Journal: bioRxiv
Article Title: CMTM6-Silencing Microbial Immunotherapy Reprograms PDAC Tumors and Restores T-cell Function
doi: 10.64898/2026.01.26.701790
Figure Lengend Snippet: ( a ) Schematic indicating treatment and imaging schedules. ( b ) Representative in vivo fluorescence imaging images showing the biodistribution of iRFP-labelled CRC2631 (CRC2631-iRFP) in C57BL/6 (BL6) tumor-free controls or in orthotopic mouse PDAC model (B6Panc02H7) one day after intravenous administration of 2.5 x 10 7 CFU and scanned. CRC2631-iRFP shows robust selective in B6Panc02H7 tumor-bearing mice compared to tumor-free controls. ( c ) Quantitative biodistribution confirms preferential accumulation of CRC2631 in primary tumors and visceral metastases ∼9 days after treatment. Collected tissues were weighed, homogenized, and dilutions were plated on chloramphenicol growth plates to selectively isolate CRC2631-iRFP counts per gram of tissue. CRC2631-iRFP counts/gr were ∼100-fold higher in tumor tissues than in the liver, a non-target clearing organs (**= p<0.0077 ). CRC2631-iRFP was undetectable in blood samples, suggesting low risk for CRC2631 translocation. ( d-g ) Representative fluorescence microscopy images of pancreases harvested from tumor-free C57BL/6J (BL6) controls or Panc02H7 mice ∼9 days after a single intravenous bolus of CRC2631-iRFP ( N= 15/group). Pancreases were cross sectioned, counterstained with DAPI to detect nuclei, and examined under a fluorescent microscope. CRC2631-RFP signal (red) was enriched in tissues derived from in B6Panc02H7 orthotopic pancreatic tumor tissue compared to tissues from tumor-free controls. ( h-k ) CRC2631-iRFP biodistribution in the genetic PDAC mouse model KPC (Kras G12D/+ ; Trp53 R172H/+ ; Pdx-1-Cre). C57BL/6J or MRI-verified tumor-positive KPC mice were treated intraperitoneally with a single or repeated (full) doses of CRC2631-iRFP (5 x 10 7 CFU) as shown in ( a ). The indicated organs were harvested and imaged 1day post the first or the 4 th bolus corresponding to single or full dose schedule ( h, i or j, k ). CRC2631-iRFP selectively and persistently home to primary pancreatic tumors after both single or repeated (4x) dosing regimens. Tumor-free controls ( k ) show negligible signal. ( l ) Normal pre-cancerous (HPNE) versus cancerous pancreatic (PANC-1/PDAC) cells were treated with CRC2631-iRFP using a 4:1 bacterial to human cell ratio or multiplicity of infection/MOI. MTT assay shows CRC2631-iRFP preferentially kills PANC-1 cells.
Article Snippet: Isolated colonies of bacteria were grown from -80°C stock aliquots frozen in 25% glycerol (Fisher, catalog# BP229-1, MA, United States) on solid or liquid LB media (Fisher/Research Products International, catalog# M342-500, MA, United States) supplemented with 200 μg/mL thymine (Arcos Pharmaceuticals) and antibiotics: 50 μg/mL kanamycin (Sigma, catalog# 60615, MO, United States), 50 μg/mL ampicillin (Sigma, catalog# A9393) or 20 μg/mL chloramphenicol (Gold Biotechnology, catalog# C-105-5, MO, United States), as required by the strain.
Techniques: Imaging, In Vivo, Fluorescence, Translocation Assay, Microscopy, Derivative Assay, Infection, MTT Assay
