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. 2019 Oct;574(7777):264-267.
doi: 10.1038/s41586-019-1608-2. Epub 2019 Oct 2.

The fungal mycobiome promotes pancreatic oncogenesis via activation of MBL

Berk Aykut #  1 , Smruti Pushalkar #  2 , Ruonan Chen  1 , Qianhao Li  2 , Raquel Abengozar  1 , Jacqueline I Kim  1 , Sorin A Shadaloey  1 , Dongling Wu  1 , Pamela Preiss  1 , Narendra Verma  3 , Yuqi Guo  2 , Anjana Saxena  4   5 , Mridula Vardhan  2 , Brian Diskin  1 , Wei Wang  1 , Joshua Leinwand  1 , Emma Kurz  1 , Juan A Kochen Rossi  1 , Mautin Hundeyin  1 , Constantinos Zambrinis  1 , Xin Li  2 , Deepak Saxena  6   7 , George Miller  8   9
Affiliations

The fungal mycobiome promotes pancreatic oncogenesis via activation of MBL

Berk Aykut et al. Nature. 2019 Oct.

Abstract

Bacterial dysbiosis accompanies carcinogenesis in malignancies such as colon and liver cancer, and has recently been implicated in the pathogenesis of pancreatic ductal adenocarcinoma (PDA)1. However, the mycobiome has not been clearly implicated in tumorigenesis. Here we show that fungi migrate from the gut lumen to the pancreas, and that this is implicated in the pathogenesis of PDA. PDA tumours in humans and mouse models of this cancer displayed an increase in fungi of about 3,000-fold compared to normal pancreatic tissue. The composition of the mycobiome of PDA tumours was distinct from that of the gut or normal pancreas on the basis of alpha- and beta-diversity indices. Specifically, the fungal community that infiltrated PDA tumours was markedly enriched for Malassezia spp. in both mice and humans. Ablation of the mycobiome was protective against tumour growth in slowly progressive and invasive models of PDA, and repopulation with a Malassezia species-but not species in the genera Candida, Saccharomyces or Aspergillus-accelerated oncogenesis. We also discovered that ligation of mannose-binding lectin (MBL), which binds to glycans of the fungal wall to activate the complement cascade, was required for oncogenic progression, whereas deletion of MBL or C3 in the extratumoral compartment-or knockdown of C3aR in tumour cells-were both protective against tumour growth. In addition, reprogramming of the mycobiome did not alter the progression of PDA in Mbl- (also known as Mbl2) or C3-deficient mice. Collectively, our work shows that pathogenic fungi promote PDA by driving the complement cascade through the activation of MBL.

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Conflict of interest statement

Competing interests

The authors declare no competing interests.

Figures

Extended Data Fig. 1.
Extended Data Fig. 1.. Fungal infiltration of the pancreas in benign disease.
Fungal DNA content was tested using qPCR in pancreata from control mice (n=5) and mice induced to develop caerulein pancreatitis (n=5). NS, non significant. Data are mean ± s.e.m. Two-tailed Student’s t-test.
Extended Data Fig. 2.
Extended Data Fig. 2.. Gut mycobial dysbiosis in murine PDA.
Hierarchical tree cladogram depicting changes in taxonomic composition of mycobiota assigned to genus level in the gut of 30 week old WT (n=12) and KC (n=14) mice based on average percent relative abundance as determined by 18S ITS sequencing.
Extended Data Fig. 3.
Extended Data Fig. 3.. Efficacy of anti-fungal treatments in pancreatic disease.
(A) Orthotopic PDA bearing WT mice were treated with vehicle (n=7) or Fluconazole (n=8) and sacrificed 3 weeks later. Tumors were harvested and weighed. Data are representative of experiments performed twice (scale bar =1cm). Data are mean ± s.e.m. Two-tailed Student’s t-test. (B) Germ-free WT mice were treated with Amphotericin B (n=6) or vehicle (n=10) and administered orthotopic KPC tumor. Mice were sacrificed 3 weeks later and tumors were harvested and weighed (scale bar = 1cm). Data are mean ± s.e.m. Two-tailed Student’s t-test. (C-E) WT mice induced to develop caerulein pancreatitis were serially treated with Amphotericin B (n=5) or vehicle (n=3). (C) Representative H&E sections of pancreata are shown, and pancreatic edema was quantified by measuring the percentage of white space area (scale bar = 100μm). (D) CD45+ inflammatory cell infiltration was determined by IHC (scale bar = 20μm). (E) Serum amylase was measured (n=5 Amphotericin B, n=3 vehicle and n=3 mock-treated animals). Data are mean ± s.e.m. Two-tailed Student’s t-test. (F) Amphotericin B-treated WT mice were repopulated with C. tropicalis (n=4) or vehicle (n=4) and sacrificed 3 weeks later. Tumors were harvested and weighed (scale bar =1cm). NS, non significant. Data are mean ± s.e.m. Two-tailed Student’s t-test.
Extended Data Fig. 4.
Extended Data Fig. 4.. Fungal dysbiosis drives PDA progression via the lectin pathway.
(A) Kaplan-Meier survival curve of human PDA patients stratified by high (n=16), medium high (n=24), medium low (n=26) and low (n=17) MBL expression based on TCGA data. Two-tailed log-rank test. (B) Vehicle (n=3) and Amphotericin B-treated (n=4) MBL-null mice were administered orthotopic KPC tumors and sacrificed 3 weeks later. Tumors were harvested and weighed. Data are representative of 3 separate experiments. NS, non significant. Data are mean ± s.e.m. Two-tailed Student’s t-test. (C) Amphotericin B-treated MBL-null mice were repopulated with M. globosa (n=5) or sham (n=4) repopulated and sacrificed 3 weeks later. Tumors were harvested and weighed. Data are representative of experiments repeated twice. NS, non significant. Data are mean ± s.e.m. Two-tailed Student’s t-test. (D) Kaplan-Meier survival curve of human PDA patients stratified by high (n=18) versus low (n=15) C3 expression based on TCGA data based on TCGA data. Two-tailed log-rank test. (E) Pancreata from 3 month-old WT, KC, and KC;MBL−/− were stained using an mAb against C3a. Representative images from two experiments are shown (scale bar = 20μm). (F) KPC tumor cells were seeded in 96-well plates with vehicle or recombinant murine C3a (n=5 per group for each time point). Cellular proliferation was measured at serial time points using the XTT assay. Data are mean ± s.e.m. Two-tailed Student’s t-test. Data are representative of experiments repeated three times.
Fig. 1.
Fig. 1.. PDA is characterized by a distinctive intra-tumoral and gut mycobiome.
(A) The abundance of intra-pancreatic fungi was compared in healthy individuals (NML) and age/gender/BMI matched PDA patients by FISH (n=3/group). Representative images are shown (scale bar = 20μm). (B) The abundance of intra-pancreatic fungi was compared in 3-month-old littermate WT and KC mice by FISH. Representative images are shown (n=3/group; scale bar = 20μm). (C) Fungal DNA content was compared in pancreata of healthy individuals and age/gender/BMI matched PDA patients using qPCR. Data are mean ± s.e.m. Two-tailed Student’s t-test. (D) Fungal DNA content was compared in pancreata of 3-month-old WT and KC mice using qPCR. Data are mean ± s.e.m. Two-tailed Student’s t-test. (E) WT (n=15) and KC (n=9) mice were administered GFP-labeled S. cerevisiae via oral gavage. Pancreata were harvested at 30 minutes and the number of GFP+ foci was determined by flow cytometry in comparison to mock-treated animals (n=6). This experiment was repeated twice. Data are mean ± s.e.m. Two-tailed Student’s t-test. (F-I) The gut (n=14 biologically independent samples) and intrapancreatic (n=11 biologically independent samples) mycobiomes in 30 week-old KC mice were analyzed by 18S ITS sequencing. (F) PCoA plots based on Bray-Curtis dissimilarity matrix. Each symbol represents a sample from gut (red) or pancreas (blue). Clusters were determined by pairwise PERMANOVA. X- and Y-axes indicate percent variation and the ellipses indicate 95% confidence interval (CI). (G) The gut and intra-pancreatic mycobiomes in 30 week-old KC mice were analyzed for alpha-diversity measures including Observed OTUs and Shannon indices. Two-sided Wilcoxon rank-sum test. Box plots show median, 25th and 75th percentiles, and whiskers extending to 1.5 interquartile ranges. (H) Taxonomic composition of mycobiota assigned to the phylum level based on average percent relative abundance. NS, non significant. Data are mean ± s.e.m. Two-tailed Student’s t-test. (I) Heatmap showing log2-transformed relative abundancies of top 20 fungal genera in gut and pancreata. (J-L) PCoA plots of fungal communities in feces of (J) 6-week, (K) 18-week, and (L) 30-week old WT and KC mice based on Bray-Curtis dissimilarity matrix. Clusters were determined by pairwise PERMANOVA. X- and Y-axes indicate percent variation and the ellipses indicate 95% CI.
Fig. 2.
Fig. 2.. Human PDA is associated with a distinct mycobiome.
(A-D) Gut (n=18) and Tumor (n=13) of specimens from PDA patients were analyzed by 18S ITS sequencing. (A) Taxonomic composition of mycobiota assigned to the phylum level based on average percent relative abundance. NS, non significant. Data are mean ± s.e.m. Two-tailed Student’s t-test. (B) Hierarchical tree cladogram depicting differences in taxonomic composition of mycobiota assigned to genus level in gut and tumor based on average percent relative abundance. (C) The gut and tumor mycobiome of PDA patients were tested for alpha-diversity measures including Observed OTUs, ACE, Chao1, Shannon, and Simpson indices. Two-sided Wilcoxon rank-sum test. Box plots show median, 25th and 75th percentiles, and whiskers extending to maximum and minimum data points. (D) PCoA plots of gut (n=18) and intra-tumoral (n=13) fungal communities in PDA patients based on Bray-Curtis dissimilarity matrix. (E) PCoA plots of fungal communities in pancreata of PDA patients (n=13) and healthy individuals (n=5) based on Bray-Curtis dissimilarity matrix. Analyses were determined by pairwise PERMANOVA. X- and Y-axes indicate percent variation and the ellipses indicate 95% CI.
Fig. 3.
Fig. 3.. Fungal dysbiosis promotes pancreatic oncogenesis.
(A) Amphotericin B and Vehicle-treated KC mice were sacrificed at 3 months of life. Pancreatic weights (n=5 and 11, respectively) were recorded. Representative H&E- and trichrome-stained sections are shown. The percentage of preserved acinar area, and the fraction of normal ducts, acinoductal metaplasia, and graded PanIN lesions were determined based on H&E staining (scale bar = 200μm). The fraction of fibrotic area per pancreas was calculated based on trichrome staining (scale bar = 200μm). Data are mean ± s.e.m. Two-tailed Student’s t-test. (B) Orthotopic PDA bearing WT mice were treated with vehicle or Amphotericin B (n=16 per group, data pooled from three independent experiments) and sacrificed 3 weeks later. Tumors were harvested and weighed. Data are representative of experiments repeated more than 5 times (scale bar =1cm). Data are mean ± s.e.m. Two-tailed Student’s t-test. (C) Orthotopic PDA bearing WT mice were treated with vehicle (n=9), Amphotericin B (n=6), Gemcitabine (n=8), or Amphotericin B + Gemcitabine (n=6). Tumor weight was recorded at 3 weeks (scale bar =1cm). Data are mean ± s.e.m. Two-tailed Student’s t-test. (D) Amphotericin B-treated WT mice were repopulated with M. globosa (n=8), S. cerevisiae (n=9), Candida sp. (n=8), or Aspergillus sp. (n=10) or vehicle (n=8) and sacrificed 3 weeks later. Tumors were harvested and weighed. Data are representative of experiments repeated twice (scale bar =1cm). NS, non significant. Data are mean ± s.e.m. Two-tailed Student’s t-test.
Fig. 4.
Fig. 4.. Fungi promote PDA progression via the MBL – C3 axis.
(A) KC (n=11) and KC;MBL-null (n=7) mice were sacrificed at 3 months of life. Tumors were weighed and stained using H&E (scale bar = 200μm), trichrome (scale bar = 200μm), and analyzed for pancreatic dysplasia and fibrosis. Data for control KC is the same as in Fig. 3. Data are mean ± s.e.m. Two-tailed Student’s t-test. (B, C) WT and MBL-null mice were administered orthotopic KPC tumor and analyzed for (B) tumor growth at 3 weeks (n=22 per arm; data represent mean ± s.e.m, two-tailed Student’s t-test) and (C) survival (n=8 WT and n=5 MBL-null animals; log-rank test). Data are representative of experiments repeated more than 5 times. (D) MBL−/− hosts were administered orthotopic KPC tumors and received intratumoral injections of recombinant C3a (n=6) or vehicle (n=6) on day 14 via laparotomy, and tumor volumes were measured. Tumors were harvested on day 21, and change in tumor volume since injection was calculated. This experiment was repeated twice. Data are mean ± s.e.m. Two-tailed Student’s t-test. (E) WT (n=10) and C3−/− (n=9) mice were administered orthotopic KPC tumor and analyzed for tumor growth at 3 weeks. Data are representative of experiments repeated 3 times. Data are mean ± s.e.m. Two-tailed Student’s t-test. (F, G) WT mice were orthotopically implanted with KPC tumors cells treated with either shRNA directed against C3aR or control scrambled shRNA. Separate shRNA vectors were used. (F) The efficacy of C3aR knockdown was measured by qPCR (n=3 per group). Data are mean ± s.e.m. Two-tailed Student’s t-test. (G) Quantitative analysis of day 21 tumor weights is shown (n=9 for scrambled and n=5 for shC3aR1 and shC3aR2). Data are mean ± s.e.m. Two-tailed Student’s t-test. (H) Vehicle (n=3–4) and Amphotericin B-treated (n=4) WT and C3−/− mice were administered orthotopic KPC tumors and sacrificed 3 weeks later. Tumors were harvested and weighed. Data are representative of experiments repeated twice. Data are mean ± s.e.m. Two-tailed Student’s t-test. (I) Schematic depicting mycobiome-MBL axis in pancreatic oncogenesis.
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