Tumor-penetrating therapy for β5 integrin-rich pancreas cancer

Pancreatic ductal adenocarcinoma (PDAC) is characterized by marked desmoplasia and drug resistance due, in part, to poor drug delivery to extravascular tumor tissue. Here, we report that carcinoma-associated fibroblasts (CAFs) induce β5 integrin expression in tumor cells in a TGF-β dependent manner, making them an efficient drug delivery target for the tumor-penetrating peptide iRGD. The capacity of iRGD to deliver conjugated and co-injected payloads is markedly suppressed when β5 integrins are knocked out in the tumor cells. Of note, β5 integrin knock-out in tumor cells leads to reduced disease burden and prolonged survival of the mice, demonstrating its contribution to PDAC progression. iRGD significantly potentiates co-injected chemotherapy in KPC mice with high β5 integrin expression and may be a powerful strategy to target an aggressive PDAC subpopulation.


Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Sample sizes for treatment study in Fig 6 were assessed using power calculation at p<0.05, power >0.80 for our initial estimates, but since we had no real preliminary data, the effect size was a bit below projected and so we increased the total N after a planned interim analysis. For the rest of the experiments 3 independent experiments were initially performed, if there was a strong trend and the p value was close to statistical significance 1-2 more experiments were performed.
One wild type mouse was excluded from the data on radiant efficiency and area of mCherry signals in Fig 5B due to the lack of mCherry signal noted during IVIS Xenogen analysis despite the presence of an mCherry positive tumor confirmed upon necropsy. The lack of mCherry signals during in vivo imaging was likely caused by internal organs overlying the tumor that interrupted with the signals.
All experiments were confirmed to be reproducible through at least 3 biological replicates.
Mice used for the study were randomly allocated into the different treatment groups. All groups had a similar average tumor size before treatment. For the rest of the experiments such as multiwell tissue culture or QPCR, the location in the plate of a given group was changed between different experiments to make sure location had no effect on the results.
-rabbit monoclonal anti-Ki67 (Clone SP6) was validated by the manufacturer in a section of formalin-fixed paraffin-embedded mouse spleen tissue. The section was pretreated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0). We independently validated it in our mouse tumor paraffin sections.
-rabbit IgG and mouse IgG1 Isotype controls were previoulay validated by us as Isotype controls for flow cytometry and functional assays (see ref 24). hBCF6008 and hBCF6011 CAFs were purchased from Asterand (Detroit, MI) mPCFAA0779 CAFs were established from mice implanted with fresh primary human PDAC tissue. hPC1356, hPCF1424, hPCF1299, and hPCF1444 CAFs were established from fresh surgical specimens of human PDAC tissue. Human cell lines were authenticated by the DNA Analysis Core Facility at the Sanford-Burnham-Prebys Medical Discovery Institute (La Jolla, CA) and by Genetica Cell Line Testing (Burlington, NC), and mouse cell lines were authenticated by American Type Culture Collection (Manassas, VA). The method used was STR profiling.

All cell lines tested negative for mycoplasma contamination
No commonly, misidentified cell lines were used in the study Mice. strains C57Bl6/129 F1 hybrids, Athymic Nude mice, sex males and females, age 6-10 weeks. Mice are kept at a maximum of 5 mice per cage, they have 12h light and dark cycles and are kept at 67 degrees Fahrenheit and 50% humidity.
No wild animals were used in this study