Week 8 journal

• 1. Fluorouracil (5FU)


  A chemotherapeutic agent used as the backbone for neoadjuvant, adjuvant, and palliative treatment of pancreatic ductal adenocarcinoma (PDAC)
• Epidermal Growth Factor Receptor (EGFR)

  A cell surface protein that is overexpressed in up to 70% of PDACs and correlates with poor prognosis and disease progression
• Aptamers


  Single-stranded oligonucleotide sequences that can recognize and bind to specific targets with high affinity and specificity
• Designing, synthesizing, and characterizing 5FU-incorporated EGFR-targeted aptamers


  1. Calibration and redesigning of previously developed E07 aptamer against EGFR

  2. In vitro transcription to generate 5FU-EGFR aptamers

  
• 5FU-EGFR aptamers


  • Achieve high target specificity for cancer cells
  • Can overcome 5FU resistance
  • Effective in reducing tumor burden in various PDAC models

  
• PDAC models used to test 5FU-EGFR aptamers

  • Syngeneic orthotopic transplantation model
  • Autochthonously growing genetically engineered PDAC model (KPC)
  • Orthotopic cell line-derived xenograft (CDX) model
  • Patient-derived organoids (PDOs)

  
• Up to 30% of PDAC specimens do not overexpress EGFR, so treatment with 5FU-EGFR aptamers may be less effective in those cases
• The 5FU-EGFR aptamers provide a blueprint for targeted cancer therapy that can overcome resistance against conventional chemotherapy and reduce adverse effects

  
• The 5FU-EGFR aptamers have the potential to become the chemotherapeutic backbone for EGFR-expressing gastrointestinal cancers currently treated with conventional 5FU as the standard of care, and provide a platform for precision medicine
• In vivo tumor models

  1. Xenogeneic orthotopic

  2. Syngeneic orthotopic

  3. KPC

  
• Treatments used in in vivo tumor models

  • Control (saline)
  • 5FU
  • 5FU equivalent to 5FU-EGFR aptamers
  • EGFR aptamers
  • 5FU-EGFR aptamers
  • 5FU-EGFR-20dFC aptamers

  
• 5FU-EGFR aptamers


  Carry 5FU at a molar ratio of 1:11, bind to EGFR, and may interfere with EGFR downstream signaling
• In the presence of EGFR-specific aptamers


  EGF-mediated phosphorylation of EGFR and extracellular signal–regulated kinase 1/2 was strongly attenuated

  
• The extracellular domains of human and mouse EGFR share 96.8% of protein sequence homology
• Aptamer binding to human EGFR


  • Modeled close to the EGF-interacting residues and should sterically interfere with EGFR
  • Binding to mouse EGFR was found to be further away from EGF-interacting residues

  
• 5FU-EGFR aptamers


  Potently inhibited the proliferation of mouse DT6606PDA and human PaTu-8988T cells, with GI50 values of <1 nmol/L
• Free formulations of 5FU

  Exhibited GI50 values of >100 nmol/L for DT6606PDA cells or >1000 nmol/L for PaTu-8988T cells
• Proliferation of the human breast cancer cell line MCF-7 with very low EGFR expression was only marginally affected upon treatment with 5FU-EGFR aptamers

  
• 5FU-EGFR aptamers


  Inhibited clonogenicity significantly more strongly than a 10-fold concentration of free 5FU
• 5FU-EGFR aptamer treatment


  Resulted in a significant accumulation of cells in the G1 phase

  
• Blocking Ab pretreatment


  Largely restored PDAC cell proliferation in the presence of aptamers
• EGFR knockout in DT6606PDA and PaTu-8988T cells

  Significantly attenuated the inhibitory effect of 5FU-EGFR aptamers on proliferation

  
• Uptake of 5FU-EGFR aptamers

  1. Clathrin-dependent endocytosis

  2. Rapid colocalization with lysosomes

  3. Significant accumulation in the cytosol

  
• Lysosomal stabilization by chloroquine pretreatment

  Showed significant reversal of reduction in proliferation on 5FU-EGFR aptamers treatment
• Aptamer uptake by lysosomes


  1. Observed in several human and murine PDAC cell lines within 10-30 minutes

  2. Inhibited by blocking Abs
• Significant accumulation of signal in the cytosol was observed as well
• Lysosomal stabilization by chloroquine pretreatment

  1. Reversal of reduction in proliferation on 5FU-EGFR aptamers treatment

  2. Increase in colocalization of Cy3-conjugated 5FU-EGFR aptamers with lysotracker within 30 minutes
• The monomeric constituents of 5FU-EGFR aptamers are released into the cytosol, where 5FUMP (5-fluorouracil monophosphate) can be phosphorylated to exert its inhibitory function on thymidylate synthase
• Tumor cell resistance to 5FU is mainly driven by alterations in drug influx/efflux, enforced drug inactivation, and mutations in drug importers

  
• Uptake of 5FU-EGFR aptamers by 5FU-resistant cells was comparable to the parental cells
• 5FU-EGFR aptamers


  Significantly inhibited cellular proliferation of 5FU-resistant cells
• 5FU-EGFR aptamers in 5FU-resistant cell-lines had 3 log phases lower GI50 than the free 5FU
• 5FU-EGFR aptamers exert pharmacologic efficacy that clearly exceeds that of equimolar free 5FU and can avert 5FU resistance
• In vivo evaluation of 5FU-EGFR aptamers


  1. Intravenous injection twice per week for 4 weeks

  2. Weekly CT scans
• Animals treated with free 5FU


  Showed significantly reduced tumor burden and delayed tumor outgrowth
• Animals treated with free 5FU


  Showed relevant treatment-associated adverse effects, including strongly compromised body conditioning score and significantly decreased body weight
• 5FU-EGFR aptamers, despite being equipotent, did not induce any obvious treatment-associated adverse effects
• In vivo evaluation of 5FU-EGFR aptamers in syngeneic mouse PDAC model

  1. Intravenous injection twice per week for 4 weeks

  2. Biweekly follow-up MRI scans

  
• Animals receiving 5FU-EGFR aptamers and 5FU-EGFR-20dFC aptamers


  Showed significantly delayed tumor growth

  
• The tumor growth delay mediated by 5FU-EGFR and 5FU-EGFR-20dFC aptamers persisted over a period of 4 weeks after treatment was interrupted