Celld imaging 3

Paraffin-embedded tissue sections of Panc1 tumors were stained with hematoxylin and eosin (H&E) or Ki67 (DAKO, Carpinteria, CA, USA), a molecule that highlights proliferating cells. Immunohistochemical reactions were performed in a DAKO Autostainer Link48 following the manufacturer’s instructions. All slides were viewed using an Olympus BX15 microscope. Images were acquired using an Olympus DP72 digital camera and processed with Olympus Cell D Imaging 3.3 software (Olympus Corporation, Tokyo, Japan); the final resolution was 1.3 microns/pixel.
Percentage intratumoral necrosis was determined by image processing using verified in-house software^{8 (link)}. To characterize the spatial distribution of necrosis inside the tumors, the fractal dimension (d_f) was calculated using the box counting method, using the same in-house software.
The proliferation rate - determined as the percentage of Ki67-positive cells (brown nuclear staining) over the total number of tumor cells - was calculated for three tumor regions: an external region accounting for 20% of the total cross sectional area, an intermediate region accounting for 30%, and an inner region accounting for 50%^{8 (link)}.

CXCR4 surface expression in AML cell lines was determined by fluorescence-activated cell sorting (FACS) analysis as previously described [28 (link)].
Additionally, IHC staining of CXCR4 expression was performed in paraffin-embedded cell blocks of OCI-AML-3, MONO-MAC-6, and HEL cell lines, using an anti-human CXCR4 antibody (1:200, ab124824, Abcam, Cambridge, UK) in an Autostainer Link 48 (Agilent Technologies, Santa Clara, CA, USA), following the manufacturer’s instructions. Representative images were taken using an Olympus DP73 digital camera (Olympus, Tokyo, Japan) at an original magnification of ×400 and processed with Olympus CellD Imaging 3.3 software.