Six-week-old male CB17 SCID mice (Janvier Labs) were subcutaneously injected with PANC-1 alone (2 × 106 cells) or co-injected with hPSCs (2 × 106 cells). In another study, animals were co-injected with PANC-1 and hPSCs (2 × 106 cells) either stably transfected with sh-ITGA5 or sh-NC. The tumor growth was followed by tumor measurement using Vernier Caliper every 2 to 3 days. In a preliminary study with AV3 peptidomimetics, animals were subcutaneously co-injected with PANC-1 (2 × 106 cells) and hPSCs (4 × 106 cells). Six tumor-bearing mice per treatment group were taken and injected with either vehicle, AV3, or sAV3. Half of the group was injected intraperitoneally (20 mg/kg) and the other three intratumorally (4 mg/kg). Injections were given twice a week starting from day 9. In the next study, animals were subcutaneously co-injected either with PANC-1/MIA PaCa-2 (2 × 106 cells) or with hPSCs (4 × 106 cells). Four tumor-bearing mice per treatment group per model were taken into consideration and each group was injected with vehicle, AV3, gemcitabine, or combined treatment (AV3 + gemcitabine). After two tumor measurements, AV3 (20 mg/kg) was injected intraperitoneally thrice a week only in AV3 treatment groups. Later in all groups, injections were given twice a week. To study the effect of AV3 on the tumor perfusion, ICG (5 mg/kg) was injected via tail vein. After 24 hours of the injection, tumors were imaged using a small animal imager (Pearl Imager, LICOR, Lincoln, NE).

A freshly excised pancreatic patient tumor piece was grafted subcutaneously into the flank of immunocompromised NOD. Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice with Matrigel as P1. After transplantation, tumor growth was monitored, and upon reaching a size of 800 to 1000 mm3, PDX tumors were harvested and transplanted into the flank of NOD/SCID animals with Matrigel as P2. After that, mice with tumors reaching around 150 mm3 were injected intraperitoneally with either vehicle, AV3 (20 mg/kg), gemcitabine (50 mg/kg), or AV3 (20 mg/kg) and gemcitabine (50 mg/kg) twice a week for 3 weeks (n = 5 mice per group).

Tumor growth was assessed with caliper every 2 to 3 days. Tumor volumes were measured using the following formula: V = L × B2/2. At the end of the experiments, animals were sacrificed under anesthesia, after which tumors were harvested and immediately snap-frozen in cold 2-methyl butane. Frozen organs were stored at −80°C until analysis. Cryosections (4 μm) were cut and fixed in acetone for 10 min before staining for collagen I and α-SMA (table S2), followed by a fluorescent secondary antibody, and the protocol was described elsewhere (37). The morphometric analysis was performed by a single observer in a blinded manner using standardized computer program with unbiased fixed settings. Whole-stained sections were scanned using a slide scanner (NanoZoomer) and analyzed. To specifically detect the stained area, the intensity was set in different sections and all samples were analyzed. These settings were kept constant during the analysis, and then data were divided into different groups.

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