Purpose: We describe the fibrotic rim formed in the desmoplastic histopathologic growth pattern (DHGP) of colorectal cancer liver metastasis (CLM) using in situ sequencing (ISS). The origin of the desmoplastic rim is still a matter of debate, and the detailed cellular organization has not yet been fully elucidated. Understanding the biology of the DHGP in CLM can lead to targeted treatment and improve survival. Experimental Design: We used ISS, targeting 150 genes, to characterize the desmoplastic rim by unsupervised clustering of gene coexpression patterns. The cohort comprised 10 chemo-naïve liver metastasis resection samples with a DHGP. Results: Unsupervised clustering of spatially mapped genes revealed molecular and cellular diversity within the desmoplastic rim. We confirmed the presence of the ductular reaction and cancer-associated fibroblasts. Importantly, we discovered angiogenesis and outer and inner zonation in the rim, characterized by nerve growth factor receptor and periostin expression. Conclusions: ISS enabled the analysis of the cellular organization of the fibrous rim surrounding CLM with a DHGP and suggests a transition from the outer part of the rim, with nonspecific liver injury response, into the inner part, with gene expression indicating collagen synthesis and extracellular matrix remodeling influenced by the interaction with cancer cells, creating a cancer cell–supportive environment. Moreover, we found angiogenic processes in the rim. Our results provide a potential explanation of the origin of the rim in DHGP and lead to exploring novel targeted treatments for patients with CLM to improve survival.

Background: Histopathological growth patterns (HGPs) have emerged as prognostic and predictive biomarkers in colorectal liver metastases (CLM). The desmoplastic/encapsulating HGP (EHGP) is associated with improved survival after surgery, whereas the replacement HGP (RHGP) is associated with poorer outcomes. However, HGPs can only be reliably assessed postoperatively, limiting their use as preoperative biomarkers. A deeper molecular understanding of HGPs could inform biomarker development and therapeutic strategies, including approaches to promote EHGP.

Methods: We applied in situ sequencing (ISS), an image-based spatial transcriptomics method, to map RNA expression in CLM tissue. Two custom gene panels (150 and 175 genes) were analysed in a chemonaïve cohort of resected CLM tissue from 19 patients with EHGP and RHGP.

Results: Distinct molecular programmes characterised the two patterns. RHGP was associated with damaged SAA1⁺ hepatocytes, KRT18⁺ neoplastic cells, and bifunctional hepatocyte-cholangiocyte cells. EHGP showed interferon-γ signalling, cytotoxic T-cell infiltration, and a fibrotic capsule with zonation-specific features: hepatic stellate cell activation, angiogenesis, and immune recruitment on the liver-facing side, and cancer-associated fibroblasts on the tumour-facing side.

Conclusions: These differences imply that growth patterns emerge from tumour-host driven interactions. They reveal new molecular features of the metastatic niche. These insights may inform the search for novel treatment strategies.

Circulating tumor DNA (ctDNA) is central to liquid biopsy-based cancer detection, yet the biological basis of its highly variable release into the bloodstream remains poorly understood. To define the tissue-level determinants of ctDNA shedding in colorectal cancer (CRC), we integrated tumor-informed plasma sequencing in a cohort of 59 patients in resectable stage IIII disease with detailed histopathology and immunophenotyping (n=49), spatial transcriptomics (n=4), and in situ mutation detection (n=6). ctDNA detectability increased with tumor burden, and ctDNA shedders exhibited a distinct architectural and microenvironmental phenotype characterized by expanded pseudolumina containing necrotic debris, frequent epithelial barrier disruption, and dense myeloid infiltration in lumina and stroma. Spatial profiling revealed stress-associated malignant programs and a myeloid-rich immune-luminal niche. In situ mutation detection localized plasma-identified mutations to necrotic material within pseudoluminal structures, establishing these compartments as potential focal reservoirs of shed DNA. These findings provide a mechanistic framework linking tissue architecture, immune remodelling, and spatially organized cell death to ctDNA release with implications for refining liquid biopsy applications. 

The neighborhood enrichment test is used to quantify spatial enrichment and depletion between spatial points withcategorical labels, which is a common data type in spatialomics. Traditionally, this test relies on a permutation-basedMonte Carlo approach, which tends to be computationallyexpensive for large datasets. In this study, we present amodified version of the test that can be computed analytically. This analytical version showed a minimum Pearson correlation of 0.95 with the conventional Monte Carlobased method across eight spatial omics datasets, but withsubstantial speed-ups. Additional experiments on a largeXenium dataset demonstrated the method’s ability to efficiently analyze large-scale data, making it a valuable toolfor analyzing spatial omics data.