SPP1 Positive Macrophage Correlation with Stage III-IV Colon Cancer Pathology Parameters

Colorectal cancer (CRC) is a frequent gastrointestinal malignancy with high rates of morbidity and mortality. Our previous studies have shown macrophages exhibited significant variations in stage III-IV colon cancer compared to other immune cells. Current study reviewed 11 stage III-IV CRC patient archived FFPE samples carried varied pathology and treatment conditions to investigate macrophage cell features. IHC control, multiplex immunofluorescence (mIF) panel control and the followed mIF staining were conducted using PN 7-Plex Detection Kit (PhenoVision Bio Co., Itd) targeted CD68, CD163, HLA-DR, panCK, SPP1 and PD-L1. The QC procedures made sure each marker from mIF results exhibited identical location and density of the same marker from IHC staining. The 11 staining slides were scanned, tumor areas were lined out by pathologist based on H&E staining from the serial section of each sample. Non-tumor region, tumor parenchyma and tumor stromal region were analyzed using PhenoVision mIF AI analysis system trained from Oncotopix Discovery system (Visiopharm). Positive cell percentage were analyzed in 11 samples. Statistical analysis exhibited correlation of CD68+/SPP1+ co-positive cell to the pathology parameter of PCR/non-PCR (p=0.05), dMMR/pMMR (p=0.024), with or without mucin pool (p=0.022) in tumor stromal region based on binary logistic regression. PD-L1 correlated to dMMR/pMMR in tumor parenchyma region. The co-positive marker did not show correlation to treatment (p=0.069) and necrosis (p=0.313) groups. There was lower CD68+/SPP1+ cell percentage in tumor stromal region in PCR vs. non-PCR group (0.46% vs. 2.01%, p=0.049) and non-cellular mucin pool group vs. with cellular mucin pool group (0.47% vs. 2.39%, p=0.022). Interestingly, there were more CD68+/SPP1+ cells in pMMR vs. dMMR group (1.94% vs. 0.24%, p=0.027). CD68+/SPP1+ co-positive cell distribution did not exhibit variations among necrosis groups (with vs. without necrosis, p=0.254), different treatment strategies (Chemoradiotherapy + Immunotherapy vs. Immunotherapy, p=0.066) and pathology stages (III vs. IV, p=0.460) in tumor, stromal, and normal region. Besides CD68+/SPP1+ co-positive marker in tumor stromal region, PD-L1 positive cell percentages were higher in tumor parenchyma region of dMMR patient samples compared to pMMR samples (10.09% vs. 1.04%, p=0.024). Current study indicates that CD68+/SPP1+ cell spatial feature correlates to PCR stage, MMR stage and mucin pool conditions of stage III-IV colon cancers. Increased sample number may lead to correlation of the marker to treatment variation which suggests the marker distribution characteristics may be utilized for stage III to IV CRCs patients' health monitoring plans. Citation Format: Shaojun Xu, Hongzhe Sun, Shuo Han, Lin Zhu, Yan Wu, Qisong Zhang, Aiwen Wu, Zhifu Zhang, Enkai Zhang, Na Li, Zhongwu Li. SPP1 positive macrophage spatial feature correlation to stage III-IV colon cancer multiple pathology parameters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7439.