDX1 involved in oxidative stress protection. In parallel, tumors in obese mice showed increase in CD133⁺ treatment refractory tumor populations compared to control animals. These observations indicated that microbial metabolite Q accumulation in high fat diet-fed mice protected tumors from chemotherapy induced oxidative stress by upregulating PRDX1. This protection could be reversed by treatment with SAM. We conclude that relative concentration of SAM and queuosine in fecal samples of pancreatic cancer patients can be developed as a potential biomarker and therapeutic target in chemotherapy refractory pancreatic cancer.

Keywords: Pancreatic cancer; chemoresistance; gut microbiome; obesity; queuosine S-adenosyl methionine.

Figure 1.

Obesity induced change in gut microbiome: Schematic diagram showing timeline of the experiment (a). High fat diet changed the gut microbiome in C57Bl6 mice (b). Differences in the microbial composition can be visualized in the heat map L1 = 1^st collection in lean mice before tumor implantation; L2 = 2^nd collection in lean mouse after 1 month of diet; L3 = 3^rd collection before start of Gem/Pac therapy in lean mouse, L4 = Final collection after sacrificing in lean mouse. O1 = 1^st collection in obese mice before tumor implantation; O2 = 2^nd collection in obese mouse after 1 month of diet; O3 = 3^rd collection before start of Gem/Pac therapy in obese mouse, O4 = Final collection after sacrificing in obese mouse (c). Dynamic changes in gut microbiome in phylum over the collection period (d,e).

Figure 2.

High fat diet fed mice show resistance to chemotherapy: KPC001 cells were implanted subcutaneously in C57BL6 mice on control and high fat diet and treated with Gem/Pac for 4 weeks. Tumor weight (a) and tumor volume (b) in mice on control (lean) diet showed significant decrease. Tumor weight (c) and tumor volume (d) in mice on high fat (obesogenic) diet showed no significant response. Schema for fecal transplant (e) is shown in which the high fat diet fed mice received the lean mice microbiome and vice versa. PCoA plots of Obese to lean transplant (f) and lean to obese transplant (g). Obese≫Lean FMT showed loss of response in the presence of chemotherapy (h) while Lean≫Obese FMT showed sensitivity to chemotherapy (i). Visible changes in histology was observed in the Lean≫Obese FMT in H&E slides (j), collagen deposition (k) and TUNEL staining (l).

Figure 3.

Metabolomic reconstruction using humaN2 pipeline was performed to determine the microbial metabolome. High fat diet fed mice showed an enrichment of Q metabolizing bacteria (a, b) while lean mice showed an enrichment of SAM metabolizing bacteria (c,d). Treatment of pancreatic cancer cells MIA-PACA2 (c) and S2VP10 (d) with paclitaxel and pre-Q showed a shift in IC50 indicating resistance. Treatment of colon cancer cell SW620 with oxaliplatin and pre-Q showed a similar shift in IC50 (e). Treatment of pancreatic cancer cells KPC001 (f) and Su86.86 (g) with SAM showed an opposite shift of IC50 indicating sensitization. Treatment of colon cancer cell RKO (h) with oxaliplatin and pre-Q showed a similar shift in IC50 indicating sensitization by SAM.

Figure 4.

Serum metabolomics of lean and obese animals using IROA: Pathway Analysis of obese vs lean animals using MetaboAnalyst showing enrichment of critical pathways (a). MSEA of obese animals (b). Relative abundance of metabolites in lean vs obese animals (c). Obese mice showed an increase in glutamic acid (d). Metacyc showed relative changes in the metabolic pathways in lean and obese animals (e). Drug detoxification pathways were enriched in obese animals (f).

Figure 5.

SAM reverted effect of obesity induced therapy resistance: SAM was increased in Lean≫Obese FMT animals in fecal (a) sample. SAM was decreased in Obese animals (b). Schema showing experimental set up SAM treatment in high fat diet fed, pancreatic tumor bearing mice (c). SAM decreased tumor volume (d) and weight (e). Treatment with SAM also decreased collagen (f) and Ki67+ cells (g).

Figure 6.

Queuosine mediated chemoresistance by upregulating PRDX1: Treatment of pancreatic cancer cell SU86.86 increased expression of PRDX1 as seen in oxidative stress PCR array analysis (a). Western blot showing upregulation of PRDX1 protein after treatment with Pre-Q (b). Silencing PRDX1 using siRNA presented Pre-Q induced resistance in MIA-PACA2 cells (c). IHC of lean and obese tumor bearing mice show upregulation of PRDX1 in obese mice (d). FMT of obese≫lean mice increased PRDX1 expression in these animals.

Figure 7.

Obesity enriched for resistant CD133+ cells in pancreatic tumors. High fat diet fed tumor bearing mice showed an increase in CD133+ cells near lipid droplets (a). CD133+ population was increased when pancreatic cancer cells were treated with adipocyte conditioned media (b). Adipocyte conditioned media also increased expression of self-renewal genes like CD133 (c), Sox2 (d), Oct4 (e) and induced resistance to paclitaxel (f). Schematic diagram showing mechanism of obesity induced resistance.