We found that the cancerous pancreas harbors a markedly more abundant microbiome compared with normal pancreas in both mice and humans, and select bacteria are differentially increased in the tumorous pancreas compared with gut. Ablation of the microbiome protects against preinvasive and invasive pancreatic ductal adenocarcinoma (PDA), whereas transfer of bacteria from PDA-bearing hosts, but not controls, reverses tumor protection. Bacterial ablation was associated with immunogenic reprogramming of the PDA tumor microenvironment, including a reduction in myeloid-derived suppressor cells and an increase in M1 macrophage differentiation, promoting TH1 differentiation of CD4 T cells and CD8 T-cell activation. Bacterial ablation also enabled efficacy for checkpoint-targeted immunotherapy by upregulating PD-1 expression. Mechanistically, the PDA microbiome generated a tolerogenic immune program by differentially activating select Toll-like receptors in monocytic cells. These data suggest that endogenous microbiota promote the crippling immune-suppression characteristic of PDA and that the microbiome has potential as a therapeutic target in the modulation of disease progression. We found that a distinct and abundant microbiome drives suppressive monocytic cellular differentiation in pancreatic cancer via selective Toll-like receptor ligation leading to T-cell anergy. Targeting the microbiome protects against oncogenesis, reverses intratumoral immune tolerance, and enables efficacy for checkpoint-based immunotherapy. These data have implications for understanding immune suppression in pancreatic cancer and its reversal in the clinic. .

Bacterial dysbiosis has emerged as an accomplice to carcinogenesis in malignancies such as colon and liver cancer, and we have recently implicated the microbiome in the pathogenesis of pancreatic ductal adenocarcinoma (PDA) 1. However, the mycobiome has not been clearly implicated in tumorigenesis. We found that fungi migrate from the gut lumen to the pancreas. PDA tumors harbored a ~3000-fold increase in fungi compared to normal pancreas in both mice and humans. The composition of the PDA mycobiome was distinct from that of gut or normal pancreas based on alpha and beta diversity indices. Specifically, the fungal community infiltrating PDA tumors was markedly enriched for Malassezia in both mice and humans. Fungal ablation was tumor-protective in slowly progressive and invasive models of PDA whereas repopulation with Malassezia-but not Candida, Saccharomyces, or Aspergillus-accelerated oncogenesis. In parallel, we discovered that ligation of mannose-binding lectin (MBL), which binds fungal wall glycans to activate the complement cascade, was required for oncogenic progression whereas MBL or C3 deletion in the extra-tumoral compartment or C3aR knockdown in tumor cells were protective. Further, reprogramming of the mycobiome did not alter PDA progression in Mbl or C3 deficient mice. Collectively, our work shows that pathogenic fungi promote PDA by driving the complement cascade via MBL activation.

The Ipl (Tssc3) gene lies in an extended imprinted region of distal mouse chromosome 7, which also contains the Igf2 gene. Expression of Ipl is highest in placenta and yolk sac, where its mRNA is derived almost entirely from the maternal allele. Ipl encodes a small cytoplasmic protein with a pleckstrin-homology (PH) domain. We constructed two lines of mice with germ-line deletions of this gene (Ipl neo and Ipl loxP ) and another line deleted for the similar but nonimprinted gene Tih1. All three lines were viable. There was consistent overgrowth of the Ipl-null placentas, with expansion of the spongiotrophoblast. These larger placentas did not confer a fetal growth advantage; fetal size was normal in Ipl nulls with the Ipl neo allele and was decreased slightly in nulls with the Ipl loxP allele. When bred into an Igf2 mutant background, the Ipl deletion partially rescued the placental but not fetal growth deficiency. Neither fetal nor placental growth was affected by deletion of Tih1. These results show a nonredundant function for Ipl in restraining placental growth. The data further indicate that Ipl can act, at least in part, independently of insulin-like growth factor-2 signaling. Thus, genomic imprinting regulates multiple pathways to control placental size. The Ipl gene, also known as Tssc3, lies on distal chromosome 7 of the mouse and human chromosome 11p15.5 (1, 2). This region contains multiple imprinted genes clustered in 1 Mb of DNA. Two of these, p57 Kip2 (Cdkn1c) and Igf2, control fetal and placental growth in mice (3-7) and humans (8). Similar to these genes, Ipl is highly expressed in the extraembryonic tissues (1), but in contrast to these genes, Ipl is expressed only weakly in the embryo proper (1, 9). Ipl encodes a cytoplasmic protein with a pleckstrin-homology (PH) domain (9), thus by analogy with other PH-domain proteins it may modulate cell signaling, intracellular trafficking, or other processes that depend on phosphatidylinositol lipid second messengers. Ipl has two close relatives: TDAG51 and Tih1. Of these genes, Tih1 is most similar to Ipl (9). Tih1 is located in a nonimprinted region of mouse chromosome 1, and it is expressed biallelically (9). To determine the function of Ipl and to accumulate data relevant to the biological rationale for imprinting, we created mice with germ-line deletions of Ipl and Tih1. Materials and MethodsGene Deletions, Genotyping, and Gene Expression. To make the Ipl neo targeting vector, a 5-kb 5Ј genomic NotI restriction fragment and a 6-kb 3Ј EcoRI͞XbaI fragment flanking Ipl were ligated upstream and downstream of Pgk-Neo in pPNT1, yielding pPNT-Ipl. To make the Ipl loxP targeting vector, the Pgk-Neo cassette of pPNT-⌬Ipl was replaced by that from pLNL, which includes flanking loxP sites. To verify homologous recombination, we used genomic PCR products upstream and downstream of the 5Ј and 3Ј cassettes. Recombination of the loxP sites was induced by crossing to HSP-Cre transgenic mice (10). The Ipl deletions (encompassing nucleotides 77,444-78,990 of GenBank a...