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Organoids for Infection Studies

Full SOP with Academic or a plan

Source Scanu et al., 2015 · The Netherlands Cancer Institute, Amsterdam · 10.1016/j.chom.2015.05.002

Authors: Tiziana Scanu, Robbert M. Spaapen, Jeroen M. Bakker, Chandra Bhan Pratap, Lin-en Wu, Ingrid Hofland, Annegien Broeks, Vijay Kumar Shukla, Mohan Kumar, Hans Janssen, Ji-Ying Song, E. Andra Neefjes-Borst, Hein te Riele, David W. Holden, Gopal Nath, Jacques Neefjes Duration: 45 days 11 phases Cell source: Mouse Embryonic Fibroblasts (MEFs) with Arf deletion and c-MYC overexpression; Murine gallbladder organoids from Ink4b-Arf-Ink4a knockout mice

Abstract

This protocol demonstrates how Salmonella enterica infection drives irreversible cellular transformation in genetically predisposed cells (TP53 inactivation and c-MYC amplification) through activation of host AKT and MAPK signaling pathways. The transformed phenotype persists even after bacterial eradication, recapitulating the molecular features observed in gallbladder carcinoma tissues from Indian patients chronically infected with Salmonella Typhi.

Cell source
Mouse Embryonic Fibroblasts (MEFs) with Arf deletion and c-MYC overexpression; Murine gallbladder organoids from Ink4b-Arf-Ink4a knockout mice
Application
Disease modeling of gallbladder carcinoma pathogenesis; mechanistic study of bacterial-induced transformation

Protocol overview

49 steps across 11 phases

Phase 1 · Preparation and culture of pre-transformed MEFs Day -3 to Day 0
  1. a Culture wild-type and Arf-deficient C57BL/6 MEFs
  2. b Prepare Salmonella culture
Phase 2 · Salmonella infection of MEFs Day 0
  1. a Infect MEFs with Salmonella
  2. b Eliminate extracellular bacteria
  3. c Recover infected cells in regular culture medium
Phase 3 · Bacterial clearance and kinetic analysis Day 0 to Day 1
  1. a Treat infected cells with ciprofloxacin at defined time points
  2. b Verify Salmonella elimination
Phase 4 · Anchorage-independent transformation assay (soft agar) Day 1 to Day 15
  1. a Prepare soft agar plates
  2. b Seed infected MEFs in soft agar suspension layer
  3. c Culture soft agar plates for colony formation
  4. d Quantify soft agar colonies
Phase 5 · Pathway inhibition studies Day 0 to Day 15
  1. a Pre-treat MEFs with kinase inhibitors
  2. b Perform infection and soft agar assay with inhibitors present
  3. c Analyze colony formation reduction
Phase 6 · Multi-generational soft agar and transcriptome analysis Day 15 to Day 45
  1. a Isolate single cells from first-generation soft agar colonies
  2. b Treat with ciprofloxacin before second-generation assay
  3. c Generate second and third-generation soft agar assays
  4. d Prepare cells for transcriptome analysis
  5. e Extract total RNA and prepare for sequencing
  6. f Perform paired-end RNA sequencing
  7. g Map reads and analyze gene expression
  8. h Identify differentially expressed genes and networks
Phase 7 · Western blot analysis of signaling pathways Day 0 to Day 10
  1. a Prepare cell lysates from infected MEFs
  2. b Separate proteins by SDS-PAGE
  3. c Transfer proteins to PVDF membranes
  4. d Block and incubate with primary antibodies
  5. e Wash membranes and incubate with secondary antibodies
  6. f Develop blots and analyze
Phase 8 · Tumor transplantation and in vivo validation Day 0 to Day 30
  1. a Select and expand transformed MEF colonies
  2. b Prepare cell suspension for transplantation
  3. c Prepare immunodeficient mice
  4. d Perform subcutaneous cell transplantation
  5. e Monitor tumor growth
  6. f Collect tumors and perform histopathology
Phase 9 · Gallbladder organoid preparation and infection Day -2 to Day 14
  1. a Isolate gallbladder tissue from transgenic mice
  2. b Generate single cells from gallbladder tissue
  3. c Prepare growth factor-reduced Matrigel
  4. d Plate organoid cultures
  5. e Culture and monitor organoid development
Phase 10 · Salmonella infection of gallbladder organoids Day 5 to Day 19
  1. a Dissociate organoids into single cells
  2. b Infect organoid-derived single cells with Salmonella
  3. c Eliminate extracellular bacteria
  4. d Culture infected organoids with antibiotic protection
  5. e Re-dissociate and re-culture infected organoids
Phase 11 · Morphological assessment of transformation in organoids Day 14
  1. a Fix organoids for histological analysis
  2. b Prepare tissue sections for immunohistochemistry
  3. c Perform immunohistochemistry staining
  4. d Analyze histological features of transformation
  5. e Perform electron microscopy of organoid ultrastructure

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Attribution

This SOP was written by Organthis based on the published method in Scanu et al., 2015. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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