Background The tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) plays a pivotal role in cancer progression and treatment resistance. Comprised of various stromal components, notably cancer-associated fibroblasts (CAFs) and immune cells such as tumor associated macrophages (TAMs), the PDAC TME fosters a suppressive milieu that hinders the efficacy of immunotherapy. This study examines the spatial distribution and interactions of TME constituents in PDAC and liver metastatic PDAC, offering insights for myeloid-targeted cancer therapies.

Methods Multiplex immunofluorescent staining was performed on human primary (n=10) and metastatic liver (n=9) PDAC formalin-fixed paraffin-embedded tissue sections with a 6-color panel including CD68, αSMA, CD3, CD19, CK19 and IL6 antibodies. Whole tissue images were acquired using the Vectra Polaris multispectral imaging system. Cell phenotypes were characterized using QuPath software, followed by spatial computational analysis using SPIAT, SpatialExperiment and spaSim software. By assessing the spatial localization of TAMs (CD68+), CAFs (αSMA+), T cells (CD3+), B cells (CD19+), and cancer cells (CK19+), we mapped tumor structures and the proximity and potential interactions of characterized cells within the TME.

Results Differential infiltration patterns emerged between primary and metastatic PDAC tumors. In metastatic PDAC tissues, TAMs were found to localize closest to cancer cells (average median distance 56 μm ± 21 μm (met) vs 105 μm ± 60 μm (pri)) and CAFs (average median distance 61 μm ± 32 μm (met) vs 120 μm ± 75 μm (pri), a pattern that was distinct from their localization in primary tumor tissue. Metastatic tumors exhibited increased infiltration of CD68+ and CD68+ IL6+ TAMs within tumor structures (7.6% ± 3.7% (met) vs 0.08% ± 0.04 (pri)), but had reduced numbers of CAFs (0.4% ± 0.2 (met) vs 0.9% ± 0.6% (pri)) and T cells (1.5% ± 1.4 (met) vs 9.9% ± 4.3% (pri)) compared to primary tumors.

Conclusions The spatial dynamics between TAMs, CAFs, and T cells within the TME suggest possible intricate intercellular communication, with TAMs displaying variable spatial associations with immune cells in metastatic as opposed to primary tumors. These results indicate a complex role for TAMs in immune modulation between primary and metastatic sites. Moreover, the distinct relationships between TAMs-CAFs and CAFs-lymphocytes lay the groundwork for future studies to dissect the identities of TAMs and CAFs through the use of single cell spatial transcriptomics, setting the stage for more precise and effective treatments for formidable cancers like PDAC.